OLIGONUCLEOTIDE COMPOSITIONS AND METHODS OF USE THEREOF

BACKGROUND

Oligonucleotides are useful in therapeutic, diagnostic, research and nanomaterials applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) for therapeutics can be limited, for example, because of their instability against extra- and intracellular nucleases and/or their poor cell penetration and distribution. There is a need for new and improved oligonucleotides and oligonucleotide compositions, such as, e.g., new oligonucleotides and oligonucleotide compositions capable of modulating exon skipping of Dystrophin for treatment of muscular dystrophy.

SUMMARY

Among other things, the present disclosure encompasses the recognition that structural elements of oligonucleotides, such as base sequence, chemical modifications (e.g., modifications of sugar, base, and/or internucleotidic linkages, and patterns thereof), and/or stereochemistry (e.g., stereochemistry of backbone chiral centers (chiral internucleotidic linkages), and/or patterns thereof), can have significant impact on oligonucleotide properties, e.g., activities, toxicities, e.g., as may be mediated by protein binding characteristics, stability, splicing-altering capabilities, etc. In some embodiments, the present disclosure demonstrates that oligonucleotide compositions comprising oligonucleotides with controlled structural elements, e.g., controlled chemical modification and/or controlled backbone stereochemistry patterns, provide unexpected properties, including but not limited to certain activities, toxicities, etc. In some embodiments, the present disclosure demonstrates that oligonucleotide properties, e.g., activities, toxicities, etc., can be modulated by chemical modifications (e.g., modifications of sugars, bases, internucleotidic linkages, etc.), chiral structures (e.g., stereochemistry of chiral internucleotidic linkages and patterns thereof, etc.), and/or combinations thereof.

In some embodiments, the present disclosure provides an oligonucleotide or an oligonucleotide composition. In some embodiments, an oligonucleotide or an oligonucleotide composition is a DMD oligonucleotide or a DMD oligonucleotide composition. In some embodiments, a DMD oligonucleotide or a DMD oligonucleotide composition is an oligonucleotide or an oligonucleotide composition capable of modulating skipping of one or more exons of the target gene Dystrophin (DMD). In some embodiments, a DMD oligonucleotide or a DMD oligonucleotide composition is useful for treatment of muscular dystrophy. In some embodiments, an oligonucleotide or oligonucleotide composition is an oligonucleotide or oligonucleotide composition which comprises a non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide or oligonucleotide composition which comprises a non-negatively charged internucleotidic linkage is capable of modulating the expression, level and/or activity of a gene target or a gene product thereof, including but not limited to, increasing or decreasing the expression, level and/or activity of a gene target or gene product thereof via any mechanism, including but not limited to: an RNase H-dependent mechanism, steric hindrance, RNA interference, modulation of skipping of one or more exon, etc. In some embodiments, the present disclosure pertains to an oligonucleotide or oligonucleotide composition which comprises a non-negatively charged internucleotidic linkage, in combination with any other structure or chemical moiety described herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide or DMD oligonucleotide composition which comprises a non-negatively charged internucleotidic linkage.

In some embodiments, the present disclosure provides technologies related to an oligonucleotide or an oligonucleotide composition for reducing levels of a transcript and/or a protein encoded thereby. In some embodiments, as demonstrated by example data described herein, provided technologies are particularly useful for reducing levels of mRNA and/or proteins encoded thereby.

In some embodiments, the present disclosure provides technologies, e.g., oligonucleotides, compositions and methods, etc., for altering gene expression, levels and/or splicing of transcripts. In some embodiments, a transcript is Dystrophin (DMD). Splicing of a transcript, such as pre-mRNA, is an essential step for the transcript to perform its biological functions in many higher eukaryotes. In some embodiments, the present disclosure recognizes that targeting splicing, especially through compositions comprising oligonucleotides having base sequences and/or chemical modifications and/or stereochemistry patterns (and/or patterns thereof) described in this disclosure, can effectively correct disease-associated mutations and/or aberrant splicing, and/or introduce and/or enhance beneficial splicing that lead to desired products, e.g., mRNA, proteins, etc. which can repair, restore, or add new desired biological functions. e.g., one or more functions of Dystrophin.

In some embodiments, the present disclosure provides compositions and methods for altering splicing of DMD transcripts, wherein altered splicing deletes or compensates for an exon(s) comprising a disease-associated mutation.

For example, in some embodiments, a Dystrophin gene can comprise an exon comprising one or more mutations associated with a disease, e.g., muscular dystrophy (including but not limited to Duchenne (Duchenne's) muscular dystrophy (DMD) and Becker (Becker's) muscular dystrophy (BMD)). In some embodiments, a disease-associated exon comprises a mutation (e.g., a missense mutation, a frameshift mutation, a nonsense mutation, a premature stop codon, etc.) in an exon. In some embodiments, the present disclosure provides compositions and methods for effectively skipping a disease-associated Dystrophin exon(s) and/or a different or an adjacent exon(s), while maintaining or restoring the reading frame so that a shorter (e.g., internally truncated) but partially functional dystrophin can be produced. A person having ordinary skill in the art appreciates that provided technologies (oligonucleotides, compositions, methods, etc.) can also be utilized for skipping of other exons, for example, those described in WO 2017/062862 and incorporated herein by reference, in accordance with the present disclosure to treat a disease and/or condition.

Among other things, the present disclosure demonstrates that chemical modifications and/or stereochemistry can be used to modulate transcript splicing by oligonucleotide compositions. In some embodiments, the present disclosure provides combinations of chemical modifications and stereochemistry to improve properties of oligonucleotides, e.g., their capabilities to alter splicing of transcripts. In some embodiments, the present disclosure provides chirally controlled oligonucleotide compositions that, when compared to a reference condition (e.g., absence of the composition, presence of a reference composition (e.g., a stereorandom composition of oligonucleotides having the same constitution (as understood by those skilled in the art, unless otherwise indicated constitution generally refers to the description of the identity and connectivity (and corresponding bond multiplicities) of the atoms in a molecular entity but omitting any distinction arising from their spatial arrangement), a different chirally controlled oligonucleotide composition, etc.), combinations thereof, etc.), provide altered splicing that can deliver one or more desired biological effects, for example, increase production of desired proteins, knockdown of a gene by producing mRNA with frameshift mutations and/or premature termination codons, knockdown of a gene expressing a mRNA with a frameshift mutation and/or premature termination codon, etc. In some embodiments, compared to a reference condition, provided chirally controlled oligonucleotide compositions are surprisingly effective. In some embodiments, desired biological effects (e.g., as measured by increased levels of desired mRNA, proteins, etc., decreased levels of undesired mRNA, proteins, etc.) can be enhanced by more than 5, 10, 15, 20, 25, 30, 40, 50, or 100 fold.

The present disclosure recognizes challenges of providing low toxicity oligonucleotide compositions and methods of use thereof. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced toxicity. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced immune responses. In some embodiments, the present disclosure recognizes that various toxicities induced by oligonucleotides are related to cytokine and/or complement activation. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced cytokine and/or complement activation. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced complement activation via the alternative pathway. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced complement activation via the classical pathway. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced drug-induced vascular injury. In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced injection site inflammation. In some embodiments, reduced toxicity can be evaluated through one or more assays widely known to and practiced by a person having ordinary skill in the art, e.g., evaluation of levels of complete activation product, protein binding, etc.

In some embodiments, the present disclosure provides oligonucleotides with enhanced antagonism of hTLR9 activity. In some embodiments, certain diseases, e.g., DMD, are associated with inflammation in, e.g., muscle tissues. In some embodiments, provided technologies (e.g., oligonucleotides, compositions, methods, etc.) provides both enhanced activities (e.g., exon-skipping activities) and hTLR9 antagonist activities which can be beneficial to one or more conditions and/or diseases associated with inflammation. In some embodiments, provided oligonucleotides and/or compositions thereof provides both exon-skipping capabilities and decreased levels of toxicity and/or inflammation. In some embodiments, the present disclosure provides an oligonucleotide which comprises one or more non-negatively charged internucleotidic linkages, wherein the oligonucleotide agonizes TLR9 activity less than another oligonucleotide which does not comprise a non-negatively charged internucleotidic linkage or which comprises fewer non-negatively charged internucleotidic linkages and which is otherwise identical. In some embodiments, the present disclosure provides an oligonucleotide which comprises one or more non-negatively charged internucleotidic linkages, wherein the oligonucleotide agonizes TLR9 activity less than an otherwise identical oligonucleotide which does not comprise a non-negatively charged internucleotidic linkage or which comprises fewer non-negatively charged internucleotidic linkages. In some embodiments, the present disclosure pertains to an oligonucleotide comprising at least one non-negatively charged internucleotidic linkage. In some embodiments, the non-negatively charged internucleotidic is selected from: n001, n002, n003 n004, n005, n006, n007 n008, n009, or n010, or a chirally controlled stereoisomer of n001 n002, n003, n004, n005, n006, n007, n008, n009, or n010. In some embodiments, the present disclosure pertains to an oligonucleotide which comprises at least two non-negatively charged internucleotidic linkages, wherein the linkages are different from each other. In some embodiments, the present disclosure pertains to an oligonucleotide comprising a CpG motif, wherein at least one internucleotidic linkage in the CpG (e.g., the p in CpG) or immediately upstream of the CpG (toward the 5′ end of the oligonucleotide) or immediately downstream of the CpG (toward the 3′ end of the oligonucleotide) is a non-negatively charged internucleotidic linkage. In some embodiments, TLR9 is a human TLR9. In some embodiments, TLR9 is a mouse TLR9.

In some embodiments, the present disclosure demonstrates that oligonucleotide properties, e.g., activities, toxicities, etc., can be modulated through chemical modifications. In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides which have a common base sequence, and comprise one or more modified internucleotidic linkages (or “non-natural internucleotidic linkages”, linkages that are not but can be utilized in place of a natural phosphate internucleotidic linkage (—OP(O)(OH)O—, which may exist as a salt form (—OP(O)(O⁻)O—) at a physiological pH) found in natural DNA and RNA), one or more modified sugar moieties, and/or one or more natural phosphate linkages. In some embodiments, provided oligonucleotides may comprise two or more types of modified internucleotidic linkages. In some embodiments, a provided oligonucleotide comprises a non-negatively charged internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a neutral internucleotidic linkage comprises a triazole, alkyne, or guanidine (e.g., cyclic guanidine) moiety. Such moieties are optionally substituted. In some embodiments, a provided oligonucleotide comprises a neutral internucleotidic linkage and another internucleotidic linkage which is not a neutral backbone. In some embodiments, a provided oligonucleotide comprises a neutral internucleotidic linkage and a phosphorothioate internucleotidic linkage. In some embodiments, provided oligonucleotide compositions comprising a plurality of oligonucleotides are chirally controlled and level of the plurality of oligonucleotides in the composition is controlled or pre-determined, and oligonucleotides of the plurality share a common stereochemistry configuration at one or more chiral internucleotidic linkages. For example, in some embodiments, oligonucleotides of a plurality share a common stereochemistry configuration at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more chiral internucleotidic linkages, each of which is independently Rp or Sp; in some embodiments, oligonucleotides of a plurality share a common stereochemistry configuration at each chiral internucleotidic linkages. In some embodiments, a chiral internucleotidic linkage where a controlled level of oligonucleotides of a composition share a common stereochemistry configuration (independently in the Rp or Sp configuration) is referred to as a chirally controlled internucleotidic linkage.

In some embodiments, a modified internucleotidic linkage is a non-negatively charged (neutral or cationic) internucleotidic linkage in that at a pH, (e.g., human physiological pH (7.4), pH of a delivery site (e.g., an organelle, cell, tissue, organ, organism, etc.), it largely (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90° %, etc.; in some embodiments, at least 30%; in some embodiments, at least 40%; in some embodiments, at least 50%; in some embodiments, at least 60%; in some embodiments, at least 70%; in some embodiments, at least 80%; in some embodiments, at least 90%; in some embodiments, at least 99%; etc.) exists as a neutral or cationic form (as compared to an anionic form (e.g., —O—P(O)(O⁻)—O— (the anionic form of natural phosphate linkage), —O—P(O)(S⁻)—O— (the anionic form of phosphorothioate linkage), etc.)), respectively. In some embodiments, a modified internucleotidic linkage is a neutral internucleotidic linkage in that at a pH, it largely exists as a neutral form. In some embodiments, a modified internucleotidic linkage is a cationic internucleotidic linkage in that at a pH, it largely exists as a cationic form. In some embodiments, a pH is human physiological pH (˜7.4). In some embodiments, a modified internucleotidic linkage is a neutral internucleotidic linkage in that at pH 7.4 in a water solution, at least 90% of the internucleotidic linkage exists as its neutral form. In some embodiments, a modified internucleotidic linkage is a neutral internucleotidic linkage in that in a water solution of the oligonucleotide, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the internucleotidic linkage exists in its neutral form. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 95%. In some embodiments, the percentage is at least 99%. In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, when in its neutral form has no moiety with a pKa that is less than 8, 9, 10, 11, 12, 13, or 14. In some embodiments, pKa of an internucleotidic linkage in the present disclosure can be represented by pKa of CH₃— the internucleotidic linkage-CH₃(i.e., replacing the two nucleoside units connected by the internucleotidic linkage with two —CH₃groups). Without wishing to be bound by any particular theory, in at least some cases, a neutral internucleotidic linkage in an oligonucleotide can provide improved properties and/or activities, e.g., improved delivery, improved resistance to exonucleases and endonucleases, improved cellular uptake, improved endosomal escape and/or improved nuclear uptake, etc., compared to a comparable nucleic acid which does not comprises a neutral internucleotidic linkage.

In some embodiments, a non-negatively charged internucleotidic linkage has the structure of e.g., of formula I-n-1, I-n-2, I-n-3, I-n-4, H, II-a-1, II-a-2, I-b-1, II-b-2, I-c-1, II-c-2, II-d-1, II-d-2, etc. In some embodiments, a non-negatively charged internucleotidic linkage comprises a triazole or alkyne moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises a guanidine moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises a cyclic guanidine moiety. In some embodiments, a modified internucleotidic linkage comprising a cyclic guanidine moiety has the structure of:

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In some embodiments, a neutral internucleotidic linkage comprising a cyclic guanidine moiety is chirally controlled. In some embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral internucleotidic linkage and at least one phosphorothioate internucleotidic linkage.

In some embodiments, a non-negatively charged internucleotidic linkage is n001, n002, n003, n004, n005, n006, n007, or n008. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled, e.g., n001R, n002R, n003R, n004R, n005R, n006R, n007R, n008R, n009R n001S, n002S, n003S, n004S, n005S, n006S, n007S, n008S, n009S, etc.

In some embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral internucleotidic linkage and at least one phosphorothioate internucleotidic linkage, wherein the phosphorothioate internucleotidic linkage is a chirally controlled internucleotidic linkage in the Sp configuration.

In some embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral internucleotidic linkage selected from a neutral internucleotidic linkage comprising an optionally substituted triazolyl group, a neutral internucleotidic linkage comprising an optionally substituted alkynyl group, and a neutral internucleotidic linkage comprising a moiety

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and at least one phosphorothioate internucleotidic linkage. In some embodiments, the present disclosure pertains to a composition comprising an oligonucleotide comprising at least one neutral internucleotidic linkage selected from a neutral internucleotidic linkage comprising an optionally substituted triazolyl group, a neutral internucleotidic linkage comprising an optionally substituted alkynyl group, and a neutral internucleotidic linkage comprising a Tmg group

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and at least one phosphorothioate internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one non-negatively charged internucleotidic linkage and at least one phosphorothioate internucleotidic linkage. In some embodiments, the non-negatively charged internucleotidic linkage is n001. In some embodiments, the non-negatively charged internucleotidic linkage and the phosphorothioate internucleotidic linkage are independently chirally controlled. In some embodiments, each of the non-negatively charged internucleotidic linkage and the phosphorothioate internucleotidic linkages are independently chirally controlled.

Various types of internucleotidic linkages differ in properties. Without wishing to be bound by any theory, the present disclosure notes that a natural phosphate linkage (phosphodiester internucleotidic linkage) is anionic and may be unstable when used by itself without other chemical modifications in vivo; a phosphorothioate internucleotidic linkage is anionic, generally more stable in vivo than a natural phosphate linkage, and generally more hydrophobic; a neutral internucleotidic linkage such as one exemplified in the present disclosure comprising a cyclic guanidine moiety is neutral at physiological pH, can be more stable in vivo than a natural phosphate linkage, and more hydrophobic.

In some embodiments, an internucleotidic linkage (e.g., a non-negatively charged internucleotidic linkage, a chirally controlled non-negatively charged internucleotidic linkage, etc.) is neutral at physiological pH, chirally controlled, stable in vivo, hydrophobic, and may increase endosomal escape.

In some embodiments, an oligonucleotide or oligonucleotide composition is: a DMD oligonucleotide or oligonucleotide composition; an oligonucleotide or oligonucleotide composition comprising a non-negatively charged internucleotidic linkage; or a DMD oligonucleotide comprising a non-negatively charged internucleotidic linkage.

In some embodiments, an oligonucleotide has, as non-limiting examples, a wing-core-wing, wing-core, core-wing, wing-wing-core-wing-wing, wing-wing-core-wing, or wing-core-wing-wing structure (in some embodiments, a wing-wing comprises or consists of a first wing and a second wing, wherein the first wing is different than the second wing, and the first and second wings are different than the core). A wing or core can be defined by any structural elements and/or patterns and/or combinations thereof. In some embodiments, a wing and core is defined by nucleoside modifications, sugar modifications, and/or internucleotidic linkages, wherein a wing comprises a nucleoside modification, sugar modification and/or internucleotidic linkage and/or pattern and/or combination thereof, that the core region does not have, or vice versa. In some embodiments, oligonucleotides of the present disclosure comprise or consist of a 5′-end region, a middle region, and a 3′-end region. In some embodiments, a 5′-end region is a 5′-wing region. In some embodiments, a 5′-wing region is a 5′-end region. In some embodiments, a 3′-end region is a 3′-wing region. In some embodiments, a 3′-wing region is a 3′-end region. In some embodiments, a core region is a middle region.

In some embodiments, each wing region (or each of the 5′-end and 3′-end regions) independently comprises one or more modified phosphate linkages and no natural phosphate linkages, and the core region (the middle region) comprises one or more modified internucleotidic linkages and one or more natural phosphate linkages. In some embodiments, each wing region (or each of the 5′-end and 3′-end regions) independently comprises one or more natural phosphate linkages and optionally one or more modified internucleotidic linkages, and the core (or the middle region) comprises one or more modified internucleotidic linkages and optionally one or more natural phosphate linkages. In some embodiments, a wing (or a 5′-end or 3′-end region) comprises modified sugar moieties. In some embodiments, a modified internucleotidic linkage is a phosphorothioate internucleotidic linkage.

Among other things, the present disclosure encompasses the recognition that stereorandom oligonucleotide preparations contain a plurality of distinct chemical entities that differ from one another, e.g., in the stereochemical structure of individual backbone chiral centers within the oligonucleotide chain. Without control of stereochemistry of backbone chiral centers, stereorandom oligonucleotide preparations provide uncontrolled (or stereorandom) compositions comprising undetermined levels of oligonucleotide stereoisomers. Even though these stereoisomers may have the same base sequence and/or chemical modifications, they are different chemical entities at least due to their different backbone stereochemistry, and they can have, as demonstrated herein, different properties, e.g., activities, toxicities, distribution etc. Among other things, the present disclosure provides chirally controlled compositions that are or contain particular stereoisomers of oligonucleotides of interest; in contrast to chirally uncontrolled compositions, chirally controlled compositions comprise controlled levels of particular stereoisomers of oligonucleotides. In some embodiments, a particular stereoisomer may be defined, for example, by its base sequence, its pattern of backbone linkages, its pattern of backbone chiral centers, and pattern of backbone phosphorus modifications, etc. As is understood in the art, in some embodiments, base sequence may refer solely to the sequence of bases and/or to the identity and/or modification status of nucleoside residues (e.g., of sugar and/or base components, relative to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) in an oligonucleotide and/or to the hybridization character (i.e., the ability to hybridize with particular complementary residues) of such residues. In some embodiments, the present disclosure demonstrates that property improvements (e.g., improved activities, lower toxicities, etc.) achieved through inclusion and/or location of particular chiral structures within an oligonucleotide can be comparable to, or even better than those achieved through use of chemical modifications, e.g., particular backbone linkages, residue modifications, etc. (e.g., through use of certain types of modified phosphates [e.g., phosphorothioate, substituted phosphorothioate, etc.], sugar modifications [e.g., 2′-modifications, etc.], and/or base modifications [e.g., methylation, etc.]). In some embodiments, the present disclosure demonstrates that chirally controlled oligonucleotide compositions of oligonucleotides comprising certain chemical modifications (e.g., 2′-F, 2′-OMe, phosphorothioate internucleotidic linkages, lipid conjugation, etc.) demonstrate unexpectedly high exon-skipping efficiency.

In some embodiments, provided oligonucleotides are blockmers. In some embodiments, a blockmer is an oligonucleotide comprising one or more blocks.

In some embodiments, a block is a portion of an oligonucleotide. In some embodiments, a block is a wing or a core. In some embodiments, a blockmer comprises one or more blocks. In some embodiments, a 5′-block is a 5′-end region or 5′-wing. In some embodiments, a 3′-block is a 3′-end region or 3′-wing.

In some embodiments, provided oligonucleotide are altmers. In some embodiments, provided oligonucleotides are altmers comprising alternating blocks. In some embodiments, a blockmer or an altmer can be defined by chemical modifications (including presence or absence), e.g., base modifications, sugar modification, internucleotidic linkage modifications, stereochemistry, etc.

In some embodiments, provided oligonucleotides comprise blocks comprising different internucleotidic linkages. In some embodiments, provided oligonucleotides comprise blocks comprising modified internucleotidic linkages and/or natural phosphate linkages.

In some embodiments, provided oligonucleotides comprise blocks comprising sugar modifications. In some embodiments, provided oligonucleotides comprise one or more blocks comprising one or more 2′-F modifications (2′-F blocks). In some embodiments, provided oligonucleotides comprise blocks comprising consecutive 2′-F modifications. In some embodiments, a block comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive 2′-F modifications.

In some embodiments, provided oligonucleotides comprises one or more blocks comprising one or more 2′-OR¹modifications (2′-OR¹blocks), wherein R¹is independently as defined and described herein and below. In some embodiments, provided oligonucleotides comprise both 2′-F and 2′-OR¹blocks. In some embodiments, provided oligonucleotides comprise alternating 2′-F and 2′-OR¹blocks. In some embodiments, provided oligonucleotides comprise a first 2′-F block at the 5′-end, and a second 2′-F block at the 3′-end, each of which independently comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive 2′-F modifications.

In some embodiments, provided oligonucleotides comprise a 5′-block wherein each sugar moiety of the 5′-block comprises a 2′-F modification. In some embodiments, provided oligonucleotides comprise a 3′-block wherein each sugar moiety of the 3′-block comprises a 2′-F modification. In some embodiments, such provided oligonucleotides comprise one or more 2′-OR¹blocks, and optionally one or more 2′-F blocks, between the 5′ and 3′ 2′-F blocks. In some embodiments, such provided oligonucleotides comprise one or more 2′-OR¹blocks, and one or more 2′-F blocks, between the 5′ and 3′ 2′-F blocks (e.g., WV-3047, WV-3048, etc.).

In some embodiments, a block is a stereochemistry block. In some embodiments, a block is an Rp block in that each internucleotidic linkage of the block is Rp. In some embodiments, a 5′-block is an Rp block. In some embodiments, a 3′-block is an Rp block. In some embodiments, a block is an Sp block in that each internucleotidic linkage of the block is Sp. In some embodiments, a 5′-block is an Sp block. In some embodiments, a 3′-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks.

In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each internucleotidic linkage in a natural phosphate linkage.

In some embodiments, a 5′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block comprises 4 or more nucleoside units.

In some embodiments, a 3′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block comprises 4 or more nucleoside units.

In some embodiments, provided oligonucleotides comprise alternating blocks comprising different modified sugar moieties and/or unmodified sugar moieties. In some embodiments, provided oligonucleotides comprise alternating blocks comprising different modified sugar moieties and unmodified sugar moieties. In some embodiments, provided oligonucleotides comprise alternating blocks comprising different modified sugar moieties. In some embodiments, provided oligonucleotides comprise alternating blocks comprising different modified sugar moieties, wherein the modified sugar moieties comprise different 2′-modifications. For example, in some embodiments, provided oligonucleotide comprises alternating blocks comprising 2′-OMe and 2′-F, respectively.

In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides which:

1) have a common base sequence complementary to a target sequence in a transcript; and

2) comprise one or more modified sugar moieties and modified internucleotidic linkages.

In some embodiments, a reference condition is absence of the composition. In some embodiments, a reference condition is presence of a reference composition. Example reference compositions comprising a reference plurality of oligonucleotides are extensively described in this disclosure. In some embodiments, oligonucleotides of the reference plurality have a different structural elements (chemical modifications, stereochemistry, etc.) compared with oligonucleotides of the plurality in a provided composition. In some embodiments, a reference composition is a stereorandom preparation of oligonucleotides having the same chemical modifications. In some embodiments, a reference composition is a mixture of stereoisomers while a provided composition is a chirally controlled oligonucleotide composition of one stereoisomer. In some embodiments, oligonucleotides of the reference plurality have the same base sequence, same sugar modifications, same base modifications, same internucleotidic linkage modifications, and/or same stereochemistry as oligonucleotide of the plurality in a provided composition but different chemical modifications, e.g., base modification, sugar modification, internucleotidic linkage modifications, etc.

Example splicing systems are widely known in the art. In some embodiments, a splicing system is an in vivo or in vitro system including components sufficient to achieve splicing of a relevant target transcript. In some embodiments, a splicing system is or comprises a spliceosome (e.g., protein and/or RNA components thereof). In some embodiments, a splicing system is or comprises an organellar membrane (e.g., a nuclear membrane) and/or an organelle (e.g., a nucleus). In some embodiments, a splicing system is or comprises a cell or population thereof. In some embodiments, a splicing system is or comprises a tissue. In some embodiments, a splicing system is or comprises an organism, e.g., an animal, e.g., a mammal such as a mouse, rat, monkey, dog, human, etc.

In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides which:

1) have a common base sequence complementary to a target sequence in a transcript; and

2) comprise one or more modified sugar moieties and modified internucleotidic linkages,

the oligonucleotide composition being characterized in that, when it is contacted with the transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

which composition is chirally controlled and it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence, for oligonucleotides of the particular oligonucleotide type,

In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

which composition is a substantially pure preparation of a single oligonucleotide in that at least about 10% of the oligonucleotides in the composition have the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers.

In some embodiments, each region (e.g., a block, wing, core, 5′-end, 3′-end, or middle region, etc.) of an oligonucleotide independently comprises 3, 4, 5, 6, 7, 8, 9, 10 or more bases. In some embodiments, each region independently comprises 3 or more bases. In some embodiments, each region independently comprises 4 or more bases. In some embodiments, each region independently comprises 5 or more bases. In some embodiments, each region independently comprises 6 or more bases. In some embodiments, each sugar moiety in a region is modified. In some embodiments, a modification is a 2′-modification. In some embodiments, each modification is a 2′-modification. In some embodiments, a modification is 2′-F. In some embodiments, each modification is 2′-F. In some embodiments, a modification is 2′-OR¹. In some embodiments, each modification is 2′-OR¹. In some embodiments, a modification is 2′-OR¹. In some embodiments, each modification is 2′-OMe. In some embodiments, each modification is 2′-OMe. In some embodiments, each modification is 2′-MOE. In some embodiments, each modification is 2′-MOE. In some embodiments, a modification is an LNA sugar modification. In some embodiments, each modification is an LNA sugar modification. In some embodiments, each internucleotidic linkage in a region is a chiral internucleotidic linkage. In some embodiments, each internucleotidic linkage in a wing, or 5′-end or 3′-end region, is an Sp chiral internucleotidic linkage. In some embodiments, a chiral internucleotidic linkage is a phosphorothioate linkage. In some embodiments, a core or middle region comprises one or more natural phosphate linkages and one or more modified internucleotidic linkages. In some embodiments, a core or middle region comprises one or more natural phosphate linkages and one or more chiral internucleotidic linkages. In some embodiments, a core region comprises one or more natural phosphate linkages and one or more Sp chiral internucleotidic linkages. In some embodiments, a core or middle region comprises one or more natural phosphate linkages and one or more Sp phosphorothioate linkages.

In some embodiments, a region (e.g., a block, wing, core, 5′-end, 3′-end, middle region, etc.) of an oligonucleotide comprises a non-negatively charged internucleotidic linkage, e.g., of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc. In some embodiments, a region comprises a neutral internucleotidic linkage. In some embodiments, a region comprises an internucleotidic linkage which comprises a triazole or alkyne moiety. In some embodiments, a region comprises an internucleotidic linkage which comprises a cyclic guanidine guanidine. In some embodiments, a region comprises an internucleotidic linkage which comprises a cyclic guanidine moiety. In some embodiments, a region comprises an internucleotidic linkage having the structure of

embedded image

In some embodiments, such internucleotidic linkages are chirally controlled.

In some embodiments, the base sequence of an oligonucleotide, e.g., the base sequence of a plurality of oligonucleotides of a particular oligonucleotide type, is or comprises a base sequence disclosed herein (e.g., a base sequence of an example oligonucleotide (e.g., those listed in the tables, examples, etc.), a target sequence, etc.) (or a portion thereof which is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases long). In some embodiments, a provided oligonucleotide has a base sequence comprising the base sequence of any example oligonucleotides or another base sequence disclosed herein, and a length of up to 30 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising the base sequence of any example oligonucleotides or another base sequence disclosed herein, and a length of up to 40 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising the base sequence of any example oligonucleotides or another base sequence disclosed herein, and a length of up to 50 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising at least 15 contiguous bases of the base sequence of an oligonucleotide example or another sequence disclosed herein, and a length of up to 30 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising at least 15 contiguous bases of the base sequence of an oligonucleotide example or another sequence disclosed herein, and a length of up to 40 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising at least 15 contiguous bases of the base sequence of an oligonucleotide example or another sequence disclosed herein, and a length of up to 50 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising a sequence having no more than 5 mismatches from the base sequence of an example oligonucleotide or another sequence disclosed herein, and a length of up to 30 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising a sequence having no more than 5 mismatches from the base sequence of an example oligonucleotide or another sequence disclosed herein, and a length of up to 40 bases. In some embodiments, a provided oligonucleotide has a base sequence comprising a sequence having no more than 5 mismatches from the base sequence of an example oligonucleotide or another sequence disclosed herein, and a length of up to 50 bases.

In some embodiments, the base sequence of a provided oligonucleotide is the base sequence of an example oligonucleotide or another sequence disclosed herein, and a pattern of backbone chiral centers comprises at least one chirally controlled center which is a Sp linkage phosphorus of a phosphorothioate linkage. In some embodiments, the base sequence of a provided oligonucleotide is the base sequence of an example oligonucleotide or another sequence disclosed herein, the oligonucleotide has a length of up to 30 bases, and a pattern of backbone chiral centers comprises at least one chirally controlled center which is a Sp linkage phosphorus of a phosphorothioate linkage. In some embodiments, the base sequence of a provided oligonucleotide is the base sequence of an example oligonucleotide or another sequence disclosed herein, the oligonucleotide has a length of up to 40 bases, and a pattern of backbone chiral centers comprises at least one chirally controlled center which is a Sp linkage phosphorus of a phosphorothioate linkage. In some embodiments, the base sequence of a provided oligonucleotide comprises at least 15 contiguous bases of any example oligonucleotides or another sequence disclosed herein, the oligonucleotide has a length of up to 30, 40, or 50 bases, and a pattern of backbone chiral centers comprises at least one chirally controlled center which is a Sp linkage phosphorus of a phosphorothioate linkage.

In some embodiments, a mismatch is a difference between the base sequence or length when two sequences are maximally aligned and compared. As a non-limiting example, a mismatch is counted if a difference exists between the base at a particular location in one sequence and the base at the corresponding position in another sequence. Thus, a mismatch is counted, for example, if a position in one sequence has a particular base (e.g., A), and the corresponding position on the other sequence has a different base (e.g., G, C or U). A mismatch is also counted, e.g., if a position in one sequence has a base (e.g., A), and the corresponding position on the other sequence has no base (e.g., that position is an abasic nucleotide which comprises a phosphate-sugar backbone but no base) or that position is skipped. A single-stranded nick in either sequence (or in the sense or antisense strand) may not be counted as mismatch, for example, no mismatch would be counted if one sequence comprises the sequence 5′-AG-3′, but the other sequence comprises the sequence 5′-AG-3′ with a single-stranded nick between the A and the G. A base modification is generally not considered a mismatch, for example, if one sequence comprises a C, and the other sequence comprises a modified C (e.g., with a 2′-modification) at the same position, no mismatch may be counted.

In some embodiments, oligonucleotides of a particular type are chemically identical in that they have the same base sequence (including length), the same pattern of chemical modifications to sugar and base moieties, the same pattern of backbone linkages (e.g., pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, non-negatively charged linkages, and combinations thereof), the same pattern of backbone chiral centers (e.g., pattern of stereochemistry (Rp/Sp) of chiral internucleotidic linkages), and the same pattern of backbone phosphorus modifications (e.g., pattern of modifications on the internucleotidic phosphorus atom, such as —S—, and -L-R¹of formula I).

In some embodiments, the present disclosure provides chirally controlled oligonucleotide compositions of oligonucleotides comprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) internucleotidic linkages, and particularly for oligonucleotides comprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) chiral internucleotidic linkages, wherein the oligonucleotides comprise at least one, and in some embodiments, more than 5, 6, 7, 8, 9, or 10 chirally controlled internucleotidic linkages. In some embodiments, in a chirally controlled composition of oligonucleotides each chiral internucleotidic linkage of the oligonucleotides is independently a chirally controlled internucleotidic linkage. In some embodiments, in a stereorandom or racemic composition of oligonucleotides, each chiral internucleotidic linkage is formed with less than 90:10, 95:5, 96:4, 97:3, or 98:2 diastereoselectivity. In some embodiments, in a stereoselective or chirally controlled composition of oligonucleotides, each chirally controlled internucleotidic linkage of the oligonucleotides independently has a diastereopurity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% at its chiral linkage phosphorus (either Rp or Sp). Among other things, the present disclosure provides technologies to prepare oligonucleotides of high diastereopurity. In some embodiments, diastereopurity of a chiral internucleotidic linkage in an oligonucleotide may be measured through a model reaction, e.g. formation of a dimer under essentially the same or comparable conditions wherein the dimer has the same internucleotidic linkage as the chiral internucleotidic linkage, the 5′-nucleoside of the dimer is the same as the nucleoside to the 5′-end of the chiral internucleotidic linkage, and the 3′-nucleoside of the dimer is the same as the nucleoside to the 3′-end of the chiral internucleotidic linkage.

As described herein, provided compositions and methods are capable of altering splicing of transcripts. In some embodiments, provided compositions and methods provide improved splicing patterns of transcripts compared to reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof. An improvement can be an improvement of any desired biological functions. In some embodiments, for example, in DMD, an improvement is production of an mRNA from which a dystrophin protein with improved biological activities is produced.

In some embodiments, the present disclosure provides a method for altering splicing of a target transcript, comprising administering a provided composition, wherein the splicing of the target transcript is altered relative to reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

In some embodiments, the present disclosure provides a method of generating a set of spliced products from a target transcript, the method comprising steps of:

contacting a splicing system containing the target transcript with an oligonucleotide composition comprising a plurality of oligonucleotides (e.g., a provided chirally controlled oligonucleotide composition), in an amount, for a time, and under conditions sufficient for a set of spliced products to be generated that is different from a set generated under reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

In some embodiments, the present disclosure provides a method for treating or preventing a disease, comprising administering to a subject an oligonucleotide composition described herein.

In some embodiments, the present disclosure provides a method for treating or preventing a disease, comprising administering to a subject an oligonucleotide composition comprising a plurality of oligonucleotides, which:

1) have a common base sequence complementary to a target sequence in a transcript; and

2) comprise one or more modified sugar moieties and modified internucleotidic linkages,

In some embodiments, the present disclosure provides a method for treating or preventing a disease, comprising administering to a subject a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers, and

4) pattern of backbone phosphorus modifications,

In some embodiments, a disease is one in which, after administering a provided composition, one or more spliced transcripts repair, restore or introduce a new beneficial function. For example, in DMD, after skipping one or more exons, functions of dystrophin can be restored, or partially restored, through a truncated but (at least partially) active version. In some embodiments, a disease is one in which, after administering a provided composition, one or more spliced transcripts repair, a gene is effectively knockdown by altering splicing of the gene transcript.

In some embodiments, a disease is muscular dystrophy, including but not limited to Duchenne (Duchenne's) muscular dystrophy (DMD) and Becker (Becker's) muscular dystrophy (BMD).

In some embodiments, a transcript is of Dystrophin gene or a variant thereof.

In some embodiments, the present disclosure provides a method of treating a disease by administering a composition comprising a plurality of oligonucleotides sharing a common base sequence comprising a nucleotide sequence, which nucleotide sequence is complementary to a target sequence in the target transcript,

the improvement that comprises using as the oligonucleotide composition a chirally controlled oligonucleotide composition characterized in that, when it is contacted with the transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

In some embodiments, a common sequence comprises a sequence (or at least 15 base long portion thereof) of any oligonucleotide in Table A1.

In some embodiments, the present disclosure provides a method of administering an oligonucleotide composition comprising a plurality of oligonucleotides having a common nucleotide sequence, the improvement that comprises:

administering an oligonucleotide composition comprising the plurality of oligonucleotides each of which independently comprises one or more negatively charged internucleotidic linkages and one or more non-negatively charged internucleotidic linkages, wherein the oligonucleotide composition is optionally chirally controlled.

administering an oligonucleotide composition comprising the plurality of oligonucleotides that is chirally controlled and that is characterized by reduced toxicity relative to a reference oligonucleotide composition of the same common nucleotide sequence.

administering an oligonucleotide composition in which each oligonucleotide in the plurality includes one or more natural phosphate linkages and one or more modified phosphate linkages;

wherein the oligonucleotide composition is characterized by reduced toxicity when tested in at least one assay that is observed with an otherwise comparable reference composition whose oligonucleotides do not comprise natural phosphate linkages.

In some embodiments, oligonucleotides can elicit proinflammatory responses. In some embodiments, the present disclosure provides compositions and methods for reducing inflammation. In some embodiments, the present disclosure provides compositions and methods for reducing proinflammatory responses. In some embodiments, the present disclosure provides methods for reducing injection site inflammation using provided compositions. In some embodiments, the present disclosure provides methods for reducing drug-induced vascular injury using provided compositions.

In some embodiments, the present disclosure provides a method, comprising administering a composition comprising a plurality of oligonucleotides of a common base sequence, which composition displays reduced injection site inflammation as compared with a reference composition comprising a plurality of oligonucleotides, each of which also has the common base sequence, but which differs structurally from the oligonucleotides of the plurality in that:

individual oligonucleotides within the reference plurality differ from one another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have a structure different from a structure represented by the plurality of oligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do not comprise a wing region and a core region.

In some embodiments, the present disclosure provides a method, comprising administering a composition comprising a plurality of oligonucleotides of a common base sequence, which composition displays altered protein binding as compared with a reference composition comprising a plurality of oligonucleotides, each of which also has the common base sequence but which differs structurally from the oligonucleotides of the plurality in that:

individual oligonucleotides within the reference plurality differ from one another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have a structure different from a structure represented by the plurality of oligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do not comprise a wing region and a core region.

administering an oligonucleotide composition comprising a plurality of oligonucleotides that is characterized by altered protein binding relative to a reference oligonucleotide composition of the same common nucleotide sequence.

In some embodiments, the present disclosure provides a method comprising administering a composition comprising a plurality of oligonucleotides of a common base sequence, which composition displays improved delivery as compared with a reference composition comprising a reference plurality of oligonucleotides, each of which also has the common base sequence but which differs structurally from the oligonucleotides of the plurality in that:

individual oligonucleotides within the reference plurality differ from one another in stereochemical structure; and/or

at least some oligonucleotides within the reference plurality have a structure different from a structure represented by the plurality of oligonucleotides of the composition; and/or

at least some oligonucleotides within the reference plurality do not comprise a wing region and a core region.

administering an oligonucleotide comprising a plurality of oligonucleotides that is characterized by improved delivery relative to a reference oligonucleotide composition of the same common nucleotide sequence.

In some embodiments, the present disclosure provides a composition comprising any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides a composition comprising any chirally controlled oligonucleotide disclosed herein.

In some embodiments, the present disclosure provides a composition comprising an oligonucleotide disclosed herein which is capable of mediating skipping of Dystrophin exon 45. In some embodiments, the present disclosure provides a composition comprising an oligonucleotide disclosed herein which is capable of mediating skipping of Dystrophin exon 51. In some embodiments, the present disclosure provides a composition comprising an oligonucleotide disclosed herein which is capable of mediating skipping of Dystrophin exon 53. In some embodiments, the present disclosure provides a composition comprising an oligonucleotide(s) disclosed herein which is capable of mediating skipping of multiple Dystrophin exons. In some embodiments, such a composition is a chirally controlled oligonucleotide composition.

In some embodiments, the present disclosure pertains to an oligonucleotide or an oligonucleotide composition capable of mediating skipping of a DMD exon or multiple DMD exons. In some embodiments, a DMD exon is exon 51. In some embodiments, a DMD exon is exon 53. In some embodiments, a DMD exon is exon 45. In some embodiments, the present disclosure pertains to an oligonucleotide composition capable of mediating skipping of a DMD exon 53, wherein the oligonucleotide composition comprises at least one chirally controlled internucleotidic linkage.

In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of DMD exon 45. In some embodiments, the present disclosure pertains to an oligonucleotide composition capable of mediating skipping of DMD exon 45, wherein the oligonucleotide composition comprises at least one chirally controlled internucleotidic linkage and comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of DMD exon 45 and comprises at least one non-negatively charged internucleotidic linkage.

In some embodiments, the present disclosure pertains to an oligonucleotide composition capable of mediating skipping of DMD exon 45, wherein the oligonucleotide composition comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of DMD exon 45 and comprises at least one non-negatively charged internucleotidic linkage.

In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of DMD exon 51. In some embodiments, the present disclosure pertains to an oligonucleotide composition capable of mediating skipping of DMD exon 51, wherein the oligonucleotide composition comprises at least one chirally controlled internucleotidic linkage and comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of DMD exon 51 and comprises at least one non-negatively charged internucleotidic linkage.

In some embodiments, the present disclosure pertains to an oligonucleotide composition capable of mediating skipping of DMD exon 51, wherein the oligonucleotide composition comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of DMD exon 51 and comprises at least one non-negatively charged internucleotidic linkage.

In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of DMD exon 53. In some embodiments, the present disclosure pertains to an oligonucleotide composition capable of mediating skipping of DMD exon 53, wherein the oligonucleotide composition comprises at least one chirally controlled internucleotidic linkage and comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of DMD exon 53 and comprises at least one non-negatively charged internucleotidic linkage.

In some embodiments, the present disclosure pertains to an oligonucleotide composition capable of mediating skipping of DMD exon 53, wherein the oligonucleotide composition comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of DMD exon 53 and comprises at least one non-negatively charged internucleotidic linkage.

In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of multiple DMD exons. In some embodiments, the present disclosure pertains to an oligonucleotide composition capable of mediating skipping of multiple DMD exons, wherein the oligonucleotide composition comprises at least one chirally controlled internucleotidic linkage and comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of multiple DMD exons and comprises at least one non-negatively charged internucleotidic linkage.

In some embodiments, the present disclosure pertains to an oligonucleotide composition capable of mediating skipping of a DMD exon, wherein the oligonucleotide composition comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of a DMD exon and comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of multiple DMD exons. In some embodiments, the present disclosure pertains to an oligonucleotide composition capable of mediating skipping of multiple DMD exons, wherein the oligonucleotide composition comprises at least one chirally controlled internucleotidic linkage and comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of multiple DMD exons and comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, a DMD exon is any DMD exon disclosed herein, including but not limited to exon 45, exon 51, exon 52, exon 53, exon 55, exon 56, and exon 57.

In some embodiments, the present disclosure pertains to an oligonucleotide composition capable of mediating skipping of multiple DMD exons, wherein the oligonucleotide composition comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a chirally controlled oligonucleotide composition, wherein the oligonucleotide is capable of mediating skipping of multiple DMD exons and comprises at least one non-negatively charged internucleotidic linkage.

In some embodiments, the present disclosure provides a chirally controlled composition of an oligonucleotide capable of mediating skipping of Dystrophin exon 51. In some embodiments, the present disclosure provides a chirally controlled composition of an oligonucleotide capable of mediating skipping of Dystrophin exon 51 and disclosed herein.

In some embodiments, the present disclosure provides a chirally controlled composition of an oligonucleotide capable of mediating skipping of Dystrophin exon 53. In some embodiments, the present disclosure provides a chirally controlled composition of an oligonucleotide capable of mediating skipping of Dystrophin exon 53 and disclosed herein.

In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9517. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9519. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9521. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9524. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9714. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9715. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9747. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9748. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9749. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9897. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9898. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9899. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9900. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9906. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-9912. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-10670. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-10671. In some embodiments, the present disclosure provides a chirally controlled composition of oligonucleotide WV-10672.

In some embodiments, the present disclosure provides a composition of an oligonucleotide having a base sequence which is, comprises, or comprises a 15-base portion of the base sequence of CUCCGGUUCUGAAGGUGUUC, wherein each U can be optionally and independently replaced by T, and wherein the composition is optionally chirally controlled. In some embodiments, the present disclosure provides a composition of an oligonucleotide having a base sequence which is CUCCGGUUCUGAAGGUGUUC, wherein each U can be optionally and independently replaced by T. and wherein the composition is optionally chirally controlled. In some embodiments, the present disclosure provides a composition of an oligonucleotide having a base sequence which comprises CUCCGGUUCUGAAGGUGUUC, wherein each U can be optionally and independently replaced by T and wherein the composition is optionally chirally controlled. In some embodiments, the present disclosure provides a composition of an oligonucleotide having a base sequence which is, comprises, or comprises a 15-base portion of CUCCGGUUCUGAAGGUGUUC, wherein each U can be optionally and independently replaced by T, and wherein the composition is optionally chirally controlled. In some embodiments, the present disclosure provides a composition of an oligonucleotide having a base sequence which is or comprises CUCCGGUUCUGAAGGUGUUCC, UCCGGUUCUGAAGGUGUUC, UCCGGUUCUGAAGGUGUUC, CCGGUUCUGAAGGUGUUC, CGGUUCUGAAGGUGUUC, GGUUCUGAAGGUGUUC. GUUCUGAAGGUGUUC, CUCCGGUUCUGAAGGUGUU, CUCCGGUUCUGAAGGUGU, CUCCGGUUCUGAAGGUG, CUCCGGUUCUGAAGGU, CUCCGGUUCUGAAGG, UCCGGUUCUGAAGGUGUU, CCGGUUCUGAAGGUGUU, UCCGGUUCUGAAGGUGU, CCGGUUCUGAAGGUGU, UCCGGUUCUGAAGGUG, CGGUUCUGAAGGUGU, UCCGGUUCUGAAGGU, CCGGUUCUGAAGGUG, CGGUUCUGAAGGUGUU, UCCGGUUCUGAAGGUGUUC, UCCGGUUCUGAAGGUG, UCCGGUUCUGAAGGU, CGGUUCUGAAGGUGUU, GGUUCUGAAGGUGUU, or GGUUCUGAAGGUGUU, wherein each U can be optionally and independently replaced by T, and wherein the composition is optionally chirally controlled. In some embodiments, the present disclosure provides a composition of an oligonucleotide having a base sequence which is, comprises, or comprises a 15-base portion of the base sequence of UUCUGAAGGUGUUCUUGUAC, wherein each U can be optionally and independently replaced by T, and wherein the composition is optionally chirally controlled. In some embodiments, the present disclosure provides a composition of an oligonucleotide having a base sequence which is UUCUGAAGGUGUUCUUGUAC, wherein each U can be optionally and independently replaced by T, and wherein the composition is optionally chirally controlled. In some embodiments, the present disclosure provides a composition of an oligonucleotide having a base sequence which comprises UUCUGAAGGUGUUCUUGUAC, wherein each U can be optionally and independently replaced by T, and wherein the composition is optionally chirally controlled. In some embodiments, the present disclosure provides a composition of an oligonucleotide having a base sequence which comprises a 15-base portion of the base sequence of UUCUGAAGGUGUUCUUGUAC, wherein each U can be optionally and independently replaced by T, and wherein the composition is optionally chirally controlled. In some embodiments, the present disclosure provides a composition of an oligonucleotide having a base sequence which is or comprises UUCUGAAGGUGUUCUUGUAC, UCUGAAGGUGUUCUUGUAC, CUGAAGGUGUUCUUGUAC, UGAAGGUGUUCUUGUAC, GAAGGUGUUCUUGUAC, AAGGUGUUCUUGUAC, UUCUGAAGGUGUUCUUGUA, UUCUGAAGGUGUUCUUGU, UUCUGAAGGUGUUCUUG, UUCUGAAGGUGUUCUU, UUCUGAAGGUGUUCU, UCUGAAGGUGUUCUUGUA, UCUGAAGGUGUUCUUGU, UCUGAAGGUGUUCUUG, UCUGAAGGUGUUCUU, CUGAAGGUGUUCUUGUA, CUGAAGGUGUUCUUGU, CUGAAGGUGUUCUUG, UGAAGGUGUUCUUGU, or UGAAGGUGUUCUUGUA, wherein each U can be optionally and independently replaced by T, and wherein the composition is optionally chirally controlled.

In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition of an oligonucleotide selected from any of the Tables. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition of an oligonucleotide selected from any of the Tables, wherein the oligonucleotide is conjugated to a lipid or a targeting moiety.

In some embodiments, an oligonucleotide is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 bases long, and optionally no more than 25, 30, 35, 40, 45, 50, 55, or 60 bases long. In some embodiments, an oligonucleotide is no more than 25 bases long. In some embodiments, an oligonucleotide is no more than 30 bases long. In some embodiments, an oligonucleotide is no more than 35 bases long. In some embodiments, an oligonucleotide is no more than 40 bases long. In some embodiments, an oligonucleotide is no more than 45 bases long. In some embodiments, an oligonucleotide is no more than 50 bases long. In some embodiments, an oligonucleotide is no more than 55 bases long. In some embodiments, an oligonucleotide is no more than 60 bases long. In some embodiments, each base is independently optionally substituted A T, C, G. or U. or an optionally substituted tautomer of A, T, C, G, or U

In some embodiments, provided oligonucleotides comprise additional chemical moieties besides their oligonucleotide chains (oligonucleotide backbones and bases), e.g., lipid moieties, targeting moieties, etc. In some embodiments, a lipid is a fatty acid. In some embodiments, an oligonucleotide is conjugated to a fatty acid. In some embodiments, a fatty acid comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more carbon atoms.

In some embodiments, a lipid is stearic acid or turbinaric acid. In some embodiments, a lipid is stearic acid acid. In some embodiments, a lipid is turbinaric acid.

In some embodiments, a lipid comprises an optionally substituted. C₁₀-C₈₀, C₁₀-C₆₀, or C₁₀-C₄₀saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—. —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)—, —S(O)₂N(R′)—, —N(R′)S(O)—. —SC(O)—, —C(O)S—, —OC(O)—, and —C(O)O—, wherein each variable is independently as defined and described herein.

In some embodiments, a lipid is selected from the group consisting of: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (DHA or cis-DHA), turbinaric acid and dilinoleyl.

In some embodiments, a lipid is conjugated to an oligonucleotide chain, optionally through one or more linker moieties. In some embodiments, a lipid is not conjugated to an oligonucleotide chain.

In some embodiments, a provided oligonucleotide is conjugated, optionally through a linker, to a chemical moiety, e.g., a lipid moiety, a peptide moiety, a targeting moiety, a carbohydrate moiety, a sulfonamide moiety, an antibody or a fragment thereof. In some embodiments, a provided compound, e.g., an oligonucleotide, has the structure of:

- A^c-[-L^LD-(R^LD)_a]_b, A^c-[-L^M-(R^D)_a]_b, [(A^c)_a-L^M]_b-R^D, (A^c)_a-L^M-(A^c)_b, or (A^c)_a-L^M-(R^D)_b,
  
  or a slat thereof, wherein:
  
  A^cis an oligonucleotide chain (e.g., H-A^c, [H]_a-A^cor [H]_b-A^cis an oligonucleotide);
  
  a is 1-1000;
  
  b is 1-1000:
  
  each of L^LDand L^Mis independently a linker moiety:
  
  R^LDis a lipid moiety; and
  
  each R^Dis independently a lipid moiety or a targeting moiety.

In some embodiments, a provided compound, e.g., an oligonucleotide, has the structure of:

- A^c-[-L^LD-(R^LD)_a]_b, A^c-[-L^M-(R^D)_a]_b, [(A^c)_a-L^M]_b-R^D, (A^c)_a-L^M-(A^c)_b, or (A^c)_a-L^M-(R^D)_b,
  
  or a salt thereof, wherein:
  
  A^cis an oligonucleotide chain (e.g., H-A^c, [H]_a-A^cor [H]_b-A^cis an oligonucleotide);
  
  a is 1-1000;
  
  b is 1-1000;
  
  each R^Dis independently R^LD, R^CDor R^TD;

R^CDis an optionally substituted, linear or branched group selected from a C_1-100aliphatic group and a C_1-100heteroaliphatic group having 1-30 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—; and one or more CH or carbon atoms are optionally and independently replaced with Cy^L;

R^LDis an optionally substituted, linear or branched C_1-100aliphatic group wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—; and one or more CH or carbon atoms are optionally and independently replaced with Cy^L;

R^TDis a targeting moiety;

each of L^LDand L^Mis independently a covalent bond, or a bivalent or multivalent, optionally substituted, linear or branched group selected from a C_1-100aliphatic group and a C_1-100heteroaliphatic group having 1-30 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C— a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—. —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—; and one or more CH or carbon atoms are optionally and independently replaced with Cy^L;

each -Cy- is independently an optionally substituted bivalent group selected from a C_3-20cycloaliphatic ring, a C_6-20aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;

each Cy^Lis independently an optionally substituted trivalent or tetravalent group selected from a C_3-20cycloaliphatic ring, a C_6-20aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;

each R′ is independently —R. —C(O)R, —C(O)OR, or —S(O)₂R; and

each R is independently —H, or an optionally substituted group selected from C_1-30aliphatic, C_1-30heteroaliphatic having 1-10 heteroatoms, C_6-30aryl, C_6-30arylaliphatic, C_6-30arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or

two R groups are optionally and independently taken together to form a covalent bond, or

two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or

two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides each having the structure of:

- A^c-[-L^LD-(R^LD)_a]_b, A^c-[-L^M-(R^D)_a]_b, [(A^c)_a-L^M]_b-R^D, (A^c)_a-L^M-(A^c)_b, or (A^c)_aL^M-(R^D)_b,
  
  or a salt thereof.

In some embodiments, [H]_b-Ac (wherein b is 1-1000) is an oligonucleotide of any one of the Tables. In some embodiments, [H]_b-Ac is an oligonucleotide of Table A1.

In some embodiments, a is 1-100. In some embodiments, a is 1-50. In some embodiments, a is 1-40. In some embodiments, a is 1-30. In some embodiments, a is 1-20. In some embodiments, a is 1-15. In some embodiments, a is 1-10. In some embodiments, a is 1-9. In some embodiments, a is 1-8. In some embodiments, a is 1-7. In some embodiments, a is 1-6. In some embodiments, a is 1-5. In some embodiments, a is 1-4. In some embodiments, a is 1-3. In some embodiments, a is 1-2. In some embodiments, a is 1. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5. In some embodiments, a is 6. In some embodiments, a is 7. In some embodiments, a is 8. In some embodiments, a is 9. In some embodiments, a is 10. In some embodiments, a is more than 10. In some embodiments, b is 1-100. In some embodiments, b is 1-50. In some embodiments, b is 1-40. In some embodiments, b is 1-30. In some embodiments, b is 1-20. In some embodiments, b is 1-15. In some embodiments, b is 1-10. In some embodiments, b is 1-9. In some embodiments, b is 1-8. In some embodiments, b is 1-7. In some embodiments, b is 1-6. In some embodiments, b is 1-5. In some embodiments, b is 1-4. In some embodiments, b is 1-3. In some embodiments, b is 1-2. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3. In some embodiments, b is 4. In some embodiments, b is 5. In some embodiments, b is 6. In some embodiments, b is 7. In some embodiments, b is 8. In some embodiments, b is 9. In some embodiments, b is 10. In some embodiments, b is more than 10. In some embodiments, an oligonucleotide has the structure of A^c-L^LD-R^LD. In some embodiments, A^cis conjugated through one or more of its sugar, base and/or internucleotidic linkage moieties. In some embodiments, A^cis conjugated through its 5′-OH (5′-O—). In some embodiments, A is conjugated through its 3′-OH (3′-O—). In some embodiments, before conjugation, A-(H)_b(b is an integer of 1-1000 depending on valency of A^c) is an oligonucleotide as described herein, for example, one of those described in any one of the Tables. In some embodiments, L^Mis -L-. In some embodiments, L^Mcomprises a phosphorothioate group. In some embodiments, L^Mis —C(O)NH—(CH₂)₆—OP(═O)(S⁻)—O—. In some embodiments, the —C(O)NH end is connected to R^LD, and the —O— end is connected to the oligonucleotide, e.g., through 5′- or 3′-end. In some embodiments, R is optionally substituted C₁₀, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, or C₂₅to C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, C₃₅, C₄₀, C₄₅, C₅₀, C₆₀, C₇₀, or C₈₀aliphatic. In some embodiments, R^LDis optionally substituted C_1-80aliphatic. In some embodiments, R^LDis optionally substituted C_20-80aliphatic. In some embodiments, R^LDis optionally substituted C_10-70aliphatic. In some embodiments, R^LDis optionally substituted C_20-70aliphatic. In some embodiments, R^LDis optionally substituted C_10-60aliphatic. In some embodiments, R^LDis optionally substituted C_20-60aliphatic. In some embodiments, R^LDis optionally substituted C_10-50aliphatic. In some embodiments, R^LDis optionally substituted C_20-50aliphatic. In some embodiments, R^LDis optionally substituted C_10-40aliphatic. In some embodiments, R^LDis optionally substituted C_20-40aliphatic. In some embodiments, R^LDis optionally substituted C_10-30aliphatic. In some embodiments, R^LDis optionally substituted C_20-30aliphatic. In some embodiments, RD is unsubstituted C₁₀, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, or C₂₅to C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, C₃₅, C₄₀, C₄₅, C₅₀, C₆₀, C₇₀, or C₈₀aliphatic. In some embodiments, R^LDis unsubstituted C_10-80aliphatic. In some embodiments, R^LDis unsubstituted C_20-80aliphatic. In some embodiments, R^LDis unsubstituted C_10-70aliphatic. In some embodiments, R^LDis unsubstituted C_20-70aliphatic. In some embodiments, R^LDis unsubstituted C_10-60aliphatic. In some embodiments, R^LDis unsubstituted C_20-60aliphatic. In some embodiments, R^LDis unsubstituted C_10-50aliphatic. In some embodiments, R^LDis unsubstituted C_20-50aliphatic. In some embodiments, R^LDis unsubstituted C_10-40aliphatic. In some embodiments, R^LDis unsubstituted C_20-40aliphatic. In some embodiments, R^LDis unsubstituted C_10-30aliphatic. In some embodiments, R^LDis unsubstituted C_20-30aliphatic.

In some embodiments, incorporation of a lipid moiety into an oligonucleotide improves at least one property of the oligonucleotide compared to an otherwise identical oligonucleotide without the lipid moiety. In some embodiments, improved properties include increased activity (e.g., increased ability to induce desirable skipping of a deleterious exon), decreased toxicity, and/or improved distribution to a tissue. In some embodiments, a tissue is muscle tissue. In some embodiments, a tissue is skeletal muscle, gastrocnemius, triceps, heart or diaphragm. In some embodiments, improved properties include reduced hTLR9 agonist activity. In some embodiments, improved properties include hTLR9 antagonist activity. In some embodiments, improved properties include increased hTLR9 antagonist activity.

In some embodiments, the present disclosure pertains to a composition comprising an a DMD oligonucleotide comprising at least one chirally controlled phosphorothioate internucleotidic linkage in the Rp or Sp configuration, at least one natural phosphate internucleotidic linkage, and at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a composition comprising an a DMD oligonucleotide comprising at least one phosphorothioate internucleotidic linkage, at least one natural phosphate internucleotidic linkage, and at least one non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a composition comprising an a DMD oligonucleotide comprising at least one phosphorothioate internucleotidic linkage, at least one natural phosphate internucleotidic linkage, and at least one chirally controlled non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure pertains to a composition comprising an a DMD oligonucleotide comprising at least one chirally controlled phosphorothioate internucleotidic linkage in the Rp or Sp configuration, at least one natural phosphate internucleotidic linkage, and at least one chirally controlled non-negatively charged internucleotidic linkage.

In some embodiments, a DMD oligonucleotide (e.g., an oligonucleotide whose base sequence contains no more than 5, 4, 3, 2, or 1 mismatches when hybridizing to a portion of a DMD transcript or a DMD genetic sequence having the same length) is capable of mediating skipping of one or more exons of the Dystrophin transcript.

In some embodiments, a DMD oligonucleotide has a base sequence which consists of the base sequence of an example oligonucleotide disclosed herein (e.g., an oligonucleotide listed in a Table), or a base sequence which comprises a 15-base portion of an example oligonucleotide nucleotide described herein. In some embodiments, a DMD oligonucleotide has a length of 15 to 50 bases.

In some embodiments, an oligonucleotide comprises a nucleobase modification, a sugar modification, and/or an internucleotidic linkage. In some embodiments, a DMD oligonucleotide has a pattern of nucleobase modifications, sugar modifications, and/or internucleotidic linkages of an example oligonucleotide described herein (or any portion thereof having a length of at least 5 bases).

In some embodiments, an oligonucleotide comprises a nucleobase modification which is BrU.

In some embodiments, an oligonucleotide comprises a sugar modification which is 2′-OMe, 2′-F, 2′-MOE, or LNA.

In some embodiments, an oligonucleotide comprises an internucleotidic linkage which is a natural phosphate linkage or a phosphorothioate internucleotidic linkage. In some embodiments, a phosphorothioate internucleotidic linkage is not chirally controlled. In some embodiments, a phosphorothioate internucleotidic linkage is a chirally controlled internucleotidic linkage (e.g., Sp or Rp).

In some embodiments, an oligonucleotide comprises a non-negatively charged internucleotidic linkage. In some embodiments, a DMD oligonucleotide comprises a neutral internucleotidic linkage. In some embodiments, a neutral internucleotidic linkage is or comprises a triazole, alkyne, or cyclic guanidine moiety.

In some embodiments, an internucleotidic linkage comprising a triazole moiety (e.g., an optionally substituted triazolyl group) in a provided oligonucleotide, e.g., a DMD oligonucleotide, has the structure of:

embedded image

In some embodiments, an internucleotidic linkage comprising a triazole moiety has the formula of

embedded image

where W is O or S. In some embodiments, an internucleotidic linkage comprising an alkyne moiety (e.g., an optionally substituted alkynyl group) has the formula of:

embedded image

wherein W is O or S. In some embodiments, an internucleotidic linkage comprises a guanidine moiety. In some embodiments, an internucleotidic linkage comprises a cyclic guanidine moiety. In some embodiments, an internucleotidic linkage comprising a cyclic guanidine moiety has the structure of:

embedded image

In some embodiments, a neutral internucleotidic linkage or internucleotidic linkage comprising a cyclic guanidine moiety is stereochemically controlled.

In some embodiments, a DMD oligonucleotide comprises a lipid moiety In some embodiments, an internucleotidic linkage comprises a Tmg group

embedded image

In some embodiments, an internucleotidic linkage comprises a Tmg group and has the structure of

embedded image

(the “Tmg internucleotidic linkage”). In some embodiments, neutral internucleotidic linkages include internucleotidic linkages of PNA and PMO, and an Tmg internucleotidic linkage.

In general, properties of oligonucleotide compositions as described herein can be assessed using any appropriate assay. Relative toxicity and/or protein binding properties for different compositions (e.g., stereocontrolled vs non-stereocontrolled, and/or different stereocontrolled compositions) are typically desirably determined in the same assay, in some embodiments substantially simultaneously and in some embodiments with reference to historical results.

Those of skill in the art will be aware of and/or will readily be able to develop appropriate assays for particular oligonucleotide compositions. The present disclosure provides descriptions of certain particular assays, for example that may be useful in assessing one or more features of oligonucleotide composition behavior e.g., complement activation, injection site inflammation, protein biding, etc.

For example, certain assays that may be useful in the assessment of toxicity and/or protein binding properties of oligonucleotide compositions may include any assay described and/or exemplified herein.

Among other things, in some embodiments, the present disclosure provides an oligonucleotide composition, comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

wherein:

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages; and

In some embodiments, the present disclosure provides a composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

which composition is chirally controlled and it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence, pattern of backbone linkages and pattern of backbone phosphorus modifications, for oligonucleotides of the particular oligonucleotide type, wherein:

the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered in that level of skipping of an exon is increased relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

In some embodiments, the present disclosure provides a composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages; and

3) pattern of backbone phosphorus modifications,

wherein:

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-negatively charged internucleotidic linkages;

In some embodiments, the present disclosure provides a composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages; and

3) pattern of backbone phosphorus modifications,

wherein:

oligonucleotides of the plurality comprise:

1) a 5′-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety;

2) a 3′-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety; and

3) a middle region between the 5′-end region and the 3′-region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotidic units comprising a phosphodiester linkage.

In some embodiments, the present disclosure provides a composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

wherein:

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages; and

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-negatively charged internucleotidic linkages.

In some embodiments, the present disclosure provides a composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

wherein:

the oligonucleotides of the plurality comprise cholesterol L-carnitine (amide and carbamate bond); Folic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; Gambogic acid; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).

In some embodiments, the present disclosure provides a pharmaceutical composition comprising an oligonucleotide or an oligonucleotide composition of the present disclosure and a pharmaceutically acceptable carrier.

In some embodiments, the present disclosure provides a method for altering splicing of a target transcript, comprising administering an oligonucleotide composition of the present disclosure. In some embodiments, the present disclosure provides a method for reducing level of a transcript or a product thereof, comprising administering an oligonucleotide composition of the present disclosure. In some embodiments, the present disclosure provides a method for increase level of a transcript or a product thereof, comprising administering an oligonucleotide composition of the present disclosure. A method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising administering to a subject susceptible thereto or suffering therefrom a composition described in the present disclosure.

In some embodiments, the present disclosure provides a method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising (a) administering to a subject susceptible thereto or suffering therefrom a composition comprising any oligonucleotide disclosed herein, and (b) administering to the subject additional treatment which is capable of preventing, treating, ameliorating or slowing the progress of muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of multiple exon skipping.

FIG. 2 shows a cartoon of a method for detecting multiple exon skipping.

FIG. 3 illustrates various strategies for multiple exon skipping.

DEFINITIONS

As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001.

Aliphatic: The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic or polycyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”), or combinations thereof. In some embodiments, aliphatic groups contain 1-100 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers to a monocyclic or bicyclic or polycyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic. In some embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers to a monocyclic C₃-C₆hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

Alkenyl: As used herein, the term “alkenyl” refers to an aliphatic group, as defined herein, having one or more double bonds.

Alkyl: As used herein, the term “alkyl” is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, an alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C₁-C₂₀for straight chain, C₂-C₂₀for branched chain), and alternatively, about 1-10. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure where such rings are monocyclic, bicyclic, or polycyclic, and alternatively about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C₁-C₄for straight chain lower alkyls).

Alkynyl: As used herein, the term “alkynyl” refers to an aliphatic group, as defined herein, having one or more triple bonds.

Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal may be a transgenic animal, a genetically-engineered animal, and/or a clone.

Approximately: As used herein, the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). In some embodiments, use of the term “about” in reference to dosages means±5 mg/kg/day.

Aryl: The term “aryl”, as used herein, used alone or as part of a larger moiety as in “aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic, bicyclic or polycyclic ring systems having a total of, e.g., five to thirty ring members, wherein at least one ring in the system is aromatic. In some embodiments, an aryl group is a monocyclic, bicyclic or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. In some embodiments, an aryl group is a biaryl group. The term “aryl” may be used interchangeably with the term “aryl ring.” In certain embodiments of the present disclosure, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl,” as it is used herein, is an aromatic ring fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

Characteristic sequence: A “characteristic sequence” is a sequence that is found in all members of a family of polypeptides or nucleic acids, and therefore can be used by those of ordinary skill in the art to define members of the family.

Comparable: The term “comparable” is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to permit comparison of results obtained or phenomena observed. In some embodiments, comparable sets of conditions or circumstances are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will appreciate that sets of conditions are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under the different sets of conditions or circumstances are caused by or indicative of the variation in those features that are varied.

Cycloaliphatic: The term “cycloaliphatic,” “carbocycle,” “carbocyclyl,” “carbocyclic radical,” and “carbocyclic ring,” are used interchangeably, and as used herein, refer to saturated or partially unsaturated, but non-aromatic, cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having, unless otherwise specified, from 3 to 30 ring members. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, a cycloaliphatic group has 3-6 carbons. In some embodiments, a cycloaliphatic group is saturated and is cycloalkyl. The term “cycloaliphatic” may also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl or 1,2,3,4-tetrahydronaphth-1-yl. In some embodiments, a cycloaliphatic group is bicyclic. In some embodiments, a cycloaliphatic group is tricyclic. In some embodiments, a cycloaliphatic group is polycyclic. In some embodiments, “cycloaliphatic” refers to C₃-C₆monocyclic hydrocarbon, or C₈-C₁₀bicyclic or polycyclic hydrocarbon, that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, or a C₉-C₁₆polycyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic.

Dosing regimen: As used herein, a“dosing regimen” or “therapeutic regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regime comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount.

Heteroaliphatic: The term “heteroaliphatic” refers to an aliphatic group wherein one or more units selected from C, CH, CH₂, and CH₃are independently replaced by one or more heteroatoms. In some embodiments, a heteroaliphatic group is heteroalkyl. In some embodiments, a heteroaliphatic group is heteroalkenyl.

Heteroaryl: The terms “heteroaryl” and “heteroar-”, as used herein, used alone or as part of a larger moiety. e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer to monocyclic, bicyclic or polycyclic ring systems having a total of, e.g., five to thirty ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, a heteroaryl group is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic or polycyclic), in some embodiments 5, 6, 9, or 10 ring atoms. In some embodiments, a heteroaryl group has 6, 10, or 14 π electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl and the like. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be monocyclic, bicyclic or polycyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring.” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted. The term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.

Heteroatom: The term “heteroatom” means an atom that is not carbon or hydrogen. In some embodiments, a heteroatom is oxygen, sulfur, nitrogen, phosphorus, boron or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring (for example, N as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as in N-substituted pyrrolidinyl); etc.). In some embodiments, a heteroatom is boron, nitrogen, oxygen, silicon, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen, silicon, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen or sulfur.

Heterocycle: As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring”, as used herein, are used interchangeably and refer to a monocyclic, bicyclic or polycyclic ring moiety (e.g., 3-30 membered) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, a hetercyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur and nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle.” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical,” are used interchangeably herein, and also include heterocyclyl rings fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl group may be monocyclic, bicyclic or polycyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

Intraperitoneal: The phrases “intraperitoneal administration” and “administered intraperitonealy” as used herein have their art-understood meaning referring to administration of a compound or composition into the peritoneum of a subject.

In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g., animal, plant, and/or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, and/or microbe).

Lower alkyl: The term “lower alkyl” refers to a C_1-4straight or branched alkyl group. Example lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

Lower haloalkyl: The term “lower haloalkyl” refers to a C_1-4straight or branched alkyl group that is substituted with one or more halogen atoms.

Optionally substituted: As described herein, compounds of the disclosure, e.g., oligonucleotides, lipids, carbohydrates, etc., may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

Suitable monovalent substituents are halogen; —(CH₂)_0-4R^o; —(CH₂)_0-4OR^o; —O(CH₂)_0-4R^o, —O—(CH₂)_0-4C(O)OR^o; —(CH₂)_0-4CH(OR^o)₂; —(CH₂)_0-4Ph, which may be substituted with R^o; —(CH₂)_0-4O(CH₂)_0-1Ph which may be substituted with R^o; —CH═CHPh, which may be substituted with R^o; —(CH₂)_0-4O(CH₂)_0-1-pyridyl which may be substituted with R^o; —NO₂; —CN; —N₃; —(CH₂)_0-4N(R^o)₂; —(CH₂)_0-4N(R^o)C(O)R^o; —N(R^o)C(S)R^o; —(CH₂)_0-4N(R^o)C(O)N(R^o)₂; —N(R^o)C(S)N(R^o)₂; —(CH₂)_0-4N(R^o)C(O)OR^o; —N(R^o)N(R^o)C(O)R^o; —N(R^o)N(R^o)C(O)N(R^o)₂; —N(R^o)N(R^o)C(O)OR^o; —(CH₂)_0-4C(O)R^o; —C(S)R^o; —(CH₂)_0-4C(O)OR^o; —(CH₂)_0-4C(O)SR^o; —(CH₂)_0-4C(O)OSi(R^o)₃; —(CH₂)_0-4OC(O)R^o; —OC(O)(CH₂)_0-4SR^o, —SC(S)SR^o; —(CH₂))_0-4SC(O)R^o; —(CH₂)_0-4C(O)N(R^o)₂; —C(S)N(R^o)₂; —C(S)SR^o; —SC(S)SR^o, —(CH₂)_0-4OC(O)N(R^o)₂; —C(O)N(OR^o)R^o; —C(O)C(O)R^o; —C(O)CH₂C(O)R^o; —C(NOR^o)R^o; —(CH₂)_0-4SSR^o; —(CH₂)_0-4(S(O)₂R^o; —(CH₂)_0-4S(O)₂OR^o; —(CH₂)_0-4OS(O)₂R^o; —S(O)₂N(R^o)₂; —(CH₂)_0-4S(O)R^o; —N(R^o)S(O)₂N(R^o)₂; —N(R^o)S(O)₂R^o; —N(OR^o)R^o; —C(NH)N(R^o)₂; —Si(R^o)₃; —OSi(R^o)₃; —P(R^o)₂; —P(OR^o)₂; —P(R^o)(OR^o); —OP(R^o)₂; —OP(OR^o)₂; —OP(R^o)(OR^o); —P[N(R^o)₂]₂; —P(R^o)[N(R^o)₂]; —P(OR^o)[N(R^o)₂]; —OP[N(R^o)₂]₂; —OP(R^o)[N(R^o)₂]; —OP(OR^o)[N(R^o)₂]; —N(R^o)P(R^o)₂; —N(R^o)P(OR^o)₂; —N(R^o)P(R^o)(OR^o); —N(R^o)P[N(R^o)₂]₂; —N(R^o)P(R^o)[N(R^o)₂]; —N(R^o)P(OR^o)[N(R^o)₂]₂; —B(R^o)₂; —B(R^o)(OR^o); —B(OR^o)₂; —OB(R^o)₂; —OB(R^o)(OR^o); —OB(OR^o)₂; —P(O)R^o)₂; —P(O)(R^o)(OR^o); —P(O)(R^o)(SR^o); —P(O)(R^o)[N(R^o)₂]; —P(O)(OR^o)₂; —P(O)(SR^o)₂; —P(O)(OR^o)[N(R^o)₂]; —P(O)(SR^o)[N(R^o)₂]; —P(O)(OR^o)(SR^o); —P(O)[N(R^o)₂]₂; —OP(O)(R^o)₂; —OP(O)(R^o)(OR^o); —OP(O)(R^o)(SR^o); —OP(O)(R^o)[N(R^o)₂]; —OP(O)(OR^o)₂; —OP(O)(SR^o)₂; —OP(O)(OR^o)[N(R^o)₂]; —OP(O)(SR^o)[N(R^o)₂]; —OP(O)(OR^o)(SR^o); —OP(O)[N(R^o)₂]₂; —SP(O)(R^o)₂; —SP(O)(R^o)(OR^o); —SP(O)(R^o)(SR^o); —SP(O)(R^o)[N(R^o)₂]; —SP(O)(OR^o)₂; —SP(O)(SR^o)₂; —SP(O)(OR^o)[N(R^o)₂]; —SP(O)(SR^o)[N(R)₂]; —SP(O)(OR^o)(SR^o); —SP(O)[N(R^o)₂]₂; —N(R^o)P(O)(R^o)₂; —N(R^o)P(O)(R^o)(OR^o); —N(R^o)P(O)(R^o)(SR^o); —N(R^o)P(O)(R^o)[N(R^o)₂]; —N(R^o)P(O)(OR^o)₂; —N(R^o)P(O)(SR^o)₂; —N(R^o)P(O)(OR^o)[N(R^o)₂]; —N(R^o)P(O)(SR^o)[N(R^o)₂]; —N(R^o)P(O)(OR^o)(SR^o); —N(R^o)P(O)[N(R^o)₂]₂; —P(R^o)₂[B(R^o)₃]; —P(OR^o)₂[B(R^o)₃]; —P(NR^o)₂[B(R^o)₃]; —P(R^o)(OR^o)[B(R^o)₃]; —P(R^o)[N(R^o)₂][B(R)₃]; —P(OR^o)[N(R^o)₂][B(R^o)₃]; —OP(R^o)₂[B(R^o)₃]; —OP(OR^o)₂[B(R^o)₃]; —OP(NR^o)₂[B(R^o)₃]; —OP(R^o)(OR^o)[B(R^o)₃]; —OP(R^o)[N(R^o)₂][B(R^o)₃]; —OP(OR^o)[N(R^o)₂][B(R^o)₃]; —N(R^o)P(R^o)₂[B(R^o)₃]; —N(R^o)P(OR^o)₂[B(R^o)₃]; —N(R^o)P(NR^o)₂[B(R^o)₃]; —N(R^o)P(R^o)(OR^o)[B(R^o)₃]; —N(R^o)P(R^o)[N(R^o)₂][B(R^o)₃]; —N(R^o)P(OR^o)[N(R^o)₂][B(R^o)₃]; —P(OR′)[B(R′)₃]—; —(C_1-4straight or branched alkylene)O—N(R^o)₂; or —(C_1-4straight or branched alkylene)C(O)O—N(R^o)₂, wherein each R^omay be substituted as defined below and is independently hydrogen, C_1-20aliphatic, C_1-20heteroaliphatic having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, —CH₂—(C_6-20aryl), —O(CH₂)_0-1(C_6-20aryl), —CH₂-(5-20 membered heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus), a 5-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, notwithstanding the definition above, two independent occurrences of R^o, taken together with their intervening atom(s), form a 3-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below.

Suitable monovalent substituents on R^o(or the ring formed by taking two independent occurrences of R^otogether with their intervening atoms), are independently halogen, —(CH₂)_0-2R^•, -(haloR^•), —(CH₂)_0-2OH, —(CH₂)_0-2OR^•, —(CH₂)_0-2CH(OR^•)₂—O(haloR^•), —CN, —N₃, —(CH₂)_0-2C(O)R^•, —(CH₂)_0-2(C(O)OH, —(CH₂)_0-2C(O)OR^•, —(CH₂)_0-2SR^•, —(CH₂)_0-2SH, —(CH₂)_0-2NH₂, —(CH₂)_0-2NHR^•, —(CH₂)_0-2NR^•₂, —NO₂, —SiR^•₃, —OSiR^•₃, —C(O)SR^•, —(C_1-4straight or branched alkylene)C(O)OR*, or —SSR^• wherein each R^• is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C₄aliphatic, —CH₂Ph, —O(CH₂)_0-1Ph, and a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R^oinclude ═O and ═S.

Suitable divalent substituents, e.g., on a suitable carbon atom, nitrogen atom, are independently the following: ═O, ═S, ═CR*₂, ═NNR*₂, ═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))_2-3O—, or —S(C(R*₂))_2-3S—, wherein each R* may be substituted as defined below and is independently hydrogen, C_1-20aliphatic, C_1-20heteroaliphatic having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, —CH₂-(C_6-20aryl), —O(CH₂)_0-1(C_6-20aryl), —CH₂-(5-20 membered heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus), a 5-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, notwithstanding the definition above, two independent occurrences of R*, taken together with their intervening atom(s), form a 3-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below. Suitable divalent substituents that are bound to vicinal substitutable atoms of an “optionally substituted” group include: —O(CR*₂)_2-3O—.

Suitable monovalent substituents on R* (or the ring formed by taking two independent occurrences of R* together with their intervening atoms), are independently halogen, —(CH₂)_0-2R^•, -(haloR^•), —(CH₂)_0-2OH, —(CH₂)_0-2OR^•, —(CH₂)_0-2CH(OR^•)₂; —O(haloR^•), —CN, —N₃, —(CH₂)_0-2C(O)R^•, —(CH₂)_0-2C(O)OH, —(CH₂)_0-2C(O)OR^•, —(CH₂)_0-2SR^•, —(CH₂)_0-2SH, —(CH₂)_0-2NH₂, —(CH₂)_0-2NHR^•, —(CH₂)_0-2NR^•₂, —NO₂, —SiR^•₃, —OSiR^•3, —C(O)SR^•, —(C_1-4straight or branched alkylene)C(O)OR^•, or —SSR^• wherein each R^• is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C₄aliphatic, —CH₂Ph, —O(CH₂)_0-1Ph, and a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R* include ═O and ═S.

In some embodiments, suitable substituents on a substitutable nitrogen of an “optionally substituted” group include —R^†, —NR^†₂, —C(O)R^†, —C(O)OR^†, —C(O)C(O)R^†, —C(O)CH₂C(O)R^†, —S(O)₂R^†, —S(O)^†₂NR^†₂, —C(S)NR^†₂, —C(NH)NR^†₂, or —N(R^†)S(O)₂R^†; wherein each R^† is independently hydrogen, C_1-6aliphatic which may be substituted as defined below, unsubstituted —OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R^†, taken together with their intervening atom(s) form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, suitable substituents on the aliphatic group of R^† are independently halogen, —R^•, -(haloR^•), —OH, —OR^•, —O(haloR^•), —CN, —C(O)OH, —C(O)OR^•, —NH₂, —NHR^•, —NR^•₂, or —NO₂, wherein each R^• is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C_1-4aliphatic, —CH₂Ph, —O(CH₂)_0-1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Oral: The phrases “oral administration” and “administered orally” as used herein have their art-understood meaning referring to administration by mouth of a compound or composition.

Parenteral: The phrases “parenteral administration” and “administered parenterally” as used herein have their art-understood meaning referring to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrastemal injection and infusion.

Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a controlled therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose, starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt”, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. In some embodiments, a provided compound comprises one or more acidic groups, e.g., an oligonucleotide, and a pharmaceutically acceptable salt is an alkali, alkaline earth metal, or ammonium (e.g., an ammonium salt of N(R)₃, wherein each R is independently as defined and described in the present disclosure) salt. Representative alkali or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, a pharmaceutically acceptable salt is a sodium salt. In some embodiments, a pharmaceutically acceptable salt is a potassium salt. In some embodiments, a pharmaceutically acceptable salt is a calcium salt. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. In some embodiments, a provided compound comprises more than one acid groups, for example, a provided oligonucleotide may comprise two or more acidic groups (e.g., in natural phosphate linkages and/or modified internucleotidic linkages). In some embodiments, a pharmaceutically acceptable salt, or generally a salt, of such a compound comprises two or more cations, which can be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or generally, a salt), each acidic group having sufficient acidity independently exists as its salt form (e.g., in an oligonucleotide comprising natural phosphate linkages and phosphorothioate internucleotidic linkages, each of the natural phosphate linkages and phosphorothioate internucleotidic linkages independently exists as its salt form). In some embodiments, a pharmaceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide. In some embodiments, a pharmaceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide, wherein each acidic linkage, e.g., each natural phosphate linkage and phosphorothioate internucleotidic linkage, exists as a sodium salt form (all sodium salt).

Protecting group: The term “protecting group,” as used herein, is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rdedition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Also included are those protecting groups specially adapted for nucleoside and nucleotide chemistry, e.g., those described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. 06/2012, the entirety of Chapter 2 is incorporated herein by reference. Suitable amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-<dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)anine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine. N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Suitably protected carboxylic acids further include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.

Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM). (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a, 4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, I-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4′-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl(DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl(TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate(levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethlene acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene ortho ester, 1-(N,N-dimethylamino)ethylidene derivative, α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

In some embodiments, a hydroxyl protecting group is acetyl, t-butyl, tbutoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl), 4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifiuoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4′-dimethoxytrityl, (DMTr) and 4,4′,4″-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4′,4″-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In some embodiments, each of the hydroxyl protecting groups is, independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and 4,4′-dimethoxytrityl. In some embodiments, the hydroxyl protecting group is selected from the group consisting of trityl, monomethoxytrityl and 4,4′-dimethoxytrityl group.

In some embodiments, a phosphorous protecting group is a group attached to the internucleotide phosphorous linkage throughout oligonucleotide synthesis. In some embodiments, the phosphorous protecting group is attached to the sulfur atom of the internucleotide phosphorothioate linkage. In some embodiments, the phosphorous protecting group is attached to the oxygen atom of the internucleotide phosphorothioate linkage. In some embodiments, the phosphorous protecting group is attached to the oxygen atom of the internucleotide phosphate linkage. In some embodiments the phosphorous protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl, 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

Protein: As used herein, the term “protein” refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). In some embodiments, proteins include only naturally-occurring amino acids. In some embodiments, proteins include one or more non-naturally-occurring amino acids (e.g., moieties that form one or more peptide bonds with adjacent amino acids). In some embodiments, one or more residues in a protein chain contain a non-amino-acid moiety (e.g., a glycan, etc). In some embodiments, a protein includes more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. In some embodiments, proteins contain L-amino acids, D-amino acids, or both: in some embodiments, proteins contain one or more amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

Subject: As used herein, the term “subject” or “test subject” refers to any organism to which a provided compound or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject may be suffering from, and/or susceptible to a disease, disorder, and/or condition.

Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and/or chemical phenomena.

Suffering from: An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with and/or displays one or more symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

Systemic: The phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” as used herein have their art-understood meaning referring to administration of a compound or composition such that it enters the recipient's system.

Tautomeric forms: The phrase “tautomeric forms,” as used herein and generally understood in the art, is used to describe different isomeric forms of organic compounds that are capable of facile interconversion. Tautomers may be characterized by the formal migration of a hydrogen atom or proton, accompanied by a switch of a single bond and adjacent double bond. In some embodiments, tautomers may result from prototropic tautomerism (i.e., the relocation of a proton). In some embodiments, tautomers may result from valence tautomerism (i.e., the rapid reorganization of bonding electrons). All such tautomeric forms are intended to be included within the scope of the present disclosure. In some embodiments, tautomeric forms of a compound exist in mobile equilibrium with each other, so that attempts to prepare the separate substances results in the formation of a mixture. In some embodiments, tautomeric forms of a compound are separable and isolatable compounds. In some embodiments of the disclosure, chemical compositions may be provided that are or include pure preparations of a single tautomeric form of a compound. In some embodiments of the disclosure, chemical compositions may be provided as mixtures of two or more tautomeric forms of a compound. In certain embodiments, such mixtures contain equal amounts of different tautomeric forms; in certain embodiments, such mixtures contain different amounts of at least two different tautomeric forms of a compound. In some embodiments of the disclosure, chemical compositions may contain all tautomeric forms of a compound. In some embodiments of the disclosure, chemical compositions may contain less than all tautomeric forms of a compound. In some embodiments of the disclosure, chemical compositions may contain one or more tautomeric forms of a compound in amounts that vary over time as a result of interconversion. In some embodiments of the disclosure, the tautomerism is keto-enol tautomerism. One of skill in the chemical arts would recognize that a keto-enol tautomer can be “trapped” (i.e., chemically modified such that it remains in the “enol” form) using any suitable reagent known in the chemical arts in to provide an enol derivative that may subsequently be isolated using one or more suitable techniques known in the art. Unless otherwise indicated, the present disclosure encompasses all tautomeric forms of relevant compounds, whether in pure form or in admixture with one another.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.

Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

Unit dose: The expression “unit dose” as used herein refers to an amount administered as a single dose and/or in a physically discrete unit of a pharmaceutical composition. In many embodiments, a unit dose contains a predetermined quantity of an active agent. In some embodiments, a unit dose contains an entire single dose of the agent. In some embodiments, more than one unit dose is administered to achieve a total single dose. In some embodiments, administration of multiple unit doses is required, or expected to be required, in order to achieve an intended effect. A unit dose may be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined quantity of one or more therapeutic agents, a predetermined amount of one or more therapeutic agents in solid form, a sustained release formulation or drug delivery device containing a predetermined amount of one or more therapeutic agents, etc. It will be appreciated that a unit dose may be present in a formulation that includes any of a variety of components in addition to the therapeutic agent(s). For example, acceptable carriers (e.g., pharmaceutically acceptable carriers), diluents, stabilizers, buffers, preservatives, etc., may be included as described infra. It will be appreciated by those skilled in the art, in many embodiments, a total appropriate daily dosage of a particular therapeutic agent may comprise a portion, or a plurality, of unit doses, and may be decided, for example, by the attending physician within the scope of sound medical judgment. In some embodiments, the specific effective dose level for any particular subject or organism may depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of specific active compound employed; specific composition employed; age, body weight, general health, sex and diet of the subject; time of administration, and rate of excretion of the specific active compound employed; duration of the treatment; drugs and/or additional therapies used in combination or coincidental with specific compound(s) employed, and like factors well known in the medical arts.

Unsaturated: The term “unsaturated,” as used herein, means that a moiety has one or more units of unsaturation.

Wild-type: As used herein, the term “wild-type” has its art-understood meaning that refers to an entity having a structure and/or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc) state or context. Those of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

Nucleic acid: The term “nucleic acid” includes any nucleotides, analogs thereof, and polymers thereof. The term “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) or analogs thereof. These terms refer to the primary structure of the molecules and include double- and single-stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated, protected and/or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and/or modified nucleobases; nucleic acids derived from sugars and/or modified sugars; and nucleic acids derived from phosphate bridges and/or modified phosphorus-atom bridges (also referred to herein as “internucleotidic linkages”). The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, natural natural phosphate internucleotidic linkages or non-natural internucleotidic linkages. Examples include, and are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix poly-refers to a nucleic acid containing 2 to about 10,000 nucleotide monomer units and wherein the prefix oligo-refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.

Nucleotide: The term “nucleotide” as used herein refers to a monomeric unit of a polynucleotide that consists of a heterocyclic base, a sugar, and one or more phosphate groups or phosphorus-containing internucleotidic linkages. Naturally occurring bases, (guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogs are also included. Naturally occurring sugars include the pentose (five-carbon sugar) deoxyribose (which is found in natural DNA) or ribose (which is found in natural RNA), though it should be understood that naturally and non-naturally occurring sugar analogs are also included, such as sugars with 2-modifications, sugars in locked nucleic acid (LNA) and phosphorodiamidate morpholino oligomer (PMO). Nucleotides are linked via internucleotidic linkages to form nucleic acids, or polynucleotides. Many internucleotidic linkages are known in the art (such as, though not limited to, natural phosphate linkage, phosphorothioate linkages, boranophosphate linkages and the like). Artificial nucleic acids include PNAs (peptide nucleic acids), phosphotriesters, phosphorothionates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates and other variants of the phosphate backbone of native nucleic acids, etc. In some embodiments, a nucleotide is a natural nucleotide comprising a naturally occurring nucleobase, a natural occurring sugar and the natural phosphate linkage. In some embodiments, a nucleotide is a modified nucleotide or a nucleotide analog, which is a structural analog that can be used in lieu of a natural nucleotide.

Modified nucleotide: The term “modified nucleotide” includes any chemical moiety which differs structurally from a natural nucleotide but is capable of performing at least one function of a natural nucleotide. In some embodiments, a modified nucleotide comprises a modification at a sugar, base and/or internucleotidic linkage. In some embodiments, a modified nucleotide comprises a modified sugar, modified nucleobase and/or modified internucleotidic linkage. In some embodiments, a modified nucleotide is capable of at least one function of a nucleotide, e.g., forming a subunit in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases.

Analog: The term “analog” includes any chemical moiety which differs structurally from a reference chemical moiety or class of moieties, but which is capable of performing at least one function of such a reference chemical moiety or class of moieties. As non-limiting examples, a nucleotide analog differs structurally from a nucleotide but performs at least one function of a nucleotide; a nucleobase analog differs structurally from a nucleobase but performs at least one function of a nucleobase; a sugar analog differs structurally from a nucleobase but performs at least one function of a sugar, etc.

Nucleoside: The term “nucleoside” refers to a moiety wherein a nucleobase or a modified nucleobase is covalently bound to a sugar or modified sugar.

Modified nucleoside: The term “modified nucleoside” refers to a chemical moiety which is chemically distinct from a natural nucleoside, but which is capable of performing at least one function of a nucleoside. In some embodiments, a modified nucleoside is derived from or chemically similar to a natural nucleoside, but which comprises a chemical modification which differentiates it from a natural nucleoside. Non-limiting examples of modified nucleosides include those which comprise a modification at the base and/or the sugar. Non-limiting examples of modified nucleosides include those with a 2′-modification at a sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (which lack a nucleobase). In some embodiments, a modified nucleoside is capable of at least one function of a nucleoside, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases.

Nucleoside analog: The term “nucleoside analog” refers to a chemical moiety which is chemically distinct from a natural nucleoside, but which is capable of performing at least one function of a nucleoside. In some embodiments, a nucleoside analog comprises an analog of a sugar and/or an analog of a nucleobase. In some embodiments, a modified nucleoside is capable of at least one function of a nucleoside, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising a complementary sequence of bases.

Sugar: The term “sugar” refers to a monosaccharide or polysaccharide in closed and/or open form. In some embodiments, sugars are monosaccharides. In some embodiments, sugars are polysaccharides. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term “sugar” also encompasses structural analogs used in lieu of conventional sugar molecules, such as glycol, polymer of which forms the backbone of the nucleic acid analog, glycol nucleic acid (“GNA”), etc. As used herein, the term “sugar” also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified sugars and nucleotide sugars. In some embodiments, a sugar is D-2-deoxyribose. In some embodiments, a sugar is beta-D-deoxyribofuranose. In some embodiments, a sugar moiety is a beta-D-deoxyribofuranose moiety. In some embodiments, a sugar is D-ribose. In some embodiments, a sugar is beta-D-ribofuranose. In some embodiments, a sugar moiety is a beta-D-ribofuranose moiety. In some embodiments, a sugar is optionally substituted beta-D-deoxyribofuranose or beta-D-ribofuranose. In some embodiments, a sugar moiety is an optionally substituted beta-D-deoxyribofuranose or beta-D-ribofuranose moiety. In some embodiments, a sugar moiety/unit in an oligonucleotide, nucleic acid, etc. is a sugar which comprises one or more carbon atoms each independently connected to an internucleotidic linkage, e.g., optionally substituted beta-D-deoxyribofuranose or beta-D-ribofuranose whose 5′-C and/or 3-C are each independently connected to an internucleotidic linkage (e.g., a natural phosphate linkage, a modified internucleotidic linkage, a chirally controlled internucleotidic linkage, etc.).

Modified sugar: The term “modified sugar” refers to a moiety that can replace a sugar. A modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of a sugar. In some embodiments, a modified sugar is substituted beta-D-deoxyribofuranose or beta-D-ribofuranose. In some embodiments, a modified sugar comprises a 2′-modification. In some embodiments, a modified sugar comprises a linker (e.g., optionally substituted bivalent heteroaliphatic) connecting two sugar carbon atoms (e.g., C2 and C4), e.g., as found in LNA. In some embodiments, a linker is —O—CH(R)—, wherein R is as described in the present disclosure. In some embodiments, a linker is —O—CH(R)—, wherein O is connected to C2, and —CH(R)— is connected to C4 of a sugar, and R is as described in the present disclosure. In some embodiments, R is methyl. In some embodiments, R is —H. In some embodiments, —CH(R)— is of S configuration. In some embodiments, —CH(R)— is of R configuration.

Nucleobase: The term “nucleobase” refers to the parts of nucleic acids that are involved in the hydrogen-bonding that binds one nucleic acid strand to another complementary strand in a sequence specific manner. The most common naturally-occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, a modified nucleobase is a substituted nucleobase which nucleobase is selected from A, T, C, G, U, and tautomers thereof. In some embodiments, the naturally-occurring nucleobases are modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the naturally-occurring nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a nucleobase is a “modified nucleobase,” e.g., a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the property of hydrogen-bonding that binds one nucleic acid strand to another in a sequence specific manner. In some embodiments, a modified nucleobase can pair with all of the five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide duplex. As used herein, the term “nucleobase” also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified nucleobases and nucleobase analogs. In some embodiments, a nucleobase is an optionally substituted A, T, C, G, or U. or a substituted nucleobase which nucleobase is selected from A, T, C, G U and tautomers thereof.

Modified nucleobase: The terms “modified nucleobase”, “modified base” and the like refer to a chemical moiety which is chemically distinct from a nucleobase, but which is capable of performing at least one function of a nucleobase. In some embodiments, a modified nucleobase is a nucleobase which comprises a modification. In some embodiments, a modified nucleobase is capable of at least one function of a nucleobase, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases. In some embodiments, a modified nucleobase is a substituted nucleobase which nucleobase is selected from A, T, C, G, U, and tautomers thereof.

Chiral ligand: The term “chiral ligand” or “chiral auxiliary” refers to a moiety that is chiral and can be incorporated into a reaction so that the reaction can be carried out with certain stereoselectivity. In some embodiments, the term may also refer to a compound that comprises such a moiety.

Blocking group: The term “blocking group” refers to a group that masks the reactivity of a functional group. The functional group can be subsequently unmasked by removal of the blocking group. In some embodiments, a blocking group is a protecting group.

Moiety: The term “moiety” refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule. In some embodiments, a moiety of a compound is a monovalent, bivalent, or polyvalent group formed from the compound by removing one or more —H and/or equivalents thereof from a compound. In some embodiments, depending on its context, “moiety” may also refer to a compound or entity from which the moiety is derived from.

Solid support: The term “solid support” when used in the context of preparation of nucleic acids, oligonucleotides, or other compounds refers to any support which enables synthesis of nucleic acids, oligonucleotides or other compounds. In some embodiments, the term refers to a glass or a polymer, that is insoluble in the media employed in the reaction steps performed to synthesize nucleic acids, and is derivatized to comprise reactive groups. In some embodiments, the solid support is Highly Cross-linked Polystyrene (HCP) or Controlled Pore Glass (CPG). In some embodiments, the solid support is Controlled Pore Glass (CPG). In some embodiments, the solid support is hybrid support of Controlled Pore Glass (CPG) and Highly Cross-linked Polystyrene (HCP).

Reading frame: The term “reading frame” refers to one of the six possible reading frames, three in each direction, of a double stranded DNA molecule. The reading frame that is used determines which codons are used to encode amino acids within the coding sequence of a DNA molecule.

Antisense: As used herein, an “antisense” nucleic acid molecule comprises a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense nucleic acid molecule can associate via hydrogen bonds to a sense nucleic acid molecule. In some embodiments, transcripts may be generated from both strands. In some embodiments, transcripts may or may not encode protein products. In some embodiments, when directed or targeted to a particular nucleic acid sequence, a “antisense” sequence may refer to a sequence that is complementary to the particular nucleic acid sequence.

Oligonucleotide: the term “oligonucleotide” refers to a polymer or oligomer of nucleotide monomers, containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, natural phosphate linkages, or non-natural internucleotidic linkages.

Oligonucleotides can be single-stranded or double-stranded. As used herein, the term “oligonucleotide strand” encompasses a single-stranded oligonucleotide. A single-stranded oligonucleotide can have double-stranded regions and a double-stranded oligonucleotide can have single-stranded regions. Example oligonucleotides include, but are not limited to structural genes, genes including control and termination regions, self-replicating systems such as viral or plasmid DNA, single-stranded and double-stranded siRNAs and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, U1 adaptors, triplex-forming oligonucleotides, G-quadruplex oligonucleotides. RNA activators, immuno-stimulatory oligonucleotides, and decoy oligonucleotides.

Double-stranded and single-stranded oligonucleotides that are effective in inducing RNA interference may also be referred to as siRNA, RNAi agent, or iRNA agent. In some embodiments, these RNA interference inducing oligonucleotides associate with a cytoplasmic multi-protein complex known as RNAi-induced silencing complex (RISC). In many embodiments, single-stranded and double-stranded RNAi agents are sufficiently long that they can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller oligonucleotides that can enter the RISC machinery and participate in RISC mediated cleavage of a target sequence, e.g. a target mRNA.

Oligonucleosides of the present disclosure can be of various lengths. In particular embodiments, oligonucleosides can range from about 2 to about 200 nucleosides in length. In various related embodiments, oligonucleosides, single-stranded, double-stranded, and triple-stranded, can range in length from about 4 to about 10 nucleosides, from about 10 to about 50 nucleosides, from about 20 to about 50 nucleosides, from about 15 to about 30 nucleosides, from about 20 to about 30 nucleosides in length. In some embodiments, the oligonucleoside is from about 9 to about 39 nucleosides in length. In some embodiments, the oligonucleoside is at least 15 nucleosides in length. In some embodiments, the oligonucleoside is at least 20 nucleosides in length. In some embodiments, the oligonucleoside is at least 25 nucleosides in length. In some embodiments, the oligonucleoside is at least 30 nucleosides in length. In some embodiments, the oligonucleoside is a duplex of complementary strands of at least 18 nucleosides in length. In some embodiments, the oligonucleoside is a duplex of complementary strands of at least 21 nucleosides in length. In some embodiments, for the purpose of oligonucleotide lengths, each nucleoside counted independently comprises an optionally substituted nucleobase selected from A, T, C, G, U and their tautomers.

Internucleotidic linkage: As used herein, the phrase “internucleotidic linkage” refers generally to a linkage, typically a phosphorus-containing linkage, between nucleotide units of a nucleic acid or an oligonucleotide, and is interchangeable with “inter-sugar linkage”, “internucleotidic linkage,” and “phosphorus atom bridge,” as used above and herein. As appreciated by those skilled in the art, natural DNA and RNA contain natural phosphate linkages. In some embodiments, an internucleotidic linkage is a natural phosphate linkage (—OP(O)(OH)O—, typically existing as its anionic form —OP(O)(O⁻)O— at pH e.g., ˜7.4), as found in naturally occurring DNA and RNA molecules. In some embodiments, an internucleotidic linkage is a modified internucleotidic linkage (or non-natural internucleotidic linkage), which is structurally different from a natural phosphate linkage but may be utilized in place of a natural phosphate linkage, e.g., phosphorothioate internucleotidic linkage. PMO linkages, etc. In some embodiments, an internucleotidic linkage is a modified internucleotidic linkage wherein one or more oxygen atoms of a natural phosphodiester linkage are independently replaced by one or more organic or inorganic moieties. In some embodiments, such an organic or inorganic moiety is selected from but not limited to ═S, ═Se, ═NR′, —SR′, —SeR′, —N(R′)₂, B(R′), —S—, —Se—, and —N(R′)—, wherein each R′ is independently as defined and described below. In some embodiments, an internucleotidic linkage is a phosphotriester linkage. In some embodiments, an internucleotidic linkage is a phosphorothioate diester linkage (phosphorothioate internucleotidic linkage,

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typically existing as its anionic form —OP(O)(S⁻)O— at pH e.g., ˜7.4). It is understood by a person of ordinary skill in the art that an internucleotidic linkage may exist as an anion or cation at a given pH due to the existence of acid or base moieties in the linkage. In some embodiments, an internucleotidic linkage is a non-negatively charged internucleotidic linkage at a given pH. In some embodiments, an internucleotidic linkage is a neutral internucleotidic linkage at a given pH. In some embodiments, a given pH is pH ˜7.4. In some embodiments, a given pH is in the range of pH about 0, 1, 2, 3, 4, 5, 6 or 7 to pH about 7, 8, 9, 10, 11, 12, 13 or 14. In some embodiments, a given pH is in the range of pH 5-9. In some embodiments, a given pH is in the range of pH 6-8. In some embodiments, an internucleotidic linkage has the structure of formula I, I-a, I-b. I-c, I-n-1, I-n-2. I-n-3, I-n-4, II, II-a-1, II-a-2. II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., as described in the present disclosure. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of formula I-n-1, i-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., as described in the present disclosure. In some embodiments, an internucleotidic linkage is one of, e.g., PNA (peptide nucleic acid) or PMO (phosphorodiamidate Morpholino oligomer) linkage. In some embodiments, an internucleotidic linkage comprises a chiral linkage phosphorus. In some embodiments, an internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, an internucleotidic linkage is selected from: s (phosphorothioate), s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17 or s18, wherein each of s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17 and s18 is independently as described in WO 2017/062862.

Unless otherwise specified, the Rp/Sp designations preceding an oligonucleotide sequence describe the configurations of linkage phosphorus in chirally controlled internucleotidic linkages sequentially from 5′ to 3′ of the oligonucleotide sequence. For instance, in (Rp, Sp)-ATsCs1GA, the phosphorus in the “s” linkage between T and C has Rp configuration and the phosphorus in “s1” linkage between C and G has Sp configuration. In some embodiments, “All-(Rp)” or “All-(Sp)” is used to indicate that all chiral linkage phosphorus atoms in chirally controlled internucleotidic linkages have the same Rp or Sp configuration, respectively. For instance, All-(Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC indicates that all the chiral linkage phosphorus atoms in the oligonucleotide have Rp configuration; All-(Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC indicates that all the chiral linkage phosphorus atoms in the oligonucleotide have Sp configuration.

Oligonucleotide type: As used herein, the phrase “oligonucleotide type” is used to define oligonucleotides that have a particular base sequence, pattern of backbone linkages (i.e., pattern of internucleotidic linkage types, for example, natural phosphate linkages, phosphorothioate internucleotidic linkages, negatively charged internucleotidic linkages, neutral internucleotidic linkages etc), pattern of backbone chiral centers (i.e. pattern of linkage phosphorus stereochemistry (Rp/Sp)), and pattern of backbone phosphorus modifications (e.g., pattern of “-X-L-R¹” groups in formula I). In some embodiments, oligonucleotides of a common designated “type” are structurally identical to one another.

One of skill in the art will appreciate that synthetic methods of the present disclosure provide for a degree of control during the synthesis of an oligonucleotide strand such that each nucleotide unit of the oligonucleotide strand can be designed and/or selected in advance to have a particular stereochemistry at the linkage phosphorus and/or a particular modification at the linkage phosphorus, and/or a particular base, and/or a particular sugar. In some embodiments, an oligonucleotide strand is designed and/or selected in advance to have a particular combination of stereocenters at the linkage phosphorus. In some embodiments, an oligonucleotide strand is designed and/or determined to have a particular combination of modifications at the linkage phosphorus. In some embodiments, an oligonucleotide strand is designed and/or selected to have a particular combination of bases. In some embodiments, an oligonucleotide strand is designed and/or selected to have a particular combination of one or more of the above structural characteristics. The present disclosure provides compositions comprising or consisting of a plurality of oligonucleotide molecules (e.g., chirally controlled oligonucleotide compositions). In some embodiments, all such molecules are of the same type. In some embodiments, all such molecules are structurally identical to one another. In some embodiments, provided compositions comprise a plurality of oligonucleotides of different types, typically in pre-determined (non-random) relative amounts.

Chiral control: As used herein, “chiral control” refers to control of the stereochemical designation of a chiral linkage phosphorus in a chiral internucleotidic linkage within an oligonucleotide. In some embodiments, a control is achieved through a chiral element that is absent from the sugar and base moieties of an oligonucleotide, for example, in some embodiments, a control is achieved through use of one or more chiral auxiliaries during oligonucleotide preparation as exemplified in the present disclosure, which chiral auxiliaries often are part of chiral phosphoramidites used during oligonucleotide preparation. In contrast to chiral control, a person having ordinary skill in the art appreciates that conventional oligonucleotide synthesis which does not use chiral auxiliaries cannot control stereochemistry at a chiral internucleotidic linkage if such conventional oligonucleotide synthesis is used to form the chiral internucleotidic linkage. In some embodiments, the stereochemical designation of each chiral linkage phosphorus in a chiral internucleotidic linkage within an oligonucleotide is controlled.

Chirally controlled oligonucleotide composition: The terms “chirally controlled (stereocontrolled or stereodefined) oligonucleotide composition”, “chirally controlled (stereocontrolled or stereodefined) nucleic acid composition”, and the like, as used herein, refers to a composition that comprises a plurality of oligonucleotides (or nucleic acids, chirally controlled oligonucleotides or chirally controlled nucleic acids) which share 1) a common base sequence, 2) a common pattern of backbone linkages; 3) a common pattern of backbone chiral centers, and 4) a common pattern of backbone phosphorus modifications (oligonucleotides of a particular type), wherein the plurality of oligonucleotides (or nucleic acids) share the same stereochemistry at one or more chiral internucleotidic linkages (chirally controlled internucleotidic linkages, whose chiral linkage phosphorus is Rp or Sp, not a random Rp and Sp mixture as non-chirally controlled internucleotidic linkages). Level of the plurality of oligonucleotides (or nucleic acids) in a chirally controlled oligonucleotide composition is non-random (pre-determined, controlled). Chirally controlled oligonucleotide compositions are typically prepared through chirally controlled oligonucleotide preparation to stereoselectively form one or more chiral internucleotidic linkages (e.g., using chiral auxiliaries as exemplified in the present disclosure, compared to non-chirally controlled (stereorandom, non-stereoselective, racemic) oligonucleotide synthesis such as traditional phosphoramidite-based oligonucleotide synthesis using no chiral auxiliaries or chiral catalysts to purposefully control stereoselectivity). A chirally controlled oligonucleotide composition is enriched, relative to a substantially racemic preparation of oligonucleotides having the common base sequence, the common pattern of backbone linkages, and the common pattern of backbone phosphorus modifications, for oligonucleotides of the plurality. In some embodiments, a chirally controlled oligonucleotide composition comprises a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications, wherein it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence, pattern of backbone linkages, and pattern of backbone phosphorus modifications, for oligonucleotides of the particular oligonucleotide type. As one having ordinary skill in the art readily appreciates, such enrichment can be characterized in that compared to a substantially racemic preparation, at each chirally controlled internucleotidic linkage, a higher level of the linkage phosphorus has the desired configuration. In some embodiments, each chirally controlled internucleotidic linkage independently has a diastereopurity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with respect to its chiral linkage phosphorus. In some embodiments, each independently has a diastereopurity of at least 90%. In some embodiments, each independently has a diastereopurity of at least 95%. In some embodiments, each independently has a diastereopurity of at least 97%. In some embodiments, each independently has a diastereopurity of at least 98%. In some embodiments, oligonucleotides of a plurality have the same constitution. In some embodiments, oligonucleotides of a plurality have the same constitution and stereochemistry, and are structurally identical.

In some embodiments, the plurality of oligonucleotides in a chirally controlled oligonucleotide composition share the same base sequence, the same, if any, nucleobase, sugar, and internucleotidic linkage modifications, and the same stereochemistry (Rp or Sp) independently at linkage phosphorus chiral centers of one or more chirally controlled internucleotidic linkages, though stereochemistry of certain linkage phosphorus chiral centers may differ. In some embodiments, about 0.1%-100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition are oligonucleotides of the plurality. In some embodiments, about 0.1%-100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-00%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition that share the common base sequence are oligonucleotides of the plurality. In some embodiments, about 0.1%-100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition that share the common base sequence, the common pattern of backbone linkages, and the common pattern of backbone phosphorus modifications are oligonucleotides of the plurality. In some embodiments, about 0.1%-100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition, or of all oligonucleotides in a composition that share a common base sequence (e.g., of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modifications (e.g., of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share a common base sequence, a common patter of base modifications, a common pattern of sugar modifications, a common pattern of internucleotidic linkage types, and/or a common pattern of internucleotidic linkage modifications (e.g., of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share the same constitution, are oligonucleotides of the plurality. In some embodiments, a percentage is at least (DP)^NCI, wherein DP is a percentage selected from 85%-100%, and NCI is the number of chirally controlled internucleotidic linkage. In some embodiments, DP is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, DP is at least 85%. In some embodiments, DP is at least 90%. In some embodiments, DP is at least 95%. In some embodiments, DP is at least 96%. In some embodiments, DP is at least 97%. In some embodiments, DP is at least 98%. In some embodiments, DP is at least 99%. In some embodiments, DP reflects diastereopurity of linkage phosphorus chiral centers chirally controlled internucleotidic linkages. In some embodiments, diastereopurity of a linkage phosphorus chiral center of an internucleotidic linkage may be typically assessed using an appropriate dimer comprising such an internucleotidic linkage and the two nucleoside units being linked by the internucleotidic linkage. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotidic linkages. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 0.1%-100% (e.g., about 1%-100%, 5%-400%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of chiral internucleotidic linkages. In some embodiments, each chiral internucleotidic linkage is a chiral controlled internucleotidic linkage, and the composition is a completely chirally controlled oligonucleotide composition. In some embodiments, not all chiral internucleotidic linkages are chiral controlled internucleotidic linkages, and the composition is a partially chirally controlled oligonucleotide composition. In some embodiments, a chirally controlled oligonucleotide composition comprises predetermined levels of individual oligonucleotide or nucleic acids types. For instance, in some embodiments a chirally controlled oligonucleotide composition comprises one oligonucleotide type at a predetermined level (e.g., as described above). In some embodiments, a chirally controlled oligonucleotide composition comprises more than one oligonucleotide type, each independently at a predetermined level. In some embodiments, a chirally controlled oligonucleotide composition comprises multiple oligonucleotide types, each independently at a predetermined level. In some embodiments, a chirally controlled oligonucleotide composition is a composition of oligonucleotides of an oligonucleotide type, which composition comprises a predetermined level of a plurality of oligonucleotides of the oligonucleotide type.

Chirally pure: as used herein, the phrase “chirally pure” is used to describe an oligonucleotide or compositions thereof, in which all or nearly all (the rest are impurities) of the oligonucleotide molecules exist in a single diastereomeric form with respect to the linkage phosphorus atoms. In many embodiments, as appreciated by those skilled in the art, a chirally pure oligonucleotide composition is substantially pure in that substantially all of the oligonucleotides in the composition are structurally identical (being the same stereoisomer).

Linkage phosphorus: as defined herein, the phrase “linkage phosphorus” is used to indicate that the particular phosphorus atom being referred to is the phosphorus atom present in an internucleotidic linkage, which phosphorus atom corresponds to the phosphorus atom of a natural phosphate linkage as occurs in naturally occurring DNA and RNA. In some embodiments, a linkage phosphorus atom is in a modified internucleotidic linkage. In some embodiments, a linkage phosphorus atom is the P of P^Lof formula I. In some embodiments, a linkage phosphorus atom is chiral.

P-modification: as used herein, the term “P-modification” refers to any modification at the linkage phosphorus other than a stereochemical modification. In some embodiments, a P-modification comprises addition, substitution, or removal of a pendant moiety covalently attached to a linkage phosphorus. In some embodiments, the “P-modification” is W, Y, Z, or -X-L-R¹of formula I.

Blockmer: the term “blockmer,” as used herein, refers to an oligonucleotide whose pattern of structural features characterizing each individual nucleotide unit is characterized by the presence of at least two consecutive nucleotide units sharing a common structural feature at the nucleobase, sugar and/or internucleotidic linkage. By common structural feature is meant common chemistry and/or stereochemistry, e.g., common modifications at nucleobases, sugars, and/or internucleotidic linkages and common stereochemistry at linkage phosphorus chiral centers. In some embodiments, the at least two consecutive nucleotide units sharing a common structural feature are referred to as a “block”.

In some embodiments, a blockmer is a “stereoblockmer,” e.g., at least two consecutive nucleotide units have the same stereochemistry at the linkage phosphorus. Such at least two consecutive nucleotide units form a “stereoblock.” For instance, (Sp, Sp)-ATsCs1GA is a stereoblockmer because at least two consecutive nucleotide units, the Ts and the Cs1, have the same stereochemistry at the linkage phosphorus (both Sp). In the same oligonucleotide (Sp, Sp)-ATsCs1GA, TsCs1 forms a block, and it is a stereoblock.

In some embodiments, a blockmer is a “P-modification blockmer,” e.g., at least two consecutive nucleotide units have the same modification at the linkage phosphorus. Such at least two consecutive nucleotide units form a “P-modification block”. For instance, (Rp, Sp)-ATsCsGA is a P-modification blockmer because at least two consecutive nucleotide units, the Ts and the Cs, have the same P-modification (i.e., both are a phosphorothioate diester). In the same oligonucleotide of (Rp, Sp)-ATsCsGA, TsCs forms a block, and it is a P-modification block.

In some embodiments, a blockmer is a “linkage blockmer,” e.g., at least two consecutive nucleotide units have identical stereochemistry and identical modifications at the linkage phosphorus. At least two consecutive nucleotide units form a “linkage block”. For instance, (Rp, Rp)-ATsCsGA is a linkage blockmer because at least two consecutive nucleotide units, the Ts and the Cs, have the same stereochemistry (both Rp) and P-modification (both phosphorothioate). In the same oligonucleotide of (Rp, Rp)-ATsCsGA, TsCs forms a block, and it is a linkage block.

In some embodiments, a blockmer is a “sugar modification blockmer,” e.g., at least two consecutive nucleotide units have identical sugar modifications. In some embodiments, a sugar modification blockmer is a 2′-F blockmer wherein at least two consecutive nucleotide units have 2′-F modification at their sugars. In some embodiments, a sugar modification blockmer is a 2′-OR blockmer wherein at lead two consecutive nucleotide units independently have 2′-OR modification at their sugars, wherein each R is independent as described in the present disclosure. In some embodiments, a sugar modification blockmer is a 2′-OMe blockmer wherein at least two consecutive nucleotide units have 2′-OMe modification at their sugars. In some embodiments, a sugar modification blockmer is a 2′-MOE blockmer wherein at lead two consecutive nucleotide units have 2′-MOE modification at their sugars. In some embodiments, a sugar modification blockmer is a LNA blockmer wherein at least two consecutive nucleotide units have LNA sugars.

In some embodiments, a blockmer comprises one or more blocks independently selected from a sugar modification block, a stereoblock, a P-modification block and a linkage block. In some embodiments, a blockmer is a stereoblockmer with respect to one block, and/or a P-modification blockmer with respect to another block, and/or a linkage blockmer with respect to yet another block.

Altmer: the term “altmer,” as used herein, refers to an oligonucleotide whose pattern of structural features characterizing each individual nucleotide unit is characterized in that no two consecutive nucleotide units of the oligonucleotide strand share a particular structural feature at the nucleobase, sugar, and/or the internucleotidic phosphorus linkage. In some embodiments, an altmer is designed such that it comprises a repeating pattern. In some embodiments, an altmer is designed such that it does not comprise a repeating pattern.

In some embodiments, an altmer is a “stereoaltmer,” e.g., no two consecutive nucleotide units have the same stereochemistry at the linkage phosphorus. For instance, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC.

Gapmer: as used herein, the term “gapmer” refers to an oligonucleotide characterized in that one or more nucleotide units (gap) do not have the structural features (e.g., nucleobase modifications, sugar modifications, internucleotidic linkage modifications, linkage phosphours stereochemistry, etc.) contained by nucleotide units flanking such one or more nucleotide units at both ends. In some embodiments, a gapmer comprises a gap of one or more natural phosphate linkages, independently flanked at both ends by non-natural internucleotidic linkages. In some embodiments, a gapmer is a sugar modification gapmer, wherein the gapmer comprises a gap of one or more nucleotide units comprising no sugar modifications which the flanking nucleotide at both ends contain. In some embodiments, a gapmer comprises a gap, wherein each nucleotide unit in the gap region contains no 2′-modification that is contained in nucleotide units flanking the gap at both ends. In some embodiments, a provided oligonucleotide comprising a gap, wherein each nucleotide unit in the gap region contains no 2′-OR modification, while nucleotide units flanking the gap at each end independently comprise a 2′-OR modification. In some embodiments, a provided oligonucleotide comprising a gap, wherein each nucleotide unit in the gap region contains no 2′-F modification, while nucleotide units flanking the gap at each end independently comprise a 2′-F modification.

Skipmer: as used herein, the term “skipmer” refers to a type of gapmer in which every other internucleotidic phosphorus linkage of the oligonucleotide strand is a phosphate diester linkage (a natural phosphate linkage), for example such as those found in naturally occurring DNA or RNA, and every other internucleotidic phosphorus linkage of the oligonucleotide strand is a modified internucleotidic linkage (a non-natural internucleotidic linkage).

For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

Unless otherwise specified, salts, such as pharmaceutically acceptable acid or base addition salts, stereoisomeric forms, and tautomeric forms, of compounds (e.g., oligonucleotides, agents, etc.) are included. Unless otherwise specified, singular forms “a” “an,” and “the” include the plural reference unless the context clearly indicates otherwise (and vice versa). Thus, for example, a reference to “a compound” may include a plurality of such compounds.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Synthetic oligonucleotides provide useful molecular tools in a wide variety of applications. For example, oligonucleotides are useful in therapeutic, diagnostic, research, and new nanomaterials applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, by their susceptibility to endo- and exo-nucleases. As such, various synthetic counterparts have been developed to circumvent these shortcomings. These include synthetic oligonucleotides that contain chemical modification. e.g., base modifications, sugar modifications, backbone modifications, etc., which, among other things, render these molecules less susceptible to degradation and improve other properties of oligonucleotides. Chemical modifications may also lead to certain undesired effects, such as increased toxicities, etc. From a structural point of view, modifications to natural phosphate linkages can introduce chirality, and certain properties of oligonucleotides may be affected by the configurations of the phosphorus atoms that form the backbone of the oligonucleotides.

In some embodiments, the chirality of the backbone (e.g. the configurations of the phosphorus atoms) or inclusion of natural phosphate linkages or non-natural internucleotidic linkages in the backbone and/or modifications of a sugar and/or nucleobase, and/or the addition of chemical moieties can affect properties and activities of oligonucleotides, e.g., the ability of a DMD oligonucleotide (e.g., an oligonucleotide antisense to a Dystrophin (DMD) transcript sequence) to skip one or more exons, and/or other properties of a DMD oligonucleotide, including but not limited to, increased stability, improved pharmacokinetics, and/or decreased immunogenicity, etc. Suitable assays for assessing properties and/or activities of provided compounds, e.g., oligonucleotides, and compositions thereof are widely known in the art and can be utilized in accordance with the present disclosure. For example, to test immunogenicity, various DMD oligonucleotides were tested in mouse serum in vivo and demonstrated minimal activation of cytokines, and various DMD oligonucleotides were tested ex vivo in human PBMC (peripheral blood mononuclear cells) for cytokine activity (e.g., IL-12p40, IL-12p70, IL-1alpha, IL-1beta, IL-6, MCP-1, MIP-1alpha, MIP-1beta, and TNF-alpha).

In some embodiments, technologies (e.g., oligonucleotides, compositions, and methods of use thereof) of the present disclosure can be utilized to target various nucleic acids (e.g., by hybridizing to a target sequence of a target nucleic acid, and/or providing level reduction, degradation, splicing modulation, transcription suppression, etc. of the target nucleic acid, etc.) In some embodiments, provided technologies are particularly useful for modulating splicing of transcripts, e.g., to increase levels of desired splicing products and/or to reduce levels of undesired splicing products. In some embodiments, provided technologies are particularly useful for reducing levels of transcripts, e.g., pre-mRNA. RNA, etc., and in many instances, reducing levels of products arising from or encoded by such transcripts such as mRNA, proteins, etc.

In some embodiments, a transcript is pre-mRNA. In some embodiments, a splicing product is mature RNA. In some embodiments, a splicing product is mRNA. In some embodiments, splicing modulation or alteration comprises skipping one or more exons. In some embodiments, splicing of a transcript is improved in that exon skipping increases levels of mRNA and proteins that have improved beneficial activities compared with absence of exon skipping. In some embodiments, an exon causing frameshift is skipped. In some embodiments, an exon comprising an undesired mutation is skipped. In some embodiments, an exon comprising a premature termination codon is skipped. An undesired mutation can be a mutation causing changes in protein sequences; it can also be a silent mutation. In some embodiments, a transcript is a transcript of Dystrophin (DMD).

In some embodiments, splicing of a transcript is improved in that exon skipping lowers levels of mRNA and proteins that have undesired activities compared with absence of exon skipping. In some embodiments, a target is knocked down through exon skipping which, by skipping one or more exons, causes premature stop codon and/or frameshift mutations. In some embodiments, provided oligonucleotides in provided compositions, e.g., oligonucleotides of a plurality, comprise base modifications, sugar modifications, and/or internucleotidic linkage modifications. In some embodiments, provided oligonucleotides comprise base modifications and sugar modifications. In some embodiments, provided oligonucleotides comprise base modifications and internucleotidic linkage modifications. In some embodiments, provided oligonucleotides comprise sugar modifications and internucleotidic modifications. In some embodiments, provided compositions comprise base modifications, sugar modifications, and internucleotidic linkage modifications. Example chemical modifications, such as base modifications, sugar modifications, internucleotidic linkage modifications, etc. are widely known in the art including but not limited to those described in this disclosure. In some embodiments, a modified base is substituted A, T, C, G or U. In some embodiments, a sugar modification is 2′-modification. In some embodiments, a 2′-modification is 2-F modification. In some embodiments, a 2′-modification is 2′-OR, wherein R¹is not hydrogen. In some embodiments, a 2′-modification is 2′-OR, wherein R¹is optionally substituted alkyl. In some embodiments, a 2′-modification is 2′-OMe. In some embodiments, a 2′-modification is 2′-MOE. In some embodiments, a modified sugar moiety is a bridged bicyclic or polycyclic ring. In some embodiments, a modified sugar moiety is a bridged bicyclic or polycyclic ring having 5-20 ring atoms wherein one or more ring atoms are optionally and independently heteroatoms. Example ring structures are widely known in the art, such as those found in BNA, LNA, etc. In some embodiments, provided oligonucleotides comprise both one or more modified internucleotidic linkages and one or more natural phosphate linkages. In some embodiments, oligonucleotides comprising both modified internucleotidic linkage and natural phosphate linkage and compositions thereof provide improved properties, e.g., activities and toxicities, etc. In some embodiments, a modified internucleotidic linkage is a chiral internucleotidic linkage. In some embodiments, a modified internucleotidic linkage is a phosphorothioate linkage. In some embodiments, a modified internucleotidic linkage is a substituted phosphorothioate linkage.

In some embodiments, provided oligonucleotides comprise one or more non-negatively charged internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic linkage is a positively charged internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a modified internucleotidic linkage (e.g., a non-negatively charged internucleotidic linkage) comprises optionally substituted triazolyl. In some embodiments, a modified internucleotidic linkage (e.g., a non-negatively charged internucleotidic linkage) comprises optionally substituted alkynyl. In some embodiments, a modified internucleotidic linkage comprises a triazole or alkyne moiety. In some embodiments, a triazole moiety, e.g., a triazolyl group, is optionally substituted. In some embodiments, a triazole moiety. e.g., a triazolyl group) is substituted. In some embodiments, a triazole moiety is unsubstituted. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted guanidine moiety. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted cyclic guanidine moiety. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted cyclic guanidine moiety and has the structure of:

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wherein W is O or S. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, a non-negatively charged internucleotidic linkage is stereochemically controlled.

In some embodiments, an internucleotidic linkage comprising an optionally substituted guanidine moiety is an internucleotidic linkage of formula I-n-2, I-n-3, I-n-4, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2 as described herein. In some embodiments, an internucleotidic linkage comprising an optionally substituted cyclic guanidine moiety is an internucleotidic linkage of formula II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2.

In some embodiments, provided oligonucleotides contain increased levels of one or more isotopes. In some embodiments, provided oligonucleotides are labeled, e.g., by one or more isotopes of one or more elements. e.g., hydrogen, carbon, nitrogen, etc. In some embodiments, provided oligonucleotides in provided compositions. e.g., oligonucleotides of a plurality, comprise base modifications, sugar modifications, and/or internucleotidic linkage modifications, wherein the oligonucleotides contain an enriched level of deuterium. In some embodiments, provided oligonucleotides are labeled with deuterium (replacing —¹H with —²H) at one or more positions. In some embodiments, one or more ¹H of an oligonucleotide or any moiety conjugated to the oligonucleotide (e.g., a targeting moiety, lipid, etc.) is substituted with ²H. Such oligonucleotides can be used in any composition or method described herein.

In some embodiments, in an oligonucleotide, a pattern of backbone chiral centers can provide improved activity(s) or characteristic(s), including but not limited to: improved skipping of one or more exons, increased stability, increased activity, increased stability and activity, low toxicity, low immune response, improved protein binding profile, increased binding to certain proteins, and/or enhanced delivery.

In some embodiments, a pattern of backbone chiral centers is or comprises S, SS, SSS. SSSS, SSSSS, SSSSSS, SSSSSSS, SOS, SSOSS, SSSOSSS, SSSSOSSSS, SSSSSOSSSSS, SSSSSSOSSSSSS, SSSSSSSOSSSSSSS, SSSSSSSSOSSSSSSSS, SSSSSSSSSOSSSSSSSSS, SOSOSOSOS, SSOSOSOSOSS, SSSOSOSOSOSOSSS, SSSSOSOSOSOSSSS, SSSSSOSOSOSOSSSSS, SSSSSSOSOSOSOSSSSSS, SOSOSSOOS, SSOSOSSOOSS, SSSOSOSSOOSSS, SSSSOSOSSOOSSSS, SSSSSOSOSSOOSSSSS, SSSSSSOSOSSOOSSSSSS, SOSOOSOOS, SSOSOOSOOSS, SSSOSOOSOOSSS, SSSSOSOOSOOSSSS, SSSSSOSOOSOOSSSSS, SSSSSSOSOOSOOSSSSSS, SOSOSSOOS, SSOSOSSOOSO, SSSOSOSSOOSOS, SSSSOSOSSOOSOSS, SSSSSOSOSSOOSOSSS, SSSSSSOSOSSOOSOSSSS, SOSOOSOOSO, SSOSOOSOOSOS, SSSOSOOSOOSOS, SSSSOSOOSOOSOSS, SSSSSOSOOSOOSOSSS, SSSSSSOSOOSOOSOSSSS, SSOSOSSOO, SSSOSOSSOOS, SSSSOSOSSOOS, SSSSSOSOSSOOSS, SSSSSSOSOSSOOSSS, OSSSSSOSOSSOOSSS, OOSSSSSSOSOSSOOS, OOSSSSSSOSOSSOOSS, OOSSSSSSOSOSSOOSSS, OOSSSSSSOSOSSOOSSSS, OOSSSSSSOSOSSOOSSSSS, and/or OOSSSSSSOSOSSOOSSSSSS, RS, SR, SRS, SRSS, SSRS, RR, RRR, RRRR, RRRRR, SRR, RRS, SRRS, SSRRS, SRRSS, SRRR, RRRS, SRRRS, SSRRRS, SSRRRS, RSRRR, SRRRSR, SSSRSSS, SSSSRSSSS, SSSSSRSSSSS, SSSSSSRSSSSSS, SSSSSSSRSSSSSSS, SSSSSSSSRSSSSSSSS, SSSSSSSSSRSSSSSSSSS, SRSRSRSRS, SSRSRSRSRSS, SSSRSRSRSRSSS, SSSSRSRSRSRSSSS, SSSSSRSRSRSRSSSSS, SSSSSSRSRSRSRSSSSSS, SRSRSSRRS, SSRSRSSRRSS, SSSRSRSSRRSSS, SSSSRSRSSRRSSSS, SSSSSRSRSSRRSSSSS, SSSSSSRSRSSRRSSSSSS, SRSRRSRRS, SSRSRRSRRSS, SSSRSRRSRRSSS, SSSSRSRRSRRSSSS, SSSSSRSRRSRRSSSSS, SSSSSSRSRRSRRSSSSSS, SRSRSSRRS, SSRSRSSRRSR, SSSRSRSSRRSRS, SSSSRSRSSRRSRSS, SSSSSRSRSSRRSRSSS, SSSSSSRSRSSRRSRSSSS, SRSRRSRRSR, SSRSRRSRRSRS, SSSRSRRSRRSRS, SSSSRSRRSRRSRSS, SSSSSRSRRSRRSRSSS, SSSSSSRSRRSRRSRSSSS, SSRSRSSRR, SSSRSRSSRRS, SSSSRSRSSRRS, SSSSSRSRSSRRSS, SSSSSSRSRSSRRSSS, RSSSSSSRSRSSRRSSS, RRSSSSSSRSRSSRRS, RRSSSSSSRSRSSRRSS, RRSSSSSSRSRSSRRSSS, RRSSSSSSRSRSSRRSSSS, RRSSSSSSRSRSSRRSSSSS, (R)_n(S)_m, (S)_t(R)_n, (O)_t(R)_n(S)_m, (S)_t(O)_m, (O)_m(S)_t, (S)_t(R)_n(S)_m, (S)_t(O)_m(S)_n, (S)_t(O)_m, wherein t, m and n are independently 1 to 20. O is a non-chiral internucleotidic linkage, R is a Rp chiral internucleotidic linkage, and S is an Sp chiral internucleotidic linkage. In some embodiments, the non-chiral center is a phosphodiester linkage. In some embodiments, the chiral center in a Sp configuration is a phosphorothioate linkage.

In some embodiments, the 5′-end region of provided oligonucleotides, e.g., a 5′-wing, comprises a stereochemistry pattern of S. SS, SSS, SSSS, SSSSS, SSSSSS, or SSSSSS. In some embodiments, each S is or represents an Sp phosphorothioate internucleotidic linkage. In some embodiments, the 5′-end region of provided oligonucleotides, e.g., a 5′-wing, comprises a stereochemistry pattern of S, SS, SSS, SSSS. SSSSS, SSSSSS, or SSSSSS, wherein the first S represents the first (the 5′-end) internucleotidic linkage of a provided oligonucleotide. In some embodiments, one or more nucleotidic units comprising an Sp internucleotidic linkage in the 5′-end region independently comprise —F. In some embodiments, each nucleotidic unit comprising an Sp internucleotidic linkage in the 5′-end region independently comprises —F. In some embodiments, one or more nucleotidic units comprising an Sp internucleotidic linkage in the Y-end region independently comprise a sugar modification. In some embodiments, each nucleotidic unit comprising an Sp internucleotidic linkage in the 5′-end region independently comprises a sugar modification. In some embodiments, each 2′-modification is the same. In some embodiments, a sugar modification is a 2′-modification. In some embodiments, a 2′-modification is 2′-OR¹. In some embodiments, a 2′-modification is 2′-F. In some embodiments, the 3′-end region of provided oligonucleotides, e.g., a 3′-wing, comprises a stereochemistry pattern of S, SS, SSS, SSSS, SSSSS, SSSSSS, or SSSSSS. In some embodiments, each S is or represents an Sp phosphorothioate internucleotidic linkage. In some embodiments, the 3′-end region of provided oligonucleotides, e.g., a 3′-wing, comprises a stereochemistry pattern of S. SS, SSS, SSSS, SSSSS, SSSSSS, or SSSSSS, wherein the last S represents the last (the 3′-end) internucleotidic linkage of a provided oligonucleotide. In some embodiments, each S represents an Sp phosphorothioate internucleotidic linkage. In some embodiments, one or more nucleotidic units comprising an Sp internucleotidic linkage in the 3′-end region independently comprise —F. In some embodiments, each nucleotidic unit comprising an Sp internucleotidic linkage in the 3′-end region independently comprises —F. In some embodiments, one or more nucleotidic units comprising an Sp internucleotidic linkage in the 3′-end region independently comprise a sugar modification. In some embodiments, each nucleotidic unit comprising an Sp internucleotidic linkage in the 3′-end region independently comprises a sugar modification. In some embodiments, each 2′-modification is the same. In some embodiments, a sugar modification is a 2′-modification. In some embodiments, a 2′-modification is 2′-OR¹. In some embodiments, a 2′-modification is 2′-F. In some embodiments, provided oligonucleotides comprise both a 5′-end region, e.g., a 5′-wing, and a 3′-end region, e.g., a 3′-end wing, as described herein. In some embodiments, the 5′-end region comprises a stereochemistry pattern of SS, wherein the first S represents the first internucleotidic linkage of a provided oligonucleotide, the 3′-end region comprises a stereochemistry pattern of SS, wherein one or more nucleotidic unit comprising an Sp internucleotidic linkage in the 5′- or 3′-end region comprise —F. In some embodiments, the 5′-end region comprises a stereochemistry pattern of SS, wherein the first S represents the first internucleotidic linkage of a provided oligonucleotide, the 3′-end region comprises a stereochemistry pattern of SS, wherein one or more nucleotidic unit comprising an Sp internucleotidic linkage in the 5′- or 3′-end region comprise a 2′-F sugar modification. In some embodiments, provided oligonucleotides further comprise a middle region between the 5-end and 3′-end regions, e.g., a core region, which comprises one or more natural phosphate linkages. In some embodiments, provided oligonucleotides further comprise a middle region between the 5′-end and 3′-end regions, e.g., a core region, which comprises one or more natural phosphate linkages and one or more internucleotidic linkages. In some embodiments, a middle region comprises one or more sugar moieties, wherein each sugar moiety independently comprises a 2′-OR modification. In some embodiments, a middle region comprises one or more sugar moieties comprising no 2′-F modification. In some embodiments, a middle region comprises one or more Sp internucleotidic linkages. In some embodiments, a middle region comprises one or more Sp internucleotidic linkages and one or more natural phosphate linkages. In some embodiments, a middle region comprises one or more Rp internucleotidic linkages. In some embodiments, a middle region comprises one or more Rp internucleotidic linkages and one or more natural phosphate linkages. In some embodiments, a middle region comprises one or more Rp internucleotidic linkages and one or more Sp internucleotidic linkages.

In some embodiments, provided oligonucleotides comprise one or more modified internucleotidic linkages. In some embodiments, provided oligonucleotides comprise one or more chiral modified internucleotidic linkages. In some embodiments, provided oligonucleotides comprise one or more chirally controlled chiral modified internucleotidic linkages. In some embodiments, provided oligonucleotides comprise one or more natural phosphate linkages. In some embodiments, provided oligonucleotides comprise one or more modified internucleotidic linkages and one or more natural phosphate linkages. In some embodiments, a modified internucleotidic linkage is a phosphorothioate linkage. In some embodiments, each modified internucleotidic linkage is a phosphorothioate linkage. In some embodiments, a modified internucleotidic linkage comprises a triazole, substituted triazole, alkyne or Tmg.

In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising a triazole or alkyne moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising an optionally substituted triazolyl or alkynyl. In some embodiments, such a nucleic acid is a siRNA, double-straned siRNA, single-stranded siRNA, oligonucleotide, gapmer, skipmer, blockmer, antisense oligonucleotide, antagomir, microRNA, pre-microRNA, antimir, supermir, ribozyme, U1 adaptor, RNA activator, RNAi agent, decoy oligonucleotide, triplex forming oligonucleotide, aptamer or adjuvant. In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a modified internucleotidic linkage comprising a triazole or alkyne moiety. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which comprises a modified internucleotidic linkage comprising a triazole or alkyne moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising a triazole moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising optionally substituted triazolyl. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising a substituted triazole moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising an alkyne moiety. In some embodiments, the present disclosure pertains to a nucleic acid or oligonucleotide which comprises, at a 5′ end, a structure of the formula:

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wherein W is O or S. In some embodiments, an oligonucleotide is a single-stranded siRNA which comprises, at a 5′ end, a structure of the formula:

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wherein W is O or S. In some embodiments, a modified internucleotidic linkage is any modified internucleotidic linkage described in Krishna et al. 2012 J. Am. Chem. Soc. 134: 11618-11631.

In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage which comprises a guanidine moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage which comprises a cyclic guanidine moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage which comprises a cyclic guanidine moiety and has the structure of:

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wherein W is O or S. In some embodiments, a neutral internucleotidic linkage or internucleotidic linkage comprising a cyclic guanidine is chirally controlled. In some embodiments, a nucleic acid comprising a non-negatively charged internucleotidic linkage or a modified internucleotidic linkage comprising a cyclic guanidine moiety is a siRNA, double-straned siRNA, single-stranded siRNA, oligonucleotide, gapmer, skipmer, blockmer, antisense oligonucleotide, antagomir, microRNA, pre-microRNA, antimir, supermir, ribozyme, U1 adaptor, RNA activator, RNAi agent, decoy oligonucleotide, triplex forming oligonucleotide, aptamer or adjuvant. In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a modified internucleotidic linkage which comprises a cyclic guanidine moiety. In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a modified internucleotidic linkage which has the structure of:

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wherein W is O or S. In some embodiments, a neutral internucleotidic linkage or internucleotidic linkage comprising a cyclic guanidine moiety is chirally controlled. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which comprises a modified internucleotidic linkage comprising a cyclic guanidine moiety. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which comprises a modified internucleotidic linkage which has the structure of:

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wherein W is O or S. In some embodiments, a neutral internucleotidic linkage or internucleotidic linkage comprising a cyclic guanidine moiety is chirally controlled. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising a cyclic guanidine moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage which has the structure of:

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wherein W is O or S. In some embodiments, the present disclosure pertains to a nucleic acid or oligonucleotide which comprises, at a 5′ end, a structure comprising a cyclic guanidine moiety. In some embodiments, the present disclosure pertains to a nucleic acid or oligonucleotide which comprises, at a 5′ end, a structure of the formula:

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wherein W is O or S. In some embodiments, the oligonucleotide is a single-stranded siRNA which comprises, at a 5′ end, a structure comprising a cyclic guanidine moiety. In some embodiments, the oligonucleotide is a single-stranded siRNA which comprises, at a 5′ end, a structure of the formula:

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wherein W is O or S. In some embodiments, the internucleotidic linkage comprise

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(wherein W is O or S) and is chirally controlled.

In some embodiments, provided oligonucleotides can bind to a transcript, and change the splicing pattern of the transcript. In some embodiments, provided oligonucleotides provides exon-skipping of an exon, with efficiency greater than a comparable oligonucleotide under one or more suitable conditions, e.g., as described herein. In some embodiments, a provided skipping efficiency is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% more than, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more fold of, that of a comparable oligonucleotide under one or more suitable conditions, e.g., as described herein. In some embodiments, a comparable oligonucleotide is an oligonucleotide which has fewer or no chirally controlled internucleotidic linkages and/or fewer or no non-negatively charged internucleotidic linkages but is otherwise identical.

In some embodiments, the present disclosure demonstrates that 2′-F modifications, among other things, can improve exon-skipping efficiency. In some embodiments, the present disclosure demonstrates that Sp internucleotidic linkages, among other things, at the 5′- and 3′-ends can improve oligonucleotide stability. In some embodiments, the present disclosure demonstrates that, among other things, natural phosphate linkages and/or Rp internucleotidic linkages can improve removal of oligonucleotides from a system. As appreciated by a person having ordinary skill in the art, various assays known in the art can be utilized to assess such properties in accordance with the present disclosure.

In some embodiments, provided oligonucleotides comprise one or more modified sugar moieties. In some embodiments, a modified sugar moiety comprises a 2′-modification. In some embodiments, a modified sugar moiety comprises a 2′-modification. In some embodiments, a 2′-modification is 2′-OR¹. In some embodiments, a 2′-modification is a 2′-OMe. In some embodiments, a 2′-modification is a 2′-MOE. In some embodiments, a 2-modification is an LNA sugar modification. In some embodiments, a 2′-modification is 2′-F. In some embodiments, each sugar modification is independently a 2′-modification. In some embodiments, each sugar modification is independently 2′-OR¹or 2′-F. In some embodiments, each sugar modification is independently 2′-OR¹or 2′-F, wherein R¹is optionally substituted C₁alkyl. In some embodiments, each sugar modification is independently 2′-OR¹or 2′-F, wherein at least one is 2′-F. In some embodiments, each sugar modification is independently 2′—OR¹or 2′-F, wherein R¹is optionally substituted C_1-6alkyl, and wherein at least one is 2′-OR¹. In some embodiments, each sugar modification is independently 2′-OR¹or 2′-F, wherein at least one is 2′-F, and at least one is 2′-OR¹. In some embodiments, each sugar modification is independently 2′-OR¹or 2′-F, wherein R¹is optionally substituted C_1-6alkyl, and wherein at least one is 2′-F, and at least one is 2′-OR¹.

In some embodiments, 5% or more of the sugar moieties of provided oligonucleotides are modified. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the sugar moieties of provided oligonucleotides are modified. In some embodiments, each sugar moiety of provided oligonucleotides is modified. In some embodiments, a modified sugar moiety comprises a 2′-modification. In some embodiments, a modified sugar moiety comprises a 2′-modification. In some embodiments, a 2′-modification is 2′-OR¹. In some embodiments, a 2′-modification is a 2′-OMe. In some embodiments, a 2′-modification is a 2′-MOE. In some embodiments, a 2′-modification is an LNA sugar modification. In some embodiments, a 2′-modification is 2′-F. In some embodiments, each sugar modification is independently a 2′-modification. In some embodiments, each sugar modification is independently 2′-OR¹or 2′-F. In some embodiments, each sugar modification is independently 2′-OR¹or 2′-F, wherein R¹is optionally substituted C_1-6alkyl. In some embodiments, each sugar modification is independently 2′-OR¹or 2′-F, wherein at least one is 2′-F. In some embodiments, each sugar modification is independently 2′-OR¹or 2′-F, wherein R¹is optionally substituted C_1-6alkyl, and wherein at least one is 2′-OR¹. In some embodiments, each sugar modification is independently 2′-OR¹or 2′-F, wherein at least one is 2′-F. and at least one is 2′-OR¹. In some embodiments, each sugar modification is independently 2′-OR¹or 2′-F, wherein R¹is optionally substituted C_1-6alkyl, and wherein at least one is 2′-F, and at least one is 2′-OR¹.

In some embodiments, provided oligonucleotides comprise one or more 2′-F. In some embodiments, provided oligonucleotides comprise two or more 2′-F.

In some embodiments, provided oligonucleotides comprise alternating 2′-F modified sugar moieties and 2′-OR¹modified sugar moieties. In some embodiments, provided oligonucleotides comprise alternating 2′-F modified sugar moieties and 2′-OMe modified sugar moieties, e.g., [(2′-F)(2′-OMe)]x, [(2′-OMe)(2′-F)]x, etc., wherein x is 1-50. In some embodiments, provided oligonucleotides comprise at least two pairs of alternating 2′-F and 2′-OMe modifications. In some embodiments, provided oligonucleotides comprises alternating phosphodiester and phosphorothioate internucleotidic linkages, e.g., [(PO)(PS)]x, [(PS)(PO)]x, etc., wherein x is 1-50. In some embodiments, provided oligonucleotides comprise at least two pairs of alternating phosphodiester and phosphorothioate internucleotidic linkages.

In some embodiments, provided oligonucleotides comprise one or more natural phosphate linkages and one or more modified internucleotidic linkages. In some embodiments, provided oligonucleotides comprise one or more natural phosphate linkages and one or more modified internucleotidic linkages and one or more non-negatively charged internucleotidic linkages.

In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides, wherein:

oligonucleotides of the plurality have the same base sequence; and

oligonucleotides of the plurality comprise one or more modified sugar moieties, or comprise one or more natural phosphate linkages and one or more modified internucleotidic linkages.

In some embodiments, oligonucleotides of a plurality comprise one or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise one or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise 2 or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise 3 or more modified sugar moieties.

In some embodiments, provided compositions alter transcript splicing so that an undesired target and/or biological function are suppressed.

In some embodiments, provided compositions alter transcript splicing so a desired target and/or biological function is enhanced.

In some embodiments, each oligonucleotide of a plurality comprises one or more modified sugar moieties and modified internucleotidic linkages.

In some embodiments, each oligonucleotide of a plurality comprises no more than about 25 consecutive unmodified sugar moieties

In some embodiments, each oligonucleotide of a plurality comprises no more than about 95% unmodified sugar moieties. In some embodiments, each oligonucleotide of a plurality comprises no more than about 90% unmodified sugar moieties. In some embodiments, each oligonucleotide of a plurality comprises no more than about 85% unmodified sugar moieties. In some embodiments, each oligonucleotide of a plurality comprises no more than about 15 consecutive unmodified sugar moieties.

In some embodiments, each oligonucleotide of a plurality comprises no more than about 95% unmodified sugar moieties.

In some embodiments, each oligonucleotide of a plurality comprises two or more modified internucleotidic linkages.

In some embodiments, about 5% of the internucleotidic linkages in each oligonucleotide of a plurality are modified internucleotidic linkages.

In some embodiments, each oligonucleotide of a plurality comprises no more than about 25 consecutive natural phosphate linkages. In some embodiments, each oligonucleotide of a plurality comprises no more than about 20 natural phosphate linkages.

In some embodiments, oligonucleotides of a plurality comprise no natural DNA nucleotide units. In some embodiments, oligonucleotides of a plurality comprise no more than 30 natural DNA nucleotides. In some embodiments, oligonucleotides of a plurality comprise no more than 30 consecutive DNA nucleotides.

In some embodiments, compared to a reference condition, provided chirally controlled oligonucleotide compositions are surprisingly effective. In some embodiments, desired biological effects (e.g., as measured by increased levels of desired mRNA, proteins, etc., decreased levels of undesired mRNA, proteins, etc.) can be enhanced by more than 5, 10, 15, 20, 25, 30, 40, 50, or 100 fold. In some embodiments, a change is measured by increase of a desired mRNA level compared to a reference condition. In some embodiments, a change is measured by decrease of an undesired mRNA level compared to a reference condition. In some embodiments, a reference condition is absence of oligonucleotide treatment. In some embodiments, a reference condition is a stereorandom composition of oligonucleotides having the same base sequence and chemical modifications.

In some embodiments, a desired biological effect is: improved skipping of one or more exons, increased stability, increased activity, increased stability and activity, low toxicity, low immune response, improved protein binding profile, increased binding to certain proteins, and/or enhanced delivery. In some embodiments, a desired biological effect is enhanced by more than 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, or 500 fold.

In some embodiments, the structure of a DMD oligonucleotide is or comprises a wing-core-wing, wing-core, or core-wing structure. In some embodiments, a 5′-wing is a 5′-end region. In some embodiments, a 3′-wing is a 3′-end region. In some embodiments, a core is a middle region. In some embodiments, a 5′-end region is a 5′-wing region. In some embodiments, a 3′-end region is a 3′-wing region. In some embodiments, a middle region is a core region.

In some embodiments, an oligonucleotide having a wing-core-wing structure is designated a gapmer. In some embodiments, a gapmer is asymmetric, in that the chemistry of one wing is different from the chemistry of the other wing. In some embodiments, a gapmer is asymmetric, in that the chemistry of one wing is different from the chemistry of the other wing, wherein the wings differ in sugar modifications and/or internucleotidic linkages, or patterns thereof. In some embodiments, a gapmer is asymmetric, in that the chemistry of one wing is different from the chemistry of the other wing, wherein the wings differ in sugar modifications, wherein one wing comprises a sugar modification not present in the other wing; or both wings each comprise a sugar modification not found in the other wing; or both wings comprise different patterns of the same types of sugar modifications; or one wing comprises only one type of sugar modification, while the other wing comprises two types of sugar modifications; etc.

In some embodiments, an internucleotidic linkage between a wing region and a core region is considered part of the wing region. In some embodiments, an internucleotidic linkage between a 5′-wing region and a core region is considered part of the wing region. In some embodiments, an internucleotidic linkage between a 3′-wing region and a core region is considered part of the wing region. In some embodiments, an internucleotidic linkage between a wing region and a core region is considered part of the core region. In some embodiments, an internucleotidic linkage between a 5′-wing region and a core region is considered part of the core region. In some embodiments, an internucleotidic linkage between a 3-wing region and a core region is considered part of the core region.

In some embodiments, provided oligonucleotides comprise two wing and one core regions. In some embodiments, provided oligonucleotides comprises a 5′-wing-core-wing-3′ structure. In some embodiments, provided oligonucleotides are of a 5′-wing-core-wing-3′ gapmer structure. In some embodiments, the two wing regions are identical. In some embodiments, the two wing regions are different. In some embodiments, the two wing regions are identical in chemical modifications. In some embodiments, the two wing regions are identical in 2′-modifications. In some embodiments, the two wing regions are identical in internucleotidic linkage modifications. In some embodiments, the two wing regions are identical in patterns of backbone chiral centers. In some embodiments, the two wing regions are identical in pattern of backbone linkages. In some embodiments, the two wing regions are identical in pattern of backbone linkage types. In some embodiments, the two wing regions are identical in pattern of backbone phosphorus modifications.

A wing region can be differentiated from a core region in that a wing region contains a different structure feature than a core region. For example, in some embodiments, a wing region differs from a core region in that they have different sugar modifications, base modifications, internucleotidic linkages, internucleotidic linkage stereochemistry, etc. In some embodiments, a wing region differs from a core region in that they have different 2′-modifications of the sugars.

In some embodiments, a region (e.g., a wing region, a core region, a 5′-end region, a middle region, a 3′-end region, etc.) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more modified internucleotidic linkages. In some embodiments, a region comprises 2 or more modified internucleotidic linkages. In some embodiments, a region comprises 3 or more modified internucleotidic linkages. In some embodiments, a region comprises 4 or more modified internucleotidic linkages. In some embodiments, a region comprises 5 or more modified internucleotidic linkages. In some embodiments, a region comprises 6 or more modified internucleotidic linkages. In some embodiments, a region comprises 7 or more modified internucleotidic linkages. In some embodiments, a region comprises 8 or more modified internucleotidic linkages. In some embodiments, a region comprises 9 or more modified internucleotidic linkages. In some embodiments, a region comprises 10 or more modified internucleotidic linkages.

In some embodiments, provided oligonucleotides comprise consecutive nucleoside units each of which comprises no 2′-OR¹modifications (wherein R¹is not hydrogen). In some embodiments, provided oligonucleotides comprise consecutive nucleoside units whose 2′-positions are independently unsubstituted or substituted with 2′-F. In some embodiments, such an oligonucleotide is a DMD oligonucleotide. In some embodiments, each of the consecutive nucleoside units is independently preceded and/or followed by a modified internucleotidic linkage. In some embodiments, each of the consecutive nucleoside units is independently preceded and/or followed by a phosphorothioate linkage. In some embodiments, each of the consecutive nucleoside units is independently preceded and/or followed by a chirally controlled modified internucleotidic linkage. In some embodiments, each of the consecutive nucleoside units is independently preceded and/or followed by a chirally controlled phosphorothioate linkage.

In some embodiments, a modified internucleotidic linkage has the structure of formula I. I-a, I-b, I-c, 1-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, etc., or a salt form thereof. In some embodiments, a modified internucleotidic linkage has a structure of formula I or a salt form thereof. In some embodiments, a modified internucleotidic linkage has a structure of formula I-a or a salt form thereof.

In some embodiments, a modified internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, a modified internucleotidic linkage is a positively-charged internucleotidic linkage. In some embodiments, a modified internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, such a heterocyclyl or heteroaryl group is of a 5-membered ring. In some embodiments, such a heterocyclyl or heteroaryl group is of a 6-membered ring.

In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a heteroaryl group is directly bonded to a linkage phosphorus. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, a non-negatively charged internucleotidic linkage comprises an unsubstituted triazolyl group, e.g.,

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In some embodiments, a non-negatively charged internucleotidic linkage comprises a substituted triazolyl group. e.g.,

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In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, at least two heteroatoms are nitrogen. In some embodiments, a heterocyclyl group is directly bonded to a linkage phosphorus. In some embodiments, a heterocyclyl group is bonded to a linkage phosphorus through a linker, e.g., ═N— when the heterocyclyl group is part of a guanidine moiety who directed bonded to a linkage phosphorus through its ═N—. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted

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group. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted

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group. In some embodiments, a non-negatively charged internucleotidic linkage comprises an substituted

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group. In some embodiments, a non-negatively charged internucleotidic linkage comprises a

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group. In some embodiments, each R¹is independently optionally substituted C_1-20alkyl. In some embodiments, each R¹is independently optionally substituted C_1-6alkyl. In some embodiments, each R¹is independently methyl. In some embodiments, the two R¹groups are different; for example, in some embodiments, one R¹is methyl, and the other is —CH₂(CH₂)₁₀CH₃.

In some embodiments, a modified internucleotidic linkage. e.g., a non-negatively charged internucleotidic linkage, comprises a triazole or alkyne moiety, each of which is optionally substituted. In some embodiments, a modified internucleotidic linkage comprises a triazole moiety. In some embodiments, a modified internucleotidic linkage comprises a unsubstituted triazole moiety. In some embodiments, a modified internucleotidic linkage comprises a substituted triazole moiety. In some embodiments, a modified internucleotidic linkage comprises an alkyl moiety. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted alkynyl group. In some embodiments, a modified internucleotidic linkage comprises an unsubstituted alkynyl group. In some embodiments, a modified internucleotidic linkage comprises a substituted alkynyl group. In some embodiments, an alkynyl group is directly bonded to a linkage phosphorus.

In some embodiments, an oligonucleotide comprising a non-negatively charged internucleotidic linkage can comprise any structure, format, or portion thereof described herein. In some embodiments, an oligonucleotide comprising a non-negatively charged internucleotidic linkage can comprise any structure, format, or portion thereof described herein as being a component of a DMD oligonucleotide. In some embodiments, any structure, format, or portion thereof described as being a component of any DMD oligonucleotide can be used in any oligonucleotide comprising a non-negatively charged internucleotidic linkage, whether or not that oligonucleotide targets DMD or not, or whether the oligonucleotide is capable of mediating skipping of a DMD exon or not. In some embodiments, an oligonucleotide comprising a non-negatively charged internucleotidic is double-stranded or single-stranded.

In some embodiments, a provided oligonucleotide composition is characterized in that, when it is contacted with the transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof. In some embodiments, a desired splicing product is increased 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 fold or more. In some embodiments, a desired splicing reference is absent (e.g., cannot be reliably detected by quantitative PCR) under reference conditions. In some embodiments, as exemplified in the present disclosure, levels of the plurality of oligonucleotides, e.g., a plurality of oligonucleotides, in provided compositions are pre-determined.

In some embodiments, provided oligonucleotides, e.g., oligonucleotides of a plurality in a provided composition, comprise two or more regions. In some embodiments, provided comprise a 5′-end region, a 3′-end region, and a middle region in between. In some embodiments, provided oligonucleotides have two wing and one core regions. In some embodiments, provided oligonucleotides are of a wing-core-wing structure. In some embodiments, the two wing regions are identical. In some embodiments, the two wing regions are different. In some embodiments, a 5′-end region is a 5′-wing region. In some embodiments, a 5′-wing region is a 5′-nd region. In some embodiments, a 3′-end region is a 3′-wing region. In some embodiments, a 3′-wing region is a 3′-end region. In some embodiments, a core region is a middle region.

In some embodiments, a region (e.g., a 5′-wing region, a 3′-wing, a core region, a 5′-end region, a middle region, etc.) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more modified internucleotidic linkages. In some embodiments, a region comprises 2 or more modified internucleotidic linkages. In some embodiments, the one or more modified internucleotidic linkages are consecutive. In some embodiments, a region comprises 2 or more consecutive modified internucleotidic linkages. In some embodiments, each internucleotidic linkage in a region is independently a modified internucleotidic linkage, wherein each chiral internucleotidic linkage is optionally and independently chirally controlled. In some embodiments, a chiral internucleotidic linkage or a modified internucleotidic linkage has the structure of formula I or a salt form thereof. In some embodiments, a chiral internucleotidic linkage or a modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, each chiral internucleotidic linkage or a modified internucleotidic linkage independently has the structure of formula I or a salt form thereof. In some embodiments, each chiral internucleotidic linkage or a modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, a region comprises 3 or consecutive modified internucleotidic linkages.

In some embodiments, a wing region comprises one or more natural phosphate linkages. In some embodiments, a core region comprises one or more natural phosphate linkages. In some embodiments, a 5′-end region comprises one or more natural phosphate linkages. In some embodiments, a 3′-end region comprises one or more natural phosphate linkages. In some embodiments, a middle region comprises one or more natural phosphate linkages. In some embodiments, the one or more natural phosphate linkages are consecutive.

In some embodiments, a natural phosphate linkage follows (e.g., connected to a 3′-position of a sugar moiety) or precedes (e.g., connected to a 5′-position of a sugar moiety) a nucleoside unit whose sugar moiety comprises a 2′-OR¹modification, wherein R¹is not hydrogen. In some embodiments, R¹is optionally substituted C_1-6aliphatic. In some embodiments, a modified internucleotidic linkage follows (e.g., connected to a 3′-position of a sugar moiety) or precedes (e.g., connected to a 5′-position of a sugar moiety) all or most (e.g., more than 55%, 60%, 70%, 80%, 90%, 95%, etc.) nucleoside units whose sugar moiety comprises no 2′-OR¹modification, wherein R¹is not hydrogen (e.g., those having two 2′-H at the 2′-position, those having a 2′-H and a 2′-F at the 2′-position (2′-F modified), etc.).

In some embodiments, a region comprises one or more nucleoside units comprising sugar modifications, e.g., 2′-F, 2′-OR¹, LNA sugar modifications, etc. In some embodiments, each sugar in a region is independently modified. In some embodiments, each sugar moiety in a wing, a 5′-end region, and/or a Y-end region is modified. In some embodiments, a modification is a 2′-modification. In some embodiments, a modification can increase stability, e.g., 2′-OR¹where in R¹is not —H (e.g., is optionally substituted C_1-6aliphatic), LNA sugar modifications, etc. In some embodiments, a region, e.g., a core region or a middle region, comprise no sugar modifications (or no 2′-OR sugar modifications/LNA modifications etc.). In some embodiments, such a core/middle region can form a duplex with a RNA for recognition/binding of a protein, e.g., RNase H, for the protein to perform one or more of its functions (e.g., in the case of RNase H, its binding and cleavage of DNA/RNA duplex).

A region and/or a provided oligonucleotide may have various patterns of backbone chiral centers. In some embodiments, each internucleotidic linkage in a region is a chirally controlled internucleotidic linkage and is Sp. In some embodiments, the 5′-end and/or the 3′-end internucleotidic linkage is a chirally controlled internucleotidic linkage and is Sp. In some embodiments, the pattern of backbone chiral centers of a wing region, a 5′-end region, and/or a Y-end region is or comprises a 5′-end and/or a 3′-end internucleotidic linkage which is a chirally controlled internucleotidic linkage and is Sp, with the other internucleotidic linkages in the region independently being an natural phosphate linkage, a modified internucleotidic linkage, or a chirally controlled internucleotidic linkage (Sp or Rp). In some embodiments, such patterns provide stability. Many example patterns of backbone chiral centers are described in the present disclosure.

In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides defined by having:

1) a common base sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is a substantially pure preparation of a single oligonucleotide in that a controlled level of the oligonucleotides in the composition have the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers.

In some embodiments, oligonucleotides having a common base sequence may have the same pattern of nucleoside modifications, e.g., sugar modifications, base modifications, etc. In some embodiments, a pattern of nucleoside modifications may be represented by a combination of locations and modifications. In some embodiments, all non-chiral linkages (e.g., PO) may be omitted. In some embodiments, oligonucleotides having the same base sequence have the same constitution.

As understood by a person having ordinary skill in the art, a stereorandom or racemic preparation of oligonucleotides is prepared by non-stereoselective and/or low-stereoselective coupling of nucleotide monomers, typically without using any chiral auxiliaries, chiral modification reagents, and/or chiral catalysts. In some embodiments, in a substantially racemic (or chirally uncontrolled) preparation of oligonucleotides, all or most coupling steps are not chirally controlled in that the coupling steps are not specifically conducted to provide enhanced stereoselectivity. An example substantially racemic preparation of oligonucleotides is the preparation of phosphorothioate oligonucleotides through sulfurizing phosphite triesters from commonly used phosphoramidite oligonucleotide synthesis with either tetraethylthiuram disulfide or (TETD) or 3H-1, 2-bensodithiol-3-one 1, 1-dioxide (BDTD), a well-known process in the art. In some embodiments, substantially racemic preparation of oligonucleotides provides substantially racemic oligonucleotide compositions (or chirally uncontrolled oligonucleotide compositions). In some embodiments, at least one coupling of a nucleotide monomer has a diastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, each internucleotidic linkage independently has a diastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, a diastereoselectivity is lower than about 60:40. In some embodiments, a diastereoselectivity is lower than about 70:30. In some embodiments, a diastereoselectivity is lower than about 80:20. In some embodiments, a diastereoselectivity is lower than about 90:10. In some embodiments, a diastereoselectivity is lower than about 91:9. In some embodiments, at least one internucleotidic linkage has a diastereoselectivity lower than about 90:10. In some embodiments, at least two internucleotidic linkages have a diastereoselectivity lower than about 90:10. In some embodiments, at least three internucleotidic linkages have a diastereoselectivity lower than about 90:10. In some embodiments, at least four internucleotidic linkages have a diastereoselectivity lower than about 90:10. In some embodiments, at least five internucleotidic linkages have a diastereoselectivity lower than about 90:10. In some embodiments, each internucleotidic linkage independently has a diastereoselectivity lower than about 90:10. In some embodiments, a non-chirally controlled internucleotidic linkage has a diastereomeric purity no more than 90%, 85%, 80%, 75%, 70%, 65%, 60%, or 55%. In some embodiments, the purity is no more than 90%. In some embodiments, the purity is no more than 85%. In some embodiments, the purity is no more than 80%.

In contrast, in chirally controlled oligonucleotide composition, at least one and typically each chirally controlled internucleotidic linkage, such as those of oligonucleotides of chirally controlled oligonucleotide compositions, independently has a diastereomeric purity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more with respect to the chiral linkage phosphors. In some embodiments, a diastereomeric purity is 95% or more. In some embodiments, a diastereomeric purity is 96% or more. In some embodiments, a diastereomeric purity is 97% or more. In some embodiments, a diastereomeric purity is 98% or more. In some embodiments, a diastereomeric purity is 99% or more. Among other things, technologies of the present disclosure routinely provide chirally controlled internucleotidic linkages with high diastereomeric purity.

As appreciated by a person having ordinary skill in the art, diastereoselectivity of a coupling or diastereomeric purity (diastereopurity) of an internucleotidic linkage can be assessed through the diastereoselectivity of a dimer formation/diasteromeric purity of the internucleotidic linkage of a dimer formed under the same or comparable conditions, wherein the dimer has the same 5′- and 3′-nucleosides and internucleotidic linkage.

In some embodiments, the present disclosure provides chirally controlled (and/or stereochemically pure) oligonucleotide compositions comprising a plurality of oligonucleotides defined by having:

1) a common base sequence;

2) a common pattern of backbone linkages; and

3) a common pattern of backbone chiral centers, which composition is a substantially pure preparation of a single oligonucleotide in that at least about 10% of the oligonucleotides in the composition have the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers.

In some embodiments, the present disclosure provides chirally controlled oligonucleotide composition of a plurality of oligonucleotides, wherein the composition is enriched, relative to a substantially racemic preparation of the same oligonucleotides, for oligonucleotides of a single oligonucleotide type. In some embodiments, the present disclosure provides chirally controlled oligonucleotide composition of a plurality of oligonucleotides wherein the composition is enriched, relative to a substantially racemic preparation of the same oligonucleotides, for oligonucleotides of a single oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications.

wherein the composition is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type.

In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides of a particular type have the same constitution. In some embodiments, oligonucleotides of an oligonucleotide type are identical.

In some embodiments, a chirally controlled oligonucleotide composition is a substantially pure preparation of an oligonucleotide type in that oligonucleotides in the composition that are not of the oligonucleotide type are impurities form the preparation process of said oligonucleotide type, in some case, after certain purification procedures.

In some embodiments, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the oligonucleotides in the composition have a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications. In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers are identical.

In some embodiments, purity of a chirally controlled oligonucleotide composition of an oligonucleotide type is expressed as the percentage of oligonucleotides in the composition that are of the oligonucleotide type. In some embodiments, at least about 10% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 20% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 30% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 40% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 50% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 60% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 70% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 80% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 90% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 92% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 94% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 95% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 96% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 97% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 98% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 99% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type.

In some embodiments, purity of a chirally controlled oligonucleotide composition can be controlled by stereoselectivity of each coupling step in its preparation process. In some embodiments, a coupling step has a stereoselectivity (e.g., diastereoselectivity) of 60% (60% of the new internucleotidic linkage formed from the coupling step has the intended stereochemistry). After such a coupling step, the new internucleotidic linkage formed may be referred to have a 60% purity. In some embodiments, each coupling step has a stereoselectivity of at least 60%. In some embodiments, each coupling step has a stereoselectivity of at least 70%. In some embodiments, each coupling step has a stereoselectivity of at least 80%. In some embodiments, each coupling step has a stereoselectivity of at least 85%. In some embodiments, each coupling step has a stereoselectivity of at least 90%. In some embodiments, each coupling step has a stereoselectivity of at least 91%. In some embodiments, each coupling step has a stereoselectivity of at least 92%. In some embodiments, each coupling step has a stereoselectivity of at least 93%. In some embodiments, each coupling step has a stereoselectivity of at least 94%. In some embodiments, each coupling step has a stereoselectivity of at least 95%. In some embodiments, each coupling step has a stereoselectivity of at least 96%. In some embodiments, each coupling step has a stereoselectivity of at least 97%. In some embodiments, each coupling step has a stereoselectivity of at least 98%. In some embodiments, each coupling step has a stereoselectivity of at least 99%. In some embodiments, each coupling step has a stereoselectivity of at least 99.5%. In some embodiments, each coupling step has a stereoselectivity of virtually 100%. In some embodiments, a coupling step has a stereoselectivity of virtually 100% in that all detectable product from the coupling step by an analytical method (e.g., NMR. HPLC, use of a nuclease which stereoselectively cleaves phosphorothioates, etc) has the intended stereoselectivity. In some embodiments, stereoselectivity of a chiral internucleotidic linkage in an oligonucleotide may be measured through a model reaction, e.g. formation of a dimer under essentially the same or comparable conditions wherein the dimer has the same internucleotidic linkage as the chiral internucleotidic linkage, the 5′-nucleoside of the dimer is the same as the nucleoside to the 5′-end of the chiral internucleotidic linkage, and the 3′-nucleoside of the dimer is the same as the nucleoside to the 3′-end of the chiral internucleotidic linkage (e.g., for fU*SfU*fC*SfU, through the dimer of fU*SfC). As appreciated by a person having ordinary skill in the art, percentage of oligonucleotides of a particular type having n chirally controlled internucleotidic linkages in a preparation may be calculated as DP¹*DP²*DP³* . . . DPⁿ, wherein each of DP¹, DP², DP³, . . . , and DPⁿis independently the diastereomeric purity of the 1^st, 2^nd, 3^rd, . . . , and n^thchirally controlled internucleotidic linkage. In some embodiments, each of DP¹, DP², DP³, . . . , and DPⁿis independently 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% or 99% or more. In some embodiments, each of DP¹, DP², DP³, . . . , and DPⁿis independently 95% or more.

In some embodiments, in provided compositions, at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% or 99% of oligonucleotides that have the base sequence of a particular oligonucleotide type (defined by 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications) are oligonucleotides of the particular oligonucleotide type. In some embodiments, at least 0.5%, 1%, 2%, 3%, 4%, 5%. 6%, 7%, 8% 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% or 99% of oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of a particular oligonucleotide type are oligonucleotides of the particular oligonucleotide type.

In some embodiments, oligonucleotides of a particular type in a chirally controlled oligonucleotide composition is enriched at least 5 fold (oligonucleotides of the particular type have a fraction of 5*(½ⁿ) of oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type, wherein n is the number of chiral internucleotidic linkages; or oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type but are not of the particular oligonucleotide type are no more than [1-(½ⁿ)]/5 of oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type) compared to a stereorandom preparation of the oligonucleotides (oligonucleotides of the particular type are typically considered to have a fraction of ½″ of oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type, wherein n is the number of chiral internucleotidic linkages, and oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type but are not of the particular oligonucleotide type are typically considered to have a fraction of [1-(½″)] of oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type). In some embodiments, the enrichment is at least 20 fold. In some embodiments, the enrichment is at least 30 fold. In some embodiments, the enrichment is at least 40 fold. In some embodiments, the enrichment is at least 50 fold. In some embodiments, the enrichment is at least 60 fold. In some embodiments, the enrichment is at least 70 fold. In some embodiments, the enrichment is at least 80 fold. In some embodiments, the enrichment is at least 90 fold. In some embodiments, the enrichment is at least 100 fold. In some embodiments, the enrichment is at least 20,000 fold. In some embodiments, the enrichment is at least (1.5)″. In some embodiments, the enrichment is at least (1.6)″. In some embodiments, the enrichment is at least (1.7)″. In some embodiments, the enrichment is at least (1.1)″. In some embodiments, the enrichment is at least (1.8)″. In some embodiments, the enrichment is at least (1.9)″. In some embodiments, the enrichment is at least 2″. In some embodiments, the enrichment is at least 3″. In some embodiments, the enrichment is at least 4″. In some embodiments, the enrichment is at least 5″ In some embodiments, the enrichment is at least 6″. In some embodiments, the enrichment is at least 7″. In some embodiments, the enrichment is at least 8″. In some embodiments, the enrichment is at least 9″. In some embodiments, the enrichment is at least 10″. In some embodiments, the enrichment is at least 15″. In some embodiments, the enrichment is at least 20″. In some embodiments, the enrichment is at least 25″. In some embodiments, the enrichment is at least 30″. In some embodiments, the enrichment is at least 40″. In some embodiments, the enrichment is at least 50″. In some embodiments, the enrichment is at least 100. In some embodiments, enrichment is measured by increase of the fraction of oligonucleotides of the particular oligonucleotide type in oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type. In some embodiments, an enrichment is measured by decrease of the fraction of oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type but are not of the particular oligonucleotide type in oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type.

In some embodiments, provided oligonucleotides are antisense oligonucleotides. In some embodiments, provided oligonucleotides are siRNA oligonucleotides. In some embodiments, a provided chirally controlled oligonucleotide composition is of oligonucleotides that can be antisense oligonucleotide, antagomir, microRNA, pre-microRNA, antimir, supermir, ribozyme, U1 adaptor. RNA activator, RNAi agent, decoy oligonucleotide, triplex forming oligonucleotide, aptamer or adjuvant. In some embodiments, a chirally controlled oligonucleotide composition is of antisense oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of siRNA oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of antagomir oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of microRNA oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of pre-microRNA oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of antimir oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of supermir oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of ribozyme oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of U1 adaptor oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of RNA activator oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of RNAi agent oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of decoy oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of triplex forming oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of aptamer oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of adjuvant oligonucleotides.

In some embodiments, a provided oligonucleotide comprises one or more chiral, modified phosphate linkages. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of oligonucleotides that include one or more modified backbone linkages, bases, and/or sugars.

In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 80%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 85%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 90%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 91%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 92%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 93%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 94%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 95%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 96%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 97%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 98%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 99%.

In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the internucleotidic linkages of an oligonucleotide are independently chiral internucleotidic linkages. In some embodiments, all chiral, modified internucleotidic linkages are chiral phosphorothioate internucleotidic linkages. In some embodiments, all chiral, modified internucleotidic linkages except non-negatively charged internucleotidic linkages are chiral phosphorothioate internucleotidic linkages. In some embodiments, each chiral internucleotidic linkage is chirally controlled. In some embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90% chiral internucleotidic linkages of an oligonucleotide are chirally controlled and are of the Sp conformation. In some embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90% phosphorothioate internucleotidic linkages of an oligonucleotide are chirally controlled and are of the Sp conformation. In some embodiments, the percentage is at least about 10%. In some embodiments, the percentage is at least about 20%. In some embodiments, the percentage is at least about 30%. In some embodiments, the percentage is at least about 40%. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 90%.

In some embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90% chiral internucleotidic linkages of an oligonucleotide are chirally controlled and are of the Rp conformation. In some embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90% chiral phosphorothioate internucleotidic linkages of an oligonucleotide are chirally controlled and are of the Rp conformation. In some embodiments, the percentage is at least about 10%. In some embodiments, the percentage is at least about 20%. In some embodiments, the percentage is at least about 30%. In some embodiments, no more than 10, 20, 30, 40, 50, 60, 70, 80, or 90% chiral internucleotidic linkages of an oligonucleotide are chirally controlled and are of the Rp conformation. In some embodiments, no more than 10, 20, 30, 40, 50, 60, 70, 80, or 90% phosphorothioate internucleotidic linkages of an oligonucleotide are of the Rp conformation. In some embodiments, the percentage is no more than 10%. In some embodiments, the percentage is no more than 20%. In some embodiments, the percentage is no more than 30%.

In some embodiments, provided chirally controlled (and/or stereochemically pure) compositions are of oligonucleotides that contain one or more modified bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) compositions are of oligonucleotides that contain no modified bases. As appreciated by those skilled in the art, many types of modified bases can be utilized in accordance with the present disclosure. Example modified bases are described herein.

In some embodiments, oligonucleotides of provided compositions comprise at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise at least one natural phosphate linkage. In some embodiments, oligonucleotides of provided compositions comprise at least two natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise at least three natural phosphate linkages.

In some embodiments, oligonucleotides of provided compositions comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise one natural phosphate linkage. In some embodiments, oligonucleotides of provided compositions comprise two natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise three natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise four natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise five natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise six natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise seven natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise eight natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise nine natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise ten natural phosphate linkages.

In some embodiments, oligonucleotides of provided compositions comprise at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise at least two consecutive natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise at least three consecutive natural phosphate linkages.

In some embodiments, oligonucleotides of the present disclosure have at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 nucleobases in length. In some embodiments, oligonucleotides of the present disclosure comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 nucleobases in length, wherein each nucleobase is independently optionally substituted A, T, C, G, U, or a tautomer thereof.

In some embodiments, provided compositions comprise oligonucleotides containing one or more residues which are modified at the sugar moiety. In some embodiments, provided compositions comprise oligonucleotides containing one or more residues which are modified at the 2′ position of the sugar moiety (referred to herein as a “2′-modification”). Examples of such modifications are described herein and include, but are not limited to, 2′-OMe, 2′-MOE, 2′-LNA, 2′-F, FRNA, FANA, S-cEt, etc. In some embodiments, provided compositions comprise oligonucleotides containing one or more residues which are 2′-modified. For example, in some embodiments, provided oligonucleotides contain one or more residues which are 2′-O-methoxyethyl (2′-MOE)-modified residues. In some embodiments, provided compositions comprise oligonucleotides which do not contain any 2′-modifications. In some embodiments, provided compositions are oligonucleotides which do not contain any 2′-MOE residues. That is, in some embodiments, provided oligonucleotides are not MOE-modified. Additional example sugar modifications are described in the present disclosure.

In some embodiments, one or more is one. In some embodiments, one or more is two. In some embodiments, one or more is three. In some embodiments, one or more is four. In some embodiments, one or more is five. In some embodiments, one or more is six. In some embodiments, one or more is seven. In some embodiments, one or more is eight. In some embodiments, one or more is nine. In some embodiments, one or more is ten. In some embodiments, one or more is at least one. In some embodiments, one or more is at least two. In some embodiments, one or more is at least three. In some embodiments, one or more is at least four. In some embodiments, one or more is at least five. In some embodiments, one or more is at least six. In some embodiments, one or more is at least seven. In some embodiments, one or more is at least eight. In some embodiments, one or more is at least nine. In some embodiments, one or more is at least ten.

In some embodiments, a base sequence, e.g., a common base sequence of a plurality of oligonucleotide, a base sequence of a particular oligonucleotide type, etc., comprises or is a sequence complementary to a gene or transcript (e.g., of Dystrophin or DMD). In some embodiments, a common base sequence comprises or is a sequence 100% complementary to a gene. In some embodiments, a common base sequence comprises or is a sequence complementary to a characteristic sequence element of a gene, which characteristic sequences differentiate the gene from a similar sequence sharing homology with the gene. In some embodiments, a common base sequence comprises or is a sequence 100% complementary to a characteristic sequence element of a gene, which characteristic sequences differentiate the gene from another allele of the gene. In some embodiments, a common base sequence comprises or is a sequence 100% complementary to a characteristic sequence element of a gene, which characteristic sequences differentiate the gene from a similar sequence sharing homology with the gene. In some embodiments, a common base sequence comprises or is a sequence complementary to characteristic sequence element of a target gene, which characteristic sequences comprises a mutation that is not found in other copies of the gene, e.g., the wild-type copy of the gene, another mutant copy the gene, etc. In some embodiments, a common base sequence comprises or is a sequence 100% complementary to characteristic sequence element of a target gene, which characteristic sequences comprises a mutation that is not found in other copies of the gene, e.g., the wild-type copy of the gene, another mutant copy the gene, etc. In some embodiments, a common base sequence comprises or is a sequence 100% complementary to a characteristic sequence element of a gene, which characteristic sequences differentiate the gene from another allele of the gene. In some embodiments, a characteristic sequence element is a mutation. In some embodiments, a characteristic sequence element is a SNP.

In some embodiments, a chiral internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, etc., or a salt form thereof. In some embodiments, linkage phosphorus of chiral internucleotidic linkages are chirally controlled. In some embodiments, a chiral internucleotidic linkage is phosphorothioate internucleotidic linkage. In some embodiments, each chiral internucleotidic linkage in an oligonucleotide of a provided composition independently has the structure of formula I. In some embodiments, each chiral internucleotidic linkage in an oligonucleotide of a provided composition independently has the structure of formula II. In some embodiments, each chiral internucleotidic linkage in an oligonucleotide of a provided composition independently has the structure of formula III. In some embodiments, each chiral internucleotidic linkage in an oligonucleotide of a provided composition is a phosphorothioate internucleotidic linkage.

As appreciated by those skilled in the art, internucleotidic linkages, e.g., those of formula I, natural phosphate linkages, phosphorothioate internucleotidic linkages, etc. may exist in their salt forms depending on pH of their environment. Unless otherwise indicated, such salt forms are included in the present application when such internucleotidic linkages are referred to.

In some embodiments, oligonucleotides of the present disclosure comprise one or more modified sugar moieties. In some embodiments, oligonucleotides of the present disclosure comprise one or more modified base moieties. As known by a person of ordinary skill in the art and described in the disclosure, various modifications can be introduced to sugar and base moieties. For example, in some embodiments, a modification is a modification described in U.S. Pat. No. 9,006,198, WO2014/012081, WO/2015/107425, and WO/2017/062862, the sugar and base modifications of each of which are incorporated herein by reference.

In some embodiments, a sugar modification is a 2′-modification. Commonly used 2′-modifications include but are not limited to 2′-OR¹, wherein R¹is not hydrogen. In some embodiments, a modification is 2′-OR, wherein R is optionally substituted aliphatic. In some embodiments, a modification is 2′-OMe. In some embodiments, a modification is 2′-O-MOE. In some embodiments, the present disclosure demonstrates that inclusion and/or location of particular chirally pure internucleotidic linkages can provide stability improvements comparable to or better than those achieved through use of modified backbone linkages, bases, and/or sugars. In some embodiments, a provided single oligonucleotide of a provided composition has no modifications on the sugars. In some embodiments, a provided single oligonucleotide of a provided composition has no modifications on 2′-positions of the sugars (i.e., the two groups at the 2′-position are either —H/—H or -H/—OH). In some embodiments, a provided single oligonucleotide of a provided composition does not have any 2′-MOE modifications.

In some embodiments, a 2′-modification is —O-L- or -L- which connects the 2′-carbon of a sugar moiety to another carbon of a sugar moiety. In some embodiments, a 2′-modification is —O-L- or -L- which connects the 2′-carbon of a sugar moiety to the 4′-carbon of a sugar moiety. In some embodiments, a 2′-modification is S-cEt. In some embodiments, a modified sugar moiety is an LNA sugar moiety.

In some embodiments, a 2′-modification is —F. In some embodiments, a 2′-modification is FANA. In some embodiments, a 2′-modification is FRNA.

In some embodiments, a sugar modification is a 5′-modification. In some embodiments, a modification is 5′-R¹, wherein R¹is not hydrogen. In some embodiments, a sugar modification is 5′-R, wherein R is not hydrogen and is otherwise as described in the present disclosure. In some embodiments, a sugar modification is 5′-R, wherein R is optionally substituted C_1-6aliphatic. In some embodiments, a sugar modification is 5′-R, wherein R is optionally substituted C_1-6alkyl. In some embodiments, a sugar modification is 5′-R, wherein R is optionally substituted methyl. In some embodiments, a sugar modification is 5′-R, wherein R is optionally substituted methyl, wherein no substituents of the methyl group comprises a carbon atom. In some embodiments, a 5′-modification is methyl. In some embodiments, each substituent is independently halogen. In some embodiments, a substituted 5′-carbon is diastereomerically pure. In some embodiments, a substituted 5-carbon has the R configuration. In some embodiments, a substituted 5-carbon has the S configuration. In some embodiments, a 5′-modification is 5′-(R)-Me. In some embodiments, a 5′-modification is 5′-(S)-Me.

In some embodiments, a sugar moiety has one and no more than one modification at a position, e.g., a 2-position, 5′-position, etc. In some embodiments, a 2′-modification takes the position corresponding to the position of the 2′-OH in a natural RNA sugar moiety. In some embodiments, a 2′-modification takes the position corresponding to the position of the 2′-H in a natural RNA sugar moiety.

In some embodiments, a sugar modification changes the size of the sugar ring. In some embodiments, a sugar modification changes the conformation of the sugar ring. In some embodiments, a sugar modification is the sugar moiety in FHNA.

In some embodiments, a sugar modification replaces a sugar moiety with another cyclic or acyclic moiety. Examples of such moieties are widely known in the art, including but not limited to those used in Morpholino, glycol nucleic acids, etc.

Certain Embodiments of Internucleotidic Linkages, Chirally Controlled Oligonucleotides and Chirally Controlled Oligonucleotide Compositions

Among other things, the present disclosure provides chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions. In some embodiments, the present disclosure provides chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions which are of high crude purity. In some embodiments, the present disclosure provides chirally controlled oligonucleotides, and chirally controlled oligonucleotide compositions which are of high diastereomeric purity. Chirally controlled oligonucleotides are oligonucleotides comprise one or more chirally controlled internucleotidic linkages, such as oligonucleotides of a plurality in chirally controlled oligonucleotide compositions. In some embodiments, chirally controlled oligonucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more chirally controlled internucleotidic linkages. In some embodiments, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more chiral internucleotidic linkages of a chirally controlled oligonucleotide are independently chirally controlled internucleotidic linkages. In some embodiments, each chiral internucleotidic linkage in a chirally controlled oligonucleotide is a chirally controlled internucleotidic linkage, and a chirally controlled oligonucleotide is diastereomerically pure.

In some embodiments, a chirally controlled oligonucleotide composition is a substantially pure composition of an oligonucleotide type in that oligonucleotides in the composition that are not of the oligonucleotide type are impurities. In some embodiments, such impurities are formed during the preparation process of oligonucleotides of said oligonucleotide type, in some case, after certain purification procedures.

In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotidic linkages with respect to the chiral linkage phosphorus (e.g., linkage phosphorus of chirally controlled internucleotidic linkages). In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotidic linkages having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, etc., or a salt form thereof. In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotidic linkages with respect to the chiral linkage phosphorus, and one or more natural phosphate linkages (unless otherwise indicated, reference in the present application to internucleotidic linkages, such as natural phosphate linkages and other types of internucleotidic linkages when applicable, includes salt forms of such linkages). Thus, diastereomerically pure internucleotidic linkages here include salt forms of diastereomerically pure internucleotidic linkages; natural phosphate linkages here include salt forms of natural phosphate linkages. A person having ordinary skill in the art appreciates that many internucleotidic linkages, such as natural phosphate linkages, exist as salt forms when at physiological pH, in many buffers (e.g., PBS buffers having a pH around 7, e.g., PH 7.4), etc.). In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotidic linkages having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, etc., or a salt form thereof, and one or more natural phosphate linkages. In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotidic linkages having the structure of formula I-c, and one or more phosphate diester linkages. In some embodiments, such oligonucleotides are prepared by using stereoselective oligonucleotide synthesis, as described in this application, to form designed diastereomerically pure internucleotidic linkages with respect to the chiral linkage phosphorus.

In some embodiments, an oligonucleotide of the present disclosure comprises at least one internucleotidic linkage, e.g., a modified (non-natural) internucleotidic linkage (e.g., non-negatively charged internucleotidic linkage) within or at the terminus (e.g. 5′ or 3′) of the oligonucleotide. In some embodiments, an oligonucleotide comprises a P-modification moiety within or at the terminus (e.g. 5′ or 3′) of the oligonucleotide.

In some embodiments, an oligonucleotide of the present disclosure comprises at least one chirally controlled internucleotidic linkage within the oligonucleotide. In some embodiments, an oligonucleotide of the present disclosure comprises at least one chirally controlled internucleotidic linkage within the oligonucleotide, and at least one natural phosphate linkage. In some embodiments, an oligonucleotide of the present disclosure comprises at least one chirally controlled internucleotidic linkage within the oligonucleotide, at least one natural phosphate linkage, and at least one phosphorothioate internucleotidic linkage. In some embodiments, an oligonucleotide of the present disclosure comprises at least one chirally controlled internucleotidic linkage within the oligonucleotide, and at least one phosphorothioate triester internucleotidic linkage. In some embodiments, an oligonucleotide of the present disclosure comprises at least one chirally controlled internucleotidic linkage within the oligonucleotide, at least one natural phosphate linkage, and at least one phosphorothioate triester internucleotidic linkage.

In some embodiments, an oligonucleotide of the present disclosure comprises at least two chirally controlled internucleotidic linkages within the oligonucleotide that have different stereochemistry and/or different P-modifications relative to one another. In some embodiments, such at least two internucleotidic linkages have different stereochemistry. In some embodiments, such at least two internucleotidic linkages have different P-modifications. In some embodiments, an oligonucleotide of the present disclosure comprises at least two chirally controlled internucleotidic linkages within the oligonucleotide that have different P-modifications relative to one another, and at least one natural phosphate linkage. In some embodiments, an oligonucleotide of the present disclosure comprises at least two chirally controlled internucleotidic linkages within the oligonucleotide that have different P-modifications relative to one another, at least one natural phosphate linkage, and at least one phosphorothioate internucleotidic linkage. In some embodiments, an oligonucleotide of the present disclosure comprises at least two chirally controlled internucleotidic linkages within the oligonucleotide that have different P-modifications relative to one another, and at least one phosphorothioate triester internucleotidic linkage. In some embodiments, an oligonucleotide of the present disclosure comprises at least two chirally controlled internucleotidic linkages within the oligonucleotide that have different P-modifications relative to one another, at least one natural phosphate linkage, and at least one phosphorothioate triester internucleotidic linkage.

In certain embodiments, an internucleotidic linkage (e.g., a modified (non-natural) internucleotidic linkage when formula I is not a natural phosphate linkage) has the structure of formula I:

embedded image

or a salt form thereof, wherein:

P^Lis P(═W), P, or P→B(R′)₃;

W is O, N(-L-R⁵), S or Se;

each of R¹and R⁵is independently —H, -L-R′, halogen, —CN, —NO₂, -L-Si(R′)₃, —OR′, —SR′, or —N(R′)₂;

each of X, Y and Z is independently —O—, —S—, —N(-L-R⁵)—, or L:

each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—. —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L;

each R′ is independently —R, —C(O)R, —C(O)OR, or —S(O)₂R;

each R is independently —H, or an optionally substituted group selected from C_1-30aliphatic, C_1-30heteroaliphatic having 1-10 heteroatoms, C_6-30aryl, C_6-30arylaliphatic, C_6-30arylheteroaliphatic having I-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or

two R groups are optionally and independently taken together to form a covalent bond, or

two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or

two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

In some embodiments, a linkage of formula I is chiral at the linkage phosphorus (P in P^L). In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotidic linkages of formula I. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotidic linkages of formula I, and wherein individual internucleotidic linkages of formula I within the oligonucleotide have different P-modifications relative to one another. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotidic linkages of formula I, and wherein individual internucleotidic linkages of formula I within the oligonucleotide have different -X-L-R¹relative to one another. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotidic linkages of formula I, and wherein individual internucleotidic linkages of formula I within the oligonucleotide have different X relative to one another. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotidic linkages of formula I, and wherein individual internucleotidic linkages of formula I within the oligonucleotide have different -L-R¹relative to one another. In some embodiments, a chirally controlled oligonucleotide is an oligonucleotide in a provided composition that is of the particular oligonucleotide type. In some embodiments, a chirally controlled oligonucleotide is an oligonucleotide in a provided composition that has the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers.

As extensively described herein, in some embodiments, -X-L-R¹is a moiety useful for oligonucleotide preparation. For example, in some embodiments, -X-L-R¹is —OCH₂CH₂CN (e.g., in non-chirally controlled internucleotidic linkages); in some embodiments. -X-L-R¹is of such a structure that H-X-L-R¹is a chiral auxiliary, optionally capped, as described herein (e.g., DPSE, PSM, etc.; particularly in chirally controlled internucleotidic linkages, although may also in non-chirally controlled internucleotidic linkages (e.g., precursors of natural phosphate linkages)).

In some embodiments, a chirally controlled oligonucleotide is an oligonucleotide in a chirally controlled composition that is of a particular oligonucleotide type, and the chirally controlled oligonucleotide is of the type. In some embodiments, a chirally controlled oligonucleotide is an oligonucleotide in a provided composition that comprises a controlled level of a plurality of oligonucleotides that share a common base sequence, a common pattern of backbone linkages, a common pattern of backbone chiral centers, and a common pattern of backbone phosphorus modifications, and the chirally controlled oligonucleotide shares the common base sequence, the common pattern of backbone linkages, the common pattern of backbone chiral centers, and the common pattern of backbone phosphorus modifications.

In some embodiments, the present disclosure provides a chirally controlled oligonucleotide, wherein at least two chirally controlled internucleotidic linkages within the oligonucleotide have different P-modifications relative to one another, in that they have different X atoms in their -XLR¹moieties, and/or in that they have different L groups in their -XLR¹moieties, and/or that they have different R¹atoms in their -XLR¹moieties, and/or in that they have different -XLR¹moieties.

In some embodiments, the present disclosure provides a chirally controlled oligonucleotide, wherein at least two of the individual internucleotidic linkages within the oligonucleotide have different stereochemistry and/or different P-modifications relative to one another and the oligonucleotide has a structure represented by the following formula:

[S^Bn1R^Bn2S^Bn3R^Bn4 . . . S^BnxR^Bny]

wherein:

each R^Bindependently represents a block of nucleotide units having the R configuration at the linkage phosphorus;

each S^Bindependently represents a block of nucleotide units having the S configuration at the linkage phosphorus;

each of n1-ny is zero or an integer, with the requirement that at least one odd n and at least one even n must be non-zero so that the oligonucleotide includes at least two individual internucleotidic linkages with different stereochemistry relative to one another; and

wherein the sum of n1-ny is between 2 and 200, and in some embodiments is between a lower limit selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more and an upper limit selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200, the upper limit being larger than the lower limit.

In some such embodiments, each n has the same value; in some embodiments, each even n has the same value as each other even n; in some embodiments, each odd n has the same value each other odd n; in some embodiments, at least two even ns have different values from one another; in some embodiments, at least two odd ns have different values from one another.

In some embodiments, at least two adjacent ns are equal to one another, so that a provided oligonucleotide includes adjacent blocks of S stereochemistry linkages and R stereochemistry linkages of equal lengths. In some embodiments, provided oligonucleotides include repeating blocks of S and R stereochemistry linkages of equal lengths. In some embodiments, provided oligonucleotides include repeating blocks of S and R stereochemistry linkages, where at least two such blocks are of different lengths from one another; in some such embodiments each S stereochemistry block is of the same length, and is of a different length from each R stereochemistry length, which may optionally be of the same length as one another.

In some embodiments, at least two skip-adjacent ns are equal to one another, so that a provided oligonucleotide includes at least two blocks of linkages of a first stereochemistry that are equal in length to one another and are separated by a block of linkages of the other stereochemistry, which separating block may be of the same length or a different length from the blocks of first stereochemistry.

In some embodiments, ns associated with linkage blocks at the ends of a provided oligonucleotide are of the same length. In some embodiments, provided oligonucleotides have terminal blocks of the same linkage stereochemistry. In some such embodiments, the terminal blocks are separated from one another by a middle block of the other linkage stereochemistry.

In some embodiments, a provided oligonucleotide of formula [S^Bn1R^Bn2S^Bn3R^Bn4 . . . S^BnxR^Bny] is a stereoblockmer. In some embodiments, a provided oligonucleotide of formula [S^Bn1R^Bn2S^Bn3R^Bn4 . . . S^BnxR^Bny] is a stereoskipmer. In some embodiments, a provided oligonucleotide of formula [S^Bn1R^Bn2S^Bn3R^Bn4 . . . S^BnxR^Bny] is a stereoaltmer. In some embodiments, a provided oligonucleotide of formula [S^Bn1R^Bn2S^Bn3R^Bn4 . . . S^BnxR^Bny] is a gapmer.

In some embodiments, a provided oligonucleotide of formula [S^Bn1R^Bn2S^Bn3R^Bn4 . . . S^BnxR^Bny] is of any of the above described patterns and further comprises patterns of P-modifications. For instance, in some embodiments, a provided oligonucleotide of formula [S^Bn1R^Bn2S^Bn3R^Bn4 . . . S^BnxR^Bny] and is a stereoskipmer and P-modification skipmer. In some embodiments, a provided oligonucleotide of formula [S^Bn1R^Bn2S^Bn3R^Bn4 . . . S^BnxR^Bny] and is a stereoblockmer and P-modification altmer. In some embodiments, a provided oligonucleotide of formula [S^Bn1R^Bn2S^Bn3R^Bn4 . . . S^BnxR^Bny] and is a stereoaltmer and P-modification blockmer.

In some embodiments, an internucleotidic linkage of formula I has the structure of:

embedded image

wherein:

P* is an asymmetric phosphorus atom and is either Rp or Sp;

W is O, S or Se;

each of X, Y and Z is independently —O—, —S—, —N(-L-R¹)—, or L;

L is a covalent bond or an optionally substituted, linear or branched C₁-C₁₀alkylene, wherein one or more methylene units of L are optionally and independently replaced by C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆heteroaliphatic moiety, —C(R′)r, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂— —SC(O)—, —C(O)S—, —OC(O)—, and —C(O)O—;
R¹is halogen, R, or an optionally substituted C₁-C₅₀aliphatic wherein one or more methylene units are optionally and independently replaced by C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—, —S—, —S—S— —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂— —SC(O)—, —C(O)S—, —OC(O)—, and —C(O)O—;
each R′ is independently —R, —C(O)R, —CO₂R or —SO₂R, or:
- two R′ are taken together with their intervening atoms to form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring;
-Cy- is an optionally substituted bivalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, and heterocyclylene;
each R is independently hydrogen, or an optionally substituted group selected from C₁-C₆aliphatic, carbocyclyl, aryl, heteroaryl, and heterocyclyl; and
each

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independently represents a connection to a nucleoside.

In some embodiments, L is a covalent bond or an optionally substituted, linear or branched C₁-C₁₀alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡—, —C(R′)—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;

R¹is halogen, R, or an optionally substituted C₁-C₅₀aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;
each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:
- two R′ on the same nitrogen are taken together with their intervening atoms to form an optionally substituted heterocyclic or heteroaryl ring, or
- two R′ on the same carbon are taken together with their intervening atoms to form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring;
-Cy- is an optionally substituted bivalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene;
each R is independently hydrogen, or an optionally substituted group selected from C₁-C₆aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and each

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independently represents a connection to a nucleoside.

In some embodiments, a chirally controlled oligonucleotide comprises one or more modified internucleotidic linkages. In some embodiments, a chirally controlled oligonucleotide comprises, e.g., a phosphorothioate or a phosphorothioate triester internucleotidic linkage. In some embodiments, a chirally controlled oligonucleotide comprises a chirally controlled phosphorothioate triester linkage. In some embodiments, a chirally controlled oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chirally controlled phosphorothioate triester internucleotidic linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chirally controlled phosphorothioate internucleotidic linkages (—O—P(O)(SH)—O— or salt forms thereof).

In some embodiments, an oligonucleotide comprises different types of internucleotidic phosphorus linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least one natural phosphate linkage and at least one modified (non-natural) internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one natural phosphate linkage and at least one phosphorothioate. In some embodiments, an oligonucleotide comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one natural phosphate linkage and at least one non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one phosphorothioate internucleotidic linkage and at least one non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one phosphorothioate internucleotidic linkage, at least one natural phosphate linkage, and at least one non-negatively charged internucleotidic linkage.

In some embodiments, an internucleotidic linkage comprises a chiral auxiliary. In some embodiments, an internucleotidic linkage of formula I, I-a, I-b, I-c. I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., comprises a chiral auxiliary, wherein P^Lis P═S. In some embodiments, an internucleotidic linkage of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, I-a-1, II-a-2, II-b-1, II-b-2, I-c-1, II-c-2, II-d-1, II-d-2, etc., comprises a chiral auxiliary, wherein P^Lis P═O. In some embodiments, a phosphorothioate triester linkage comprises a chiral auxiliary, which, for example, is used to control the stereoselectivity of a reaction. In some embodiments, a phosphorothioate triester linkage does not comprise a chiral auxiliary. Example chiral auxiliaries that can be utilized in accordance with the present disclosure include those described in U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, US 20130178612, US 20150211006, U.S. Pat. No. 9,598,458. US 20170037399, WO 2017/015555, WO 2017/062862, WO 2018/237194, WO 2019/055951, the chiral auxiliaries of each of which is incorporated herein by reference. In some embodiments, one or more -X-L-R¹independently comprise or are an optionally substituted chiral auxiliary. In some embodiments, one or more -X-L-R¹are each independently of such a structure that H-X-L-R¹is a chiral reagent/chiral auxiliary described herein (e.g., one having the structure of formula 3-I, formula 3-AA, etc.). In some embodiments, H-X-L-R¹is a capped chiral reagent/chiral auxiliary described herein (e.g., one having the structure of formula 3-1, formula 3-AA, etc.), which is capped in that an amino group of the chiral reagent/chiral auxiliary (e.g., H-W¹and H-W²is or comprises H-NG⁵-) is capped (e.g., forming R¹-NG⁵-(e.g., R¹C(O)-NG⁵-, RS(O)₂—NG⁵-, etc.)). In some embodiments, R′ is optionally substituted C_1-6alkyl. In some embodiments. R′ is methyl. In some embodiments one or more -X-L-R¹are each independently of such a structure that H-X-L-R¹is

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In some embodiments, one or more -X-L-R¹are each independently of such a structure that H-X-L-R¹is

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In some embodiments one or more -X-L-R¹are each independently of such a structure that H-X-L-R¹is

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In some embodiments, one or more -X-L-R¹are each independently of such a structure that H-X-L-R¹is a compound selected from Tables CA-1, CA-2, CA-3, CA-4, CA-5, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, CA-12, or CA-13, or a related (having the same constitution) diastereomer or enantiomer thereof. In some embodiments, one or more -X-L-R¹are each independently of such a structure that H-X-L-R¹is

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In some embodiments, one or more -X-L-R¹are each independently of such a structure that H-X-L-R¹is

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In some embodiments, one or more -X-L-R¹are each independently of such a structure that H-X-L-R¹is

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In some embodiments, one or more -X-L-R¹are each independently of such a structure that H-X-L-R¹is a compound selected from Tables CA-1, CA-2, CA-3, CA-4, CA-5, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, CA-12, or CA-13, or a related (having the same constitution) diastereomer or enantiomer thereof, wherein the —NH— of the 5-membered pyrrolidinyl is replaced with —N(R¹)—. In some embodiments, one or more -X-L-R¹are independently

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In some embodiments, one or more -X-L-R¹are independently

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In some embodiments, one or more -X-L-R¹are independently

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In some embodiments, one or more -X-L-R¹are each independently of such a structure that H-X-L-R¹is a compound selected from Tables CA-1, CA-2, CA-3, CA-4, CA-5, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, CA-12, or CA-13, or a related (having the same constitution) diastereomer or enantiomer thereof, wherein the connection to the linkage phosphorus is through the alcohol hydroxyl group. In some embodiments, one or more -X-L-R¹are independently,

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In some embodiments, one or more -X-L-R¹are independently

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In some embodiments, one or more -X-L-R¹are independently

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In some embodiments, one or more -X-L-R¹are each independently of such a structure that H-X-L-R¹is a compound selected from Tables CA-1, CA-2, CA-3, CA-4, CA-5, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, CA-12, or CA-13, or a related (having the same constitution) diastereomer or enantiomer thereof, wherein the —NH— of the 5-membered pyrrolidinyl is replaced with —N(R¹)—, and wherein the connection to the linkage phosphorus is through the alcohol hydroxyl group. In some embodiments, one or more -X-L-R¹are independently

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and one or more -X-L-R¹are independently

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In some embodiments, one or more -X-L-R¹are independently

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and one or more -X-L-R¹are independently

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In some embodiments, one or more -X-L-R¹are independently

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and one or more -X-L-R¹are independently

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In some embodiments, R¹is a capping group utilized in oligonucleotide synthesis. In some embodiments, R¹is —C(O)—R′. In some embodiments, R¹is —C(O)—R′, wherein R′ is optionally substituted C_1-6aliphatic. In some embodiments, R¹is —C(O)CH₃.

In some embodiments, an oligonucleotide, e.g., a chirally controlled oligonucleotide, an oligonucleotide of a plurality, etc. is linked to a solid support. In some embodiments, an oligonucleotide is not linked to a solid support.

In some embodiments, an oligonucleotide comprises at least one natural phosphate linkage and at least two consecutive chirally controlled modified internucleotidic linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least one natural phosphate linkage and at least two consecutive chirally controlled phosphorothioate internucleotidic linkages.

In some embodiments, a chirally controlled oligonucleotide is a blockmer. In some embodiments, a chirally controlled oligonucleotide is a stereoblockmer. In some embodiments, a chirally controlled oligonucleotide is a P-modification blockmer. In some embodiments, a chirally controlled oligonucleotide is a linkage blockmer.

In some embodiments, a chirally controlled oligonucleotide is an altmer. In some embodiments, a chirally controlled oligonucleotide is a stereoaltmer. In some embodiments, a chirally controlled oligonucleotide is a P-modification altmer. In some embodiments, a chirally controlled oligonucleotide is a linkage altmer.

In some embodiments, a chirally controlled oligonucleotide is a unimer.

In some embodiments, in a unimer, all nucleotide units within a strand share at least one common structural feature at the internucleotidic phosphorus linkage. In some embodiments, a common structural feature is a common stereochemistry at the linkage phosphorus or a common modification at the linkage phosphorus. In some embodiments, a chirally controlled oligonucleotide is a stereounimer. In some embodiments, a chirally controlled oligonucleotide is a P-modification unimer. In some embodiments, a chirally controlled oligonucleotide is a linkage unimer.

In some embodiments, a chirally controlled oligonucleotide is a gapmer.

In some embodiments, a chirally controlled oligonucleotide is a skipmer.

In some embodiments, the present disclosure provides oligonucleotides comprising one or more modified internucleotidic linkages independently having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, I-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, I-d-2, III, or a salt form thereof.

In some embodiments, L is a covalent bond or an optionally substituted, linear or branched C₁-C₁₀alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;

R¹is halogen, R, or an optionally substituted C₁-C₅₀aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)—, —S(O)₂N(R′)—, —N(R′)S(O)—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;
each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R, or:
- two R′ on the same nitrogen are taken together with their intervening atoms to form an optionally substituted heterocyclic or heteroaryl ring, or
- two R′ on the same carbon are taken together with their intervening atoms to form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring
-Cy- is an optionally substituted bivalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene;
each R is independently hydrogen, or an optionally substituted group selected from C₁-C₆aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and
each

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independently represents a connection to a nucleoside.

In some embodiments, a chirally controlled oligonucleotide comprises one or more modified internucleotidic phosphorus linkages. In some embodiments, a chirally controlled oligonucleotide comprises, e.g., a phosphorothioate or a phosphorothioate triester linkage. In some embodiments, a chirally controlled oligonucleotide comprises a phosphorothioate triester linkage. In some embodiments, a chirally controlled oligonucleotide comprises at least two phosphorothioate triester linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least three phosphorothioate triester linkages. Example modified internucleotidic phosphorus linkages are described further herein. In some embodiments, a chirally controlled oligonucleotide comprises different internucleotidic phosphorus linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least one phosphate diester internucleotidic linkage and at least one modified internucleotidic linkage. In some embodiments, a chirally controlled oligonucleotide comprises at least one phosphate diester internucleotidic linkage and at least one phosphorothioate triester linkage. In some embodiments, a chirally controlled oligonucleotide comprises at least one phosphate diester internucleotidic linkage and at least two phosphorothioate triester linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least one phosphate diester internucleotidic linkage and at least three phosphorothioate triester linkages.

In some embodiments, P* is an asymmetric phosphorus atom and is either Rp or Sp. In some embodiments, P* is Rp. In other embodiments, P* is Sp. In some embodiments, an oligonucleotide comprises one or more internucleotidic linkages of formula I wherein each P* is independently Rp or Sp. In some embodiments, an oligonucleotide comprises one or more internucleotidic linkages of formula I wherein each P* is Rp. In some embodiments, an oligonucleotide comprises one or more internucleotidic linkages of formula I wherein each P* is Sp. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein P* is Rp. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein P* is Sp. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein P* is Rp, and at least one internucleotidic linkage of formula I wherein P* is Sp.

In some embodiments, W is O, S, or Se. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, W is Se. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein W is O. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein W is S. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein W is Se.

In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein W is O. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein W is S.

In some embodiments, X is —O—. In some embodiments, X is —S—. In some embodiments, X is —O— or —S—. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein X is —O—. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein X is —S—. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein X is —O—, and at least one internucleotidic linkage of formula I wherein X is —S—. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein X is —O—, and at least one internucleotidic linkage of formula I wherein X is —S—, and at least one internucleotidic linkage of formula I wherein L is an optionally substituted, linear or branched C₁-C₁₀alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—. —OC(O)—, or —C(O)O—.

In some embodiments, X is —N(-L-R¹)—. In some embodiments, X is —N(R′)—. In some embodiments, X is —N(R′)—. In some embodiments, X is —N(R)—. In some embodiments, X is —NH—.

In some embodiments, X is L. In some embodiments, X is a covalent bond. In some embodiments, X is or an optionally substituted, linear or branched C₁-C₁₀alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In some embodiments, X is an optionally substituted C₁-C₁alkylene or C₁-C₁₀alkenylene. In some embodiments, X is methylene.

In some embodiments, Y is —O—. In some embodiments, Y is —S—.

In some embodiments, Y is —N(-L-R¹)—. In some embodiments, Y is —N(R′)—. In some embodiments, Y is —N(R′)—. In some embodiments, Y is —N(R)—. In some embodiments, Y is —NH—.

In some embodiments, Y is L. In some embodiments, Y is a covalent bond. In some embodiments, Y is or an optionally substituted, linear or branched C₁-C₀alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene. —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—. —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In some embodiments, Y is an optionally substituted C₁-C₁₀alkylene or C₁-C₁₀alkenylene. In some embodiments, Y is methylene.

In some embodiments, Z is —O—. In some embodiments, Z is —S—.

In some embodiments, Z is —N(-L-R¹)—. In some embodiments, Z is —N(R¹)—. In some embodiments, Z is —N(R′)—. In some embodiments, Z is —N(R)—. In some embodiments, Z is —NH—.

In some embodiments, Z is L. In some embodiments, Z is a covalent bond. In some embodiments, Z is or an optionally substituted, linear or branched C₁-C₁₀alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—. In some embodiments. Z is an optionally substituted C₁-C₁₀alkylene or C₁-C₁₀alkenylene. In some embodiments, Z is methylene.

In some embodiments, L is a covalent bond or an optionally substituted, linear or branched C₁-C₁₀alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡—C—, —C(R′)₂, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—.

In some embodiments, L is a covalent bond. In some embodiments, L is an optionally substituted, linear or branched C₁-C₁₀alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—.

In some embodiments, L has the structure of -L¹-V-, wherein:

L¹is an optionally substituted group selected from

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C₁-C₆alkylene, C₁-C₆alkenylene, carbocyclylene, arylene, C₁-C₆heteroalkylene, heterocyclylene, and heteroarylene;

V is selected from —O—, —S—, —NR′—, C(R′)₂, —S—S—, —B—S—S—C—,

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or an optionally substituted group selected from C₁-C₆alkylene, arylene, C₁-C₆heteroalkylene, heterocyclylene, and heteroarylene;

A is ═O, ═S, ═NR′, or ═C(R′)₂;

each of B and C is independently —O—, —S—, —NR′—, —C(R′)—, or an optionally substituted group selected from C₁-C₆alkylene, carbocyclylene, arylene, heterocyclylene, or heteroarylene; and

each R′ is independently as defined above and described herein.

In some embodiments, L¹is

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In some embodiments, L¹is,

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wherein Ring Cy′ is an optionally substituted arylene, carbocyclylene, heteroarylene, or heterocyclylene. In some embodiments, L¹is optionally substitute

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In some embodiments, L¹is

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In some embodiments, L¹is connected to X. In some embodiments, L¹is an optionally substituted group selected from

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and the sulfur atom is connect to V. In some embodiments, L¹is an optionally substituted group selected from

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and the carbon atom is connect to X.

In some embodiments, L has the structure of:

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wherein:

E is —O—, —S—, —NR′— or —C(R′)₂;

custom-character is a single or double bond; the two R^L1are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, carbocyclic, heteroaryl or heterocyclic ring; and each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

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wherein:

G is —O—, —S—, or —NR′;

custom-character is a single or double bond; and

the two R^L1taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, L has the structure of:

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wherein:

E is —O—, —S—, —NR′— or —C(R′)₂—;
D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—, ═C(CO₂—(C₁-C₆aliphatic))-, or ═C(CF₃)—; and

each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

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wherein:

G is —O—, —S—, or —NR′;
D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—, ═C(CO₂—(C₁-C₆aliphatic))-, or ═C(CF₃)—.

In some embodiments, L has the structure of:

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wherein:

E is —O—, —S—, —NR′— or —C(R)₂—;
D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—, ═C(CO₂—(C₁-C₆aliphatic))-, or ═C(CF₃)—; and

each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

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wherein:

G is —O—, —S—, or —NR′;
D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)— ═C(I)—, ═C(CN)— ═C(NO₂)—, ═C(CO₂—(C₁-C₆aliphatic))-, or ═C(CF₃)—.

In some embodiments, L has the structure of:

embedded image

wherein:

E is —O—, —S—, —NR′— or —C(R′)₂—;

custom-character is a single or double bond;

the two R^L1are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring;

and each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

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wherein:

G is —O—, —S—, or —NR′;

custom-character is a single or double bond;

the two R^L1already taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring:

and each R′ is independently as defined above and described herein.

In some embodiments, L las the structure of:

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wherein:

E is —O—, —S—, —NR′— or —C(R′)₂—;
D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO)—, ═C(CO₂—(C₁-C₆aliphatic))-, or ═C(CF₃— and

each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

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wherein:

G is —O—, —S—, or —NR′;
D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—, ═C(CO₂—(C₁-C₆aliphatic))-, or ═C(CF₃)—; and

each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

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wherein:

E is —O—, —S—, —NR′— or —C(R′)₂—;
D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—, ═C(CO₂—(C₁-C₆aliphatic))-, or ═C(CF₃)—; and

each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

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wherein:

G is —O—, —S—, or —NR′;
D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—, ═C(CO₂—(C₁-C₆(aliphatic))-, or ═C(CF₃)—; and

each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

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wherein:

E is —O—, —S—, —NR′— or —C(R′)₂-;

custom-character is a single or double bond;

the R^L1are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring; and each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

embedded image

wherein:

G is —O—, —S—, or —NR′;

custom-character is a single or double bond;

the two R^L1are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring; and each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

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wherein:

E is —O—, —S—, —NR′— or —C(R′)₂—;
D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—, ═C(CO₂—(C₁-C₆aliphatic))-, or ═C(CF₃)—; and

each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

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wherein:

G is —O—, —S—, or —NR′;
D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—, ═C(CO₂—(C₁-C₆aliphatic))-, or ═C(CF₃)—; and

R′ is as defined above and described herein.

In some embodiments, L has the structure of:

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wherein:

E is —O—, —S—, —NR′— or —C(R′)₂—;
D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(I)—, ═C(CN)—, ═C(NO₂)—, ═C(CO₂—(C₁-C₆(aliphatic))-, or ═C(CF₃)—; and

each R′ is independently as defined above and described herein.

In some embodiments, L has the structure of:

embedded image

wherein:

G is —O—, —S—, or —NR′;
D is ═N—, ═C(F)—, ═C(Cl)—, ═C(Br)—, ═C(O)—, ═C(CN)—, ═C(NO₂)—, ═C(CO₂—(C₁-C₆aliphatic))-, or ═C(CF₃)—; and

R′ is as defined above and described herein.

In some embodiments, L has the structure of:

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wherein the phenyl ring is optionally substituted. In some embodiments, the phenyl ring is not substituted. In some embodiments, the phenyl ring is substituted.

In some embodiments, L has the structure of:

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wherein the phenyl ring is optionally substituted. In some embodiments, the phenyl ring is not substituted. In some embodiments, the phenyl ring is substituted.

In some embodiments, L has the structure of:

embedded image

wherein:

custom-character is a single or double bond; and

the two R^L1are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, L has the structure of:

embedded image

wherein:

G is —O—, —S—, or —NR′;
is a single or double bond; and
the two R^L1are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C₃-C₁₀carbocyclic, heteroaryl or heterocyclic ring.

In some embodiments, E is —O—, —S—, —NR′— or —C(R′)₂—, wherein each R′ independently as defined above and described herein. In some embodiments, E is —O—, —S—, or —NR′—. In some embodiments, E is —O—, —S—, or —NH—. In some embodiments, E is —O—. In some embodiments, E is —S—. In some embodiments, E is —NH—.

In some embodiments, G is —O—, —S—, or —NR′, wherein each R′ independently as defined above and described herein. In some embodiments, G is —O—, —S—, or —NH—. In some embodiments, G is —O—. In some embodiments, G is —S—. In some embodiments, G is —NH—.

In some embodiments, L is -L³-G-, wherein:

L³is an optionally substituted C₁-C₅alkylene or alkenylene, wherein one or more methylene units are optionally and independently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —S(O)—, —S(O)₂—, or

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and

wherein each of G, R′ and Ring Cy′ is independently as defined above and described herein.

In some embodiments, L is -L³-S—, wherein L³is as defined above and described herein. In some embodiments, L is -L³-O—, wherein L³is as defined above and described herein. In some embodiments, L is -L³-N(R′)—, wherein each of L³and R′ is independently as defined above and described herein. In some embodiments, L is -L³-NH—, wherein each of L³and R′ is independently as defined above and described herein.

In some embodiments, L³is an optionally substituted C₅alkylene or alkenylene, wherein one or more methylene units are optionally and independently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —S(O)—, —S(O)₂—, or

embedded image

and each of R′ and Ring Cy′ is independently as defined above and described herein. In some embodiments, L³is an optionally substituted C₅alkylene. In some embodiments, -L³-G- is

embedded image

In some embodiments, L³is an optionally substituted C₄alkylene or alkenylene, wherein one or more methylene units are optionally and independently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —S(O)—, —S(O)—, or

embedded image

and each of R′ and Cy′ is independently as defined above and described herein.

In some embodiments, -L³-G- is

embedded image

In some embodiments, L³is an optionally substituted C₃alkylene or alkenylene, wherein one or more methylene units are optionally and independently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —S(O)—, —S(O)₂, or

embedded image

and each of R′ and Cy′ is independently as defined above and described herein.

In some embodiments -L³-G- is

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In some embodiments, L is

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In some embodiments, L is

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In some embodiments, L is

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In some embodiments, L³is an optionally substituted C₂alkylene or alkenylene, wherein one or more methylene units are optionally and independently replaced by —O—, —S—, —N(R′)—, —C(O)— —C(S)—, —C(NR′)—, —S(O)—, —S(O)₂—, or

embedded image

and each of R′ and Cy′ is independently as defined above and described herein.

In some embodiments, -L³-G- is

embedded image

wherein each of G and Cy′ is independently as defined above and described herein. In some embodiments, L is

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In some embodiments, L is -L⁴-G-, wherein L⁴is an optionally substituted C₁-C₂alkylene; and G is as defined above and described herein. In some embodiments, L is -L⁴-G-, wherein L⁴is an optionally substituted C₁-C₂alkylene; G is as defined above and described herein; and G is connected to R¹. In some embodiments, L is -L⁴-G-, wherein L⁴is an optionally substituted methylene; G is as defined above and described herein; and G is connected to R¹. In some embodiments, L is -L⁴-G-, wherein L⁴is methylene; G is as defined above and described herein; and G is connected to R¹. In some embodiments, L is -L⁴-G-, wherein L⁴is an optionally substituted —(CH₂)₂—; G is as defined above and described herein; and G is connected to R¹. In some embodiments, L is -L⁴-G-, wherein L⁴is —(CH₂)₂—; G is as defined above and described herein; and G is connected to R¹.

In some embodiments, L is

embedded image

wherein G is as defined above and described herein, and G is connected to R¹. In some embodiments, L is

embedded image

wherein G is as defined above and described herein, and G is connected to R¹. In some embodiments, L is

embedded image

wherein G is as defined above and described herein, and G is connected to R¹. In some embodiments, L is

embedded image

wherein the sulfur atom is connected to R¹. In some embodiments, L is

embedded image

wherein the oxygen atom is connected to R¹.

In some embodiments, L is

embedded image

wherein G is as defined above and described herein.

In some embodiments, L is —S—R^L3— or —S—C(O)—R^L3—, wherein R^L3is an optionally substituted, linear or branched, C₁-C₉, alkylene, wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each of R′ and -Cy- is independently as defined above and described herein. In some embodiments, L is —S—R^L3— or —S—C(O)—R^L3—, wherein R^L3is an optionally substituted C₁-C₆alkylene. In some embodiments, L is —S—R^L3- or —S—C(O)—R^L3—, wherein R^L3is an optionally substituted C₁-C₆alkenylene. In some embodiments, L is —S—R^L3— or —S—C(O)—R^L3—, wherein R^L3is an optionally substituted C₁-C₆alkylene wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₁-C₆alkenylene, arylene, or heteroarylene. In some embodiments, In some embodiments, R^L3is an optionally substituted —S—(C₁-C₆alkenylene)-, —S—(C₁-C₆alkylene)-, —S—(C₁-C₆alkylene)-arylene-(C₁-C₆alkylene)-, —S—CO-arylene-(C₁-C₆alkylene)-, or —S—CO—(C₁-C₆alkylene)-arylene-(C₁-C₆alkylene)-.

In some embodiments, L is

embedded image

In some embodiments, L is

embedded image

In some embodiments, L is

embedded image

In some embodiments,

embedded image

In some embodiments, the sulfur atom in the L embodiments described above and herein is connected to X. In some embodiments, the sulfur atom in the L embodiments described above and herein is connected to R¹.

In some embodiments, R¹is halogen, R, or an optionally substituted C₁-C₅₀aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable is independently as defined above and described herein. In some embodiments, R¹is halogen, R, or an optionally substituted C₁-C₁₀aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable is independently as defined above and described herein.

In some embodiments, R¹is hydrogen. In some embodiments, R¹is halogen. In some embodiments, R¹is —F. In some embodiments, R¹is —Cl. In some embodiments, R¹is —Br. In some embodiments, R¹is —I.

In some embodiments, R¹is R wherein R is as defined above and described herein.

In some embodiments, R¹is hydrogen. In some embodiments, R¹is an optionally substituted group selected from C₁-C₅₀aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl.

In some embodiments, R¹is an optionally substituted C₁-C₅₀aliphatic. In some embodiments, R¹is an optionally substituted C₁-C₁₀aliphatic. In some embodiments, R¹is an optionally substituted C₁-C₆aliphatic. In some embodiments, R¹is an optionally substituted C₁-C₆alkyl. In some embodiments, R¹is optionally substituted, linear or branched hexyl. In some embodiments, R¹is optionally substituted, linear or branched pentyl. In some embodiments, R¹is optionally substituted, linear or branched butyl. In some embodiments, R¹is optionally substituted, linear or branched propyl. In some embodiments, R¹is optionally substituted ethyl. In some embodiments, R¹is optionally substituted methyl.

In some embodiments, R¹is optionally substituted phenyl. In some embodiments, R¹is substituted phenyl. In some embodiments, R¹is phenyl.

In some embodiments, R¹is optionally substituted carbocyclyl. In some embodiments, R¹is optionally substituted C₃-C₁₀carbocyclyl. In some embodiments, R¹is optionally substituted monocyclic carbocyclyl. In some embodiments, R¹is optionally substituted cycloheptyl. In some embodiments, R¹is optionally substituted cyclohexyl. In some embodiments, R is optionally substituted cyclopentyl. In some embodiments, R¹is optionally substituted cyclobutyl. In some embodiments, R¹is an optionally substituted cyclopropyl. In some embodiments, R¹is optionally substituted bicyclic carbocyclyl.

In some embodiments, R¹is an optionally substituted C₁-C₅₀polycyclic hydrocarbon. In some embodiments, R¹is an optionally substituted C₁-C₅₀polycyclic hydrocarbon wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)₂, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable is independently as defined above and described herein. In some embodiments, R¹is optionally substituted

embedded image

In some embodiments, R¹is

embedded image

In some embodiments, R¹is optionally substituted

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In some embodiments, R¹is an optionally substituted C₁-C₅₀aliphatic comprising one or more optionally substituted polycyclic hydrocarbon moieties. In some embodiments, R¹is an optionally substituted C₁-C₅₀aliphatic comprising one or more optionally substituted polycyclic hydrocarbon moieties, wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable is independently as defined above and described herein. In some embodiments. R¹is an optionally substituted C₁-C₅₀aliphatic comprising one or more optionally substituted

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In some embodiments, R¹is

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In some embodiments, R¹is

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In some embodiments, R¹is

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In some embodiments, R¹is

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In some embodiments, R¹is

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In some embodiments, R¹is an optionally substituted aryl. In some embodiments, R¹is an optionally substituted bicyclic aryl ring.

In some embodiments, R¹is an optionally substituted heteroaryl. In some embodiments, R¹is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, or oxygen. In some embodiments, R¹is a substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is an unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, or oxygen.

In some embodiments, R¹is an optionally substituted 5 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, R¹is an optionally substituted 6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R¹is an optionally substituted 5-membered monocyclic heteroaryl ring having 1 heteroatom selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is selected from pyrrolyl, furanyl, or thienyl.

In some embodiments, R¹is an optionally substituted 5-membered heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R¹is an optionally substituted 5-membered heteroaryl ring having 1 nitrogen atom, and an additional heteroatom selected from sulfur or oxygen. Example R groups include optionally substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl.

In some embodiments, R¹is a 6-membered heteroaryl ring having 1-3 nitrogen atoms. In other embodiments, R¹is an optionally substituted 6-membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments, R¹is an optionally substituted 6-membered heteroaryl ring having 2 nitrogen atoms. In certain embodiments, R¹is an optionally substituted 6-membered heteroaryl ring having 1 nitrogen. Example R¹groups include optionally substituted pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.

In certain embodiments, R¹is an optionally substituted 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is an optionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In other embodiments, R¹is an optionally substituted 5,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R¹is an optionally substituted 5,6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is an optionally substituted indolyl. In some embodiments, R¹is an optionally substituted azabicyclo[3.2.1]octanyl. In certain embodiments, R¹is an optionally substituted 5,6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is an optionally substituted azaindolyl. In some embodiments, R¹is an optionally substituted benzimidazolyl. In some embodiments, R¹is an optionally substituted benzothiazolyl. In some embodiments, R¹is an optionally substituted benzoxazolyl. In some embodiments, R¹is an optionally substituted indazolyl. In certain embodiments, R¹is an optionally substituted 5,6-fused heteroaryl ring having 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R¹is an optionally substituted 6,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is an optionally substituted 6,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In other embodiments, R¹is an optionally substituted 6,6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is an optionally substituted quinolinyl. In some embodiments, R¹is an optionally substituted isoquinolinyl. According to one aspect, R¹is an optionally substituted 6,6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is a quinazoline or a quinoxaline.

In some embodiments, R¹is an optionally substituted heterocyclyl. In some embodiments, R¹is an optionally substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is a substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is an unsubstituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, R¹is an optionally substituted heterocyclyl. In some embodiments. R¹is an optionally substituted 6 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is an optionally substituted 6 membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is an optionally substituted 6 membered partially unsaturated heterocyclic ring having 2 oxygen atoms.

In certain embodiments, R¹is a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R¹is oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl, aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl, thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl, piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl, oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl, tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl, azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl, oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl, dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl, thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl, tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl, oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl, oxathiolanedionyl, piperazinedionyl, morpholinedionyl, thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, R¹is an optionally substituted 5 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In certain embodiments, R¹is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R¹is an optionally substituted tetrahydropyridinyl, dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R¹is an optionally substituted 8-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R¹is an optionally substituted indolinyl. In some embodiments, R¹is an optionally substituted isoindolinyl. In some embodiments, R¹is an optionally substituted 1, 2, 3, 4-tetrahydroquinoline. In some embodiments, R¹is an optionally substituted 1, 2, 3, 4-tetrahydroisoquinoline.

In some embodiments, R¹is an optionally substituted C₁-C₁₀aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, wherein each variable is independently as defined above and described herein. In some embodiments, R¹is an optionally substituted C₁-C₁₀aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally-Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —OC(O)—, or —C(O)O—, wherein each R′ is independently as defined above and described herein. In some embodiments, R¹is an optionally substituted C₁-C₁₀aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally-Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —OC(O)—, or —C(O)O—, wherein each R′ is independently as defined above and described herein.

In some embodiments, R¹is

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In some embodiments, R¹is CH₃—,

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In some embodiments, R¹comprises a terminal optionally substituted —(CH₂)₂-moiety which is connected to L. Examples of such R¹groups are depicted below:

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In some embodiments, R¹comprises a terminal optionally substituted —(CH₂)— moiety which is connected to L. Example such R¹groups are depicted below:

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In some embodiments, R¹is —S—R^L2, wherein R^L2is an optionally substituted C₁-C₉aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—. —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, and each of R′ and -Cy- is independently as defined above and described herein. In some embodiments, R^L2is —S—R^L2, wherein the sulfur atom is connected with the sulfur atom in L group.

In some embodiments, R¹is —C(O)—R^L2, wherein R^L2is an optionally substituted C₁-C₉aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, and each of R′ and -Cy- is independently as defined above and described herein. In some embodiments, R¹is —C(O)—R^L2, wherein the carbonyl group is connected with G in L group. In some embodiments, R¹is —C(O)—R^L2, wherein the carbonyl group is connected with the sulfur atom in L group.

In some embodiments, R^L2is optionally substituted C₁-C₉aliphatic. In some embodiments, R^L2is optionally substituted C₁-C₉alkyl. In some embodiments, R^L2is optionally substituted C₁-C₉alkenyl. In some embodiments, R^L2is optionally substituted C₁-C₉alkynyl. In some embodiments, R^L2is an optionally substituted C₁-C₉aliphatic wherein one or more methylene units are optionally and independently replaced by -Cy- or —C(O)—. In some embodiments, R^L2is an optionally substituted C₁-C₉aliphatic wherein one or more methylene units are optionally and independently replaced by -Cy-. In some embodiments, R^L2is an optionally substituted C₁-C₉aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted heterocycylene. In some embodiments, R^L2is an optionally substituted C₁-C₉aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted arylene. In some embodiments, R^L2is an optionally substituted C₁-C₉aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted heteroarylene. In some embodiments, Ru is an optionally substituted C₁-C₉aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₃-C₁₀carbocyclylene. In some embodiments, R^L2is an optionally substituted C₁-C₉aliphatic wherein two methylene units are optionally and independently replaced by -Cy- or —C(O)—. In some embodiments, R is an optionally substituted C₁-C₉, aliphatic wherein two methylene units are optionally and independently replaced by -Cy- or —C(O)—. Example R^L2groups are depicted below:

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In some embodiments R¹is hydrogen, or an optionally substituted group selected from

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—S—(C₁-C₁₀aliphatic), C₁-C₁₀aliphatic, aryl, C₁-C₆heteroalkyl, heteroaryl and heterocyclyl. In some embodiments, R¹is

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or —S—(C₁-C₁₀aliphatic). In some embodiments, R is

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In some embodiments, R¹is an optionally substituted group selected from —S—(C₁-C₆aliphatic), C₁-C₁₀aliphatic, C₁-C₆heteroaliphatic, aryl, heterocyclyl and heteroaryl.

In some embodiments, R¹is

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In some embodiments, the sulfur atom in the R¹embodiments described above and herein is connected with the sulfur atom, G. E. or —C(O)— moiety in the L embodiments described above and herein. In some embodiments, the —C(O)— moiety in the R¹embodiments described above and herein is connected with the sulfur atom, G, E, or —C(O)— moiety in the L embodiments described above and herein.

In some embodiments, -L-R¹is any combination of the L embodiments and R¹embodiments described above and herein.

In some embodiments, -L-R¹is -L³-G-R¹wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹is -L⁴-G-R¹wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹is -L³-G-S—R^L2, wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹is -L³-G-C(O)—R^L2, wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹is

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wherein R^L2is an optionally substituted C₁-C₉aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, —C(R′)₂—, -Cy-, —O—, —S— —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—, and each G is independently as defined above and described herein.

In some embodiments, -L-R¹is —R^L3—S—S—R^L2, wherein each variable is independently as defined above and described herein. In some embodiments, -L-R¹is —R^L3—C(O)—S—S—R^L2, wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -L-R¹has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, L has the structure of:

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wherein each variable is independently as defined above and described herein.

In some embodiments, -X-L-R¹has the structure of:

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wherein:

the phenyl ring is optionally substituted, and

each of R and X is independently as defined above and described herein.

In some embodiments, -L-R¹is

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In some embodiments, -L-R¹is:

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In some embodiments, -L-R¹is CH₃—,

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In some embodiments, -L-R¹is

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In some embodiments, -L-R¹comprises a terminal optionally substituted —(CH₂)₂-moiety which is connected to X. In some embodiments, -L-R¹comprises a terminal —(CH₂)₂-moiety which is connected to X. Examples of such -L-R¹moieties are depicted below:

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In some embodiments, -L-R¹comprises a terminal optionally substituted —(CH₂)-moiety which is connected to X. In some embodiments, -L-R¹comprises a terminal —(CH₂)— moiety which is connected to X. Examples of such -L-R¹moieties are depicted below:

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In some embodiments, -L-R¹is

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In some embodiments, -L-R¹is CH₃—,

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and X is —S—.

In some embodiments, -L-R¹is CH₃—,

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X is —S—. W is O, Y is —O—, and Z is —O—.

In some embodiments, R¹is

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or —S—(C₁-C₁₀aliphatic).

In some embodiments R¹is

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In some embodiments, X is —O— or —S—, and R¹is

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or —S—(C₁-C₁₀aliphatic).

In some embodiments, X is —O— or —S—, and R¹is

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—S—(C₁-C₁₀aliphatic) or —S—(C₁-C₅₀aliphatic).

In some embodiments, L is a covalent bond and -L-R¹is R¹.

In some embodiments, -L-R¹is not hydrogen.

In some embodiments, -X-L-R¹is R¹is

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—S—(C₁-C₁₀aliphatic) or —S—(C₁-C₅₀aliphatic).

In some embodiments, -X-L-R¹has the structure of

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wherein the

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moiety is optionally substituted. In some embodiments, -X-L-R¹is

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In some embodiments, -X-L-R¹is

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In some embodiments, -X-L-R¹is

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In some embodiments, -X-L-R¹has the structure of

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wherein X′ is O or S, Y′ is —O—, —S— or —NR′—, and the

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moiety is optionally substituted. In some embodiments, Y′ is —O—, —S— or —NH—. In some embodiments,

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In some embodiments,

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In some embodiments,

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In some embodiments, -X-L-R¹has the structure of

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wherein X′ is O or S, and the

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moiety is optionally substituted. In some embodiments,

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In some embodiments, -X-L-R¹is

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wherein the

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is optionally substituted. In some embodiments, -X-L-R¹is

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wherein the

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is substituted. In some embodiments, -X-L-R¹is

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wherein the

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is unsubstituted.

In some embodiments, -X-L-R¹is R¹—C(O)—S-L^x-S— wherein L^xis an optionally substituted group selected from

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In some embodiments, L^xis

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In some embodiments, -X-L-R¹is (CH₃)₃C—S—S-L^x-S—. In some embodiments, -X-L-R¹is R¹—C(═X′)—Y′—C(R)₂—S-L^x-S—. In some embodiments, -X-L-R¹is R—C(═X′)—Y′—CH₂-L^x-S—. In some embodiments. -X-L-R¹is

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As will be appreciated by a person skilled in the art, many of the -X-L-R¹groups described herein are cleavable and can be converted to -X⁻ after administration to a subject. In some embodiments, -X-L-R¹is cleavable. In some embodiments, -X-L-R¹is —S-L-R¹, and is converted to —S⁻ after administration to a subject. In some embodiments, the conversion is promoted by an enzyme of a subject. As appreciated by a person skilled in the art, methods of determining whether the -S-L-R¹group is converted to -S⁻ after administration is widely known and practiced in the art, including those used for studying drug metabolism and pharmacokinetics.

In some embodiments, the internucleotidic linkage having the structure of formula I is

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In some embodiments, the internucleotidic linkage of formula I has the structure of formula I-a:

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wherein each variable is independently as defined above and described herein.

In some embodiments, the internucleotidic linkage of formula I has the structure of formula I-b:

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wherein each variable is independently as defined above and described herein.

In some embodiments, the internucleotidic linkage of formula I is an phosphorothioate triester linkage having the structure of formula I-c:

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wherein R is not —H when L is a covalent bond.

In some embodiments, the internucleotidic linkage having the structure of formula I is

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In some embodiments, the internucleotidic linkage having the structure of formula I-c is

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In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more natural phosphate linkages, and one or more modified internucleotidic linkages having the formula of I-a, I-b, or I-c.

In some embodiments, a modified internucleotidic linkage has the structure of I. In some embodiments, a modified internucleotidic linkage has the structure of I-a. In some embodiments, a modified internucleotidic linkage has the structure of I-b. In some embodiments, a modified internucleotidic linkage has the structure of I-c.

In some embodiments, a modified internucleotidic linkage is phosphorothioate internucleotidic linkage. Examples of internucleotidic linkages having the structure of formula I that can be utilized in accordance with the present disclosure include those described in U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, US 20130178612, US 20150211006, U.S. Pat. No. 9,598,458, US 20170037399, WO 2017/015555, WO 2017/062862, the internucleotidic linkages of each of which is incorporated herein by reference.

Non-limiting examples of internucleotidic linkages that can be utilized in accordance with the present disclosure also include those described in the art, including, but not limited to, those described in any of: Gryaznov, S.; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143, Jones et al. J. Org. Chem. 1993, 58, 2983, Koshkin et al. 1998 Tetrahedron 54: 3607-3630, Lauritsen et al. 2002 Chem. Comm. 5: 530-531, Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256, Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226, Petersen et al. 2003 TRENDS Biotech. 21: 74-81, Schultz et al. 1996 Nucleic Acids Res. 24: 2966, Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507, 220, and Vasseur et al. J. Am. Chem. Soc. 1992, 114, 4006.

In some embodiments, oligonucleotides comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more non-negatively charged internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic linkage is not negatively charged in that at a given pH in an aqueous solution less than 50%, 40%, 40%, 30%, 20%, 10%, 5%, or 1% of the internucleotidic linkage exists in a negatively charged salt form. In some embodiments, a pH is about pH 7.4. In some embodiments, a pH is about 4-9. In some embodiments, the percentage is less than 10%. In some embodiments, the percentage is less than 5%. In some embodiments, the percentage is less than 1%. In some embodiments, an internucleotidic linkage is a non-negatively charged internucleotidic linkage in that the neutral form of the internucleotidic linkage has no pKa that is no more than about 1, 2, 3, 4, 5, 6, or 7 in water. In some embodiments, no pKa is 7 or less. In some embodiments, no pKa is 6 or less. In some embodiments, no pKa is 5 or less. In some embodiments, no pKa is 4 or less. In some embodiments, no pKa is 3 or less. In some embodiments, no pKa is 2 or less. In some embodiments, no pKa is 1 or less. In some embodiments, pKa of the neutral form of an internucleotidic linkage can be represented by pKa of the neutral form of a compound having the structure of CH₃—the internucleotidic linkage-CH₃. For example, pKa of the neutral form of an internucleotidic linkage having the structure of formula I may be represented by the pKa of the neutral form of a compound having the structure of

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pKa of

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can be represented by pKa

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In some embodiments, a non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage is a positively-charged internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage comprises a guanidine moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises a heteroaryl base moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises a triazole moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises an alkynyl moiety.

In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises —P^L(—N═)—, wherein P^Lis as described in the present disclosure. In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises —P(—N═)—. In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises —P(═)(—N═)—. In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises —P(═O)(—N═)—. In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises —P(═S)(—N═)—.

In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises

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wherein P^Lis as described in the present disclosure. For example, in some embodiments, P^Lis P; in some embodiments, P^Lis P(O); in some embodiments, P^Lis P(S); etc. In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2 II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof (not negatively charged). In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, has the structure of formula I-n-1 or a salt form thereof:

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In some embodiments, X is a covalent bond and -X-Cy-R¹is -Cy-R. In some embodiments, -Cy- is an optionally substituted bivalent group selected from a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms. In some embodiments. -Cy- is an optionally substituted bivalent 5-20 membered heteroaryl ring having 1-10 heteroatoms. In some embodiments, -Cy-R¹is optionally substituted 5-20 membered heteroaryl ring having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, -Cy-R¹is optionally substituted 5-membered heteroaryl ring having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, -Cy-R¹is optionally substituted 6-membered heteroaryl ring having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, -Cy-R¹is optionally substituted triazolyl.

In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, has the structure of formula I-n-2 or a salt form thereof:

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In some embodiments, R¹is R′. In some embodiments, L is a covalent bond. In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, has the structure of formula I-n-3 or a salt form thereof:

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In some embodiments, two R′ on different nitrogen atoms are taken together to form a ring as described. In some embodiments, a formed ring is 5-membered. In some embodiments, a formed ring is 6-membered. In some embodiments, a formed ring is substituted. In some embodiments, the two R′ group that are not taken together to form a ring are each independently R. In some embodiments, the two R′ group that are not taken together to form a ring are each independently hydrogen or an optionally substituted C_1-6aliphatic. In some embodiments, the two R′ group that are not taken together to form a ring are each independently hydrogen or an optionally substituted C_1-6alkyl. In some embodiments, the two R′ group that are not taken together to form a ring are the same. In some embodiments, the two R′ group that are not taken together to form a ring are different. In some embodiments, both of them are —CH₃.

In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, has the structure of formula I-n-4 or a salt form thereof:

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wherein each of L^aand L^bis independently L or —N(R¹)—, and each other variable is independently as described in the present disclosure. In some embodiments, L is a covalent bond, and an internucleotidic linkage of formula I-n-4 has the structure of:

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or a salt form thereof, wherein each variable is independently as described in the present disclosure.

In some embodiments, L^ais —N(R¹)—. In some embodiments, L^ais L as described in the present disclosure. In some embodiments, L^ais a covalent bond. In some embodiments, L^ais —N(R′)—. In some embodiments, L^ais —N(R)—. In some embodiments, L^ais —O—. In some embodiments, L^ais —S—. In some embodiments, L^ais —S(O)—. In some embodiments, L^ais —S(O)₂—. In some embodiments, L^ais —S(O)₂N(R′)—. In some embodiments, L^bis —N(R′)—. In some embodiments, L^bis L as described in the present disclosure. In some embodiments, L^bis a covalent bond. In some embodiments, L^bis —N(R′)—. In some embodiments, L^bis —N(R)—. In some embodiments, L^bis —O—. In some embodiments, L^bis —S—. In some embodiments, L^bis —S(O)—. In some embodiments, L^bis —S(O)₂—. In some embodiments, L^bis —S(O)₂N(R′)—. In some embodiments, L^aand L^bare the same. In some embodiments, L^aand L^bare different. In some embodiments, at least one of L^aand L^bis —N(R′)—. In some embodiments, at least one of L^aand L^bis —O—. In some embodiments, at least one of L^aand L^bis —S—. In some embodiments, at least one of L^aand L^bis a covalent bond. In some embodiments, as described herein, R¹is R. In some embodiments, R¹is —H. In some embodiments, R¹is optionally substituted C_1-10aliphatic. In some embodiments, R¹is optionally substituted C_1-10alkyl. In some embodiments, a structure of formula I-n-4 is a structure of formula I-n-2. In some embodiments, a structure of formula I-n-4 is a structure of formula I-n-3. In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, has the structure of formula I. In some embodiments, X, e.g., in formula I, II, etc., is —N(-L-R⁵)—, wherein R⁵is R as described herein. In some embodiments, X is —NH—. In some embodiments, L. e.g., in -X-L- of formula I. II, etc., comprises —SO₂—. In some embodiments, L is —SO₂—. In some embodiments, L is a covalent bond. In some embodiments. L is —C(O)O—(C_1-4alkylene)- wherein the alkylene is optionally substituted. In some embodiments, L is —C(O)OCH₂—. In some embodiments, R¹, e.g., in formula I, III, etc., comprise an optionally substituted ring. In some embodiments, R¹is R as described herein. In some embodiments, R¹is optionally substituted phenyl. In some embodiments, R¹is 4-methylphenyl. In some embodiments, R¹is 4-methoxyphenyl. In some embodiments, R¹is 4-aminophenyl. In some embodiments, R¹is an optionally substituted heteroaliphatic ring. In some embodiments, R¹is an optionally substituted 3-10 (e.g., 3, 4, 5, 6, 7, or 8) membered heteroaliphatic ring. In some embodiments, R¹is an optionally substituted 5- or 6-membered saturated monocyclic heteroaliphatic ring having 1-3 heteroatoms. In some embodiments, the ring is 5-membered. In some embodiments, the ring is 6-membered. In some embodiments, the number of ring heteroatom(s) is 1. In some embodiments, the number of ring heteroatoms is 2. In some embodiments, a heteroatom is oxygen. In some embodiments, R¹is optionally substituted

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In some embodiments, R¹is optionally substituted

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In some embodiments, R¹is

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In some embodiments, R¹is optionally substituted C_1-30aliphatic. In some embodiments, R¹is optionally substituted C_1-10alkyl.

In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, has the structure of formula II or a salt form thereof:

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or a salt form thereof, wherein:

P^Lis P(═W), P, or P→B(R′)₃;

W is O, N(-L-R⁵), S or Se;

each of X, Y and Z is independently —O—, —S—, —N(-L-R⁵)—, or L;

R⁵is —H, -L-R′, halogen, —CN, —NO₂, -L-Si(R′)₃, —OR′, —SR′, or —N(R′)₂;

Ring A^Lis an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms;

each R^sis independently —H, halogen, —CN, —N₃, —NO, —NO₂, -L-R′, -L-Si(R)₃, -L-OR′, -L-SR′, -L-N(R′)₂, —O-L-R′, —O-L-Si(R)₃, —O-L-OR′, —O-L-SR′, or —O-L-N(R′)₂;

g is 0-20;

each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C alkenylene, —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃], —OP(O)(OR′)O—, —OP(O)(SR′)O—, —P(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L;

each R′ is independently —R, —C(O)R, —C(O)OR, or —S(O)₂R;

each R is independently —H, or an optionally substituted group selected from C_1-30aliphatic, C_1-30heteroaliphatic having 1-10 heteroatoms, C_1-30aryl, C_6-30arylaliphatic, C_6-30arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or

two R groups are optionally and independently taken together to form a covalent bond, or,

In some embodiments, Ring A^Lin various structures of the present disclosure is an optionally substituted aryl ring. In some embodiments, Ring A^Lis an optionally substituted phenyl ring. In some embodiments, Ring A^Lis an optionally substituted 3-10 (e.g., 3, 4, 5, 6, 7, or 8) membered heteroaliphatic ring. In some embodiments, Ring A^Lis an optionally substituted 5- or 6-membered saturated monocyclic heteroaliphatic ring having 1-3 heteroatoms. In some embodiments, the ring is 5-membered. In some embodiments, the ring is 6-membered. In some embodiments, the number of ring heteroatom(s) is 1. In some embodiments, the number of ring heteroatoms is 2. In some embodiments, a heteroatom is oxygen. In some embodiments, R^sis optionally substituted C₁-C₆alkyl group. In some embodiments, R^sis Me. In some embodiments, R^sis OR, wherein R is hydrogen or C₁-C₆alkyl group. In some embodiments, R^sis OH. In some embodiments, R^sis OMe. In some embodiments, R^sis —N(R′)₂. In some embodiments, R^sis —NH₂. In some embodiments,

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In some embodiments,

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In some embodiments,

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In some embodiments,

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In some embodiments, an internucleotidic linkage, e.g. a neutral internucleotidic linkage of formula I or II, is n002

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which, as one skilled in the art will appreciate, can exist under certain conditions in the form of

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In some embodiments, an internucleotidic linkage, e.g. a neutral internucleotidic linkage of formula I or II, is n005(

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which, as one skilled in the art will appreciate, can exist under certain conditions in the form of

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In some embodiments, an internucleotidic linkage, e.g. a neutral internucleotidic linkage of formula I or II, is n006

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which, as one skilled in the art will appreciate, can exist under certain conditions in the form of

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In some embodiments, an internucleotidic linkage, e.g. a neutral internucleotidic linkage of formula I or II, is n007

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which, as one skilled in the art will appreciate, can exist under certain conditions in a form of

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In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II, has the structure of formula II-a-1 or a salt form thereof:

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or a salt form thereof.

In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II, has the structure of formula II-a-2 or a salt form thereof:

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or a salt form thereof.

In some embodiments, A^Lis bonded to —N═ or L through a carbon atom. In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II or II-a-1, II-a-2, has the structure of formula II-b-1 or a salt form thereof:

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In some embodiments, a structure of formula II-a-1 or II-a-2 may be referred to a structure of formula II-a. In some embodiments, a structure of formula II-b-1 or II-b-2 may be referred to a structure of formula II-b. In some embodiments, a structure of formula II-c-1 or II-c-2 may be referred to a structure of formula II-c. In some embodiments, a structure of formula II-d-1 or II-d-2 may be referred to a structure of formula II-d.

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In some embodiments, Ring A^Lis an optionally substituted 3-20 membered monocyclic ring having 0-10 heteroatoms (in addition to the two nitrogen atoms for formula I-b). In some embodiments, Ring A^Lis an optionally substituted 5-membered monocyclic saturated ring.

In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II, II-a, or II-b, has the structure of formula II-c-1 or a salt form thereof:

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In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II, II-a, or II-b, has the structure of formula II-c-2 or a salt form thereof:

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In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II, II-a, II-b, or II-c has the structure of formula II-d-1 or a salt form thereof:

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In some embodiments, each R′ is independently optionally substituted C_1-6aliphatic. In some embodiments, each R′ is independently optionally substituted C_1-6alkyl. In some embodiments, each R′ is independently —CH₃. In some embodiments, each R^sis —H.

In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

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In some embodiments, W is O. In some embodiments, W is S. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled. In some embodiments, the linkage phosphorus is Rp. In some embodiments, the linkage phosphorus is Sp.

In some embodiments, each non-negatively charged internucleotidic linkage or neutral internucleotidic linkage (e.g., those of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2) is independently Rp at its linkage phosphorus. In some embodiments, each negatively charged chiral internucleotidic linkage is Sp at its linkage phosphorus. In some embodiments, each phosphorothioate internucleotidic linkages is Sp at its linkage phosphorus. In some embodiments, each natural phosphate linkage is independently bonded to a sugar comprising a 2′-OR modification, wherein R is not —H. In some embodiments, each natural phosphate linkage is independently bonded to a sugar comprising a 2′-OR modification, wherein R is not —H, at a 3′-position. In some embodiments, each sugar that contains no 2′-OR modification wherein R is not —H is independently bonded to at least one non-natural phosphate linkages, in many cases, two non-natural natural phosphate linkages. In some embodiments, each 2′-F modified sugar is independently bonded to at least one non-natural phosphate linkages, in many cases, two non-natural natural phosphate linkages. In some embodiments, each non-natural phosphate linkage is a phosphorothioate internucleotidic linkage. In some embodiments, each non-natural phosphate linkage is a Sp phosphorothioate internucleotidic linkage. In some embodiments, each sugar bonded to non-negatively charged internucleotidic linkage or neutral internucleotidic linkage (e.g., those of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2) independently contains no 2′-OR. In some embodiments, each sugar bonded to non-negatively charged internucleotidic linkage or neutral internucleotidic linkage (e.g., those of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2) is a 2′-F modified sugar.

In some embodiments, the present disclosure provides a compound, e.g., an oligonucleotide, a chirally controlled oligonucleotide, an oligonucleotide of a provided composition (e.g., of a plurality of oligonucleotides), having the structure of formula O-I:

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or a salt thereof, wherein:

R^5sis independently R′ or —OR′;

each BA is independently an optionally substituted group selected from C_3-30cycloaliphatic, C_6-30aryl, C_5-30heteroaryl having 1-10 heteroatoms, C_3-30heterocyclyl having 1-10 heteroatoms, a natural nucleobase moiety, and a modified nucleobase moiety;

each s is independently 0-20;

each L^sis independently —C(R^5s)₂—, or L;

each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L.

each Ring A is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each L^Pis independently an internucleotidic linkage;

z is 1-1000;

L^3Eis L or -L-L-;

R^3Eis —R′, -L-R′, —OR′, or a solid support;

each R′ is independently —R, —C(O)R, —C(O)OR, or —S(O)₂R;

two R groups are optionally and independently taken together to form a covalent bond, or

In some embodiments, each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, III, I-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, each internucleotidic linkage independently has the structure of formula I, I-a, I-b. I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, each internucleotidic linkage independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, each internucleotidic linkage independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

In some embodiments, each BA is independently an optionally substituted group selected from C_5-30, heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and C_3-30heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon;

each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2. II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

In some embodiments, each BA is independently an optionally substituted C_5-30heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein the heteroaryl comprises one or more heteroatoms selected from oxygen and nitrogen;

each Ring A is independently an optionally substituted 5-10 membered monocyclic or bicyclic saturated ring having 0-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein the ring comprises at least one oxygen atom; and

each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

In some embodiments, each BA is independently an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U;

each Ring A is independently an optionally substituted 5-7 membered monocyclic or bicyclic saturated ring having one or more oxygen atoms; and

each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, I-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

In some embodiments, each BA is independently an optionally substituted or protected nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil and tautomers thereof;

each Ring A is independently an optionally substituted 5-7 membered monocyclic or bicyclic saturated ring having one or more oxygen atoms; and

In some embodiments, BA is an optionally substituted group selected from C_3-30cycloaliphatic, C_6-30aryl, C_5-30heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C_3-3heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. In some embodiments, BA is an optionally substituted group selected from C_5-3heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C_3-30heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. In some embodiments, BA is an optionally substituted group selected from C_5-30heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. In some embodiments, BA is optionally substituted C_5-30heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, BA is optionally substituted natural nucleobases and tautomers thereof. In some embodiments, BA is protected natural nucleobases and tautomers thereof. Various nucleobase protecting groups for oligonucleotide synthesis are known and can be utilized in accordance with the present disclosure. In some embodiments, BA is an optionally substituted nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil, and tautomers thereof. In some embodiments, BA is an optionally protected nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil, and tautomers thereof.

In some embodiments, BA is optionally substituted C_3-30cycloaliphatic. In some embodiments, BA is optionally substituted C_6-30aryl. In some embodiments, BA is optionally substituted C_3-30heterocyclyl. In some embodiments, BA is optionally substituted C_5-30heteroaryl. In some embodiments, BA is an optionally substituted natural base moiety. In some embodiments, BA is an optionally substituted modified base moiety. BA is an optionally substituted group selected from C_3-30cycloaliphatic, C_6-30aryl, C_3-30heterocyclyl, and C_5-30heteroaryl. In some embodiments, BA is an optionally substituted group selected from C_3-30cycloaliphatic, C_6-30aryl, C_3-30heterocyclyl, C_5-30heteroaryl, and a natural nucleobase moiety.

In some embodiments, BA is connected through an aromatic ring. In some embodiments, BA is connected through a heteroatom. In some embodiments, BA is connected through a ring heteroatom of an aromatic ring. In some embodiments, BA is connected through a ring nitrogen atom of an aromatic ring.

In some embodiments, BA is a natural nucleobase moiety. In some embodiments, BA is an optionally substituted natural nucleobase moiety. In some embodiments, BA is a substituted natural nucleobase moiety. In some embodiments, BA is optionally substituted, or an optionally substituted tautomer of, A, T, C, U, or G. In some embodiments, BA is natural nucleobase A, T, C, U, or G. In some embodiments, BA is an optionally substituted group selected from natural nucleobases A, T, C, U, and G.

In some embodiments, BA is an optionally substituted purine base residue. In some embodiments, BA is a protected purine base residue. In some embodiments, BA is an optionally substituted adenine residue. In some embodiments, BA is a protected adenine residue. In some embodiments, BA is an optionally substituted guanine residue. In some embodiments, BA is a protected guanine residue. In some embodiments, BA is an optionally substituted cytosine residue. In some embodiments, BA is a protected cytosine residue. In some embodiments, BA is an optionally substituted thymine residue. In some embodiments, BA is a protected thymine residue. In some embodiments, BA is an optionally substituted uracil residue. In some embodiments, BA is a protected uracil residue. In some embodiments, BA is an optionally substituted 5-methylcytosine residue. In some embodiments, BA is a protected 5-methylcytosine residue.

In some embodiments, BA is a protected base residue as used in oligonucleotide preparation. In some embodiments, BA is a base residue illustrated in US 2011/0294124, US 2015/0211006, US 2015/0197540, and WO 2015/107425, each of which is incorporated herein by reference.

In some embodiments, R^5s-L^s- is —CH₂OH. In some embodiments, R^5s-L^s- is —CH(R^5s)—OH, wherein R^5sis as described in the present disclosure. In some embodiments, L^sis —CH₂—. In some embodiments, L^sis —CH(R^5s)- wherein R^5sis not —H. In some embodiments, L^sis —CH(R^5s)—wherein R^5sis not —H and is otherwise R. In some embodiments, R is optionally substituted C₁-C₆aliphatic. In some embodiments, R is optionally substituted C₁-C₆alkyl. In some embodiments, R is methyl. In some embodiments, —CH(R^5s)— wherein R^5sis not —H has is R. In some embodiments, —CH(R^5s)— wherein R^5sis not —H has is S.

Example embodiments for variables, e.g., variables of each of the formulae, are additionally described in the present disclosure, and may be independently and optionally combined.

In some embodiments, the present disclosure provides oligonucleotides and oligonucleotide compositions that are chirally controlled. For instance, in some embodiments, a provided composition contains controlled levels of one or more individual oligonucleotide types, wherein an oligonucleotide type is defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone P-modifications. In some embodiments, oligonucleotides of the same oligonucleotide type are identical.

In some embodiments, a provided oligonucleotide is an altmer. In some embodiments, a provided oligonucleotide is a P-modification altmer. In some embodiments, a provided oligonucleotide is a stereoaltmer.

In some embodiments, a provided oligonucleotide is a blockmer. In some embodiments, a provided oligonucleotide is a P-modification blockmer. In some embodiments, a provided oligonucleotide is a stereoblockmer.

In some embodiments, a provided oligonucleotide is a gapmer.

In some embodiments, a provided oligonucleotide is a skipmer.

In some embodiments, a provided oligonucleotide is a hemimer. In some embodiments, a hemimer is an oligonucleotide wherein the 5′-end or the 3′-end has a sequence that possesses a structure feature that the rest of the oligonucleotide does not have. In some embodiments, the 5′-end or the 3′-nd has or comprises 2 to 20 nucleotides. In some embodiments, a structural feature is a base modification. In some embodiments, a structural feature is a sugar modification. In some embodiments, a structural feature is a P-modification. In some embodiments, a structural feature is stereochemistry of the chiral internucleotidic linkage. In some embodiments, a structural feature is or comprises a base modification, a sugar modification, a P-modification, or stereochemistry of the chiral internucleotidic linkage, or combinations thereof. In some embodiments, a hemimer is an oligonucleotide in which each sugar moiety of the 5′-end sequence shares a common modification. In some embodiments, a hemimer is an oligonucleotide in which each sugar moiety of the 3′-nd sequence shares a common modification. In some embodiments, a common sugar modification of the 5′ or 3′ end sequence is not shared by any other sugar moieties in the oligonucleotide. In some embodiments, an example hemimer is an oligonucleotide comprising a sequence of substituted or unsubstituted 2′-O-alkyl sugar modified nucleosides, bicyclic sugar modified nucleosides. β-D-ribonucleosides or 3-D- deoxyribonucleosides (for example 2′-MOE modified nucleosides, and LNA™ or ENA™ bicyclic sugar modified nucleosides) at one terminus and a sequence of nucleosides with a different sugar moiety (such as a substituted or unsubstituted 2′-O-alkyl sugar modified nucleosides, bicyclic sugar modified nucleosides or natural ones) at the other terminus. In some embodiments, a provided oligonucleotide is a combination of one or more of unimer, altmer, blockmer, gapmer, hemimer and skipmer. In some embodiments, a provided oligonucleotide is a combination of one or more of unimer, altmer, blockmer, gapmer, and skipmer. For instance, in some embodiments, a provided oligonucleotide is both an altmer and a gapmer. In some embodiments, a provided nucleotide is both a gapmer and a skipmer. One of skill in the chemical and synthetic arts will recognize that numerous other combinations of patterns are available and are limited only by the commercial availability and/or synthetic accessibility of constituent parts required to synthesize a provided oligonucleotide in accordance with methods of the present disclosure. In some embodiments, a hemimer structure provides advantageous benefits. In some embodiments, provided oligonucleotides are 5′-hemimers that comprises modified sugar moieties in a 5′-end sequence. In some embodiments, provided oligonucleotides are 5′-hemimers that comprises modified 2′-sugar moieties in a 5′-end sequence.

In some embodiments, a provided oligonucleotide comprises one or more optionally substituted nucleotides. In some embodiments, a provided oligonucleotide comprises one or more modified nucleotides. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted nucleosides. In some embodiments, a provided oligonucleotide comprises one or more modified nucleosides. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted nucleosides or sugars of LNAs.

In some embodiments, a provided oligonucleotide comprises one or more optionally substituted nucleobases. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted natural nucleobases. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted modified nucleobases. In some embodiments, a provided oligonucleotide comprises one or more 5-methylcytidine; 5-hydroxymethylcytidine, 5-formylcytosine, or 5-carboxylcytosine. In some embodiments, a provided oligonucleotide comprises one or more 5-methylcytidine.

In some embodiments, a provided oligonucleotide comprises one or more optionally substituted sugars. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted sugars found in naturally occurring DNA and RNA. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted ribose or deoxyribose. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted ribose or deoxyribose, wherein one or more hydroxyl groups of the ribose or deoxyribose moiety is optionally and independently replaced by halogen, R′, —N(R′)₂, —OR′, or —SR′, wherein each R′ is independently as defined above and described herein. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2′ position of the deoxyribose is optionally and independently substituted with R, halogen, R′, —N(R′)₂, —OR′, or —SR′, wherein each R′ is independently as defined above and described herein. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2′ position of the deoxyribose is optionally and independently substituted with halogen. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2′ position of the deoxyribose is optionally and independently substituted with one or more —F, halogen. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2′ position of the deoxyribose is optionally and independently substituted with —OR′, wherein each R′ is independently as defined above and described herein. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2′ position of the deoxyribose is optionally and independently substituted with —OR′, wherein each R′ is independently an optionally substituted C₁-C₆aliphatic. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2′ position of the deoxyribose is optionally and independently substituted with —OR′, wherein each R′ is independently an optionally substituted C₁-C₆alkyl. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2′ position of the deoxyribose is optionally and independently substituted with -OMe. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2′ position of the deoxyribose is optionally and independently substituted with —O-methoxyethyl.

In some embodiments, a provided oligonucleotide is single-stranded oligonucleotide. In some embodiments, a provided oligonucleotide is a hybridized oligonucleotide strand. In certain embodiments, a provided oligonucleotide is a partially hybridized oligonucleotide strand. In certain embodiments, a provided oligonucleotide is a completely hybridized oligonucleotide strand. In certain embodiments, a provided oligonucleotide is a double-stranded oligonucleotide. In certain embodiments, a provided oligonucleotide is a triple-stranded oligonucleotide (e.g., a triplex).

In some embodiments, a provided oligonucleotide is chimeric. For example, in some embodiments, a provided oligonucleotide is DNA-RNA chimera, DNA-LNA chimera, etc.

In some embodiments, an oligonucleotide is a chirally controlled oligonucleotide variant of an oligonucleotide described in WO2012/030683. For example, in some embodiments, a chirally controlled oligonucleotide variant comprises a chirally controlled version of a chiral internucleotidic linkage which is not chirally controlled in WO2012/030683. In some embodiments, a chirally controlled oligonucleotide variant comprises one or more chirally controlled internucleotidic linkages which independently replace one or more natural phosphate linkages or non-chirally controlled modified internucleotidic linkages in WO2012/030683.

In some embodiments, a provided oligonucleotide is or comprises a portion of GNA, LNA, PNA, TNA or Morpholino.

In some embodiments, a provided oligonucleotide is from about 15 to about 25 nucleotide units in length. In some embodiments, a provided oligonucleotide is from about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide units in length.

In some embodiments, the present disclosure provides oligonucleotides comprising one or more modified internucleotidic linkage, which can be chiral at linkage phosphorus and chirally controlled. In some embodiments, an oligonucleotide comprises one or more linkages L^PO, L^PAor L^PB, wherein:

each L^POis independently

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or a salt form thereof;

each L^PAis independently an internucleotidic linkage having the structure of

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or a salt form thereof;

each L^PBis independently an internucleotidic linkage having the structure of

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or a salt form thereof;

N^xis —N(-L-R⁵)-L-R¹,

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and

W^Nis ═N-L-R⁵,

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wherein each other variable is independently as described herein.

In some embodiments, each L^POis independently

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or a salt form thereof.

In some embodiments, —O-L-R¹is —OH. In some embodiments, -X-L-R¹, e.g., in L^POis —OCH₂CH₂CN. In some embodiments, —S-L-R¹is —SH. In some embodiments, L^PAis a phosphorothioate internucleotidic linkage with the specified stereochemistry. In some embodiments, L^PBis a phosphorothioate internucleotidic linkage with the specified stereochemistry. In some embodiments, X is-O—, and -X-L-R¹is as described in the present disclosure, e.g., -X-L-R¹is

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wherein each variable is independently in accordance with the present disclosure, or H-X-L-R¹is a chiral auxiliary as described herein. In some embodiments, -X-L-R¹is

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wherein G⁴and G⁵are taken together to form an optionally substituted ring as described herein. In some embodiments, -X-L-R¹is

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In some embodiments, G²is —CH₂Si(R)₃as described herein. In some embodiments, G²is —CH₂Si(Ph)₂Me. In some embodiments, G²comprises an electron-withdrawing group as described herein, for example, in some embodiments, G²is —CH₂SO₂R as described herein. In some embodiments, G²is —CH₂SO₂Ph.

In some embodiments, N^xis —N(-L-R⁵)-L-R¹, and an internucleotidic linkage having such a N^xgroup is an internucleotidic linkage having the structure of formula I wherein P^Lis P═O, Y and Z are —O—, and X is —N(-L-R⁵)— linkage phosphorus stereochemistry is as specified. In some embodiments, N^xis

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and an internucleotidic linkage having such a N^xgroup is an internucleotidic linkage having the structure of formula II, wherein P^Lis P═O, Y and Z are —O—, and X is —N(-L-R⁵)—, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, N^xis

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In some embodiments, N^xis

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In some embodiments, N^xis

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In some embodiments, N^xis

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In some embodiments, N^xis

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and an internucleotidic linkage having such a N^xgroup is an internucleotidic linkage having the structure of formula I-n-3, wherein P^Lis P═O, and Y and Z are —O—, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, R¹is optionally substituted alkyl. In some embodiments, R¹is methyl. In some embodiments, N^x

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In some embodiments, two R¹on the same nitrogen independently are taken together to form an optionally substituted ring as described herein, e.g., an optionally substituted 5- or 6-membered ring which in addition to the nitrogen atom, has 1-3 heteroatoms. In some embodiments the ring is saturated. In some embodiments, the ring is monocyclic. In some embodiments N^xis

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In some embodiments, N^xis

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In some embodiments, N^xis

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Those skilled in the art will appreciate that two —N(R¹)₂groups, in any, in a structure or formula can either be the same or different. In some embodiments, N^xis

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and an internucleotidic linkage having such a N^xgroup is an internucleotidic linkage having the structure of formula I-n4, wherein P^Lis P═O. L is a covalent bond, and Y and Z are —O—, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, N^xis

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and an internucleotidic linkage having such a N^xgroup is an internucleotidic linkage having the structure of formula II-a-1, wherein P^Lis P═O, L is a covalent bond, and Y and Z are —O—, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, N^xis

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and an internucleotidic linkage having such a N^xgroup is an internucleotidic linkage having the structure of formula II-b-1, wherein PL is P═O, L is a covalent bond, and Y and Z are —O—, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, N^xis

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and an internucleotidic linkage having such a N^xgroup is an internucleotidic linkage having the structure of formula -c-1, wherein P^Lis P═O, L is a covalent bond, and Y and Z are —O—, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, N^xis

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and an internucleotidic linkage having such a N^xgroup is an internucleotidic linkage having the structure of formula II-d-1, wherein P^Lis P═O, L is a covalent bond, and Y and Z are —O—, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, R′ or R^sis optionally substituted alkyl. In some embodiments, R′ or R^sis —CH₃. In some embodiments, R′ or R^sis —CH₂(CH₂)₁₀CH₃In some embodiments, R^sis —H. In some embodiments, N^xis

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In some embodiments, N^xis

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In some embodiments P=W^Nis a P^Ngroup as described herein. In some embodiments, W^Nis

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wherein each variable is as described herein (for example, in N^x). In some embodiments, W^Nis

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In some embodiments, as described herein R′ or R^sis optionally substituted alkyl or —H. In some embodiments, R′ is —CH₃. In some embodiments, R′ is —CH₂(CH₂)₁₀CH₃. In some embodiments, R^sis —H In some embodiments, W^Nis

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In some embodiments, W^Nis

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In some embodiments, W^Nis ═N-L-R⁵wherein each variable is as described herein. For example, in some embodiments. L is —SO₂—. In some embodiments, L is —C(O)OCH₂—. In some embodiments, as described herein, R⁵is or comprise an optionally substituted ring. In some embodiments, R⁵is R as described herein. In some embodiments, R⁵is optionally substituted phenyl. In some embodiments, R⁵is 4-methylphenyl. In some embodiments, R⁵is 4-methoxyphenyl. In some embodiments, R⁵is 4-aminophenyl. In some embodiments, R⁵is an optionally substituted heteroaliphatic ring. In some embodiments, R⁵is an optionally substituted 3-10 (e.g., 3, 4, 5, 6, 7, or 8) membered heteroaliphatic ring. In some embodiments, R⁵is an optionally substituted 5- or 6-membered saturated monocyclic heteroaliphatic ring having 1-3 heteroatoms. In some embodiments, the ring is 5-membered. In some embodiments, the ring is 6-membered. In some embodiments, the number of ring heteroatom(s) is 1. In some embodiments, the number of ring heteroatoms is 2. In some embodiments, a heteroatom is oxygen. In some embodiments, R⁵is optionally substituted

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In some embodiments, R⁵is optionally substituted

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In some embodiments, R⁵is

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In some embodiments, R⁵is optionally substituted C_1-30aliphatic. In some embodiments, R⁵is optionally substituted C_1-10alkyl. In some embodiments, W^Nis

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In some embodiments, W^Nis

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In some embodiments, W^Nis

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In some embodiments, W^Nis

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In some embodiments, W^Nis

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In some embodiments, W^Nis

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In some embodiments, W^Nis

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In some embodiments, W^Nis

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In some embodiments, W^Nis

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In some embodiments, W^Nis

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In some embodiments, Q⁻ is PF₆⁻.

In some embodiments, -X-L-R¹in

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In some embodiments, -X-L-R¹in

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In some embodiments, G²is —CH₂Si(R)₃described herein. In some embodiments, G²is —CH₂Si(Ph)₂Me. In some embodiments, -X-L-R¹in

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In some embodiments, -X-L-R¹in

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In some embodiments, G²comprises an electron-withdrawing group as described herein. In some embodiments, G²is —CH₂SO₂R, wherein R is not —H. In some embodiments, R is optionally substituted phenyl. In some embodiments, G²is —CH₂SO₂Ph. In some embodiments, R is optionally substituted C_1-6aliphatic, e.g., t-butyl. In some embodiments, as described herein, R¹is —C(O)R′. In some embodiments, R¹is —C(O)CH₃. In some embodiments, R¹is —H.

In some embodiments, L^POis a natural phosphate linkage. In some embodiments, L^PAis a Rp phosphorothioate internucleotidic linkage. In some embodiments, L^PAis a Rp non-negatively charged internucleotidic linkage. e.g., n001. In some embodiments, L^PBis a Sp phosphorothioate internucleotidic linkage. In some embodiments, L^PBis a Sp non-negatively charged internucleotidic linkage, e.g., n001. In some embodiments, an oligonucleotide comprises one or more linkages L. In some embodiments, an oligonucleotide comprises one or more linkages L^PA. In some embodiments, an oligonucleotide comprises one or more linkages L^PB. In some embodiments, an oligonucleotide comprises one or more internucleotidic linkages independently selected from L^PO, L^PAand L^PB. In some embodiments, each internucleotidic linkage is independently selected from L^PO, L^PAand L^PB. In some embodiments, each internucleotidic linkage is independently selected from L^PAand L^PB. In some embodiments, at least one internucleotidic linkage is L^PAor L^PB. In some embodiments, each chirally controlled internucleotidic linkage is independently selected from L^PAand L^PB.

In some embodiments, the present disclosure provides oligonucleotides (e.g., chirally controlled oligonucleotides) and compositions thereof (e.g., chirally controlled oligonucleotide compositions), wherein the internucleotidic linkages of the oligonucleotides or regions thereof are or comprise the following consecutive internucleotidic linkages (from 5′ to 3′):

(L^PX/L^PO)t[(L^PA)n(L^PB)m]y, (L^PX/L^PO)t[(L^PO)n(L^PB)m]y, (L^PX/L^PO)t[(L^PO/L^PA)n(L^PB)m]y, [(L^PA)n(L^PB)m]y, [(L^PO)n(L^PB)m]y, ((L^PB)t[(L^PA)n(L^PB)m]y, (L^PB)t[(L^PO)(L^PB)m]y, (L^PB)t[(L^PO/L^PA)n(L^PB)m]y, [(L^PA)n(L^PB)m]y, [(L^PO)n(L^PB)m]y, [(L^PO/L^PA)n(L^PB)m]y, (L^PA)t(L^PX)n(L^PA)m, (L^PX/L^PO)t(L^PX)n(L^PX/L^PO)m, (L^PX/L^PO)t(L^PB)n(L^PX/L^PO)m, (L^PX/L^PO)t[(L^PX/L^PO)n]y(L^PX/L^PO)m, (L^PX/L^PO)t[(L^PB/L^PO)n]y(L^PX/L^PO)m, (L^PX/L^PO)t[(L^PB/L^PO)n]y(L^PX/L^PO)m, (L^PA/L^PO)t(L^PX)n(L^PA/L^PO)m, (L^PA/L^PO)t(L^PB)n(L^PA/L^PO)m, (L^PA/L^PO)t[(L^PX/L^PO)n]y(L^PA/L^PO)m, (L^PA/L^PO)t[(L^PB/L^PO)n]y(L^PA/L^PO)m, or (L^PA/L^PO)t[(L^PB/L^PO)n]y(L^PA/L^PO)m, or a combination thereof, wherein:

each L^PXis independently L^PAor L^PB; and

each other variable is independently as described herein.

In some embodiments, internucleotidic linkages of an provided oligonucleotides or regions thereof comprise or are consecutive internucleotidic linkages [(L^PA)n(L^PB)m]y, [(L^PO)n(L^PB)m]y, (L^PB)t[(L^PA)n(L^PB)m]y, or (L^PB)t[(L^PO)n(L^PB)m]y. In some embodiments, internucleotidic linkages of an provided oligonucleotides or regions thereof comprise or are consecutive internucleotidic linkages (L^PA)(L^PB)m. In some embodiments, internucleotidic linkages of an provided oligonucleotides or regions thereof comprise or are consecutive internucleotidic linkages [(L^PA)(L^PB)m]y. In some embodiments, internucleotidic linkages of an provided oligonucleotides or regions thereof comprise or are consecutive internucleotidic linkages (L^PB)t(L^PA)(L^PB)m. In some embodiments, each sugar between two of the consecutive internucleotidic linkages independently contains no 2′-modification. In some embodiments, each sugar between two of the consecutive internucleotidic linkages is independently

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In some embodiments, n is 1. In some embodiments, y is 1. In some embodiments, y is 2-10. In some embodiments, t is 1. In some embodiments, t is 2-10. In some embodiments, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is 1, and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is 1, and m is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, t is 2-10, n is 1 and m is 2-10. In some embodiments, each L^PAis independently

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or a salt form thereof. In some embodiments, each L^PBis independently

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or a salt form thereof. In some embodiments, each L^PAis independently

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or a salt form thereof, and each L^PBis independently

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or a salt form thereof.

In some embodiments, internucleotidic linkages of an provided oligonucleotides or regions thereof comprise or are consecutive internucleotidic linkages (from 5′ to 3′) (L^PO)m(L^PA/L^PB)n, L^PO(L^PA/L^PB)n, (L^PO)m(L^PB)n, L^PO(L^PB)n, [(L^PO)m(L^PA/L^PB)n]y, [L^PO(L^PA/L^PB)n]y, [(L^PO)m(L^PB)n]y, [L^PO(L^PB)n]y, (L^PA/L^PB)t(L^PO)m(L^PA/L^PB)_n, (L^PA/L^PB)_tL^PO(L^PA/L^PB)n, (L^PA/L^PB)t(L^PO)m(L^PB)n, (L^PA/L^PB)tL^PO(L^PB)n, (L^PA/L^PB)t[(L^PO)m(L^PA/L^PB)n]y, (L^PA/L^PB)t[L^PO(L^PA/L^PB)n]y, (L^PA/L^PB)t[(L^PO)m(L^PB)n]y, (L^PA/L^PB)t[L^PO(L^PB)n]y, (L^PO)m(L^PA/L^PB)n(L^PA/L^PB)t, L^PO(L^PA/L^PB)n(L^PA/L^PB)t, (L^PO)m(L^PB)n(L^PA/L^PB)t, L^PO(L^PB)n(L^PA/L^PB)t, [(L^PO)m(L^PA/L^PB)n]y(L^PA/L^PB)t, [L^PO(L^PA/L^PB)n]y(L^PA/L^PB)t, [(L^PO)m(L^PB)n]y(L^PA/L^PB)t, [L^PO(L^PB)n]y(L^PA/L^PB)t, (L^PA/L^PB)t[(L^PO)m(L^PA/L^PB)n]y(L^PA/L^PB)t, L^PB(L^PA/L^PB)t[(L^PO)m(L^PA/L^PB)n]y(L^PA/L^PB)tL^PB, (L^PA/L^PB)t[(L^PO)m(L^PB)n]y(L^PA/L^PB)t, L^PB(L^PA/L^PB)t[(L^PO)m(L^PB)n]y(L^PA/L^PB)tL^PB, (L^PA/L^PB)t[(L^PO)(L^PA/L^PB)]y(L^PA/L^PB)t, L^PB(L^PA/L^PB)t[(L^PO)(L^PA/L^PB)]y(L^PA/L^PB)tL^PB, (L^PA/L^PB)t[(L^PO)(L^PB)]y(L^PA/L^PB)t, L^PB(L^PA/L^PB)t[(L^PO)(L^PB)]y(L^PA/L^PB)tL^PB, or a combination thereof, wherein each variable is independently as described herein. In some embodiments, at least one L^PA/L^PBof (L^PA/L^PB)t is L^PA. In some embodiments, at least one L^PA/L^PBof (L^PA/L^PB)t is L^PB. In some embodiments, at least one L^PA/L^PBof (L^PA/L^PB)t is L^PA, and at least one L^PA/L^PBof (L^PA/L^PB)t is L^PB. In some embodiments, at least one L^PA/L^PBof (L^PA/L^PB)m is L^PA. In some embodiments, at least one L^PA/L^PBof (L^PA/L^PB)m is L^PA. In some embodiments, at least one L^PA/L^PBof (L^PA/L^PB)m is L^PA, and at least one L^PA/L^PBof (L^PA/L^P)m is L^PB. In some embodiments, each L^PA/L^PBof (L^PA/L^PB)m is L^PB. In some embodiments, a sugar bonded to a L^POlinkage at its 3′-carbon comprises a 2-modification, wherein the T-modification is not 2′-F. In some embodiments, a sugar bonded to a L^POlinkage at its 3′-carbon is independently

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wherein R^2sis not —H or —OH. In some embodiments, each sugar bonded to a L^POlinkage at its 3′-carbon is independently

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wherein R^2sis not —H or —OH. In some embodiments, each sugar bonded to a L^POlinkage at its 3′-carbon is independently

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wherein R^2sis not —H or —OH. In some embodiments, R^4sis —H. In some embodiments. R^2sis not —H, —F or —OH. In some embodiments, each sugar bonded to a L^POlinkage at its 3′-carbon is independently

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wherein R^2sis not —H, —F or —OH. In some embodiments, R^2sis —OR, wherein R is optionally substituted C_1-6aliphatic. In some embodiments, R is optionally substituted C_1-6alkyl. In some embodiments, R^2sis -OMe. In some embodiments, a 5′-end sugar, a 3′-nd sugar, and/or a sugar between L^PA/L^PBand L^PA/L^PBcomprises a 2′-F modification. In some embodiments, a 5′-end sugar, a 3-end sugar, and/or a sugar between L^PA/L^PBand L^PA/L^PBis

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wherein R^2sis —F. In some embodiments, each sugar comprises a 2′-F is bonded to a modified internucleotidic linkage. e.g., at its 3′-carbon. In some embodiments, a modified internucleotidic linkage is L^PAor L^PB. In some embodiments, each L^PAis independently

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or a salt form thereof. In some embodiments, each L^PBis independently

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or a salt form thereof. In some embodiments, t is 2-10. In some embodiments, each L^PAis independently

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or a salt form thereof, and each L^PBis independently

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or a salt form thereof. In some embodiments, each modified internucleotidic linkage in a provided oligonucleotide is independently L^PO(wherein -X-L-R¹is not —H),

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or a salt form thereof. In some embodiments, each modified internucleotidic linkage is independently

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or a salt form thereof. In some embodiments, each modified internucleotidic linkage is independently

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or a salt form thereof. In some embodiments, m is 1. In some embodiments, each m is 1. In some embodiments, n is 2 or more. In some embodiments, each n is 2 or more. In some embodiments, t is 1. In some embodiments, t is 2 or more. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5. In some embodiments, t is 6. In some embodiments, t is 7. In some embodiments, t is 8. In some embodiments, t is 9. In some embodiments, t is 10. In some embodiments, each t is independently 2 or more. In some embodiments, each t is independently 3 or more. In some embodiments, each t is independently 4 or more. In some embodiments, each t is independently 5 or more.

In some embodiments, each of L^PO, L^PAand L^PBindependently bonds to a 5′-sugar through its 3′-carbon, and to a 3′-sugar through its 5′-carbon, e.g., each L^PAis independently an internucleotidic linkage having the structure of

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or a salt form thereof; each L^PBis independently an internucleotidic linkage having the structure of

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or a salt form thereof. Example sugar structures are described herein, e.g., in some embodiments, each sugar moiety independently has the structure of

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wherein each variable is independently as described m the present disclosure.

In some embodiments, L^POhas a pattern, location, number, percentage, etc. as described herein for a natural phosphate linkage. In some embodiments, L^PAhas a pattern, location, number, percentage. etc. as described herein for a Rp internucleotidic linkage. In some embodiments, a Rp internucleotidic linkage is a Rp phosphorothioate internucleotidic linkage. In some embodiments, a Rp internucleotidic linkage is a Rp non-negatively charged internucleotidic linkage (e.g., n001). In some embodiments, L^PBhas a pattern, location, number, percentage, etc. as described herein for a Sp internucleotidic linkage. In some embodiments, a Sp internucleotidic linkage is a Sp phosphorothioate internucleotidic linkage. In some embodiments, a Sp internucleotidic linkage is a Sp non-negatively charged internucleotidic linkage (e.g., n001).

In some embodiments, the present disclosure provides an oligonucleotide, wherein the first internucleotidic linkage from the 5′-end is an internucleotidic linkage of O^SP, and each other internucleotidic linkage is independently selected from O^P, *^PD, *^PDS, *^PDR, *^N, *^NS, *^NR, wherein:

O^5Pis

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L^PO, L^PA, L^PB, or a salt form thereof;

each O^Pis independently L^PO; each *^PDis independently

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or a salt form thereof;

each *^PDS is independently

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or a salt form thereof;

each *^PDR is independently

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or a salt form thereof;

each *^Nis independently

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or a salt form thereof;

each *^NS is independently

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or a salt form thereof; and

each *^NR is independently

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or a salt form thereof;

wherein each variable in independently as described herein, wherein -X-L-R¹is not —OH.

In some embodiments, O^5Pis independently

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L^PO, L^PA, L^PB, or a salt form thereof. In some embodiments, each O^Pis independently L^PO. In some embodiments, each *^PDis independently

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or a salt form thereof. In some embodiments, each *^PDS is independently

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or a salt form thereof. In some embodiments, each *^PDR is independently

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or a salt form thereof. In some embodiments, each *^Nis independently

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or a salt form thereof. In some embodiments, each *^NS is independently

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or a salt form thereof. In some embodiments, each *^NR is independently

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or a salt form thereof.

In some embodiments, X is —O—. In some embodiments, -L-R¹contains an electron-withdrawing group. In some embodiments, -L-R¹is —CH₂G², wherein the methylene unit is optionally substituted. In some embodiments, -L-R¹is —CH(R′)G². In some embodiments, G²does not comprise a chiral element, and G²comprises an electron-withdrawing group as described herein, e.g., in some embodiments. G²is —CH₂CN (e.g., in O^5P, O^P, *^PD, or *^N, wherein linkage phosphorus is not chirally controlled). In some embodiments, G²comprises a chiral element, e.g., wherein linkage phosphorus is chirally controlled. In some embodiments, -X-L-R¹is of such a structure that H-X-L-R¹is a chiral reagent described herein, or a capped chiral reagent described herein wherein an amino group of the chiral reagent (typically of -W¹—H or —W²—H, which comprises an amino group -NHG⁵-) is capped, e.g., with —C(O)R′ (replacing a —H, e.g., —N[—C(O)R′]G⁵-). In some embodiments, -X-L-R¹is

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wherein each variable is independently in accordance with the present disclosure. In m embodiments. -X-L-R¹is

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wherein each variable is independently in accordance with the present disclosure. In some embodiments, R¹is —H or —C(O)R′. In some embodiments, wherein R¹is —H, e.g., in O^5P. In some embodiments, R¹is —C(O)R′ (e.g., in O^5P, O^P, *^PDS, *^PDR, *^NS *^NR, etc.). In some embodiments, R¹is CH₃C(O)—. In some embodiments, as described herein, G²is In some embodiments, G²is —C(R)₂Si(R)₃, wherein —C(R)₂— is optionally substituted —CH₂—, and each R of —Si(R)₃is independently an optionally substituted group selected from C_1-10aliphatic, heterocyclyl, heteroaryl and aryl. In some embodiments, G²is —CH₂Si(Me)(Ph)₂. In some embodiments, e.g., in *^PS, *^DR, etc., G²is —CH₂Si(Me)(Ph)₂. In some embodiments, G²comprises an electron-withdrawing group as described herein. In some embodiments, G²is —C(R)₂SO₂R′, wherein —C(R)₂— is optionally substituted —CH₂—, and R′ is an optionally substituted group selected from C_1-10aliphatic, heterocyclyl, heteroaryl and aryl. In some embodiments, R′ is phenyl. In some embodiments, e.g., in *^NS, *^NR, etc., G²is —CH₂SO₂Ph.

In some embodiments, the present disclosure provides an oligonucleotide (“a first oligonucleotide”), which has an identical structure as an oligonucleotide described in a Table herein or an oligonucleotide described in e.g., US 20150211006, US 20170037399, US 20180216107, US 20180216108, US 20190008986, WO 2017/015555, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/022473, WO 2018/067973, WO 2018/098264, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/032612, etc., the oligonucleotide of each of which is incorporated herein by reference (“a second oligonucleotide”), which second oligonucleotide comprises modified internucleotidic linkages, except that compared to the second oligonucleotide, in the first oligonucleotide:

the first internucleotidic linkage from the 5′-end is an internucleotidic linkage of O^5P; and for the rest linkages:

at each location where there is a phosphate linkage in the second oligonucleotide, there is independently a linkage of O^Pin the first oligonucleotide;

at each location where there is a stereorandom phosphorothioate linkages in the second oligonucleotide, there is independently a linkage of *^PDin the first oligonucleotide;

at each location where there is a Sp phosphorothioate linkage in the second oligonucleotide, there is independently a linkage of *^PDS in the first oligonucleotide;

at each location where there is a Rp phosphorothioate linkage in the second oligonucleotide, there is independently a linkage of *^PDR in the first oligonucleotide;

at each location where there is a stereorandom non-negatively charged internucleotidic linkage in the second oligonucleotide, there is independently a linkage of *^Nin the first oligonucleotide;

at each location where there is a Sp non-negatively charged internucleotidic linkage in the second oligonucleotide, there is independently a linkage of *^NS in the first oligonucleotide;

at each location where there is a Rp non-negatively charged internucleotidic linkage in the second oligonucleotide, there is independently a linkage of *^NR in the first oligonucleotide, and

each nucleobase in the first oligonucleotide is optionally and independently protected (e.g., as in oligonucleotide synthesis), and each additional chemical moiety, if any, in the first oligonucleotide is optionally and independently protected (e.g., —OH in a carbohydrate moiety protected as -OAc).

In some embodiments, at each location where there is a phosphate linkage in the second oligonucleotide, there is independently a linkage of O^Pin the first oligonucleotide; at each location where there is a stereorandom phosphorothioate linkages in the second oligonucleotide, there is independently a linkage of *^PDin the first oligonucleotide; at each location where there is a Sp phosphorothioate linkage in the second oligonucleotide, there is independently a linkage of *^PDS in the first oligonucleotide; at each location there is a Rp phosphorothioate linkage in the second oligonucleotide, there is independently a linkage of *^PDR in the first oligonucleotide; at each location there is a stereorandom non-negatively charged internucleotidic linkage in the second oligonucleotide, there is independently a linkage of *^Nin the first oligonucleotide; at each location there is a Sp non-negatively charged internucleotidic linkage in the second oligonucleotide, there is independently a linkage of *^NS in the first oligonucleotide; at each location there is a Rp non-negatively charged internucleotidic linkage in the second oligonucleotide, there is independently a linkage of *^NR in the first oligonucleotide, and each nucleobase in the first oligonucleotide is optionally and independently protected (e.g., as in oligonucleotide synthesis), and each additional chemical moiety, if any, in the first oligonucleotide is optionally and independently protected (e.g., —OH in a carbohydrate moiety protected as -OAc); wherein each of O^5P, O^P, *^PDS, *^PDR, *^N, *^NS and *^NR is independently as described herein. In some embodiments, such an oligonucleotide is linked to a support optionally through a linker, e.g., a CNA linker to CPG. In some embodiments, as appreciated by those skilled in the art, after a removal process of -X-L-R, a linkage of O^5P, O^P, *^PD, *^PDS, *^PDR, *^N, *^NS or *^NR becomes a linkage it replaces. In some embodiments, such oligonucleotides (e.g., first oligonucleotides) are useful intermediates for preparing their corresponding oligonucleotides (e.g., second oligonucleotides). In some embodiments, the present disclosure provides chirally controlled oligonucleotide composition of a provided first oligonucleotide or a stereoisomer thereof.

In some embodiments, as appreciated by those skilled in the art, W^Nis of such a structure that its N-moiety has the same non-hydrogen atoms and connections of non-hydrogen atoms as the N-moiety of the non-negatively charged internucleotidic linkage it replaces (without considering single, double, or triple bond etc.). For example, in some embodiments, P^Nin *^Nis

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(such a *^Nis n001^P), and its corresponding non-negatively charged internucleotidic linkage is n001.

In some embodiments, a provided oligonucleotide has the same “Description” as an oligonucleotide listed in a Table herein (e.g., Table A1), except that:

the oligonucleotide comprises at least one linkage of O^P, and/or at each location in the oligonucleotide where there is a phosphate linkage, there is independently a linkage of O^P, wherein O^Pis

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at each location where there is a stereorandom phosphorothioate linkages, there is independently a linkage of *^PD, wherein *^PDis

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at each location where there is a Sp phosphorothioate linkage, there is independently a linkage of *^PDS, wherein *^PDS is

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at each location where there is a Rp phosphorothioate linkage, there is independently a linkage of *^PDR, wherein *^PDR is

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at each location where there is a stereorandom n001, there is independently a linkage of *^N, wherein *^Nis

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(as appreciated by those skilled in the art, it is associated with an anion (e.g., Q⁻ such as PF₆⁻ (which can be an anion in a modification step)));

at each location where there is a Sp n001, there is independently a linkage of *^NS, wherein *^NS is

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(as appreciated by those skilled in the art, it is associated with an anion (e.g., Q⁻ such as PF₆⁻ (which can be an anion in a modification step))); and

at each location where there is a Rp n001, there is independently a linkage of *^NR, wherein *^NR is

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(as appreciated by those skilled in the art, it is associated with an anion (e.g., Q⁻ such as PF₆⁻ (which can be an anion in a modification step))); and

the oligonucleotide is optionally connected to a solid support, optionally through a linker. In some embodiments, the oligonucleotide is connected to a solid support, e.g., CPG, polystyrene support, etc. In some embodiments, the oligonucleotide is connected to a solid support through a linker, e.g., a CNA linker. In some embodiments, such an oligonucleotide is an oligonucleotide of formula O-I or a salt form thereof.

Certain Embodiments of Stereochemistry and Pattern of Backbone Chiral Centers

Among other things, the present disclosure provides oligonucleotides comprising one or more chirally controlled internucleotidic linkages. In some embodiments, the present disclosure provides chirally controlled oligonucleotide compositions. In some embodiments, each chiral linkage phosphorus of provided oligonucleotides is independently chirally controlled (stereocontrolled) (e.g., each independently having a stereopurity (diastereopurity) of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (e.g., as typically assessed using an appropriate dimer comprising an internucleotidic linkage containing the linkage phosphorus, and the two nucleoside units being linked by the internucleotidic linkage)). In some embodiments, a stereopurity is at least 90%. In some embodiments, a stereopurity is at least 95%. In some embodiments, a stereopurity is at least 96%. In some embodiments, a stereopurity is at least 97%. In some embodiments, a stereopurity is at least 98%. In some embodiments, a stereopurity is at least 99%. With the capability to fully control stereochemistry and other modifications (e.g., base modifications, sugar modifications, internucleotidic linkage modifications, etc.), the present disclosure provides technologies of improved properties and/or activities compared to corresponding non-chirally controlled technologies.

In some embodiments, pattern of backbone chiral centers of a region, particularly a core region or a middle region, or of an oligonucleotide (e.g., an oligonucleotide of a plurality of oligonucleotides) is or comprises (Np/Op)t[(Rp)n(Sp)m]y, (Np/Op)t[(Op)n(Sp)m]y, (Np/Op)t[(Op/Rp)n(Sp)m]y, (Sp)t[(Rp)n(Sp)m]y, (Sp)t[(Op)n(Sp)m]y, (Sp)t[(Op/Rp)n(Sp)m]y, [(Rp)n(Sp)m]y, [(Op)n(Sp)m]y, [(Op/Rp)n(Sp)m]y, (Rp)t(Np)n(Rp)m. (Rp)t(Sp)n(Rp)m, (Rp)t[(Np/Op)n]y(Rp)m, (Rp)t[(Sp/Np)n]y(Rp)m, (Rp)t[(Sp/Op)n]y(Rp)m, (Np/Op)t(Np)n(Np/Op)m, (Np/Op)t(Sp)n(Np/Op)m, (Np/Op)t[(Np/Op)n]y(Np/Op)m, (Np/Op)t[(Sp/Op)n]y(Np/Op)m, (Np/Op)t[(Sp/Op)n]y(Np/Op)m, (Rp/Op)t(Np)n(Rp/Op)m, (Rp/Op)t(Sp)n(Rp/Op)m, (Rp/Op)t[(Np/Op)n]y(Rp/Op)m, (Rp/Op)t[(Sp/Op)n]y(Rp/Op)m, or (Rp/Op)t[(Sp/Op)n]y(Rp/Op)m (unless otherwise specified, description of patterns of modifications and stereochemistry are from 5′ to 3′ as typically used in the art), wherein Sp indicates S configuration of a chiral linkage phosphorus of a chiral modified internucleotidic linkage, Rp indicates R configuration of a chiral linkage phosphorus of a chiral modified internucleotidic linkage, Op indicates an achiral linkage phosphorus of a natural phosphate linkage, each Np is independently Rp, or Sp, and each of m, n, t and y is independently 1-50 as described in the present disclosure. In some embodiments, a pattern of backbone chiral centers is or comprises [(Rp/Op)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises [(Rp)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises [(Op)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t[(Rp/Op)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t[(Rp)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t[(Op)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Sp)t[(Rp/Op)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Sp)t[(Rp)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Sp)t[(Op)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp)t(Np)n(Rp)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp)t(Sp)n(Rp)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp)t[(Np/Op)n]y(Rp)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp)t[(Sp/Np)n]y(Rp)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp)t[(Sp/Op)n]y(Rp)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t(Np)n(Np/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t(Sp)n(Np/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t[(Np/Op)n]y(Np/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t[(Sp/Op)n]y(Np/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t[(Sp/Op)n]y(Np/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp/Op)t(Np)n(Rp/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp/Op)t(Sp)n(Rp/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp/Op)t[(Np/Op)n]y(Rp/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp/Op)t[(Sp/Op)n]y(Rp/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp)(Rp/Op)t[(Sp/Op)n]y(Rp/Op)m(Rp). In some embodiments, n is 1. For example, in some embodiments, a pattern of backbone chiral centers is or comprises (Sp)t[Op(Sp)m]y; in some embodiments, a pattern of backbone chiral centers is or comprises (Sp)t[Rp(Sp)m]y. In some embodiments, y is 1. In some embodiments, m is 2 or more. In some embodiments, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is 1, and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is 1, and m is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 internucleotidic linkages preceding, and there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 internucleotidic linkages after the Rp or Op. In some embodiments, there are at least 2 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 3 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 4 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 5 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 6 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 7 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 8 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 9 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 10 internucleotidic linkages preceding and/or following. In some embodiments, y is 1. In some embodiments, y is 2 or more. In some embodiments, y is 2, 3, 4, or 5. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, a region having such a pattern of backbone chiral centers contains no 2′-modifications on its sugar moieties, wherein the 2′-modification is 2′-OR¹or 2′-O-L-, wherein R¹is not hydrogen and L comprises a carbon atom and connects to another carbon atom of the sugar moiety. In some embodiments, each sugar moiety of a region having such a pattern of backbone chiral centers is independently a natural DNA sugar moiety

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As appreciated by a person having ordinary skill in the art, for a natural DNA sugar moiety in natural DNA, C1 is connected to a base, C3 and C5 are each independently connected to internucleotidic linkages or —OH (when at the 5′- or 3′-end)). Certain benefits/advantages provided by such patterns of backbone chiral centers are described in US 20170037399, WO 2017/015555, and WO 2017/062862.

In some embodiments, y, t, n and m each are independently 1-20 as described in the present disclosure. In some embodiments, y is 1. In some embodiments, y is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, y is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, y is 6. In some embodiments, y is 7. In some embodiments, y is 8. In some embodiments, y is 9. In some embodiments, y is 10.

In some embodiments, n is 1. In some embodiments, n is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, n is 1-10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, n is 1. In some embodiments, n is 2, 3, 4, 5, 6, 7 or 8. In some embodiments, n is 3, 4, 5, 6, 7 or 8. In some embodiments, n is 4, 5, 6, 7 or 8. In some embodiments, n is 5, 6, 7 or 8. In some embodiments, n is 6, 7 or 8. In some embodiments, n is 7 or 8. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

In some embodiments, m is 0-50. In some embodiments, m is 1-50. In some embodiments, m is 1. In some embodiments, m is 2-50. In some embodiments, m is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, m is 2, 3, 4, 5, 6, 7 or 8. In some embodiments, m is 3, 4, 5, 6, 7 or 8. In some embodiments, m is 4, 5, 6, 7 or 8. In some embodiments, m is 5, 6, 7 or 8. In some embodiments, m is 6, 7 or 8. In some embodiments, m is 7 or 8. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11. In some embodiments, m is 12. In some embodiments, m is 13. In some embodiments, m is 14. In some embodiments, m is 15. In some embodiments, m is 16. In some embodiments, m is 17. In some embodiments, m is 18. In some embodiments, m is 19. In some embodiments, m is 20. In some embodiments, m is 21. In some embodiments, m is 22. In some embodiments, m is 23. In some embodiments, m is 24. In some embodiments, m is 25. In some embodiments, m is at least 2. In some embodiments, m is at least 3. In some embodiments, m is at least 4. In some embodiments, m is at least 5. In some embodiments, m is at least 6. In some embodiments, m is at least 7. In some embodiments, m is at least 8. In some embodiments, m is at least 9. In some embodiments, m is at least 10. In some embodiments, m is at least 11. In some embodiments, m is at least 12. In some embodiments, m is at least 13. In some embodiments, m is at least 14. In some embodiments, m is at least 15. In some embodiments, m is at least 16. In some embodiments, m is at least 17. In some embodiments, m is at least 18. In some embodiments, m is at least 19. In some embodiments, m is at least 20. In some embodiments, m is at least 21. In some embodiments, m is at least 22. In some embodiments, m is at least 23. In some embodiments, m is at least 24. In some embodiments, m is at least 25. In some embodiments, m is at least greater than 25.

In some embodiments, t is 1-20. In some embodiments, t is 1. In some embodiments, t is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, t is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, t is 1-5. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5. In some embodiments, t is 6. In some embodiments, t is 7. In some embodiments, t is 8. In some embodiments, t is 9. In some embodiments, t is 10. In some embodiments, t is 11. In some embodiments, t is 12. In some embodiments, t is 13. In some embodiments, t is 14. In some embodiments, t is 15. In some embodiments, t is 16. In some embodiments, t is 17. In some embodiments, t is 18. In some embodiments, t is 19. In some embodiments, t is 20.

In some embodiments, each of t and m is independently at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, each of t and m is independently at least 3. In some embodiments, each of t and m is independently at least 4. In some embodiments, each of t and m is independently at least 5. In some embodiments, each of t and m is independently at least 6. In some embodiments, each of t and m is independently at least 7. In some embodiments, each of t and m is independently at least 8. In some embodiments, each of t and m is independently at least 9. In some embodiments, each oft and m is independently at least 10.

In some embodiments, provided oligonucleotides comprises a block, e.g., a first block, a 5′-wing, etc., that has a pattern of backbone chiral centers of or comprising a t-section, e.g., (Sp)t, (Rp)t, (Np/Op)t, (Rp/Op)t, etc., a block, e.g., a second block, a core, etc., that has a pattern of backbone chiral centers of or comprising a y- or n-section, e.g., (Np)n, (Sp)n, [(Np/Op)n]y, [(Rp/Op)n]y, [(Sp/Op)n]y, etc., and a block, e.g., a third block, a 3′-wing, etc., that has a pattern of backbone chiral centers of or comprising a m-section, e.g., (Sp)m, (Rp)m, (Np/Op)m, (Rp/Op)m, etc.

In some embodiments, a t-, y-, n-, or m-section that comprises Np or Rp, e.g., (Rp)t, (Np/Op)t, (Rp/Op)t, (Np)n, [(Np/Op)n]y, [(Rp/Op)n]y, (Rp)m, (Np/Op)m, (Rp/Op)m, etc. independently comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or 100% Rp. In some embodiments, a t- or in-section that comprises Np or Rp independently comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or 100% Rp. In some embodiments, provided oligonucleotides comprise at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or 100% Rp. In some embodiments, a percentage is at least 10%. In some embodiments, a percentage is at least 20%. In some embodiments, a percentage is at least 30%. In some embodiments, a percentage is at least 40%. In some embodiments, a percentage is at least 50%. In some embodiments, a percentage is at least 60%. In some embodiments, a percentage is at least 70%. In some embodiments, a percentage is at least 75%. In some embodiments, a percentage is at least 80%. In some embodiments, a percentage is at least 85%. In some embodiments, a percentage is at least 901%. In some embodiments, a percentage is at least 95%. In some embodiments, a percentage is 100%.

In some embodiments, each sugar moiety bonded to a Rp or Op linkage phosphorus at 3′ independently comprises a modification. In some embodiments, each sugar moiety bonded to a Rp or Op linkage phosphorus at 5′ independently comprises a modification. In some embodiments, each sugar moiety bonded to a Rp linkage phosphorus at 3′ independently comprises a modification. In some embodiments, each sugar moiety bonded to a Rp linkage phosphorus at 5′ independently comprises a modification. In some embodiments, each sugar moiety bonded to an Op linkage phosphorus at 3′ independently comprises a modification. In some embodiments, each sugar moiety bonded to an Op linkage phosphorus at 5′ independently comprises a modification. In some embodiments, each sugar moiety bonded to a Sp linkage phosphorus at 3′ independently comprises a modification. In some embodiments, each sugar moiety bonded to a Sp linkage phosphorus at 5′ independently comprises a modification. In some embodiments, each sugar moiety independently comprises a modification. In some embodiments, a modification is a 2′-modification. In some embodiments, a modification is 2′-OR, wherein R is not hydrogen. In some embodiments, a modification is 2′-OR wherein R is optionally substituted C_1-6alkyl. In some embodiments, a modification is 2′-OR, wherein R is substituted C_1-6alkyl. In some embodiments, a modification is 2′-OR, wherein R is optionally substituted C₁-C₆alkyl. In some embodiments, a modification is 2′-OR, wherein R is substituted C_2-6alkyl. In some embodiments, R is —CH₂CH₂OMe. In some embodiments, a modification is or comprises -L- connecting two sugar carbons, e.g., those found in LNA. In some embodiments, a modification is -L- connecting C2 and C4 of a sugar moiety. In some embodiments, L is —CH₂—CH(R)—, wherein R is as described in the present disclosure. In some embodiments, L is —CH₂—CH(R)—, wherein R is as described in the present disclosure and is not hydrogen. In some embodiments, L is —CH₂—(R)—CH(R)—, wherein R is as described in the present disclosure and is not hydrogen. In some embodiments, L is —CH₂—(S)—CH(R)—, wherein R is as described in the present disclosure and is not hydrogen. In some embodiments, a block, a wing, a core, or an oligonucleotide has sugar modifications as described in the present disclosure.

In some embodiments, a provided pattern of backbone chiral centers is or comprises (Rp/Sp)-(All Rp or All Sp)-(Rp/Sp), wherein each Rp/Sp is independently Rp or Sp. In some embodiments, a provided pattern of backbone chiral centers is or comprises (Rp)-(All Sp)-(Rp). In some embodiments, a provided pattern of backbone chiral centers is or comprises (Sp)-(All Sp)-(Sp). In some embodiments, a provided pattern of backbone chiral centers is or comprises (Sp)-(All Rp)-(Sp). In some embodiments, a provided pattern of backbone chiral centers is or comprises (Rp/Sp)-(repeating (Sp)m(Rp)n)-(Rp/Sp). In some embodiments, a provided pattern of backbone chiral centers is or comprises(Rp/Sp)-(repeating SpSpRp)-(Rp/Sp).

Blocks

In some embodiments, provided oligonucleotides comprise one or more blocks, characterized by base modifications, sugar modifications, types of internucleotidic linkages, stereochemistry of linkage phosphorus, etc. In some embodiments, provided oligonucleotides comprises or are of a 5′-first block-second block-third block-3′ structure. In some embodiments, a first block is a 5′-wing. In some embodiments, a first block is 5′-end region. In some embodiments, a second block is a core. In some embodiments, a second block is a middle region between a 5′-end and a 3′-end region. In some embodiments, a third block a 3′-wing. In some embodiments, a third block is a 3′-end region. Each of a 5′-wing, 5′-end region, core, middle region, 3′-wing, and 3′-end region can independently be a block.

In some embodiments, provided oligonucleotides comprises or are of a 5′-wing-core-wing-3′, 5′-wing-core-3′ or 5′-core-wing-3′ structures. In some embodiments, a first block, a second block, a third block, a wing (e.g., a 5′-wing, a 3′-wing) and/or a core of provided oligonucleotides are each independently a block or comprise one or more blocks as described in the present disclosure.

Various blocks, 5′-wings, 3′-wings and cores can be utilized in accordance with the present disclosure, including those described in US 20150211006, US 20150211006, WO 2017015555, WO 2017015575, WO 2017062862, WO 2017160741, blocks, 5′-wings, 3′-wings and cores of each of which are incorporated herein by reference.

In some embodiments, a block is a linkage phosphorus stereochemistry block. For example, in some embodiments, a block comprises only Rp, Sp, or Op linkage phosphorus. In some embodiments, a block is a Rp block comprising only Rp linkage phosphorus. In some embodiments, a block is a Rp/Op block comprising only Rp/Op linkage phosphorus. In some embodiments, a block is a Sp/Op block comprising only Sp/Op linkage phosphorus. In some embodiments, a block is an Op block. In some embodiments, an oligonucleotide, or a region thereof (a first block, a second block, a third block, a wing, a core, etc.) comprises one or more of a Rp block, a Sp block and/or an Op block. In some embodiments, a block comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, linkage phosphorus.

In some embodiments, a block is a sugar modification block. In some embodiments, a block is a 2′-modification block wherein each sugar moiety of the block independently comprises the 2′-modification. In some embodiments, a 2′-modification is 2′-OR wherein R is as described in the present disclosure. In some embodiments, a 2′-modification is a 2′-OR wherein R is not hydrogen. In some embodiments, a 2′-modification is 2′-OMe. In some embodiments, a 2′-modification is 2′-MOE. In some embodiments, a modification is a LNA modification. In some embodiments, an oligonucleotide, or a region thereof (a first block, a second block, a third block, a wing, a core, etc.) comprises one or more sugar modification blocks, each independently of its own sugar modification. In some embodiments, a block comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, sugar moieties.

As illustrated herein, a block can be of various lengths. In some embodiments, a block is of 1-30, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length. In some embodiments, a 5′-first block-second-block-third block-3′, or a 5′-wing-core-wing-3′ is of 5-10-5, 3-10-4, 3-10-6.4-12-4, etc.

In some embodiments, an oligonucleotide or a block or region thereof (e.g., a 5′-end region, a 5′-wing, a middle region, a core region, a 3′-end region, a 3′-ring, etc.) comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, non-negatively charged internucleotidic linkages as described in the present disclosure. In some embodiments, a provided oligonucleotide comprises two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, consecutive non-negatively charged internucleotidic linkages. In some embodiments, a block or region comprises two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, consecutive non-negatively charged internucleotidic linkages. In some embodiments, the number is 1. In some embodiments, the number is 2. In some embodiments, the number is 3. In some embodiments, the number is 4. In some embodiments, the number is 5. In some embodiments, the number is 6. In some embodiments, the number is 7. In some embodiments, the number is 8. In some embodiments, the number is 9. In some embodiments, the number is 10 or more. In some embodiments, each internucleotidic linkage between nucleoside units in a block, e.g., a 5′-end region, a 5′-wing, is a non-negatively charged internucleotidic linkage except the first internucleotidic linkage between two nucleoside units of the block from the 5′-end of the block. In some embodiments, each internucleotidic linkage between nucleoside units in a block, e.g., a 3′-end region, a 3′-wing, is a non-negatively charged internucleotidic linkage except the first internucleotidic linkage between two nucleoside units of the block from the 3′-end of the block. In some embodiments, each internucleotidic linkage between nucleoside units in a region, e.g., a 5′-end region, a 5′-wing, is a non-negatively charged internucleotidic linkage except the first internucleotidic linkage between two nucleoside units of the region from the 5′-end of the region. In some embodiments, each internucleotidic linkage between nucleoside units in a region, e.g., a 3′-end region, a 3′-wing, is a non-negatively charged internucleotidic linkage except the first internucleotidic linkage between two nucleoside units of the region from the Y-end of the region. In some embodiments, each internucleotidic linkage in a region or block, e.g., a 5′-end region, a 5′-wing, a middle region, a core region, a 3′-end region, a 3′-ring, etc., is independently a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage or a Rp chiral internucleotidic linkage. In some embodiments, each internucleotidic linkage in a region or block is independently a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage or a Rp phosphorothioate internucleotidic linkage. In some embodiments, about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of internucleotidic linkages of an oligonucleotide or a region or block, e.g., a 5′-end region, a 5′-wing, a middle region, a core region, a 3′-end region, a 3′-ring, etc., is independently a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage or a Rp chiral internucleotidic linkage. In some embodiments, about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of internucleotidic linkages of an oligonucleotide or a region or block is independently a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage or a Rp phosphorothioate internucleotidic linkage. In some embodiments, about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of internucleotidic linkages of an oligonucleotide or a region or block is independently a non-negatively charged internucleotidic linkage or a natural phosphate internucleotidic linkage. In some embodiments, about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 901%, 95% or more of internucleotidic linkages of an oligonucleotide or a region or block is independently a non-negatively charged internucleotidic linkage. In some embodiments, the percentage is 45% or more. In some embodiments, the percentage is 50% or more. In some embodiments, the percentage is 60% or more. In some embodiments, the percentage is 70% or more. In some embodiments, the percentage is 80% or more. In some embodiments, the percentage is 90% or more. In some embodiments, a region or block is a wing. In some embodiments, a region or block is a 5′-wing. In some embodiments, a region or block is a 3′-wing. In some embodiments, a region or block is a core. As described herein, a region or block, e.g., a wing, a core, etc., can have various lengths, e.g., comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleobases. In some embodiments, each nucleobase is independently optionally substituted A, T, C, G, U or an optionally substituted tautomer of A, T, C, G, or U.

Length

As described in the present disclosure, provided oligonucleotides can be of various lengths. e.g., 2-200, 10-15, 10-25, 15-20, 15-25, 15-40, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 60, 70, 80, 90, 100, 150, nucleobases in length, wherein each nucleobase is independently optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C. G, or U. In some embodiments, provided oligonucleotides, e.g., oligonucleotide of a plurality in chirally controlled oligonucleotide compositions, are 15 nucleobases in length. In some embodiments, provided oligonucleotides are 16 nucleobases in length. In some embodiments, provided oligonucleotides are 17 nucleobases in length. In some embodiments, provided oligonucleotides are 18 nucleobases in length. In some embodiments, provided oligonucleotides are 19 nucleobases in length. In some embodiments, provided oligonucleotides are 20 nucleobases in length. In some embodiments, provided oligonucleotides are 21 nucleobases in length. In some embodiments, provided oligonucleotides are 22 nucleobases in length. In some embodiments, provided oligonucleotides are 23 nucleobases in length. In some embodiments, provided oligonucleotides are 24 nucleobases in length. In some embodiments, provided oligonucleotides are 25 nucleobases in length.

As described in the present disclosure, provided oligonucleotides, oligonucleotides of a plurality in chirally controlled oligonucleotide compositions, may comprise various modifications, e.g., base modifications, sugar modifications, internucleotidic linkage modifications, etc. In some embodiments, the oligonucleotide composition comprises at least one modified nucleotide, at least one modified sugar moiety, at least one morpholino moiety, at least one 2′-deoxy ribonucleotide, at least one locked nucleotide, and/or at least one bicyclic nucleotide.

Nucleobases

In some embodiments, a nucleobase is a natural nucleobase. In some embodiments, a nucleobase is a modified nucleobase (non-natural nucleobase). In some embodiments, a nucleobase, e.g., BA, in provided oligonucleotides is a natural nucleobase (e.g., adenine, cytosine, guanosine, thymine, or uracil) or a modified nucleobase derived from a natural nucleobase, e.g., optionally substituted adenine, cytosine, guanosine, thymine, or uracil, or tautomeric forms thereof. Examples include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine, and tautomeric forms thereof, having their respective amino groups protected by protecting groups, e.g., one or more of —R, —C(O)R, etc. Example protecting groups, including those useful for oligonucleotide synthesis, are widely known in the art and can be utilized in accordance with the present disclosure. In some embodiments, a protected nucleobase and/or derivative is selected from nucleobases with one or more acyl protecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil and other modified nucleobases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two being the natural degradation products). Example modified nucleobases are also disclosed in Chiu and Rana. RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313. In some embodiments, a modified nucleobase is substituted uracil, thymine, adenine, cytosine, or guanine. In some embodiments, a modified nucleobase is a functional replacement, e.g., in terms of hydrogen bonding and/or base pairing, of uracil, thymine, adenine, cytosine, or guanine. In some embodiments, a nucleobase is optionally substituted uracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine. In some embodiments, a nucleobase is uracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine.

In some embodiments, a modified base is optionally substituted adenine, cytosine, guanine, thymine, or uracil. In some embodiments, a modified nucleobase is independently adenine, cytosine, guanine, thymine or uracil, modified by one or more modifications by which:

(1) a nucleobase is modified by one or more optionally substituted groups independently selected from acyl, halogen, amino, azide, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heteroaryl, carboxyl, hydroxyl, biotin, avidin, streptavidin, substituted silyl, and combinations thereof:

(2) one or more atoms of a nucleobase are independently replaced with a different atom selected from carbon, nitrogen or sulfur;

(3) one or more double bonds in a nucleobase are independently hydrogenated; or

(4) one or more optionally substituted aryl or heteroaryl rings are independently inserted into a nucleobase.

Modified nucleobases also include expanded-size nucleobases in which one or more aryl rings, such as phenyl rings, have been added. Nucleic base replacements described in the Glen Research catalog (available at the Glen Research website); Krueger A T et al, Ace. Chem. Res., 2007, 40, 141-150; Kool, ET, Acc. Chem. Res., 2002, 35, 936-943; Benner S. A., et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F. E., et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin. Chem. Biol., 2006, 10, 622-627, are contemplated as useful for oligonucleotides of the present disclosure.

In some embodiments, modified nucleobases include structures such as, but not limited to, corrin- or porphyrin-derived rings. Porphyrin-derived base replacements have been described in Morales-Rojas, H and Kool, E T, Org. Lett., 2002, 4, 4377-4380. Shown below is an example of a porphyrin-derived ring which can be used as a nucleobase replacement:

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In some embodiments, a modified nucleobase is fluorescent. Examples of such fluorescent modified nucleobases include phenanthrene, pyrene, stillbene, isoxanthine, isozanthopterin, terphenyl, terthiophene, benzoterthiophene, coumarin, lumazine, tethered stillbene, benzo-uracil, and naphtho-uracil.

In some embodiments, a modified nucleobase is a universal base or a degenerate base, e.g., 3-nitropyrrole, 5′-nitroindole, P, K, etc.

In some embodiments, other nucleosides can also be used in technologies disclosed in the present disclosure and include nucleosides that incorporate modified nucleobases, or nucleobases covalently bound to modified sugars. Some examples of nucleosides that incorporate modified nucleobases include 4-acetylcytidine; 5-(carboxyhydroxylmethyl)uridine; 2′-O-methylcytidine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; dihydrouridine; 2′-O-methylpseudouridine; beta,D-galactosylqueosine; 2′-O-methylguanosine; N⁶-isopentenyladenosine; 1-methyladenosine; 1-methylpseudouridine; 1-methylguanosine; 1-methylinosine; 2,2-dimethylguanosine; 2-methyladenosine; 2-methylguanosine; N⁷-methylguanosine; 3-methyl-cytidine; 5-methylcytidine; 5-hydroxymethylcytidine; 5-formylcytosine; 5-carboxylcytosine; M-methyladenosine; 7-methylguanosine; 5-methylaminoethyluridine; 5-methoxyaminomethyl-2-thiouridine; beta,D-mannosylqueosine; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 2-methylthio-N⁶-isopentenyladenosine; N-((9-beta,D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine; N-((9-beta,D-ribofuranosylpurine-6-yl)-N-methylcarbamoyl)threonine; uridine-5-oxyacetic acid methylester; uridine-5-oxyacetic acid (v); pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine; 2-thiouridine; 4-thiouridine; 5-methyluridine; 2′-O-methyl-5-methyluridine; and 2′-O-methyluridine.

In some embodiments, a nucleobase is optionally substituted A, T, C, G or U, wherein one or more —NH₂are independently and optionally replaced with —C(-L-R¹)₃, one or more —NH— are independently and optionally replaced with —C(-L-R¹)₂—, one or more ═N— are independently and optionally replaced with —C(-L-R¹)₂—, one or more ═CH— are independently and optionally replaced with ═N—, and one or more ═O are independently and optionally replaced with ═S, ═N(-L-R¹), or ═C(-L-R¹)₂, wherein two or more -L-R¹are optionally taken together with their intervening atoms to form a 3-30 membered bicyclic or polycyclic ring having 0-10 heteroatom ring atoms. In some embodiments, a modified nucleobase is optionally substituted A, T, C, G or U, wherein one or more —NH₂are independently and optionally replaced with —C(-L-R¹)₃, one or more —NH— are independently and optionally replaced with —C(-L-R¹)₂—, one or more ═N— are independently and optionally replaced with —C(-L-R)—, one or more ═CH— are independently and optionally replaced with ═N—, and one or more ═O are independently and optionally replaced with ═S, ═N(-L-R), or ═C(-L-R¹)₂, wherein two or more -L-R¹are optionally taken together with their intervening atoms to form a 3-30 membered bicyclic or polycyclic ring having 0-10 heteroatom ring atoms, wherein the modified base is different than the natural A, T, C, G and U. In some embodiments, a nucleobase is optionally substituted A, T. C. G or U. In some embodiments, a modified base is substituted A, T, C. G or U, wherein the modified base is different than the natural A, T, C. G and U.

In some embodiments, a modified nucleobase may be optionally substituted. In some embodiments, a modified nucleobase contains one or more, e.g., heteroatoms, alkyl groups, or linking moieties connected to fluorescent moieties, biotin or avidin moieties, or other proteins or peptides. In some embodiments, a nucleobase or modified nucleobase comprises or is conjugated with one or more biomolecule binding moieties such as e.g., antibodies, antibody fragments, biotin, avidin, streptavidin, receptor ligands, or chelating moieties. In some embodiments, a modified nucleobase is modified by substitution with a fluorescent or biomolecule binding moiety. In some embodiments, a substituent on a nucleobase or modified nucleobase is a fluorescent moiety. In some embodiments, a substituent on a nucleobase or modified nucleobase is biotin or avidin.

Example nucleobases are also described in US 20110294124, US 20120316224, US 20140194610, US 20150211006, US 20150197540, WO 2015107425, WO/2017/015555, WO/2017/015575, and WO/2017/062862, the nucleobases of each of which is incorporated herein by reference.

Sugars

In some embodiments, oligonucleotides comprise one or more modified sugar moieties beside the natural sugar moieties. In some embodiments, a sugar is a natural sugar. In some embodiments, a sugar is a modified sugar (non-natural sugar). The most common naturally occurring nucleotides are comprised of ribose sugars linked to the nucleobases adenosine (A), cytosine (C), guanine (G), and thymine (T) or uracil (U). Also included in the present disclosure are modified nucleotides wherein an internucleotidic linkage is linked to various positions of a sugar or modified sugar. As non-limiting examples, an internucleotidic linkage can be linked to the 2′, 3′, 4′ or 5′ position of a sugar.

In some embodiments, a sugar moiety is

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wherein each variable is independently as described in the present disclosure. In some embodiments, a sugar moiety is

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wherein L^sis —C(R^5s)₂—, wherein each R^5sis independently as described in the present disclosure. In some embodiments, a sugar moiety has the structure of

embedded image

wherein each variable is independently as described in the present disclosure. In some embodiments, a sugar moiety has the structure of

embedded image

wherein each variable is independently as described in the present disclosure. In some embodiments, a sugar has or is derived from the structure of

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wherein each variable is independently as described in the present disclosure. In some embodiments, a nucleoside has the structure of

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wherein each variable is independently as described in the present disclosure. In some embodiments, a nucleoside moiety has or comprises the structure of

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wherein each variable is independently as described in the present disclosure. In some embodiments, L^sis —CH(R)—, wherein R is as described in the present disclosure. In some embodiments, R is —H. In some embodiments, R is not —H, and L^sis —(R)—CH(R)—. In some embodiments, R is not —H, and L^sis —(S)—CH(R)—. In some embodiments, R, as described in the present disclosure, is optionally substituted C_1-6alkyl. In some embodiments, R is methyl.

Various types of sugar modifications are known and can be utilized in accordance with the present disclosure. In some embodiments, a sugar modification is a 2′-modification (e.g. R^2s(e.g., in

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In some embodiments, a 2′-modification is 2′-F. In some embodiments, a 2′-modification is 2′-OR, wherein R is not hydrogen. In some embodiments, a 2′-modification is 2′-OR, wherein R is optionally substituted C_1-6aliphatic. In some embodiments, a 2′-modification is 2′-OR, wherein R is optionally substituted C_1-6alkyl. In some embodiments, a 2′-modification is 2′-OMe. In some embodiments, a 2′-modification is 2′-MOE. In some embodiments, a 2′-modification is a LNA sugar modification (C2-O—CH₂—C4). In some embodiments, a 2′-modification is (C2-O—C(R)₂—C4), wherein each R is independently as described in the present disclosure. In some embodiments, a 2′-modification is (C2-O—CHR—C4), wherein R is as described in the present disclosure. In some embodiments, a 2′-modification is (C2-O—(R)—CHR—C4), wherein R is as described in the present disclosure and is not hydrogen. In some embodiments, a 2′-modification is (C2-O—(S)—CHR—C4), wherein R is as described in the present disclosure and is not hydrogen. In some embodiments, R is optionally substituted C_1-6aliphatic. In some embodiments, R is optionally substituted C_1-6alkyl. In some embodiments, R is unsubstituted C_1-6alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, a 2′-modification is (C2-O—CHR—C4), wherein R is optionally substituted C_1-6aliphatic. In some embodiments, a 2′-modification is (C2-O—CHR—C4), wherein R is optionally substituted C_1-6alkyl. In some embodiments, a 2′-modification is (C2-O—CHR—C4), wherein R is methyl. In some embodiments, a 2′-modification is (C2-O—CHR—C4), wherein R is ethyl. In some embodiments, a 2′-modification is (C2-O—(R)—CHR—C4), wherein R is optionally substituted C_1-6aliphatic. In some embodiments, a 2′-modification is (C2-O—(R)—CHR—C4), wherein R is optionally substituted C_1-6alkyl. In some embodiments, a 2′-modification is (C2-O—(R)—CHR—C4), wherein R is methyl. In some embodiments, a 2′-modification is (C2-O—(R)—CHR—C4), wherein R is ethyl. In some embodiments, a 2′-modification is (C2-O—(S)—CHR—C4), wherein R is optionally substituted C_1-6aliphatic. In some embodiments, a 2′-modification is (C2-O—(S)—CHR—C4), wherein R is optionally substituted C_1-6alkyl. In some embodiments, a 2′-modification is (C2-O—(S)—CHR—C4), wherein R is methyl. In some embodiments, a 2′-modification is (C2-O—(S)—CHR—C4), wherein R is ethyl. In some embodiments, a 2′-modification is C2-O—(R)—CH(CH₂CH₃)—C4. In some embodiments, a 2′-modification is C2-O—(S)H(CH₂CH₃)—C4. In some embodiments, a sugar moiety is a natural DNA sugar moiety. In some embodiments, a sugar moiety is a natural DNA sugar moiety modified at 2′ (2′-modification). In some embodiments, a sugar moiety is an optionally substituted natural DNA sugar moiety. In some embodiments, a sugar moiety is an 2′-substituted natural DNA sugar moiety.

Many modified sugars can be incorporated within oligonucleotides of the present disclosure. In some embodiments, a modified sugar contains one or more substituents at the 2′ position including one of the following: —F; —CF₃, —CN, —N, —NO, —NO₂, —OR′, —SR′, or —N(R′)₂, wherein each R′ is independently as described in the present disclosure; —O—(C₁-C₁₀alkyl), —S—(C₁-C₁₀alkyl), —NH—(C₁-C₁₀alkyl), or —N(C₁-C₁₀alkyl)₂; —O—(C₂-C₁₀alkenyl), —S—(C₂-C₁₀alkenyl), —NH—(C₁-C₁₀alkenyl), or —N(C₂-C₁₀alkenyl)₂; —O—(C₂-C₁₀alkynyl). —S—(C₂-C₁₀alkynyl), —NH—(C₂-C₁₀alkynyl), or —N(C₂-C₁₀alkynyl)₂; or —O—(C₁-C₁₀alkylene)-O—(C₁-C₁₀alkyl), —O—(C₁-C₁₀alkylene)-NH—(C₁-C₁₀alkyl) or —O—(C₁-C₁₀alkylene)-NH(C₁-C₁₀alkyl)₂, —NH—(C₁-C₁₀alkylene)-O—(C₁-C₁₀alkyl), or —N(C₁-C₁₀alkyl)-(C₁-C₁₀alkylene)-O—(C₁-C₁₀alkyl), wherein the alkyl, alkylene, alkenyl and alkynyl may be substituted or unsubstituted. Examples of substituents include, and are not limited to, —O(CH₂)_nOCH₃, and —O(CH₂)_nNH₂, wherein n is from 1 to about 10, MOE, DMAOE, and DMAEOE. Certain modified sugars are described in WO 2001/088198, WO/2017/062862, and Martin et al., Helv. Chim. Acta, 1995, 78, 486-504. In some embodiments, a modified sugar comprises one or more groups selected from a substituted silyl group, an RNA cleaving group, a reporter group, a fluorescent label, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, a group for improving the pharmacodynamic properties of an oligonucleotide, or other substituents having similar properties. In some embodiments, modifications are made atone or more of the 2′, 3′, 4′, 5′, or 6′ positions of a sugar, including the 3′ position of a sugar on the 3′-terminal nucleoside or in the 5′ position of the 5′-terminal nucleoside. In some embodiments, a RNA comprises a sugar which has, at the 2′ position, a 2′-OH, or 2∝—OR¹, wherein OR¹is optionally substituted alkyl, including 2′-OMe.

In some embodiments, a 2′-modification is 2′-F.

In some embodiments, the 2′-OH of a ribose is replaced with a substituent (e.g., R^2s) including one of the following: —H, —F; —CF₃, —CN, —N₃, —NO, —NO₂, —OR′, —SR′, or —N(R′)₂, wherein each R′ is independently as defined above and described herein; —O—(C₁-C₁₀alkyl), —S—(C₁-C₁₀alkyl), —NH—(C₁-C₁₀alkyl), or —N(C₁-C₁₀alkyl)₂; —O—(C₂-C₁₀alkenyl), —S—(C₁-C₁₀alkenyl), —NH—(C₁-C₁₀alkenyl), or —N(C₁-C₁₀alkenyl)₂; —O—(C₂-C₁₀alkynyl), —S—(C₂-C₁₀alkynyl), —NH—(C₂-C₁₀alkynyl), or —N(C₂-C₁₀alkynyl)₂; or —O—(C₁-C₁₀alkylene)-O—(C₁-C₁₀alkyl), —O—(C₁-C₁₀alkylene)-NH—(C₁-C₁₀alkyl) or —O—(C₁-C₁₀alkylene)-NH(C₁-C₁₀alkyl)₂, —NH—(C₁-C₁₀alkylene)-O—(C₁-C₁₀alkyl), or —N(C₁-C₁₀alkyl)-(C₁-C₁₀alkylene)-O—(C₁-C₁₀alkyl), wherein the alkyl, alkylene, alkenyl and alkynyl may be substituted or unsubstituted. In some embodiments, the 2′-OH is replaced with —H (deoxyribose). In some embodiments, the 2′-OH is replaced with —F. In some embodiments, the 2′-OH is replaced with —OR′. In some embodiments, the 2′-OH is replaced with -OMe. In some embodiments, the 2′-OH is replaced with —OCH₂CH₂OMe.

In some embodiments, a modified sugars is a sugar in locked nucleic acids (LNAs). In some embodiments, two substituents on sugar carbon atoms are taken together to form a bivalent moiety. In some embodiments, two substituents are on two different sugar carbon atoms. In some embodiments, a formed bivalent moiety has the structure of -L- as defined herein. In some embodiments, -L- is —O—CH₂—, wherein —CH₂— is optionally substituted. In some embodiments, -L- is —O—CH₂—. In some embodiments, -L- is —O—CH(Me)-. In some embodiments, -L- is —O—CH(Et)-. In some embodiments, -L- is between C2 and C4 of a sugar moiety. In some embodiments, a locked nucleic acid sugar has the structure indicated below, wherein R^2sis —OCH₂C4′-:

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In some embodiments, a modified sugar is an ENA sugar or modified ENA sugar such as those described in, e.g., Seth et al., J Am Chem Soc. 2010 Oct. 27; 132(42): 14942-14950. In some embodiments, a modified sugar is any of those found in an XNA (xenonucleic acid), for instance, arabinose, anhydrohexitol, threose, 2′fluoroarabinose, or cyclohexene.

In some embodiments, a modified sugar is one described in WO 2017/062862.

In some embodiments, modified sugars are sugar mimetics such as cyclobutyl or cyclopentyl moieties in place of pentofuranosyl. Representative United States patents that teach preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; and 5,359,044. In some embodiments, modified sugars are sugars in which the oxygen atom within the ribose ring is replaced by nitrogen, sulfur, selenium, or carbon. In some embodiments, a modified sugar is a modified ribose wherein the oxygen atom within the ribose ring is replaced with nitrogen, and wherein the nitrogen is optionally substituted with an alkyl group (e.g., methyl, ethyl, isopropyl, etc).

Non-limiting examples of modified sugars include glycerol, which form glycerol nucleic acid (GNA) analogues. In some embodiments, an GNA analogue is described in Zhang, R et al., J. Am. Chem. Soc., 2008, 130, 5846-5847; Zhang L, et al., J. Am. Chem. Soc., 2005, 127, 4174-4175 and Tsai C H et al., PNAS. 2007, 14598-14603.

In some embodiments, another example of a GNA derived analogue, flexible nucleic acid (FNA) based on the mixed acetal aminal of formyl glycerol, is described in Joyce G F et al., PNAS, 1987, 84, 4398-4402 and Heuberger B D and Switzer C, J. Am. Chem. Soc., 2008, 130, 412-413.

Additional non-limiting examples of modified sugars include hexopyranosyl (6′ to 4′), pentopyranosyl (4′ to 2′), pentopyranosyl (4′ to 3′), or tetrofuranosyl (3′ to 2′) sugars.

In some embodiments, one or more hydroxyl group in a sugar moiety is optionally and independently replaced with halogen, R′—N(R′)₂, —OR′, or —SR′, wherein each R′ is independently as defined above and described herein.

In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more), inclusive, of the sugars in an oligonucleotide, e.g., a chirally controlled oligonucleotide, an oligonucleotide of a plurality of oligonucleotide of an oligonucleotide composition, etc. are modified. In some embodiments, sugars of purine nucleosides and in some embodiments, only purine nucleosides, are modified (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more] of the purine nucleosides are modified). In some embodiments, sugars of pyrimidine nucleosides and in some embodiments, only pyrimidine nucleosides, are modified (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more] of the pyrimidine nucleosides are modified). In some embodiments, both purine and pyrimidine nucleosides are modified.

In some embodiments, modified sugars include those described in: A. Eschenmoser, Science (1999), 284:2118; M. Bohringer et al, Helv. Chim. Acta (1992), 75:1416-1477; M. Egli et al, J. Am. Chem. Soc. (2006), 128(33):10847-56; A. Eschenmoser in Chemical Synthesis: Gnosis to Prognosis, C. Chatgilialoglu and V. Sniekus, Ed., (Kluwer Academic, Netherlands, 1996), p. 293; K.-U. Schoning et al, Science (2000), 290:1347-1351; A. Eschenmoser et al. Helv. Chim. Acta (1992), 75:218; J. Hunziker et al. Helv. Chim. Acta (1993), 76:259; G. Otting et al, Helv. Chim. Acta (1993), 76:2701; K. Groebke et al, Helv. Chim. Acta (1998), 81:375; and A. Eschenmoser, Science (1999), 284:2118. Modifications to the 2′ modifications can be found in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein. In some embodiments, a modified sugar is one described in WO2012/030683. In some embodiments, a modified sugar is any modified sugar described in any of: Gryaznov, S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Hendrix et al. 1997 Chem. Eur. J. 3: 110; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5; Jepsen et al. 2004 Oligo. 14: 130-146; Jones et al. J. Org. Chem. 1993, 58, 2983; Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273; Koshkin et al. 1998 Tetrahedron 54: 3607-3630; Kumar et al. 1998 Bioo. Med. Chem. Let. 8: 2219-2222; Lauritsen et al. 2002 Chem. Comm. 5: 530-531; Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256; Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226; Morita et al. 2001 Nucl. Acids Res. Supp. 1: 241-242; Morita et al. 2002 Bioo. Med. Chem. Lett. 12: 73-76; Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226; Nielsen et al. 1997 Chem. Soc. Rev. 73: Nielsen et al. 1997 J. Chem. Soc. Perkins Transl. 1: 3423-3433; Obika et al. 1997 Tetrahedron Lett. 38 (50): 8735-8; Obika et al. 1998 Tetrahedron Lett. 39: 5401-5404; Pallan et al. 2012 Chem. Comm. 48: 8195-8197; Petersen et al. 2003 TRENDS Biotech. 21: 74-81; Rajwanshi et al. 1999 Chem. Commun. 1395-1396; Schultz et al. 1996 Nucleic Acids Res. 24: 2966: Seth et al. 2009 J. Med. Chem. 52: 10-13; Seth et al. 2010 J. Med. Chem. 53: 8309-8318; Seth et al. 2010 J. Org. Chem. 75: 1569-1581; Seth et al. 2012 Bioo. Med. Chem. Lett. 22: 296-299; Seth et al. 2012 Mol. Ther-Nuc. Acids. 1, e47; Seth, Punit P; Siwkowski, Andrew; Allerson, Charles R; Vasquez, Guillermo; Lee, Sam; Prakash, Thazha P; Kinberger, Garth; Migawa, Michael T; Gaus, Hans; Bhat, Balkrishen; et al. From Nucleic Acids Symposium Series (2008), 52(1), 553-554; Singh et al. 1998 Chem. Comm. 1247-1248; Singh et al. 1998 J. Org. Chem. 63: 10035-39; Singh et al. 1998 J. Org. Chem. 63: 6078-6079; Sorensen 2003 Chem. Comm. 2130-2131; Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507, 220; Van Aerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34: 1338; Vasseur et al. J. Am. Chem. Soc. 1992, 114, 4006; WO 20070900071; WO 20070900071; or WO 2016/079181.

In some embodiments, a modified sugar moiety is an optionally substituted pentose or hexose moiety. In some embodiments, a modified sugar moiety is an optionally substituted pentose moiety. In some embodiments, a modified sugar moiety is an optionally substituted hexose moiety. In some embodiments, a modified sugar moiety is an optionally substituted ribose or hexitol moiety. In some embodiments, a modified sugar moiety is an optionally substituted ribose moiety. In some embodiments, a modified sugar moiety is an optionally substituted hexitol moiety.

In some embodiments, a sugar is D-2-deoxyribose. In some embodiments, a sugar is beta-D-deoxyribofuranose. In some embodiments, a sugar moiety is a beta-D-doxyribofuranose moiety. In some embodiments, a sugar is D-ribose. In some embodiments, a sugar is beta-D-ribofuranose. In some embodiments, a sugar moiety is a beta-D-ribofuranose moiety. In some embodiments, a sugar is optionally substituted beta-D-deoxyribofuranose or beta-D-ribofuranose. In some embodiments, a sugar moiety is an optionally substituted beta-D-deoxyribofuranose or beta-D-ribofuranose moiety. In some embodiments, a sugar moiety/unit in an oligonucleotide, nucleic acid, etc. is a sugar which comprises one or more carbon atoms each independently connected to an internucleotidic linkage, e.g., optionally substituted beta-D-deoxyribofuranose or beta-D-ribofuranose whose 5′-C and/or 3′-C are each independently connected to an internucleotidic linkage (e.g., a natural phosphate linkage, a modified internucleotidic linkage, a chirally controlled internucleotidic linkage, etc.).

In some embodiments, each nucleoside of a provided oligonucleotide comprises a 2′-O-methoxyethyl sugar modification.

In some embodiments, the oligonucleotide composition comprises at least one locked nucleic acid (LNA) nucleotide. In some embodiments, the oligonucleotide composition comprises at least one modified nucleotide comprising a modified sugar moiety which is modified at the 2′-position.

In some embodiments, the oligonucleotide composition comprises modified sugar moiety which comprises a 2′-substituent selected from the group consisting of: H, OR R, halogen, SH, SR, NH₂, NHR, NR₂, and ON, wherein R is an optionally substituted C₁-C₆alkyl, alkenyl, or alkynyl and halogen is F, Cl, Br or I.

In some embodiments, a modified nucleobase, sugar, nucleoside, nucleotide, and/or modified internucleotidic linkage is selected from those described in Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507, 220; Gryaznov, S.; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226; Jones et al. J. Org. Chem. 1993, 58, 2983; Vasseur et al. J. Am. Chem. Soc. 1992, 114, 4006; Van Aerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34: 1338; Hendrix et al. 1997 Chem. Eur. J. 3: 110; Koshkin et al. 1998 Tetrahedron 54: 3607-3630; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5; Nielsen et al. 1997 Chem. Soc. Rev. 73; Schultz et al. 1996 Nucleic Acids Res. 24: 2966; Obika et al. 1997 Tetrahedron Lett. 38 (50): 8735-8; Obika et al. 1998 Tetrahedron Lett. 39: 5401-5404; Singh et al. 1998 Chem. Comm. 1247-1248; Kumar et al. 1998 Bioo. Med. Chem. Let. 8: 2219-2222; Nielsen et al. 1997 J. Chem. Soc. Perkins Transl. 1: 3423-3433; Singh et al. 1998 J. Org. Chem. 63: 6078-6079; Seth et al. 2010 J. Org. Chem. 75: 1569-1581; Singh et al. 1998 J. Org. Chem. 63: 10035-39; Sorensen 2003 Chem. Comm. 2130-2131; Petersen et al. 2003 TRENDS Biotech. 21: 74-81; Rajwanshi et al. 1999 Chem. Commun. 1395-1396; Jepsen et al. 2004 Oligo. 14: 130-146; Morita et al. 2001 Nucl. Acids Res. Supp. 1: 241-242; Morita et al. 2002 Bioo. Med. Chem. Lett. 12: 73-76; Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226; Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273; Lauritsen et al. 2002 Chem. Comm. 5: 530-531; Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256; WO 20070900071; Seth et al., Nucleic Acids Symposium Series (2008), 52(1), 553-554; Seth et al. 2009 J. Med. Chem. 52: 10-13; Seth et al. 2012 Mol. Ther-Nuc. Acids. 1, e47; Pallan et al. 2012 Chem. Comm. 48: 8195-8197; Seth et al. 2010 J. Med. Chem. 53: 8309-8318; Seth et al. 2012 Bioo. Med. Chem. Lett. 22: 296-299; WO 2016/079181; U.S. Pat. Nos. 6,326,199; 6,066,500; and 6,440,739.

In some embodiments, sugars and nucleosides include 6′-modified bicyclic sugars and nucleosides, respectively, that have either (R) or (S)-chirality at the 6′-position, e.g., those described in U.S. Pat. No. 7,399,845. In other embodiments, sugars and nucleosides include 5′-modified bicyclic sugars and nucleosides, respectively, that have either (R) or (S)-chirality at the 5′-position, e.g., those described in US Patent Application Publication No. 20070287831.

In some embodiments, modified sugars, nucleobases, nucleosides, nucleotides, and/or internucleotidic linkages are described in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191; 5,459.255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the sugars, nucleobases, nucleosides, nucleotides, and internucleotidic linkages of each of which are incorporated by reference.

In some embodiments, modified sugars, nucleobases, nucleosides, nucleotides, and/or internucleotidic linkages are those described in any of: Gryaznov, S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Hendrix et al. 1997 Chem. Eur. J. 3: 110; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5; Jepsen et al. 2004 Oligo. 14: 130-146; Jones et al. J. Org. Chem. 1993, 58, 2983; Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273; Koshkin et al. 1998 Tetrahedron 54: 3607-3630; Kumar et al. 1998 Bioo. Med. Chem. Let. 8: 2219-2222; Lauritsen et al. 2002 Chem. Comm. 5: 530-531; Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256; Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226: Morita et al. 2001 Nucl. Acids Res. Supp. 1: 241-242; Morita et al. 2002 Bioo. Med. Chem. Lett. 12: 73-76; Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226; Nielsen et al. 1997 Chem. Soc. Rev. 73; Nielsen et al. 1997 J. Chem. Soc. Perkins Transl. 1: 3423-3433; Obika et al. 1997 Tetrahedron Lett. 38 (50): 8735-8; Obika et al. 1998 Tetrahedron Lett. 39: 5401-5404; Pallan et al. 2012 Chem. Comm. 48: 8195-8197; Petersen et al. 2003 TRENDS Biotech. 21: 74-81; Rajwanshi et al. 1999 Chem. Commun. 1395-1396; Schultz et al. 1996 Nucleic Acids Res. 24: 2966; Seth et al. 2009 J. Med. Chem. 52: 10-13; Seth et al. 2010 J. Med. Chem. 53: 8309-8318; Seth et al. 2010 J. Org. Chem. 75: 1569-1581; Seth et al. 2012 Bioo. Med. Chem. Lett. 22: 296-299; Seth et al. 2012 Mol. Ther-Nuc. Acids. 1, e47; Seth, Punit P; Siwkowski, Andrew; Allerson, Charles R; Vasquez. Guillermo; Lee, Sam; Prakash, Thazha P; Kinberger, Garth; Migawa, Michael T; Gaus, Hans; Bhat, Balkrishen; et al. From Nucleic Acids Symposium Series (2008). 52(1), 553-554; Singh et al. 1998 Chem. Comm. 1247-1248; Singh et al. 1998 J. Org. Chem. 63: 10035-39; Singh et al. 1998 J. Org. Chem. 63: 6078-6079; Sorensen 2003 Chem. Comm. 2130-2131: Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507, 220; Van Aerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34: 1338; Vasseur et al. J. Am. Chem. Soc. 1992, 114, 4006; WO 20070900071; WO 20070900071; and WO 2016/079181.

In some embodiments, modified sugars, nucleobases, nucleosides, nucleotides, and/or internucleotidic linkages include, or include those in, HNA, PNA, 2′-Fluoro N3′-P5′-phosphoramidate, LNA, beta-D-oxy-LNA, 2′-0,3′-C-linked bicyclic, PS-LNA, beta-D-thio-LNA, beta-D-amino-LNA, xylo-LNA [c], alpha-L-LNA, ENA, beta-D-ENA, amide-linked LNA, methylphosphonate-LNA, (R S)-cEt, (R, S)-cMOE, (R. S)-5′-Me-LNA, S-Me cLNA, Methylene-cLNA, 3′-Me-alpha-L-LNA, R-6′-Me-alpha-L-LNA, S-5′-Me-alpha-L-LNA, or R-5′-Me-alpha-L-LNA. Certain modified sugars, nucleobases, nucleosides, nucleotides, and/or internucleotidic linkages are described in U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, US 20130178612, US 20150211006, U.S. Pat. No. 9,598,458, US 20170037399, WO 2017/015555, WO 2017/062862, the modified sugars, nucleobases, nucleosides, nucleotides, and internucleotidic linkages of each of which are incorporated herein by reference.

Dystrophin

In some embodiments, the present disclosure provides technologies, e.g., oligonucleotides, compositions, methods, etc., related to the dystrophin (DMD) gene or a product encoded thereby (a transcript, a protein (e.g., various variants of the dystrophin protein), etc.). In some embodiments, the base sequence of an oligonucleotide is or comprise a sequence which sequence is, or is complementary (e.g., 85%, 90%, 95%, 100%; in many embodiments, 100%) to, a sequence in the DMD gene or a product thereof (e.g., a transcript, mRNA, etc.) (such an oligonucleotide-DMD oligonucleotide). In some embodiments, such a sequence in the DMD gene or a product thereof comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20, 31, 32, 33, 34, 35 or more nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 10 nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 15 nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 16 nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 17 nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 18 nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 19 nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 20 nucleobases. In some embodiments, the present disclosure provides technologies, including DMD oligonucleotides and compositions and methods of use thereof, for treatment of muscular dystrophy, including but not limited to, Duchenne Muscular Dystrophy (also abbreviated as DMD) and Becker Muscular Dystrophy (BMD). In some embodiments, DMD comprises one or more mutations. In some embodiments, such mutations are associated with reduced biological functions of dystrophin protein in a subject suffering from or susceptible to muscular dystrophy.

In some embodiments, the dystrophin (DMD) gene or a product thereof, or a variant or portion thereof, may be referred to as DMD, BMD, CMD3B, DXS142, DXS164, DXS206, DXS230, DXS239, DXS268, DXS269, DXS270, DXS272, MRX85, or dystrophin; External IDs: OMIM: 300377 MGI: 94909; HomoloGene: 20856; GeneCards: DMD; In Human: Entrez: 1756; Ensembl: ENSG00000198947; UniProt: P11532; RefSeq (mRNA): NM_000109; NM_004006; NM_004007; NM_004009; NM_004010; RefSeq (protein): NP_000100; NP_003997 NP_004000; NP_004001; NP_004002; Location (UCSC): Chr X: 31.1-33.34 Mb; In Mouse: Entrez: 13405; Ensembl: ENSMUSG00000045103; UniProt: P11531; RefSeq (mRNA): NM_007868; NM_001314034; NM_001314035; NM_001314036; NM_001314037; RefSeq (protein); NP_001300963: NP_001300964; NP_001300965; NP_001300966; NP_001300967; Location (UCSC): Chr X: 82.95-85.21 Mb.

The DMD gene reportedly contains 79 exons distributed over 2.3 million bp of genetic real estate on the X chromosome; however, only approximately 14,000 bp (<1%) is reported to be used for translation into protein (coding sequence). It is reported that about 99.5% of the genetic sequence, the intronic sequences, is spliced out of the 2.3 million bp initial heteronuclear RNA transcript to provide a mature 14,000 bp mRNA that includes all key information for dystrophin protein production. In some embodiments, patients with DMD have mutation(s) in the DMD gene that prevent the appropriate construction of the wild-type DMD mRNA and/or the production of the wild-type dystrophin protein, and patients with DMD often show marked dystrophin deficiency in their muscle.

In some embodiments, a dystrophin transcript, e.g., mRNA, or protein encompasses those related to or produced from alternative splicing. For example, sixteen alternative transcripts of the dystrophin gene were reported following an analysis of splicing patterns of the DMD gene in skeletal muscle, brain and heart tissues. Sironi et al. 2002 FEBS Letters 517: 163-166.

It is reported that dystrophin has several isoforms. In some embodiments, dystrophin refers to a specific isoform. At least three full-length dystrophin isoforms have been reported, each controlled by a tissue-specific promoter. Klamut et al. 1990 Mol. Cell. Biol. 10: 193-205; Nudel et al. 1989 Nature 337: 76-78; Gorecki et al. 1992 Hum. Mol. Genet. 1: 505-510. The muscle isoform is reportedly mainly expressed in skeletal muscle but also in smooth and cardiac muscles [Bies, R D., Phelps, S. F., Cortez. M. D., Roberts, R., Caskey, C. T. and Chamberlain, J. S. 1992 Nucleic Acids Res. 20: 1725-1731], the brain dystrophin is reportedly specific for cortical neurons but can also be detected in heart and cerebellar neurons, while the Purkinje-cell type reportedly accounts for nearly all cerebellar dystrophin [Gorecki et al. 1992 Hum. Mol. Genet. 1: 505-510]. Alternative splicing reportedly provides a means for dystrophin diversification: the 3′ region of the gene reportedly undergoes alternative splicing resulting in tissue-specific transcripts in brain neurons, cardiac Purkinje fibers, and smooth muscle cells [Bies et al. 1992 Nucleic Acids Res. 20: 1725-1731; and Feener et al. 1989 Nature 338: 509-511] while 12 patterns of alternative splicing have been reported in the 5′ region of the gene in skeletal muscle [Surono et al. 1997 Biochem. Biophys. Res. Commun. 239: 895-899].

In some embodiments, a dystrophin mRNA, gene or protein is a revertant version. Among others, revertant dystrophins were reported in, for example: Hoffman et al. 1990 J. Neurol. Sci. 99:9-25; Klein et al. 1992 Am. J. Hum. Genet. 50: 950-959; and Chelly et al. 1990 Cell 63: 1239-1348; Arahata et al. 1998 Nature 333: 861-863; Bonilla et al. 1988 Cell 54: 447-452: Fanin et al. 1992 Neur. Disord. 2: 41-45; Nicholson et al. 1989 J. Neurol. Sci. 94: 137-146; Shimizu et al. 1988 Proc. Jpn. Acad. Sci. 64: 205-208; Sicinzki t al. 1989 Science 244: 1578-1580; and Sherratt et al. Am. J. Hum. Genet. 53: 1007-1015.

Various mutations in the DMD gene can and/or were reported to cause muscular dystrophy.

Muscular Dystrophy

Compositions comprising one or more DMD oligonucleotides described herein can be used to treat muscular dystrophy. In some embodiments, muscular dystrophy (MD) is any of a group of muscle conditions, diseases, or disorders that results in (increasing) weakening and breakdown of skeletal muscles over time. The conditions, diseases, or disorders differ in which muscles are primarily affected, the degree of weakness, when symptoms begin, and how quickly symptoms worsen. Many MD patients will eventually become unable to walk. In many cases muscular dystrophy is fatal. Some types are also associated with problems in other organs, including the central nervous system. In some embodiments, the muscular dystrophy is Duchenne (Duchenne's) Muscular Dystrophy (DMD) or Becker (Becker's) Muscular Dystrophy (BMD).

In some embodiments, a symptom of Duchenne Muscular Dystrophy is muscle weakness associated with muscle wasting, with the voluntary muscles being first affected, especially those of the hips, pelvic area, thighs, shoulders, and calves. Muscle weakness can also occur later, in the arms, neck, and other areas. Calves are often enlarged. Symptoms usually appear before age six and may appear in early infancy. Other physical symptoms are: awkward manner of walking, stepping, or running (in some cases, patients tend to walk on their forefeet, because of an increased calf muscle tone), frequent falls, fatigue, difficulty with motor skills (e.g., running, hopping, jumping), lumbar hyperordosis, possibly leading to shortening of the hip-flexor muscles, unusual overall posture and/or manner of walking, stepping, or running, muscle contractures of Achilles tendon and hamstrings impair functionality, progressive difficulty walking, muscle fiber deformities, pseudohypertrophy (enlarging) of tongue and calf muscles, higher risk of neurobehavioral disorders (e.g., ADHD), learning disorders (e.g., dyslexia), and non-progressive weaknesses in specific cognitive skills (e.g., short-term verbal memory), which are believed to be the result of absent or dysfunctional dystrophin in the brain, eventual loss of ability to walk (usually by the age of 12), skeletal deformities (including scoliosis in some cases), and trouble getting up from lying or sitting position.

In some embodiments, Becker muscular dystrophy (BMD) is caused by mutations that give rise to shortened but in-frame transcripts resulting in the production of truncated but partially functional protein(s). Such partially functional protein(s) were reported to retain the critical amino terminal, cysteine rich and C-terminal domains but usually lack elements of the central rod domains which were reported to be of less functional significance. England et al. 1990 Nature, 343, 180-182.

In some embodiments, BMD phenotypes range from mild DMD to virtually asymptomatic, depending on the precise mutation and the level of dystrophin produced. Yin et al. 2008 Hum. Mol. Genet. 17: 3909-3918.

In some embodiments, dystrophy patients with out-of-frame mutations are generally diagnosed with the more severe Duchenne Muscular Dystrophy, and dystrophy patients with in-frame mutations are generally diagnosed with the less severe Becker Muscular Dystrophy. However, a minority of patients with in-frame deletions are diagnosed with Duchenne Muscular Dystrophy, including those with deletion mutations starting or ending in exons 50 or 51, which encode part of the hinge region, such as deletions of exons 47 to 51, 48 to 51, and 49 to 53. Without wishing to be bound by any particular theory, the present disclosure notes that the patient-to-patient variability in disease severity despite the presence of the same exon deletion reportedly may be related to the effect of the specific deletion breakpoints on mRNA splicing efficiency and/or patterns; translation or transcription efficiency after genome rearrangement; and stability or function of the truncated protein structure. Yokota et al. 2009 Arch. Neurol. 66: 32.

Exon Skipping as a Treatment for Muscular Dystrophy

In some embodiments, a treatment for muscular dystrophy comprises the use of a DMD oligonucleotide which is capable of mediating skipping of one or more Dystrophin exons. In some embodiments, the present disclosure provides methods for treatment of muscular dystrophy comprising administering to a subject suffering therefrom or susceptible thereto an DMD oligonucleotide, or a composition comprising a DMD oligonucleotide. Particularly, among other things, the present disclosure demonstrates that chirally controlled oligonucleotide/chirally controlled oligonucleotide compositions are unexpectedly effective for modulating exon skipping compared to otherwise identical but non-chirally controlled oligonucleotide/oligonucleotide compositions. In some embodiments, the present disclosure demonstrates incorporation of one or more non-negatively charged internucleotidic linkage can greatly improve delivery and/or overall exon skipping efficiency.

In some embodiments, a treatment for muscular dystrophy employs the use of a DMD oligonucleotide, wherein the oligonucleotide is capable of providing skipping of one or more exons. Skipping of one or more (e.g., multiple) DMD exons can, for example, remove a mutated exon(s), or compensate for a mutation(s) (e.g., restoring the reading frame if the mutation is a frameshift mutation) in an exon which is not skipped. In some embodiments, a DMD oligonucleotide is capable of mediating the skipping of an exon which comprises a mutation (e.g., a frameshift, insertion, deletion, missense, or nonsense mutation, or other mutation), wherein the skipping of the exon maintains (or restores) the proper reading frame of the DMD gene, and translation produces a truncated but functional (or largely functional) DMD protein. In some embodiments, a DMD oligonucleotide compensates for an exon comprising a frameshift mutation by providing skipping of a different exon (not the one comprising the frameshift mutation), and thus restoring the reading frame of the DMD gene. In some embodiments, a patient having muscular dystrophy has a frameshift mutation in one exon of the DMD gene; and this patient is treated with a DMD oligonucleotide which does not cause skipping of the exon having the mutation, but causes skipping of a different exon, which restores the reading frame of the DMD gene, so that a functional DMD protein is produced (and, if the deleted exon is 3′ to the exon which has the frameshift mutation, this functional DMD protein will generally have an amino acid of a normal DMD protein, except for a sequence of amino acids not normally found in DMD, spanning from the frameshift mutation to the exon which is 3′ to the deleted exon).

In some embodiments, a composition comprising a DMD oligonucleotide is useful for treatment of a Dystrophin-related disorder of the central nervous system. In some embodiments, the present disclosure pertains to a method of treatment of a Dystrophin-related disorder of the central nervous system, wherein the method comprises the step of administering a therapeutically effective amount of a DMD oligonucleotide to a patient suffering from a Dystrophin-related disorder of the central nervous system. In some embodiments, a DMD oligonucleotide is administered outside the central nervous system (as non-limiting examples, intravenously or intramuscularly) to a patient suffering from a Dystrophin-related disorder of the central nervous system, and the DMD oligonucleotide is capable of passing through the blood-brain barrier into the central nervous system. In some embodiments, a DMD oligonucleotide is administered directly into the central nervous system (as non-limiting example, via intrathecal, intraventricular, intracranial, etc., delivery).

In some embodiments, a Dystrophin-related disorder of the central nervous system, or a symptom thereof, can be any one or more of: decreased intelligence, decreased long term memory, decreased short term memory, language impairment, epilepsy, autism spectrum disorder, attention deficit hyperactivity disorder (ADHD), obsessive-compulsive disorder, learning problem, behavioral problem, a decrease in brain volume, a decrease in grey matter volume, lower white matter fractional anisotropy, higher white matter radial diffusivity, an abnormality of skull shape, or a deleterious change in the volume or structure of the hippocampus, globus pallidus, caudate putamen, hypothalamus, anterior commissure, periaqueductal gray, internal capsule, amygdala, corpus callosum, septal nucleus, nucleus accumbens, fimbria, ventricle, or midbrain thalamus. In some embodiments, a patient exhibiting muscle-related symptoms of muscular dystrophy also exhibits symptoms of a Dystrophin-related disorder of the central nervous system.

In some embodiments, a Dystrophin-related disorder of the central nervous system is related to, associated with and/or caused by an abnormality in the level, activity, expression and/or distribution of a gene product of the Dystrophin gene, such as full-length Dystrophin or a smaller isoform of Dystrophin, including, but not limited to, Dp260, Dp140, Dp116, Dp71 or Dp40. In some embodiments, a DMD oligonucleotide is administered into the central nervous system of a muscular dystrophy patient in order to ameliorate one or more systems of a Dystrophin-related disorder of the central nervous system. In some embodiments, a Dystrophin-related disorder of the central nervous system is related to, associated with and/or caused by an abnormality in the level, activity, expression and/or distribution of a gene product of the Dystrophin gene, such as full-length Dystrophin or a smaller isoform of Dystrophin, including, but not limited to, Dp260, Dp140, Dp116, Dp71 or Dp40. In some embodiments, administration of a DMD oligonucleotide to a patient suffering from a Dystrophin-related disorder of the central nervous system increases the level, activity, and/or expression and/or improves the distribution of a gene product of the Dystrophin gene.

In some embodiments, the present disclosure provides technologies for modulating dystrophin pre-mRNA splicing, whereby selected exons are excised to either remove nonsense mutations or restore the reading frame around frameshifting mutations from the mature mRNA. In some embodiments, a DMD oligonucleotide capable of skipping an exon is capable of restoring the reading frame.

As a non-limiting example, in a patient with Duchenne Muscular Dystrophy who has a deletion of exon 50, an out-of-frame transcript is generated in which exon 49 is spliced to exon 51. As a result, a stop codon is generated in exon 51, which prematurely aborts dystrophin synthesis. In some embodiments, the present disclosure provides oligonucleotides that can mediate skipping of exon 51, restore the open reading frame of the transcript, and allow the production of a truncated dystrophin similar to that in patients with Becker muscular dystrophy (BMD).

In some embodiments, in a DMD patient, a DMD gene comprises an exon comprising a mutation, and the disorder is at least partially treated by skipping of one or more exons (e.g., the exon comprising the mutation, or an exon adjacent to the exon comprising the mutation, or a set of consecutive exons, including the exon comprising the mutation).

In some embodiments, in a DMD patient, a DMD gene or transcript has a mutation in an exon(s), which is a missense or nonsense mutation and/or deletion, insertion, inversion, translocation or duplication. In some embodiments, in a DMD patient, a DMD gene or transcript has a mutation in an exon(s) which results in a frameshift, premature stop codon, or otherwise perturbation of the proper reading frame.

In some embodiments, in a treatment for muscular dystrophy, an exon of DMD is skipped, wherein the exon encodes a string of amino acids not essential for DMD protein function, or whose skipping can provide a fully or partially functional DMD protein. In some embodiments, in a treatment for muscular dystrophy, an exon of DMD is skipped, wherein the exon(s) skipped include an exon which comprises a mutation or is adjacent to (e.g., flanking) an exon comprising a mutation, or wherein multiple exons are skipped, the skipped exons optionally include an exon comprising a mutation. In some embodiments, in a treatment for muscular dystrophy, two or more exons are skipped, wherein the exons skipped include an exon which comprises a mutation or is adjacent to (e.g., flanking) an exon comprising a mutation. In some embodiments, in a treatment for muscular dystrophy, an exon comprises a frameshift mutation, and the skipping of a different exon (while leaving the exon with the frameshift mutation in place) restores the proper reading frame.

In some embodiments, in a treatment for muscular dystrophy, a DMD oligonucleotide is capable of mediating skipping of one or more DMD exons, thereby either restoring or maintaining the proper reading frame, and/or creating an artificially internally truncated DMD which provides at least partially improved or fully restored biological activity.

In some embodiments, an DMD oligonucleotide skips an exon(s) which is not exon 64 and exon 70, portions of which are reportedly important for protein function, and/or which is not first or the last exon. In some embodiments, an DMD oligonucleotide skips an exon(s), but skipping of the exon(s) does not cause deletion of one or more or all actin-binding sites in the N-terminal region.

In some embodiments, an internally truncated DMD protein produced from a dystrophin transcript with a skipped exon(s) is more functional than a terminally truncated DMD protein e.g., produced from a dystrophin transcript with an out-of-frame deletion.

In some embodiments, an internally truncated DMD protein produced from a dystrophin transcript with a skipped exon(s) is more resistant to nonsense-mediated decay, which can degrade a terminally truncated DMD protein, e.g., produced from a dystrophin transcript with an out-of-frame deletion.

In some embodiments, the present disclosure encompasses the recognition that the nature and location of a DMD mutation may be utilized to design exon-skipping strategy. In some embodiments, if a DMD patient has a mutation in an exon, skipping of the mutated exon can produce an internally truncated (internally shortened) but at least partially functional DMD protein product.

In some embodiments, a DMD patient has a mutation which alters splicing of a DMD transcript, e.g., by inactivating a site required for splicing, or activating a cryptic site so that it becomes active for splicing, or by creating an alternative (e.g., unnatural) splice site. In some embodiments, such a mutation causes production of proteins with low or no activities. In some embodiments, splicing modulation, e.g., exon skipping, suppression of such a mutation, etc., can be employed to remove or reduce effects of such a mutation, e.g., by restoring proper splicing to produce proteins with restored activities, or producing an internally truncated dystrophin protein with improved or restored activities, etc.

In some embodiments, a DMD patient has a mutation which is a duplication of one or several exons, and the present disclosure provides exon skipping technologies to delete the duplication and/or to restore the reading frame.

In some embodiments, a DMD patient has a mutation which causes the skipping of an exon, which in turn can cause a frameshift. In some embodiments, the present disclosure provides technologies that can provide skipping of an additional exon(s) to restore the reading frame. For example, deletion of exon 51, which causes a frame shift, may be addressed by skipping of exon 50 or 52, which restores the reading frame. In some embodiments, a DMD patient has a mutation in one exon which causes a frame shift, and a deletion of a different exon(s) (e.g., a different exon, or an adjacent or flanking exon(s) immediately 5′ or 3′ to the mutated exon) restores the reading frame.

In some embodiments, restoring the reading frame can convert an out-of-frame mutation to an in-frame mutation; in some embodiments, in humans, such a change can transform severe Duchenne Muscular Dystrophy into milder Becker Muscular Dystrophy.

In some embodiments, a DMD patient or a patient suspected to have DMD is analyzed for DMD genotype prior to administration of a composition comprising a DMD oligonucleotide.

In some embodiments, a DMD patient or a patient suspected to have DMD is analyzed for DMD phenotype prior to administration of a composition comprising a DMD oligonucleotide.

In some embodiments, a DMD patient is analyzed for genotype and phenotype to determine the relationship of DMD genotype and DMD phenotype prior to administration of a composition comprising a DMD oligonucleotide.

In some embodiments, a patient is genetically verified to have dystrophy prior to administration of a composition comprising a DMD oligonucleotide.

In some embodiments, analysis of DMD genotype or genetic verification of DMD or a patient comprises determining if the patient has one or more deleterious mutations in DMD.

In some embodiments, analysis of DMD genotype or genetic verification of DMD or a patient comprises determining if the patient has one or more deleterious mutations in DMD and/or analyzing DMD splicing and/or detecting splice variants of DMD, wherein a splice variant is produced by an abnormal splicing of DMD.

In some embodiments, analysis of DMD genotype or genetic verification of DMD informs the selection of a composition comprising a DMD oligonucleotide useful for treatment.

In some embodiments, an abnormal or mutant DMD gene or a portion thereof is removed or copied from a patient or a patient's cell(s) or tissue(s) and the abnormal or mutant DMD gene, or a portion thereof comprising the abnormality or mutation, or a copy thereof, is inserted into a cell. In some embodiments, this cell can be used to test various compositions comprising a DMD oligonucleotide to predict if such a composition would be useful as a treatment for the patient. In some embodiments, the cell is a myoblast or myotubule.

In some embodiments, an individual or patient can produce, prior to treatment with a DMD oligonucleotide, one or more splice variants of DMD, often each variant being produced at a very low level. In some embodiments, a method such as that described in Example 20 can be used to detect low levels of splice variants being produced in a patient prior to, during or after administration of a DMD oligonucleotide.

In some embodiments, a patient and/or the tissues thereof are analyzed for production of various splicing variants of a DMD gene prior to administration of a composition comprising a DMD oligonucleotide.

In some embodiments, the present disclosure provides methods for designing a DMD oligonucleotide (e.g., an oligonucleotide capable of mediating skipping of one or more exons of DMD). In some embodiments, the present disclosure utilizes rationale design described herein and optionally sequence walks to design oligonucleotides, e.g., for testing exon skipping in one or more assays and/or conditions. In some embodiments, an efficacious oligonucleotide is developed following rational design, including using various information of a given biological system.

In some embodiments, in a method for developing DMD oligonucleotides, oligonucleotides are designed to anneal to one or more potential splicing-related motifs and then tested for their ability to mediate exon skipping. In some embodiments, splicing-related motifs include, but are not limited to, any one or more of: an acceptor, exon recognition sequence (ERS), exonic splice enhancer (ESE) site, splicing enhancer sequence (SES), branch point sequence, and donor splice site of a target exon. Certain sequences that may be involved in splicing were reported in, for example: Disset et al. 2006 Human Mol. Gen. 15: 999-1013.

In some embodiments, software packages, such as RESCUE-ESE, ESEfinder, and the PESX server, may be utilized to predict putative ESE sites (Fairbrother et al. 2002 Science 297: 1007-1013; Cartegni et al. 2003 Nat. Struct. Biol. 120-125; Zhang and Chasin 2004 Gen. Dev. 18: 1241-1250; Smith et al. 2006 Hum. Mol. Genet. 15: 2490-2508).

In some embodiments, a DMD oligonucleotide which targets or interacts with an acceptor, exon recognition sequence (ERS), exonic splice enhancer (ESE) site, or donor splice site of a DMD exon does not interact or significantly interact with a sequence in another (e.g., off-target) gene.

In some embodiments, in a rational approach to DMD oligonucleotide design, oligonucleotides are designed with consideration of secondary structures of dystrophin transcripts, e.g., mRNA. Designed oligonucleotide can then be assessed for exon skipping. A number of effective DMD oligonucleotides have been designed using rational approaches described in the present disclosure.

In some embodiments, alternatively or additionally, sequence walk, e.g., of an exon sequence can be performed to search for efficacious DMD oligonucleotide sequences.

In some embodiments, provided methods comprise sequence walking. In some embodiments, a set of overlapping oligonucleotides is generated. In some embodiments, oligonucleotides in a set have the same length, and the 5′ ends of the oligonucleotides in the set are evenly spaced apart. In some embodiments, a set of overlapping oligonucleotides encompasses an entire exon or a portion(s) thereof. The 5′ ends of the oligonucleotides in a walk can be evenly spaced at a suitable distance, e.g., 1 base apart, 2 bases apart, 3 bases apart, etc. Among other things, the present disclosure demonstrates that sequences can be optimized and in combination with chemistry and/or stereochemistry technologies of the present disclosure, highly effective oligonucleotides (and compositions and methods of use thereof) can be prepared.

Example Technologies for Assessing Oligonucleotides and Oligonucleotide Compositions

Various technologies for assessing properties and/or activities of oligonucleotides can be utilized in accordance with the present disclosure, e.g., US 20170037399, WO 2017/015555, WO 2017/015575, WO 2017/192664, WO 2017/062862, WO 2017/192679, WO 2017/210647, etc.

For example, DMD oligonucleotides can be evaluated for their ability to mediate exon skipping in various assays, including in vitro and in vivo assays, in accordance with the present disclosure. In vitro assays can be performed in various test cells described herein or known in the art, including but not limited to, A48-50 Patient-Derived Myoblast Cells. In vivo tests can be performed in test animals described herein or known in the art, including but not limited to, a mouse, rat, cat, pig, dog, monkey, or non-human primate.

As non-limiting examples, a number of assays are described below for assessing properties/activities of DMD oligonucleotides. Various other suitable assays are available and may be utilized to assess oligonucleotide properties/activities, including those of oligonucleotides not designed for exon skipping (e.g., for oligonucleotides that may involve RNase H for reducing levels of target transcripts, assays described in US 20170037399, WO 2017/015555, WO 2017/015575, WO 2017/192664, WO 2017/192679, WO 2017/210647, etc.).

A DMD oligonucleotide can be evaluated for its ability to mediate skipping of an exon in the Dystrophin RNA, which can be tested, as non-limiting examples, using nested PCR, qRT-PCR, and/or sequencing.

A DMD oligonucleotide can be evaluated for its ability to mediate protein restoration (e.g., production of an internally truncated protein lacking the amino acids corresponding to the codons encoded in the skipped exon, which has improved functions compared to proteins (if any) produced prior to exon skipping), which can be evaluated by a number of methods for protein detection and/or quantification, such as western blot, immunostaining, etc. Antibodies to dystrophin are commercially available or if desired, can be developed for desired purposes.

A DMD oligonucleotide can be evaluated for its ability to mediate production of a stable restored protein. Stability of restored protein can be tested, in non-limiting examples, in assays for serum and tissue stability.

A DMD oligonucleotide can be evaluated for its ability to bind protein, such as albumin. Example related technologies include those described, e.g., in WO 2017/015555, WO 2017/015575, etc.

A DMD oligonucleotide can be evaluated for immuno activity, e.g., through assays for cytokine activation, complement activation. TLR9 activity, etc. Example related technologies include those described, e.g., in WO 2017/015555, WO 2017/015575, WO 2017/192679, WO 2017/210647, etc.

In some embodiments, efficacy of a DMD oligonucleotide can be tested, e.g., in in silico analysis and prediction, a cell-free extract, a cell transfected with artificial constructs, an animal such as a mouse with a human Dystrophin transgene or portion thereof, normal and dystrophic human myogenic cell lines, and/or clinical trials. It may be desirable to utilize more than one assay, as normal and dystrophic human myogenic cell lines may sometimes produce different efficacy results under certain conditions (Mitrpant et al. 2009 Mol. Ther. 17: 1418).

In some embodiments, DMD oligonucleotides can be tested in vitro in cells. In some embodiments, testing in vitro in cells involves gymnotic delivery of the oligonucleotide(s), or delivery using a delivery agent or transfectant, many of which are known in the art and may be utilized in accordance with the present disclosure.

In some embodiments, DMD oligonucleotides can be tested in vitro in normal human skeletal muscle cells (hSkMCs). See, for example, Arechavala et al. 2007 Hum. Gene Ther. 18: 798-810.

In some embodiments, DMD oligonucleotides can be tested in a muscle explant from a DMD patient. Muscle explants from DMD patients are reported in, for example, Fletcher et al. 2006 J. Gene Med. 8: 207-216; McClorey et al. 2006 Neur. Dis. 16: 583-590; and Arechavala et al. 2007 Hum. Gene Ther. 18: 798-810.

In some embodiments, cells are or comprise cultured muscle cells from DMD patients. See, for example: Aartsma-Rus et al. 2003 Hum. Mol. Genet. 8: 907-914.

In some embodiments, an individual DMD oligonucleotide may demonstrate experiment-to-experiment variability in its ability to skip an exon under certain circumstances. In some embodiments, an individual DMD oligonucleotide can demonstrate variability in its ability to skip an exon(s) depending on which cells are used, the growth conditions, and other experimental factors. To control variations, typically oligonucleotides to be tested and control oligonucleotides are assayed under the same or substantially the same conditions.

In vitro experiments also include those conducted with patient-derived myoblasts. Certain results from such experiments were described herein. In certain such experiments, cells were cultured in skeletal growth media to keep them in a dividing/immature myoblast state. The media was then changed to ‘differentiation’ media (containing insulin and 2% horse serum) concurrent with spiking oligonucleotides in the media for dosing. The cells differentiated into myotubes as they were getting dosed for a suitable period of time, e.g., a total of 4d for RNA experiments and 6d for protein experiments (such conditions referenced as ‘Od pre-differentiation’ (0d+4d for RNA, 0d+6d for protein)).

Without wishing to be bound by any particular theory, the present disclosure notes that it may be desirable to know if DMD oligonucleotides are able to enter mature myotubes and induce skipping in these cells as well as ‘immature’ cells. In some embodiments, the present disclosure provided assays to test effects of DMD oligonucleotides in myotubes. In some embodiments, a dosing schedule different from the ‘Od pre-differentiation’ was used, wherein the myoblasts were pre-differentiated into myotubes in differentiation media for several days (4d or 7d or 10d) and then DMD oligonucleotides were administered. Certain related protocols are described in Example 19.

In some embodiments, the present disclosure demonstrated that, in the pre-differentiation experiments, DMD oligonucleotides (excluding those which are PMOs) usually give about the same level of RNA skipping and dystrophin protein restoration, regardless of the number of days cells were cultured in differentiation media prior to dosing. In some embodiments, the present disclosure provides oligonucleotides that may be able to enter and be active in myoblasts and in myotubes. In some embodiments, a DMD oligonucleotide is tested in vitro in Δ45-52 DMD patient cells (also designated D45-52 or de145-52) or Δ52 DMD patient cells (also designated D52 or de152) with 0, 4 or 7 days of pre-differentiation.

In some embodiments, DMD oligonucleotides can be tested in any one or more of various animal models, including non-mammalian and mammalian models; including, as non-limiting examples, Caenorhabditis, Drosophila, zebrafish, mouse, rat, cat, dog and pig. See, for example, a review in McGreevey et al. 2015 Dis. Mod. Mech. 8: 195-213.

Example use of mdx mice is reported in, for example: Lu et al. 2003 Nat. Med. 9: 1009; Jearawiriyapaisarn et al. 2008 Mol. Ther., 16, 1624-1629; Yin et al. 2008 Hum. Mol. Genet., 17, 3909-3918; Wu et al. 2009 Mol. Ther., 17, 864-871: Wu et al. 2008 Proc. Nat Acad. Sci. USA, 105, 14814-14819; Mann et al. 2001 Proc. Nat. Acad. Sci. USA 98: 42-47; and Gebski et al. 2003 Hum. Mol. Gen. 12:1801-1811.

Efficacy of DMD oligonucleotides can be tested in dogs, such as the Golden Retriever Muscular Dystrophy (GRMD) animal model. Lu et al. 2005 Proc. Natl. Acad. Sci. USA 102:198-203; Alter et al. 2006 Nat. Med. 12:175-7; McClorey et al. 2006 Gene Ther. 13:1373-81; and Yokota et al. 2012 Nucl. Acid Ther. 22: 306.

A DMD oligonucleotide can be evaluated in vivo in a test animal for efficient delivery to various tissues (e.g., skeletal, heart and/or diaphragm muscle); this can be tested, in non-limiting examples, by hybridization ELISA and tests for distribution in animal tissue.

A DMD oligonucleotide can be evaluated in vivo in a test animal for plasma PK: this can be tested, as non-limiting examples, by assaying for AUC (area under the curve) and half-life.

In some embodiments, DMD oligonucleotides can be tested in vivo, via an intramuscular administration a muscle of a test animal.

In some embodiments, DMD oligonucleotides can be tested in vivo, via an intramuscular administration into the gastrocnemius muscle of a test animal.

In some embodiments, DMD oligonucleotides can be tested in vivo, via an intramuscular administration into the gastrocnemius muscle of a mouse.

In some embodiments, DMD oligonucleotides can be tested in vivo, via an intramuscular administration into the gastrocnemius muscle of a mouse model transgenic for the entire human dystrophin locus. See, for example: Bremmer-Bout et al. 2004 Mol. Ther. 10, 232-240.

Additional tests which can be performed to evaluate the efficacy of DMO oligonucleotides include centrally nucleated fiber counts and dystrophin-positive fiber counts, and functional grip strength analysis. See, as non-limiting examples, experimental protocols reported in: Yin et al. 2009 Hum. Mol. Genet. 18: 4405-4414.

Additional methods of testing DMD oligonucleotides include, as non-limiting example, methods reported in; Kinali et al. 2009 Lancet 8: 918; Bertoni et al. 2003 Hum. Mol. Gen. 12: 1087-1099.

Certain Embodiments of Oligonucleotides and Compositions Thereof

Among other things, the present disclosure provides oligonucleotides, and compositions and methods of use thereof, useful for targeting various genes, including products encoded thereby and/or conditions, diseases and/or disorders associated therewith. In some embodiments, the present disclosure provides oligonucleotides, and compositions and methods of use thereof, for DMD. In some embodiments, the present disclosure provides a DMD oligonucleotide, wherein the base sequence of the DMD oligonucleotide is or comprises at least 15 contiguous bases of the sequence of any DMD oligonucleotide listed herein. In some embodiments, the present disclosure provides a DMD oligonucleotide, wherein the base sequence of the DMD oligonucleotide is or comprises at least 15 contiguous bases of the sequence of any DMD oligonucleotide listed herein, and wherein the DMD oligonucleotide is less than about 50 bases long. In some embodiments, the present disclosure provides an oligonucleotide or an oligonucleotide composition which comprises a non-negatively charged internucleotidic linkage.

In some embodiments, the present disclosure provides a chirally controlled composition of a DMD oligonucleotide (a plurality of DMD oligonucleotides), wherein the base sequence of the DMD oligonucleotide is or comprises at least 15 contiguous bases of the sequence of any DMD oligonucleotide listed herein. In some embodiments, the present disclosure provides a chirally controlled composition of a DMD oligonucleotide, wherein the base sequence of the DMD oligonucleotide is or comprises at least 15 contiguous bases of the sequence of any DMD oligonucleotide listed herein, and wherein the DMD oligonucleotide is less than about 50 bases long.

In some embodiments, the present disclosure provides a chirally controlled oligonucleotide having a sequence consisting of or comprising a sequence or a 15 base portion thereof found in any oligonucleotide listed in Table A1, wherein one or more U may be optionally and independently replaced with T or vice versa.

In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU, CUCCGGUUCUGAAGGUGUUC, or UUCUGAAGGUGUUCUUGUAC, or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein at least one internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU, CUCCGGUUCUGAAGGUGUUC, or UUCUGAAGGUGUUCUUGUAC, or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein at least one chirally controlled internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU, CUCCGGUUCUGAAGGUGUUC, or UUCUGAAGGUGUUCUUGUAC, or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein at least one chirally controlled internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU, CUCCGGUUCUGAAGGUGUUC, or UUCUGAAGGUGUUCUUGUAC, or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein each internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, I-b-1, II-b-2, I-c-1, II-c-2, I-d-1, II-d-2, or a salt form thereof. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU, CUCCGGUUCUGAAGGUGUUC, or UUCUGAAGGUGUUCUUGUAC, or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein at least one internucleotidic linkage has the structure of formula I-c or a salt form thereof. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU, CUCCGGUUCUGAAGGUGUUC, or UUCUGAAGGUGUUCUUGUAC, or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein at least one internucleotidic linkage has the structure of formula I-c or a salt form thereof, and at least one internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU, CUCCGGUUCUGAAGGUGUUC, or UUCUGAAGGUGUUCUUGUAC, or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein at least one internucleotidic linkage is a chirally controlled phosphorothioate internucleotidic linkage, and at least one internucleotidic linkage is a non-negatively charged internucleotidic linkage having the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU, CUCCGGUUCUGAAGGUGUUC, or UUCUGAAGGUGUUCUUGUAC, or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein each internucleotidic linkage is a phosphodiester.

In some embodiments, an oligonucleotide comprises one or more internucleotidic linkages which comprise a phosphorus modification prone to “autorelease” under certain conditions. That is, under certain conditions, a particular phosphorus modification is designed such that it self-cleaves from the oligonucleotide to provide, e.g., a phosphate diester such as those found in naturally occurring DNA and RNA. In some embodiments, such a phosphorus modification has a structure of —O-L-R¹, wherein each of L and R¹is independently as described in the present disclosure.

In some embodiments, a provided oligonucleotide of the present disclosure comprises chemical modifications and/or stereochemistry that delivers desirable properties, e.g., delivery to target cells/tissues/organs, pharmacodynamics, pharmacokinetics, etc.

In some embodiments, an oligonucleotide comprises a modification at a linkage phosphorus which can be transformed to a natural phosphate linkage by one or more esterases, nucleases, and/or cytochrome P450 enzymes, including but not limited to: CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1, CYP3A4, CYP3A5, CYP3A7, CYP3A43, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, CYP4X1, CYP4Z1, CYP5A1, CYP7A1, CYP7B1, CYP8A1 (prostacyclin synthase), CYP8B1 (bile acid biosynthesis), CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP19A1, CYP20A1, CYP21A2, CYP24A1, CYP26A1, CYP26B1, CYP26C1. CYP27A1 (bile acid biosynthesis), CYP27B1 (vitamin D3 1-alpha hydroxylase, activates vitamin D3), CYP27C1 (unknown function), CYP39A1 CYP46A1, and CYP51 A1 (lanosterol 14-alpha demethylase).

In some embodiments, an oligonucleotide comprises a modification at a linkage phosphorus that is a pro-drug moiety, e.g., a P-modification moiety facilitates delivery of an oligonucleotide to a desired location prior to removal. For instance, in some embodiments, a P-modification moiety results from PEGylation at the linkage phosphorus. One of skill in the relevant arts will appreciate that various PEG chain lengths are useful and that the selection of chain length will be determined in part by the result that is sought to be achieved by PEGylation. For instance, in some embodiments, PEGylation is effected in order to reduce RES uptake and extend in vivo circulation lifetime of an oligonucleotide.

In some embodiments, a PEGylation reagent for use in accordance with the present disclosure is of a molecular weight of about 300 g/mol to about 100,000 g/mol. In some embodiments, a PEGylation reagent is of a molecular weight of about 300 g/mol to about 10,000 g/mol. In some embodiments, a PEGylation reagent is of a molecular weight of about 300 g/mol to about 5,000 g/mol. In some embodiments, a PEGylation reagent is of a molecular weight of about 500 g/mol. In some embodiments, a PEGylation reagent of a molecular weight of about 1000 g/mol. In some embodiments, a PEGylation reagent is of a molecular weight of about 3000 g/mol. In some embodiments, a PEGylation reagent is of a molecular weight of about 5000 g/mol.

In certain embodiments, a PEGylation reagent is PEG500. In certain embodiments, a PEGylation reagent is PEG1000. In certain embodiments, a PEGylation reagent is PEG3000. In certain embodiments, a PEGylation reagent is PEG5000.

In some embodiments, an oligonucleotide comprises a P-modification moiety that acts as a PK enhancer, e.g., lipids, PEGylated lipids, etc.

In some embodiments, oligonucleotides of the present disclosure, e.g., DMD oligonucleotides, comprise a P-modification moiety that promotes cell entry and/or endosomal escape, such as a membrane-disruptive lipid or peptide.

In some embodiments, an oligonucleotide comprises a P-modification moiety that acts as a targeting moiety. In some embodiments, a P-modification moiety is or comprises a targeting moiety. In some embodiments, a target moiety is an entity that is associates with a payload of interest (e.g., with an oligonucleotide or oligonucleotide composition) and also interacts with a target site of interest so that the payload of interest is targeted to the target site of interest when associated with the targeting moiety to a materially greater extent than is observed under otherwise comparable conditions when the payload of interest is not associated with the targeting moiety. A targeting moiety may be, or comprise, any of a variety of chemical moieties, including, for example, small molecule moieties, nucleic acids, polypeptides, carbohydrates, etc. Targeting moieties are described, e.g., in Adarsh et al., “Organelle Specific Targeted Drug Delivery—A Review,” International Journal of Research in Pharmaceutical and Biomedical Sciences, 2011, p. 895.

Examples of such targeting moieties include, but are not limited to, proteins (e.g. Transferrin), oligopeptides (e.g., cyclic and acyclic RGD-containing oligopeptides), antibodies (monoclonal and polyclonal antibodies, e.g. IgG, IgA, IgM, IgD, IgE antibodies), sugars/carbohydrates (e.g., monosaccharides and/or oligosaccharides (mannose, mannose-6-phosphate, galactose, and the like)), vitamins (e.g., folate), or other small biomolecules. In some embodiments, a targeting moiety is a steroid molecule (e.g., bile acids including cholic acid, deoxycholic acid, dehydrocholic acid, cortisone; digoxigenin; testosterone; cholesterol; cationic steroids such as cortisone having a trimethylaminomethyl hydrazide group attached via a double bond at the 3-position of the cortisone ring, etc.). In some embodiments, a targeting moiety is a lipophilic molecule (e.g., alicyclic hydrocarbons, saturated and unsaturated fatty acids, waxes, terpenes, and polyalicyclic hydrocarbons such as adamantine and buckminsterfullerenes). In some embodiments, a lipophilic molecule is a terpenoid such as vitamin A, retinoic acid, retinal, or dehydroretinal. In some embodiments, a targeting moiety is a peptide.

In some embodiments, a P-modification moiety is a targeting moiety having the structure of -X-L-R¹wherein each of X, L, and R¹is independently as described in the present disclosure.

In some embodiments, a P-modification moiety facilitates cell specific delivery.

In some embodiments, a P-modification moiety may perform one or more than one functions. For instance, in some embodiments, a P-modification moiety acts as a PK enhancer and a targeting ligand. In some embodiments, a P-modification moiety acts as a pro-drug and an endosomal escape agent. Numerous other such combinations are possible and are included in the present disclosure.

Certain Examples of Oligonucleotides and Compostions

In some embodiments, the present disclosure provides oligonucleotides and/or oligonucleotide compositions that are useful for various purposes. e.g., modulating skipping, reducing levels of transcripts, improving levels of beneficial proteins, treating conditions, diseases and disorders, etc. In some embodiments, the present disclosure provides oligonucleotide compositions with improved properties, e.g., increased activities, reduced toxicities, etc. Among other things, oligonucleotides of the present disclosure comprise chemical modifications, stereochemistry, and/or combinations thereof which can improve various properties and activities of oligonucleotides. Non-limiting examples are listed in Table A1. In some embodiments, an oligonucleotide type is a type as defined by the base sequence, pattern of backbone linkages, pattern of backbone chiral centers and pattern of backbone phosphorus modifications of an oligonucleotide in Table A1, wherein the oligonucleotide comprises at least one chirally controlled internucleotidic linkage (at least one R or S in “Stereochemistry/Linkage”). In some embodiments, a plurality of oligonucleotides of a particular oligonucleotide type is a plurality of an oligonucleotide in Table A1 (e.g., a plurality of oligonucleotides is a plurality of WV-1095). In some embodiments, a plurality of oligonucleotides in a chirally controlled oligonucleotide composition is a plurality of an oligonucleotide in Table A1 (e.g., a plurality of oligonucleotides is a plurality of WV-1095), wherein the oligonucleotide comprises at least one chirally controlled internucleotidic linkage (at least one R or S in “Stereochemistry/Linkage”).

Table A1 lists non-limiting examples of DMD oligonucleotides. All of the oligonucleotides in Table A1 are DMD oligonucleotides, except for WV-12915 WV-12914 WV-12913, WV-12912, WV-12911, WV-12910, WV-12909, WV-12908, WV-12907, WV-12906. WV-12905. WV-12904, WV-15887, WV-24100, WV-24101, WV-24102, WV-24103, WV-24104, WV-24105, WV-24106, WV-24107, WV-24108, WV-24109, WV-24110, WV-XBD108, WV-XBD 109, WV-XBD 110, WV-XKCD108, WV-XKCD 109, WV-XKCD 110, which all target Malat-1, which is a gene target different than DMD.

In some embodiments, the present disclosure pertains to an oligonucleotide or oligonucleotide composition, wherein the base sequence of the oligonucleotide comprises at least 15 contiguous bases, with 1-3 mismatches, of the base sequence of a DMD oligonucleotide disclosed in Table A1. In some embodiments, the present disclosure pertains to an oligonucleotide or oligonucleotide composition, wherein the base sequence of the oligonucleotide comprises at least 15 contiguous bases of the base sequence of a DMD oligonucleotide disclosed in Table A1. In some embodiments, the present disclosure pertains to an oligonucleotide or oligonucleotide composition, wherein the base sequence of the oligonucleotide comprises the base sequence of a DMD oligonucleotide disclosed in Table A1. In some embodiments, the present disclosure pertains to an oligonucleotide or oligonucleotide composition, wherein the base sequence of the oligonucleotide is the base sequence of a DMD oligonucleotide disclosed in Table A1.

In some embodiments, the present disclosure pertains to an oligonucleotide or oligonucleotide composition, wherein the base sequence of the oligonucleotide comprises at least 15 contiguous bases, with 1-3 mismatches, of the base sequence of a DMD oligonucleotide disclosed in Table A1, or wherein the base sequence of the oligonucleotide comprises at least 15 contiguous bases of the base sequence of a DMD oligonucleotide disclosed in Table A1, or wherein the base sequence of the oligonucleotide comprises the base sequence of a DMD oligonucleotide disclosed in Table A1, or wherein the base sequence of the oligonucleotide is the base sequence of a DMD oligonucleotide disclosed in Table A1; and wherein the oligonucleotide is stereorandom (e.g., not chirally controlled), or the oligonucleotide is chirally controlled, and/or the oligonucleotide comprises at least one internucleotidic linkage which is chirally controlled, and/or the oligonucleotide optionally comprises a sugar modification which is a LNA, and/or the oligonucleotide comprises a sugar which is a natural deoxyribose, a 2′-OMe or a 2′-MOE. In some embodiments, the present disclosure pertains to an oligonucleotide capable of mediating skipping of a DMD exon, wherein the oligonucleotide comprises at least one LNA.

In the following table ID indicates identification or oligonucleotide number; and Description indicates the modified sequence.

TABLE A1

Example Oligonucleotides

ID
Description
Naked Base Sequence
Linkage / Stereochemistry

ONT
mU*S mC*S mA*S mA*S mG*S mG*S mA*S mA*S mG*S mA*S mU*S
UCAAGGAAGAUGGCA
SSSSSSSSSSSSSSS

-395
mG*S mG*S mC*S mA*S mU*S mU*S mU*S mC*S mU
UUUCU
SSSS

WV-
G * G * C * C * A * A * A * C * C * T * C * G * G * C * T * T * A * C * C * T
GGCCAAACCTCGGCT
XXXXX XXXXX

1093

TACCT
XXXXX XXXX

WV-
mG mG mC mC mA mA mA mC mC mU mC mG mG mC mU mU mA mC mC
GGCCAAACCUCGGCU
OOOOO OOOOO

1094
mU
UACCU
OOOOOOOOO

WV-
G * RG * RC * RC * SfA * SfA * SfA * RC * RC * fG * RC * RG * RG *
GGCCAAACCUCGGCU
RRRRRRRRRRRRR

1095
RC * fG * fG * SfA * RC * RC * fG
TACCT
RRRRRR

WV-
G * SG * SC * SC * SA * SA * SA * SC * SC * SfU * SC * SG * SG * SC * SfU *
GGCCAAACCTCGGCT
SSSSSSSSSSSSSSS

1096
SfU * SA * SC * SC * SfU
TACCT
SSSS

WV-
G * SG * SC * SC * SA * S mA mA mC mC mU mC mG mG mCT * SfU * SA *
GGCCAAACCUCGGCT
SSSSSOOOOOOOO

1097
Sc * SC * SfU
TACCT
OSSSSS

WV-
mG mG mC mCA * SA * SA * S mCC * SfU * SC * SG * S mGC * SfU * SfU * S
GGCCAAACCUCGGCT
OOOOSSSOSSSSOS

1098
mA mC mC mU
TACCU
SSOOO

WV-
G * S mGC * S mCA * S mAA * S mCC * S mUC * S mGG * S mCT * S mUA
GGCCAAACCUCGGCT
SOSOSOSOSOSOS

1099
* S mCC * S mU
UACCU
OSOSOS

WV-
mGG * S mCC * S mAA * S mAC * S mCT * S mCG * S mGC * S mUT * S
GGCCAAACCTCGGCU
OSOSOSOSOSOSO

1100
mAC * S mC mU
TACCU
SOSOSO

WV-
G * SG * S mC mCA * SA * S mA mCC * SfU * SC * S mG mGC * SfU * S mU
GGCCAAACCTCGGCT
SSOOSSOOSSSOOS

1101
mAC * SC * S mU
UACCU
SOOSS

WV-
G * SG * SC * S mC mA mAA * SC * S mC mU mCG * SG * S mC mU mUA *
GGCCAAACCUCGGCU
SSSOOOSSOOOSS

1102
SC * SC * S mU
UACCU
OOOSSS

WV-
G * SG * SC * SC * S mA mA mA mCC * SfU * SC * S mG mG mC mUT * SA
GGCCAAACCTCGGCU
SSSSOOOOSSSOO

1103
* SC * SC * S mU
TACCU
OOSSSS

WV-
G * SG * SC * S mCA * SA * SA * S mCC * SfU * SC * S mGG * SC * SfU * S
GGCCAAACCTCGGCT
SSSOSSSOSSSOSS

1104
mUA * SC * SC * S mU
UACCU
SOSSS

WV-
mG mG mC mCA * SA * SA * SC * SC * S mU mC mG mG mCT * SfU * SA *
GGCCAAACCUCGGCT
OOOOSSSSSOOOO

1105
SC * SC * S mU
TACCU
OSSSSS

WV-
G * SG * S mC mC mA mA mA mC mC mUC * S mG mGC * S mUT * SA *
GGCCAAACCUCGGCU
SSOOOOOOOOSO

1106
SC * SC * S mU
TACCU
OSOSSSS

WV-
T * C * A * A * G * G * A * A * G * A * T * G * G * C * A * T * T * T * C * T
TCAAGGAAGATGGCA
XXXXX XXXXX

1107

TTTCT
XXXXX XXXX

WV-
mU mC mA mA mG mG mA mA mG mA mU mG mG mC mA mU mU mU
UCAAGGAAGAU
OOOOO OOOOO O

1108
mC mU
GGCAUUUCU
OOOOOOOO

WV-
T * RC * SfA * SfA * RG * RG * SfA * SfA * RG * SfA * fG * RG * RG *
TCAAGGAAGATGGCA
RRRRRRRRRRRRR

1109
RC * SfA * fG * fG * fG* RC * fG
TTTCT
RRRRRR

WV-
T * SC * SA * SA * SG * SG * SA * SA * SG * SA * SfU * SG * SG * SC * SA *
TCAAGGAAGATGGCA
SSSSSSSSSSSSSSS

1110
SfU * SfU * SfU * SC * SfU
TTTCT
SSSS

WV-
T * SC * SA * SA * SG * S mG mA mA mG mA mU mG mG mCA * SfU * SfU *
TCAAGGAAGAUGGCA
SSSSSOOOOOOOO

1111
SfU * SC * SfU
TTTCT
OSSSSS

WV-
mU mC mA mAG * SG * SA * S mAG * SA * SfU * SG * S mGC * SA * SfU * S
UCAAGGAAGATGGCA
OOOOSSSOSSSSOS

1112
mU
mU mC mU
UUUCUSSOOO

WV-
T * S mCA * S mAG * S mGA * S mAG * S mAT * S mGG * S mCA * S mUT
TCAAGGAAGATGGCA
SOSOSOSOSOSOS

1113
* S mUC * S mU
UTUCU
OSOSOS

WV-
mUC * S mAA * S mGG * S mAA * S mGA * S mUG * S mGC * S mAT * S
UCAAGGAAGAUGGCA
OSOSOSOSOSOSO

1114
mUT * S mC mU
UUTCU
SOSOSO

WV-
T * SC * S mA mAG * SG * S mA mAG * SA * SfU * S mG mGC * SA * S mU
TCAAGGAAGATGGCA
SSOOSSOOSSSOOS

1115
mUT * SC * S mU
UUTCU
SOOSS

WV-
T * SC * SA * S mA mG mGA * SA * S mG mA mUG * SG * S mC mA mUT *
TCAAGGAAGAUGGCA
SSSOOOSSOOOSS

1116
SfU * SC * S mU
UTTCU
OOOSSS

WV-
T * SC * SA * SA * S mG mG mA mAG * SA * SfU * S mG mG mC mAT * SfU
TCAAGGAAGATGGCA
SSSSOOOOSSSOO

1117
* SfU * SC * S mU
UTTCU
OOSSSS

WV-
T * SC * SA * S mAG * SG * SA * S mAG * SA * SfU * S mGG * SC * SA * S
TCAAGGAAGATGGCA
SSSOSSSOSSSOSS

1118
mUT * SfU * SC * S mU
UTTCU
SOSSS

WV-
mU mC mA mAG * SG * SA * SA * SG * S mA mU mG mG mCA * SfU * SfU *
UCAAGGAAGAUGGCA
OOOOSSSSSOOOO

1119
SfU * SC * S mU
TTTCU
OSSSSS

WV-
T * SC * S mA mA mG mG mA mA mG mAT * S mG mGC * S mAT * SfU * SfU
TCAAGGAAGATGGCA
SSOOOOOOOOSO

1120
* SC * S mU
TTTCU
OSOSSSS

WV-
G * G * C * C * A * mA mA mC mC mU mC mG mG mCT * T * A * C * C * T
GGCCAAACCUCGGCT
XXXXXOOOOOOO

1121

TACCT
OOXXXXX

WV-
mG mG mC mCA * A * A * mCC * T * C * G * mGC * T * T * mA mC mC
GGCCAAACCTCGGCT
OOOOXXXOXXXX

1122
mU
TACCU
OXXXOOO

WV-
G * mGC * mCA * mAA * mCC * mUC * mGG * mCT * mUA * mCC *
GGCCAAACCUCGGCT
XOXOXOXOXOXO

1123
mU
UACCU
XOXOXOX

WV-
mGG * mCC * mAA * mAC * mCT * mCG * mGC * mUT * mAC * mC
GGCCAAACCTCGGCU
OXOXOXOXOXOX

1124
mU
TACCU
OXOXOXO

WV-
G * G * mC mCA * A * mA mC mCT * C * mG mGC * T * mU mAC * C *
GGCCAAACCTCGGCT
XXOOXXOOOXXO

1125
mU
UACCU
OXXOOXX

WV-
G * G * C * mC mA mAA * C * mC mU mCG * G * mC mU mUA * C * C *
GGCCAAACCUCGGCU
XXXOOOXXOOOX

1126
mU
UACCU
XOOOXXX

WV-
G * G * C * C * mA mA mA mCC * T * C * mG mG mC mUT * A * C * C *
GGCCAAACCTCGGCU
XXXXOOOOXXXO

1127
mU
TACCU
OOOXXXX

WV-
G * G * C * mCA * A * A * mCC * T * C * mGG * C * T * mUA * C * C *
GGCCAAACCTCGGCT
XXXOXXXOXXXO

1128
mU
UACCU
XXXOXXX

WV-
mG mG mC mCA * A * A * C * C * mU mC mG mG mCT * T * A * C * C *
GGCCAAACCUCGGCT
OOOOXXXXXOOO

1129
mU
TACCU
OOXXXXX

WV-
G * G * mC mC mA mA mA mC mC mUC * mG mGC * mUT * A * C * C *
GGCCAAACCUCGGCU
XXOOOOOOOOXO

1130
mU
TACCU
OXOXXXX

WV-
T * C * A * A * G * mG mA mA mG mA mU mG mG mCA * T * T * T * C * T
TCAAGGAAGAUGGCA
XXXXXOOOOOOO

1131

TTTCT
OOXXXXX

WV-
mU mC mA mAG * G * A * mAG * A * T * G * mGC * A * T * mU mU mC
UCAAGGAAGATGGCA
OOOOXXXOXXXX

1132
mU
UUUCU
OXXXOOO

WV-
T * mCA * mAG * mGA * mAG * mAT * mGG * mCA * mUT * mUC *
TCAAGGAAGATGGCA
XOXOXOXOXOXO

1133
mU
UTUCU
XOXOXOX

WV-
mUC * mAA * mGG * mAA * mGA * mUG * mGC * mAT * mUT * mC
UCAAGGAAGAUGGCA
OXOXOXOXOXOX

1134
mU
UUTCU
OXOXOXO

WV-
T * C * mA mAG * G * mA mAG * A * T * mG mGC * A * mU mUT * C *
TCAAGGAAGATGGCA
XXOOXXOOXXXO

1135
mU
UUTCU
OXXOOXX

WV-
T * C * A * mA mG mGA * A * mG mA mUG * G * mC mA mUT * T * C *
TCAAGGAAGAUGGCA
XXXOOOXXOOOX

1136
mU
UTTCU
XOOOXXX

WV-
T * C * A * A * mG mG mA mAG * A * T * mG mG mC mAT * T * T * C *
TCAAGGAAGATGGCA
XXXXOOOOXXXO

1137
mU
TTTCU
OOOXXXX

WV-
T * C * A * mAG * G * A * mAG * A * T * mGG * C * A * mUT * T * C *
TCAAGGAAGATGGCA
XXXOXXXOXXXO

1138
mU
UTTCU
XXXOXXX

WV-
mU mC mA mAG * G * A * A * G * mA mU mG mG mCA * T * T * T * C *
UCAAGGAAGAUGGCA
OOOOXXXXXOOO

1139
mU
TTTCU
OOXXXXX

WV-
T * C * mA mA mG mG mA mA mG mAT * mG mGC * mAT * T * T * C *
TCAAGGAAGATGGCA
XXOOOOOOOOXO

1140
mU
TTTCU
OXOXXXX

WV-
mG * mG * mC * mC * mA * mA mA mC mC mU mC mG mG mC mU *
GGCCAAACCUCGGCU
XXXXXOOOOOOO

1141
mU * mA * mC * mC * mU
UACCU
OOXXXXX

WV-
mG mG mC mC mA * mA * mA * mC mC * mU * mC * mG * mG mC *
GGCCAAACCUCGGCU
OOOOXXXOXXXX

1142
mU * mU * mA mC mC mU
UACCU
OXXXOOO

WV-
mG * mG mC * mC mA * mA mA * mC mC * mU mC * mG mG * mC mU
GGCCAAACCUCGGCU
XOXOXOXOXOXO

1143
* mU mA * mC mC * mU
UACCU
XOXOXOX

WV-
mG mG * mC mC * mA mA * mA mC * mC mU * mC mG * mG mC * mU
GGCCAAACCUCGGCU
OXOXOXOXOXOX

1144
mU * mA mC * mC mU
UACCU
OXOXOXO

WV-
mG * mG * mC mC mA * mA * mA mC mC mU * mC * mG mG mC * mU
GGCCAAACCUCGGCU
XXOOXXOOOXXO

1145
* mU mA mC * mC * mU
UACCU
OXXOOXX

WV-
mG * mG * mC * mC mA mA mA * mC * mC mU mC mG * mG * mC mU
GGCCAAACCUCGGCU
XXXOOOXXOOOX

1146
mU mA * mC * mC * mU
UACCU
XOOOXXX

WV-
mG * mG * mC * mC * mA mA mA mC mC * mU * mC * mG mG mC mU
GGCCAAACCUCGGCU
XXXXOOOOXXXO

1147
mU * mA * mC * mC * mU
UACCU
OOOXXXX

WV-
mG * mG * mC * mC mA * mA * mA * mC mC * mU * mC * mG mG *
GGCCAAACCUCGGCU
XXXOXXXOXXXO

1148
mC * mU * mU mA * mC * mC * mU
UACCU
XXXOXXX

WV-
mG mG mC mC mA * mA * mA * mC * mC * mU mC mG mG mC mU *
GGCCAAACCUCGGCU
OOOOXXXXXOOO

1149
mU * mA * mC * mC * mU
UACCU
OOXXXXX

WV-
mG * mG * mC mC mA mA mA mC mC mU mC * mG mG mC * mU mU *
GGCCAAACCUCGGCU
XXOOOOOOOOXO

1150
mA * mC * mC * mU
UACCU
OXOXXXX

WV-
mU * mC * mA * mA * mG * mG mA mA mG mA mU mG mG mC mA *
UCAAGGAAGAUGGCA
XXXXXOOOOOOO

1151
mU * mU * mU * mC * mU
UUUCU
OOXXXXX

WV-
mU mC mA mA mG * mG * mA * mA mG * mA * mU * mG * mG mC *
UCAAGGAAGAUGGCA
OOOOXXXOXXXX

1152
mA * mU * mU mU mC mU
UUUCU
OXXXOOO

WV-
mU * mC mA * mA mG * mG mA * mA mG * mA mU * mG mG * mC
UCAAGGAAGAUGGCA
XOXOXOXOXOXO

1153
mA * mU mU * mU mC * mU
UUUCU
XOXOXOX

WV-
mU mC * mA mA * mG mG * mA mA * mG mA * mU mG * mG mC *
UCAAGGAAGAUGGCA
OXOXOXOXOXOX

1154
mA mU * mU mU * mC mU
UUUCU
OXOXOXO

WV-
mU * mC * mA mA mG * mG * mA mA mG * mA * mU * mG mG mC *
UCAAGGAAGAUGGCA
XXOOXXOOXXXO

1155
mA * mU mU mU * mC * mU
UUUCU
OXXOOXX

WV-
mU * mC * mA * mA mG mG mA * mA * mG mA mU mG * mG * mC
UCAAGGAAGAUGGCA
XXXOOOXXOOOX

1156
mA mU mU * mU * mC * mU
UUUCU
XOOOXXX

WV-
mU * mC * mA * mA * mG mG mA mA mG * mA * mU * mG mG mC
UCAAGGAAGAUGGCA
XXXXOOOOXXXO

1157
mA mU * mU * mU * mC * mU
UUUCU
OOOXXXX

WV-
mU * mC * mA * mA mG * mG * mA * mA mG * mA * mU * mG mG *
UCAAGGAAGAUGGCA
XXXOXXXOXXXO

1158
mC * mA * mU mU * mU * mC * mU
UUUCU
XXXOXXX

WV-
mU mC mA mA mG * mG * mA * mA * mG * mA mU mG mG mC mA *
UCAAGGAAGAUGGCA
OOOOXXXXXOOO

1159
mU * mU * mU * mC * mU
UUUCU
OOXXXXX

WV-
mU * mC * mA mA mG mG mA mA mG mA mU * mG mG mC * mA mU *
UCAAGGAAGAUGGCA
XXOOOOOOOOXO

1160
mU * mU * mC * mU
UUUCU
OXOXXXX

WV-
fG * fG * fC * fC * fA * fA * fA * fC * fC * fU * fC * fG * fG * fC * fU * fU *
GGCCAAACCUCGGCU
XXXXX XXXXX

1678
fA * fC * fC * fU
UACCU
XXXXX XXXX

WV-
mG * mG * fC * fC * mA * mA * mA * fC * fC * fU * fC * mG * mG * fC
GGCCAAACCUCGGCU
XXXXX XXXXX

1679
* fU * fU * mA * fC * fC * fU
UACCU
XXXXX XXXX

WV-
fG * fG * mC * mC * fA * fA * fA * mC * mC * mU * mC * fG * fG * mC
GGCCAAACCUCGGCU
XXXXX XXXXX

1680
* mU * mU * fA * mC * mC * mU
UACCU
XXXXX XXXX

WV-
mG * fG * mC * fC * mA * fA * mA * fC * mC * fU * mC * fG * mG * fC
GGCCAAACCUCGGCU
XXXXX XXXXX

1681
* mU * fU * mA * fC * mC * fU
UACCU
XXXXX XXXX

WV-
mG * mG * mC * mC * mA * mA * fA * fC * fC * fU * fC * fG * fG * fC *
GGCCAAACCUCGGCU
XXXXX XXXXX

1682
mU * mU * mA * mC * mC * mU
UACCU
XXXXX XXXX

WV-
fG * fG * fC * fC * fA * fA * mA * mC * mC * mU * mC * mG * mG * mC
GGCCAAACCUCGGCU
XXXXX XXXXX

1683
* fU * fU * fA * fC * fC * fU
UACCU
XXXXX XXXX

WV-
fG * fU * fC * fC * mA * mA * mA * fC * fC * mU * fC * fG * fG * fC * mU
GGCCAAACCUCGGCU
XXXXX XXXXX

1684
* mU * mA * fC * fC * mU
UACCU
XXXXX XXXX

WV-
mG * mG * mC * mC * fA * fA * fA * mC * mC * fu * mC * mG * mG *
GGCCAAACCUCGGCU
XXXXX XXXXX

1685
mC * fU * fU * fA * mC * mC * fU
UACCU
XXXXX XXXX

WV-
rA rG rA rA rA rU rG rC rC rA rU rC rU rU rC rC rU rU rG rA
AGAAAUGCCAUCUUC
OOOOO OOOOO

1687

CUUGA
OOOOOOOOO

WV-
fU * fC * fA * fA * fG * fG * fA * fA * fG * fA * fU * fG * fG * fC * fA * fU *
UCAAGGAAGAUGGCA
XXXXX XXXXX

1709
fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * mA * mA * mG * mG * mA * mA * mG * mA * fU * mG * mG
UCAAGGAAGAUGGCA
XXXXX XXXXX

1710
* fC * mA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * fA * fA * fG * fG * fA * fA * fG * fA * mU * fG * fG * mC * fA
UCAAGGAAGAUGGCA
XXXXX XXXXX

1711
* mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
mU * fC * mA * fA * mG * fG * mA * fA * mG * fA * mU * fG * mG * fC
UCAAGGAAGAUGGCA
XXXXX XXXXX

1712
* mA * fU * mU * fU * mC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * fA * fA * fG * fA * fU * fG * fG * fC *
UCAAGGAAGAUGGCA
XXXXX XXXXX

1713
mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * mA * mA * mG * mA * mU * mG * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

1714
mC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * fC * mA * mA * fG * fG * mA * mA * fG * mA * mU * fG * fG * fC
UCAAGGAAGAUGGCA
XXXXX XXXXX

1715
* mA * mU * mU * mU * fC * mU
UUUCU
XXXXX XXXX

WV-
fU * mC * fA * fA * mG * mG * fA * fA * mG * fA* fU * mG * mG * mC
UCAAGGAAGAUGGCA
XXXXX XXXXX

1716
* fA * fU * fU * fU * mC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * mG * mA * mA * mG * mA * mU * mG * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2095
mC * mA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * mG * mG * mA * mA * mG * mA * mU * mG * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2096
mC * mA * mU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * mA * mG * mG * mA * mA * mG * mA * mU * mG * mG
UCAAGGAAGAUGGCA
XXXXX XXXXX

2097
* mC * mA * mU * mU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * mA * mA * mG * mG * mA * mA * mG * mA * mU * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2098
mG * mC * mA * mU * mU * mU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * mC * mA * mA * mG * mG * mA * mA * mG * mA * mU * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2099
mG * mC * mA * mU * mU * mU * mC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG mA * mA * mG * mA * mU * mG * mG *
UCAAGGAAGAUGGCA
XXXXXOXXXXXX

2100
mCfA * fU * fU * fU * fC * fU
UUUCU
XOXXXXX

WV-
fU * fC * fA * fA * fGfG mA * mA * mG * mA * mU * mG * mG *
UCAAGGAAGAUGGCA
XXXXOOXXXXXX

2101
mCfAfU * fU * fU * fC * fU
UUUCU
XOOXXXX

WV-
fU * fC * fA * fAfGfG mA * mA * mG * mA * mU * mG * mG *
UCAAGGAAGAUGGCA
XXXOOOXXXXXX

2102
mCfAfUfU * fU * fC * fU
UUUCU
XOOOXXX

WV-
fU * fC * fAfAfGfG mA * mA * mG * mA * mU * mG * mG *
UCAAGGAAGAUGGCA
XXOOOOXXXXXX

2103
mCfAfUfUfU * fC * fU
UUUCU
XOOOOXX

WV-
fU * fCfAfAfGfG mA * mA * mG * mA * mU * mG * mG *
UCAAGGAAGAUGGCA
XOOOOOXXXXXX

2104
mCfAfUfUfUfC * fU
UUUCU
XOOOOOX

WV-
fUfCfAfAfGfG mA * mA * mG * mA * mU * mG * mG *
UCAAGGAAGAUGGCA
OOOOOOXXXXXX

2105
mCfAfUfUfUfCfU
UUUCU
XOOOOOO

WV-
fU * fC * fA * fA * fG * fG * fA * fA * fG * fA * mU * mG * mG *mC *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2106
mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * fU * fG * fG
UCAAGGAAGAUGGCA
XXXXX XXXXX

2107
* fC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * mA * mA * mG * mA * mU * mG * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2108
mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * mU * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2109
mG * mC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mC * mU * mC * mC * mA * mA * mC * mA * mU * mC * mA * mA *
CUCCAACAUCAAGGA
XXXXX XXXXX

2165
mG * mG * mA * mA * mG * mA * mU * mG * mG * mC * mA * mU *
AG
XXXXX XXXXX

mU * mU * mC * mU * mA * mG
AUGGCAUUUCUAG
XXXXX XXXX

WV-
mA * mC * mC * mA * mG * mA * mG * mU * mA * mA * mC * mA *
ACCAGAGUAACAG
XXXXX XXXXX

2179
mG * mU * mC * mU * mG * mA * mG * mU * mA * mG * mG * mA *
UCUGAGUAGGAG
XXXXX XXXXX

mG

XXXX

WV-
mC * mA * mC * mC * mA * mG * mA * mG * mU * mA * mA * mC *
CACCAGAGUAACAG
XXXXX XXXXX

2180
mA * mG * mU * mC * mU * mG * mA * mG * mU * mA * mG * mG *
UCUGAGUAGGA
XXXXX XXXXX

mA

XXXX

WV-
mU * mC * mA * mC * mC * mA * mG * mA * mG * mU * mA * mA *
UCACCAGAGUAACA
XXXXX XXXXX

2181
mC * mA * mG * mU * mC * mU * mG * mA * mG * mU * mA * mG *
GUCUGAGUAGG
XXXXX XXXXX

mG

XXXX

WV-
mG * mU * mC * mA * mC * mC * mA * mG * mA * mG * mU * mA *
GUCACCAGAGUAAC
XXXXX XXXXX

2182
mA * mC * mA * mG * mU * mC * mU * mG * mA * mG * mU * mA *
AGUCUGAGUAG
XXXXX XXXXX

mG

XXXX

WV-
mG * mU * mU * mG * mU * mG * mU * mC * mA * mC * mC * mA *
GUUGUGUCACCAGA
XXXXX XXXXX

2183
mG * mA * mG * mU * mA * mA * mC * mA * mG * mU * mC * mU *
GUAACAGUCUG
XXXXX XXXXX

mG

XXXX

WV-
mG * mG * mU * mU * mG * mU * mG * mU * mC * mA * mC * mC *
GGUUGUGUCACCAG
XXXXX XXXXX

2184
mA * mG * mA * mG * mU * mA * mA * mC * mA * mG * mU * mC *
AGUAACAGUCU
XXXXX XXXXX

mU

XXXX

WV-
mA * mG * mG * mU * mU * mG * mU * mG * mU * mC * mA * mC *
AGGUUGUGUCAC
XXXXX XXXXX

2185
mC * mA * mG * mA * mG * mU * mA * mA * mC * mA * mG * mU *
CAGAGUAACAGUC
XXXXX XXXXX

mC

XXXX

WV-
mC * mA * mG * mG * mU * mU * mG * mU * mG * mU * mC * mA *
CAGGUUGUGUCA
XXXXX XXXXX

2186
mC * mC * mA * mG * mA * mG * mU * mA * mA * mC * mA * mG *
CCAGAGUAACAGU
XXXXX XXXXX

mU

XXXX

WV-
mA * mC * mA * mG * mG * mU * mU * mG * mU * mG * mU * mC *
ACAGGUUGUGUC
XXXXX XXXXX

2187
mA * mC * mC * mA * mG * mA * mG * mU * mA * mA * mC * mA *
ACCAGAGUAACAG
XXXXX XXXXX

mG

XXXX

WV-
mC * mC * mA * mC * mA * mG * mG * mU * mU * mG * mU * mG *
CCACAGGUUGUG
XXXXX XXXXX

2188
mU * mC * mA * mC * mC * mA * mG * mA * mG * mU * mA * mA *
UCACCAGAGUAAC
XXXXX XXXXX

mC

XXXX

WV-
mA * mC * mC * mA * mC * mA * mG * mG * mU * mU * mG * mU *
ACCACAGGUUGUG
XXXXX XXXXX

2189
mG * mU * mC * mA * mC * mC * mA * mG * mA * mG * mU * mA *
UCACCAGAGUAA
XXXXX XXXXX

mA

XXXX

WV-
mA * mA * mC * mC * mA * mC * mA * mG * mG * mU * mU * mG *
AACCACAGGUUGU
XXXXX XXXXX

2190
mU * mG * mU * mC * mA * mC * mC * mA * mG * mA * mG * mU *
GUCACCAGAGUA
XXXXX XXXXX

mA

XXXX

WV-
mU * mA * mA * mC * mC * mA * mC * mA * mG * mG * mU * mU *
UAACCACAGGUUG
XXXXX XXXXX

2191
mG * mU * mG * mU * mC * mA * mC * mC * mA * mG * mA * mG *
UGUCACCAGAGU
XXXXX XXXXX

mU

XXXX

WV-
mG * mU * mA * mA * mC * mC * mA * mC * mA * mG * mG * mU *
GUAACCACAGGUU
XXXXX XXXXX

2192
mU * mG * mU * mG * mU * mC * mA * mC * mC * mA * mG * mA *
GUGUCACCAGAG
XXXXX XXXXX

mG

XXXX

WV-
mA * mG * mU * mA * mA * mC * mC * mA * mC * mA * mG * mG *
AGUAACCACAGGU
XXXXX XXXXX

2193
mU * mU * mG * mU * mG * mU * mC * mA * mC * mC * mA * mG *
UGUGUCACCAGA
XXXXX XXXXX

mA

XXXX

WV-
mU * mA * mG * mU * mA * mA * mC * mC * mA * mC * mA * mG *
UAGUAACCACAGG
XXXXX XXXXX

2194
mG * mU * mU * mG * mU * mG * mU * mC * mA * mC * mC * mA *
UUGUGUCACCAG
XXXXX XXXXX

mG

XXXX

WV-
mU * mU * mA * mG * mU * mA * mA * mC * mC * mA * mC * mA *
UUAGUAACCACAG
XXXXX XXXXX

2195
mG * mG * mU * mU * mG * mU * mG * mU * mC * mA * mC * mC *
GUUGUGUCACCA
XXXXX XXXXX

mA

XXXX

WV-
mC * mU * mU * mA * mG * mU * mA * mA * mC * mC * mA * mC *
CUUAGUAACCACA
XXXXX XXXXX

2196
mA * mG * mG * mU * mU * mG * mU * mG * mU * mC * mA * mC *
GGUUGUGUCACC
XXXXX XXXXX

mC

XXXX

WV-
mC * mC * mU * mU * mA * mG * mU * mA * mA * mC * mC * mA *
CCUUAGUAACCACA
XXXXX XXXXX

2197
mC * mA * mG * mG * mU * mU * mG * mU * mG * mU * mC * mA *
GGUUGUGUCAC
XXXXX XXXXX

mC

XXXX

WV-
mU * mC * mC * mU * mU * mA * mG * mU * mA * mA * mC * mC *
UCCUUAGUAACCAC
XXXXX XXXXX

2198
mA * mC * mA * mG * mG * mU * mU * mG * mU * mG * mU * mC *
AGGUUGUGUCA
XXXXX XXXXX

mA

XXXX

WV-
mG * mU * mU * mU * mC * mC * mU * mU * mA * mG * mU * mA *
GUUUCCUUAGUAAC
XXXXX XXXXX

2199
mA * mC * mC * mA * mC * mA * mG * mG * mU * mU * mG * mU *
CACAGGUUGUG
XXXXX XXXXX

mG

XXXX

WV-
mA * mG * mU * mU * mU * mC * mC * mU * mU * mA * mG * mU *
AGUUUCCUUAGUAA
XXXXX XXXXX

2200
mA * mA * mC * mC * mA * mU * mA * mG * mG * mU * mU * mG *
CCACAGGUUGU
XXXXX XXXXX

mU

XXXX

WV-
mC * mA * mG * mU * mU * mU * mC * mC * mU * mU * mA * mG *
CAGUUUCCUUAGU
XXXXX XXXXX

2201
mU * mA * mA * mC * mC * mA * mC * mA * mG * mG * mU * mU *
AACCACAGGUUG
XXXXX XXXXX

mG

XXXX

WV-
mG * mC * mA * mG * mU * mU * mU * mC * mC * mU * mU * mA *
GCAGUUUCCUUAGU
XXXXX XXXXX

2202
mG * mU * mA * mA * mC * mC * mA * mC * mA * mG * mG * mU *
AACCACAGGUU
XXXXX XXXXX

mU

XXXX

WV-
mG * mG * mC * mA * mG * mU * mU * mU * mC * mC *mU * mU *
GGCAGUUUCCUUAG
XXXXX XXXXX

2203
mA * mG * mU * mA * mA * mC * mC * mA * mC * mA * mG * mG *
UAACCACAGGU
XXXXX XXXXX

mU

XXXX

WV-
mU * mG * mG * mC * mA * mG * mU * mU * mU * mC * mC * mU *
UGGCAGUUUCCUUA
XXXXX XXXXX

2204
mU * mA * mG * mU * mA * mA * mC * mC * mA * mC * mA * mG *
GUAACCACAGG
XXXXX XXXXX

mG

XXXX

WV-
mA * mU * mG * mG * mC * mA * mG * mU * mU * mU * mC * mC *
AUGGCAGUUUCCUU
XXXXX XXXXX

2205
mU * mU * mA * mG * mU * mA * mA * mC * mC * mA * mC * mA *
AGUAACCACAG
XXXXX XXXXX

mG

XXXX

WV-
mA * mG * mA * mU * mG * mG * mC * mA * mG * mU * mU * mU *
AGAUGGCAGUUUCCU
XXXXX XXXXX

2206
mC * mC * mU * mU * mA * mG * mU * mA * mA * mC * mC * mA *
UAGUAACCAC
XXXXX XXXXX

mC

XXXX

WV-
mG * mA * mG * mA * mU * mG * mG * mC * mA * mG * mU * mU *
GAGAUGGCAGUUUCC
XXXXX XXXXX

2207
mU * mC * mC * mU * mU * mA * mG * mU * mA * mA * mC * mC *
UUAGUAACCA
XXXXX XXXXX

mA

XXXX

WV-
mG * mG * mA * mG * mA * mU * mG * mG * mC * mA * mG * mU *
GGAGAUGGCAGUUUC
XXXXX XXXXX

2208
mU * mU * mC * mC * mU * mU * mA * mG * mU * mA * mA * mC *
CUUAGUAACC
XXXXX XXXXX

mC

XXXX

WV-
mU * mG * mG * mA * mG * mA * mU * mG * mG * mC * mA * mG *
UGGAGAUGGCAGUUU
XXXXX XXXXX

2209
mU * mU * mU * mC * mC * mU * mU * mA * mG * mU * mA * mA *
CCUUAGUAAC
XXXXX XXXXX

mC

XXXX

WV-
mU * mU * mG * mG * mA * mG * mA * mU * mG * mG * mC * mA *
UUGGAGAUGGCAGUU
XXXXX XXXXX

2210
mG * mU * mU * mU * mC * mC * mU * mU * mA * mG * mU * mA *
UCCUUAGUAA
XXXXX XXXXX

mA

XXXX

WV-
mU * mU * mU * mG * mG * mA * mG * mA * mU * mG * mG * mC *
UUUGGAGAUGGCAGU
XXXXX XXXXX

2211
mA * mG * mU * mU * mU * mC * mC * mU * mU * mA * mG * mU *
UUCCUUAGUA
XXXXX XXXXX

mA

XXXX

WV-
mA * mG * mU * mU * mU * mG * mG * mA * mG * mA * mU * mG *
AGUUUGGAGAUGGCA
XXXXX XXXXX

2212
mG * mC * mA * mG * mU * mU * mU * mC * mC * mU * mU * mA *
GUUUCCUUAG
XXXXX XXXXX

mG

XXXX

WV-
mU * mA * mG * mU * mU * mU * mG * mG * mA * mG * mA * mU *
UAGUUUGGAGAUGGC
XXXXX XXXXX

2213
mG * mG * mC * mA * mG * mU * mU * mU * mC * mC * mU * mU *
AGUUUCCUUA
XXXXX XXXXX

mA

XXXX

WV-
mC * mU * mA * mG * mU * mU * mU * mG * mG * mA * mG * mA *
CUAGUUUGGAGAUGG
XXXXX XXXXX

2214
mU * mG * mG * mC * mA * mG * mU * mU * mU * mC * mC * mU *
CAGUUUCCUU
XXXXX XXXXX

mU

XXXX

WV-
mU * mC * mU * mA * mG * mU * mU * mU * mG * mG * mA * mG *
UCUAGUUUGGAGAUG
XXXXX XXXXX

2215
mA * mU * mG * mG * mC * mA * mG * mU * mU * mU * mC * mC *
GCAGUUUCCU
XXXXX XXXXX

mU

XXXX

WV-
mU * mU * mC * mU * mA * mG * mU * mU * mU * mG * mG * mA *
UUCUAGUUUGGAGAU
XXXXX XXXXX

2216
mG * mA * mU * mG * mG * mC * mA * mG * mU * mU * mU * mC *
GGCAGUUUCC
XXXXX XXXXX

mC

XXXX

WV-
mC * mA * mU * mU * mU * mC * mU * mA * mG * mU * mU * mU *
CAUUUCUAGUUUGGA
XXXXX XXXXX

2217
mG * mG * mA * mG * mA * mU * mG * mG * mC * mA * mG * mU *
GAUGGCAGUU
XXXXX XXXXX

mU

XXXX

WV-
mG * mC * mA * mU * mU * mU * mC * mU * mA * mG * mU * mU *
GCAUUUCUAGUUUGG
XXXXX XXXXX

2218
mU * mG * mG * mA * mG * mA * mU * mG * mG * mC * mA * mG *
AGAUGGCAGU
XXXXX XXXXX

mU

XXXX

WV-
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU * mA *
AUGGCAUUUCUAGUU
XXXXX XXXXX

2219
mG * mU * mU * mU * mG * mG * mA * mG * mA * mU * mG * mG *
UGGAGAUGGC
XXXXX XXXXX

mC

XXXX

WV-
mG * mA * mA * mG * mA * mU * mG * mG * mC * mA * mU * mU *
GAAGAUGGCAUUUCU
XXXXX XXXXX

2220
mU * mC * mU * mA * mG * mU * mU * mU * mG * mG * mA * mG *
AGUUUGGAGA
XXXXX XXXXX

mA

XXXX

WV-
mA * mG * mG * mA * mA * mG * mA * mU * mG * mG * mC * mA *
AGGAAGAUGGCAUUU
XXXXX XXXXX

2221
mU * mU * mU * mC * mU * mA * mG * mU * mU * mU * mG * mG *
CUAGUUUGGA
XXXXX XXXXX

mA

XXXX

WV-
mA * mA * mG * mG * mA * mA * mG * mA * mU * mG * mG * mC *
AAGGAAGAUGGCAUU
XXXXX XXXXX

2222
mA * mU * mU * mU * mC * mU * mA * mG * mU * mU * mU * mG *
U CUAGUUUGG
XXXXX XXXXX

mG

XXXX

WV-
mC * mA * mA * mG * mG * mA * mA * mG * mA * mU * mG * mG *
CAAGGAAGAUGGCAU
XXXXX XXXXX

2223
mC * mA * mU * mU * mU * mC * mU * mA * mG * mU * mU * mU *
UU CUAGUUUG
XXXXX XXXXX

mG

XXXX

WV-
mC * mA * mU * mC * mA * mA * mG * mG * mA * mA * mG * mA *
CAUCAAGGAAGAUGG
XXXXX XXXXX

2224
mU * mG * mG * mC * mA * mU * mU * mU * mC * mU * mA * mG *
CAU UUCUAGU
XXXXX XXXXX

mU

XXXX

WV-
mA * mC * mA * mU * mC * mA * mA * mG * mG * mA * mA * mG *
ACAUCAAGGAAGAUG
XXXXX XXXXX

2225
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU * mA *
GCA UUUCUAG
XXXXX XXXXX

mG

XXXX

WV-
mA * mA * mC * mA * mU * mC * mA * mA * mG * mG * mA * mA *
AACAUCAAGGAAGAU
XXXXX XXXXX

2226
mG * mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU *
GGC AUUUCUA
XXXXX XXXXX

mA

XXXX

WV-
mC * mA * mA * mC * mA * mU * mC * mA * mA * mG * mG * mA *
CAACAUCAAGGAAGA
XXXXX XXXXX

2227
mA * mG * mA * mU * mG * mG * mC * mA * mU * mU * mU * mC *
UGG CAUUUCU
XXXXX XXXXX

mU

XXXX

WV-
mC * mU * mC * mC * mA * mA * mC * mA * mU * mC * mA * mA *
CUCCAACAUCAAGGA
XXXXX XXXXX

2228
mG * mG * mA * mA * mG * mA * mU * mG * mG * mC * mA * mU *
AGAU GGCAUU
XXXXX XXXXX

mU

XXXX

WV-
mA * mC * mC * mU * mC * mC * mA * mA * mC * mA * mU * mC *
ACCUCCAACAUCAAG
XXXXX XXXXX

2229
mA * mA * mG * mG * mA * mA * mG * mA * mU * mG * mG * mC *
GAAGAUGGCA
XXXXX XXXXX

mA

XXXX

WV-
mG * mU * mA * mC * mC * mU * mC * mC * mA * mA * mC * mA *
GUACCUCCAACAUCA
XXXXX XXXXX

2230
mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * mU * mG *
AGGAAGAUGG
XXXXX XXXXX

mG

XXXX

WV-
mA * mG * mG * mU * mA * mC * mC * mU * mC * mC * mA * mA *
AGGUACCUCCAACAU
XXXXX XXXXX

2231
mC * mA * mU * mC * mA * mA * mG * mG * mA * mA * mG * mA *
CAAGGAAGAU
XXXXX XXXXX

mU

XXXX

WV-
mA * mG * mA * mG * mC * mA * mG * mG * mU * mA * mC * mC *
AGAGCAGGUACCUCC
XXXXX XXXXX

2232
mU * mC * mC * mA * mA * mC * mA * mU * mC * mA * mA * mG *
AACAUCAAGG
XXXXX XXXXX

mG

XXXX

WV-
mC * mA * mG * mA * mG * mC * mA * mG * mG * mU * mA * mC *
CAGAGCAGGUACCUC
XXXXX XXXXX

2233
mC * mU * mC * mC * mA * mA * mC * mA * mU * mC * mA * mA *
CAACAUCAAG
XXXXX XXXXX

mG

XXXX

WV-
mC * mU * mG * mC * mC * mA * mG * mA * mG * mC * mA * mG *
CUGCCAGAGCAGGUA
XXXXX XXXXX

2234
mG * mU * mA * mC * mC * mU * mC * mC * mA * mA * mC * mA *
CCUCCAACAU
XXXXX XXXXX

mU

XXXX

WV-
mU * mC * mU * mG * mC * mC * mA * mG * mA * mG * mC * mA *
UCUGCCAGAGCAGGU
XXXXX XXXXX

2235
mG * mG * mU * mA * mC * mC * mU * mC * mC * mA * mA * mC *
ACCUCCAACA
XXXXX XXXXX

mA

XXXX

WV-
mA * mU * mC * mU * mG * mC * mC * mA * mG * mA * mG * mC *
AUCUGCCAGAGCAGG
XXXXX XXXXX

2236
mA * mG * mG * mU * mA * mC * mC * mU * mC * mC * mA * mA *
UACCUCCAAC
XXXXX XXXXX

mC

XXXX

WV-
mA * mA * mU * mC * mU * mG * mC * mC * mA * mG * mA * mG *
AAUCUGCCAGAGCAG
XXXXX XXXXX

2237
mC * mA * mG * mG * mU * mA * mC * mC * mU * mC * mC * mA *
GUACCUCCAA
XXXXX XXXXX

mA

XXXX

WV-
mA * mA * mA * mU * mC * mU * mG * mC * mC * mA * mG * mA *
AAAUCUGCCAGAGCA
XXXXX XXXXX

2238
mG * mC * mA * mG * mG * mU * mA * mC * mC * mU * mC * mC *
GGUACCUCCA
XXXXX XXXXX

mA

XXXX

WV-
mG * mA * mA * mA * mU * mC * mU * mG * mC * mC * mA * mG *
GAAAUCUGCCAGAGC
XXXXX XXXXX

2239
mA * mG * mC * mA * mG * mG * mU * mA * mC * mC * mU * mC *
AGGUACCUCC
XXXXX XXXXX

mC

XXXX

WV-
mU * mG * mA * mA * mA * mU * mC * mU * mG * mC * mC * mA *
UGAAAUCUGCCAGAG
XXXXX XXXXX

2240
mG * mA * mG * mC * mA * mG * mG * mU * mA * mC * mC * mU *
CAGGUACCUC
XXXXX XXXXX

mC

XXXX

WV-
mU * mU * mG * mA * mA * mA * mU * mC * mU * mG * mC * mC *
UUGAAAUCUGCCAGA
XXXXX XXXXX

2241
mA * mG * mA * mG * mC * mA * mG * mG * mU * mA * mC * mC *
GCAGGUACCU
XXXXX XXXXX

mU

XXXX

WV-
mC * mC * mC * mG * mG * mU * mU * mG * mA * mA * mA * mU *
CCCGGUUGAAAUCUG
XXXXX XXXXX

2242
mC * mU * mG * mC * mC * mA * mG * mA * mG * mC * mA * mG *
CCAGAGCAGG
XXXXX XXXXX

mG

XXXX

WV-
mC * mC * mA * mA * mG * mC * mC * mC * mG * mG * mU * mU *
CCAAGCCCGGUUGAA
XXXXX XXXXX

2243
mG * mA * mA * mA * mU * mC * mU * mG * mC * mC * mA * mG *
AUCUGCCAGA
XXXXX XXXXX

mA

XXXX

WV-
mU * mC * mC * mA * mA * mG * mC * mC * mC * mG * mG * mU *
UCCAAGCCCGGUUGA
XXXXX XXXXX

2244
mU * mG * mA * mA * mA * mU * mC * mU * mG * mC * mC * mA *
AAUCUGCCAG
XXXXX XXXXX

mG

XXXX

WV-
mG * mU * mC * mC * mA * mA * mG * mC * mC * mC * mG * mG *
GUCCAAGCCCGGUU
XXXXX XXXXX

2245
mU * mU * mG * mA * mA * mA * mU * mC * mU * mG * mC * mC *
GAAAUCUGCCA
XXXXX XXXXX

mA

XXXX

WV-
mU * mC * mU * mG * mU * mC * mC * mA * mA * mG * mC * mC *
UCUGUCCAAGCCCGG
XXXXX XXXXX

2246
mC * mG * mG * mU * mU * mG * mA * mA * mA * mU * mC * mU *
UUGAAAUCUG
XXXXX XXXXX

mG

XXXX

WV-
mU * mU * mC * mU * mG * mU * mC * mC * mA * mA * mG * mC *
UUCUGUCCAAGCCCG
XXXXX XXXXX

2247
mC * mC * mG * mG * mU * mU * mG * mA * mA * mA * mU * mC *
GUUGAAAUCU
XXXXX XXXXX

mU

XXXX

WV-
mG * mU * mU * mC * mU * mG * mU * mC * mC * mA * mA * mG *
GUUCUGUCCAAGCCC
XXXXX XXXXX

2248
mC * mC * mC * mG * mG * mU * mU * mG * mA * mA * mA * mU *
GGUUGAAAUC
XXXXX XXXXX

mC

XXXX

WV-
mA * mG * mU * mU * mC * mU * mG * mU * mC * mC * mA * mA *
AGUUCUGUCCAAGC
XXXXX XXXXX

2249
mG * mC * mC * mC * mG * mG * mU * mU * mG * mA * mA * mA *
CCGGUUGAAAU
XXXXX XXXXX

mU

XXXX

WV-
mA * mA * mG * mU * mU * mC * mU * mG * mU * mC * mC * mA *
AAGUUCUGUCCAA
XXXXX XXXXX

2250
mA * mG * mC * mC * mC * mG * mG * mU * mU * mG * mA * mA *
GCCCGGUUGAAA
XXXXX XXXXX

mA

XXXX

WV-
mU * mA * mA * mG * mU * mU * mC * mU * mG * mU * mC * mC *
UAAGUUCUGUCC
XXXXX XXXXX

2251
mA * mA * mG * mC * mC * mC * mG * mG * mU * mU * mG * mA *
AGCCCGGUUGAA
XXXXX XXXXX

mA

XXXX

WV-
mG * mU * mA * mA * mG * mU * mU * mC * mU * mG * mU * mC *
GUAAGUUCUGU
XXXXX XXXXX

2252
mC * mA * mA * mG * mC * mC * mC * mG * mG * mU * mU * mG *
CCAAGCCCGGUUGA
XXXXX XXXXX

mA

XXXX

WV-
mG * mG * mU * mA * mA * mG * mU * mU * mC * mU * mG * mU *
GGUAAGUUCUGUCCA
XXXXX XXXXX

2253
mC * mC * mA * mA * mG * mC * mC * mC * mG * mG * mU * mU *
AGCCCGGUUG
XXXXX XXXXX

mG

XXXX

WV-
mC * mG * mG * mU * mA * mA * mG * mU * mU * mC * mU * mG *
CGGUAAGUUCUGUCC
XXXXX XXXXX

2254
mU * mC * mC * mA * mA * mG * mC * mC * mC * mG * mG * mU *
AAGCCCGGUU
XXXXX XXXXX

mU

XXXX

WV-
mU * mC * mG * mG * mU * mA * mA * mG * mU * mU * mC * mU *
UCGGUAAGUUCUGUC
XXXXX XXXXX

2255
mG * mU * mC * mC * mA * mA * mG * mC * mC * mC * mG * mG *
CAAGCCCGGU
XXXXX XXXXX

mU

XXXX

WV-
mG * mU * mC * mG * mG * mU * mA * mA * mG * mU * mU * mC *
GUCGGUAAGUUCUGU
XXXXX XXXXX

2256
mU * mG * mU * mC * mC * mA * mA * mG * mC * mC * mC * mG *
CCAAGCCCGG
XXXXX XXXXX

mG

XXXX

WV-
mA * mG * mU * mC * mG * mG * mU * mA * mA * mG * mU * mU *
AGUCGGUAAGUUCUG
XXXXX XXXXX

2257
mC * mU * mG * mU * mC * mC * mA * mA * mG * mC * mC * mC *
UCCAAGCCCG
XXXXX XXXXX

mG

XXXX

WV-
mC * mA * mG * mU * mC * mG * mG * mU * mA * mA * mG * mU *
CAGUCGGUAAGUUCU
XXXXX XXXXX

2258
mU * mC * mU * mG * mU * mC * mC * mA * mA * mG * mC * mC *
GUCCAAGCCC
XXXXX XXXXX

mC

XXXX

WV-
mA * mA * mA * mG * mC * mC * mA * mG * mU * mC * mG * mG *
AAAGCCAGUCGGUAA
XXXXX XXXXX

2259
mU * mA * mA * mG * mU * mU * mC * mU * mG * mG * mC * mC *
GUUCUGUCCA
XXXXX XXXXX

mA

XXXX

WV-
mG * mA * mA * mA * mG * mC * mC * mA * mG * mU * mC * mG *
GAAAGCCAGUCGGUA
XXXXX XXXXX

2260
mG * mU * mA * mA * mG * mU * mU * mC * mU * mG * mU * mC *
AGUUCUGUCC
XXXXX XXXXX

mC

XXXX

WV-
mG * mU * mC * mA * mC * mC * mC * mA * mC * mC * mA * mU *
GUCACCCACCAUCAC
XXXXX XXXXX

2261
mC * mA * mC * mC * mC * mU * mC * mU * mG * mU * mG * mA *
CCUCUGUGAU
XXXXX XXXXX

mU

XXXX

WV-
mG * mG * mU * mC * mA * mC * mC * mC * mA * mC * mC * mA *
GGUCACCCACCAUCA
XXXXX XXXXX

2262
mU * mC * mA * mC * mC * mC * mU * mC * mU * mG * mU * mG *
CCCUCUGUGA
XXXXX XXXXX

mA

XXXX

WV-
mA * mA * mG * mG * mU * mC * mA * mC * mC * mC * mA * mC *
AAGGUCACCCACCAU
XXXXX XXXXX

2263
mC * mA * mU * mC * mA * mC * mC * mC * mU * mC * mU * mG *
CACCCUCUGU
XXXXX XXXXX

mU

XXXX

WV-
mC * mA * mA * mG * mG * mU * mC * mA * mC * mC * mC * mA *
CAAGGUCACCCACCA
XXXXX XXXXX

2264
mC * mC * mA * mU * mC * mA * mC * mC * mC * mU * mC * mU *
UCACCCUCUG
XXXXX XXXXX

mG

XXXX

WV-
mU * mC * mA * mA * mG * mG * mU * mC * mA * mC * mC * mC *
UCAAGGUCACCCACC
XXXXX XXXXX

2265
mA * mC * mC * mA * mU * mC * mA * mC * mC * mC * mU * mC *
AUCACCCUCU
XXXXX XXXXX

mU

XXXX

WV-
mC * mU * mC * mA * mA * mG * mG * mU * mC * mA * mC * mC *
CUCAAGGUCACCCAC
XXXXX XXXXX

2266
mC * mA * mC * mC * mA * mU * mC * mA * mC * mC * mC * mU *
CAUCACCCUC
XXXXX XXXXX

mC

XXXX

WV-
mC * mU * mU * mG * mA * mU * mC * mA * mA * mG * mC * mA *
CUUGAUCAAGCAGAG
XXXXX XXXXX

2267
mG * mA * mG * mA * mA * mA * mG * mC * mC * mA * mG * mU *
AAAGCCAGUC
XXXXX XXXXX

mC

XXXX

WV-
mA * mU * mA * mA * mC * mU * mU * mG * mA * mU * mC * mA *
AUAACUUGAUCAAGC
XXXXX XXXXX

2268
mA * mG * mC * mA * mG * mA * mG * mA * mA * mA * mG * mC *
AGAGAAAGCC
XXXXX XXXXX

mC

XXXX

WV-
mA * mG * mU * mA * mA * mC * mA * mG * mU * mC * mU * mG *
AGUAACAGUCUGAGU
XXXXX XXXXX

2273
mA * mG * mU * mA * mG * mG * mA * mG
AGGAG
XXXXX XXXX

WV-
mG * mA * mG * mU * mA * mA * mC * mA * mG * mU * mC * mU *
GAGUAACAGUCUGAG
XXXXX XXXXX

2274
mG * mA * mG * mU * mA * mG * mG * mA
UAGGA
XXXXX XXXX

WV-
mA * mG * mA * mG * mU * mA * mA * mC * mA * mG * mU * mC *
AGAGUAACAGUCUGA
XXXXX XXXXX

2275
mU * mG * mA * mG * mU * mA * mG * mG
GUAGG
XXXXX XXXX

WV-
mC * mA * mG * mA * mG * mU * mA * mA * mC * mA * mG * mU *
CAGAGUAACAGUCUG
XXXXX XXXXX

2276
mC * mU * mG * mA * mG * mU * mA * mG
AGUAG
XXXXX XXXX

WV-
mG * mU * mC * mA * mC * mC * mA * mG * mA * mG * mU * mA
GUCACCAGAGUAACA
XXXXX XXXXX

2277
mA * mC * mA * mG * mU * mC * mU * mG
GUCUG
XXXXX XXXX

WV-
mU * mG * mU * mC * mA * mC * mC * mA * mG * mA * mG * mU *
UGUCACCAGAGUAAC
XXXXX XXXXX

2278
mA * mA * mC * mA * mG * mU * mC * mU
AGUCU
XXXXX XXXX

WV-
mG * mU * mG * mU * mC * mA * mC * mC * mA * mG * mA * mG *
GUGUCACCAGAGUAA
XXXXX XXXXX

2279
mU * mA * mA * mC * mA * mG * mU *mC
CAGUC
XXXXX XXXX

WV-
mU * mG * mU * mG * mU * mC * mA * mC * mC * mA * mG * mA *
UGUGUCACCAGAGUA
XXXXX XXXXX

2280
mG * mU * mA * mA * mC * mA * mG * mU
ACAGU
XXXXX XXXX

WV-
mU * mU * mG * mU * mG * mU * mC * mA * mC * mC * mA * mG *
UUGUGUCACCAGAGU
XXXXX XXXXX

2281
mA * mG * mU * mA * mA * mC * mA * mG
AACAG
XXXXX XXXX

WV-
mG * mG * mU * mU * mG * mU * mG * mU * mC * mA * mC * mC *
GGUUGUGUCACCAGA
XXXXX XXXXX

2282
mA * mG * mA * mG * mU * mA * mA * mC
GUAAC
XXXXX XXXX

WV-
mA * mG * mG * mU * mU * mG * mU * mG * mU * mC * mA * mC *
AGGUUGUGUCACCAG
XXXXX XXXXX

2283
mC * mA * mG * mA * mG * mU * mA * mA
AGUAA
XXXXX XXXX

WV-
mC * mA * mG * mG * mU * mU * mG * mU * mG * mU * mC * mA *
CAGGUUGUGUCACCA
XXXXX XXXXX

2284
mC * mC * mA * mG * mA * mG * mU * mA
GAGUA
XXXXX XXXX

WV-
mA * mC * mA * mG * mG * mU * mU * mG * mU * mG * mU * mC *
ACAGGUUGUGUCACC
XXXXX XXXXX

2285
mA * mC * mC * mA * mG * mA * mG * mU
AGAGU
XXXXX XXXX

WV-
mC * mA * mC * mA * mG * mG * mU * mU * mG * mU * mG * mU *
CACAGGUUGUGUCAC
XXXXX XXXXX

2286
mC * mA * mC * mC * mA * mG * mA * mG
CAGAG
XXXXX XXXX

WV-
mC * mC * mA * mC * mA * mG * mG * mU * mU * mG * mU * mG *
CCACAGGUUGUGUCA
XXXXX XXXXX

2287
mU * mC * mA * mC * mC * mA * mG * mA
CCAGA
XXXXX XXXX

WV-
mA * mC * mC * mA * mC * mA * mG * mG * mU * mU * mG * mU *
ACCACAGGUUGUGUC
XXXXX XXXXX

2288
mG * mU * mC * mA * mC * mC * mA * mG
ACCAG
XXXXX XXXX

WV-
mA * mA * mC * mC * mA * mC * mA * mG * mG * mU * mU * mG *
AACCACAGGUUGUGU
XXXXX XXXXX

2289
mU * mG * mU * mC * mA * mC * mC * mA
CACCA
XXXXX XXXX

WV-
mU * mA * mA * mC * mC * mA * mC * mA * mG * mG * mU * mU *
UAACCACAGGUUGUG
XXXXX XXXXX

2290
mG * mU * mG * mU * mC * mA * mC * mC
UCACC
XXXXX XXXX

WV-
mG * mU * mA * mA * mC * mC * mA * mC * mA * mG * mG * mU *
GUAACCACAGGUUGU
XXXXX XXXXX

2291
mU * mG * mU * mG * mU * mC * mA * mC
GUCAC
XXXXX XXXX

WV-
mA * mG * mU * mA * mA * mC * mC * mA * mC * mA * mG * mG *
AGUAACCACAGGUUG
XXXXX XXXXX

2292
mU * mU * mG * mU * mG * mU * mC * mA
UGUCA
XXXXX XXXX

WV-
mC * mU * mU * mA * mG * mU * mA * mA * mC * mC * mA * mC *
CUUAGUAACCACAGG
XXXXX XXXXX

2293
mA * mG * mG * mU * mU * mG * mU * mG
UUGUG
XXXXX XXXX

WV-
mC * mC * mU * mU * mA * mG * mU * mA * mA * mC * mC * mA *
CCUUAGUAACCACAG
XXXXX XXXXX

2294
mC * mA * mG * mG * mU * mU * mG * mU
GUUGU
XXXXX XXXX

WV-
mU * mC * mC * mU * mU * mA * mG * mU * mA * mA * mC * mC *
UCCUUAGUAACCACA
XXXXX XXXXX

2295
mA * mC * mA * mG * mG * mU * mU * mG
GGUUG
XXXXX XXXX

WV-
mU * mU * mC * mC * mU * mU * mA * mG * mU * mA * mA * mC *
UUCCUUAGUAACCAC
XXXXX XXXXX

2296
mC * mA * mC * mA * mG * mG * mU * mU
AGGUU
XXXXX XXXX

WV-
mU * mU * mU * mC * mC * mU * mU * mA * mG * mU * mA * mA *
UUUCCUUAGUAACCA
XXXXX XXXXX

2297
mC * mC * mA * mC * mA * mG * mG * mU
CAGGU
XXXXX XXXX

WV-
mG * mU * mU * mU * mC * mC * mU * mU * mA * mG * mU * mA *
GUUUCCUUAGUAACC
XXXXX XXXXX

2298
mA * mC * mC * mA * mC * mA * mG * mG
ACAGG
XXXXX XXXX

WV-
mA * mG * mU * mU * mU * mC * mC * mU * mU * mA * mG * mU *
AGUUUCCUUAGUAAC
XXXXX XXXXX

2299
mA * mA * mC * mC * mA * mC * mA * mG
CACAG
XXXXX XXXX

WV-
mG * mC * mA * mG * mU * mU * mU * mC * mC * mU * mU * mA *
GCAGUUUCCUUAGUA
XXXXX XXXXX

2300
mG * mU * mA * mA * mC * mC * mA * mC
ACCAC
XXXXX XXXX

WV-
mG * mG * mC * mA * mG * mU * mU * mU * mC * mC * mU * mU *
GGCAGUUUCCUUAGU
XXXXX XXXXX

2301
mA * mG * mU * mA * mA * mC * mC * mA
AACCA
XXXXX XXXX

WV-
mU * mG * mG * mC * mA * mG * mU * mU * mU * mC * mC * mU *
UGGCAGUUUCCUUAG
XXXXX XXXXX

2302
mU * mA * mG * mU * mA * mA * mC * mC
UAACC
XXXXX XXXX

WV-
mA * mU * mG * mG * mC * mA * mG * mU * mU * mU * mC * mC *
AUGGCAGUUUCCUUA
XXXXX XXXXX

2303
mU * mU * mA * mG * mU * mA * mA * mC
GUAAC
XXXXX XXXX

WV-
mG * mA * mU * mG * mG * mC * mA * mG * mU * mU * mU * mC *
GAUGGCAGUUUCCUU
XXXXX XXXXX

2304
mC * mU * mU * mA * mG * mU * mA * mA
AGUAA
XXXXX XXXX

WV-
mA * mG * mA * mU * mG * mG * mC * mA * mG * mU * mU * mU *
AGAUGGCAGUUUCCU
XXXXX XXXXX

2305
mC * mC * mU * mU * mA * mG * mU * mA
UAGUA
XXXXX XXXX

WV-
mG * mG * mA * mG * mA * mU * mG * mG * mC * mA * mG * mU *
GGAGAUGGCAGUUUC
XXXXX XXXXX

2306
mU * mU * mC * mC * mU * mU * mA * mG
CUUAG
XXXXX XXXX

WV-
mU * mG * mG * mA * mG * mA * mU * mG * mG * mC * mA * mG *
UGGAGAUGGCAGUU
XXXXX XXXXX

2307
mU * mU * mU * mC * mC * mU * mU * mA
UCCUUA
XXXXX XXXX

WV-
mU * mU * mG * mG * mA * mG * mA * mU * mG * mG * mC * mA *
UUGGAGAUGGCAGU
XXXXX XXXXX

2308
mG * mU * mU * mU * mC * mC * mU * mU
UUCCUU
XXXXX XXXX

WV-
mU * mU * mU * mG * mG * mA * mG * mA * mU * mG * mG * mC *
UUUGGAGAUGGCAG
XXXXX XXXXX

2309
mA * mG * mU * mU * mU * mC * mC * mU
UUUCCU
XXXXX XXXX

WV-
mG * mU * mU * mU * mG * mG * mA * mG * mA * mU * mG * mG *
GUUUGGAGAUGGCA
XXXXX XXXXX

2310
mC * mA * mG * mU * mU * mU * mC * mC
GUUUCC
XXXXX XXXX

WV-
mC * mU * mA * mG * mU * mU * mU * mG * mG * mA * mG * mA *
CUAGUUUGGAGAUG
XXXXX XXXXX

2311
mU * mG * mG * mC * mA * mG * mU * mU
GCAGUU
XXXXX XXXX

WV-
mU * mC * mU * mA * mG * mU * mU * mU * mG * mG * mA * mG *
UCUAGUUUGGAGAU
XXXXX XXXXX

2312
mA * mU * mG * mG * mC * mA * mG * mU
GGCAGU
XXXXX XXXX

WV-
mA * mU * mU * mU * mC * mU * mA * mG * mU * mU * mU * mG *
AUUUCUAGUUUGGA
XXXXX XXXXX

2313
mG * mA * mG * mA * mU * mG * mG * mC
GAUGGC
XXXXX XXXX

WV-
mU * mG * mG * mC * mA * mU * mU * mU * mC * mU * mA * mG *
UGGCAUUUCUAGUUU
XXXXX XXXXX

2314
mU * mU * mU * mG * mG * mA * mG * mA
GGAGA
XXXXX XXXX

WV-
mG * mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU *
GAUGGCAUUUCUAGU
XXXXX XXXXX

2315
mA * mG * mU * mU * mU * mG * mG * mA
UUGGA
XXXXX XXXX

WV-
mA * mG * mA * mU * mG * mG * mC * mA * mU * mU * mU * MC *
AGAUGGCAUUUCUAG
XXXXX XXXXX

2316
mU * mA * mG * mU * mU * mU * mG * mG
UUUGG
XXXXX XXXX

WV-
mA * mA * mG * mA * mU * mG * mG * mC * mA * mU * mU * mU *
AAGAUGGCAUUUCUA
XXXXX XXXXX

2317
mC * mU * mA * mG * mU * mU * mU * mG
GUUUG
XXXXX XXXX

WV-
mA * mG * mG * mA * mA * mG * mA * mU * mG * mG * mC * mA *
AGGAAGAUGGCAUU
XXXXX XXXXX

2318
mU * mU * mU * mC * mU * mA * mG * mU
UCUAGU
XXXXX XXXX

WV-
mA * mA * mG * mG * mA * mA * mG * mA * mU * mG * mG * mC *
AAGGAAGAUGGCAU
XXXXX XXXXX

2319
mA * mU * mU * mU * mC * mU * mA * mG
UUCUAG
XXXXX XXXX

WV-
mC * mA * mA * mG * mG * mA * mA * mG * mA * mU * mG * mG *
CAAGGAAGAUGGCAU
XXXXX XXXXX

2320
mC * mA * mU * mU * mU * mC * mU * mA
UUCUA
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * mU * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2321
mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
mA * mC * mA * mU * mC * mA * mA * mG * mG * mA * mA * mG *
ACAUCAAGGAAGAUG
XXXXX XXXXX

2322
mA * mU * mG * mG * mC * mA * mU * mU
GCAUU
XXXXX XXXX

WV-
mC * mA * mA * mC * mA * mU * mC * mA * mA * mG * mG * mA *
CAACAUCAAGGAAGA
XXXXX XXXXX

2323
mA * mG * mA * mU * mG * mG * mC * mA
UGGCA
XXXXX XXXX

WV-
mU * mC * mC * mA * mA * mC * mA * mU * mC * mA * mA * mG *
UCCAACAUCAAGGAA
XXXXX XXXXX

2324
mG * mA * mA * mG * mA * mU * mG * mG
GAUGG
XXXXX XXXX

WV-
mC * mC * mU * mC * mC * mA * mA * mC * mA * mU * mC * mA *
CCUCCAACAUCAAGG
XXXXX XXXXX

2325
mA * mG * mG * mA * mA * mG * mA * mU
AAGAU
XXXXX XXXX

WV-
mA * mG * mG * mU * mA * mC * mC * mU * mC * mC * mA * mA *
AGGUACCUCCAACAU
XXXXX XXXXX

2326
mC * mA * mU * mC * mA * mA * mG * mG
CAAGG
XXXXX XXXX

WV-
mC * mA * mG * mG * mU * mA * mC * mC * mU * mC * mC * mA *
CAGGUACCUCCAACA
XXXXX XXXXX

2327
mA * mC * mA * mU * mC * mA * mA * mG
UCAAG
XXXXX XXXX

WV-
mA * mG * mA * mG * mC * mA * mG * mG * mU * mA * mC * mC *
AGAGCAGGUACCUCC
XXXXX XXXXX

2328
mU * mC * mC * mA * mA * mC * mA * mU
AACAU
XXXXX XXXX

WV-
mC * mA * mG * mA * mG * mC * mA * mG * mG * mU * mA * mC *
CAGAGCAGGUACCUC
XXXXX XXXXX

2329
mC * mU * mC * mC * mA * mA * mC * mA
CAACA
XXXXX XXXX

WV-
mC * mC * mA * mG * mA * mG * mC * mA * mG * mG * mU * mA *
CCAGAGCAGGUACCU
XXXXX XXXXX

2330
mC * mC * mU * mC * mC * mA * mA * mC
CCAAC
XXXXX XXXX

WV-
mG * mC * mC * mA * mG * mA * mG * mC * mA * mG * mG * mU *
GCCAGAGCAGGUACC
XXXXX XXXXX

2331
mA * mC * mC * mU * mC * mC * mA * mA
UCCAA
XXXXX XXXX

WV-
mU * mG * mC * mC * mA * mG * mA * mG * mC * mA * mG * mG *
UGCCAGAGCAGGUAC
XXXXX XXXXX

2332
mU * mA * mC * mC * mU * mC * mC * mA
CUCCA
XXXXX XXXX

WV-
mC * mU * mG * mC * mC * mA * mG * mA * mG * mC * mA * mG *
CUGCCAGAGCAGGUA
XXXXX XXXXX

2333
mG * mU * mA * mC * mC * mU * mC * mC
CCUCC
XXXXX XXXX

WV-
mU * mC * mU * mG * mC * mC * mA * mG * mA * mG * mC * mA *
UCUGCCAGAGCAGGU
XXXXX XXXXX

2334
mG * mG * mU * mA * mC * mC * mU * mC
ACCUC
XXXXX XXXX

WV-
mA * mU * mC * mU * mG * mC * mC * mA * mG * mA * mG * mC *
AUCUGCCAGAGCAGG
XXXXX XXXXX

2335
mA * mG * mG * mU * mA * mC * mC * mU
UACCU
XXXXX XXXX

WV-
mU * mU * mG * mA * mA * mA * mU * mC * mU * mG * mC * mC *
UUGAAAUCUGCCAGA
XXXXX XXXXX

2336
mA * mG * mA * mG * mC * mA * mG * mG
GCAGG
XXXXX XXXX

WV-
mC * mC * mC * mG * mG * mU * mU * mG * mA * mA * mA * mU *
CCCGGUUGAAAUCUG
XXXXX XXXXX

2337
mC * mU * mG * mC * mC * mA * mG * mA
CCAGA
XXXXX XXXX

WV-
mG * mC * mC * mC * mG * mG * mU * mU * mG * mA * mA * mA *
GCCCGGUUGAAAUCU
XXXXX XXXXX

2338
mU * mC * mU * mG * mC * mC * mA * mG
GCCAG
XXXXX XXXX

WV-
mA * mG * mC * mC * mC * mG * mG * mU * mU * mG * mA * mA *
AGCCCGGUUGAAAUC
XXXXX XXXXX

2339
mA * mU * mC * mU * mG * mC * mC * mA
UGCCA
XXXXX XXXX

WV-
mC * mC * mA * mA * mG * mC * mC * mC * mG * mG * mU * mU *
CCAAGCCCGGUUGAA
XXXXX XXXXX

2340
mG * mA * mA * mA * mU * mC * mU * mG
AUCUG
XXXXX XXXX

WV-
mU * mC * mC * mA * mA * mG * mC * mC * mC * mG * mG * mU *
UCCAAGCCCGGUUGA
XXXXX XXXXX

2341
mU * mG * mA * mA * mA * mU * mC * mU
AAUCU
XXXXX XXXX

WV-
mG * mU * mC * mC * mA * mA * mG * mC * mC * mC * mG * mG *
GUCCAAGCCCGGUUG
XXXXX XXXXX

2342
mU * mU * mG * mA * mA * mA * mU * mC
AAAUC
XXXXX XXXX

WV-
mU * mG * mU * mC * mC * mA * mA * mG * mC * mC * mC * mG *
UGUCCAAGCCCGGUU
XXXXX XXXXX

2343
mG * mU * mU * mG * mA * mA * mA * mU
GAAAU
XXXXX XXXX

WV-
mC * mU * mG * mU * mC * mC * mA * mA * mG * mC * mC * mC *
CUGUCCAAGCCCGGU
XXXXX XXXXX

2344
mG * mG * mU * mU * mG * mA * mA * mA
UGAAA
XXXXX XXXX

WV-
mU * mC * mU * mG * mU * mC * mC * mA * mA * mG * mC * mC *
UCUGUCCAAGCCCGG
XXXXX XXXXX

2345
mC * mG * mG * mU * mU * mG * mA * mA
UUGAA
XXXXX XXXX

WV-
mU * mU * mC * mU * mG * mU * mC * mC * mA * mA * mG * mC *
UUCUGUCCAAGCCCG
XXXXX XXXXX

2346
mC * mC * mG * mG * mU * mU * mG * mA
GUUGA
XXXXX XXXX

WV-
mG * mU * mU * mC * mU * mG * mU * mC * mC * mA * mA * mG *
GUUCUGUCCAAGCCC
XXXXX XXXXX

2347
mC * mC * mC * mG * mG * mU * mU * mG
GGUUG
XXXXX XXXX

WV-
mA * mG * mU * mU * mC * mU * mG * mU * mC * mC * mA * mA *
AGUUCUGUCCAAGCC
XXXXX XXXXX

2348
mG * mC * mC * mC * mG * mG * mU * mU
CGGUU
XXXXX XXXX

WV-
mA * mA * mG * mU * mU * mC * mU * mG * mU * mC * mC * mA *
AAGUUCUGUCCAAGC
XXXXX XXXXX

2349
mA * mG * mC * mC * mC * mG * mG * mU
CCGGU
XXXXX XXXX

WV-
mU * mA * mA * mG * mU * mU * mC * mU * mG * mU * mC * mC *
UAAGUUCUGUCCAAG
XXXXX XXXXX

2350
mA * mA * mG * mC * mC * mC * mG * mG
CCCGG
XXXXX XXXX

WV-
mG * mU * mA * mA * mG * mU * mU * mC * mU * mG * mU * mC *
GUAAGUUCUGUCCAA
XXXXX XXXXX

2351
mC * mA * mA * mG * mC * mC * mC * mG
GCCCG
XXXXX XXXX

WV-
mG * mG * mU * mA * mA * mG * mU * mU * mC * mU * mG * mU *
GGUAAGUUCUGUCCA
XXXXX XXXXX

2352
mC * mC * mA * mA * mG * mC * mC * mC
AGCCC
XXXXX XXXX

WV-
mC * mA * mG * mU * mC * mG * mG * mU * mA * mA * mG * mU *
CAGUCGGUAAGUUCU
XXXXX XXXXX

2353
mU * mC * mU * mG * mU * mC * mC * mA
GUCCA
XXXXX XXXX

WV-
mC * mC * mA * mG * mU * mC * mG * mG * mU * mA * mA * mG *
CCAGUCGGUAAGUUC
XXXXX XXXXX

2354
mU * mU * mC * mU * mG * mU * mC * mC
UGUCC
XXXXX XXXX

WV-
mC * mC * mA * mC * mC * mA * mU * mC * mA * mC * mC * mC *
CCACCAUCACCCUCU
XXXXX XXXXX

2355
mU * mC * mU * mG * mU * mG * mA * mU
GUGAU
XXXXX XXXX

WV-
mC * mC * mC * mA * mC * mC * mA * mU * mC * mA * mC * mC *
CCCACCAUCACCCUC
XXXXX XXXXX

2356
mC * mU * mC * mU * mG * mU * mG * mA
UGUGA
XXXXX XXXX

WV-
mC * mA * mC * mC * mC * mA * mC * mC * mA * mU * mC * mA *
CACCCACCAUCACCC
XXXXX XXXXX

2357
mC * mC * mC * mU * mC * mU * mG * mU
UCUGU
XXXXX XXXX

WV-
mU * mC * mA * mC * mC * mC * mA * mC * mC * mA * mU * mC *
UCACCCACCAUCACC
XXXXX XXXXX

2358
mA * mC * mC * mC * mU * mC * mU * mG
CUCUG
XXXXX XXXX

WV-
mG * mU * mC * mA * mC * mC * mC * mA * mC * mC * mA * mU *
GUCACCCACCAUCAC
XXXXX XXXXX

2359
mC * mA * mC * mC * mC * mU * mC * mU
CCUCU
XXXXX XXXX

WV-
mG * mG * mU * mC * mA * mC * mC * mC * mA * mC * mC * mA *
GGUCACCCACCAUCA
XXXXX XXXXX

2360
mU * mC * mA * mC * mC * mC * mU * mC
CCCUC
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mC * mA * mG * mA * mG * mA * mA *
UCAAGCAGAGAAAGC
XXXXX XXXXX

2361
mA * mG * mC * mC * mA * mG * mU * mC
CAGUC
XXXXX XXXX

WV-
mU * mU * mG * mA * mU * mC * mA * mA * mG * mC * mA * mG *
UUGAUCAAGCAGAGA
XXXXX XXXXX

2362
mA * mG * mA * mA * mA * mG * mC * mC
AAGCC
XXXXX XXXX

WV-
mU * S mC * S mA * R mA * R mG * R mG * R mA * R mA * R mG * R
UCAAGGAAGAUGGCA
SSRRRRRRRRRRR

2363
mA * R mU * R mG * R mG * R mC * R mA * R mU * R mU * R mU * S
UUUCU
RRRRSS

mC * S mU

WV-
mU * S mC * S mA * S mA * S mG * R mG * R mA * R mA * R mG * R
UCAAGGAAGAUGGCA
SSSSRRRRRRRRR

2364
mA * R mU * R mG * R mG * R mC * R mA * R mU * S mU * S mU * S
UUUCU
RRSSSS

mC * S mU

WV-
mU * S mC * S mA * S mA * S mG * S mG * R mA * R mA * R mG * R mA
UCAAGGAAGAUGGCA
SSSSSRRRRRRRR

2365
* R mU * R mG * R mG * R mC * R mA * S mU * S mU * S mU * S mC * S
UUUCU
RSSSSS

mU

WV-
mU * S mC mA mA mG mG mA mA mG mA mU mG mG mC mA mU mU mU
UCAAGGAAGAUGGCA
SOOOOO OOOOO

2366
mC * S mU
UUUCU
OOOOOOOS

WV-
mU * S mC * S mA mA mG mG mA mA mG mA mU mG mG mC mA mU mU
UCAAGGAAGAUGGCA
SSOOOOO OOOOO

2367
mU * S mC * S mU
UUUCU
OOOOOSS

WV-
mU * S mC * S mA * S mA mG mG mA mA mG mA mU mG mG mC mA mU
UCAAGGAAGAUGGCA
SSSOOOOO

2368
mU * S mU * S mC * S mU
UUUCU
OOOOO OOOSSS

WV-
mU * S mC * S mA * S mA * S mG mG mA mA mG mA mU mG mG mC mA
UCAAGGAAGAUGGCA
SSSSOOOOO

2369
mU * S mU * S mU * S mC * S mU
UUUCU
OOOOO OSSSS

WV-
mU * S mC * S mA * S mA * S mG * S mG mA mA mG mA mU mG mG mC
UCAAGGAAGAUGGCA
SSSSSOOOOOOOO

2370
mA * S mU * S mU * S mU * S mC * S mU
UUUCU
OSSSSS

WV-
mU * mC mA mA mG mG mA mA mG mA mU mG mG mC mA mU mU mU
UCAAGGAAGAUGGCA
XOOOOO OOOOO

2381
mC * mU
UUUCU
OOOOOOOX

WV-
mU * mU * mA mA mG mG mA mA mG mA mU mG mG mC mA mU mU
UCAAGGAAGAUGGCA
XXOOOOO

2382
mU * mC * mU
UUUCU
OOOOO

OOOOOXX

WV-
mU * mC * mA * mA mG mG mA mA mG mA mU mG mG mC mA mU mU
UCAAGGAAGAUGGCA
XXXOOOOO

2383
* mU * mC * mU
UUUCU
OOOOO OOOXXX

WV-
mU * mC * mA * mA * mG mG mA mA mG mA mU mG mG mC mA mU *
UCAAGGAAGAUGGCA
XXXXOOOOO

2384
mU * mU * mC * mU
UUUCU
OOOOO OXXXX

WV-
mU * mC * mA * mA * mG * mG mA mA mG mA mU mG mG mC mA *
UCAAGGAAGAUGGCA
XXXXXOOOOOOO

2385
mU * mU * mU * mC * mU
UUUCU
OOXXXXX

WV-
fU * fC * fA * fA * fG * fG * mA mA mG mA mU mG mG mC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOOOOOO

2432
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * mG mA mA mG mA mU mG mG mC mA * fU * fU *
UCAAGGAAGAUGGCA
XXXXXOOOOOOO

2433
fU * fC * fU
UUUCU
OOXXXXX

WV-
fU * fC * fA * fA * mG mG mA mA mG mA mU mG mG mC mA mU * fU *
UCAAGGAAGAUGGCA
XXXXOOOOO

2434
fU * fC * fU
UUUCU
OOOOO OXXXX

WV-
fU * fC * fA * mA mG mG mA mA mG mA mU mG mG mC mA mU mU * fU
UCAAGGAAGAUGGCA
XXXOOOOO

2435
* fC * fU
UUUCU
OOOOO OOOXXX

WV-
fU * fC * mA mA mG mG mA mA mG mA mU mG mG mC mA mU mU mU *
UCAAGGAAGAUGGCA
XXOOOOO

2436
fC * fU
UUUCU
OOOOO

OOOOOXX

WV-
fU * mC mA mA mG mG mA mA mG mA mU mG mG mC mA mU mU mU
UCAAGGAAGAUGGCA
XOOOOO OOOOO

2437
mC * fU
UUUCU
OOOOOOOX

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA mA mG mA mU mG mG mC * SfA *
UCAAGGAAGAUGGCA
SSSSSSOOOOOOO

2438
SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * S mG mA mA mG mA mU mG mG mC mA *
UCAAGGAAGAUGGCA
SSSSSOOOOOOOO

2439
SfU * SfU * SfU * SfC * SfU
UUUCU
OSSSSS

WV-
fU * SfC * SfA * SfA * S mG mG mA mA mG mA mU mG mG mC mA mU *
UCAAGGAAGAUGGCA
SSSSOOOOO

2440
SfU * SfU * SfC * SfU
UUUCU
OOOOO OSSSS

WV-
fU * SfC * SfA * S mA mG mG mA mA mG mA mU mG mG mC mA mU mU *
UCAAGGAAGAUGGCA
SSSOOOOO

2441
SfU * SfC * SfU
UUUCU
OOOOO OOOSSS

WV-
fU * SfC * S mA mA mG mG mA mA mG mA mU mG mG mC mA mU mU
UCAAGGAAGAUGGCA
SSOOOOO OOOOO

2442
mU * SfC * SfU
UUUCU
OOOOOSS

WV-
fU * S mC mA mA mG mG mA mA mG mA mU mG mG mC mA mU mU mU
UCAAGGAAGAUGGCA
SOOOOO OOOOO

2443
mC * SfU
UUUCU
OOOOOOOS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA * R mA * R mG * R mA * R mU *
UCAAGGAAGAUGGCA
SSSSSSRRRRRRRS

2444
R mG * R mG * R mC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * S mG * R mA * R mA * R mG * R mA * R
UCAAGGAAGAUGGCA
SSSSSRRRRRRRR

2445
mU * R mG * R mG * R mC * R mA * SfU * SfU * SfU * SfC * SfU
UUUCU
RSSSSS

WV-
fU * SfC * SfA * SfA * S mG * R mG * R mA * R mA * R mG * R mA * R
UCAAGGAAGAUGGCA
SSSSRRRRRRRRR

2446
mU * R mG * R mG * R mC * R mA * R mU * SfU * SfU * SfC * SfU
UUUCU
RRSSSS

WV-
fU * SfC * SfA * S mA * R mG * R mG * R mA * R mA * R mG * R mA *
UCAAGGAAGAUGGCA
SSSRRRRRRRRRR

2447
R mU * R mG * R mG * R mC * R mA * R mU * R mU * SfU * SfC * SfU
UUUCU
RRRSSS

WV-
fU * SfC * S mA * R mA * R mG * R mG * R mA * R mA * R mG * R mA
UCAAGGAAGAUGGCA
SSRRRRRRRRRRR

2448
* R mU * R mG * R mG * R mC * R mA * R mU * R mU * R mU * SfC *
UUUCU
RRRRSS

SfU

WV-
fU * S mC * R mA * R mA * R mG * R mG * R mA * R mA * R mG * R
UCAAGGAAGAUGGCA
SRRRRRRRRRRRR

2449
mA * R mU * R mG * R mG * R mC * R mA * R mU * R mU * R mU * R
UUUCU
RRRRRS

mC * SfU

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * R mG * R mA * R mU * R
UCAAGGAAGAUGGCA
SSSSSSSRRRRRSS

2526
mG * R mG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * S mG * R mA * R mU * R
UCAAGGAAGAUGGCA
SSSSSSSSRRRSSSS

2527
mG * SfG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mA * R mU * SfG *
UCAAGGAAGAUGGCA
SSSSSSSSSRSSSSS

2528
SfG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA mG mA mU mG mG * SfC *
UCAAGGAAGAUGGCA
SSSSSSSOOOOOSS

2529
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * S mG mA mU mG * SfG *
UCAAGGAAGAUGGCA
SSSSSSSSOOOSSS

2530
SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mA mU * SfG * SfG *
UCAAGGAAGAUGGCA
SSSSSSSSSOSSSSS

2531
SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * mA * mG * mA * mU * mG *
UCAAGGAAGAUGGCA
SSSSSSXXXXXXX

2532
mG * fC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
mU * S mC * S mA * S mA * S mG * S mG * S mA * R mA * R mG * R mA
UCAAGGAAGAUGGCA
SSSSSSRRRRRRRS

2533
* R mU * R mG * R mG * R mC * S mA * S mU * S mU * S mU * S mC * S
UUUCU
SSSSS

mU

WV-
mU * S mC * S mA * S mA * S mG * S mG * S mA * S mA * R mG * R mA *
UCAAGGAAGAUGGCA
SSSSSSSRRRRRSS

2534
R mU * R mG * R mG * S mC * S mA * S mU * S mU * S mU * S mC * S mU
UUUCU
SSSSS

WV-
mU * S mC * S mA * S mA * S mG * S mG * S mA * S mA * S mG * R mA *
UCAAGGAAGAUGGCA
SSSSSSSSRRRSSSS

2535
R mU * R mG * S mG * S mC * S mA * S mU * S mU * S mU * S mC * S mU
UUUCU
SSSS

WV-
mU * S mC * S mA * S mA * S mG * S mG * S mA * S mA * S mG * S mA *
UCAAGGAAGAUGGCA
SSSSSSSSSRSSSSS

2536
R mU * S mG * S mG * S mC * S mA * S mU * S mU * S mU * S mC * S mU
UUUCU
SSSS

WV-
mU * S mC * S mA * S mA * S mG * S mG * S mA * mA * mG * mA * mU
UCAAGGAAGAUGGCA
SSSSSSXXXXXXX

2537
* mG * mG * mC * S mA * S mU * S mU * S mU * S mC * S mU
UUUCU
SSSSSS

WV-
L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * mU
UCAAGGAAGAUGGCA
XXXXX XXXXX

2538
* mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
Mod013L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2578
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
Mod014L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2579
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
Mod005L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2580
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
Mod015L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2581
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
Mod016L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2582
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
Mod017L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2583
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
Mod018L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2584
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
Mod019L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2585
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
Mod006L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2586
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
Mod020L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2587
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
Mod021 * mU * mC * mA * mA * mG * mG * mA * mA * mG * mA *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2588
mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
mC * mA * mA * mA * mG * mA * mA * mG * mA * mU * mG * mG *
CAAAGAAGAUGGCAU
XXXXX XXXXX

2625
mC * mA * mU * mU * mU * mC * mU * mA * mG * mU * mU * mU *
UUCUA GUUUG
XXXXX XXXXX

mG

XXXX

WV-
mG * mC * mA * mA * mA * mG * mA * mA * mG * mA * mU * mG *
GCAAAGAAGAUGGCA
XXXXX XXXXX

2627
mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fG * fC * fA * fA * fA * fG * mA * mA * mG * mA * mU * mG * mG *
GCAAAGAAGAUGGCA
XXXXX XXXXX

2628
mC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * mA mA mG mA mU mG mG mC *
UCAAGGAAGAUGGCA
XXXXXXOOOOOO

2660
mA * mU * mU * mU * mC * mU
UUUCU
OXXXXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA mG mA mU mG mG * mC
UCAAGGAAGAUGGCA
XXXXXXXOOOOO

2661
* mA * mU * mU * mU * mC * mU
UUUCU
XXXXXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * mG mA mU mG * mG *
UCAAGGAAGAUGGCA
XXXXXXXXOOOX

2662
mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * mG * mA mU * mG *
UCAAGGAAGAUGGCA
XXXXX

2663
mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXOXXXXX

XXXX

WV-
mU * S mC * S mA * S mA * S mG * S mG * S mA mA mG mA mU mG mG
UCAAGGAAGAUGGCA
SSSSSSOOOOOOO

2664
mC * S mA * S mU * S mU * S mU * S mC * S mU
UUUCU
SSSSSS

WV-
mU * S mC * S mA * S mA * S mG * S mG * S mA * S mA mG mA mU mG
UCAAGGAAGAUGGCA
SSSSSSSOOOOOSS

2665
mG * S mC * S mA * S mU * S mU * S mU * S mC * S mU
UUUCU
SSSSS

WV-
mU * S mC * S mA * S mA * S mG * S mG * S mA * S mA * S mG mA mU
UCAAGGAAGAUGGCA
SSSSSSSSOOOSSS

2666
mG * S mG * S mC * S mA * S mU * S mU * S mU * S mC * S mU
UUUCU
SSSSS

WV-
mU * S mC * S mA * S mA * S mG * S mG * S mA * S mA * S mG * S mA
UCAAGGAAGAUGGCA
SSSSSSSSSOSSSSS

2667
mU * S mG * S mG * S mC * S mA * S mU * S mU * S mU * S mC * S mU
UUUCU
SSSS

WV-
fU * fC * fA * fA * fG * fG * fA * mA mG mA mU mG mG * fC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXXOOOOO

2668
fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * fG * fG * fA * fA * mG mA mU mG * fG * fC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXXXOOOX

2669
fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * fG * fG * fA * fA * fG * mA mU * fG * fG * fC * fA * fU *
UCAAGGAAGAUGGCA
XXXXX

2670
fU * fU * fC * fU
UUUCU
XXXXOXXXXX

XXXX

WV-
L001 * mG * mG * mC * mC * mA * mA * mA * mC * mC * mU * mC *
GGCCAAACCUCGGCU
XXXXX XXXXX

2733
mG * mG * mC * mU * mU * mA * mC * mC * mU
UACCU
XXXXX XXXXX

WV-
L001 * mG * mG * mC * mC * mA * mA * mA * mC * mC * mU * mC *
GGCCAAACCUC
XXXXX XXXXX

2734
mG * mG * mC * mU * mU * mA * mC * mC * mU * mG * mA * mA *
GGCUUACCUGAAAU
XXXXX XXXXX

mA * mU

XXXXX

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA mA mG mA * R mU mG mG mC *
UCAAGGAAGAUGGCA
SSSSSSOOOROOO

2737
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA mA mG * R mA * R mU * R mG
UCAAGGAAGAUGGCA
SSSSSSOORRROO

2738
mG mC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA mA * R mG * R mA * R mU * R
UCAAGGAAGAUGGCA
SSSSSSORRRRROS

2739
mG * R mG mC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA * R mA * R mG mA mU mG * R
UCAAGGAAGAUGGCA
SSSSSSRROOORRS

2740
mG * R mC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA * R mA mG mA mU mG mG * R
UCAAGGAAGAUGGCA
SSSSSSROOOOOR

2741
mC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA * S mA * S mG mA mU mG * S mG
UCAAGGAAGAUGGCA
SSSSSSSSOOOSSS

2742
* S mC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA * S mA mG mA mU mG mG * S mC
UCAAGGAAGAUGGCA
SSSSSSSOOOOOSS

2743
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA * S mA * S mG * S mA * S mU * S
UCAAGGAAGAUGGCA
SSSSSSSSSSSSSSS

2744
mG * S mG * S mC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA mA mG mA mAfU * S mG mG * SfC *
UCAAGGAAGAUGGCA
SSSSSSOOOOSOSS

2745
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA * R mA * R mG * R mA * RfU * S
UCAAGGAAGAUGGCA
SSSSSSRRRRSRSS

2746
mG * R mG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * S mG * S mG * SfAfA mG mAfU * S mG mG * SfC *
UCAAGGAAGAUGGCA
SSSSSSOOOOSOSS

2747
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * S mG * S mG * SfA * RfA * R mG * R mA * RfU * S
UCAAGGAAGAUGGCA
SSSSSSRRRRSRSS

2748
mG * R mG * SfC * SfA * SfU * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * S mA mG mA mU mG mG * SfC *
UCAAGGAAGAUGGCA
SSSSSSSOOOOOSS

2749
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * S mA * R mG * R mA * R mU * R
UCAAGGAAGAUGGCA
SSSSSSSRRRRRSS

2750
mG * R mG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
TCAAGGAAGATGGCATTTCT
TCAAGGAAGATGGCA
OOOOO OOOOO

2752

TTTCT
OOOOOOOOO

WV-
mU * S mC * S mA * S mA * SfG * SfG * S mA * R mA * R mG * R mA *
UCAAGGAAGAUGGCA
SSSSSSRRRRRRRS

2783
R mU * R mG * R mG * R mC * SfA * SfU * S mU * S mU * S mC * S mU
UUUCU
SSSSS

WV-
mU * S mC * S mA * S mA * SfG * SfG * SfA * S mA * R mG * R mA * R
UCAAGGAAGAUGGCA
SSSSSSSRRRRRSS

2784
mU * R mG * R mG * SfC * SfA * SfU * S mU * S mU * S mC * S mU
UUUCU
SSSSS

WV-
mU * S mC * S mA * S mA * SfG * SfG * SfA * SfA * S mG * R mA * R mU
UCAAGGAAGAUGGCA
SSSSSSSSRRRSSSS

2785
* R mG * SfG * SfC * SfA * SfU * S mU * S mU * S mC * S mU
UUUCU
SSSS

WV-
mU * S mC * S mA * S mA * SfG * SfG * SfA * SfA * SfG * S mA * R mU *
UCAAGGAAGAUGGCA
SSSSSSSSSRSSSSS

2786
SfG * SfG * SfC * SfA * SfU * S mU * S mU * S mC * S mU
UUUCU
SSSS

WV-
mU * S mC * S mA * S mA * SfG * SfG * S mA mA mG mA mU mG mG mC
UCAAGGAAGAUGGCA
SSSSSSOOOOOOO

2787
* SfA * SfU * S mU * S mU * S mC * S mU
UUUCU
SSSSSS

WV-
mU * S mC * S mA * S mA * SfG * SfG * SfA * S mA mG mA mU mG mG *
UCAAGGAAGAUGGCA
SSSSSSSOOOOOSS

2788
SfC * SfA * SfU * S mU * S mU * S mC * S mU
UUUCU
SSSSS

WV-
mU * S mC * S mA * S mA * SfG * SfG * SfA * SfA * S mG mA mU mG *
UCAAGGAAGAUGGCA
SSSSSSSSOOOSSS

2789
SfU * SfC * SfA * SfU * S mU * S mU * S mC * S mU
UUUCU
SSSSS

WV-
mU * S mC * S mA * S mA * SfG * SfG * SfA * SfA * SfG * S mA mU * SfG
UCAAGGAAGAUGGCA
SSSSSSSSSOSSSSS

2790
* SfG * SfC * SfA * SfU * S mU * S mU * S mC * S mU
UUUCU
SSSS

WV-
mU * S mC * S mA * SfA * SfG * SfG * S mA * R mA * R mG * R mA * R
UCAAGGAAGAUGGCA
SSSSSSRRRRRRRS

2791
mU * R mG * R mG * R mC * SfA * SfU * SfU * S mU * S mC * S mU
UUUCU
SSSSS

WV-
mU * S mC * S mA * SfA * SfG * SfG * SfA * S mA * R mG * R mA * R
UCAAGGAAGAUGGCA
SSSSSSSRRRRRSS

2792
mU * R mG * R mG * SfC * SfA * SfU * SfU * S mU * S mC * S mU
UUUCU
SSSSS

WV-
mU * S mC * S mA * SfA * SfG * SfG * SfA * SfA * S mG * R mA * R mU *
UCAAGGAAGAUGGCA
SSSSSSSSRRRSSSS

2793
R mG * SfG * SfC * SfA * SfU * SfU * S mU * S mC * S mU
UUUCU
SSSS

WV-
mU * S mC * S mA * SfA * SfG * SfG * SfA * SfA * SfG * S mA * R mU *
UCAAGGAAGAUGGCA
SSSSSSSSSRSSSSS

2794
SfG * SfG * SfC * SfA * SfU * SfU * S mU * S mC * S mU
UUUCU
SSSS

WV-
mU * S mC * S mA * SfA * SfG * SfG * SfA * SfA * S mG mA mU mG * SfG
UCAAGGAAGAUGGCA
SSSSSSSSOOOSSS

2795
* SfU * SfA * SfU * SfU * S mU * S mC * S mU
UUUCU
SSSSS

WV-
mU * S mC * S mA * SfA * SfG * SfG * SfA * SfA * SfG * S mA mU * SfG *
UCAAGGAAGAUGGCA
SSSSSSSSSOSSSSS

2796
SfG * SfC * SfA * SfU * SfU * S mU * S mC * S mU
UUUCU
SSSS

WV-
fU * fC * fA * fA * fG * fG * fA * fA * mG * mA * mU * mG * mG * fC *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2797
fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * fA * fA * mG * mA * mU * mG * fG * fC * fA
UCAAGGAAGAUGGCA
XXXXX XXXXX

2798
* fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * fA * fA * fG * mA * mU * fG * fG * fC * fA *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2799
fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * fA * mA * mG * mA * mU * mG * mG * fC *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2800
fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * fA * fG * fG * mA * mA * mG * mA * mU * mG * mG
UCAAGGAAGAUGGCA
XXXXX XXXXX

2801
* mC * fA * fU * fU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * fA * fG * fG * fA * mA * mG * mA * mU * mG * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2802
fC * fA * fU * fU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * fA * fG * fG * fA * fA * mG * mA * mU * mG * fG * fC
UCAAGGAAGAUGGCA
XXXXX XXXXX

2803
* fA * fU * fU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * fA * fG * fG * fA * fA * fG * mA * mU * fG * fG * fC *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2804
fA * fU * fU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * fA * fG * fG * fA * fA * mG mA mU mG * fG * fC * fA *
UCAAGGAAGAUGGCA
XXXXXXXXOOOX

2805
fU * fU * mU * mC * mU
UUUCU
XXXXXXX

WV-
mU * mC * mA * fA * fG * fG * fA * fA * fG * mA mU * fG * fG * fC * fA *
UCAAGGAAGAUGGCA
XXXXX

2806
fU * fU * mU * mC * mU
UUUCU
XXXXOXXXXX

XXXX

WV-
Mod024L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2807
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
Mod026L001 * mU * mC * mA * mA * mG * mG * mA * mA * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

2808
mA * mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXXX

WV-
fU * fC * fA * fA * fG * fG * mA * mA * mG * mA * BrdU * mG * mG *
UCAAGGAAGATGGCA
XXXXX XXXXX

2812
mC * fA * fU * fU * fU * fC * fC
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * fA * fA * fG * mA * BrdU * fG * fG * fC * fA *
UCAAGGAAGATGGCA
XXXXX XXXXX

2813
fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * BrdU * mG
UCAAGGAAGATGGCA
XXXXX XXXXX

2814
* mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * S mG mA BrdU mG * SfG *
UCAAGGAAGATGGCA
SSSSSSSSOOOSSS

3017
SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * fC * fA * fA * fG * fG * fA * fA * mG mA BrdU mG * fG * fC * fA * fU
UCAAGGAAGATGGCA
XXXXXXXXOOOX

3018
* fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA mA mG mA BrdU mG mG mC * SfA
UCAAGGAAGATGGCA
SSSSSSOOOOOOO

3019
* SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * fC * fA * fA * fG * fG * mA mA mG mA BrdU mG mG mC * fA * fU *
UCAAGGAAGATGGCA
XXXXXXOOOOOO

3020
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
L001 * fU * SfC * SfA * SfA * SfG * SfG * S mA mA mG mA mU mG mG mC
UCAAGGAAGAUGGCA
XSSSSSSOOOOOO

3022
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
OSSSSSS

WV-
Mod015L001 * fU * SfC * SfA * SfA * SfG * SfG * S mA mA mG mA mU mG
UCAAGGAAGAUGGCA
XSSSSSSOOOOOO

3023
mG mC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
OSSSSSS

WV-
Mod006L001 * fU * SfC * SfA * SfA * SfG * SfG * S mA mA mG mA mU mG
UCAAGGAAGAUGGCA
XSSSSSSOOOOOO

3024
mG mC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
OSSSSSS

WV-
L001 * fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * S mG mA mU mG *
UCAAGGAAGAUGGCA
XSSSSSSSSOOOSS

3025
SfG * SfC * SfA * SfU * SfU * SfU * SfC * sfU
UUUCU
SSSSSS

WV-
Mod015L001 * fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * S mG mA mU
UCAAGGAAGAUGGCA
XSSSSSSSSOOOSS

3026
mG * SfG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod006L001 * fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * S mG mA mU
UCAAGGAAGAUGGCA
XSSSSSSSSOOOSS

3027
mG * SfG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * S mG mA mU mG mG * SfC *
UCAAGGAAGAUGGCA
SSSSSSSSOOOOSS

3028
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
L001 * fU * fC * fA * fA * fG * fG * mA * mA * mG * mA * mU * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

3029
mG * mC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXXX

WV-
Mod015L001 * fU * fC * fA * fA * fG * fG * mA * mA * mG * mA * mU *
UCAAGGAAGAUGGCA
XXXXX XXXXX

3030
mG * mG * mC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXXX

WV-
Mod006L001 * fU * fC * fA * fA * fG * fG * mA * mA * mG * mA * mU *
UCAAGGAAGAUGGCA
XXXXX XXXXX

3031
mG * mG * mC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXXX

WV-
Mod020L001 * fU * fC * fA * fA * fG * fG * mA * mA * mG * mA * mU *
UCAAGGAAGAUGGCA
XXXXX XXXXX

3032
mG * mG * mC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXXX

WV-
Mod019L001 * fU * fC * fA * fA * fG * fG * mA * mA * mG * mA * mU *
UCAAGGAAGAUGGCA
XXXXX XXXXX

3033
mG * mG * mC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXXX

WV-
L001 * fU * fC * fA * fA * fG * fG * fA * fA * mG mA mU mG * fG * fC * fA
UCAAGGAAGAUGGCA
XXXXX

3034
* fU * fU * fU * fC * fU
UUUCU
XXXXOOOXXXXX

XXX

WV-
Mod015L001 * fU * fC * fA * fA * fG * fG * fA * fA * mG mA mU mG * fG *
UCAAGGAAGAUGGCA
XXXXX

3035
fC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXOOOXXXXX

XXX

WV-
Mod006L001 * fU * fC * fA * fA * fG * fG * fA * fA * mG mA mU mG * fG *
UCAAGGAAGAUGGCA
XXXXX

3036
fC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXOOOXXXXX

XXX

WV-
Mod020L001 * fU * fC * fA * fA * fG * fG * fA * fA * mG mA mU mG * fG *
UCAAGGAAGAUGGCA
XXXXX

3037
fC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXOOOXXXXX

XXX

WV-
Mod019L001 * fU * fC * fA * fA * fG * fG * fA * fA * mG mA mU mG * fG *
UCAAGGAAGAUGGCA
XXXXX

3038
fC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXOOOXXXXX

XXX

WV-
fU * fC * fA * fA * fG * fG * mA mA mG mA * mU mG mG mC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOOOXOO

3039
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mA mA mG * mA * mU * mG mG mC * fA *
UCAAGGAAGAUGGCA
XXXXXXOOXXXO

3040
fU * fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mA mA * mG * mA * mU * mG * mG mC *
UCAAGGAAGAUGGCA
XXXXXXOXXXXX

3041
fA * fU * fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mA * mA * mG mA mU mG * mG * mC * fA
UCAAGGAAGAUGGCA
XXXXXXXXOOOX

3042
* fU * fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mA * mA mG mA mU mG mG * mC * fA * fU
UCAAGGAAGAUGGCA
XXXXXXXOOOOO

3043
* fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mA * mA * mG mA mU mG * mG * mC * fA
UCAAGGAAGAUGGCA
XXXXXXXXOOOX

3044
* fU * fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mA * mA mG mA mU mG mG * mC * fA * fU
UCAAGGAAGAUGGCA
XXXXXXXOOOOO

3045
* fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mA mA mG mAfU * mG mG * fC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOOOOXO

3046
fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mA * mA * mG * mA * fU * mG * mG * fC *
UCAAGGAAGAUGGCA
XXXXX XXXXX

3047
fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * mG * mG * fAfA mG mAfU * mG mG * fC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOOOOXO

3048
fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * mG * mG * fA * fA * mG * mA * fU * mG * mG * fC *
UCAAGGAAGAUGGCA
XXXXX XXXXX

3049
fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * mA * mA * mG * mA * fU * mG * mG * fC *
UCAAGGAAGAUGGCA
XXXXX XXXXX

3050
fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * mG * mG * mG * fA * fA * mG * mA * fU * mG * mG * fC *
UCAAGGAAGAUGGCA
XXXXX XXXXX

3051
fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * mG * mG * fA * fA * mG mA mU mG * mG * fC * fA *
UCAAGGAAGAUGGCA
XXXXXXXXOOOX

3052
fU * fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * mG * mG * mA * mA * mG mAfU mG * mG * fC * fA
UCAAGGAAGAUGGCA
XXXXXXXXOOOX

3053
* fU * fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * mG * mG * fA * fA * mG * mA * mU * mG * mG * fC
UCAAGGAAGAUGGCA
XXXXX XXXXX

3054
* fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * mG * mG * mA * mA * mG * mA * fU * mG * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

3055
fC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * mG * mG * fAfA mG mA * fU * mG mG * fC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOOOXXO

3056
fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * mG * mG * fA * fA * mG * mA * fU * mG * mG * fC *
UCAAGGAAGAUGGCA
XXXXX XXXXX

3057
fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * mG * mG * fA * fA * mG * fA * fU * mG * mG * fC *
UCAAGGAAGAUGGCA
XXXXX XXXXX

3058
fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * mG * mG * fA * fA * mG mA mU mG mG * fC * fA * fU
UCAAGGAAGAUGGCA
XXXXXXXXOOOO

3059
* fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * mG * mG * fA * fA * mG mAfU * mG mG * fC * fA * fU
UCAAGGAAGAUGGCA
XXXXXXXXOOXO

3060
* fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * fC * fA * fA * mG * mG * mA * mA * mG mAfU * mG mG * fC * fA
UCAAGGAAGAUGGCA
XXXXXXXXOOXO

3061
* fU * fU * fU * fC * fU
UUUCU
XXXXXXX

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA mA mG mA mU: mG mG mC * SfA
UCAAGGAAGAUGGCA
SSSSSSOOOODOO

3070
* SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA mA: mG mA: mU mG: mG mC *
UCAAGGAAGAUGGCA
SSSSSSODODODO

3071
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA: mA mG: mA mU: mG mG: mC *
UCAAGGAAGAUGGCA
SSSSSSDODODOD

3072
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA: mA mG mA mU: mG mG: mC *
UCAAGGAAGAUGGCA
SSSSSSDOOODOD

3073
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * fG:fG: mA mA mG mA mU: mG mG mC * SfA * SfU *
UCAAGGAAGAUGGCA
SSSXDDOOOODO

3074
SfU * SfU * SfC * SfU
UUUCU
OSSSSSS

WV-
fU * SfC * SfA * SfA * mG: mG: mA mA mG mA mU: mG mG mC * SfA *
UCAAGGAAGAUGGCA
SSSXDDOOOODO

3075
SfU * SfU * SfU * SfC * SfU
UUUCU
OSSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * S mA mG mA mU: mG mG * SfC *
UCAAGGAAGAUGGCA
SSSSSSSOOODOSS

3076
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * fG:fG:fA * S mA mG mA mU: mG mG * SfC * SfA *
UCAAGGAAGAUGGCA
SSSXDDSOOODOS

3077
SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * mG: mG:fA * S mA mG mA mU: mG mG * SfC * SfA
UCAAGGAAGAUGGCA
SSSXDDSOOODOS

3078
* SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * S mG mA mU: mG * SfG *
UCAAGGAAGAUGGCA
SSSSSSSSOODSSS

3079
SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * S mG: mA: mU: mG * SfG *
UCAAGGAAGAUGGCA
SSSSSSSSDDDSSS

3080
SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * S mG: mA mU: mG * SfG *
UCAAGGAAGAUGGCA
SSSSSSSSDODSSS

3081
SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * fG:fG:fA * SfA * S mG mA mU: mG * SfG * SfC * SfA
UCAAGGAAGAUGGCA
SSSXDDSSOODSS

3082
* SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * mG: mG:fA * SfA * S mG mA mU: mG * SfG * SfC *
UCAAGGAAGAUGGCA
SSSXDDSSOODSS

3083
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod015L001 mU * mC * mA * mA * mG * mG * mA * mA * mG * mA *
UCAAGGAAGAUGGCA
OXXXXX XXXXX

3084
mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
Mod019L001 mU * mC * mA * mA * mG * mG * mA * mA * mG * mA *
UCAAGGAAGAUGGCA
OXXXXX XXXXX

3085
mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
Mod020L001 mU * mC * mA * mA * mG * mG * mA * mA * mG * mA *
UCAAGGAAGAUGGCA
OXXXXX XXXXX

3086
mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
Mod015L001: mU * mC * mA * mA * mG * mG * mA * mA * mG * mA
UCAAGGAAGAUGGCA
DXXXXX XXXXX

3087
* mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
Mod019L001: mU * mC * mA * mA * mG * mG * mA * mA * mG * mA
UCAAGGAAGAUGGCA
DXXXXX XXXXX

3088
* mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
Mod020L001: mU * mC * mA * mA * mG * mG * mA * mA * mG * mA
UCAAGGAAGAUGGCA
DXXXXX XXXXX

3089
* mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fU * SfC * SfA * SfA * SfG:fG: mA mA mG mA mU: mG mG mC * SfA * SfU
UCAAGGAAGAUGGCA
SSSSDDOOOODOO

3113
* SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * S mG: mG: mA mA mG mA mU: mG mG mC * SfA *
UCAAGGAAGAUGGCA
SSSSDDOOOODOO

3114
SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG:fG:fA * S mA mG mA mU: mG * SfC * SfA *
UCAAGGAAGAUGGCA
SSSSDDSOOODOS

3115
SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * S mG: mG:fA * S mA mG mA mU: mG mG * SfC *
UCAAGGAAGAUGGCA
SSSSDDSOOODOS

3116
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG:fG:fA * SfA * S mG mA mU: mG * SfG * SfC *
UCAAGGAAGAUGGCA
SSSSDDSSOODSSS

3117
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * S mG: mG:fA * SfA * S mG mA mU: mG * SfG * SfC *
UCAAGGAAGAUGGCA
SSSSDDSSOODSSS

3118
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mA mU mG * SfG *
UCAAGGAAGAUGGCA
SSSSSSSSSOOSSSS

3120
SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSS

WV-
fU * fC * fA * fA * fG * fG * fA * fA * fG * mA mU mG * fG * fC * fA * fU *
UCAAGGAAGAUGGCA
XXXXX

3121
fU * fU * fC * fU
UUUCU
XXXXOOXXXXXX

XX

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * S mUfG * S mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSOSOS

3152
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * S mGfA * S mUfG * S mG *
UCAAGGAAGAUGGCA
SSSSSSSSOSOSSSS

3153
SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSS

WV-
L001 mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * mU *
UCAAGGAAGAUGGCA
OXXXXX XXXXX

3357
mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
L001fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mA mU * SfG *
UCAAGGAAGAUGGCA
OSSSSSSSSSOSSSS

3358
SfG * SfC * SfA* SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
Mod013L001 mU * mC * mA * mA * mG * mG * mA * mA * mG * mA *
UCAAGGAAGAUGGCA
OXXXXX XXXXX

3359
mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
Mod013L001fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mA mU
UCAAGGAAGAUGGCA
OSSSSSSSSSOSSSS

3360
* SfG * SfG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
Mod014L001fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mA mU
UCAAGGAAGAUGGCA
OSSSSSSSSSOSSSS

3361
* SfG * SfG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
Mod005L001fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mA mU
UCAAGGAAGAUGGCA
OSSSSSSSSSOSSSS

3362
* SfG * SfG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
Mod015L001fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mA mU
UCAAGGAAGAUGGCA
OSSSSSSSSSOSSSS

3363
* SfG * SfG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
Mod020L001fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mA mU
UCAAGGAAGAUGGCA
OSSSSSSSSSOSSSS

3364
* SfG * SfG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
Mod027L001fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mA mU
UCAAGGAAGAUGGCA
OSSSSSSSSSOSSSS

3365
* SfG * SfG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
Mod029L001fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mA mU
UCAAGGAAGAUGGCA
OSSSSSSSSSOSSSS

3366
* SfG * SfG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfGfA * S mAfG * S mAfU * S mGfGfC * SfA *
UCAAGGAAGAUGGCA
SSSSSOSOSOSOOS

3463
SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfAfG * S mAfU * S mG * S mG *
UCAAGGAAGAUGGCA
SSSSSSSOSOSSSSS

3464
SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mAfU * S mG * SfG *
UCAAGGAAGAUGGCA
SSSSSSSSSOSSSS5

3465
SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * S mG mAfU * S mG mG *
UCAAGGAAGAUGGCA
SSSSSSSSOOSOSS

3466
SfC * SfA * SfU * SfU * SfU * SfC * SfG
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SfA * SfA * SfG * S mAfU * S mGfG *
UCAAGGAAGAUGGCA
SSSSSSSSSOSOSSS

3467
SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * mA mA mG mAfU * S mG mG * SfC *
UCAAGGAAGAUGGCA
SSSSSXOOOOSOS

3468
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mA * S mA * S mG * S mA * SfU * S
UCAAGGAAGAUGGCA
SSSSSSSSSSSSSSS

3469
mG * S mG * SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * SfUfG * S mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSOSOS

3470
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * SfU mG mGfC * SfA
UCAAGGAAGAUGGCA
SSSSSSOSOSOOOS

3471
* SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * SfU * S mG mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSSOOS

3472
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSSOOS

3473
SfA * SfG * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfAfU * S mG mGfC * SfA
UCAAGGAAGAUGGCA
SSSSSSOSOOSOOS

3506
* SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mAfU * S mG mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOOSOOS

3507
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * SfU * S mG mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSSOOS

3508
SfAfU * SfU * SfU * SfC * SfU
UUUCU
OSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSSOOS

3509
SfAfU * SfU * SfU * SfC * SfU
UUUCU
OSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfAfU * S mG mGfC * S
UCAAGGAAGAUGGCA
SSSSSSOSOOSOOS

3510
mA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG* SfG* S mAfA * S mG mAfU * S mG mGfC * S
UCAAGGAAGAUGGCA
SSSSSSOSOOSOOS

3511
mA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfAfU * S mG mGfC * S
UCAAGGAAGAUGGCA
SSSSSSOSOOSOOS

3512
mAfU * SfU * SfU * SfC * SfU
UUUCU
OSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mAfU * S mG mGfC * S
UCAAGGAAGAUGGCA
SSSSSSOSOOSOOS

3513
mAfU * SfU * SfU * SfC * SfU
UUUCU
OSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfAfU * S mG mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOOSOOS

3514
SfAfU * SfU * SfU * SfC * SfU
UUUCU
OSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mAfU * S mG mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOOSOOS

3515
SfAfU * SfU * SfU * SfC * SfU
UUUCU
OSSSS

WV-
fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXOX

3516
* fU * fC * fU
UUUCU
OXXXXXX

WV-
Mod030fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA *
UCAAGGAAGAUGGCA
OXXXXXXOXOXO

3517
fU * fU * fU * fC * fU
UUUCU
XOXXXXXX

WV-
Mod031fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA *
UCAAGGAAGAUGGCA
OXXXXXXOXOXO

3518
fU * fU * fU * fC * fU
UUUCU
XOXXXXXX

WV-
Mod032fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA *
UCAAGGAAGAUGGCA
OXXXXXXOXOXO

3519
fU * fU * fU * fC * fU
UUUCU
XOXXXXXX

WV-
Mod033fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA *
UCAAGGAAGAUGGCA
OXXXXXXOXOXO

3520
fU * fU * fU * fC * fU
UUUCU
XOXXXXXX

WV-
Mod013L001fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU *
UCAAGGAAGAUGGCA
OSSSSSSOSOSSOO

3543
S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod005L001fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU *
UCAAGGAAGAUGGCA
OSSSSSSOSOSSOO

3544
S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod015L001fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU *
UCAAGGAAGAUGGCA
OSSSSSSOSOSSOO

3545
S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod020L001fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfG *
UCAAGGAAGAUGGCA
OSSSSSSOSOSSOO

3546
S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod027L001fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU *
UCAAGGAAGAUGGCA
OSSSSSSOSOSSOO

3547
S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod029L001fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU *
UCAAGGAAGAUGGCA
OSSSSSSOSOSSOO

3548
S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod030fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S
UCAAGGAAGAUGGCA
OSSSSSSOSOSSOO

3549
mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod032fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S
UCAAGGAAGAUGGCA
OSSSSSSOSOSSOO

3550
mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod033fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S
UCAAGGAAGAUGGCA
OSSSSSSOSOSSOO

3551
mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod020L001 * fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfG
UCAAGGAAGAUGGCA
OXSSSSSSOSOSSO

3552
* S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
OSSSSSS

WV-
Mod005L001 * fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU
UCAAGGAAGAUGGCA
OXSSSSSSOSOSSO

3553
* S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
OSSSSSS

WV-
Mod014L00lfU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfG *
UCAAGGAAGAUGGCA
OOSSSSSSOSOSSO

3554
S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
OSSSSSS

WV-
Mod030 * fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S
UCAAGGAAGAUGGCA
XSSSSSSOSOSSOO

3555
mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod032 * fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S
UCAAGGAAGAUGGCA
XSSSSSSOSOSSOO

3556
mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod033 * fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfG * S
UCAAGGAAGAUGGCA
XSSSSSSOSOSSOO

3557
mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod033 * fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC *
UCAAGGAAGAUGGCA
XXXXXXXOXOXO

3558
fA * fU * fU * fU * fC * fU
UUUCU
XOXXXXXX

WV-
Mod020L001fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC *
UCAAGGAAGAUGGCA
OXXXXXXOXOXO

3559
fA * fU * fU * fU * fC * fU
UUUCU
XOXXXXXX

WV-
Mod020L001 * fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG *
UCAAGGAAGAUGGCA
XXXXXXXOXOXO

3560
mGfC * fA * fU * fU * fU * fC * fU
UUUCU
XOXXXXXX

WV-
L001 * fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG
UCAAGGAAGAUGGCA
XSSSSSSOSOSSOO

3753
mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
L00lfU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG
UCAAGGAAGAUGGCA
OSSSSSSOSOSSOO

3754
mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
L001 * fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA *
UCAAGGAAGAUGGCA
XXXXXXXOXOXO

3820
fU * fU * fU * fC * fU
UUUCU
XOXXXXXX

WV-
L001fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA * fU
UCAAGGAAGAUGGCA
OXXXXXXOXOXO

3821
* fU * fU * fC * fU
UUUCU
XOXXXXXX

WV-
Mod015L001 * fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG *
UCAAGGAAGAUGGCA
XXXXXXXOXOXO

3855
mGfC * fA * fU * fU * fU * fC * fU
UUUCU
XOXXXXXX

WV-
Mod015L001fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC *
UCAAGGAAGAUGGCA
OXXXXXXOXOXO

3856
fA * fU * fU * fU * fC * fU
UUUCU
XOXXXXXX

WV-
Mod033L001 * fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU
UCAAGGAAGAUGGCA
XSSSSSSOSOSSOO

3971
* S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod015L001 * fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU
UCAAGGAAGAUGGCA
XSSSSSSOSOSSOO

4106
* S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod015L001 * SfU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA *
UCAAGGAAGAUGGCA
SSSSSSSOSOSSOO

4107
SfG * S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfG
UUUCU
SSSSSS

WV-
L001 * SfU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S
UCAAGGAAGAUGGCA
SSSSSSSOSOSSOO

4191
mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSSOOS

4231
SfA * SfU * SfU * SfU * SfC
UUUC
SSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSSOOS

4232
SfA * SfU * SfU * SfU
UUU
SSS

WV-
fC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG mGfC * SfA *
CAAGGAAGAUGGCAU
SSSSSOSOSSOOSS

4233
SfU * SfU * SfU * SfC * SfU
UUCU
SSSS

WV-
Mod020L001 mG * mG * mC * mC * mA * mA * mA * mC * mC * mU *
GGCCAAACCUCGGCU
OXXXXX XXXXX

4610
mC * mG * mG * mC * mU * mU * mA * mC * mC * mU
UACCU
XXXXX XXXX

WV-
Mod015L001 mG * mG * mC * mC * mA * mA * mA * mC * mC * mU *
GGCCAAACCUCGGCU
OXXXXX XXXXX

4611
mC * mG * mG * mC * mU * mU * mA * mC * mC * mU
UACCU
XXXXX XXXX

WV-
fU * fU * fC * fU * fG * fU * mA * mA * mG * mG * mU * mU * mU *
UUCUGUAAGGUUUU
XXXXX XXXXX

4614
mU * fU * fA * fU * fG * fU * fG
UAUGUG
XXXXX XXXX

WV-
fA * fU * fU * fU * fC * fU * mG * mU * mA * mA * mG * mG * mU *
AUUUCUGUAAGGUU
XXXXX XXXXX

4615
mU * fU * fU * fU * fA * fU * fU
UUUAUG
XXXXX XXXX

WV-
fC * fC * fA * fU * fU * fU * mC * mU * mG * mU * mA * mA * mG *
CCAUUUCUGUAAGGU
XXXXX XXXXX

4616
mG * fU * fU * fU * fU * fU * fA
UUUUA
XXXXX XXXX

WV-
fA * fU * fU * fC * fA * fU * mU * mU * mC * mU * mG * mU * mA *
AUCCAUUUCUGUAAG
XXXXX XXXXX

4617
mA * fG * fG * fU * fU * fU * fU
GUUUU
XXXXX XXXX

WV-
fC * fA * fU * fC * fC * fA * mU * mU * mU * mC * mU * mG * mU *
CAUCCAUUUCUGUAA
XXXXX XXXXX

4618
mA * fA * fG * fG * fU * fU * fU
GGUUU
XXXXX XXXX

WV-
fC * fC * fA * fU * fC * fC * mA * mU * mU * mU * mC * mU * mG *
CCAUCCAUUUCUGUA
XXXXX XXXXX

4619
mU * fA * fA * fG * fG * fU * fU
AGGUU
XXXXX XXXX

WV-
fG * fC * fC * fA * fU * fC * mC * mA * mU * mU * mU * mC * mU *
GCCAUCCAUUUCUGU
XXXXX XXXXX

4620
mG * fU * fA * fA * fG * fG * fU
AAGGU
XXXXX XXXX

WV-
fA * fG * fC * fC * fA * fU * mC * mC * mA * mU * mU * mU * mC *
AGCCAUCCAUUUCUG
XXXXX XXXXX

4621
mU * fG * fU * fA * fA * fG * fG
UAAGG
XXXXX XXXX

WV-
fC * fA * fG * fC * fC * fA * mU * mC * mC * mA * mU * mU * mU *
CAGCCAUCCAUUUCU
XXXXX XXXXX

4622
mC * fU * fG * fU * fA * fA * fG
GUAAG
XXXXX XXXX

WV-
fU * fC * fA * fG * fC * fC * mA * mU * mC * mC * mA * mU * mU *
UCAGCCAUCCAUUUC
XXXXX XXXXX

4623
mU * fC * fU * fG * fU * fA * fA
UGUAA
XXXXX XXXX

WV-
fU * fU * fC * fA * fG * fC * mC * mA * mU * mC * mC * mA * mU *
UUCAGCCAUCCAUUU
XXXXX XXXXX

4624
mU * fU * fU * fU * fG * fU * fA
CUGUA
XXXXX XXXX

WV-
fC * fU * fU * fC * fA * fG * mC * mC * mA * mU * mC * mC * mA *
CUUCAGCCAUCCAUU
XXXXX XXXXX

4625
mU * fU * fU * fC * fU * fG * fU
UCUGU
XXXXX XXXX

WV-
fA * fC * fU * fU * fC * fA * mG *mC * mC * mA * mU * mC * mC *
ACUUCAGCCAUCCAU
XXXXX XXXXX

4626
mA * fU * fU * fU * fC * fU * fG
UUCUG
XXXXX XXXX

WV-
fA * fA * fC * fU * fU * fC * mA * mG * mC * mC * mA * mU * mC *
AACUUCAGCCAUCCA
XXXXX XXXXX

4627
mC * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fC * fA * fA * fC * fU * fU * mC * mA * mG * mC * mC * mA * mU *
CAACUUCAGCCAUCC
XXXXX XXXXX

4628
mC * fC * fA * fU * fU * fU * fC
AUUUC
XXXXX XXXX

WV-
fU * fC * fA * fA * fC * fU * mU * mC * mA * mG * mC * mC * mA *
UCAACUUCAGCCAUC
XXXXX XXXXX

4629
mU * fC * fC * fA * fU * fU * fU
CAUUU
XXXXX XXXX

WV-
fA * fU * fC * fA * fA * fC * mU * mU * mC * mA * mG * mC * mC *
AUCAACUUCAGCCAU
XXXXX XXXXX

4630
mA * fU * fC * fC * fA * fU * fU
CCAUU
XXXXX XXXX

WV-
fC * fA * fU * fC * fA * fA * mC * mU * mU * mC * mA * mG * mC *
CAUCAACUUCAGCCA
XXXXX XXXXX

4631
mC * fA * fU * fC * fC * fA * fU
UCCAU
XXXXX XXXX

WV-
fA * fC * fA * fU * fC * fA * mA * mC * mU * mU * mC * mA * mG *
ACAUCAACUUCAGCC
XXXXX XXXXX

4632
mC * fC * fA * fU * fC * fC * fA
AUCCA
XXXXX XXXX

WV-
fA * fA * fC * fA * fU * fC * mA * mA * mC * mU * mU * mC * mA *
AACAUCAACUUCAGC
XXXXX XXXXX

4633
mG * fC * fC * fA * fU * fC * fC
CAUCC
XXXXX XXXX

WV-
fG * fA * fA * fA * fA * fC * mA * mU * mC * mA * mA * mC * mU *
GAAAACAUCAACUUC
XXXXX XXXXX

4634
mU * fC * fA * fG * fC * fC * fA
AGCCA
XXXXX XXXX

WV-
fC * fA * fG * fG * fA * fA * mA * mA * mC * mA * mU * mC * mA *
CAGGAAAACAUCAAC
XXXXX XXXXX

4635
mA * fC * fU * fU * fC * fA * fG
UUCAG
XXXXX XXXX

0

WV-
fU * fU * fU * fC * fA * fG * mG * mA * mA * mA * mA * mC * mA *
UUUCAGGAAAACAUG
XXXXX XXXXX

4636
mU * fC * fA * fA * fC * fU * fU
AACUU
XXXXX XXXX

WV-
fC * fU * fC * fU * fU * fU * mC * mA * mG * mG * mA * mA * mA *
CUCUUUCAGGAAAAC
XXXXX XXXXX

4637
mA * fC * fA * fU * fC * fA * fA
AUCAA
XXXXX XXXX

WV-
fU * fU * fC * fC * fU * fC * mU * mU * mU * mC * mA * mG * mG *
UUCCUCUUUCAGGAA
XXXXX XXXXX

4638
mA * fA * fA * fA * fC * fA * fU
AACAU
XXXXX XXXX

WV-
fG * fC * fC * fA * fU * fU * mC * mC * mU * mC * mU * mU * mU *
GCCAUUCCUCUUUCA
XXXXX XXXXX

4639
mC * fA * fG * fG * fA * fA * fA
GGAAA
XXXXX XXXX

WV-
fG * fG * fC * fC * fA * fU * mU * mC * mC * mU * mC * mU * mU *
GGCCAUUCCUCUUUC
XXXXX XXXXX

4640
mU * fC * fA * fG * fG * fA * fA
AGGAA
XXXXX XXXX

WV-
fA * fG * fG * fC * fC * fA * mU * mU * mC * mC * mU * mC * mU *
AGGCCAUUCCUCUUU
XXXXX XXXXX

4641
mU * fU * fC * fA * fG * fG * fA
CAGGA
XXXXX XXXX

WV-
fC * fA * fG * fG * fC * fU * mA * mU * mU * mC * mC * mU * mC *
CAGGCCAUUCCUCUU
XXXXX XXXXX

4642
mU * fU * fU * fC * fA * fG * fG
UCAGG
XXXXX XXXX

WV-
fG * fC * fA * fG * fG * fC * mC * mA * mU * mU * mC * mC * mU *
GCAGGCCAUUCCUCU
XXXXX XXXXX

4643
mC * fU * fU * fU * fC * fA * fG
UUCAG
XXXXX XXXX

WV-
fG * fG * fC * fA * fG * fG * mC * mC * mA * mU * mU * mC * mC *
GGCAGGCCAUUCCUC
XXXXX XXXXX

4644
mU * fC * fU * fU * fU * fC * fA
UUUCA
XXXXX XXXX

WV-
fG * fG * fG * fC * fA * fG * mG * mC * mC * mA * mU * mU * mC *
GGGCAGGCCAUUCCU
XXXXX XXXXX

4645
mC * fU * fC * fU * fU * fU * fC
CUUUC
XXXXX XXXX

WV-
fA * fG * fG * fG * fC * fA * mG * mG * mC * mC * mA * mU * mU *
AGGGCAGGCCAUUCC
XXXXX XXXXX

4646
mC * fC * fU * fC * fU * fU * fU
UCUUU
XXXXX XXXX

WV-
fC * fA * fG * fG * fG * fC * mA * mG * mG * mC * mC * mA * mU *
CAGGGCAGGCCAUUC
XXXXX XXXXX

4647
mU * fC * fC * fU * fC * fU * fU
CUCUU
XXXXX XXXX

WV-
fC * fC * fA * fG * fG * fG * mC * mA * mG * mG * mC * mC * mA *
CCAGGGCAGGCCAUU
XXXXX XXXXX

4648
mU * fU * fC * fC * fU * fC * fU
CCUCU
XXXXX XXXX

WV-
fC * fC * fC * fA * fG * fG * mG * mC * mA * mG * mG * mC * mC *
CCCAGGGCAGGCCAU
XXXXX XXXXX

4649
mA * fU * fU * fC * fC * fU * fC
UCCUC
XXXXX XXXX

WV-
fC * fC * fC * fC * fA * fG * mG * mG * mC * mA * mG * mG * mC * mC
CCCCAGGGCAGGCCA
XXXXX XXXXX

4650
* fA * fU * fU * fC * fC * fU
UUCCU
XXXXX XXXX

WV-
fC * fC * fC * fC * fC * fA * mG * mG * mG * mC * mA * mG * mG * mC
CCCCCAGGGCAGGCC
XXXXX XXXXX

4651
* fC * fA * fU * fU * fC * fC
AUUCC
XXXXX XXXX

WV-
fU * fC * fC * fC * fC * fC * mA * mG * mG * mG * mC * mA * mG *
UCCCCCAGGGCAGGC
XXXXX XXXXX

4652
mG * fC * fC * fA * fU * fU * fC
CAUUC
XXXXX XXXX

WV-
fA * fU * fC * fC * fC * fC * mC * mA * mG * mG * mG * mC * mA *
AUCCCCCAGGGCAGG
XXXXX XXXXX

4653
mG * fG * fU * fC * fA * fU * fU
CCAUU
XXXXX XXXX

WV-
fC * fA * fU * fC * fC * fC * mC * mC * mA * mG * mG * mG * mC * mA
CAUCCCCCAGGGCAG
XXXXX XXXXX

4654
* fG * fG * fC * fC * fA * fU
GCCAU
XXXXX XXXX

WV-
fG * fC * fA * fU * fC * fC * mC * mC * mC * mA * mG * mG * mG * mC
GCAUCCCCCAGGGCA
XXXXX XXXXX

4655
* fA * fG * fG * fC * fC * fA
GGCCA
XXXXX XXXX

WV-
fA * fG * fC * fA * fU * fC * mC * mC * mC * mC * mA * mG * mG *
AGCAUCCCCCAGGGC
XXXXX XXXXX

4656
mG * fC * fA * fG * fG * fC * fC
AGGCC
XXXXX XXXX

WV-
fC * fA * fG * fC * fA * fU * mC * mC * mC * mC * mC * mA * mG * mG
CAGCAUCCCCCAGGG
XXXXX XXXXX

4657
* fG * fC * fA * fG * fG * fC
CAGGC
XXXXX XXXX

WV-
fU * fC * fA * fG * fC * fA * mU * mC * mC * mC * mC * mC * mA * mG
UCAGCAUCCCCCAGG
XXXXX XXXXX

4658
* fG * fG * fC * fA * fG * fG
GCAGG
XXXXX XXXX

WV-
fU * fU * fC * fA * fG * fC * mA * mU * mC * mC * mC * mC * mC * mA
UUCAGCAUCCCCCAG
XXXXX XXXXX

4659
* fG * fG * fG * fC * fA * fG
GGCAG
XXXXX XXXX

WV-
fU * fU * fU * fC * fA * fG * mC * mA * mU * mC * mC * mC * mC * mC
UUUCAGCAUCCCCCA
XXXXX XXXXX

4660
* fA * fG * fG * fG * fC * fA
GGGCA
XXXXX XXXX

WV-
fU * fU * fU * fU * fC * fA * mG * mC * mA * mU * mC * mC * mC *
AUUUCAGCAUCCCCC
XXXXX XXXXX

4661
mC * fC * fA * fG * fG * fG * fC
AGGGC
XXXXX XXXX

WV-
fG * fA * fU * fU * fU * fC * mA * mG * mC * mA * mU * mC * mC *
GAUUUCAGCAUCCCC
XXXXX XXXXX

4662
mC * fC * fC * fA * fG * fG * fG
CAGGG
XXXXX XXXX

WV-
fG * fG * fA * fU * fU * fU * mC * mA * mG * mC * mA * mU * mC *
GGAUUUCAGCAUCCC
XXXXX XXXXX

4663
mC * fC * fC * fC * fA * fG * fG
CCAGG
XXXXX XXXX

WV-
fA * fG * fG * fA * fU * fU * mU * mC * mA * mG * mC * mA * mU *
AGGAUUUCAGCAUCC
XXXXX XXXXX

4664
mC * fC * fC * fC * fC * fA * fG
CCCAG
XXXXX XXXX

WV-
fC * fA * fG * fG * fA * fU * mU * mU * mC * mA * mG * mC * mA *
CAGGAUUUCAGCAUC
XXXXX XXXXX

4665
mU * fC * fC * fC * fC * fC * fA
CCCCA
XXXXX XXXX

WV-
fU * fC * fA * fG * fG * fA * mU * mU * mU * mC * mA * mG * mC *
UCAGGAUUUCAGCAU
XXXXX XXXXX

4666
mA * fU * fC * fC * fC * fC * fC
CCCCC
XXXXX XXXX

WV-
fU * fU * fC * fA * fG * fG * mA * mU * mU * mU * mC * mA * mG *
UUCAGGAUUUCAGCA
XXXXX XXXXX

4667
mC * fA * fU * fC * fC * fC * fC
UCCCC
XXXXX XXXX

WV-
fU * fU * fU * fC * fA * fG * mG * mA * mU * mU * mU * mC * mA *
UUUCAGGAUUUCAGC
XXXXX XXXXX

4668
mG * fC * fA * fU * fC * fC * fC
AUCCC
XXXXX XXXX

WV-
fU * fU* fU * fU * fC * fA * mG * mG * mA * mU * mU * mU * mC *
UUUUCAGGAUUUCAG
XXXXX XXXXX

4669
mA * fG * fC * fA * fU * fC * fC
CAUCC
XXXXX XXXX

WV-
fU * fU * fU * fU * fU * fC * mA * mG * mG * mA * mU * mU * mU *
UUUUUCAGGAUUUCA
XXXXX XXXXX

4670
mC * fA * fG * fC * fA * fU * fC
GCAUC
XXXXX XXXX

WV-
fU * fU * fU * fU * fU * fU * mC * mA * mG * mG * mA * mU * mU *
UUUUUUCAGGAUUUC
XXXXX XXXXX

4671
mU * fC * fA * fG * fC * fA * fU
AGCAU
XXXXX XXXX

WV-
fG * fU * fU * fU * fU * fU * mU * mC * mA * mG * mG * mA * mU *
GUUUUUUCAGGAUU
XXXXX XXXXX

4672
mU * fU * fC * fA * fG * fC * fA
UCAGCA
XXXXX XXXX

WV-
fU * fG * fU * fU * fU * fU * mU * mU * mC * mA * mG * mG * mA *
UGUUUUUUCAGGAU
XXXXX XXXXX

4673
mU * fU * fU * fC * fA * fG * fC
UUCAGC
XXXXX XXXX

WV-
fC * fU * fG * fU * fU * fU * mU * mU * mU * mC * mA * mG * mG *
CUGUUUUUUCAGGAU
XXXXX XXXXX

4674
mA * fU * fU * fU * fC * fA * fG
UUCAG
XXXXX XXXX

WV-
fG * fC * fU * fG * fU * fU * mU * mU * mU * mU * mC * mA * mG *
GCUGUUUUUUCAGGA
XXXXX XXXXX

4675
mG * fA * fU * fU * fU * fC * fA
UUUCA
XXXXX XXXX

WV-
fA * fG * fC * fU * fG * fU * mU * mU * mU * mU * mU * mC * mA *
AGCUGUUUUUUCAGG
XXXXX XXXXX

4676
mG * fG * fA * fU * fU * fU * fC
AUUUC
XXXXX XXXX

WV-
fG * fA * fG * fC * fU * fG * mU * mU * mU * mU * mU * mU * mC *
GAGCUGUUUUUUCAG
XXXXX XXXXX

4677
mA * fG * fG * fA * fU * fU * fU
GAUUU
XXXXX XXXX

WV-
fU * fG * fA * fG * fC * fU * mG * mU * mU * mU * mU * mU * mU *
UGAGCUGUUUUUUCA
XXXXX XXXXX

4678
mC * fA * fG * fG * fA * fU * fU
GGAUU
XXXXX XXXX

WV-
fU * fU * fG * fA * fG * fC * mU * mG * mU * mU * mU * mU * mU *
UUGAGCUGUUUUUUC
XXXXX XXXXX

4679
mU * fC * fA * fG * fG * fA * fU
AGGAU
XXXXX XXXX

WV-
fU * fU * fU * fG * fA * fG * mC * mU * mG * mU * mU * mU * mU *
UUUGAGCUGUUUUU
XXXXX XXXXX

4680
mU * fU * fC * fA * fG * fG * fA
UCAGGA
XXXXX XXXX

WV-
fG * fU * fU * fU * fG * fA * mG * mC * mU * mG * mU * mU * mU *
GUUUGAGCUGUUUU
XXXXX XXXXX

4681
mU * fU * fU * fC * fA * fG * fG
UUCAGG
XXXXX XXXX

WV-
fU * fU * fG * fU * fU * fU * mG * mA * mG * mC * mU * mG * mU *
UUGUUUGAGCUGUU
XXXXX XXXXX

4682
mU * fU * fU * fU * fU * fC * fA
UUUUCA
XXXXX XXXX

WV-
fC * fA * fU * fU * fG * fU * mU * mU * mG * mA * mG * mC * mU *
CAUUGUUUGAGCUGU
XXXXX XXXXX

4683
mG * fU * fU * fU * fU * fU * fU
UUUUU
XXXXX XXXX

WV-
fG * fC * fA * fU * fU * fG * mU * mU * mU * mG * mA * mG * mC *
GCAUUGUUUGAGCUG
XXXXX XXXXX

4684
mU * fG * fU * fU * fU * fU * fU
UUUUU
XXXXX XXXX

WV-
fU * fG * fC * fA * fU * fU * mG * mU * mU * mU * mG * mA * mG *
UGCAUUGUUUGAGCU
XXXXX XXXXX

4685
mC * fU * fG * fU * fU * fU * fU
GUUUU
XXXXX XXXX

WV-
fC * fU * fG * fC * fA * fU * mU * mG * mU * mU * mU * mG * mA *
CUGCAUUGUUUGAGC
XXXXX XXXXX

4686
mG * fC * fU * fG * fU * fU * fU
UGUUU
XXXXX XXXX

WV-
fU * fC * fU * fG * fC * fA * mU * mU * mG * mU * mU * mU * mG *
UCUGCAUUGUUUGAG
XXXXX XXXXX

4687
mA * fG * fC * fU * fG * fU * fU
CUGUU
XXXXX XXXX

WV-
fC * fU * fC * fU * fG * fC * mA * mU * mU * mG * mU * mU * mU *
CUCUGCAUUGUUUGA
XXXXX XXXXX

4688
mG * fA * fG * fC * fU * fG * fU
GCUGU
XXXXX XXXX

WV-
fA * fC * fU * fC * fU * fG * mC * mA * mU * mU * mG * mU * mU *
ACUCUGCAUUGUUUG
XXXXX XXXXX

4689
mU * fG * fA * fG * fC * fU * fG
AGCUG
XXXXX XXXX

WV-
fU * fA * fC * fU * fC * fU * mG * mC * mA * mU * mU * mG * mU *
UACUCUGCAUUGUUU
XXXXX XXXXX

4690
mU * fU * fG * fA * fG * fC * fU
GAGCU
XXXXX XXXX

WV-
fG * fU * fA * fC * fU * fC * mU * mG * mC * mA * mU * mU * mG *
UUACUCUGCAUUGUU
XXXXX XXXXX

4691
mU * fU * fU * fG * fA * fG * fC
UGAGC
XXXXX XXXX

WV-
fC * fU * fU * fA * fC * fU * mC * mU * mG * mC * mA * mU * mU *
CUUACUCUGCAUUGU
XXXXX XXXXX

4692
mG * fU * fU * fU * fG * fA * fG
UUGAG
XXXXX XXXX

WV-
fU * fC * fU * fU * fA * fC * mU * mC * mU * mG * mC * mA * mU *
UCUUACUCUGCAUUG
XXXXX XXXXX

4693
mU * fG * fU * fU * fU * fG * fA
UUUGA
XXXXX XXXX

WV-
fA * fU * fC * fU * fU * fA * mC * mU * mC * mU * mG * mC * mA *
AUCUUACUCUGCAUU
XXXXX XXXXX

4694
mU * fU * fG * fU * fU * fU * fG
GUUUG
XXXXX XXXX

WV-
fA * fA * fU * fC * fU * fU * mA * mC * mU * mC * mU * mG * mC *
AAUCUUACUCUGCAU
XXXXX XXXXX

4695
mA * fU * fU * fG * fU * fU * fU
UGUUU
XXXXX XXXX

WV-
fC * fA * fA * fA * fU * fC * mU * mU * mA * mC * mU * mC * mU *
CAAAUCUUACUCUGC
XXXXX XXXXX

4696
mG * fC * fA * fU * fU * fG * fU
AUUGU
XXXXX XXXX

WV-
fG * fA * fU * fA * fC * fA * mA * mA * mU * mC * mU * mU * mA *
GAUACAAAUCUUACU
XXXXX XXXXX

4697
mC * fU * fC * fU * fG * fC * fA
CUGCA
XXXXX XXXX

WV-
fA * fA * fU * fU * fC * fU * mU * mU * mC * mA * mA * mC * mU *
AAUUCUUUCAACUAG
XXXXX XXXXX

4698
mA * fG * fA * fA * fU * fA * fA
AAUAA
XXXXX XXXX

WV-
fU * fG * fA * fA * fU * fU * mC * mU * mU * mU * mC * mA * mA *
UGAAUUCUUUCAACU
XXXXX XXXXX

4699
mC * fU * fA * fG * fA * fA * fU
AGAAU
XXXXX XXXX

WV-
fU * fC * fU * fG * fA * fA * mU * mU * mC * mU * mU * mU * mC *
UCUGAAUUCUUUCAA
XXXXX XXXXX

4700
mA * fA * fC * fU * fA * fG * fA
CUAGA
XXXXX XXXX

WV-
fA * fU * fU * fC * fU * fG * mA * mA * mU * mU * mC * mU * mU *
AUUCUGAAUUCUUUC
XXXXX XXXXX

4701
mU * fC * fA * fA * fC * fU * fA
AACUA
XXXXX XXXX

WV-
fU * fG * fA * fU * fU * fC * mU * mG * mA * mA * mU * mU * mC *
UGAUUCUGAAUUCUU
XXXXX XXXXX

4702
mU * fU * fU * fC * fA * fA * fC
UCAAC
XXXXX XXXX

WV-
fA * fC * fU * fG * fA * fU * mU * mC * mU * mG * mA * mA * mU *
ACUGAUUCUGAAUUC
XXXXX XXXXX

4703
mU * fC * fU * fU * fU * fC * fA
UUUCA
XXXXX XXXX

WV-
fC * fC * fA * fC * fU * fG * mA * mU * mU * mC * mU * mG * A *
CCACUGAUUCUGAAU
XXXXX XXXXX

4704
mA * fU * fU * fC * fU * fU * fU
UCUUU
XXXXX XXXX

WV-
fU * fC * fC * fC * fA * fC * mU * mG * mA * mU * mU * mC * mU *
UCCCACUGAUUCUGA
XXXXX XXXXX

4705
mG * fA * fA * fU * fU * fC * fU
AUUCU
XXXXX XXXX

WV-
fC * fA * fU * fC * fC * fC * mA * mC * mU * mG * mA * mU * mU *
CAUCCCACUGAUUCU
XXXXX XXXXX

4706
mC * fU * fG * fA * fA * fU * fU
GAAUU
XXXXX XXXX

WV-
fU * fU * fC * fA * fU * fC * mC * mC * mA * mC * mU * mG * mA *
UUCAUCCCACUGAUU
XXXXX XXXXX

4707
mU * fU * fC * fU *fG * fA * fA
CUGAA
XXXXX XXXX

WV-
fA * fC * fU * fU * fC * fA * mU * mC * mC * mC * mA * mC * mU *
ACUUCAUCCCACUGA
XXXXX XXXXX

4708
mG * fA * fU * fU * fC * fU * fG
UUCUG
XXXXX XXXX

WV-
fG * fU * fA * fC * fU * fU * mC * mA * mU * mC * mC * mC * mA *
GUACUUCAUCCCACU
XXXXX XXXXX

4709
mC * fU * fG * fA * fU * fU * fC
GAUUC
XXXXX XXXX

WV-
fU * fU * fG * fU * fA * fC * mU * mU * mC * mA * mU * mC * mC *
UUGUACUUCAUCCCA
XXXXX XXXXX

4710
mC * fA * fC * fU * fG * fA * fU
CUGAU
XXXXX XXXX

WV-
fU * fC * fU * fU * fG * fU * mA * mC * mU * mU * mC * mA * mU *
UCUUGUACUUCAUCC
XXXXX XXXXX

4711
mC * fC * fC * fA * fC * fU * fG
CACUG
XXXXX XXXX

WV-
fG * fU * fU * fC * fU * fU * mG * mU * mA * mC * mU * mU * mC *
GUUCUUGUACUUCAU
XXXXX XXXXX

4712
mA * fU * fC * fC * fC * fA * fC
CCCAC
XXXXX XXXX

WV-
fG * fU * fG * fU * fU * fC * mU * mU * mG * mU * mA *mC * mU *
GUGUUCUUGUACUUC
XXXXX XXXXX

4713
mU * fC * fA * fU * fC * fC * fC
AUCCC
XXXXX XXXX

WV-
fA * fG * fG * fU * fG * fU * mU * mC * mU * mU * mG * mU * mA *
AGGUGUUCUUGUACU
XXXXX XXXXX

4714
mC * fU * fU * fC * fA * fU * fC
UCAUC
XXXXX XXXX

WV-
fG * fA * fA * fG * fG * fU * mG * mU * mU * mC * mU * mU * mG *
GAAGGUGUUCUUGU
XXXXX XXXXX

4715
mU * fA * fC * fU * fU * fC * fA
ACUUCA
XXXXX XXXX

WV-
fC * fU * fG * fA * fA * fG * mG * mU * mG * mU * mU * mC * mU *
CUGAAGGUGUUCUUG
XXXXX XXXXX

4716
mU * fG * fU * fA * fC * fU * fU
UACUU
XXXXX XXXX

WV-
fU * fU * fC * fU * fG * fA * mA * mG * mG * mU * mG * mU * mU *
UUCUGAAGGUGUUCU
XXXXX XXXXX

4717
mC * fU * fU * fG * fU * fA * fC
UGUAC
XXXXX XXXX

WV-
fG * fG * fU * fU * fC * fU * mG * mA * mA * mG * mG * mU * mG *
GGUUCUGAAGGUGU
XXXXX XXXXX

4718
mU * fU * fU * fU * fU * fG * fU
UCUUGU
XXXXX XXXX

WV-
fC * fC * fG * fG * fU * fU * mC * mU * mG * mA * mA * mG * mG *
CCGGUUCUGAAGGUG
XXXXX XXXXX

4719
mU * fG * fU * fU * fC * fU * fU
UUCUU
XXXXX XXXX

WV-
fC * fU * fC * fC * fG * fG * mU * mU * mC * mU * mG * mA * mA *
CUCCGGUUCUGAAGG
XXXXX XXXXX

4720
mG * fG * fU * fG * fU * fU * fC
UGUUC
XXXXX XXXX

WV-
fG * fC * fC * fU * fC * fC * mG * mG * mU * mU * mC * mU * mG *
GCCUCCGGUUCUGAA
XXXXX XXXXX

4721
mA * fA * fG * fG * fU * fG * fU
GGUGU
XXXXX XXXX

WV-
fU * fU * fG * fC * fC * fU * mC * mC * mG * mG * mU * mU * mC *
UUGCCUCCGGUUCUG
XXXXX XXXXX

4722
mU * fG * fA * fA * fG * fG * fU
AAGGU
XXXXX XXXX

WV-
fU * fG * fU * fU * fG * fC * mC * mU * mC * mC * mG * mG * mU *
UGUUGCCUCCGGUUC
XXXXX XXXXX

4723
mU * fC * fU * fG * fA * fA * fG
UGAAG
XXXXX XXXX

WV-
fA * fC * fU * fG * fU * fU * mG * mC * mC * mU * mC * mC * mG *
ACUGUUGCCUCCGGU
XXXXX XXXXX

4724
mG * fU * fU * fC * fU * fG * fA
UCUGA
XXXXX XXXX

WV-
fC * fA * fA * fC * fU * fG * mU * mU * mG * mC * mC * mU * mC *
CAACUGUUGCCUCCG
XXXXX XXXXX

4725
mC * fG * fG * fU * fU * fC * fU
GUUCU
XXXXX XXXX

WV-
fU * fU * fC * fA * fA * fC * mU * mG * mU * mU * mG * mC * mC *
UUCAACUGUUGCCUC
XXXXX XXXXX

4726
mU * fC * fC * fG * fG * fU * fU
CGGUU
XXXXX XXXX

WV-
fC * fA * fU * fU * fC * fA * mA * mC * mU * mG * mU * mU * mG *
CAUUCAACUGUUGCC
XXXXX XXXXX

4727
mC * fC * fU * fC * fC * fG * fG
UCCGG
XXXXX XXXX

WV-
fU * fU * fC * fA * fU * fU * mC * mA * mA * mC * mU * mG * mU *
UUCAUUCAACUGUUG
XXXXX XXXXX

4728
mU * fG * fC * fC * fU * fC * fC
CCUCC
XXXXX XXXX

WV-
fA * fU * fU * fU * fC * fA * mU * mU * mC * mA * mA * mC * mU *
AUUUCAUUCAACUGU
XXXXX XXXXX

4729
mG * fU * fU * fG * fC * fC * fU
UGCCU
XXXXX XXXX

WV-
fA * fU * fC * fC * fU * fU * mU * mA * mA * mC * mA * mU * mU *
AUCCUUUAACAUUUC
XXXXX XXXXX

4730
mU * fC * fA * fU * fU * fC * fA
AUUCA
XXXXX XXXX

WV-
fG * fA * fA * fU * fC * fC * mU * mU * mU * mA * mA * mC * mA *
GAAUCCUUUAACAUU
XXXXX XXXXX

4731
mU * fU * fU * fC * fA * fU * fU
UCAUU
XXXXX XXXX

WV-
fU * fU * fG * fA * fA * fU * mC * mC * mU * mU * mU * mA * mA *
UUGAAUCCUUUAACA
XXXXX XXXXX

4732
mC * fA * mU * fU * fU * fC * fA
UUUCA
XXXXX XXXX

WV-
fU * fG * fU * fU * fG * fA * mA * mU * mC * mC * mU * mU * mU *
UGUUGAAUCCUUUAA
XXXXX XXXXX

4733
mA * fA * fC * fA * fU * fU * fU
CAUUU
XXXXX XXXX

WV-
fU * fG * fU * fG * fU * fU * mG * mA * mA * mU * mC * mC * mU *
UGUGUUGAAUCCUUU
XXXXX XXXXX

4734
mU * fU * fA * fA * fC * fA * fU
AACAU
XXXXX XXXX

WV-
fA * fU * fU * fG * fU * fG * mU * mU * mG * mA * mA * mU * mC *
AUUGUGUUGAAUCCU
XXXXX XXXXX

4735
mC * fU * fU * fU * fA * fA * fC
UUAAC
XXXXX XXXX

WV-
fC * fC * fA * fU * fU * fG * mU * mG * mU * mU * mG * mA * mA *
CCAUUGUGUUGAAUC
XXXXX XXXXX

4736
mU * fC * fC * fU * fU * fU * fA
CUUUA
XXXXX XXXX

WV-
fA * fG * fC * fC * fA * fU * mU * mG * mU * mG * mU * mU * mG *
AGCCAUUGUGUUGAA
XXXXX XXXXX

4737
mA * fA * fU * fC * fC * fU * fU
UCCUU
XXXXX XXXX

WV-
fC * fC * fA * fG * fC * fC * mA * mU * mU * mG * mU * mG * mU *
CCAGCCAUUGUGUUG
XXXXX XXXXX

4738
mU * fG * fA * fA * fU * fC * fC
AAUCC
XXXXX XXXX

WV-
fU * fU * fC * fC * fA * fG * mC * mC * mA * mU * mU * mG * mU *
UUCCAGCCAUUGUGU
XXXXX XXXXX

4739
mG * fU * fU * fG * fA * fA * fU
UGAAU
XXXXX XXXX

WV-
fG * fC * fU * fU * fC * fC * mA * mG * mC * mC * mA * mU * mU *
GCUUCCAGCCAUUGU
XXXXX XXXXX

4740
mG * fU * fG * fU * fU * fG * fA
GUUGA
XXXXX XXXX

WV-
fU * fA * fG * fC * fU * fU * mC * mC * mA * mG * mC * mC * mA *
UAGCUUCCAGCCAUU
XXXXX XXXXX

4741
mU * fU * fG * fU * fG * fU * fU
GUGUU
XXXXX XXXX

WV-
fC * fU * fU * fA * fG * fC * mU * mU * mC * mC * mA * mG * mC *
CUUAGCUUCCAGCCA
XXXXX XXXXX

4742
mC * fA * fU * fU * fU * fU * fG
UUGUG
XXXXX XXXX

WV-
fU * fC * fC * fU * fU * fA * mG * mC * mU * mU * mC * mC * mA *
UCCUUAGCUUCCAGC
XXXXX XXXXX

4743
mG * fC * fC * fA * fU * fU * fG
CAUUG
XXXXX XXXX

WV-
fC * fU * fU * fC * fC * fU * mU * mA * mG * mC * mU * mU * mC *
CUUCCUUAGCUUCCA
XXXXX XXXXX

4744
mC * fA * fG * fC * fC * fA * fU
GCCAU
XXXXX XXXX

WV-
fU * fU * fC * fU * fU * fC * mC * mU * mU * mA * mG * mC * mU *
UUCUUCCUUAGCUUC
XXXXX XXXXX

4745
mU * fC * fC * fA * fG * fC * fC
CAGCC
XXXXX XXXX

WV-
fG * fC * fU * fU * fC * fU * mU * mC * mC * mU * mU * mA * mG *
GCUUCUUCCUUAGCU
XXXXX XXXXX

4746
mC * fU * fU * fC * fC * fA * fG
UCCAG
XXXXX XXXX

WV-
fC * fA * fG * fC * fU * fU * mC * mU * mU * mC * mC * mU * mU *
CAGCUUCUUCCUUAG
XXXXX XXXXX

4747
mA * fG * fC * fU * fU * fC * fC
CUUCC
XXXXX XXXX

WV-
fC * fU * fC * fA * fG * fC * mU * mU * mC * mU * mU * mC * mC *
CUCAGCUUCUUCCUU
XXXXX XXXXX

4748
mU * fU * fA * fG * fC * fU * fU
AGCUU
XXXXX XXXX

WV-
fC * fU * fG * fC * fU * fC * mA * mG * mC * mU * mU * mC * mU *
CUGCUCAGCUUCUUC
XXXXX XXXXX

4749
mU * fC * fC * fU * fU * fA * fG
CUUAG
XXXXX XXXX

WV-
fA * fC * fC * fU * fG * fC * mU * mC * mA * mG * mC * mU * mU *
ACCUGCUCAGCUUCU
XXXXX XXXXX

4750
mC * fU * fU * fC * fC * fU * fU
UCCUU
XXXXX XXXX

WV-
fA * fG * fA * fC * fC * fU * mG * mC * mU * mC * mA * mG * mC *
AGACCUGCUCAGCUU
XXXXX XXXXX

4751
mU * fU * fC * fU * fU * fC * fC
CUUCC
XXXXX XXXX

WV-
fU * fA * fA * fG * fA * fC * mC * mU * mG * mC * mU * mC * mA *
UAAGACCUGCUCAGC
XXXXX XXXXX

4752
mG * fC * fU * fU * fC * fU * fU
UUCUU
XXXXX XXXX

WV-
fC * fC * fU * fA * fA * fG * mA * mC * mC * mU * mG * mC * mU *
CCUAAGACCUGCUCA
XXXXX XXXXX

4753
mC * fA * fG * fC * fU * fU * fC
GCUUC
XXXXX XXXX

WV
fG * fU * fC * fC * fU * fA * mA * mG * mA * mC * mC * mU * mG *
GUCCUAAGACCUGCU
XXXXX XXXXX

4754
mC * fU * fC * fA * fG * fC * fU
CAGCU
XXXXX XXXX

WV-
fC * fU * fG * fU * fC * fC * mU * mA * mA * mG * mA * mC * mC *
CUGUCCUAAGACCUG
XXXXX XXXXX

4755
mU * fG * fC * fU * fC * fA * fG
CUCAG
XXXXX XXXX

WV-
fG * fG * fC * fC * fU * fG * mU * mC * mC * mU * mA * mA * mG *
GGCCUGUCCUAAGAC
XXXXX XXXXX

4756
mA * fC * fC * fU * fG * fC * fU
CUGCU
XXXXX XXXX

WV-
fU * fU * fG * fG * fC * fC * mU * mG * mU * mC * mC * mU * mA *
CUGGCCUGUCCUAAG
XXXXX XXXXX

4757
mA * fG * fA * fC * fC * fU * fG
ACCUG
XXXXX XXXX

WV-
fC * fU * fC * fU * fG * fG * mC * mC * mU * mG * mU * mC * mC *
CUCUGGCCUGUCCUA
XXXXX XXXXX

4758
mU * fA * fA * fG * fA * fC * fC
AGACC
XXXXX XXXX

WV-
fG * fG * fC * fU * fC * fU * mG * mG * mC * mC * mU * mG * mU *
GGCUCUGGCCUGUCC
XXXXX XXXXX

4759
mC * fC * fU * fA * fA * fG * fA
UAAGA
XXXXX XXXX

WV-
fU * fU * fG * fG * fC * fU * mC * mU * mG * mG * mC * mC * mU *
UUGGCUCUGGCCUGU
XXXXX XXXXX

4760
mG * fU * fC * fC * fU * fA * fA
CCUAA
XXXXX XXXX

WV-
fG * fC * fU * fU * fG * fG * mC * mU * mC * mU * mG * mG * mC *
GCUUGGCUCUGGCCU
XXXXX XXXXX

4761
mC * fU * fG * fU * fC * fC * fU
GUCCU
XXXXX XXXX

WV-
fA * fA * fG * fC * fU * fU * mG * mG * mC * mU * mC * mU * mG *
AAGCUUGGCUCUGGC
XXXXX XXXXX

4762
mG * fC * fC * fU * fG * fU * fC
CUGUC
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fC * mU * mU * mG * mG * mC * mU * mC *
UCAAGCUUGGCUCUG
XXXXX XXXXX

4763
mU * fG * fG * fC * fC * fU * fG
GCCUG
XXXXX XXXX

WV-
fU * fC * fC * fU * fU * fC * mC * mA * mU * mG * mA * mC * mU *
UCCUUCCAUGACUCA
XXXXX XXXXX

4764
mC * fA * fA * fG * fC * fU * fU
AGCUU
XXXXX XXXX

WV-
fC * fC * fU * fC * fC * fU * mU * mC * mC * mA * mU * mG * mA * mC
CCUCCUUCCAUGACU
XXXXX XXXXX

4765
* fU * fC * fA * fA * fG * fC
CAAGC
XXXXX XXXX

WV-
fA * fC * fC * fC * fU * fC * mC * mU * mU * mC * mC * mA * mU * mG
ACCCUCCUUCCAUGA
XXXXX XXXXX

4766
* fA * fC * fU * fC * fA * fA
CUCAA
XXXXX XXXX

WV-
fG * fG * fA * fC * fC * fC * mU * mC * mC * mU * mU * mC * mC * mA
GGACCCUCCUUCCAU
XXXXX XXXXX

4767
* fU * fG * fA * fC * fU * fC
GACUC
XXXXX XXXX

WV-
fA * fG * fG * fG * fA * fC * mC * mC * mU * mC * mC * mU * mU *
AGGGACCCUCCUUCC
XXXXX XXXXX

4768
mC * fC * fA * fU * fG * fA * fC
AUGAC
XXXXX XXXX

WV-
fA * fU * fA * fG * fG * fG * mA * mC * mC * mC * mU * mC * mC *
AUAGGGACCCUCCUU
XXXXX XXXXX

4769
mU * fU * fC * fC * fA * fU * fG
CCAUG
XXXXX XXXX

WV-
fG * fU * fA * fU * fA * fG * mG * mG * mA * mC * mC * mC * mU *
GUAUAGGGACCCUCC
XXXXX XXXXX

4770
mC * fC * fU * fU * fC * fC * fA
UUCCA
XXXXX XXXX

WV-
fC * fU * fG * fU * fA * fU * mA * mG * mG * mG * mA * mC * mC *
CUGUAUAGGGACCCU
XXXXX XXXXX

4771
mC * fU * fC * fC * fU * fU * fC
CCUUC
XXXXX XXXX

WV-
fU * fA * fC * fU * fG * fU * mA * mU * mA * mG * mG * mG * mA *
UACUGUAUAGGGACC
XXXXX XXXXX

4772
mC * fC * fC * fU * fC * fU * fU
CUCCU
XXXXX XXXX

WV-
fU * fC * fU * fA * fC * fU * mG * mU * mA * mU * mA * mG * mG *
UCUACUGUAUAGGGA
XXXXX XXXXX

4773
mG * fA * fC * fC * fC * fU * fC
CCCUC
XXXXX XXXX

WV-
fC * fA * fU * fC * fU * fA * mC * mU * mG * mU * mA * mU * mA *
CAUCUACUGUAUAGG
XXXXX XXXXX

4774
mG * fG * fG * fA * fC * fC * fC
GACCC
XXXXX XXXX

WV-
fU * fG * fC * fA * fU * fC * mU * mA * mC * mU * mG * mU * mA *
UGCAUCUACUGUAUA
XXXXX XXXXX

4775
mU * fA * fG * fG * fG * fA * fC
GGGAC
XXXXX XXXX

WV-
fA * fU * fU * fG * fC * fA * mU * mC * mU * mA * mC * mU * mG *
AUUGCAUCUACUGUA
XXXXX XXXXX

4776
mU * fA * fU * fA * fG * fG * fG
UAGGG
XXXXX XXXX

WV-
fG * fG * fA * fU * fU * fG * mC * mA * mU * mC * mU * mA * mC *
GGAUUGCAUCUACUG
XXXXX XXXXX

4777
mU * fG * fU * fA * fU * fA * fG
UAUAG
XXXXX XXXX

WV-
fU * fU * fG * fG * fA * fU * mU * mG * mC * mA * mU * mC * mU *
UUGGAUUGCAUCUAC
XXXXX XXXXX

4778
mA * fC * fU * fG * fU * fA * fU
UGUAU
XXXXX XXXX

WV-
fU * fU * fU * fU * fG * fG * mA * mU * mU * mG * mC * mA * mU *
UUUUGGAUUGCAUCU
XXXXX XXXXX

4779
mC * fU * fA * fC * fU * fG * fU
ACUGU
XXXXX XXXX

WV-
fU * fC * fU * fU * fU * fU * mG * mG * mA * mU * mU * mG * mC *
UCUUUUGGAUUGCAU
XXXXX XXXXX

4780
mA * fU * fC * fU * fA * fC * fU
CUACU
XXXXX XXXX

WV-
fU * fU * fU * fC * fU * fU * mU * mU * mG * mG * mA * mU * mU *
UUUCUUUUGGAUUGC
XXXXX XXXXX

4781
mG * fC * fA * fU * fC * fU * fA
AUCUA
XXXXX XXXX

WV-
fA * fU * fU * fU * fU * fC * mU * mU * mU * mU * mG * mG * mA *
AUUUUCUUUUGGAU
XXXXX XXXXX

4782
mU * fU * fG * fC * fA * fU * fC
UGCAUC
XXXXX XXXX

WV-
fU * fG * fA * fU * fU * fU * mU * mC * mU * mU * mU * mU * mG *
UGAUUUUCUUUUGG
XXXXX XXXXX

4783
mG * fA * fU * fU * fG * fC * fA
AUUGCA
XXXXX XXXX

WV-
fU * fG * fU * fG * fA * fU * mU * mU * mU * mC * mU * mU * mU *
UGUGAUUUUCUUUU
XXXXX XXXXX

4784
mU * fG * fG * fA * fU * fU * fG
GGAUUG
XXXXX XXXX

WV-
fU * fC * fU * fG * fU * fG * mA * mU * mU * mU * mU * mC * mU *
UCUGUGAUUUUCUUU
XXXXX XXXXX

4785
mU * fU * fU * fG * fG * fA * fU
UGGAU
XXXXX XXXX

WV-
fU * fU * fU * fC * fU * fG * mU * mG * mA * mU * mU * mU * mU *
UUUCUGUGAUUUUCU
XXXXX XXXXX

4786
mC * fU * fU * fU * fU * fG * fG
UUUGG
XXXXX XXXX

WV-
fG * fG * fU * fU * fU * fC * mU * mG * mU * mG * mA * mU * mU *
GGUUUCUGUGAUUU
XXXXX XXXXX

4787
mU * fU * fC * fU * fU * fU * fU
UCUUUU
XXXXX XXXX

WV-
fU * fU * fG * fG * fU * fU * mU * mC * mU * mG * mU * mG * mA *
UUGGUUUCUGUGAU
XXXXX XXXXX

4788
mU * fU * fU * fU * fC * fU * fU
UUUCUU
XXXXX XXXX

WV-
fC * fC * fU * fU * fG * fG * mU * mU * mU * mC * mU * mG * mU *
CCUUGGUUUCUGUGA
XXXXX XXXXX

4789
mG * fA * fU * fU * fU * fU * fC
UUUUC
XXXXX XXXX

WV-
fA * fA* fC * fC * fU * fU * mG * mG * mU * mU * mU * mC * mU *
AACCUUGGUUUCUGU
XXXXX XXXXX

4790
mG * fU * fG * fA * fU * fU * fU
GAUUU
XXXXX XXXX

WV-
fC * fG * fA * fA * fC * fC * mU * mU * mG * mG * mU * mU * mU *
CUAACCUUGGUUUCU
XXXXX XXXXX

4791
mC * fU * fG * fU * fG * fA * fU
GUGAU
XXXXX XXXX

WV-
fU * fA * fC * fU * fA * fA * mC * mC * mU * mU * mG * mG * mU *
UACUAACCUUGGUUU
XXXXX XXXXX

4792
mU * fU * fC * fU * fG * fU * fG
CUGUG
XXXXX XXXX

WV-
fG * fA * fU * fA * fC * fU * mA * mU * mC * mC * mU * mU * mG *
GAUACUAACCUUGGU
XXXXX XXXXX

4793
mG * fU * fU * fU * fC * fU * fG
UUCUG
XXXXX XXXX

WV-
ChTEGfU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG
UCAAGGAAGAUGGCA
OSSSSSSOSOSSOO

4890
mGfC * SfA * SfU * SfU * SfU * SfC * SfU

UUUCUSSSSSS

WV-
L001 mG * mG * mC * mC * mA * mA * mA * mC * mC * mU * mC *
GGCCAAACCUCGGCU
OXXXXX XXXXX

6010
mG * mG * mC * mU * mU * mA * mC * mC * mU
UACCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

6137
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
Mod012L001fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * S mUfG
UCAAGGAAGAUGGCA
OSSSSSSOSOSOSO

6409
* S mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod012L001fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC *
UCAAGGAAGAUGGCA
OXXXXXXOXOXO

6410
fA * fU * fU * fU * fC * fU
UUUCU
XOXXXXXX

WV-
L001fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * S mUfG * S
UCAAGGAAGAUGGCA
OSSSSSSOSOSOSO

6560
mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod012L001 mU * S mC * S mA * S mA * S mG * S mG * S mA mA * S mG
UCAAGGAAGAUGGCA
OSSSSSSOSOSOSO

6826
mA * S mU mG * S mG mC * S mA * S mU * S mU * S mU * S mC * S mU
UUUCU
SSSSSS

WV-
Mod012L001 mU * mC * mA * mA * mG * mG * mA mA * mG mA * mU
UCAAGGAAGAUGGCA
OXXXXXXOXOXO

6827
mG * mG mC * mA * mU * mU * mU * mC * mU
UUUCU
XOXXXXXX

WV-
Mod012L001 mU * mC * mA * mA * mG * mG * mA * mA * mG * mA *
UCAAGGAAGAUGGCA
OXXXXX XXXXX

6828
mU * mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
Mod012L001fC * fC * fU * fU * fC * fC * mCfU * mGfA * mAfG * mGfU *
CCUUCCCUGAAGGUU
OXXXXXXOXOXO

6829
fU * fC * fC * fU * fC * fC
CCUCC
XOXXXXXX

WV-
Mod012L001 mC * mC * mU * mU * mC * mC * mC mU * mG mA * mA
CCUUCCCUGAAGGUU
OXXXXXXOXOXO

6830
mG * mG mU * mU * mC * mC * mU * mC * mC
CCUCC
XOXXXXXX

WV-
L001 mU * S mC * S mA * S mA * S mG * S mG * S mA mA * S mG mA * S
UCAAGGAAGAUGGCA
OSSSSSSOSOSOSO

7109
mU mG * S mG mC * S mA * S mU * S mU * S mU * S mC * S mU
UUUCU
SSSSSS

WV-
L001 mU * mC * mA * mA * mG * mG * mA mA * mG mA * mU mG *
UCAAGGAAGAUGGCA
OXXXXXXOXOXO

7110
mG mC * mA * mU * mU * mU * mC * mU
UUUCU
XOXXXXXX

WV-
L00lfC * fC * fU * fU * fC * fC * mCfU * mGfA * mAfU * mGfU * fU * fC
CCUUCCCUGAAGGUU
OXXXXXXOXOXO

7111
* fC * fU * fC * fC
CCUCC
XOXXXXXX

WV-
L001 mC * mC * mU * mU * mC * mC * mC mU * mG mA * mA mG *
CCUUCCCUGAAGGUU
OXXXXXXOXOXO

7112
mG mU * mU * mC * mC * mU * mC * mC
CCUCC
XOXXXXXX

WV-
fU * fC * fAfAfGfG mAfA * mG mA * fU * mG mGfC * fA * fU * fU * fU *
UCAAGGAAGAUGGCA
XXOOOOOXOXXO

7333
fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fAfA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXOXXXOXOXXO

7334
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fAfG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXOXXOXOXXO

7335
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fGfG * mAfA * mG mA * fU * mG mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXOXOXOXXO

7336
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG mAfA * mG mA * fU * mG mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXOOXOXXO

7337
* fU * fC * fU
UUUCU
OXXXXXX

WV-
Mod020L001fU * fC * fAfAfGfG mAfA * mG mA * fU * mG mGfC * fA *
UCAAGGAAGAUGGCA
OXXOOOOOXOXX

7338
fU * fU * fU * fC * fU
UUUCU
OOXXXXXX

WV-
Mod020L001fU * fC * fAfA * fG * fG * mAfA * mG mA * fU * mG mGfC *
UCAAGGAAGAUGGCA
OXXOXXXOXOXX

7339
fA * fU * fU * fU * fC * fU
UUUCU
OOXXXXXX

WV-
Mod020L001fU * fC * fA * fAfG * fG * mAfA * mG mA * fU * mG mGfC *
UCAAGGAAGAUGGCA
OXXXOXXOXOXX

7340
fA * fU * fU * fU * fC * fU
UUUCU
OOXXXXXX

WV-
Mod020L001fU * fC * fA * fA * fGfG * mAfA * mG mA * fU * mG mGfC *
UCAAGGAAGAUGGCA
OXXXXOXOXOXX

7341
fA * fU * fU * fU * fC * fU
UUUCU
OOXXXXXX

WV-
Mod020L001fU * fC * fA * fA * fG * fG mAfA * mG mA * fU * mG mGfC *
UCAAGGAAGAUGGCA
OXXXXXOOXOXX

7342
fA * fU * fU * fU * fC * fU
UUUCU
OOXXXXXX

WV-
T * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU * fU
TCAAGGAAGAUGGCA
XXXXXXOXOXXO

7343
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * C * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7344
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * A * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7345
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * A * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7346
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * G * fG * mAfA * mG mA * fU * mG mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7347
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * G * mAfA * mG mA * fU * mG mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7348
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAA * mG mA * fU * mG mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7349
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * T * mG mGfC * fA * fU * fU
UCAAGGAAGATGGCA
XXXXXXOXOXXO

7350
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7351
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * A * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7352
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * T * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7353
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fG * T
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7354
* fU * fC * fU
UTUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7355
fU * T * fC * fU
UUTCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7356
fU * fU * C * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * mAfA * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7357
fU * fU * fC * T
UUUCT
OXXXXXX

WV-
fU * fC * A * fA * fG * G * mAfA mG mA * fU * mG mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7358
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * C * fA * fA * G * fG * mAfA * mG mA * fU * mG mGfC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7359
* fU * fC * fU
UUUCU
OXXXXXX

WV-
T * fC * fA * A * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU * fU
TCAAGGAAGAUGGCA
XXXXXXOXOXXO

7360
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * T * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7361
* fU * T * fU
UUUTU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * A * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7362
* T * fC * fU
UUTCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGC * fA * fU * T
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7363
* fU * fC * T
UTUCT
OXXXXXX

WV-
fU * fC * A * fA * fG * G * mAfA * mG mA * fU * mG mGfC * fA * T * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7364
fU * T * fU
TUUTU
OXXXXXX

WV-
fU * fC * A * fA * fG * G * mAfA * mG mA * fU * mG mGfC * A * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7365
* T * fC * fU
UUTCU
OXXXXXX

WV-
fU * fC * A * fA * fG * G * mAfA * mG mA * fU * mG mGC * fA * fU * T *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7366
fU * fC * T
UTUCT
OXXXXXX

WV-
fU * C * fA * fA * G * fG * mAfA * mG mA * fU * mG mGfC * fA * T * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7367
fU * T * fU
TUUTU
OXXXXXX

WV-
fU * C * fA * fA * G * fG * mAfA * mG mA * fU * mG mGfC * A * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7368
* T * fC * fU
UUTCU
OXXXXXX

WV-
fU * C * fA * fA * G * fG * mAfA * mG mA * fU * mG mGC * fA * fU * T *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7369
fU * fC * T
UTUCT
OXXXXXX

WV-
T * fC * fA * A * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * T * fU *
TCAAGGAAGAUGGCA
XXXXXXOXOXXO

7370
fU * T * fU
TUUTU
OXXXXXX

WV-
T * fC * fA * A * fG * fG * mAfA * mG mA * fU * mG mGfC * A * fU * fU *
TCAAGGAAGAUGGCA
XXXXXXOXOXXO

7371
T * fC * fU
UUTCU
OXXXXXX

WV-
T * fC * fA * A * fG * fG * mAfA * mG mA * fU * mG mGC * fA * fU * T *
TCAAGGAAGAUGGCA
XXXXXXOXOXXO

7372
fU * fC * T
UTUCT
OXXXXXX

WV-
Teo * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU *
TCAAGGAAGAUGGCA
XXXXXXOXOXXO

7373
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * m5Ceo * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7374
fU * fU * fG * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * Aeo * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7375
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * Aeo * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7376
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * Geo * fG * mAfA * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7377
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * Geo * mAfA * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7378
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAAeo * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7379
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * Teo * mG mGfC * fA * fU *
UCAAGGAAGATGGCA
XXXXXXOXOXXO

7380
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mG m5Ceo * fA *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7381
fU * fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * Aeo * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7382
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * Teo *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7383
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7384
Teo * fU * fC * fU
UTUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7385
fU * Teo * fC * fU
UUTCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7386
fU * fU * m5Ceo * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7387
fU * fU * fC * Teo
UUUCT
OXXXXXX

WV-
fU * fC * Aeo * fA * fG * Geo * mAfA * mG mA * fU * mG mGfC * fA * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7388
* fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * m5Ceo * fA * fA * Geo * fG * mAfA * mG mA * fU * mG mGfC * fA *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7389
fU * fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
Teo * fC * fA * Aeo * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU
TCAAGGAAGAUGGCA
XXXXXXOXOXXO

7390
* fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * Teo *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7391
fU * fU * Teo * fU
TUUTU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * mAfA * mG mA * fU * mG mGfC * Aeo * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7392
fU * Teo * fC * fU
UUTCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * mAfA * mG mA * fU * mG mG m5Ceo * fA *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7393
fU * Teo * fU * fC * Teo
UTUCT
OXXXXXX

WV-
fU * fC * Aeo * fA * fG * Geo * mAfA * mG mA * fU * mG mGfC * fA * Teo
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7394
* fU * fU * Teo * fU
TUUTU
OXXXXXX

WV-
fU * fC * Aeo * fA * fG * Geo * mAfA * mG mA * fU * mG mGfC * Aeo *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7395
fU * fU * Teo * fC * fU
UUTCU
OXXXXXX

WV-
fU * fC * Aeo * fA * fG * Geo * mAfA * mG mA * fU * mG mG m5Ceo * fA
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7396
* fU * Teo * fU * fC * Teo
UTUCT
OXXXXXX

WV-
fU * m5Ceo * fA * fA * Geo * fG * mAfA * mG mA * fU * mG mGfC * fA *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7397
Teo * fU * fU * Teo * fU
TUUTU
OXXXXXX

WV-
fU * m5Ceo * fA * fA * Geo * fG * mAfA * mG mA * fU * mG mGfC * Aeo
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7398
* fU * fU * Teo * fC * fU
UUTCU
OXXXXXX

WV-
fU * m5Ceo * fA * fA * Geo * fG * mAfA * mG mA * fU * mG mG m5Ceo *
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

7399
fA * fU * Teo * fU * fC * Teo
UTUCT
OXXXXXX

WV-
Teo * fC * fA * Aeo * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * Teo
TCAAGGAAGAUGGCA
XXXXXXOXOXXO

7400
* fU * fU * Teo * fU
TUUTU
OXXXXXX

WV-
Teo * fC * fA * Aeo * fG * fG * mAfA * mG mA * fU * mG mGfC * Aeo * fU
TCAAGGAAGAUGGCA
XXXXXXOXOXXO

7401
* fU * Teo * fC * fU
UUTCU
OXXXXXX

WV-
Teo * fC * fA * Aeo * fG * fG * mAfA * mG mA * fU * mG mG m5Ceo * fA
TCAAGGAAGAUGGCA
XXXXXXOXOXXO

7402
* fU * Teo * fU * fC * Teo
UTUCT
OXXXXXX

WV-
BrfU * SfC * SfA * SfA * SfG * SfU * S mAfA * S mGfA * S mUfG * S mGfC
UCAAGGAAGAUGGCA
SSSSSSOSOSOSOS

7410
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
Acet5fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * S mUfG * S
UCAAGGAAGAUGGCA
SSSSSSOSOSOSOS

7411
mGfC * SfA * SfU * SfU * SfG * SfU * SfU
UUUCU
SSSSS

WV-
BrfU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA * fU *
UCAAGGAAGAUGGCA
XXXXXXOXOXOX

7412
fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
Acet5fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXOX

7413
* fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
BrmU * mC * mA * mA * mG * mG * mA * mA * mG * mA * mU * mG
UCAAGGAAGAUGGCA
XXXXX XXXXX

7414
* mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
Acet5 mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * mU *
UCAAGGAAGAUGGCA
XXXXX XXXXX

7415
mG * mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fC * fU * fU * fU * fA * fA * mC * mA * mU * mU * mU * mC * mA *
CUUUAACAUUUCAUU
XXXXX XXXXX

7436
mU * fU * fC * fA * fA * fC * fU
CAACU
XXXXX XXXX

WV-
fU * fU * fA * fA * fC * fA * mU * mU * mU * mC * mA * mU * mU *
UUAACAUUUCAUUCA
XXXXX XXXXX

7437
mC * fA * fA * fC * fU * fG * fU
ACUGU
XXXXX XXXX

WV-
fA * fA * fC * fA * fU * fU * mU * mC * mA * mU * mU * mC * mA *
AACAUUUCAUUCAAC
XXXXX XXXXX

7438
mA * fC * fU * fG * fU * fU * fG
UGUUG
XXXXX XXXX

WV-
fC * fA * fU * fU * fU * fC * mA * mU * mU * mC * mA * mA * mC *
CAUUUCAUUCAACUG
XXXXX XXXXX

7439
mU * fG * fU * fU * fG * fU * fC
UUGUC
XXXXX XXXX

WV-
fU * fU * fU * fC * fA * fU * mU * mC * mA * mA * mC * mU * mG *
UUUCAUUCAACUGUU
XXXXX XXXXX

7440
mU * fU * fG * fU * fC * fU * fC
GUCUC
XXXXX XXXX

WV-
fU * fC * fA * fU * fU * fC * mA * mA * mC * mU * mG * mU * mU *
UCAUUCAACUGUUGU
XXXXX XXXXX

7441
mG * fU * fC * fU * fC * fC * fU
CUCCU
XXXXX XXXX

WV-
fA * fU * fU * fC * fA * fA * mC * mU * mG * mU * mU * mG * mU *
AUUCAACUGUUGUCU
XXXXX XXXXX

7442
mC * fU * fC * fC * fU * fG * fU
CCUGU
XXXXX XXXX

WV-
fU * fC * fA * fA * fC * fU * mG * mU * mU * mG * mU * mC * mU *
UCAACUGUUGUCUCC
XXXXX XXXXX

7443
mC * fC * fU * fG * fU * fU * fC
UGUUC
XXXXX XXXX

WV-
fA * fA * fC * fU * fG * fU * mU * mG * mU * mC * mU * mC * mC *
AACUGUUGUCUCCUG
XXXXX XXXXX

7444
mU * fG * fU * fU * fC * fU * fG
UUCUG
XXXXX XXXX

WV-
fC * fU * fG * fU * fU * fG * mU * mC * mU * mC * mC * mU * mG *
CUGUUGUCUCCUGUU
XXXXX XXXXX

7445
mU * fU * fC * fU * fG * fC * fA
CUGCA
XXXXX XXXX

WV-
fG * fU * fU * fG * fU * fC * mU * mC * mC * mU * mG * mU * mU *
GUUGUCUCCUGUUCU
XXXXX XXXXX

7446
mC * fU * fG * fC * fA * fG * fC
GCAGC
XXXXX XXXX

WV-
fU * fG * fU * fC * fU * fC * mC * mU * mG * mU * mU * mC * mU *
UGUCUCCUGUUCUGC
XXXXX XXXXX

7447
mG * fC * fA * fG * fC * fU * fG
AGCUG
XXXXX XXXX

WV-
fU * fC * fU * fC * fC * fU * mG * mU * mU * mC * mU * mG * mC *
UCUCCUGUUCUGCAG
XXXXX XXXXX

7448
mA * fG * fC * fU * fG * fU * fU
CUGUU
XXXXX XXXX

WV-
fU * fC * fC * fU * fG * fU * mU * mC * mU * mG * mC * mA * mG *
UCCUGUUCUGCAGCU
XXXXX XXXXX

7449
mC * fU * fG * fU * fU * fU * fU
GUUCU
XXXXX XXXX

WV-
fC * fU * fG * fU * fU * fC * mU * mG * mC * mA * mG * mC * mU *
CUGUUCUGCAGCUGU
XXXXX XXXXX

7450
mG * fU * fU * fC * fU * fU * fG
UCUUG
XXXXX XXXX

WV-
fG * fU * fU * fC * fU * fG * mC * mA * mG * mC * mU * mG * mU *
GUUCUGCAGCUGUUC
XXXXX XXXXX

7451
mU * fC * fU * fU * fG * fA * fA
UUGAA
XXXXX XXXX

WV-
fU * fC * fU * fG * fC * fA * mG * mC * mU * mG * mU * mU * mC *
UCUGCAGCUGUUCUU
XXXXX XXXXX

7452
mU * fU * fG * fA * fA * fC * fC
GAACC
XXXXX XXXX

WV-
fU * fG * fC * fA * fG * fC * mU * mG * mU * mU * mC * mU * mU *
UGCAGCUGUUCUUA
XXXXX XXXXX

7453
mG * fA * fA * fC * fC * fU * fC
ACCUC
XXXXX XXXX

WV-
fU * fG * fU * fU * fC * fU * mU * mG * mA * mA * mC * mC * mU *
UGUUCUUGAACCUCA
XXXXX XXXXX

7454
mC * fA * fU * fC * fC * fC * fA
UCCCA
XXXXX XXXX

WV-
fC * fA * fG * fC * fU * fG * mU * mU * mC * mU * mU * mG * mA *
CAGCUGUUCUUGAAC
XXXXX XXXXX

7455
mA * fC * fC * fU * fC * fA * fU
CUCAU
XXXXX XXXX

WV-
fG * fC * fU * fG * fU * fU * mC * mU * mU * mG * mA * mA * mC *
GCUGUUCUUGAACCU
XXXXX XXXXX

7456
mC * fU * fC * fA * fU * fC * fC
CAUCC
XXXXX XXXX

WV-
L001fU * fC * fAfAfGfG mAfA * mG mA * fU * mG mGfC * fA * fU * fU *
UCAAGGAAGAUGGCA
OXXOOOOOXOXX

7457
fU * fC * fU
UUUCU
OOXXXXXX

WV-
L001fU * fC * fAfA * fG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU
UCAAGGAAGAUGGCA
OXXOXXXOXOXX

7458
* fU * fU * fC * fU
UUUCU
OOXXXXXX

π

WV-
L001fU * fC * fA * fAfG * fG * mAfA * mG mA * fU * mG mGfC * fA * fU
UCAAGGAAGAUGGCA
OXXXOXXOXOXX

7459
* fU * fU * fC * fU
UUUCU
OOXXXXXX

WV-
L001fU * fC * fA * fA * fGfG * mAfA * mG mA * fU * mG mGfC * fA * fU
UCAAGGAAGAUGGCA
OXXXXOXOXOXX

7460
* fU * fU * fC * fU
UUUCU
OOXXXXXX

WV-
L001fU * fC * fA * fA * fG * fG mAfA * mG mA * fU * mG mGfC * fA * fU
UCAAGGAAGAUGGCA
OXXXXXOOXOXX

7461
* fU * fU * fC * fU
UUUCU
OOXXXXXX

WV-
mU * mC * mA * mA * mG * mG * mA mA * mG mA * mU mG * mG
UCAAGGAAGAUGGCA
XXXXXXOXOXOX

7506
mC * mA * mU * mU * mU * mC * mU
UUUCU
OXXXXXX

WV-
fC * fC * fU * fU * fC * fC * mCfU * mGfA * mAfG * mGfU * fU * fC * fC
CCUUCCCUGAAGGUU
XXXXXXOXOXOX

7507
* fU * fC * fC
CCUCC
OXXXXXX

WV-
mC * mC * mU * mU * mC * mC * mC mU * mG mA * mA mG * mG
CCUUCCCUGAAGGUU
XXXXXXOXOXOX

7508
mU * mU * mC * mC * mU * mC * mC
CCUCC
OXXXXXX

WV-
fU * RfC * RfA * RfA * RfG * RfG * R mAfA * R mGfA * R mUfG * R
UCAAGGAAGAUGGCA
RRRRRROROROR

7596
mGfC * RfA * RfU * RfU * RfU * RfC * RfU
UUUCU
ORRRRRR

WV-
fG * fC * fC * fA * fU * fU * mU * mU * mG * mU * mU * mG * mC *
GCCAUUUUGUUGCUC
XXXXX XXXXX

7677
mU * fC * fU * fU * fU * fC * fA
UUUCA
XXXXX XXXX

WV-
fA * fG * fC * fC * fA * fU * mU * mU * mU * mG * mU * mU * mG *
AGCCAUUUUGUUGCU
XXXXX XXXXX

7678
mC * fU * fC * fU * fU * fU * fC
CUUUC
XXXXX XXXX

WV-
fA * fA * fG * fC * fC * fA * mU * mU * mU * mU * mG * mU * mU *
AAGCCAUUUUGUUGC
XXXXX XXXXX

7679
mG * fC * fU * fC * fU * fU * fU
UCUUU
XXXXX XXXX

WV-
fU * fU * fG * fA * fA * fG * mC * mC * mA * mU * mU * mU * mU *
UUGAAGCCAUUUUGU
XXXXX XXXXX

7680
mG * fU * fU * fG * fC * fU * fC
UGCUC
XXXXX XXXX

WV-
fU * fA * fG * fU * fU * fG * mA * mA * mG * mC * mC * mA * mU *
UAGUUGAAGCCAUUU
XXXXX XXXXX

7681
mU * fU * fU * fG * fU * fU * fG
UGUUG
XXXXX XXXX

WV-
fA * fG * fA * fU * fA * fG * mU * mU * mG * mA * mA * mG * mC *
AGAUAGUUGAAGCCA
XXXXX XXXXX

7682
mC * fA * fU * fU * fU * fU * fG
UUUUG
XXXXX XXXX

WV-
fC * fU * fC * fA * fG * fA * mU * mA * mG * mU * mU * mG * mA *
CUCAGAUAGUUGAAG
XXXXX XXXXX

7683
mA * fG * fC * fC * fA * fU * fU
CCAUU
XXXXX XXXX

WV-
fU * fC * fA * fC * fU * fC * mA * mG * mA * mU * mA * mG * mU *
UCACUCAGAUAGUUG
XXXXX XXXXX

7684
mU * fG * fA * fA * fG * fC * fC
AAGCC
XXXXX XXXX

WV-
fG * fU * fG * fU * fC * fA * mC * mU * mC * mA * mG * mA * mU *
GUGUCACUCAGAUAG
XXXXX XXXXX

7685
mA * fG * fU * fU * fG * fA * fA
UUGAA
XXXXX XXXX

WV-
fA * fC * fA * fG * fU * fG * mU * mC * mA * mC * mU * mC * mA *
ACAGUGUCACUCAGA
XXXXX XXXXX

7686
mG * fA * fU * fA * fG * fU * fU
UAGUU
XXXXX XXXX

WV-
fC * fA * fC * fA * fG * fU * mG * mU * mC * mA * mC * mU * mC *
CACAGUGUCACUCAG
XXXXX XXXXX

7687
mA * fG * fA * fU * fA * fG * fU
AUAGU
XXXXX XXXX

WV-
fC * fU * fU * fC * fA * fC * mA * mG * mU * mG * mU * mC * mA *
CUUCACAGUGUCACU
XXXXX XXXXX

7688
mC * fU * fC * fA * fG * fA * fU
CAGAU
XXXXX XXXX

WV-
fC * fC * fU * fU * fC * fA * mC * mA * mG * mU * mG * mU * mC *
CCUUCACAGUGUCAC
XXXXX XXXXX

7689
mA * fC * fU * fC * fA * fG * fA
UCAGA
XXXXX XXXX

WV-
fC * fU * fC * fC * fU * fU * mC * mA * mC * mA * mG * mU * mG *
CUCCUUCACAGUGUC
XXXXX XXXXX

7690
mU * fC * fA * fC * fU * fC * fA
ACUCA
XXXXX XXXX

WV-
fA * fU * fC * fU * fC * fC * mU * mU * mC * mA * mC * mA * mG *
AUCUCCUUCACAGUG
XXXXX XXXXX

7691
mU * fG * fU * fC * fA * fC * fU
UCACU
XXXXX XXXX

WV-
fC * fC * fA * fU * fC * fU * mC * mC * mU * mU * mC * mA * mC * mA
CCAUCUCCUUCACAG
XXXXX XXXXX

7692
* fG * fU * fG * fU * fC * fA
UGUCA
XXXXX XXXX

WV-
fG * fG * fC * fC * fA * fU * mC * mU * mC * mC * mU * mU * mC *
GGCCAUCUCCUUCAC
XXXXX XXXXX

7693
mA * fC * fA * fG * fU * fG * fU
AGUGU
XXXXX XXXX

WV-
fU * fU * fG * fG * fC * fC * mA * mU * mC * mU * mC * mC * mU *
UUGGCCAUCUCCUUC
XXXXX XXXXX

7694
mU * fC * fA * fC * fA * fG * fU
ACAGU
XXXXX XXXX

WV-
fU * fC * fU * fU * fG * fG * mC * mC * mA * mU * mC * mU * mC *
UCUUGGCCAUCUCCU
XXXXX XXXXX

7695
mC * fU * fU * fC * fA * fC * fA
UCACA
XXXXX XXXX

WV-
fU * fU * fU * fC * fU * fU * mG * mG * mC * mC * mA * mU * mC *
UUUCUUGGCCAUCUC
XXXXX XXXXX

7696
mU * fC * fC * fU * fU * fC * fA
CUUCA
XXXXX XXXX

WV-
fG * fC * fU * fU * fU * fC * mU * mU * mG * mG * mC * mC * mA *
GCUUUCUUGGCCAUC
XXXXX XXXXX

7697
mU * fC * fU * fC * fC * fU * fU
UCCUU
XXXXX XXXX

WV-
fG * fU * fG * fC * fU * fU * mU * mC * mU * mU * mG * mG * mC *
GUGCUUUCUUGGCCA
XXXXX XXXXX

7698
mC * fA * fU * fC * fU * fC * fC
UCUCC
XXXXX XXXX

WV-
fA * fG * fG * fU * fG * fC * mU * mU * mU * mC * mU * mU * mG *
AGGUGCUUUCUUGGC
XXXXX XXXXX

7699
mG * fC * fC * fA * fU * fC * fU
CAUCU
XXXXX XXXX

WV-
fG * fA * fA * fG * fG * fU * mG * mC * mU * mU * mU * mC * mU *
GAAGGUGCUUUCUUG
XXXXX XXXXX

7700
mU * fG * fG * fC * fC * fA * fU
GCCAU
XXXXX XXXX

WV-
fC * fU * fG * fA * fA * fG * mG * mU * mG * mC * mU * mU * mU *
CUGAAGGUGCUUUCU
XXXXX XXXXX

7701
mC * fU * fU * fG * fG * fC * fC
UGGCC
XXXXX XXXX

WV-
fU * fU * fC * fU * fG * fA * mA * mG * mG * mU * mG * mC * mU *
UUCUGAAGGUGCUUU
XXXXX XXXXX

7702
mU * fU * fC * fU * fU * fG * fG
CUUGG
XXXXX XXXX

WV-
fU * fA * fU * fU * fU * fC * mU * mG * mA * mA * mG * mG * mU *
UAUUUCUGAAGGUGC
XXXXX XXXXX

7703
mG * fC * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
fA * fU * fA * fU * fU * fU * mC * mU * mG * mA * mA * mG * mG *
AUAUUUCUGAAGGU
XXXXX XXXXX

7704
mU * fG * fC * fU * fU * fU * fC
GCUUUC
XXXXX XXXX

WV-
fG * fG * fC * fA * fU * fA * mU * mU * mU * mC * mU * mG * mA *
GGCAUAUUUCUGAAG
XXXXX XXXXX

7705
mA * fG * fG * fU * fG * fC * fU
GUGCU
XXXXX XXXX

WV-
fU * fG * fG * fC * fA * fU * mA * mU * mU * mU * mC * mU * mG *
UGGCAUAUUUCUGAA
XXXXX XXXXX

7706
mA * fA * fG * fG * fU * fG * fC
GGUGC
XXXXX XXXX

WV-
fU * fC * fU * fG * fG * fC * mA * mU * mA * mU * mU * mU * mC *
UCUGGCAUAUUUCUG
XXXXX XXXXX

7707
mU * fG * fA * fA * fG * fG * fU
AAGGU
XXXXX XXXX

WV-
fU * fC * fU * fG * fA * fC * mA * mG * mA * mU * mA * mU * mU *
UCUGACAGAUAUUUC
XXXXX XXXXX

7708
mU * fC * fU * fG * fG * fC * fA
UGGCA
XXXXX XXXX

WV-
fA * fU * fU * fC * fU * fG * mA * mC * mA * mG * mA * mU * mA *
AUUCUGACAGAUAUU
XXXXX XXXXX

7709
mU * fU * fU * fC * fU * fG * fG
UCUGG
XXXXX XXXX

WV-
fC * fA * fA * fA * fU * fU * mC * mU * mG * mA * mC * mA * mG *
CAAAUUCUGACAGAU
XXXXX XXXXX

7710
mA * fU * fA * fU * fU * fU * fC
AUUUC
XXXXX XXXX

WV-
fU * fC * fU * fC * fU * fU * mC * mA * mA * mA * mU * mU * mC *
UCUCUUCAAAUUCUG
XXXXX XXXXX

7711
mU * fG * fA * fC * fA * fG * fA
ACAGA
XXXXX XXXX

WV-
fC * fU * fU * fC * fA * fA * mU * mC * mU * mC * mU * mU * mC *
CCUCAAUCUCUUCAA
XXXXX XXXXX

7712
mA * fA * fA * fU * fU * fC * fU
AUUCU
XXXXX XXXX

WV-
fG * fC * fC * fC * fC * fU * mC * mA * mA * mU * mC * mU * mC * mU
GCCCCUCAAUCUCUU
XXXXX XXXXX

7713
* fU * fC * fA * fA * fA * fU
CAAAU
XXXXX XXXX

WV-
fU * fG * fC * fC * fC * fC * mU * mC * mA * mA * mU * mC * mU * mC
UGCCCCUCAAUCUCU
XXXXX XXXXX

7714
* fU * fU * fC * fA * fA * fA
UCAAA
XXXXX XXXX

WV-
fG * fU * fG * fC * fC * fC * mC * mU * mC * mA * mA * mU * mC *
GUGCCCCUCAAUCUC
XXXXX XXXXX

7715
mU * fC * fU * fU * fC * fA * fA
UUCAA
XXXXX XXXX

WV-
fA * fG * fU * fG * fC * fC * mC * mC * mU * mC * mA * mA * mU *
AGUGCCCCUCAAUCU
XXXXX XXXXX

7716
mC * fU * fC * fU * fU * fC * fA
CUUCA
XXXXX XXXX

WV-
fC * fC * fA * fG * fU * fG * mC * mC * mC * mC * mU * mC * mA * mA
CCAGUGCCCCUCAAU
XXXXX XXXXX

7717
* fU * fC * fU * fC * fU * fU
CUCUU
XXXXX XXXX

WV-
fU * fU * fC * fC * fA * fU * mU * mG * mC * mC * mC * mC * mU * mC
UUCCAGUGCCCCUCA
XXXXX XXXXX

7718
* fA * fA * fU * fC * fU * fC
AUCUC
XXXXX XXXX

WV-
fU * fC * fU * fU * fC * fC * mA * mG * mU * mG * mC * mC * mC * mC
UCUUCCAGUGCCCCU
XXXXX XXXXX

7719
* fU * fC * fA * fA * fU * fC
CAAUC
XXXXX XXXX

WV-
fU * fU * fU * fC * fU * fU * mC * mC * mA * mG * mU * mG * mC *
UUUCUUCCAGUGCCC
XXXXX XXXXX

7720
mC * fC * fC * fU * fC * fA * fA
CUCAA
XXXXX XXXX

WV-
fA * fG * fU * fU * fU * fC * mU * mC * mC * mC * mA * mG * mU *
AGUUUCUUCCAGUGC
XXXXX XXXXX

7721
mG * fC * fC * fC * fC * fU * fC
CCCUC
XXXXX XXXX

WV-
fA * fA * fA * fG * fU * fU * mC * mC * mU * mU * mC * mC * mA *
AAAGUUUCUUCCAGU
XXXXX XXXXX

7722
mG * fU * fG * fC * fC * fC * fC
GCCCC
XXXXX XXXX

WV-
fA * fG * fG * fA * fA * fA * mG * mU * mU * mU * mC * mU * mU *
AGGAAAGUUUCUUCC
XXXXX XXXXX

7723
mC * fC * fA * fG * fU * fG * fC
AGUGC
XXXXX XXXX

WV-
fG * fG * fA * fG * fG * fA * mA * mA * mG * mU * mU * mU * mC *
GGAGGAAAGUUUCU
XXXXX XXXXX

7724
mU * fU * fC * fC * fA * fG * fU
UCCAGU
XXXXX XXXX

WV-
fC * fU * fG * fG * fG * fA * mG * mG * mA * mA * mA * mG * mU *
CUGGGAGGAAAGUU
XXXXX XXXXX

7725
mU * fU * fC * fU * fU * fC * fC
UCUUCC
XXXXX XXXX

WV-
fA * fC * fU * fG * fG * fG * mA * mG * mG * mA * mA * mA * mG *
ACUGGGAGGAAAGU
XXXXX XXXXX

7726
mU * fU * fU * fC * fU * fU * fC
UUCUUC
XXXXX XXXX

WV-
fC * fC * fA * fA * fC * fU * mG * mG * mG * mA * mG * mG * mA *
CCAACUGGGAGGAAA
XXXXX XXXXX

7727
mA * fA * fG * fU * fU * fU * fC
GUUUC
XXXXX XXXX

WV-
fC * fC * fA * fC * fC * fA * mA * mC * mU * mG * mG * mG * mA *
CCACCAACUGGGAGG
XXXXX XXXXX

7728
mG * fG * fA * fA * fA * fG * fU
AAAGU
XXXXX XXXX

WV-
fU * fU * fU * fC * fC * fA * mC * mC * mA * mA * mC * mU * mG *
UUUCCACCAACUGGG
XXXXX XXXXX

7729
mG * fG * fA * fG * fG * fA * fA
AGGAA
XXXXX XXXX

WV-
fC * fU * fU * fU * fC * fC * mA * mC * mC * mA * mA * mC * mU *
CUUUCCACCAACUGG
XXXXX XXXXX

7730
mG * fG * fG * fA * fG * fG * fA
GAGGA
XXXXX XXXX

WV-
fG * fC * fU * fU * fU * fC * mC * mA * mC * mC * mA * mA * mC *
GCUUUCCACCAACUG
XXXXX XXXXX

7731
mU * fG * fG * fG * fA * fG * fG
GGAGG
XXXXX XXXX

WV-
fC * fA * fG * fC * fU * fU * mU * mC * mC * mA * mC * mC * mA *
CAGCUUUCCACCAAC
XXXXX XXXXX

7732
mA * fC * fU * fG * fG * fG * fA
UGGGA
XXXXX XXXX

WV-
fG * fG * fC * fA * fG * fC * mU * mU * mU * mC * mC * mA * mC *
GGCAGCUUUCCACCA
XXXXX XXXXX

7733
mC * fA * fA * fC * fU * fG * fG
ACUGG
XXXXX XXXX

WV-
fU * fU * fG * fG * fC * fA * mG * mC * mU * mU * mU * mC * mC *
UUGGCAGCUUUCCAC
XXXXX XXXXX

7734
mA * fC * fC * fA * fA * fC * fU
CAACU
XXXXX XXXX

WV-
fU * fU * fU * fU * fG * fG * mC * mA * mG * mC * mU * mU * mU *
UUUUGGCAGCUUUCC
XXXXX XXXXX

7735
mC * fC * fA * fC * fC * fA * fA
ACCAA
XXXXX XXXX

WV-
fG * fC * fU * fU * fU * fU * mG * mG * mC * mA * mG * mC * mU *
GCUUUUGGCAGCUUU
XXXXX XXXXX

7736
mU * fU * fC * fC * fA * fC * fC
CCACC
XXXXX XXXX

WV-
fU * fA * fG * fC * fU * fU * mU * mU * mG * mG * mC * mA * mG *
UAGCUUUUGGCAGCU
XXXXX XXXXX

7737
mC * fU * fU * fU * fC * fC * fA
UUCCA
XXXXX XXXX

WV-
fU * fC * fU * fA * fG * fC * mU * mU * mU * mU * mG * mG * mC *
UCUAGCUUUUGGCAG
XXXXX XXXXX

7738
mA * fG * fC * fU * fU * fU * fC
CUUUC
XXXXX XXXX

WV-
fC * fU * fU * fC * fU * fA * mG * mC * mU * mU * mU * mU * mG *
CUUCUAGCUUUUGGC
XXXXX XXXXX

7739
mG * fC * fA * fG * fC * fU * fU
AGCUU
XXXXX XXXX

WV-
fU * fU * fC * fU * fU * fC * mU * mA * mG * mC * mU * mU * mU *
UUCUUCUAGCUUUUG
XXXXX XXXXX

7740
mU * fG * fG * fC * fA * fG * fC
GCAGC
XXXXX XXXX

WV-
fU * fG * fU * fU * fC * fU * mU * mC * mU * mA * mG * mC * mU *
UGUUCUUCUAGCUUU
XXXXX XXXXX

7741
mU * fU * fU * fG * fG * fC * fA
UGGCA
XXXXX XXXX

WV-
fU * fA * fU * fG * fU * fU * mC * mU * mU * mC * mU * mA * mG *
UAUGUUCUUCUAGCU
XXXXX XXXXX

7742
mC * fU * fU * fU * fU * fG * fG
UUUGG
XXXXX XXXX

WV-
fC * fA * fU * fA * fU * fG * mU * mU * mC * mU * mU * mC * mU *
CAUAUGUUCUUCUAG
XXXXX XXXXX

7743
mA * fG * fC * fU * fU * fU * fU
CUUUU
XXXXX XXXX

WV-
fU * fU * fC * fA * fU * fA * mU * mG * mU * mU * mC * mU * mU *
UUCAUAUGUUCUUCU
XXXXX XXXXX

7744
mC * fU * fA * fG * fC * fU * fU
AGCUU
XXXXX XXXX

WV-
fA * fU * fU * fC * fA * fU * mA * mU * mG * mU * mU * mC * mU *
AUUCAUAUGUUCUUC
XXXXX XXXXX

7745
mU * fC * fU * fA * fG * fC * fU
UAGCU
XXXXX XXXX

WV-
fU * fA * fU * fU * fC * fA * mU * mA * mU * mG * mU * mU * mC *
UAUUCAUAUGUUCUU
XXXXX XXXXX

7746
mU * fU * fC * fU * fA * fG * fC
CUAGC
XXXXX XXXX

WV-
fG * fU * fU * fU * fA * fU * mU * mC * mA * mU * mA * mU * mG *
GUUUAUUCAUAUGU
XXXXX XXXXX

7747
mU * fU * fC * fU * fU * fC * fU
UCUUCU
XXXXX XXXX

WV-
fA * fG * fU * fU * fU * fA * mU * mU * mC * mA * mU * mA * mU *
AGUUUAUUCAUAUG
XXXXX XXXXX

7748
mG * fU * fU * fC * fU * fU * fC
UUCUUC
XXXXX XXXX

WV-
fG * fA * fA * fG * fU * fU * mU * mA * mU * mU * mC * mA * mU *
GAAGUUUAUUCAUA
XXXXX XXXXX

7749
mA * fU * fG * fU * fU * fC * fU
UGUUCU
XXXXX XXXX

WV-
fU * fC * fG * fA * fA * fG * mU * mU * mU * mA * mU * mU * mC *
UCGAAGUUUAUUCAU
XXXXX XXXXX

7750
mA * fU * fA * fU * fG * fU * fU
AUGUU
XXXXX XXXX

WV-
fU * fU * fC * fG * fA * fA * mG * mU * mU * mU * mA * mU * mU *
UUCGAAGUUUAUUCA
XXXXX XXXXX

7751
mC * fA * fU * fA * fU * fG * fU
UAUGU
XXXXX XXXX

WV-
fU * fU * fU * fC * fG * fA * mA * mG * mU * mU * mU * mA * mU *
UUUCGAAGUUUAUUC
XXXXX XXXXX

7752
mU * fC * fA * fU * fA * fU * fG
AUAUG
XXXXX XXXX

WV-
fA * fA * fU * fU * fU * fU * mC * mG * mA * mA * mG * mU * mU *
AAUUUUCGAAGUUU
XXXXX XXXXX

7753
mU * fA * fU * fU * fC * fA * fU
AUUCAU
XXXXX XXXX

WV-
fU * fG * fA * fA * fA * fG * mU * mU * mU * mC * mG * mA * mA *
UGAAAUUUUCGAAG
XXXXX XXXXX

7754
mG * fU * fU * fU * fA * fU * fU
UUUAUU
XXXXX XXXX

WV-
fA * fC * fC * fU * fG * fA * mA * mA * mU * mU * mU * mU * mC *
ACCUGAAAUUUUCGA
XXXXX XXXXX

7755
mG * fA * fA * fG * fU * fU * fU
AGUUU
XXXXX XXXX

WV-
fG * fU * fA * fC * fC * fU * mG * mA * mA * mA * mU * mU * mU *
UUACCUGAAAUUUUC
XXXXX XXXXX

7756
mU * fC * fG * fA * fA * fG * fU
GAAGU
XXXXX XXXX

WV-
fG * fC * fU * fU * fA * fC * mC * mU * mG * mA * mA * mA * mU *
GCUUACCUGAAAUUU
XXXXX XXXXX

7757
mU * fU * fU * fC * fG * fA * fA
UCGAA
XXXXX XXXX

WV-
fC * fG * fG * fC * fU * fU * mA * mC * mC * mU * mG * mA * mA *
CGGCUUACCUGAAAU
XXXXX XXXXX

7758
mA * fU * fU * fU * fU * fC * fG
UUUCG
XXXXX XXXX

WV-
fC * fU * fC * fG * fG * fC * mU * mU * mA * mC * mC * mU * mG *
CUCGGCUUACCUGAA
XXXXX XXXXX

7759
mA * fA * fA * fU * fU * fU * fU
AUUUU
XXXXX XXXX

WV-
fA * fC * fC * fU * fC * fG * mG * mC * mU * mU * mA * mC * mC *
ACCUCGGCUUACCUG
XXXXX XXXXX

7760
mU * fG * fA * fA * fA * fU * fU
AAAUU
XXXXX XXXX

WV-
fA * fA * fA * fC * fC * fU * mC * mG * mG * mC * mU * mU * mA *
AAACCUCGGCUUACC
XXXXX XXXXX

7761
mC * fC * fU * fG * fA * fA * fA
UGAAA
XXXXX XXXX

WV-
fC * fC * fA * fA * fA * fC * mC * mU * mC * mG * mG * mC * mU *
CCAAACCUCGGCUUA
XXXXX XXXXX

7762
mU * fA * fC * fC * fU * fU * fA
CCUGA
XXXXX XXXX

WV-
fG * fC * fC * fA * fA * fA * mC * mC * mU * mC * mG * mG * mC *
GCCAAACCUCGGCUU
XXXXX XXXXX

7763
mU * fU * fA * fC * fC * fU * fG
ACCUG
XXXXX XXXX

WV-
fA * fG * fG * fC * fC * fA * mA * mA * mC * mC * mU * mC * mG *
AGGCCAAACCUCGGC
XXXXX XXXXX

7764
mG * fC * fU * fU * fA * fC * fC
UUACC
XXXXX XXXX

WV-
fA * fA * fA * fG * fG * fC * mC * mA * mA * mA * mC * mC * mU *
AAAGGCCAAACCUCG
XXXXX XXXXX

7765
mC * fG * fG * fC * fU * fU * fA
GCUUA
XXXXX XXXX

WV-
fU * fU * fA * fA * fA * fG * mG * mC * mC * mA * mA * mA * mC *
UUAAAGGCCAAACCU
XXXXX XXXXX

7766
mC * fU * fC * fG * fG * fC * fU
CGGCU
XXXXX XXXX

WV-
fG * fU * fU * fU * fA * fA * mA * mG * mG * mC * mC * mA * mA *
GUUUAAAGGCCAAAC
XXXXX XXXXX

7767
mA * fC * fC * fU * fC * fG * fG
CUCGG
XXXXX XXXX

WV-
fU * fA * fG * fU * fU * fU * mA * mA * mA * mG * mG * mC * mC *
UAGUUUAAAGGCCAA
XXXXX XXXXX

7768
mA * fA * fA * fC * fC * fU * fC
ACCUC
XXXXX XXXX

WV-
fU * fA * fU * fA * fG * fU * mU * mU * mA * mA * mA * mG * mG *
UAUAGUUUAAAGGCC
XXXXX XXXXX

7769
mC * fC * fA * fA * fA * fC * fC
AAACC
XXXXX XXXX

WV-
fA * fA * fU * fA * fU * fA * mG * mU * mU * mU * mA * mA * mA *
AAUAUAGUUUAAAG
XXXXX XXXXX

7770
mG * fG * fC * fC * fA * fA * fA
GCCAAA
XXXXX XXXX

WV-
fA * fA * fA * fA * fU * fA * mU * mA * mG * mU * mU * mU * mA *
AAAAUAUAGUUUAA
XXXXX XXXXX

7771
mA * fA * fG * fG * fC * fC * fA
AGGCCA
XXXXX XXXX

WV-
Mod028L001 * fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU
UCAAGGAAGAUGGCA
XSSSSSSOSOSSOO

8130
* S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
Mod028L001fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU *
UCAAGGAAGAUGGCA
OSSSSSSOSOSSOO

8131
S mG mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SAeofA * SGeoAeo * SfU * SGeoGeofC *
UCAAGGAAGAUGGCA
SSSSSSOSOSSOOS

8230
SfA * SfG * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SAeofA * SGeoAeofU * SGeoGeofC * SfA
UCAAGGAAGAUGGCA
SSSSSSOSOOSOOS

8231
* SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SAeoAeoGeoAeoTeoGeoGeofC * SfA *
UCAAGGAAGATGGCA
SSSSSSOOOOOOO

8232
SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSSS

WV-
fU * RfC * RfA * RfA * RfG * RfG * R mAfA * R mG mA * RfU * R mG
UCAAGGAAGAUGGCA
RRRRRRORORRO

8449
mGfC * RfA * RfU * RfU * RfU * RfC * RfU
UUUCU
ORRRRRR

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * Geo * Aeo * Teo * Geo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8478
m5Ceo * Aeo * Teo * Teo * Teo * m5Ceo * Teo
TTTCT
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * Geo * Aeo * Teo * Geo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8479
m5Ceo * Aeo * Teo * Teo * Teo * m5Ceo * mU
TTTCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * Geo * Aeo * Teo * Geo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8480
m5Ceo * Aeo * Teo * Teo * Teo * mC * mU
TTTCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * Geo * Aeo * Teo * Geo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8481
m5Ceo * Aeo * Teo * Teo * mU * mC * mU
TTUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * Geo * Aeo * Teo * Geo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8482
m5Ceo * Aeo * Teo * mU * mU * mC * mU
TUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * Geo * Aeo * Teo * Geo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8483
m5Ceo * Aeo * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * Geo * Aeo * Teo * Geo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8484
m5Ceo * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * Geo * Aeo * Teo * Geo * Geo * mC
UCAAGGAAGATGGCA
XXXXX XXXXX

8485
* mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * Geo * Aeo * Teo * Geo * mG * mC
UCAAGGAAGATGGCA
XXXXX XXXXX

8486
* mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * Geo * Aeo * Teo * mG * mG * mC
UCAAGGAAGATGGCA
XXXXX XXXXX

8487
* mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * Geo * Aeo * mU * mG * mG * mC
UCAAGGAAGAUGGCA
XXXXX XXXXX

8488
* mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * Geo * mA * mU * mG * mG * mC
UCAAGGAAGAUGGCA
XXXXX XXXXX

8489
* mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * Aeo * mG * mA * mU * mG * G *
UCAAGGAAGAUGGCA
XXXXX XXXXX

8490
mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fU * fC * fA * fA * fG * fG * Aeo * mA * mG * mA * mU * mG * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

8491
mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
Teo * m5Ceo * Aeo * Aeo * Geo * Geo * Aeo * Aeo * Geo * Aeo * Teo * Geo
TCAAGGAAGATGGCA
XXXXX XXXXX

8492
* Geo * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * m5Ceo * Aeo * Aeo * Geo * Geo * Aeo * Aeo * Geo * Aeo * Teo * Geo
UCAAGGAAGATGGCA
XXXXX XXXXX

8493
* Geo * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * Aeo * Aeo * Geo * Geo * Aeo * Aeo * Geo * Aeo * Teo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8494
Geo * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * Aeo * Geo * Geo * Aeo * Aeo * Geo * Aeo * Teo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8495
Geo * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * Geo * Geo * Aeo * Aeo * Geo * Aeo * Teo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8496
Geo * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * Geo * Aeo * Aeo * Geo * Aeo * Teo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8497
Geo * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * Aeo * Aeo * Geo * Aeo * Teo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8498
Geo * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * mA * Aeo * Geo * Aeo * Teo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8499
Geo * m5Ceo * fA * fU * fU * fU * fC *fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * Geo * Aeo * Teo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8500
Geo * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * mG * Aeo * Teo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8501
Geo * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * Teo * Geo *
UCAAGGAAGATGGCA
XXXXX XXXXX

8502
Geo * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * mU * Geo *
UCAAGGAAGAUGGCA
XXXXX XXXXX

8503
Geo * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * mU * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

8504
Geo * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * mU * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

8505
mG * m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
XXXXX XXXX

WV-
Teo * m5Ceo * Aeo * Aeo * Geo * Geo * Aeo * Aeo * Geo * Aeo * Teo * Geo
TCAAGGAAGATGGCA
XXXXX XXXXX

8506
* Geo * m5Ceo * Aeo * Teo * Teo * Teo * m5Ceo * Teo
TTTCT
XXXXX XXXX

WV-
CTCCAACATCAAGGAAGATGGCATTTCTAG +all PMO
CTCCAACATCAAGGA
XXXXX XXXXX

8806

AGATGG CATTTCTAG
XXXXX XXXXX

WV-
mU * R mC * R mA * R mA * R mG * R mG * R mA * R mA * R mG * R
UCAAGGAAGAUGGCA
RRRRRRRRRRRRR

884
mA * R mU * R mG * R mG * R mC * R mA * R mU * R mU * R mU * R
UUUCU
RRRRRR

mC * R mU

WV-
mU * S mC * R mA * S mA * R mG * S mG * R mA * S mA * R mG * S mA
UCAAGGAAGAUGGCA
SRSRSRSRSRSRSR

885
* R mU * S mG * R mG * S mC * R mA * S mU * R mU * S mU * R mC * S
UUUCU
SRSRS

mU

WV-
mU * R mC * R mA * R mA * S mG * S mG * S mA * S mA * S mG * S mA
UCAAGGAAGAUGGCA
RRRSSSSSSSSSSSS

886
* S mU * S mG * S mG * S mC * S mA * S mU * S mU * R mU * R mC * R
UUUCU
SRRR

mU

WV-
mU * S mC * S mA * S mA * R mG * R mG * R mA * R mA * R mG * R
UCAAGGAAGAUGGCA
SSSRRRRRRRRRR

887
mA * R mU * R mG * R mG * R mC * R mA * R mU * R mU * S mU * S
UUUCU
RRRSSS

mC * S mU

WV-
mU * R mC * R mA * R mA * R mG * R mG * S mA * S mA * R mG * S
UCAAGGAAGAUGGCA
RRRRRSSRSSRSSR

888
mA * S mU * R mG * S mG * S mC * R mA * R mU * R mU * R mU * R
UUUCU
RRRRR

mC * R mU

WV-
mU * S mC * S mA * S mA * S mG * S mG * R mA * R mA * S mG * R mA
UCAAGGAAGAUGGCA
SSSSSRRSRRSRRS

889
* R mU * S mG * R mG * R mC * S mA * S mU * S mU * S mU * S mC * S
UUUCU
SSSSS

mU

WV-
mU * R mC * R mA * R mA * S mG * S mG * R mA * R mA * S mG * R
UCAAGGAAGAUGGCA
RRRSSRRSRRRSR

890
mA * R mU * R mG * S mG * R mC * R mA * S mU * S mU * R mU * R
UUUCU
RSSRRR

mC * R mU

WV-
mU * S mC * S mA * S mA * R mG * R mG * S mA * S mA * R mG * S mA
UCAAGGAAGAUGGCA
SSSRRSSRSSSRSS

891
* S mU * S mG * R mG * S mC * S mA * R mU * R mU * S mU * S mC * S
UUUCU
RRSSS

mU

WV-
mU * S mC * R mA * R mA * R mG * R mG * R mA * R mA * R mG * R
UCAAGGAAGAUGGCA
SRRRRRRRRRRRR

892
mA * R mC * R mG * R mG * R mC * R mA * R mU * R mU * R mU * R
UUUCU
RRRRRS

mC * S mU

WV-
mU * R mC * S mA * S mA * S mG * S mG * S mA * S mA * S mG * S mA *
UCAAGGAAGAUGGCA
RSSSSSSSSSSSSSS

893
S mU * S mG * S mG * S mC * S mA * S mU * S mU * S mU * S mC * R mU
UUUCU
SSSR

WV-
fA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG mGfC * SfA * SfU *
AAGGAAGAUGGCAU
SSSSOSOSSOOSSS

8937
SfU * SfU * SfC * SfU
UUCU
SSS

WV-
mU * S mC * R mA * S mA * S mG * R mG * R mA * S mA * S mG * R mA
UCAAGGAAGAUGGCA
SRSSRRSSRSSRRR

894
* S mU * S mG * R mG * R mC * R mA * S mU * S mU * S mU * S mC * R
UUUCU
SSSSR

mU

WV-
mU * R mC * S mA * R mA * R mG * S mG * S mA * R mA * R mG * S
UCAAGGAAGAUGGCA
RSRRSSRRSRRSSS

895
mA * R mU * R mG * S mG * S mC * S mA * R mU * R mU * R mU * R
UUUCU
RRRRS

mC * S mU

WV-
mU * S mC * S mA * R mA * R mG * R mG * R mA * R mA * R mG * R
UCAAGGAAGAUGGCA
SSRRRRRRRRSRR

896
mA * R mU * S mG * R mG * R mC * S mA * R mU * S mU * S mU * S mC
UUUCU
SRSSSS

* S mU

WV-
mU * R mC * R mA * S mA * S mG * S mG * S mA * S mA * S mG * S mA *
UCAAGGAAGAUGGCA
RRSSSSSSSSRSSR

897
S mU * R mG * S mG * S mC * R mA * S mU * R mU * R mU * R mC * R
UUUCU
SRRRR

mU

WV-
fG * fU * fA * fC * fU * fU * m5Ceo * Aeo * Teo * m5Ceo * m5Ceo *
GUACUUCATCCCACU
XXXXX XXXXX

9067
m5Ceo * Aeo * m5Ceo * fU * fG * fA * fU * fU * fC
GAUUC
XXXXX XXXX

WV-
fG * fU * fA * fC * fU * fU * m5Ceo * AeoTeo * m5Ceo m5Ceo * m5CeoAeo
GUACUUCATCCCACU
XXXXXXXOXOXO

9068
* m5CeofU * fG * fA * fU * fU * fC
GAUUC
XOXXXXX

WV-
fG * fU * fA * fC * fU * fU * m5CeoAeo * Teo m5Ceo * m5Ceo m5Ceo * Aeo
GUACUUCATCCCACU
XXXXXXOXOXOX

9069
m5Ceo * fU * fG * fA * fU * fU * fC
GAUUC
OXXXXXX

WV-
fG * fU * fA * fC * fU * fU * m5Ceo * mA * Teo * mC * m5Ceo * mC * Aeo
GUACUUCATCCCACU
XXXXX XXXXX

9070
* mC * fU * fG * fA * fU * fU * fC
GAUUC
XXXXX XXXX

WV-
fG * fU * fA * fC * fU * fU * m5Ceo * mATeo * mC m5Ceo * mCAeo *
GUACUUCATCCCACU
XXXXXXXOXOXO

9071
mCfU * fG * fA * fU * fU * fC
GAUUC
XOXXXXX

WV-
fG * fU * fA * fC * fU * fU * m5Ceo mA * Teo mC * m5Ceo mC * Aeo mC *
GUACUUCATCCCACU
XXXXXXOXOXOX

9072
fU * fG * fA * fU * fU * fC
GAUUC
OXXXXXX

WV-
fG * fU * fA * fC * fU * fU * mC * Aeo * mU * m5Ceo * mC * m5Ceo *
GUACUUCAUCCCACU
XXXXX XXXXX

9073
mA * m5Ceo * fU * fG * fA * fU * fU * fC
GAUUC
XXXXX XXXX

WV-
fG * fU * fA * fC * fU * fU * mC * Aeo mU * m5Ceo mC * m5Ceo mA *
GUACUUCAUCCCACU
XXXXXXXOXOXO

9074
m5CeofU * fG * fA * fU * fU * fU
GAUUC
XOXXXXX

WV-
fG * fU * fA * fC * fU * fU * mCAeo * mU m5Ceo * mC m5Ceo * mA
GUACUUCAUCCCACU
XXXXXXOXOXOX

9075
m5Ceo * fU * fG * fA * fU * fU * fC
GAUUC
OXXXXXX

WV-
fG * fU * fA * fC * fU * fU * m5Ceo * fA * Teo * fC * m5Ceo * fC * Aeo * fC
GUACUUCATCCCACU
XXXXX XXXXX

9076
* fU * fG * fA * fU * fU * fC
GAUUC
XXXXX XXXX

WV-
fG * fU * fA * fC * fU * fU * m5Ceo * fATeo * fC m5Ceo * fCAeo * fCfU * fG
GUACUUCATCCCACU
XXXXXXXOXOXO

9077
* fA * fU * fU * fC
GAUUC
XOXXXXX

WV-
fG * fU * fA * fC * fU * fU * m5CeofA * TeofC * m5CeofC * AeofC * fU * fG
GUACUUCATCCCACU
XXXXXXOXOXOX

9078
* fA * fU * fU * fC
GAUUC
OXXXXXX

WV-
fG * fU * fA * fC * fU * fU * fC * Aeo * fU * m5Ceo * fC * m5Ceo * fA *
GUACUUCAUCCCACU
XXXXX XXXXX

9079
m5Ceo * fU * fG * fA * fU * fU * fC
GAUUC
XXXXX XXXX

WV-
fG * fU * fA * fC * fU * fU * fC * AeofU * m5CeofC * m5CeofA * m5CeofU
GUACUUCAUCCCACU
XXXXXXXOXOXO

9080
* fG * fA * fU * fU * fC
GAUUC
XOXXXXX

WV-
fG * fU * fA * fC * fU * fU * fCAeo * fU m5Ceo * fC m5Ceo * fA m5Ceo * fU
GUACUUCAUCCCACU
XXXXXXOXOXOX

9081
* fG * fA * fU * fU * fC
GAUUC
OXXXXXX

WV-
fG * fU * fA * fC * fU * fU * mC * fA * mU * fC * mC * fC * mA * fC * fU
GUACUUCAUCCCACU
XXXXX XXXXX

9082
* fG * fA * fU * fU * fC
GAUUC
XXXXX XXXX

WV-
fG * fU * fA * fC * fU * fU * mC * fA mU * fC mC * fC mA * fCfU * fG * fA
GUACUUCAUCCCACU
XXXXXXXOXOXO

9083
* fU * fU * fC
GAUUC
XOXXXXX

WV-
fG * fU * fA * fC * fU * fU * mCfA * mUfC * mCfC * mAfC * fU * fG * fA
GUACUUCAUCCCACU
XXXXXXOXOXOX

9084
* fU * fU * fC
GAUUC
OXXXXXX

WV-
fG * fU * fA * fC * fU * fU * fC * mA * fU * mC * fC * mC * fA * mC * fU
GUACUUCAUCCCACU
XXXXX XXXXX

9085
* fG * fA * fU * fU * fC
GAUUC
XXXXX XXXX

WV-
fG * fU * fA * fC * fU * fU * fC * mAfU * mCfC * mCfA * mCfU * fG * fA
GUACUUCAUCCCACU
XXXXXXXOXOXO

9086
* fU * fU * fC
GAUUC
XOXXXXX

WV-
fG * fU * fA * fC * fU * fU * fC mA * fU mC * fC mC * fA mC * fU * fG * fA
GUACUUCAUCCCACU
XXXXXXOXOXOX

9087
* fU * fU * fC
GAUUC
OXXXXXX

WV-
Geo * Teo * Aeo * m5Ceo * Teo * Teo * m5Ceo * Aeo * Teo * m5Ceo *
GTACTTCATCCCACU
XXXXX XXXXX

9088
m5Ceo * m5Ceo * Aeo * m5Ceo * fU * fG * fA * fU * fU * fC
GAUUC
XXXXX XXXX

WV-
mG * mU * mA * mC * mU * Teo * m5Ceo * Aeo * Teo * m5Ceo * m5Ceo
GUACUTCATCCCACU
XXXXX XXXXX

9089
* m5Ceo * Aeo * m5Ceo * fU * fG * fA * fU * fU * fC
GAUUC
XXXXX XXXX

WV-
mG * mU * mA * mC * mU * mU * m5Ceo * Aeo * Teo * m5Ceo
GUACUUCATCCCACU
XXXXX XXXXX

9090
m5Ceo * m5Ceo * Aeo * m5Ceo * fU * fG * fA * fU * fU * fC
GAUUC
XXXXX XXXX

WV-
fG * fU * fG * fU * fU * fC * Teo * Teo * Geo * Teo * Aeo * m5Ceo * Teo *
GUGUUCTTGTACTTC
XXXXX XXXXX

9091
Teo * fC * fA * fU * fC * fC * fC
AUCCC
XXXXX XXXX

WV-
fG * fU * fG * fU * fU * fC * Teo * TeoGeo * TeoAeo * m5CeoTeo * TeofC *
GUGUUCTTGTACTTC
XXXXXXXOXOXO

9092
fA * fU * fC * fC * fC
AUCCC
XOXXXXX

WV-
fG * fU * fG * fU * fU * fC * TeoTeo * GeoTeo * Aeo m5Ceo * TeoTeo * fC *
GUGUUCTTGTACTTC
XXXXXXOXOXOX

9093
fA * fU * fC * fC * fC
AUCCC
OXXXXXX

WV-
fG * fU * fG * fU * fU * fc * Teo * mU * Geo * mU * Aeo * mC * Teo * mU
GUGUUCTUGUACTUC
XXXXX XXXXX

9094
* fC * fA * fU * fC * fC * fC
AUCCC
XXXXX XXXX

WV-
fG * fU * fG * fU * fU * fC * Teo * mUGeo * mUAeo * mCTeo * mUfC * fA
GUGUUCTUGUACTUC
XXXXXXXOXOXO

9095
* fU * fC * fC * fC
AUCCC
XOXXXXX

WV-
fG * fU * fG * fU * fU * fC * Teo mU * Geo mU * Aeo mC * Teo mU * fC * fA
GUGUUCTUGUACTUC
XXXXXXOXOXOX

9096
* fU * fC * fC * fC
AUCCC
OXXXXXX

WV-
fU * fU * fG * fU * fU * fC * mU * Teo * mG * Teo * mA * m5Ceo * mU *
GUGUUCUTGTACUTC
XXXXX XXXXX

9097
Teo * fC * fA * fU * fC * fC * fC
AUCCC
XXXXX XXXX

WV-
fG * fU * fG * fU * fU * fC * mU * Teo mG * Teo mA * m5Ceo mU * TeofC *
GUGUUCUTGTACUTC
XXXXXXXOXOXO

9098
fA * fU * fC * fC * fC
AUCCC
XOXXXXX

WV-
fG * fU * fG * fU * fU * fC * mUTeo * mGTeo * mA m5Ceo * mUTeo * fC *
GUGUUCUTGTACUTC
XXXXXXOXOXOX

9099
fA * fU * fC * fC * fC
AUCCC
OXXXXXX

WV-
fU * fU * fG * fU * fU * fC * Teo * fU * Geo * fU * Aeo * fC * Teo * fU * fC *
GUGUUCTUGUACTUC
XXXXX XXXXX

9100
fA * fU * fC * fC * fC
AUCCC
XXXXX XXXX

WV-
fG * fU * fG * fU * fU * fC * Teo * fUGeo * fUAeo * fCTeo * fUfC * fA * fU *
GUGUUCTUGUACTUC
XXXXXXXOXOXO

9101
fC * fC * fC
AUCCC
XOXXXXX

WV-
fG * fU * fG * fU * fU * fC * TeofU * GeofU * AeofC * TeofU * fC * fA * fU *
GUGUUCTUGUACTUC
XXXXXXOXOXOX

9102
fC * fC * fC
AUCCC
OXXXXXX

WV-
fG * fU * fG * fU * fU * fC * fU * Teo * fG * Teo * fA * m5Ceo * fU * Teo *
GUGUUCUTGTACUTC
XXXXX XXXXX

9103
fC * fA * fU * fC * fC * fC
AUCCC
XXXXX XXXX

WV-
fG * fU * fG * fU * fU * fC * fU * TeofG * TeofA * m5CeofU * TeofC * fA *
GUGUUCUTGTACUTC
XXXXXXXOXOXO

9104
fG * fC * fC * fC
AUCCC
XOXXXXX

WV-
fG * fU * fG * fU * fU * fC * fUTeo * fGTeo * fA m5Ceo * fUTeo * fC * fA *
GUGUUCUTGTACUTC
XXXXXXOXOXOX

9105
fU * fC * fC * fC
AUCCC
OXXXXXX

WV-
fG * fU * fG * fU * fU * fC * mU * fU * mG * fU * mA * fC * mU * fU * fC
GUGUUCUUGUACUUC
XXXXX XXXXX

9106
* fA * fU * fC * fC * fC
AUCCC
XXXXX XXXX

WV-
fG * fU * fG * fU * fU * fC * mU * fU mG * fU mA * fC mU * fUfC * fA * fU
GUGUUCUUGUACUUC
XXXXXXXOXOXO

9107
* fC * fC * fC
AUCCC
XOXXXXX

WV-
fG * fU * fG * fU * fU * fC * mUfU * mGfU * mAfC * mUfU * fC * fA * fU
GUGUUCUUGUACUUC
XXXXXXOXOXOX

9108
* fC * fC * fC
AUCCC
OXXXXXX

WV-
fG * fU * fG * fU * fU * fC * fU * mU * fG * mC * fA * mC * fU * mU * fC
GUGUUCUUGUACUUC
XXXXX XXXXX

9109
* fA * fU * fC * fC * fC
AUCCC
XXXXX XXXX

WV-
fG * fU * fG * fU * fU * fC * fU * mUfG * mUfA * mCfU * mUfC * fA * fU
GUGUUCUUGUACUUC
XXXXXXXOXOXO

9110
* fC * fC * fC
AUCCC
XOXXXXX

WV-
fG * fU * fG * fU * fU * fC * fU mU * fG mU * fA mC * fU mU * fC * fA * fU
GUGUUCUUGUACUUC
XXXXXXOXOXOX

9111
* fC * fC * fC
AUCCC
OXXXXXX

WV-
Geo * Teo * Geo * Teo * Teo * m5Ceo * Teo * Teo * Geo * Teo * Aeo *
GTGTTCTTGTACTTCA
XXXXX XXXXX

9112
m5Ceo * Teo * Teo * fC * fA * fU * fC * fC * fC

UCCC XXXXX XXXX

WV-
mG * mU * mG * mU * mU * m5Ceo * Teo * Teo * Geo * Teo * Aeo *
GUGUUCTTGTACTTC
XXXXX XXXXX

9113
m5Ceo * Teo * Teo * fC * fA * fU * fC * fC * fC
AUCCC
XXXXX XXXX

WV-
mG * mU * mG * mU * mU * mC * Teo * Teo * Geo * Teo * Aeo * m5Ceo
GUGUUCTTGTACTTC
XXXXX XXXXX

9114
* Teo * Teo * fC * fA * fU * fC * fC * fC
AUCCC
XXXXX XXXX

WV-
fU * fU * fC * fU * fG * fA * Aeo * Geo * Geo * Teo * Geo * Teo * Teo *
UUCUGAAGGTGTTCU
XXXXX XXXXX

9115
m5Ceo * fU * fU * fG * fU * fA * fC
UGUAC
XXXXX XXXX

WV-
fU * fU * fC * fU * fG * fA * Aeo * GeoGeo * TeoGeo * TeoTeo * m5CeofU *
UUCUGAAGGTGTTCU
XXXXXXXOXOXO

9116
fU * fG * fU * fA * fC
UGUAC
XOXXXXX

WV-
fU * fU * fC * fU * fG * fA * AeoGeo * GeoTeo * GeoTeo * Teo m5Ceo * fU *
UUCUGAAGGTGTTCU
XXXXXXOXOXOX

9117
fU * fG * fU * fA * fC
UGUAC
OXXXXXX

WV-
fU * fU * fC * fU * fG * fA * Aeo * mG * Geo * mU * Geo * mU * Teo * mC
UUCUGAAGGUGUTCU
XXXXX XXXXX

9118
* fU * fU * fG * fU * fA * fC
UGUAC
XXXXX XXXX

WV-
fU * fU * fC * fU * fG * fA * Aeo * mGGeo * mUGeo * mUTeo * mCfU * fU
UUCUGAAGGUGUTCU
XXXXXXXOXOXO

9119
* fG * fU * fA * fC
UGUAC
XOXXXXX

WV-
fU * fU * fC * fU * fG * fA * Aeo mG * Geo mU * Geo mU * Teo mC * fU * fU
UUCUGAAGGUGUTCU
XXXXXXOXOXOX

9120
* fG * fU * fA * fC
UGUAC
OXXXXXX

WV-
fU * fU * fC * fU * fG * fA * mA * Geo * mG * Teo * mG * Teo * mU *
UUCUGAAGGTGTUCU
XXXXX XXXXX

9121
m5Ceo * fU * fU * fG * fU * fA * fC
UGUAC
XXXXX XXXX

WV-
fU * fU * fC * fU * fG * fA * mA * Geo mG * Teo mG * Teo mU * m5CeofU
UUCUGAAGGTGTUCU
XXXXXXXOXOXO

9122
* fU * fG * fU * fA * fC
UGUAC
XOXXXXX

WV-
fU * fU * fC * fU * fG * fA * mAGeo * mGTeo * mGTeo * mU m5Ceo * fU
UUCUGAAGGTGTUCU
XXXXXXOXOXOX

9123
* fU * fG * fU * fA * fC
UGUAC
OXXXXXX

WV-
fU * fU * fC * fU * fG * fA * Aeo * fG * Geo * fU * Geo * fU * Teo * fC * fU *
UUCUGAAGGUGUTCU
XXXXX XXXXX

9124
fU * fG * fU * fA * fC
UGUAC
XXXXX XXXX

WV-
fU * fU * fC * fG * fG * fA * Aeo * fGGeo * fUGeo * fUTeo * fCfU * fU * fG *
UUCUGAAGGUGUTCU
XXXXXXXOXOXO

9125
fU * fA * fC
UGUAC
XOXXXXX

WV-
fU * fU * fC * fU * fG * fA * AeofG * GeofU * GeofU * TeofC * fU * fU * fG *
UUCUGAAGGUGUTCU
XXXXXXOXOXOX

9126
fU * fA * fC
UGUAC
OXXXXXX

WV-
fU * fU * fC * fU * fG * fA * fA * Geo * fG * Teo * fG * Teo * fU * m5Ceo *
UUCUGAAGGTGTUCU
XXXXX XXXXX

9127
fU * fU * fG * fU * fA * fC
UGUAC
XXXXX XXXX

WV-
fU * fU * fC * fU * fG * fA * fA * GeofG * TeofG * TeofU * m5CeofU * fU *
UUCUGAAGGTGTUCU
XXXXXXXOXOXO

9128
fG * fU * fA * fC
UGUAC
XOXXXXX

WV-
fU * fU * fC * fU * fG * fA * fAGeo * fGTeo * fGTeo * fU m5Ceo * fU * fU *
UUCUGAAGGTGTUCU
XXXXXXOXOXOX

9129
fG * fU * fA * fC
UGUAC
OXXXXXX

WV-
fU * fU * fC * fU * fG * fA * mA * fG * mG * fU * mG * fU * mU * fC * fU
UUCUGAAGGUGUUCU
XXXXX XXXXX

9130
* fU * fG * fU * fA * fC
UGUAC
XXXXX XXXX

WV-
fU * fU * fC * fU * fG * fA * mA * fG mG * fU mG * fU mU * fCfU * fU * fG
UUCUGAAGGUGUUCU
XXXXXXXOXOXO

9131
* fU * fA * fC
UGUAC
XOXXXXX

WV-
fU * fU * fC * fU * fG * fA * mAfG * mGfU * mGfU * mUfC * fU * fU * fG
UUCUGAAGGUGUUCU
XXXXXXOXOXOX

9132
* fU * fA * fC
UGUAC
OXXXXXX

WV-
fU * fU * fC * fU * fG * fA * fA * mG * fG * mU * fG * mU * fU * mC * fU
UUCUGAAGGUGUUCU
XXXXX XXXXX

9133
* fU * fG * fU * fA * fC
UGUAC
XXXXX XXXX

WV-
fU * fU * fC * fU * fG * fA * fA * mGfG * mUfG * mUfU * mCfU * fG * fG
UUCUGAAGGUGUUCU
XXXXXXXOXOXO

9134
* fU * fA * fC
UGUAC
XOXXXXX

WV-
fU * fU * fC * fU * fG * fA * fA mG * fG mU * fG mU * fU mC * fU * fU * fG
UUCUGAAGGUGUUCU
XXXXXXOXOXOX

9135
* fU * fA * fC
UGUAC
OXXXXXX

WV-
Teo * Teo * m5Ceo * Teo * Geo * Aeo * Aeo * Geo * Geo * Teo * Geo * Teo *
TTCTGAAGGTGTTCU
XXXXX XXXXX

9136
Teo * m5Ceo * fU * fU * fG * fU * fA * fC
UGUAC
XXXXX XXXX

WV-
mU * mU * mC * mU * mG * Aeo * Aeo * Geo * Geo * Teo * Geo * Teo *
UUCUGAAGGTGTTCU
XXXXX XXXXX

9137
Teo * m5Ceo * fU * fU * fG * fU * fA * fC
UGUAC
XXXXX XXXX

WV-
mU * mU * mC * mU * mG * mA * Aeo * Geo * Geo * Teo * Geo * Teo *
UUCUGAAGGTGTTCU
XXXXX XXXXX

9138
Teo * m5Ceo * fU * fU * fG * fU * fA * fC
UGUAC
XXXXX XXXX

WV-
fC * fU * fC * fC * fG * fG * Teo * Teo * m5Ceo * Teo * Geo * Aeo * Aeo *
CUCCGGTTCTGAAGG
XXXXX XXXXX

9139
Geo * fG * fU * fG * fU * fU * fC
UGUUC
XXXXX XXXX

WV-
fC * fU * fC * fC * fG * fG * Teo * Teo m5Ceo * TeoGeo * AeoAeo * GeofG *
CUCCGGTTCTGAAGG
XXXXXXXOXOXO

9140
fU * fG * fU * fU * fC
UGUUC
XOXXXXX

WV-
fC * fU * fC * fC * fG * fG * TeoTeo * m5CeoTeo * GeoAeo * AeoGeo * fG *
CUCCGGTTCTGAAGG
XXXXXXOXOXOX

9141
fU * fG * fU * fU * fC
UGUUC
OXXXXXX

WV-
fC * fU * fC * fC * fG * fG * Teo * mU * m5Ceo * mU * Geo * mA * Aeo *
CUCCGGTUCUGAAGG
XXXXX XXXXX

9142
mG * fG * fU * fG * fU * fU * fC
UGUUC
XXXXX XXXX

WV-
fC * fU * fC * fC * fG * fG * Teo * mU m5Ceo * mUGeo * mAAeo * mGfG
CUCCGGTUCUGAAGG
XXXXXXXOXOXO

9143
* fU * fG * fU * fU * fU
UGUUC
XOXXXXX

WV-
fC * fU * fC * fC * fG * fG * Teo mU * m5Ceo mU * Geo mA * Aeo mG * fG
CUCCGGTUCUGAAGG
XXXXXXOXOXOX

9144
* fU * fG * fU * fU * fC
UGUUC
OXXXXXX

WV-
fC * fU * fC * fC * fG * fG * mU * Teo * mC * Teo * mG * Aeo * mA * Geo
CUCCGGUTCTGAAGG
XXXXX XXXXX

9145
* fG * fU * fG * fU * fU * fU
UGUUC
XXXXX XXXX

+p

WV-
fC * fU * fC * fC * fG * fG * mU * Teo mC * Teo mG * Aeo mA * GeofG * fU
CUCCGGUTCTGAAGG
XXXXXXXOXOXO

9146
* fG * fU * fU * fC
UGUUC
XOXXXXX

WV-
fC * fU * fC * fC * fG * fG * mUTeo * mCTeo * mGAeo * mAGeo * fG * fU
CUCCGGUTCTGAAGG
XXXXXXOXOXOX

9147
* fG * fU * fU * fC
UGUUC
OXXXXXX

WV-
fC * fU * fC * fC * fG * fG * Teo * fU * m5Ceo * fU * Geo * fA * Aeo * fG *
CUCCGGTUCUGAAGG
XXXXX XXXXX

9148
fG * fU * fG * fU * fU * fC
UGUUC
XXXXX XXXX

WV-
fC * fU * fC * fC * fG * fG * Teo * fU m5Ceo * fUGeo * fAAeo * fGfG * fU *
CUCCGGTUCUGAAGG
XXXXXXXOXOXO

9149
fG * fU * fU * fC
UGUUC
XOXXXXX

WV-
fC * fU * fC * fC * fG * fG * TeofU * m5CeofU * GeofA * AeofG * fG * fU *
CUCCGGTUCUGAAGG
XXXXXXOXOXOX

9150
fG * fU * fU * fC
UGUUC
OXXXXXX

WV-
fC * fU * fC * fC * fG * fG * fU * Teo * fC * Teo * fG * Aeo * fA * Geo * fG *
CUCCGGUTCTGAAGG
XXXXX XXXXX

9151
fU * fG * fU * fU * fC
UGUUC
XXXXX XXXX

WV-
fC * fU * fC * fC * fG * fG * fU * TeofC * TeofG * AeofA * GeofG * fU * fG *
CUCCGGUTCTGAAGG
XXXXXXXOXOXO

9152
fU * fU * fC
UGUUC
XOXXXXX

WV-
fC * fU * fC * fC * fG * fG * fUTeo * fCTeo * fGAeo * fAGeo * fG * fU * fG *
CUCCGGUTCTGAAGG
XXXXXXOXOXOX

9153
fU * fU * fC
UGUUC
OXXXXXX

WV-
fC * fU * fC * fC * fG * fG * mU * fU * mC * fU * mG * fA * mA * fG * fG
CUCCGGUUCUGAAGG
XXXXX XXXXX

9154
* fU * fG * fU * fU * fC
UGUUC
XXXXX XXXX

WV-
fC * fU * fC * fC * fG * fG * mU * fU mC * fU mG * fA mA * fGfG * fU * fG
CUCCGGUUCUGAAGG
XXXXXXXOXOXO

9155
* fU * fU * fC
UGUUC
XOXXXXX

WV
fC * fU * fC * fC * fG * fG * mUfU * mCfU * mGfA * mAfG * fG * fU * fG
CUCCGGUUCUGAAGG
XXXXXXOXOXOX

9156
* fU * fU * fC
UGUUC
OXXXXXX

WV-
fC * fU * fC * fC * fG * fG * fU * mU * fC * mU * fG * mA * fA * mG * fG
CUCCGGUUCUGAAGG
XXXXX XXXXX

9157
* fU * fG * fU * fU * fC
UGUUC
XXXXX XXXX

WV-
fC * fU * fC * fC * fG * fG * fU * mUfC * mUfG * mAfA * mGfG * fU * fG
CUCCGGUUCUGAAGG
XXXXXXXOXOXO

9158
* fU * fU * fC
UGUUC
XOXXXXX

WV-
fC * fU * fC * fC * fG * fG * fU mU * fC mU * fG mA * fA mG * fG * fU * fG
CUCCGGUUCUGAAGG
XXXXXXOXOXOX

9159
* fU * fU * fC
UGUUC
OXXXXXX

WV-
m5Ceo * Teo * m5Ceo * m5Ceo * Geo * Geo * Teo * Teo * 5Ceo * Teo *
CTCCGGTTCTGAAGG
XXXXX XXXXX

9160
Geo * Aeo * Aeo * Geo * fG * fU * fG * fU * fU * fC
UGUUC
XXXXX XXXX

WV-
mC * mU * mC * mC * mG * Geo * Teo * Teo * m5Ceo * Teo * Geo * Aeo
CUCCGGTTCTGAAGG
XXXXX XXXXX

9161
* Aeo * Geo * fG * fU * fG * fU * fU * fC
UGUUC
XXXXX XXXX

WV-
mC * mU * mC * mC * mG * mG * Teo * Teo * m5Ceo * Teo * Geo * Aeo
CUCCGGTTCTGAAGG
XXXXX XXXXX

9162
* Aeo * Geo * fG * fU * fG * fU * fU * fC
UGUUC
XXXXX XXXX

WV-
fU * fC * fU * fU * fG * fG * m5Ceo * m5Ceo * Aeo * Teo * m5Ceo * Teo *
UCUUGGCCATCTCCU
XXXXX XXXXX

9163
m5Ceo * m5Ceo * fU * fU * fC * fA * fC * fA
UCACA
XXXXX XXXX

WV-
fU * fC * fU * fG * fG * fG * m5Ceo * m5CeoAeo * Teo m5Ceo * Teo m5Ceo
UCUUGGCCATCTCCU
XXXXXXXOXOXO

9164
* m5CeofU * fU * fC * fA * fC * fA
UCACA
XOXXXXX

WV-
fU * fC * fU * fU * fG * fG * m5Ceo m5Ceo * AeoTeo * m5CeoTeo * m5Ceo
UCUUGGCCATCTCCU
XXXXXXOXOXOX

9165
m5Ceo * fU * fU * fC * fA * fC * fA
UCACA
OXXXXXX

WV-
fU * fC * fU * fU * fG * fG * m5Ceo * mC * Aeo * mU * m5Ceo * mU *
UCUUGGCCAUCUCCU
XXXXX XXXXX

9166
m5Ceo * mC * fU * fU * fC * fA * fC * fA
UCACA
XXXXX XXXX

WV-
fU * fC * fU * fU * fG * fG * m5Ceo * mCAeo * mU m5Ceo * mU m5Ceo *
UCUUGGCCAUCUCCU
XXXXXXXOXOXO

9167
mCfU * fU * fC * fA * fC * fA
UCACA
XOXXXXX

WV-
fU * fC * fU * fU * fG * fG * m5Ceo mC * Aeo mU * m5Ceo mU * m5Ceo
UCUUGGCCAUCUCCU
XXXXXXOXOXOX

9168
mC * fU * fU * fC * fA * fC * fA
UCACA
OXXXXXX

WV-
fU * fC * fU * fU * fg * fG * mC * m5Ceo * mA * Teo * mC * Teo * mC *
UCUUGGCCATCTCCU
XXXXX XXXXX

9169
m5Ceo * fU * fU * fC * fA * fC * fA
UCACA
XXXXX XXXX

WV-
fU * fC * fU * fU * fG * fG * mC * m5Ceo mA * Teo mC * Teo mC *
UCUUGGCCATCTCCU
XXXXXXXOXOXO

9170
m5CeofU * fU * fC * fA * fC * fA
UCACA
XOXXXXX

WV-
fU * fC * fU * fU * fG * fG * mC m5Ceo * mATeo * mcTeo * mC m5Ceo *
UCUUGGCCATCTCCU
XXXXXXOXOXOX

9171
fU * fU * fC * fA * fC * fA
UCACA
OXXXXXX

WV-
fU * fC * fU * fU * fG * fG * m5Ceo * fC * Aeo * fU * m5Ceo * fU * m5Ceo
UCUUGGCCAUCUCCU
XXXXX XXXXX

9172
* fC * fU * fU * fC * fA * fC * fA
UCACA
XXXXX XXXX

WV-
fU * fC * fU * fU * fG * fG * m5Ceo * fCAeo * fU m5Ceo * fU m5Ceo * fCfU
UCUUGGCCAUCUCCU
XXXXXXXOXOXO

9173
* fU * fC * fA * fC * fA
UCACA
XOXXXXX

WV-
fU * fC * fU * fU * fG * fG * m5CeofC * AeofU * m5CeofU * m5CeofC * fU
UCUUGGCCAUCUCCU
XXXXXXOXOXOX

9174
* fU * fC * fA * fC * fA
UCACA
OXXXXXX

WV-
fU * fC * fU * fU * fG * fG * fC * m5Ceo * fA * Teo * fC * Teo * fC * m5Ceo
UCUUGGCCATCTCCU
XXXXX XXXXX

9175
* fU * fU * fC * fA * fC * fA
UCACA
XXXXX XXXX

WV-
fU * fC * fU * fU * fG * fG * fC * m5CeofA * TeofC * TeofC * m5CeofU * fU
UCUUGGCCATCTCCU
XXXXXXXOXOXO

9176
* fC * fA * fC * fA
UCACA
XOXXXXX

WV-
fU * fC * fU * fU * fG * fG * fC m5Ceo * fATeo * fCTeo * fC m5Ceo * fU * fU
UCUUGGCCATCTCCU
XXXXXXOXOXOX

9177
* fC * fA * fC * fA
UCACA
OXXXXXX

WV-
fU * fC * fU * fU * fG * fG * mC * fC * mA * fU * mC * fU * mC * fC * fU
UCUUGGCCAUCUCCU
XXXXX XXXXX

9178
* fU * fC * fA * fC * fA
UCACA
XXXXX XXXX

WV-
fU * fC * fU * fU * fG * fG * mC * fC mA * fG mC * fU mC * fCfU * fU * fC
UCUUGGCCAUCUCCU
XXXXXXXOXOXO

9179
* fA * fC * fA
UCACA
XOXXXXX

WV-
fU * fC * fU * fU * fG * fG * mCfC * mAfU * mCfU * mCfC * fU * fG * fC
UCUUGGCCAUCUCCU
XXXXXXOXOXOX

9180
* fA * fC * fA
UCACA
OXXXXXX

WV-
fU * fC * fU * fu * fG * fG * fC * mC * fA * mU * fC * mU * fC * mC * fU
UCUUGGCCAUCUCCU
XXXXX XXXXX

9181
* fU * fC * fA * fC * fA
UCACA
XXXXX XXXX

WV-
fU * fC * fU * fU * fG * fG * fC * mCfA * mUfC * mUfC * mCfU * fU * fC
UCUUGGCCAUCUCCU
XXXXXXXOXOXO

9182
* fA * fC * fA
UCACA
XOXXXXX

WV-
fU * fC * fU * fU * fG * fG * fC mC * fA mU * fC mU * fC mC * fU * fU * fC
UCUUGGCCAUCUCCU
XXXXXXOXOXOX

9183
* fA * fC * fA
UCACA
OXXXXXX

WV-
Teo * m5Ceo * Teo * Teo * Geo * Geo * m5Ceo * m5Ceo * Aeo * Teo *
TCTTGGCCATCTCCUU
XXXXX XXXXX

9184
m5Ceo * Teo * m5Ceo * m5Ceo * fU * fU * fC * fA * fC * fA
CACA
XXXXX XXXX

WV-
mU * mC * mU * mU * mG * Geo * m5Ceo * m5Ceo * Aeo * Teo *
UCUUGGCCATCTCCU
XXXXX XXXXX

9185
m5Ceo * Teo * m5Ceo * m5Ceo * fU * fU * fC * fA * fC * fA
UCACA
XXXXX XXXX

WV-
mU * mC * mU * mU * mG * mG * m5Ceo * m5Ceo * Aeo * Teo *
UCUUGGCCATCTCCU
XXXXX XXXXX

9186
m5Ceo * Teo * m5Ceo * m5Ceo * fU * fU * fC * fA * fC * fA
UCACA
XXXXX XXXX

WV-
fU * fU * fU * fC * fU * fU * Geo * Geo * m5Ceo * m5Ceo * Aeo * Teo *
UUUCUUGGCCATCTC
XXXXX XXXXX

9187
m5Ceo * Teo * fC * fC * fU * fU * fC * fA
CUUCA
XXXXX XXXX

WV-
fU * fU * fU * fC * fU * fU * Geo * Geo m5Ceo * m5CeoAeo * Teo m5Ceo *
UUUCUUGGCCATCTC
XXXXXXXOXOXO

9188
TeofC * fC * fU * fU * fC * fA
CUUCA
XOXXXXX

WV-
fU * fU * fU * fC * fU * fU * GeoGeo * m5Ceo m5Ceo * AeoTeo * m5CeoTeo
UUUCUUGGCCATCTC
XXXXXXOXOXOX

9189
* fC * fC * fU * fU * fC * fA
CUUCA
OXXXXXX

WV-
fU * fU * fU * fC * fU * fU * Geo * mG * m5Ceo * mC * Aeo * mU *
UUUCUUGGCCAUCUC
XXXXX XXXXX

9190
m5Ceo * mU * fC * fC * fU * fU * fC * fA
CUUCA
XXXXX XXXX

WV-
fU * fU * fU * fC * fU * fU * Geo * mG m5Ceo * mCAeo * mU m5Ceo *
UUUCUUGGCCAUCUC
XXXXXXXOXOXO

9191
mUfC * fC * fU * fU * fC * fA
CUUCA
XOXXXXX

WV-
fU * fU * fU * fC * fU * fU * Geo mG * m5Ceo mC * Aeo mU * m5Ceo mU *
UUUCUUGGCCAUCUC
XXXXXXOXOXOX

9192
fC * fC * fU * fU * fC * fA
CUUCA
OXXXXXX

WV-
fU * fU * fU * fC * fU * fU * mG * Geo * mC * m5Ceo * mA * Teo * mC *
UUUCUUGGCCATCTC
XXXXX XXXXX

9193
Teo * fC * fC * fU * fU * fC * fA
CUUCA
XXXXX XXXX

WV-
fU * fU * fU * fC * fU * fG * mG * Geo mC * m5Ceo mA * Teo mC * TeofC *
UUUCUUGGCCATCTC
XXXXXXXOXOXO

9194
fC * fU * fU * fC * fA
CUUCA
XOXXXXX

WV-
fU * fU * fU * fC * fU * fU * mGGeo * mC m5Ceo * mATeo * mCTeo * fC *
UUUCUUGGCCATCTC
XXXXXXOXOXOX

9195
fC * fU * fU * fC * fA
CUUCA
OXXXXXX

WV-
fU * fU * fU * fC * fU * fU * Geo * fG * m5Ceo * fC * Aeo * fU * m5Ceo *
UUUCUUGGCCAUCUC
XXXXX XXXXX

9196
fU * fC * fC * fU * fU * fC * fA
CUUCA
XXXXX XXXX

WV-
fU * fU * fU * fC * fU * fU * Geo * fG m5Ceo * fCAeo * fU m5Ceo * fUfC *
UUUCUUGGCCAUCUC
XXXXXXXOXOXO

9197
fC * fU * fU * fC * fA
CUUCA
XOXXXXX

WV-
fU * fU * fU * fC * fU * fU * GeofG * m5CeofC * AeofU * m5CeofU * fC *
UUUCUUGGCCAUCUC
XXXXXXOXOXOX

9198
fC * fU * fU * fC * fA
CUUCA
OXXXXXX

WV-
fU * fU * fU * fC * fU * fU * fG * Geo * fC * m5Ceo * fA * Teo * fC * Teo *
UUUCUUGGCCATCTC
XXXXX XXXXX

9199
fC * fC * fU * fU * fC * fA
CUUCA
XXXXX XXXX

WV-
fU * fU * fU * fC * fU * fU * fG * GeofC * m5CeofA * TeofC * TeofC * fC *
UUUCUUGGCCATCTC
XXXXXXXOXOXO

9200
fU * fU * fC * fA
CUUCA
XOXXXXX

WV-
fU * fU * fU * fC * fU * fU * fGGeo * fC m5Ceo * fATeo * fCTeo * fC * fC *
UUUCUUGGCCATCTC
XXXXXXOXOXOX

9201
fU * fU * fC * fA
CUUCA
OXXXXXX

WV-
fU * fU * fU * fC * fU * fU * mG * fG * mC * fC * mA * fU * mC * fU * fC
UUUCUUGGCCAUCUC
XXXXX XXXXX

9202
* fC * fU * fU * fC * fA
CUUCA
XXXXX XXXX

WV-
fU * fU * fU * fC * fU * fU * mG * fG mC * fC mA * fU mC * fUfC * fC * fU
UUUCUUGGCCAUCUC
XXXXXXXOXOXO

9203
* fU * fC * fA
CUUCA
XOXXXXX

WV-
fU * fU * fU * fC * fU * fU * mGfG * mCfC * mAfU * mCfU * fC * fC * fU
UUUCUUGGCCAUCUC
XXXXXXOXOXOX

9204
* fU * fC * fA
CUUCA
OXXXXXX

WV-
fU * fU * fU * fC * fU * fU * fG * mG * fC * mC * fA * mU * fC * mU * fC
UUUCUUGGCCAUCUC
XXXXX XXXXX

9205
* fC * fU * fU * fC * fA
CUUCA
XXXXX XXXX

WV-
fU * fU * fU * fC * fU * fU * fG * mGfC * mCfA * mUfC * mUfC * fC * fU
UUUCUUGGCCAUCUC
XXXXXXXOXOXO

9206
* fU * fC * fA
CUUCA
XOXXXXX

WV-
fU * fU * fU * fC * fU * fU * fG mG * fC mC * fA mU * fC mU * fC * fC * fU
UUUCUUGGCCAUCUC
XXXXXXOXOXOX

9207
* fU * fC * fA
CUUCA
OXXXXXX

WV-
Teo * Teo * Teo * m5Ceo * Teo * Teo * Geo * Geo * m5Ceo * m5Ceo * Aeo *
TTTCTTGGCCATCTCC
XXXXX XXXXX

9208
Teo * m5Ceo * Teo * fC * fC * fU * fU * fC * fA
UUCA
XXXXX XXXX

WV-
mU * mU * mU * mC * mU * Teo * Geo * Geo * m5Ceo * m5Ceo * Aeo *
UUUCUTGGCCATCTC
XXXXX XXXXX

9209
Teo * m5Ceo * Teo * fC * fC * fU * fU * fC * fA
CUUCA
XXXXX XXXX

WV-
mU * mU * mU * mC * mU * mU * Geo * Geo * m5Ceo * m5Ceo * Aeo *
UUUCUUGGCCATCTC
XXXXX XXXXX

9210
Teo * m5Ceo * Teo * fC * fC * fU * fU * fC * fA
CUUCA
XXXXX XXXX

WV-
Teo * S m5Ceo * SAeo * SAeo * SGeo * SGeo * SAeofA * SGeoAeo * SfU *
TCAAGGAAGAUGGCA
SSSSSSOSOSSOOS

9222
SGeoGeofC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
Teo * S m5Ceo * SAeo * SAeo * SGeo * SGeo * SAeoAeo * SGeoAeo * STeo *
TCAAGGAAGATGGCA
SSSSSSOSOSSOOS

9223
SGeoGeo m5Ceo * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
Teo * S m5Ceo * SAeo * SAeo * SGeo * SGeo * SAeo * SAeo * SGeo * SAeo *
TCAAGGAAGATGGCA
SSSSSSSSSSSSSSS

9224
STeo * SGeo * SGeo * S m5Ceo * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSS

WV-
Teo * m5Ceo * Aeo * Aeo * Geo * Geo * AeofA * GeoAeo * fU * GeoGeofC *
TCAAGGAAGAUGGCA
XXXXXXOXOXXO

9225
fA * fU * fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
Teo * m5Ceo * Aeo * Aeo * Geo * Geo * AeoAeo * GeoAeo * Teo * GeoGeo
TCAAGGAAGATGGCA
XXXXXXOXOXXO

9226
m5Ceo * fA * fU * fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fC * fA * fA * fG * fG * AeofA * GeoAeo * fU * GeoGeofC * fA * fU * fU
UCAAGGAAGAUGGCA
XXXXXXOXOXXO

9227
* fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * SfU * SfU * SfU * SfG * SfG * S mC * S mA * S mG * S mC * S mU * S
UUUUGGCAGCUUUCC
SSSSSSSSSSSSSSS

9408
mU * S mU * S mC * SfC * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSS

WV-
fU * SfU * SfU * SfU * SfG * SfG * S mC * SfA * S mG * S mC * SfU * S mU
UUUUGGCAGCUUUCC
SSSSSSSSSSSSSSS

9409
* S mU * SfC * SfC * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSS

WV-
fU * SfU * SfU * SfU * SfG * SfG * S m5Ceo * SfA * SGeo * S m5Ceo * SfU *
UUUUGGCAGCUTTCC
SSSSSSSSSSSSSSS

9410
STeo * STeo * SfC * SfC * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSS

WV-
fU * SfU * SfU * SfU * SfG * SfG * S mCfA * S mG mC * SfU * S mU mUfC *
UUUUGGCAGCUUUCC
SSSSSSOSOSSOOS

9411
SfC * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSSS

WV-
fU * SfU * SfU * SfU * SfG * SfG * S m5CeofA * SGeo m5Ceo * SfU *
UUUUGGCAGCUTTCC
SSSSSSOSOSSOOS

9412
STeoTeofC * SfC * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSSS

WV-
fU * SfU * SfU * SfU * SfG * SfG * S m5CeofA * S mG m5Ceo * SfU *
UUUUGGCAGCUTTCC
SSSSSSOSOSSOOS

9413
STeoTeofC * SfC * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSSS

WV-
fU * SfU * SfU * SfU * SfG * SfG * S m5CeofA * S mG mC * SfU *
UUUUGGCAGCUTTCC
SSSSSSOSOSSOOS

9414
STeoTeofC * SfC * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSSS

WV-
fU * fU * fU * fU * fG * fG * mC * fA * mG * mC * fU * mU * mU * fC *
UUUUGGCAGCUUUCC
XXXXX XXXXX

9415
fC * fA * fC * fC * fA * fA
ACCAA
XXXXX XXXX

WV-
fU * fU * fU * fU * fG * fG * m5Ceo * fA * Geo * m5Ceo * fU * Teo * Teo *
UUUUGGCAGCUTTCC
XXXXX XXXXX

9416
fC * fC * fA * fC * fC * fA * fA
ACCAA
XXXXX XXXX

WV-
fU * fU * fU * fU * fG * fG * mCfA * mG mC * fU * mU mUfC * fC * fA *
UUUUGGCAGCUUUCC
XXXXXXOXOXXO

9417
fC * fC * fA * fA
ACCAA
OXXXXXX

WV-
fU * fU * fU * fU * fG * fG * m5CeofA * Geo m5Ceo * fU * TeoTeofC * fC *
UUUUGGCAGCUTTCC
XXXXXXOXOXXO

9418
fA * fC * fC * fA * fA
ACCAA
OXXXXXX

WV-
fU * fU * fU * fU * fG * fG * m5CeofA * mG m5Ceo * fU * TeoTeofC * fC *
UUUUGGCAGCUTTCC
XXXXXXOXOXXO

9419
fA * fC * fC * fA * fA
ACCAA
OXXXXXX

WV-
mU * mC * mA * mA * mG * mG * mA * mA * mG * mA * mU * mG *
UCAAGGAAGAUGGCA
XXXXX XXXXX

942
mG * mC * mA * mU * mU * mU * mC * mU
UUUCU
XXXXX XXXX

WV-
fU * fU * fU * fU * fG * fG * m5CeofA * mG mC * fU * TeoTeofC * fC * fA *
UUUUGGCAGCUTTCC
XXXXXXOXOXXO

9420
fC * fC * fA * fA
ACCAA
OXXXXXX

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mC mU * SfG * S mA mAfG *
CUCCGGUUCUGAAGG
SSSSSSOSOSSOOS

9422
SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * STeofU * S m5CeoTeo * SfG * SAeoAeofG
CUCCGGTUCTGAAGG
SSSSSSOSOSSOOS

9423
* SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * STeofU * S m5CeoTeo * SfG * S mA
CUCCGGTUCTGAAGG
SSSSSSOSOSSOOS

9424
mAfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * STeofU * S m5Ceo mU * SfG * S mA
CUCCGGTUCUGAAGG
SSSSSSOSOSSOOS

9425
mAfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * fU * fC * fC * fG * fG * mUfU * mC mU * fG * mA mAfG * fG * fU *
CUCCGGUUCUGAAGG
XXXXXXOXOXXO

9426
fG * fU * fU * fC
UGUUC
OXXXXXX

WV-
fC * fU * fC * fC * fG * fG * TeofU * m5CeoTeo * fG * AeoAeofG * fG * fU *
CUCCGGTUCTGAAGG
XXXXXXOXOXXO

9427
fG * fU * fU * fC
UGUUC
OXXXXXX

WV-
fC * fU * fC * fC * fG * fG * TeofU * m5CeoTeo * fG * mA mAfG * fG * fU *
CUCCGGTUCTGAAGG
XXXXXXOXOXXO

9428
fG * fU * fU * fC
UGUUC
OXXXXXX

WV-
fC * fU * fC * fC * fG * fG * TeofU * m5Ceo mU * fG * mA mAfG * fG * fU
CUCCGGTUCUGAAGG
XXXXXXOXOXXO

9429
* fG * fU * fU * fC
UGUUC
OXXXXXX

WV-
mG * mG * mC * mC * mA * mA * mA * mC * mC * mU * mC * mG *
GGCCAAACCUCGGCU
XXXXX XXXXX

943
mG * mC * mU * mU * mA * mC * mC * mU
UACCU
XXXXX XXXX

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SfC * SfU * S mG mA mA
CUCCGGUUCUGAAGG
SSSSSSSSSSOOOO

9511
mGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfU * SfC * SfG * SfG * SfU * SfU * S mCfU * S mGfA * S mA mG
CUCCGGUUCUGAAGG
SSSSSSSSOSOSOO

9512
mGfU * SfG * SfU * SfU * SfC
UGUUC
OSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * S mGfA * S mA
CUCCGGUUCUGAAGG
SSSSSSSSOSOSOO

9513
mGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * S mGfA * S mAfG *
CUCCGGUUCUGAAGG
SSSSSSSSOSOSOS

9514
S mGfU * SfG * SfU * SfU * SfC
UGUUC
OSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * S mGfA * SfA * S
CUCCGGUUCUGAAGG
SSSSSSSSOSOSSO

9515
mG mGfU * SfG * SfU * SfU * SfC
UGUUC
OSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * S mG * SfA * S mA
CUCCGGUUCUGAAGG
SSSSSSSSOSSSOO

9516
mG mGfU * SfG * SfU * SfU * SfC
UGUUC
OSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * S mG * SfA * S mA
CUCCGGUUCUGAAGG
SSSSSSSSOSSSOO

9517
mGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * S mG * SfA * S
CUCCGGUUCUGAAGG
SSSSSSSSOSSSOS

9518
mAfG * S mGfU * SfG * SfU * SfU * SfC
UGUUC
OSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * S mG * SfA * SfA *
CUCCGGUUCUGAAGG
SSSSSSSSOSSSSO

9519
S mG mGfU * SfG * SfU * SfU * SfC
UGUUC
OSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * SfG * SfA * S mA
CUCCGGUUCUGAAGG
SSSSSSSSOSSSOO

9520
mG mGfU * SfG * SfU * SfU * SfC
UGUUC
OSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * SfG * SfA * S mA
CUCCGGUUCUGAAGG
SSSSSSSSOSSSOO

9521
mGfG * SfU * SfG * SfU * SfU * SfU
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * SfG * SfA * S
CUCCGGUUCUGAAGG
SSSSSSSSOSSSOS

9522
mAfG * S mGfU * SfG * SfU * SfU * SfC
UGUUC
OSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * SfG * SfA * SfA * S
CUCCGGUUCUGAAGG
SSSSSSSSOSSSSO

9523
mG mGfU * SfG * SfU * SfU * SfC
UGUUC
OSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mCfU * S mGfA * S mAfG *
CUCCGGUUCUGAAGG
SSSSSSOSOSOSOS

9524
SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * mUfC * mUfG * mAfA * mGfG *
CUCCGGUUCUGAAGG
SSSSSSXOXOXOX

9525
SfU * SfG * SfU * SfU * SfC
UGUUC
OSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * S mUfC * S mUfG * S mAfA * S
CUCCGGUUCUGAAGG
SSSSSSSOSOSOSO

9534
mGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * S mG * SfU * SfU * S mCfU * SfG * SfA * S mA
CUCCGGUUCUGAAGG
SSSSSSSSOSSSOO

9535
mG mGfU * SfG * SfU * SfU * SfC
UGUUC
OSSSS

WV-
fC * SfU * SfC * SfC * SfG * S mG * SfU * SfU * S mCfU * SfG * SfA * S mA
CUCCGGUUCUGAAGG
SSSSSSSSOSSSOO

9536
mGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * S mG * SfU * SfU * S mCfU * SfG * SfA * S
CUCCGGUUCUGAAGG
SSSSSSSSOSSSOS

9537
mAfG * S mGfU * SfG * SfU * SfU * SfC
UGUUC
OSSSS

WV-
fC * SfU * SfC * SfC * SfG * S mG * SfU * SfU * S mCfU * SfG * SfA * SfA *
CUCCGGUUCUGAAGG
SSSSSSSSOSSSSO

9538
S mG mGfU * SfG * SfU * SfU * SfC
UGUUC
OSSSS

WV-
fC * SfU * SfC * SfC * SfG * S mG * SfU * SfU * SfC * SfU * S mG mA mA
CUCCGGUUCUGAAGG
SSSSSSSSSSOOOO

9539
mGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
Teo * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG mGfC
TCAAGGAAGAUGGCA
SSSSSSOSOSSOOS

9540
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
Teo * RfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG
TCAAGGAAGAUGGCA
RSSSSSOSOSSOOS

9541
mGfU * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fA * fA * fU * fA * fU * fU * mC * mU * mU * mC * mU * mA * mA *
AAUAUUCUUCUAAA
XXXXX XXXXX

9594
mA * mG * mA * mA * mA * mG * fC * fU * fU * fA * fA * fA
GAAAGCUUAAA
XXXXX XXXXX

XXXX

WV-
fU * fC * fU * fU * fC * fU * mA * mA * mA * mG * mA * mA * mA *
UCUUCUAAAGAAAG
XXXXX XXXXX

9595
mG * mC * mU * mU * mA * mA * fA * fA * fA * fG * fU * fC
CUUAAAAAGUC
XXXXX XXXXX

XXXX

WV-
fU * fA * fA * fA * fG * fA * mA * mA * mG * mC * mU * mU * mA *
UAAAGAAAGCUUAA
XXXXX XXXXX

9596
mA * mA * mA * mA * mG * mU * fC * fU * fG * fC * fU * fA
AAAGUCUGCUA
XXXXX XXXXX

XXXX

WV-
fA * fA * fA * fG * fC * fU * mU * mA * mA * mA * mA * mA * mG *
AAAGCUUAAAAAGUC
XXXXX XXXXX

9597
mU * mC * mU * mG * mC * mU * fA * fA * fA * fA * fU * fG
UGCUAAAAUG
XXXXX XXXXX

XXXX

WV-
fU * fU * fA * fA * fA * fA * mA * mG * mU * mC * mU * mG * mC *
UUAAAAAGUCUGCUA
XXXXX XXXXX

9598
mU * mA * mA * mA * mA * mU * fG * fU * fU * fU * fU * fC
AAAUGUUUUC
XXXXX XXXXX

XXXX

WV-
fA * fA * fG * fU * fC * fU * mG * mC * mU * mA * mA * mA * mA *
AAGUCUGCUAAAAUG
XXXXX XXXXX

9599
mU * mG * mU * mU * mU * mU * fC * fA * fU * fU * fC * fC
UUUUCAUUCC
XXXXX XXXXX

XXXX

WV-
fU * fG * fC * fU * fA * fA * mA * mA * mU * mG * mU * mU * mU *
UGCUAAAAUGUUUUC
XXXXX XXXXX

9600
mU * mC * mA * mU * mU * mC * fC * fU * fA * fU * fU * fA
AUUCCUAUUA
XXXXX XXXXX

XXXX

WV-
fA * fA * fA * fU * fG * fU * mU * mU * mU * mC * mA * mU * mU *
AAAUGUUUUCAUUCC
XXXXX XXXXX

9601
mC * mC * mU * mA * mU * mU * fA * fG * fA * fU * fC * fU
UAUUAGAUCU
XXXXX XXXXX

XXXX

WV-
fU * fU * fU * fU * fC * fA * mU * mU * mC * mC * mU * mA * mU *
UUUUCAUUCCUAUUA
XXXXX XXXXX

9602
mU * mA * mG * mA * mU * mC * fU * fG * fU * fC * fG * fC
GAUCUGUCGC
XXXXX XXXXX

XXXX

WV-
fA * fU * fU * fC * fC * fU * mA * mU * mU * mA * mG * mA * mU *
AUUCCUAUUAGAUCU
XXXXX XXXXX

9603
mC * mU * mG * mU * mC * mG * fC * fC * fC * fU * fA * fC
GUCGCCCUAC
XXXXX XXXXX

XXXX

WV-
fU * fA * fU * fU * fA * fG * mA * mU * mC * mU * mG * mU * mC *
UAUUAGAUCUGUCGC
XXXXX XXXXX

9604
mG * mC * mC * mC * mU * mA * fC * fC * fU * fC * fU * fU
CCUACCUCUU
XXXXX XXXXX

XXXX

WV-
fG * fA * fU * fC * fU * fG * mU * mC * mG * mC * mC * mC * mU *
GAUCUGUCGCCCUAC
XXXXX XXXXX

9605
mA * mC * mC * mU * mC * mU * fU * fU * fU * fU * fU * fC
CUCUUUUUUC
XXXXX XXXXX

XXXX

WV-
fG * fU * fC * fG * fC * fC * mC * mU * mA * mC * mC * mU * mC * mU
GUCGCCCUACCUCUU
XXXXX XXXXX

9606
* mU * mU * mU * mU * mU * fC * fU * fG * fU * fC * fU
UUUUCUGUCU
XXXXX XXXXX

XXXX

WV-
fC * fC * fU * fA * fC * fC * mU * mC * mU * mU * mU * mU * mU *
CCUACCUCUUUUUUC
XXXXX XXXXX

9607
mU * mC * mU * mG * mU * mC * fU * fG * fA * fC * fA * fG
UGUCUGACAG
XXXXX XXXXX

XXXX

WV-
fC * fU * fC * fU * fU * fU * mU * mU * mU * mC * mU * mG * mU *
CUCUUUUUUCUGUCU
XXXXX XXXXX

9608
mC * mU * mG * mA * mC * mA * fG * fC * fU * fG * fU * fU
GACAGCUGUU
XXXXX XXXXX

XXXX

WV-
fU * fU * fU * fU * fC * fU * mG * mU * mC * mU * mG * mA * mC *
UUUUCUGUCUGACAG
XXXXX XXXXX

9609
mA * mG * mC * mU * mG * mU * fU * fU * fG * fC * fA * fG
CUGUUUGCAG
XXXXX XXXXX

XXXX

WV-
fU * fG * fU * fC * fU * fG * mA * mC * mA * mG * mC * mU * mG *
UGUCUGACAGCUGUU
XXXXX XXXXX

9610
mU * mU * mU * mG * mC * mA * fG * fA * fC * fC * fU * fC
UGCAGACCUC
XXXXX XXXXX

XXXX

WV-
fG * fA * fC * fA * fG * fC * mU * mG * mU * mU * mU * mG * mC *
GACAGCUGUUUGCAG
XXXXX XXXXX

9611
mA * mG * mA * mC * mC * mU * fC * fC * fU * fG * fC * fC
ACCUCCUGCC
XXXXX XXXXX

XXXX

WV-
fC * fU * fG * fU * fU * fU * mG * mC * mA * mG * mA * mC * mC *
CUGUUUGCAGACCUC
XXXXX XXXXX

9612
mU * mC * mC * mU * mG * mC * fC * fA * fC * fC * fG * fC
CUGCCACCGC
XXXXX XXXXX

XXXX

WV-
fU * fG * fC * fA * fG * fA * mC * mC * mU * mC * mC * mU * mG *
UGCAGACCUCCUGCC
XXXXX XXXXX

9613
mC * mC * mA * mC * mC * mG * fC * fA * fG * fA * fU * fU
ACCGCAGAUU
XXXXX XXXXX

XXXX

WV-
fA * fC * fC * fU * fC * fC * mU * mG * mC * mC * mA * mC * mC * mG
ACCUCCUGCCACCGC
XXXXX XXXXX

9614
* mC * mA * mG * mA * mU * fU * fC * fA * fG * fG * fC
AGAUUCAGGC
XXXXX XXXXX

XXXX

WV-
fC * fU * fG * fC * fC * fA * mC * mC * mG * mC * mA * mG * mA *
CUGCCACCGCAGAUU
XXXXX XXXXX

9615
mU * mU * mC * mA * mG * mG * fC * fU * fU * fC * fC * fC
CAGGCUUCCC
XXXXX XXXXX

XXXX

WV-
fA * fC * fC * fG * fC * fA * mG * mA * mU * mU * mC * mA * mG *
ACCGCAGAUUCAGGC
XXXXX XXXXX

9616
mG * mC * mU * mU * mC * mC * fC * fA * fA * fU * fU * fU
UUCCCAAUUU
XXXXX XXXXX

XXXX

WV-
fA * fG * fA * fU * fU * fC * mA * mG * mG * mC * mU * mU * mC *
AGAUUCAGGCUUCCC
XXXXX XXXXX

9617
mC * mC * mA * mA * mU * mU * fU * fU * fU * fC * fC * fU
AAUUUUUCCU
XXXXX XXXXX

XXXX

WV-
fC * fA * fG * fG * fC * fU * mU * mC * mC *mC * mA * mA * mU *
CAGGCUUCCCAAUUU
XXXXX XXXXX

9618
mU * mU * mU * mU * mC * mC * fU * fG * fU * fA * fG * fA
UUCCUGUAGA
XXXXX XXXXX

XXXX

WV-
fU * fU * fC * fC * fC * fA * mA * mU * mU * mU * mU * mU * mC *
UUCCCAAUUUUUCCU
XXXXX XXXXX

9619
mC * mU * mG * mU * mA * mG * fA * fA * fU * fA * fC * fU
GUAGAAUACU
XXXXX XXXXX

XXXX

WV-
fA * fA * fU * fU * fU * fU * mU * mC * mC * mU * mG * mU * mA *
AAUUUUUCCUGUAGA
XXXXX XXXXX

9620
mG * mA * mA * mU * mA * mC * fU * fG * fG * fC * fA * fU
AUACUGGCAU
XXXXX XXXXX

XXXX

WV-
fU * fU * fC * fC * fU * fG * mU * mA * mG * mA * mA * mU * mA *
UUCCUGUAGAAUACU
XXXXX XXXXX

9621
mC * mU * mG * mG * mC * mA * fU * fC * fU * fG * fU * fU
GGCAUCUGUU
XXXXX XXXXX

XXXX

WV-
fG * fU * fA * fG * fA * fA * mU * mA * mC * mU * mG * mG * mC *
GUAGAAUACUGGCAU
XXXXX XXXXX

9622
mA * mU * mC * mU * mG * mU * fU * fU * fU * fU * fG * fA
CUGUUUUUGA
XXXXX XXXXX

XXXX

WV-
fA * fU * fA * fC * fU * fG * mG * mC * mA * mU * mC * mU * mG *
AUACUGGCAUCUGUU
XXXXX XXXXX

9623
mU * mU * mU * mU * mU * mG * fA * fG * fG * fA * fU * fU
UUUGAGGAUU
XXXXX XXXXX

XXXX

WV-
fG * fG * fC * fA * fU * fC * mU * mG * mU * mU * mU * mU * mU *
GGCAUCUGUUUUUGA
XXXXX XXXXX

9624
mG * mA * mG * mG * mA * mU * fU * fG * fC * fU * fG * fA
GGAUUGCUGA
XXXXX XXXXX

XXXX

WV-
fC * fU * fG * fU * fU * fU * mU * mU * mG * mA * mG * mG * mA *
CUGUUUUUGAGGAU
XXXXX XXXXX

9625
mU * mU * mG * mC * mU * mG * fA * fA * fU * fU * fA * fU
UGCUGAAUUAU
XXXXX XXXXX

XXXX

WV-
fU * fU * fU * fG * fA * fG * mG * mA * mU * mU * mG * mC * mU *
UUUGAGGAUUGCUG
XXXXX XXXXX

9626
mG * mA * mA * mU * mU * mA * fU * fU * fU * fC * fU * fU
AAUUAUUUCUU
XXXXX XXXXX

XXXX

WV-
fG * fG * fA * fU * fU * fG * mC * mU * mG * mA * mA * mU * mU *
GGAUUGCUGAAUUA
XXXXX XXXXX

9627
mA * mU * mU * mU * mC * mU * fU * fC * fU * fC * fC * fA
UUUCUUCCCCA
XXXXX XXXXX

XXXX

WV-
fG * fC * fU * fG * fA * fA * mU * mU * mA * mU * mU * mU * mC *
GCUGAAUUAUUUCUU
XXXXX XXXXX

9628
mU * mU * mC * mC * mC * mC * fA * fG * fU * fU * fG * fC
CCCCAGUUGC
XXXXX XXXXX

XXXX

WV-
fA * fU * fU * fA * fU * fU * mU * mC * mU * mU * mC * mC * mC *
AUUAUUUCUUCCCCA
XXXXX XXXXX

9629
mC * mA * mG * mU * mU * mG * fC * fA * fU * fU * fC * fA
GUUGCAUUCA
XXXXX XXXXX

XXXX

WV-
fU * fU * fC * fU * fU * fC * mC * mC * mC * mA * mG * mU * mU *
UUCUUCCCCAGUUGC
XXXXX XXXXX

9630
mG * mC * mA * mU * mU * mC * fA * fA * fU * fG * fU * fU
AUUCAAUGUU
XXXXX XXXXX

XXXX

WV-
fC * fC * fC * fC * fA * fG * mU * mU * mG * mC * mA * mU * mU *
CCCCAGUUGCAUUCA
XXXXX XXXXX

9631
mC * mA * mA * mU * mG * mU * fU * fC * fU * fG * fA * fC
AUGUUCUGAC
XXXXX XXXXX

XXXX

WV-
fG * fU * fU * fG * fC * fA * mU * mU * mC * mA * mA * mU * mG *
GUUGCAUUCAAUGUU
XXXXX XXXXX

9632
mU * mU * mC * mU * mG * mA * fC * fA * fA * fC * fA * fG
CUGACAACAG
XXXXX XXXXX

XXXX

WV-
fA * fU * fU * fC * fA * fA * mU * mG * mU * mU * mC * mU * mG *
AUUCAAUGUUCUGAC
XXXXX XXXXX

9633
mA * mC * mA * mA * mC * mA * fG * fU * fU * fU * fG * fC
AACAGUUUGC
XXXXX XXXXX

XXXX

WV-
fA * fU * fG * fU * fU * fC * mU * mG * mA * mC * mA * mA * mC *
AUGUUCUGACAACAG
XXXXX XXXXX

9634
mA * mG * mU * mU * mU * mG * fC * fC * fG * fC * fU * fG
UUUGCCGCUG
XXXXX XXXXX

XXXX

WV-
fC * fU * fG * fA * fC * fA * mA * mC * mA * mG * mU * mU * mU *
CUGACAACAGUUUGC
XXXXX XXXXX

9635
mG * mC * mC * mG * mC * mU * fG * fC * fC * fC * fA * fA
CGCUGCCCAA
XXXXX XXXXX

XXXX

WV-
fA * fA * fC * fA * fG * fU * mU * mU * mG * mC * mC * mG * mC *
AACAGUUUGCCGCUG
XXXXX XXXXX

9636
mU * mG * mC * mC * mC * mA * fA * fU * fG * fC * fC * fA
CCCAAUGCCA
XXXXX XXXXX

XXXX

WV-
fU * fU * fU * fG * fC * fC * mG * mC * mU * mG * mC * mC * mC *
UUUGCCGCUGCCCAA
XXXXX XXXXX

9637
mA * mA * mU * mG * mC * mC * fA * fU * fU * fC * fU * fG
UGCCAUCCUG
XXXXX XXXXX

XXXX

WV-
fC * fG * fC * fU * fG * fC * mC * mC * mA * mA * mU * mG * mC * mC
CGCUGCCCAAUGCCA
XXXXX XXXXX

9638
* mA * mU * mC * mC * mU * fG * fG * fA * fG * fU * fU
UCCUGGAGUU
XXXXX XXXXX

XXXX

WV-
fC * fC * fC * fA * fA * fU * mG * mC * mC * mA * mU * mC * mC * mU
CCCAAUGCCAUCCUG
XXXXX XXXXX

9639
* mG * mG * mA * mG * mU * fU * fC * fC * fU * fG * fU
GAGUUCCUGU
XXXXX XXXXX

XXXX

WV-
fU * fG * fC * fC * fA * fU * mC * mC * mU * mG * mG * mA * mG *
UGCCAUCCUGGAGUU
XXXXX XXXXX

9640
mU * mU * mC * mC * mU * mG * fU * fA * fA * fG * fA * fU
CCUGUAAGAU
XXXXX XXXXX

XXXX

WV-
fU * fC * fC * fU * fG * fG * mA * mG * mU * mU * mC * mC * mU *
UCCUGGAGUUCCUGU
XXXXX XXXXX

9641
mG * mU * mA * mA * mG * mA * fU * fA * fC * fC * fA * fA
AAGAUACCAA
XXXXX XXXXX

XXXX

WV-
fG * fA * fG * fU * fU * fC * mC * mU * mG * mU * mA * mA * mG *
GAGUUCCUGUAAGAU
XXXXX XXXXX

9642
mA * mU * mA * mC * mC * mA * fA * fA * fA * fA * fG * fG
ACCAAAAAGG
XXXXX XXXXX

XXXX

WV-
fC * fC * fU * fG * fU * fA * mA * mG * mA * mU * mA * mC * mC *
CCUGUAAGAUACCAA
XXXXX XXXXX

9643
mA * mA * mA * mA * mA * mG * fG * fC * fA * fA * fA * fA
AAAGGCAAAA
XXXXX XXXXX

XXXX

WV-
fA * fA * fG * fA * fU * fA * mC * mC * mA * mA * mA * mA * mA *
AAGAUACCAAAAAGG
XXXXX XXXXX

9644
mG * mG * mC * mA * mA * mA * fA * fC * fA * fA * fA * fA
CAAAACAAAA
XXXXX XXXXX

XXXX

WV-
fA * fC * fC * fA * fA * fA * mA * mA * mG * mG * mC * mA * mA *
ACCAAAAAGGCAAAA
XXXXX XXXXX

9645
mA * mA * mC * mA * mA * mA * fA * fA * fU * fG * fA * fA
CAAAAAUGAA
XXXXX XXXXX

XXXX

WV-
fA * fA * fA * fG * fG * fC * mA * mA * mA * mA * mC * mA * mA *
AAAGGCAAAACAAAA
XXXXX XXXXX

9646
mA * mA * mA * mU * mG * mA * fA * fG * fC * fC * fC * fC
AUGAAGCCCC
XXXXX XXXXX

XXXX

WV-
fC * fA * fA * fA * fA * fC * mA * mA * mA * mA * mA * mU * mG *
CAAAACAAAAAUGAA
XXXXX XXXXX

9647
mA * mA * mG * mC * mC * mC * fC * fA * fU * fG * fU * fC
GCCCCAUGUC
XXXXX XXXXX

XXXX

WV-
fC * fA * fA * fA * fA * fA * mU * mG * mA * mA * mG * mC * mC *
CAAAAAUGAAGCCCC
XXXXX XXXXX

9648
mC * mC * mA * mU * mG * mU * fC * fU * fU * fU * fU * fU
AUGUCUUUUU
XXXXX XXXXX

XXXX

WV-
fA * fU * fG * fA * fA * fG * mC * mC * mC * mC * mA * mU * mG *
AUGAAGCCCCAUGUC
XXXXX XXXXX

9649
mU * mC * mU * mU * mU * mU * fU * fA * fU * fU * fU * fG
UUUUUAUUUG
XXXXX XXXXX

XXXX

WV-
fG * fC * fC * fC * fC * fA * mU * mG * mU * mC * mU * mU * mU *
GCCCCAUGUCUUUUU
XXXXX XXXXX

9650
mU * mU * mA * mU * mU * mU * fG * fA * fG * fA * fA * fA
AUUUGAGAAA
XXXXX XXXXX

XXXX

WV-
fA * fU * fG * fU * fC * fU * mU * mU * mU * mU * mA * mU * mU *
AUGUCUUUUUAUUU
XXXXX XXXXX

9651
mU * mG * mA * mG * mA * mA * fA * fA * fG * fA * fU * fU
GAGAAAAGAUU
XXXXX XXXXX

XXXX

WV-
fU * fU * fU * fU * fU * fA * mU * mU * mU * mG * mA * mG * mA *
UUUUUAUUUGAGAA
XXXXX XXXXX

9652
mA * mA * mA * mG * mA * mU * fU * fA * fA * fA * fC * fA
AAGAUUAAACA
XXXXX XXXXX

XXXX

WV-
fA * fU * fU * fU * fG * fA * mG * mA * mA * mA * mA * mG * mA *
AUUUGAGAAAAGAU
XXXXX XXXXX

9653
mU * mU * mA * mA * mA * mC * fA * fG * fU * fG * fU * fG
UAAACAGUGUG
XXXXX XXXXX

XXXX

WV-
fA * fG * fA * fA * fA * fA * mG * mA * mU * mU * mA * mA * mA *
AGAAAAGAUUAAAC
XXXXX XXXXX

9654
mC * mA * mG * mU * mG * mU * fG * fC * fU * fA * fC * fC
AGUGUGCUACC
XXXXX XXXXX

XXXX

WV-
fA * fG * fA * fU * fU * fA * mA * mA * mC * mA * mG * mU * mG *
AGAUUAAACAGUGU
XXXXX XXXXX

9655
mU * mG * mC * mU * mA * mC * fC * fA * fC * fA * fU * fG
GCUACCACAUG
XXXXX XXXXX

XXXX

WV-
fA * fA * fA * fC * fA * fG * mU * mG * mU * mG * mC * mU * mA *
AAACAGUGUGCUACC
XXXXX XXXXX

9656
mC * mC * mA * mC * mA * mU * fG * fC * fA * fG * fU * fU
ACAUGCAGUU
XXXXX XXXXX

XXXX

WV-
fG * fU * fG * fU * fG * fC * mU * mA * mC * mC * mA * mC * mA *
GUGUGCUACCACAUG
XXXXX XXXXX

9657
mU * mG * mC * mA * mG * mU * fU * fG * fU * fA * fC * fU
CAGUUGUACU
XXXXX XXXXX

XXXX

WV-
fU * fU * fG * fC * fC * fG * mC * mU * mG * mC * mC * mC * mA *
UUGCCGCUGCCCAAU
XXXXX XXXXX

9658
mA * mU * mG * mC * mC * mA * fU * fC * fC * fU * fG * fG
GCCAUCCUGG
XXXXX XXXXX

XXXX

WV-
fG * fC * fC * fC * fA * fA * mU * mG * mC * mC * mA * fU * fC * fC * fU
GCCCAAUGCCAUCCU
XXXXX XXXXX

9659
*fG * fG
GG
XXXXXX

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mA mG mGfU * S mGfU * SfU * SfC *
UUCUGAAGGUGUUCU
SSSSSSOOOSOSSS

9680
SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mA mG mGfU * S mG * SfU * SfU *
UUCUGAAGGUGUUCU
SSSSSSOOOSSSSS

9681
SfC * SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mA mG mG mU * SfG * SfU * SfU *
UUCUGAAGGUGUUCU
SSSSSSOOOSSSSS

9682
SfC * SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * SfA * S mG mGfU * S mG * SfU * SfU * S
UUCUGAAGGUGUUCU
SSSSSSSOOSSSSO

9683
mCfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mAfG * S mGfU * S mG * SfU * SfU * S
UUCUGAAGGUGUUCU
SSSSSSOSOSSSSO

9684
mCfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fG * SfU * SfC * SfU * SfG * SfA * S mA mGfG * SfU * S mG * SfU * SfU * S
UUCUGAAGGUGUUCU
SSSSSSOOSSSSSO

9685
mCfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * S mAfA * S mG mGfU * S mG * SfU * SfU * S
UUCUGAAGGUGUUCU
SSSSSOSOOSSSSO

9686
mCfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * S mA mAfG * S mGfU * S mG * SfU * SfU * S
UUCUGAAGGUGUUCU
SSSSSOOSOSSSSO

9687
mCfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * S mA mA mGfG * SfU * S mG * SfU * SfU * S
UUCUGAAGGUGUUCU
SSSSSOOOSSSSSO

9688
mCfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * S mAfA * S mG mGfU * S mG * SfU * SfU *
UUCUGAAGGUGUUCU
SSSSSOSOOSSSSS

9689
SfC * SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * S mA mAfG * S mGfU * S mG * SfU * SfU *
UUCUGAAGGUGUUCU
SSSSSOOSOSSSSS

9690
SfC * SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * S mA mA mGfG * SfU * S mG * SfU * SfU *
UUCUGAAGGUGUUCU
SSSSSOOOSSSSSS

9691
SfC * SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fC * fU * fC * fC * fG * fG * fU * fU * mCfU * mG * fA * mA mGfG * fG *
CUCCGGUUCUGAAGG
XXXXXXXXOXXX

9699
fG * fU * fU * fC
UGUUC
OOXXXXX

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mCfU * S mG * SfA mAfG *
CUCCGGUUCUGAAGG
SSSSSSOSOSSOOS

9700
SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mU * SfU * S mCfU * S mfG * SfA mAfG
CUCCGGUUCUGAAGG
SSSSSSSSOSSOOS

9701
* SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mC * SfU * S mG * SfA mAfG
CUCCGGUUCUGAAGG
SSSSSSOSSSSOOS

9702
* SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mCfU * S mG * SfA * S mAfG
CUCCGGUUCUGAAGG
SSSSSSOSOSSSOS

9703
* SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mCfU * S mG * SfA mA * SfG
CUCCGGUUCUGAAGG
SSSSSSOSOSSOSS

9704
* SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * S mG * S mUfU * S mCfU * S mG * SfA mAfG *
CUCCGGUUCUGAAGG
SSSSSSOSOSSOOS

9709
SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfUfU * S mCfU * S mG * SfA mAfG *
CUCCGGUUCUGAAGG
SSSSSSOSOSSOOS

9710
SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * SfCfU * S mG * SfA mAfG *
CUCCGGUUCUGAAGG
SSSSSSOSOSSOOS

9711
SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mCfU * SfG * SfA mAfG *
CUCCGGUUCUGAAGG
SSSSSSOSOSSOOS

9712
SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mCfU * S mG * SfAfAfG *
CUCCGGUUCUGAAGG
SSSSSSOSOSSOOS

9713
SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mU * S mU * S mC * S mU * S mG * S
CUCCGGUUCUGAAGG
SSSSSSSSSSSSSSS

9714
mA * S mA * S mG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mU * SfU * S mC * SfU * S mG * SfA *
CUCCGGUUCUGAAGG
SSSSSSSSSSSSSSS

9715
S mA * SfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * SBrmUfG * S mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSOSOS

9737
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * S mUfG * S mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSOSOS

9738
SfA * S BrfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * S mUfG * S mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSOSOS

9739
SfA * SfU * S BrfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * S mUfG * S mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSOSOS

9740
SfA * SfU * SfU * S BrfU * SfC * SfU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * S mUfG * S mGfC *
UCAAGGAAGAUGGCA
SSSSSSOSOSOSOS

9741
SfA * SfU * SfU * SfU * SfC * S BrfU
UUUCU
SSSSS

WV-
BrfU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mGfA * SBrmUfG * S
UCAAGGAAGAUGGCA
SSSSSSOSOSOSOS

9742
mGfC * SfA * S BrfU * S BrfU * S BrfU * SfC * S BrfU
UUUCU
SSSSS

WV-
5 MSfC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mC mU * SfG * S mA
CUCCGGUUCUGAAGG
SSSSSSOSOSSOOS

9743
mAfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mC mU * SfG * S mA mAfG *
CUCCGGUUCUGAAGG
SSSSSSOSOSSOOS

9744
SfG * SfU * SfG * SfU * SfU * S 5 MSfC
UGUUC
SSSSS

WV-
5 MSfC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mC mU * SfG * S mA
CUCCGGUUCUGAAGG
SSSSSSOSOSSOOS

9745
mAfG * SfG * SfU * SfG * SfU * SfU * S 5 MSfC
UGUUC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mAfG * S mGfU * S mG * SfU mUfC *
UUCUGAAGGUGUUCU
SSSSSSOSOSSOOS

9746
SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mA * SfG * S mGfU * S mG * SfU
UUCUGAAGGUGUUCU
SSSSSSSSOSSOOS

9747
mUfC * SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mAfG * S mG * SfU * S mG * SfU
UUCUGAAGGUGUUCU
SSSSSSOSSSSOOS

9748
mUfC * SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mAfG * S mGfU * S mG * SfU * S
UUCUGAAGGUGUUCU
SSSSSSOSOSSSOS

9749
mUfC * SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mAfG * S mGfU * S mG * SfU mU *
UUCUGAAGGUGUUCU
SSSSSSOSOSSOSS

9750
SfC * SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * S mA * S mAfG * S mGfU * S mG * SfU mUfC *
UUCUGAAGGUGUUCU
SSSSSSOSOSSOOS

9751
SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * SfA * SfG * S mGfU * S mG * SfU mUfC
UUCUGAAGGUGUUCU
SSSSSSSSOSSOOS

9752
* SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mAfG * SfG * SfU * S mG * SfU mUfC
UUCUGAAGGUGUUCU
SSSSSSOSSSSOOS

9753
* SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mAfG * S mGfU * SfG * SfU * S mUfC
UUCUGAAGGUGUUCU
SSSSSSOSOSSSOS

9754
* SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mAfG * S mGfU * S mG * SfUfU * SfU
UUCUGAAGGUGUUCU
SSSSSSOSOSSOSS

9755
* SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mA * S mG * S mG * S mU * S mG * S
UUCUGAAGGUGUUCU
SSSSSSSSSSSSSSS

9756
mU * S mU * S mC * SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mA * SfG * S mG * SfU * S mG * SfU *
UUCUGAAGGUGUUCU
SSSSSSSSSSSSSSS

9757
S mU * SfC * SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * SfAfG * S mGfU * S mG * SfU mUfC *
UUCUGAAGGUGUUCU
SSSSSSOSOSSOOS

9758
SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mAfG * SfGfU * S mG * SfU mUfC *
UUCUGAAGGUGUUCU
SSSSSSOSOSSOOS

9759
SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fG * SfU * SfC * SfU * SfG * SfA * S mAfG * S mGfU * SfG * SfU mUfC *
UUCUGAAGGUGUUCU
SSSSSSOSOSSOOS

9760
SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fU * SfU * SfC * SfU * SfG * SfA * S mAfG * S mGfU * S mG * SfUfUfC *
UUCUGAAGGUGUUCU
SSSSSSOSOSSOOS

9761
SfU * SfU * SfG * SfU * SfA * SfC
UGUAC
SSSSS

WV-
fA * fA * fU * fA * fU * fU * fU * fU * mU * mC * mU * mA * mA * mA *
AAUAUUCUUCUAAAG
XXXXX XXXXX

9762
mG * mA * fA * fA * fG * fC * fU * fU * fA * fA * fA
AAAGCUUAAA
XXXXX XXXXX

XXXX

WV-
fU * fC * fU * fU * fC * fU * fA * fA * mA * mG * mA * mA * mG *
UCUUCUAAAGAAAGC
XXXXX XXXXX

9763
mC * mU * fU * fA * fA * fA * fA * fA * fG * fU * fC
UUAAAAAGUC
XXXXX XXXXX

XXXX

WV-
fU * fA * fA * fA * fG * fA * fA * fA * mG * mC * mU * mU * mA * mA *
UAAAGAAAGCUUAA
XXXXX XXXXX

9764
mA * mA * fA * fG * fU * fC * fU * fG * fC * fU * fA
AAAGUCUGCUA
XXXXX XXXXX

XXXX

WV-
fA * fA * fA * fG * fC * fU * fU * fA * mA * mA * mA * mA * mG * mU *
AAAGCUUAAAAAGUC
XXXXX XXXXX

9765
mC * mU * fG * fC * fU * fA * fA * fA * fA * fU * fG
UGCUAAAAUG
XXXXX XXXXX

XXXX

WV-
fU * fU * fA * fA * fA * fA * fA * fG * mU * mC * mU * mG * mC * mU *
UUAAAAAGUCUGCUA
XXXXX XXXXX

9766
mA * mA * fA * fA * fU * fG * fU * fU * fU * fU * fC
AAAUGUUUUC
XXXXX XXXXX

XXXX

WV-
fA * fA * fG * fU * fC * fU * fG * fC * mU * mA * mA * mA * mA * mU *
AAGUCUGCUAAAAUG
XXXXX XXXXX

9767
mG * mU * fU * fU * fU * fC * fA * fU * fU * fC * fC
UUUUCAUUCC
XXXXX XXXXX

XXXX

WV-
fU * fG * fC * fU * fA * fA * fA * fA * mU * mG * mU * mU * mU * mU *
UGCUAAAAUGUUUUC
XXXXX XXXXX

9768
mC * mA * fU * fU * fC * fC * fU * fA * fU * fU * fA
AUUCCUAUUA
XXXXX XXXXX

XXXX

WV-
fA * fA * fA * fU * fG * fU * fU * fU * mU * mC * mA * mU * mU * mC *
AAAUGUUUUCAUUCC
XXXXX XXXXX

9769
mC * mU * fA * fU * fU * fA * fG * fA * fU * fC * fU
UAUUAGAUCU
XXXXX XXXXX

XXXX

WV-
fU * fU * fU * fU * fC * fA * fU * fU * mC * mC * mU * mA * mU * mU *
UUUUCAUUCCUAUUA
XXXXX XXXXX

9770
mA * mG * fA * fU * fC * fU * fG * fG * fC * fG * fC
GAUCUGUCGC
XXXXX XXXXX

XXXX

WV-
fA * fU * fU * fC * fC * fU * fA * fU * mU * mA * mG * mA * mU * mC *
AUUCCUAUUAGAUCU
XXXXX XXXXX

9771
mU * mG * fU * fC * fG * fC * fC * fC * fU * fA * fC
GUCGCCCUAC
XXXXX XXXXX

XXXX

WV-
fU * fA * fU * fU * fA * fG * fA * fU * mC * mU * mG * mU * mC * mG *
UAUUAGAUCUGUCGC
XXXXX XXXXX

9772
mC * mC * fC * fU * fA * fC * fC * fU * fC * fU * fU
CCUACCUCUU
XXXXX XXXXX

XXXX

WV-
fG * fA * fU * fC * fU * fG * fU * fC * mG * mC * mC * mC * mU * mA *
GAUCUGUCGCCCUAC
XXXXX XXXXX

9773
mC * mC * fU * fC * fU * fU * fU * fU * fU * fU * fC
CUCUUUUUUC
XXXXX XXXXX

XXXX

WV-
fG * fU * fC * fG * fC * fC * fC * fU * mA * mC * mC * mU * mC * mU *
GUCGCCCUACCUCUU
XXXXX XXXXX

9774
mU * mU * fU * fU * fU * fC * fU * fG * fU * fC * fU
UUUUCUGUCU
XXXXX XXXXX

XXXX

WV-
fC * fC * fU * fA * fC * fC * fU * fC * mU * mU * mU * mU * mU * mU *
CCUACCUCUUUUUUC
XXXXX XXXXX

9775
mC * mU * fG * fU * fC * fU * fG * fA * fC * fA * fG
UGUCUGACAG
XXXXX XXXXX

XXXX

WV-
fC * fU * fC * fU * fU * fU * fU * fU * mU * mC * mU * mG * mU * mC *
CUCUUUUUUCUGUCU
XXXXX XXXXX

9776
mU * mG * fA * fC * fA * fG * fC * fU * fG * fU * fU
GACAGCUGUU
XXXXX XXXXX

XXXX

WV-
fU * fU * fU * fU * fC * fU * fG * fU * mC * mU * mG * mA * mC * mA *
UUUUCUGUCUGACAG
XXXXX XXXXX

9777
mG * mC * fU * fG * fU * fU * fU * fG * fC * fA * fG
CUGUUUGCAG
XXXXX XXXXX

XXXX

WV-
fU * fG * fU * fC * fU * fG * fA * fC * mA * mG * mC * mU * mG * mU *
UGUCUGACAGCUGUU
XXXXX XXXXX

9778
mU * mU * fG * fC * fA * fG * fA * fC * fC * fU * fC
UGCAGACCUC
XXXXX XXXXX

XXXX

WV-
fG * fA * fC * fA * fG * fC * fU * fG * mU * mU * mU * mG * mC * mA *
GACAGCUGUUUGCAG
XXXXX XXXXX

9779
mG * mA * fC * fC * fU * fC * fC * fU * fG * fC * fC
ACCUCCUGCC
XXXXX XXXXX

XXXX

WV-
fC * fU * fG * fU * fU * fU * fG * fC * mA * mG * mA * mC * mC * mU *
CUGUUUGCAGACCUC
XXXXX XXXXX

9780
mC * mC * fU * fG * fC * fC * fA * fC * fC * fG * fC
CUGCCACCGC
XXXXX XXXXX

XXXX

WV-
fU * fG * fC * fA * fG * fA * fC * fC * mU * mC * mC * mU * mG * mC *
UGCAGACCUCCUGCC
XXXXX XXXXX

9781
mC * mA * fC * fC * fG * fC * fA * fG * fA * fU * fU
ACCGCAGAUU
XXXXX XXXXX

XXXX

WV-
fA * fC * fC * fU * fC * fC * fU * fG * mC * mC * mA * mC * mC * mG *
ACCUCCUGCCACCGC
XXXXX XXXXX

9782
mC * mA * fG * fA * fU * fU * fC * fA * fG * fG * fC
AGAUUCAGGC
XXXXX XXXXX

XXXX

WV-
fC * fU * fG * fC * fC * fA * fC * fC * mG * mC * mA * mG * mA * mU *
CUGCCACCGCAGAUU
XXXXX XXXXX

9783
mU * mC * fA * fG * fG * fC * fU * fU * fC * fC * fC
CAGGCUUCCC
XXXXX XXXXX

XXXX

WV-
fA * fC * fC * fG * fC * fA * fG * fA * mU * mU * mC * mA * mG * mG *
ACCGCAGAUUCAGG
XXXXX XXXXX

9784
mC * mU * fU * fC * fC * fC * fA * fA * fU * fU * fU
UUCCCAAUUU
XXXXX XXXXX

XXXX

WV
fA * fG * fA * fU * fU * fC * fA * fG * mG * mC * mU * mU * mC * mC *
AGAUUCAGGCUUCC
XXXXX XXXXX

9785
mC * mA * fA * fU * fU * fU * fU * fU * fC * fC * fU
AAUUUUUCCU
XXXXX XXXXX

XXXX

WV-
fC * fA * fG * fG * fC * fU * fU * fC * mC * mC * mA * mA * mU * mU *
CAGGCUUCCCAAUUU
XXXXX XXXXX

9786
mU * mU * fU * fC * fC * fU * fG * fU * fA * fG * fA
UUCCUGUAGA
XXXXX XXXXX

XXXX

WV-
fU * fU * fC * fC * fC * fA * fA * fU * mU * mU * mU * mU * mC * mC *
UUCCCAAUUUUUCCU
XXXXX XXXXX

9787
mU * mG * fU * fA * fG * fA * fA * fU * fA * fC * fU
GUAGAAUACU
XXXXX XXXXX

XXXX

WV-
fA * fA * fU * fU * fU * fU * fU * fC * mC * mU * mG * mU * mA * mG *
AAUUUUUCCUGUAGA
XXXXX XXXXX

9788
mA * mA * fU * fA * fC * fU * fG * fG * fC * fA * fU
AUACUGGCAU
XXXXX XXXXX

XXXX

WV-
fU * fU * fC * fC * fU * fG * fU * fA * mG * mA * mA * mU * mA * mC *
UUCCUGUAGAAUACU
XXXXX XXXXX

9789
mU * mG * fG * fC * fA * fU * fC * fU * fG * fU * fU
GGCAUCUGUU
XXXXX XXXXX

XXXX

WV-
fG * fU * fA * fG * fA * fA * fU * fA * mC * mU * mG * mG * mC * mA *
GUAGAAUACUGGCAU
XXXXX XXXXX

9790
mU * mC * fU * fG * fU * fU * fU * fU * fU * fG * fA
CUGUUUUUGA
XXXXX XXXXX

XXXX

WV-
fA * fU * fA * fC * fU * fG * fG * fC * mA * mU * mC * mU * mG * mU *
AUACUGGCAUCUGUU
XXXXX XXXXX

9791
mU * mU * fU * fU * fG * fA * fG * fG * fA * fU * fU
UUUGAGGAUU
XXXXX XXXXX

XXXX

WV-
fG * fG * fC * fA * fU * fC * fU * fG * mU * mU * mU * mU * mU * mG *
GGCAUCUGUUUUUGA
XXXXX XXXXX

9792
mA * mG * fG * fA * fU * fU * fG * fC * fU * fG * fA
GGAUUGCUGA
XXXXX XXXXX

XXXX

WV-
fC * fU * fG * fU * fU * fU * fU * fU * mG * mA * mG * mG * mA * mU *
CUGUUUUUGAGGAU
XXXXX XXXXX

9793
mU * mG * fC * fU * fG * fA * fA * fU * fU * fA * fU
UGCUGAAUUAU
XXXXX XXXXX

XXXX

WV-
fU * fU * fU * fG * fA * fG * fG * fA * mU * mU * mG * mC * mU * mG *
UUUGAGGAUUGCUG
XXXXX XXXXX

9794
mA * mA * fU * fU * fA * fU * fU * fU * fC * fU * fU
AAUUAUUUCUU
XXXXX XXXXX

XXXX

WV-
fG * fG * fA * fU * fU * fG * fC * fU * mG * mA * mA * mU * mU * mA *
GGAUUGCUGAAUUA
XXXXX XXXXX

9795
mU * mU * fU * fC * fU * fU * fC * fC * fC * fC * fA
UUUCUUCCCCA
XXXXX XXXXX

XXXX

WV-
fG * fC * fU * fG * fA * fA * fU * fU * mA * mU * mU * mU * mC * mU *
GCUGAAUUAUUUCUU
XXXXX XXXXX

9796
mU * mC * fC * fC * fC * fA * fG * fU * fU * fG * fC
CCCCAGUUGC
XXXXX XXXXX

XXXX

WV-
fA * fU * fU * fA * fU * fU * fU * fC * mU * mU * mC * mC * mC * mC *
AUUAUUUCUUCCCCA
XXXXX XXXXX

9797
mA * mG * fU * fU * fG * fC * fA * fU * fU * fC * fA
GUUGCAUUCA
XXXXX XXXXX

XXXX

WV-
fU * fU * fC * fU * fU * fC * fC * fC * mC * mA * mG * mU * mU * mG *
UUCUUCCCCAGUUGC
XXXXX XXXXX

9798
mC * mA * fU * fU * fC * fA * fA * fU * fG * fU * fU
AUUCAAUGUU
XXXXX XXXXX

XXXX

WV-
fC * fC * fC * fC * fA * fG * fU * fU * mG * mC * mA * mU * mU * mC *
CCCCAGUUGCAUUCA
XXXXX XXXXX

9799
mA * mA * fU * fG * fU * fU * fC * fU * fG * fA * fC
AUGUUCUGAC
XXXXX XXXXX

XXXX

WV-
fG * fU * fU * fG * fC * fA * fU * fU * mC * mA * mA * mU * mG * mU *
GUUGCAUUCAAUGUU
XXXXX XXXXX

9800
mU * mC * fU * fG * fA * fC * fA * fA * fC * fA * fG
CUGACAACAG
XXXXX XXXXX

XXXX

WV-
fA * fU * fU * fC * fA * fA * fU * fG * mU * mU * mC * mU * mG * mA *
AUUCAAUGUUCUGAC
XXXXX XXXXX

9801
mC * mA * fA * fC * fA * fG * fU * fU * fU * fG * fC
AACAGUUUGC
XXXXX XXXXX

XXXX

WV-
fA * fU * fG * fU * fU * fC * fU * fG * mA * mC * mA * mA * mC * mA *
AUGUUCUGACAACAG
XXXXX XXXXX

9802
mG * mU * fU * fU * fG * fC * fC * fG * fC * fU * fG
UUUGCCGCUG
XXXXX XXXXX

XXXX

WV-
fC * fU * fG * fA * fC * fA * fA * fC * mA * mG * mU * mU * mU * mG *
CUGACAACAGUUUGC
XXXXX XXXXX

9803
mC * mC * fG * fC * fU * fG * fC * fC * fC * fA * fA
CGCUGCCCAA
XXXXX XXXXX

XXXX

WV-
fA * fA * fC * fA * fG * fU * fU * fU * mG * mC * mC * mG * mC * mU *
AACAGUUUGCCGCUG
XXXXX XXXXX

9804
mG * mC * fC * fC * fA * fA * fU * fG * fC * fC * fA
CCCAAUGCCA
XXXXX XXXXX

XXXX

WV-
fU * fU * fU * fG * fC * fC * fG * fC * mU * mG* mC * mC * mC * mA *
UUUGCCGCUGCCCAA
XXXXX XXXXX

9805
mA * mU * fG * fC * fC * fA * fU * fC * fC * fU * fG
UGCCAUCCUG
XXXXX XXXXX

XXXX

WV-
fC * fG * fC * fU * fG * fC * fC * fC * mA * mA * mU * mG * mC * mC *
CGCUGCCCAAUGCCA
XXXXX XXXXX

9806
mA * mU * fC * fC * fU * fG * fG * fA * fG * fU * fU
UCCUGGAGUU
XXXXX XXXXX

XXXX

WV-
fC * fC * fC * fA * fA * fU * fG * fC * mC * mA * mU * mC * mC * mU *
CCCAAUGCCAUCCU
XXXXX XXXXX

9807
mG * mG * fA * fG * fU * fU * fC * fC * fU * fG * fU
GAGUUCCUGU
XXXXX XXXXX

XXXX

WV-
fU * fG * fC * fC * fA * fU * fC * fC * mU * mG * mG * mA * mG * mU *
UGCCAUCCUGGAGUU
XXXXX XXXXX

9808
mU * mC * fC * fU * fG * fU * fA * fA * fA * fG * fA * fU
CCUGUAAGAU
XXXXX XXXXX

XXXX

WV-
fU * fC * fC * fU * fG * fG * fA * fG * mU * mU * mC * mC * mU * mG *
UCCUGGAGUUCCUGU
XXXXX XXXXX

9809
mU * mA * fA * fG * fA * fU * fA * fC * fC * fA * fA
AAGAUACCAA
XXXXX XXXXX

XXXX

WV-
fG * fA * fG * fU * fU * fC * fC * fU * mG * mU * mA * mA * mG * mA *
GAGUUCCUGUAAGAU
XXXXX XXXXX

9810
mU * mA * fC * fC * fA * fA * fA * fA * fA * fG * fG
ACCAAAAAGG
XXXXX XXXXX

XXXX

WV-
fC * fC * fU * fG * fU * fA * fA * fG * mA * mU * mA * mC * mC * mA *
CCUGUAAGAUACCAA
XXXXX XXXXX

9811
mA * mA * fA * fA * fG * fG * fC * fA * fA * fA * fA
AAAGGCAAAA
XXXXX XXXXX

XXXX

WV-
fA * fA * fG * fA * fU * fA * fC * fC * mA * mA * mA * mA * mA * mG *
AAGAUACCAAAAAGG
XXXXX XXXXX

9812
mG * mC * fA * fA * fA * fA * fC * fA * fA * fA * fA
CAAAACAAAA
XXXXX XXXXX

XXXX

WV-
fA * fC * fC * fA * fA * fA * fA * fA * mG * mG * mC * mA * mA * mA *
ACCAAAAAGGCAAAA
XXXXX XXXXX

9813
mA * mC * fA * fA * fA * fA * fA * fU * fG * fA * fA
CAAAAAUGAA
XXXXX XXXXX

XXXX

WV-
fA * fA * fA * fG * fG * fC * fA * fA * mA * mA * mC * mA * mA * mA *
AAAGGCAAAACAAAA
XXXXX XXXXX

9814
mA * mA * fU * fG * fA * fA * fG * fC * fC * fC * fC
AUGAAGCCCC
XXXXX XXXXX

XXXX

WV-
fC * fA * fA * fA * fA * fC * fA * fA * mA * mA * mA * mU * mG * mA *
CAAAACAAAAAUGAA
XXXXX XXXXX

9815
mA * mG * fC * fC * fC * fC * fA * fU * fG * fU * fC
GCCCCAUGUC
XXXXX XXXXX

XXXX

WV-
fC * fA * fA * fA * fA * fA * fU * fG * mA * mA * mG * mC * mC * mC *
CAAAAAUGAAGCCCC
XXXXX XXXXX

9816
mC * mA * fU * fG * fU * fC * fU * fU * fU * fU * fU
AUGUCUUUUU
XXXXX XXXXX

XXXX

WV-
fA * fU * fG * fA * fA * fG * fC * fC * mC * mC * mA * mU * mG * mU *
AUGAAGCCCCAUGUC
XXXXX XXXXX

9817
mC * mU * fU * fU * fU * fU * fA * fU * fU * fU * fG
UUUUUAUUUG
XXXXX XXXXX

XXXX

WV-
fG * fC * fC * fC * fC * fA * fU * fG * mU * mC * mU * mU * mU * mU *
GCCCCAUGUCUUUUU
XXXXX XXXXX

9818
mU * mA * fU * fU * fU * fG * fA * fG * fA * fA * fA
AUUUGAGAAA
XXXXX XXXXX

XXXX

WV-
fA * fU * fG * fU * fC * fU * fU * fU * mU * mU * mA * mU * mU * mU *
AUGUCUUUUUAUUU
XXXXX XXXXX

9819
mG * mA * fG * fA * fA * fA * fA * fG * fA * fU * fU
GA GAAAAGAUU
XXXXX XXXXX

XXXX

WV-
fU * fU * fU * fU * fU * fA * fU * fU * mU * mG * mA * mG * mA * mA *
UUUUUAUUUGAGAA
XXXXX XXXXX

9820
mA * mA * fG * fA * fU * fU * fA * fA * fA * fC * fA
AA GAUUAAACA
XXXXX XXXXX

XXXX

WV-
fA * fU * fU * fU * fG * fA * fG * fA * mA * mA * mA * mG * mA * mU *
AUUUGAGAAAAGAU
XXXXX XXXXX

9821
mU * mA * fA * fA * fC * fA * fG * fU * fG * fU * fG
UAA ACAGUGUG
XXXXX XXXXX

XXXX

WV-
fA * fG * fA * fA * fA * fA * fG * fA * mU * mU * mA * mA * mA * mC *
AGAAAAGAUUAAAC
XXXXX XXXXX

9822
mA * mG * fU * fG * fU * fG * fC * fU * fA * fC * fC
AGU GUGCUACC
XXXXX XXXXX

XXXX

WV-
fA * fG * fA * fU * fU * fA * fA * fA * mC * mA * mG * mU * mG * mU *
AGAUUAAACAGUGU
XXXXX XXXXX

9823
mG * mC * fU * fA * fC * fC * fA * fC * fA * fU * fG
GCU ACCACAUG
XXXXX XXXXX

XXXX

WV-
fA * fA * fA * fC * fA * fG * fU * fG * mU * mG * mC * mU * mA * mC *
AAACAGUGUGCUACC
XXXXX XXXXX

9824
mC * mA * fC * fA * fU * fG * fC * fA * fG * fU * fU
ACA UGCAGUU
XXXXX XXXXX

XXXX

WV-
fG * fU * fG * fU * fG * fC * fU * fA * mC * mC * mA * mC * mA * mU *
GUGUGCUACCACAUG
XXXXX XXXXX

9825
mG * mC * fA * fG * fU * fU * fG * fU * fA * fU * fU
CAG UUGUACU
XXXXX XXXXX

XXXX

WV-
fG * fC * fC * fC * fA * fA * fU * fG * fC * fC * fA * fU * fC * fC * fU * fG *
GCCCAAUGCCAUCCU
XXXXX XXXXX

9826
fG
GG
XXXXXX

WV-
fC * fC * fA * fC * fA * fG * mG * mU * mU * mG * mU * mG * mU *
CCACAGGUUGUGUCA
XXXXX XXXXX

9827
mC * mA * mC * mC * mA * mG * mA * mG * mU * mA * mA * fC * fA
CC
XXXXX XXXXX

* fG * fU * fC * fU
AGAGUAACAGUCU
XXXXX XXXX

WV-
fG * fU * fG * fU * fC * fA * mC * mC * mA * mG * mA * mG * mU *
GUGUCACCAGAGUAA
XXXXX XXXXX

9828
mA * mA * mC * mA * mG * mU * mC * mU * mG * mA * mG * fU *
CA
XXXXX XXXXX

fA * fG * fG * fA * fG
GUCUGAGUAGGAG
XXXXX XXXX

WV-
fA * fG * fG * fU * fU * fG * mU * mG * mU * mC * mA * mC * mC *
AGGUUGUGUCACCAG
XXXXX XXXXX

9829
mA * mG * mA * mG * mU * mA * mA * mC * mA * mG * mU * fC *
AG
XXXXX XXXXX

fU * fG * fA * fG * fU
UAACAGUCUGAGU
XXXXX XXXX

WV-
fG * fG * fC * fA * fG * fU * mU * mU * mC * mC * mU * mU * mA *
GGCAGUUUCCUUAGU
XXXXX XXXXX

9830
mG * mU * mA * mA * mC * mC * mA * mC * mA * mG * mG * fU * fU
AACCACAGGUUGUGU
XXXXX XXXXX

* fG * fG * fG * fU

XXXXX XXXX

WV-
fA * fG * fA * fU * fG * fG * mC * mA * mG * mU * mU * mU * mC *
AGAUGGCAGUUUCCU
XXXXX XXXXX

9831
mC * mU * mU * mA * mG * mU * mA * mA * mC * mC * mA * fC * fA
U
XXXXX XXXXX

* fG * fG * fU * fU
AGUAACCACAGGUU
XXXXX XXXX

WV-
fA * fU * fG * fG * fC * fA * mU * mU * mU * mC * mU * mA * mG *
AUGGCAUUUCUAGUU
XXXXX XXXXX

9832
mU * mU * mU * mG * mG * mA * mG * mA * mU * mG * mG * fC *
UG
XXXXX XXXXX

fA * fG * fU * fU * fU
GAGAUGGCAGUUU
XXXXX XXXX

WV-
fU * fU * fA * fU * fA * fA * mC * mU * mU * mG * mA * mU * mC *
UUAUAACUUGAUCAA
XXXXX XXXXX

9833
mA * mA * mG * mC * mA * mG * mA * mG * mA * mA * mA * fG *
GCA
XXXXX XXXXX

fC * fC * fA * fG * fU
GAGAAAGCCAGU
XXXXX XXXX

WV-
fA * fU * fA * fC * fC * fU * fU * mC * mU * mG * mC * mU * mU * mG
AUACCUUCUGCUUGA
XXXXX XXXXX

9834
* mA * mU * mG * mA * mU * mC * mA * mU * mC * mU * fC * fG *
UGA
XXXXX XXXXX

fU * fU * fG * fA
UCAUCUCGUUGA
XXXXX XXXX

WV-
fU * fG * fU * fC * fA * fC * mC * mA * mG * mA * mG * mU * mA *
UGUCACCAGAGUAAC
XXXXX XXXXX

9835
mA * mC * mA * mG * mU * mC * mU * mG * mA * mG * fU * fA * fG
AGU CUGAGUAGGAG
XXXXX XXXXX

* fG * fA * fG

XXXXXXXX

WV-
fG * fU * fC * fA * fC * fC * mA * mG * mA * mG * mU * mA * mA *
GUCACCAGAGUAACA
XXXXX XXXXX

9836
mC * mA * mG * mU * mC * mU * mG * mA * mG * fU * fA * fG * fG *
GUC UGAGUAGGAG
XXXXX XXXXX

fA * fG

XXXXXXX

WV-
fU * fC * fA * fC * fC * fA * mG * mA * mG * mU * mA * mA * mC *
UCACCAGAGUAACAG
XXXXX XXXXX

9837
mA * mG * mU * mC * mU * mG * mA * mG * fU * fA * fG * fG * fA *
UCU GAGUAGGAG
XXXXX XXXXX

fG

XXXXXX

WV-
fC * fA * fC * fC * fA * fG * fA * mG * mU * mA * mA * mC * mA * mG
CACCAGAGUAACAGU
XXXXX XXXXX

9838
* mU * mC * mU * mG * mA * mG * fU * fA * fG * fG * fA * fG
CUG AGUAGGAG
XXXXX XXXXX

XXXXX

WV-
fA * fC * fC * fA * fG * fA * mG * mU * mA * mA * mC * mA * mG *
ACCAGAGUAACAGUC
XXXXX XXXXX

9839
mU * mC * mU * mG * mA * mG * fU * fA * fG * fG * fA * fG
UGA GUAGGAG
XXXXX XXXXX

XXXX

WV-
fC * fC * fA * fC * fA * fG * fG * fU * fU * fG * fU * mG * mU * mC * mA
CCACAGGUUGUGUCA
XXXXX XXXXX

9840
* mC * mC * mA * mG * fA * fG * fU * fA * fA * fC * fA * fG * fU * fC *
CCAGAGUAACAGUCU
XXXXX XXXXX

fU

XXXXX XXXX

WV-
fG * fU * fG * fU * fC * fA * fC * fC * fA * fG * fA * mG * mU * mA * mA
GUGUCACCAGAGUAA
XXXXX XXXXX

984
* mC * mA * mG * mU * fC * fU * fG * fA * fG * fU * fA * fG * fG * fA *
C
XXXXX XXXXX

fG
AGUCUGAGUAGGAG
XXXXX XXXX

WV-
fA * fG * fG * fU * fU * fG * fU * fG * fU * fC * fA * mC * mC * mA * mG
AGGUUGUGUCACCAG
XXXXX XXXXX

9842
* mA * mG * mU * mA * fA * fC * fA * fG * fU * fC * fU * fG * fA * fG *
A
XXXXX XXXXX

fu
GUAACAGUCUGAGU
XXXXX XXXX

WV-
fG * fG * fC * fA * fG * fU * fU * fU * fC * fC * fU * mU * mA * mG * mU
GGCAGUUUCCUUAGU
XXXXX XXXXX

9843
* mA * mA * mC * mC * fA * fC * fA * fG * fG * fU * fU * fG * fU * fG *
A
XXXXX XXXXX

fU
ACCACAGGUUGUGU
XXXXX XXXX

WV-
fA * fG * fA * fU * fG * fG * fC * fA * fG * fU * fU * mU * mC * mC * mU
AGAUGGCAGUUUCCU
XXXXX XXXXX

9844
* mU * mA * mG * mU * fA * fA * fC * fC * fA * fC * fA * fG * fG * fU *
UA
XXXXX XXXXX

fU
GUAACCACAGGUU
XXXXX XXXX

WV-
fA * fU * fG * fG * fC * fA * fU * fU * fU * fC * fU * mA * mG * mU * mU
AUGGCAUUUCUAG
XXXXX XXXXX

9845
* mU * mG * mG * mA * fG * fA * fU * fG * fG * fC * fA * fG * fU * fU *
UUUGGAGAUGGCAG
XXXXX XXXXX

fu
UUU
XXXXX XXXX

WV-
fU * fU * fA * fU * fA * fA * fC * fU * fU * fG * fA * mU * mC * mA * mA
UUAUAACUUGAUCA
XXXXX XXXXX

9846
* mG * mC * mA * mG * fA * fG * fA * fA * fA * fG * fC * fC * fA * fG *
AGCAGAGAAAGCCAG
XXXXX XXXXX

fU
U
XXXXX XXXX

WV-
fA * fU * fA * fC * fC * fU * fU * fC * fU * fG * fC * mU * mU * mG * mA
AUACCUUCUGCUUGA
XXXXX XXXXX

9847
* mU * mG * mA * mU * fC * fA * fU * fC * fU * fC * fG * fU * fU * fG *
UGAUCAUCUCGUUGA
XXXXX XXXXX

fA

XXXXX XXXX

WV-
fU * fG * fU * fC * fA * fC * fC * fA * fG * fA * mG * mU * mA * mA *
UGUCACCAGAGUAAC
XXXXX XXXXX

9848
mC * mA * mG * mU * fC * fU * fG * fA * fG * fU * fA * fG * fG * fA * fG
A GUCUGAGUAGGAG
XXXXX XXXXX

XXXXXXXX

WV-
fG * fU * fC * fA * fC * fC * fA * fG * fA * mG * mU * mA * mA * mC *
GUCACCAGAGUAACA
XXXXX XXXXX

9849
mA * mG * mU * fC * fU * fG * fA * fG * fU * fA * fG * fG * fA * fG
G UCUGAGUAGGAG
XXXXX XXXXX

XXXXXXX

WV-
fU * fC * fA * fC * fC * fA * fG * fA * mG * mU * mA * mA * mC * mA *
UCACCAGAGUAACAG
XXXXX XXXXX

9850
mG * mU * fC * fU * fG * fA * fG * fU * fA * fG * fG * fA * fG
U CUGAGUAGGAG
XXXXX XXXXX

XXXXXX

WV-
fC * fA * fC * fC * fA * fG * fA * mG * mU * mA * mA * mC * mA * mG
CACCAGAGUAACAGU
XXXXX XXXXX

9851
* mU * fC * fU * fG * fA * fG * fU * fA * fG * fG * fA * fG
CU GAGUAGGAG
XXXXX XXXXX

XXXXX

WV-
fA * fC * fC * fA * fG * fA * mG * mU * mA * mA * mC * mA * mG *
ACCAGAGUAACAGUC
XXXXX XXXXX

9852
mU * fC * fU * fG * fA * fG * fU * fA * fG * fG * fA * fG
U GAGUAGGAG
XXXXX XXXXX

XXXX

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG
UCAAGGAAGAUGGCA
SSSSSSOSOSSOOS

9858
mGfC * SfA * SfU * SfU * SfU * SfC * SfUL004
UUUCU
SSSSSO

WV-
fU * SfU * SfU * SfU * SfG * S mGfC * S mA mG mC * SfU * SfU * SfU *
UUUUGGCAGCUUUCC
SSSSSOSOOSSSSS

9875
SfC * SfC * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSSS

WV-
fU * SfU * SfU * SfU * SfG * SfG * SfC * SfA * S mG mC * SfU * S mU
UUUUGGCAGCUUUCC
SSSSSSSSOSSOOS

9876
mUfC * SfC * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSSS

WV-
fU * SfU * SfU * SfU * SfG * SfG * S mCfA * SfG * S mC * SfU * S mU
UUUUGGCAGCUUUCC
SSSSSSOSSSSOOS

9877
mUfC * SfC * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSSS

WV-
fU * SfU * SfU * SfU * SfG * SfG * S mCfA * S mG mC * SfU * SfU * S
UUUUGGCAGCUUUCC
SSSSSSOSOSSSOS

9878
mUfC * SfC * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSSS

WV-
fU * SfU * SfU * SfU * SfG * SfG * S mCfA * S mG mC * SfU * S mUfU *
UUUUGGCAGCUUUCC
SSSSSSOSOSSOSS

9879
SfC * SfC * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * S mG * SfA * S
CUCCGGUUCUGAAGG
SSSSSSSSOSSSOSS

9897
mAfG * SfG * SfG * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mCfU * S mG * SfA * S
CUCCGGUUCUGAAGG
SSSSSSSSOSSSOSS

9898
mA mG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SfC * SfU * S mG * SfA *
CUCCGGUUCUGAAGG
SSSSSSSSSSSSOOS

9899
S mA mGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * S mC * SfU * S mG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSSSSSOOS

9900
* S mA mGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * S mGfU * SfU * SfC * SfU * S mG * SfA * S
CUCCGGUUCUGAAGG
SSSSSOSSSSSSOO

9901
mA mGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * S mGfU * SfU * S mC * SfU * S mG * SfA * S
CUCCGGUUCUGAAGG
SSSSSOSSSSSSOO

9902
mA mGfU * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * SfC * SfU * S mG * SfA * S
CUCCGGUUCUGAAGG
SSSSSSOSSSSSOO

9903
mA mGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mC * SfU * S mG * SfA * S
CUCCGGUUCUGAAGG
SSSSSSOSSSSSOO

9904
mA mGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG mU * SfU * S mC * SfU * S mG * SfA * S
CUCCGGUUCUGAAGG
SSSSSOSSSSSSSSS

9905
mA * SfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mUfU * S mC * SfU * S mG * SfA * S
CUCCGGUUCUGAAGG
SSSSSSOSSSSSSSS

9906
mA * SfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mU * SfU mC * SfU * S mG * SfA * S
CUCCGGUUCUGAAGG
SSSSSSSOSSSSSSS

9907
mA * SfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mU * SfU * S mCfU * S mG * SfA * S
CUCCGGUUCUGAAGG
SSSSSSSSOSSSSSS

9908
mA * SfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mU * SfU * S mC * SfU mG * SfA * S
CUCCGGUUCUGAAGG
SSSSSSSSSOSSSSS

9909
mA * SfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mU * SfU * S mC * SfU * S mGfA * S
CUCCGGUUCUGAAGG
SSSSSSSSSSOSSSS

9910
mA * SfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mU * SfU * S mC * SfU * S mG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSSSSOSSS

9911
mA * SfG * SfG * SfU * SfG * SfU * SfG * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mU * SfU * S mC * SfG * S mG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSSSSSOSS

9912
* S mAfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfG * SfC * SfC * SfG * SfG * S mU * SfG * S mC * SfU * S mG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSSSSSSOS

9913
* S mA * SfGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * S mU * SfU * S mC * SfU * S mG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSSSSSSSO

9914
* S mA * SfG * SfGfU * SfG * SfU * SfU * SfC
UGUUC
SSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * S mAfA * S mG mA * SfU * S mG
UCAAGGAAGAUGGCA
SSSSSSOSOSSOOS

10255
mGfC * SfA * SfU * SfU * SfU * SfC * S mU
UUUCU
SSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * S mAfG * S mA mU * SfA * S mG
UCACUCAGAUAGUUG
SSSSSSOSOSSOOS

10256
mUfU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
SSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SfA * SfG * S mA mU * SfA * S mG
UCACUCAGAUAGUUG
SSSSSSSSOSSOOS

10257
mUfU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
SSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * S mAfG * SfA * S mU * SfA * S mG
UCACUCAGAUAGUUG
SSSSSSOSSSSOOS

10258
mUfU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
SSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * S mAfG * S mA mU * SfA * SfG * S
UCACUCAGAUAGUUG
SSSSSSOSOSSSOS

10259
mUfU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
SSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * S mAfG * S mA mU * SfA * S mGfU *
UCACUCAGAUAGUUG
SSSSSSOSOSSOSS

10260
SfU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
SSSSS

WV-
fG * SfC * SfA * SfA * SfA * SfG * S mAfA * S mG mA * SfU * S mG
GCAAAGAAGAUGGCA
SSSSSSOSOSSOOS

10261
mGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSSSS

WV-
fG * fC * fA * fA * fA * fG * mAfA * mG mA * fU * mG mGfC * fA * fU
GCAAAGAAGAUGGCA
XXXXXXOXOXXO

10262
* fU * fU * fC * fU
UUUCU
OXXXXXX

WV-
fU * fU * fC * fU * fU * fG * fU * fA * fC * mU * mU * mC * mA * mU *
UUCUUGUACUUCAUC
XXXXX XXXXX

10439
mC * mC * mC * mA * mC * mU * mG * fA * fU * fU * fC * fU * fG * fA *
CCACU
XXXXX XXXXX

fA * fU
GAUUCUGAAU
XXXXX XXXX

WV-
fG * fU * fG * fU * fU * fC * fU * fU * fG * mU * mA * mC * mU * mU *
GUGUUCUUGUACUUC
XXXXX XXXXX

10440
mC * mA * mU * mC * mC * mC * mA * fC * fU * fG * fA * fU * fU * fC *
AUCCC
XXXXX XXXXX

fU * fG
ACUGAUUCUG
XXXXX XXXX

WV-
fA * fA * fU * fG * fU * fG * fU * fU * fC * mU * mU * mG * mU * mA *
AAGGUGUUCUUGUAC
XXXXX XXXXX

10441
mC * mU * mU * mC * mA * mU * mC * fC * fC * fA * fC * fU * fG * fA *
UUCAU
XXXXX XXXXX

fU * fU
CCCACUGAUU
XXXXX XXXX

WV-
fC * fU * fG * fA * fA * fG * fG * fU * fG * mU * mU * mC * mU * mU *
CUGAAGGUGUUCUUG
XXXXX XXXXX

10442
mG * mU * mA * mC * mU * mU * mC * fA * fU * fC * fC * fC * fA * fC *
UACUU
XXXXX XXXXX

fU * fG
CAUCCCACUG
XXXXX XXXX

WV-
fG * fU * fU * fC * fU * fG * fA * fA * fG * mG * mU * mG * mU * mU *
GUUCUGAAGGUGUUC
XXXXX XXXXX

10443
mC * mU * mU * mG * mU * mA * mC * fU * fU * fC * fA * fU * fC * fC *
UUGUA
XXXXX XXXXX

fC * fA
CUUCAUCCCA
XXXXX XXXX

WV-
fC * fC * fG * fG * fU * fU * fC * fU * fG * mA * mA * mG * mG * mU *
CCGGUUCUGAAGGUG
XXXXX XXXXX

10444
mG * mU * mU * mC * mU * mU * mG * fU * fA * fC * fU * fU * fC * fA *
UUCUU
XXXXX XXXXX

fU * fC
GUACUUCAUC
XXXXX XXXX

WV-
fC * fC * fU * fC * fC * fG * fG * fU * fU * mC * mU * mG * mA * mA *
CCUCCGGUUCUGAAG
XXXXX XXXXX

10445
mG * mG * mU * mG * mU * mU * mC * fU * fU * fG * fU * fA * fC * fU *
GUGUU
XXXXX XXXXX

fU * fC
CUUGUACUUC
XXXXX XXXX

WV-
fU * fU * fG * fC * fC * fU * fC * fC * fG * mG * mU * mU * mC * mU *
UUGCCUCCGGUUCUG
XXXXX XXXXX

10446
mG * mA * mA * mG * mG * mU * mG * fU * fU * fC * fU * fU * fG * fU *
AAGGU
XXXXX XXXXX

fA * fC
GUUCUUGUAC
XXXXX XXXX

WV-
fC * fU * fG * fU * fU * fG * fC * fC * fU * mC * mC * mG * mG * mU *
CUGUUGCCUCCGGUU
XXXXX XXXXX

10447
mU * mC * mU * mG * mA * mA * mG * fG * fU * fG * fU * fU * fC * fU *
CUGAA
XXXXX XXXXX

fU * fG
GGUGUUCUUG
XXXXX XXXX

WV-
fC * fA * fA * fC * fU * fG * fU * fU * fG * mC * mC * mU * mC * mC *
CAACUGUUGCCUCCG
XXXXX XXXXX

10448
mG * mG * mU * mU * mC * mU * mG * fA * fA * fG * fG * fU * fG * fU *
GUUCU
XXXXX XXXXX

fU * fC
GAAGGUGUUC
XXXXX XXXX

WV-
fA * fU * fU * fC * fA * fA * fC * fU * fG * mU * mU * mG * mC * mC *
AUUCAACUGUUGCCU
XXXXX XXXXX

10449
mU * mC * mC * mG * mG * mU * mU * fC * fU * fG * fA * fA * fG * fG *
CCGGU
XXXXX XXXXX

fU * fG
UCUGAAGGUG
XXXXX XXXX

WV-
fU * fU * fC * fA * fU * fU * fC * fA * fA * mC * mU * mG * mU * mU *
UUCAUUCAACUGUUG
XXXXX XXXXX

10450
mG * mC * mC * mU * mC * mC * mG * fG * fU * fU * fC * fU * fG * fA *
CCUCC
XXXXX XXXXX

fA * fG
GGUUCUGAAG
XXXXX XXXX

WV-
fC * fA * fU * fU * fU * fC * fA * fU * fU * mC * mA * mA * mC * mU *
CAUUUCAUUCAACUG
XXXXX XXXXX

10451
mG * mU * mU * mG * mC * mC * mU * fC * fC * fG * fG * fU * fU * fC *
UUGCC
XXXXX XXXXX

fU *fG
UCCGGUUCUG
XXXXX XXXX

WV-
fU * fA * fA * fC * fA * fU * fU * fU * fC * mA * mU * mU * mC * mA *
UAACAUUUCAUUCAA
XXXXX XXXXX

10452
mA * mC * mU * mG * mU * mU * mG * fC * fC * fU * fC * fC * fG * fG *
CUGUU
XXXXX XXXXX

fU * fU
GCCUCCGGUU
XXXXX XXXX

WV-
fC * fU * fU * fU * fA * fA * fC * fA * fU * mU * mU * mC * mA * mU *
CUUUAACAUUUCAUU
XXXXX XXXXX

10453
mU * mC * mA * mA * mC * mU * mG * fU * fU * fG * fC * fC * fU * fC *
CAACU
XXXXX XXXXX

fC * fG
GUUGCCUCCG
XXXXX XXXX

WV-
fU * fA * fC * fU * fU * fC * fA * mU * mC * mC * mC * mA * mC * mU *
UACUUCAUCCCACUG
XXXXX XXXXX

10454
mG * mA * mU * fU * fC * fU * fG * fA * fA * fU * fU
AUUCU GAAUU
XXXXX XXXXX

XXXX

WV-
fU * fU * fG * fU * fA * fC * fU * mU * mC * mA * mU * mC * mC * mC *
UUGUACUUCAUCCCA
XXXXX XXXXX

10455
mA * mC * mU * fG * fA * fU * fU * fC * fU * fG * fA
CUGAU UCUGA
XXXXX XXXXX

XXXX

WV-
fU * fU * fC * fU * fU * fG * fU * mA * mC * mU * mU * mC * mA * mU *
UUCUUGUACUUCAUC
XXXXX XXXXX

10456
mC * mC * mC * fA * fC * fU * fG * fA * fU * fU * fC
CCACU GAUUC
XXXXX XXXXX

XXXX

WV-
fG * fU * fG * fU * fU * fC * fU * mU * mG * mU * mA * mC * mU * mU *
GUGUUCUUGUACUUC
XXXXX XXXXX

10457
mC * mA * mU * fC * fC * fC * fA * fC * fU * fG * fA
AUCCC ACUGA
XXXXX XXXXX

XXXX

WV-
fA * fA * fG * fG * fU * fG * fU * mU * mC * mU * mU * mG * mU * mA *
AAGGUGUUCUUGUAC
XXXXX XXXXX

10458
mC * mU * mU * fC * fA * fU * fC * fC * fC * fA * fC
UUCAU CCCAC
XXXXX XXXXX

XXXX

WV-
fC * fU * fG * fA * fA * fG * fG * mU * mG * mU * mU * mC * mU * mU *
CUGAAGGUGUUCUUG
XXXXX XXXXX

10459
mG * mU * mA * fC * fU * fU * fC * fA * fU * fC * fC
UACUU CAUCC
XXXXX XXXXX

XXXX

WV-
fG * fU * fU * fC * fU * fG * fA * mA * mG * mG * mU * mG * mU * mU *
GUUCUGAAGGUGUUC
XXXXX XXXXX

10460
mC * mU * mU * fG * fU * fA * fC * fU * fU * fC * fA
UUGUA CUUCA
XXXXX XXXXX

XXXX

WV-
fC * fC * fG * fG * fU * fU * fC * mU * mG * mA * mA * mG * mG * mU *
CCGGUUCUGAAGGUG
XXXXX XXXXX

10461
mG * mU * mU * fC * fU * fU * fG * fU * fA * fC * fU
UUCUU GUACU
XXXXX XXXXX

XXXX

WV-
fC * fC * fU * fC * fC * fG * fG * mU * mU * mC * mU * mG * mA * mA *
CCUCCGGUUCUGAAG
XXXXX XXXXX

10462
mG * mG * mU * fG * fU * fU * fC * fU * fU * fG * fU
GUGUU CUUGU
XXXXX XXXXX

XXXX

WV-
fU * fU * fG * fC * fC * fU * fC * mC * mG * mG * mU * mU * mC * mU *
UUGCCUCCGGUUCUG
XXXXX XXXXX

10463
mG * mA * mA * fG * fG * fU * fG * fU * fU * fC * fU
AAGGU GUUCU
XXXXX XXXXX

XXXX

WV-
fC * fU * fG * fU * fU * fG * fC * mC * mU * mC * mC * mG * mG * mU *
CUGUUGCCUCCGGUU
XXXXX XXXXX

10464
mU * mC * mU * fG * fA * fA * fG * fG * fU * fG * fU
CUGAA GGUGU
XXXXX XXXXX

XXXX

WV-
fC * fA * fA * fC * fU * fG * fU * mU * mG * mC * mC * mU * mC * mC *
CAACUGUUGCCUCCG
XXXXX XXXXX

10465
mG * mG * mU * fU * fC * fU * fG * fA * fA * fG * fG
GUUCU GAAGG
XXXXX XXXXX

XXXX

WV-
fA * fU * fU * fC * fA * fA * fC * mU * mG * mU * mU * mG * mC * mC *
AUUCAACUGUUGCCU
XXXXX XXXXX

10466
mU * mC * mC * fG * fG * fU * fU * fC * fU * fG * fA
CCGGU UCUGA
XXXXX XXXXX

XXXX

WV-
fU * fU * fC * fA * fU * fU * fC * mA * mA * mC * mU * mG * mU * mU *
UUCAUUCAACUGUUG
XXXXX XXXXX

10467
mG * mC * mC * fU * fC * fC * fG * fG * fU * fU * fC
CCUCC GGUUC
XXXXX XXXXX

XXXX

WV-
fC * fA * fU * fU * fU * fC * fA * mU * mU * mC * mA * mA * mC * mU *
CAUUUCAUUCAACUG
XXXXX XXXXX

10468
mG * mU * mU * fG * fC * fC * fU * fC * fC * fG * fG
UUGCC UCCGG
XXXXX XXXXX

XXXX

WV-
fU * fA * fA * fC * fA * fU * fU * mU * mC * mA * mU * mU * mC * mA *
UAACAUUUCAUUCAA
XXXXX XXXXX

10469
mA * mC * mU * fG * fU * fU * fG * fC * fC * fU * fC
CUGUU GCCUC
XXXXX XXXXX

XXXX

WV-
fC * fU * fU * fU * fA * fA * fC * mA * mU * mU * mU * mC * mA * mU *
CUUUAACAUUUCAUU
XXXXX XXXXX

10470
mU * mC * mA * fA * fC * fU * fG * fU * fU * fG * fC
CAACU GUUGC
XXXXX XXXXX

XXXX

WV-
fA * fU * fC * fC * fA * fC * fC * fU * fG * mC * mC * mU * mC * mG *
AUCCACCUGCCUCGG
XXXXX XXXXX

10487
mG * mC * mC * mU * mC * mC * mC * fA * fA * fA * fG * fU * fG * fC *
CCUCC
XXXXX XXXXX

fU * fG
CAAAGUGCUG
XXXXX XXXX

WV-
fC * fC * fU * fC * fA * fG * fG * fU * fG * mA * mU * mC * mC * mA *
CCUCAGGUGAUCCAC
XXXXX XXXXX

10488
mC * mC * mU * mG * mC * mC * mU * fC * fG * fG * fC * fC * fU * fC *
CUGCC UCGGCCUCCC
XXXXX XXXXX

fC * fC

XXXXX XXXX

WV-
fA * fA * fA * fC * fU * fC * fC * fU * fG * mA * mC * mC * mU * mC *
AAACUCCUGACCUCA
XXXXX XXXXX

10489
mA * mG * mG * mU * mG * mA * mU * fC * fC * fA * fC * fC * fU * fG *
GGUGA
XXXXX XXXXX

fC * fC
UCCACCUGCC
XXXXX XXXX

WV-
fA * fU * fU * fU * fU * fU * fA * fA * fU * mA * mG * mA * mG * mA *
AUUUUUAAUAGAGA
XXXXX XXXXX

10490
mC * mA * mG * mG * mG * mU * mU * fU * fC * fA * fC * fC * fA * fU *
CAGGGU
XXXXX XXXXX

fG * fU
UUCACCAUGU
XXXXX XXXX

WV-
fC * fU * fA * fC * fA * fG * fG * fC * fA * mC * mG * mU * mG * mC *
CUACAGGCACGUGCC
XXXXX XXXXX

10491
mC * mA * mU * mC * mA * mU * mG * fC * fC * fC * fA * fG * fC * fU *
AUCAU
XXXXX XXXXX

fA * fA
GCCCAGCUAA
XXXXX XXXX

WV-
fC * fC * fU * fC * fC * fU * fG * fU * fC * mU * mC * mA * mG * mC *
CCUCCUGUCUCAGCC
XXXXX XXXXX

10492
mC * mC * mC * mC * mC * mG * mA * fG * fU * fA * fG * fC * fA * fG *
UCCCG
XXXXX XXXXX

fG * fA
AGUAGCAGGA
XXXXX XXXX

WV-
fU * fC * fC * fG * fC * fU * fC * fA * fC * mU * mG * mC * mA * mA *
UCCGCUCACUGCAAC
XXXXX XXXXX

10493
mC * mC * mU * mC * mC * mG * mC * fC * fU * fC * fC * fC * fG * fG *
CUCCG CCUCCCGGGU
XXXXX XXXXX

fG * fU

XXXXX XXXX

WV-
fU * fC * fU * fU * fG * fU * fA * fA * fC * mC * mC * mA * mG * mG *
UCUUGUAACCCAGGC
XXXXX XXXXX

10494
mC * mU * mG * mG * mA * mG * mU * fG * fC * fA * fA * fU * fG * fG *
UGGAG
XXXXX XXXXX

fU * fG
UGCAAUGGUG
XXXXX XXXX

WV-
fA * fG * fU * fG * fA * fA * fC * fC * fC * mA * mA * mG * mG * mG *
AGUGAACCCAAGGGA
XXXXX XXXXX

10495
mA * mA * mG * mA * mU * mA * mA * fG * fU * fG * fU * fA * fU * fU *
AGAUA
XXXXX XXXXX

fA * fG
AGUGUAUUAG
XXXXX XXXX

WV-
fU * fG * fA * fU * fU * fA * fA * fU * fU * mU * mA * mC * mC * mC *
UGAUUAAUUUACCCC
XXXXX XXXXX

10496
mC * mC * mC * mA * mA * mA * mU * fA * fA * fA * fU * fC * fA * fC *
CCAAA
XXXXX XXXXX

fU * fU
UAAAUCACUU
XXXXX XXXX

WV-
fA * fC * fU * fG * fG * fC * fU * fG * fC * mC * mU * mU * mG * mC *
ACUGGCUGCCUUGCC
XXXXX XXXXX

10497
mC * mU * mC * mA * mC * mC * mU * fG * fG * fC * fU * fC * fA * fU *
UCACC
XXXXX XXXXX

fU * fU
UGUCUCAUUU
XXXXX XXXX

WV-
fG * fG * fG * fA * fU * fA * fA * fA * fG * mC * mU * mC * mC * mA *
GGGAUAAAGCUCCAG
XXXXX XXXXX

10498
mG * mU * mG * mA * mC * mC * mC * fA * fC * fA * fA * fC * fA *fG *
UGACC
XXXXX XXXXX

fC * fA
CACAACAGCA
XXXXX XXXX

WV-
fU * fU * fC * fC * fA * fG * fA * fG * fU * mU * mU * mC * mC * mC *
UUCCAGAGUUUCCCA
XXXXX XXXXX

10499
mA * mA * mG * mG * mG * mA * mU * fA * fA * fA * fG * fC * fU * fC *
AGGGA
XXXXX XXXXX

fC * fA
UAAAGCUCCA
XXXXX XXXX

WV-
fG * fG * fG * fG * fA * fA * fA * fU * fA * mA * mC * mU * mC * mU *
GGGGAAAUAACUCUG
XXXXX XXXXX

10500
mG * mA * mG * mG * mC * mA * mU * fG * fU * fA * fU * fU * fU * fU *
AGGCA
XXXXX XXXXX

fA * fC
UGUAUUUUAC
XXXXX XXXX

WV-
fC * fU * fU * fG * fA * fU * fG * fC * fU * mA * mG * mG * mG * mG *
CUUGAUGCUAGGGGA
XXXXX XXXXX

10501
mA * mA * mA * mU * mA * mA * mC * fU * fC * fU * fG * fA * fG * fG *
AAUAA
XXXXX XXXXX

fC * fA
CUCUGAGGCA
XXXXX XXXX

WV-
fA * fC * fU * fA * fG * fC * fU * fC * fC * mC * mU * mU * mG * mA *
ACUAGCUCCCUUGAU
XXXXX XXXXX

10502
mU * mG * mC * mU * mA * mG * mG * fG * fG * fA * fA * fA * fU * fA *
GCUAG
XXXXX XXXXX

fA * fC
GGGAAAUAAC
XXXXX XXXX

WV-
fC * fA * fG * fA * fG * fG * fC * fA * fG * mC * mC * mU * mG * mU *
CAGAGGCAGCCUGUA
XXXXX XXXXX

10503
mA * mU * mA * mU * mA * mA * mU * fG * fA * fC * fU * fA * fA * fG *
UAUAA
XXXXX XXXXX

fU * fG
UGACUAAUUG
XXXXX XXXX

WV-
fC * fU * fC * fC * fA * fG * fC * fU * fC * mC * mC * mA * mG * mA *
CUCCAGCUCCCAGAG
XXXXX XXXXX

10504
mG * mG * mC * mA * mG * mC * mC * fU * fG * fU * fA * fU * fA * fU *
GCAGC
XXXXX XXXXX

fA * fA
CUGUAUAUAA
XXXXX XXXX

WV-
fA * fU * fG * fC * fC * fU * fC * fC * fC * mC * mU * mC * mC * mA *
AUGCCUCCCCUCCAG
XXXXX XXXXX

10505
mG * mC * mU * mC * mC * mC * mA * fG * fA * fG * fG * fC * fA * fG *
CUCCC AGAGGCAGCC
XXXXX XXXXX

fC * fC

XXXXX XXXX

WV-
fC * fA * fG * fG * fC * fA * fA * fC * fU * mG * mA * mU * mG * mC *
CAGGCAACUGAUGCC
XXXXX XXXXX

10506
mC * mU * mC * mC * mC * mC * mU * fC * fC * fA * fG * fC * fU * fC *
UCCCC UCCAGCUCCC
XXXXX XXXXX

fC * fC

XXXXX XXXX

WV-
fA * fU * fG * fU * fG * fA * fC * fA * fG * mG * mC * mU * mA * mG *
AUGUGACAGGCUAGA
XXXXX XXXXX

10507
mA * mC * mA * mU * mA * mC * mC * fA * fG * fG * fC * fA * fA * fC *
CAUAC
XXXXX XXXXX

fU * fG
CAGGCAACUG
XXXXX XXXX

WV-
fA * fG * fU * fG * fC * fC * fA * fG * fC * mA * mU * mU * mU * mC *
AGUGCCAGCAUUUCA
XXXXX XXXXX

10508
mA * mU * mU * mG * mC * mC * mU * fG * fA * fA * fG * fG * fC * fU *
UUGCC
XXXXX XXXXX

fU * fU
UGAAGGCUUU
XXXXX XXXX

WV-
fA * fC * fC * fC * fA * fU * fC * fA * fG * mC * mC * mU * mG * mA *
ACCCAUCAGCCUGAU
XXXXX XXXXX

10509
mU * mU * mU * mC * mC * mC * mA * fG * fU * fG * fC * fC * fA * fG *
UUCCC
XXXXX XXXXX

fC * fA
AGUGCCAGCA
XXXXX XXXX

WV-
fC * fC * fA * fC * fU * fU * fC * fA * fG * mC * mA * mC * mC * mC *
CCACUUCAGCACCCA
XXXXX XXXXX

10510
mA * mU * mC * mA * mG * mC * mC * fU * fG * fA * fU * fU * fU * fC *
UCAGC
XXXXX XXXXX

fC * fC
CUGAUUUCCC
XXXXX XXXX

WV-
fU * fC * fC * fA * fU * fA * fU * fC * fC * mC * mC * mU * mC * mA *
UCCAUAUCCCCUCAU
XXXXX XXXXX

10511
mU * mC * mC * mU * mU * mG * mC * fC * fA * fC * fU * fU * fC * fA *
CCUUG CCACUUCAGC
XXXXX XXXXX

fG * fC

XXXXX XXXX

WV-
fA * fA * fU * fU * fC * fU * fU * fG * fA * mU * mC * mC * mC * mU *
AAUUCUUGAUCCCUA
XXXXX XXXXX

10512
mA * mG * mA * mA * mC * mC * mA * fA * fA * fU * fA * fU * fG * fA *
GAACC
XXXXX XXXXX

fA * fU
AAAUAUGAAU
XXXXX XXXX

WV-
fA * fA * fC * fA * fU * fC * fA * fA * fC * mA * mU * mA * mU * mA *
AACAUCAACAUAUAU
XXXXX XXXXX

10513
mU * mA * mU * mA * mA * mA * mA * fU * fU * fU * fU * fA * fA * fC *
AUAAA
XXXXX XXXXX

fU * fC
AUUUUAACUC
XXXXX XXXX

WV-
fU * fU * fA * fU * fG * fG * fC * fU * fA * mG * mG * mA * mU * mG *
UUAUGGCUAGGAUG
XXXXX XXXXX

10514
mA * mU * mG * mA * mA * mC * mA * fA * fC * fA * fG * fG * fA * fU *
AUGAAC
XXXXX XXXXX

fU * fC
AACAGGAUUC
XXXXX XXXX

WV-
fG * fU * fA * fA * fA * fU * fG * fC * fU * mA * mG * mU * mC * mU *
GUAAAUGCUAGUCUG
XXXXX XXXXX

10515
mG * mG * mA * mG * mG * mA * mG * fA * fC * fA * fU * fU * fU * fU *
GAGGA
XXXXX XXXXX

fA * fA
GACAUUUUAA
XXXXX XXXX

WV-
fG * fG * fA * fA * fA * fA * fA * fU * fA * mA * mA * mU * mA * mU *
GGAAAAAUAAAUAU
XXXXX XXXXX

10516
mA * mU * mA * mG * mU * mA * mG * fU * fA * fA * fA * fU * fG * fC *
AUAGUA
XXXXX XXXXX

fU * fA
GUAAAUGCUA
XXXXX XXXX

WV-
fG * fG * fC * fC * fA * fA * fC * fU * fU * mC * mU * mU * mU * mU *
GGCCAACUUCUUUUA
XXXXX XXXXX

10517
mA * mA * mC * mA * mA * mU * mA * fC * fC * fU * fA * fA * fG * fA *
ACAAU
XXXXX XXXXX

fA * fU
ACCUAAGAAU
XXXXX XXXX

WV-
fA * fU * fG * fU * fU * fG * fC * fU * fU * mA * mU * mU * mU * mA *
AUGUUGCUUAUUUA
XXXXX XXXXX

10518
mA * mA * mA * mA * mA * mU * mU * fA * fU * fU * fC * fA * fU * fU *
AAAAAU
XXXXX XXXXX

fG * fU
UAUUCAUUGU
XXXXX XXXX

WV-
fC * fA * fA * fA * fC * fG * fU * fU * fA * mU * mC * mU * mC * mA *
CAAACGUUAUCUCAC
XXXXX XXXXX

10519
mC * mA * mU * mU * mU * mA * mU * fG * fU * fU * fG * fC * fU * fU *
AUUUA
XXXXX XXXXX

fA * fU
UGUUGCUUAU
XXXXX XXXX

WV-
fA * fG * fA * fC * fA * fU * fU * fU * fU * mA * mA * mA * mC * mG *
AGACAUUUUAAAUG
XXXXX XXXXX

10520
mU * mA * mA * mC * mU * mU * mC * fC * fA * fA * fA * fC * fG * fU *
UAACUU
XXXXX XXXXX

fU * fA
CCAAACGUUA
XXXXX XXXX

WV-
fC * fU * fA * fG * fA * fA * fU * fA * fA * mA * mA * mG * mG * mA *
CUAGAAUAAAAGGA
XXXXX XXXXX

10521
mA * mA * mA * mA * mU * mA * mA * fA * fU * fA * fU * fA * fU * fA *
AAAAUA
XXXXX XXXXX

fG * fU
AAUAUAUAGU
XXXXX XXXX

WV-
fU * fU * fA * fU * fU * fU * fU * fA * fA * mA * mA * mA * mG * mG *
UUAUUUUAAAAAGG
XXXXX XXXXX

10522
mU * mA * mU * mC * mU * mU * mU * fG * fA * fU * fA * fC * fU * fA *
UAUCUU
XXXXX XXXXX

fA * fC
UGAUACUAAC
XXXXX XXXX

WV-
fU * fA * fU * fC * fA * fA * fA * fU * fG * mU * mA * mA * mC * mC *
UAUCAAAUGUAACCA
XXXXX XXXXX

10523
mA * mG * mU * mA * mU * mU * mU * fU * fA * fU * fU * fU * fU * fA *
GUAUU
XXXXX XXXXX

fA * fA
UUAUUUUAAA
XXXXX XXXX

WV-
fU * fA * fC * fA * fA * fU * fC * fU * fA * mU * mG * mG * mU * mA *
UACAAUCUAUGGUAU
XXXXX XXXXX

10524
mU * mA * mA * mU * mU * mU * mU * fA * fU * fC * fA * fA * fA * fU *
AAUUU
XXXXX XXXXX

fG * fU
UAUCAAAUGU
XXXXX XXXX

WV-
fU * fA * fC * fA * fU * fU * fA * fA * fA * mC * mA * mU * mC * mA *
UACAUUAAACAUCAU
XXXXX XXXXX

10525
mU * mU * mA * mA * mA * mU * mU * fA * fC * fA * fA * fU * fC * fU *
UAAAU
XXXXX XXXXX

fA * fU
UACAAUCUAU
XXXXX XXXX

WV-
fU * fG * fA * fU * fU * fU * fU * fC * fU * mG * mU * mU * mA * mA *
UGAUUUUCUGUUAA
XXXXX XXXXX

10526
mU * mA * mA * mC * mU * mU * mU * fA * fC * fA * fU * fU * fA * fA *
UAACUU
XXXXX XXXXX

fA * fC
UACAUUAAAC
XXXXX XXXX

WV-
fA * fU * fA * fA * fA * fU * fA * fU * fA * mC * mA * mA * mA * mG *
AUAAAUAUACAAAG
XXXXX XXXXX

10527
mU * mC * mU * mA * mC * mU * mG * fU * fU * fC * fA * fU * fU * fU *
UCUACU
XXXXX XXXXX

fC * fA
GUUCAUUUCA
XXXXX XXXX

WV-
fG * fG * fG * fU * fG * fA * fC * fA * fG * mU * mG * mA * mG * mA *
GGGUGACAGUGAGAC
XXXXX XXXXX

10528
mC * mU * mC * mU * mG * mU * mC * fU * fC * fU * fA * fA * fG * fA *
UCUGU
XXXXX XXXXX

fA * fA
CUCUAAGAAA
XXXXX XXXX

WV-
fA * fC * fU * fU * fU * fA * fG * fC * fC * mU * mG * mG * mG * mU *
ACUUUAGCCUGGGUG
XXXXX XXXXX

10529
mG * mA * mC * mA * mG * mU * mG * fA * fG * fA * fC * fU * fC * fU *
ACAGU
XXXXX XXXXX

fG * fU
GAGACUCUGU
XXXXX XXXX

WV-
fA * fG * fC * fC * fU * fG * fG * fG * fU * mG * mA * mC * mA * mG *
AGCCUGGGUGACAGU
XXXXX XXXXX

10530
mU * mG * mA * mG * mA * mC * mU * fC * fU * fG * fU * fC * fU * fC *
GAGAC
XXXXX XXXXX

fU * fA
UCUGUCUCUA
XXXXX XXXX

WV-
fG * fA * fU * fU * fG * fU * fG * fC * fC * mA * mC * mU * mG * mC *
GAUUGUGCCACUGCA
XXXXX XXXXX

10531
mA * mC * mU * mU * mU * mA * mG * fC * fC * fU * fG * fG * fG * fU *
CUUUA
XXXXX XXXXX

fG * fA
GCCUGGGUGA
XXXXX XXXX

WV-
fA * fG * fG * fC * fU * fC * fA * fG * fU * mG * mA * mG * mC * mU *
AGGCUCAGUGAGCUA
XXXXX XXXXX

10532
mA * mU * mG * mA * mU * mU * mG * fU * fG * fC * fC * fA * fC * fU *
UGAUU
XXXXX XXXXX

fG * fC
GUGCCACUGC
XXXXX XXXX

WV
fG * fC * fA * fG * fG * fA * fG * fG * fA * mC * mU * mG * mC * mU *
GCAGGAGGACUGCUU
XXXXX XXXXX

10533
mU * mG * mA * mG * mC * mC * mC * fC * fA * fG * fA * fG * fU * fU *
GAGCC
XXXXX XXXXX

fC * fA
CCAGAGUUCA
XXXXX XXXX

WV-
fG * fG * fA * fG * fG * fC * fU * fG * fA * mG * mG * mC * mA * mG *
GGAGGCUGAGGCAGG
XXXXX XXXXX

10534
mG * mA * mG * mG * mA * mC * mU * fG * fC * fU * fU * fG * fA * fG *
AGGAC
XXXXX XXXXX

fC * fC
UGCUUGAGCC
XXXXX XXXX

WV-
fU * fA * fC * fU * fA * fG * fG * fG * fA * mG * mG * mC * mU * mG *
UACUAGGGAGGCUGA
XXXXX XXXXX

10535
mA * mG * mG * mC * mA * mG * mG * fA * fG * fG * fA * fC * fU * fG *
GGCAG
XXXXX XXXXX

fC * fU
GAGGACUGCU
XXXXX XXXX

WV-
fA * fC * fA * fC * fG * fC * fC * fU * fG * mG * mC * mU * mA * mG *
ACACGCCUGGCUAGU
XXXXX XXXXX

10536
mU * mA * mG * mU * mC * mC * mC * fA * fG * fC * fU * fA * fC * fU *
AGUCC
XXXXX XXXXX

fA * fG
CAGCUACUAG
XXXXX XXXX

WV-
fG * fC * fG * fU * fG * fG * fU * fG * fG * mU * mA * mC * mA * mC *
GCGUGGUGGUACACG
XXXXX XXXXX

10537
mG * mC * mC * mU * mG * mG * mC * fU * fA * fG * fU * fA * fG * fU *
CCUGG
XXXXX XXXXX

fC * fC
CUAGUAGUCC
XXXXX XXXX

WV-
fA * fG * fG * fC * fC * fA * fA * fG * fA * mG * mU * mU * mC * mA *
AGGCCAAGAGUUCAA
XXXXX XXXXX

10538
mA * mG * mA * mA * mC * mC * mC * fA * fU * fC * fU * fC * fU * fA *
GAACC
XXXXX XXXXX

fC * fA
CAUCUCUACA
XXXXX XXXX

WV-
fC * fA * fA * fG * fG * fA * fA * fG * fG * mA * mG * mA * mA * mU *
CAAGGAAGGAGAAU
XXXXX XXXXX

10539
mU * mG * mC * mU * mU * mG * mA * fG * fG * fC * fC * fA * fA * fG *
UGCUUG
XXXXX XXXXX

fA * fG
AGGCCAAGAG
XXXXX XXXX

WV-
fU * fU * fU * fG * fG * fG * fA * fG * fG * mC * mC * mA * mA * mG *
UUUGGGAGGCCAAGG
XXXXX XXXXX

10540
mG * mA * mA * mG * mG * mA * mG * fA * fA * fU * fU * fG * fC * fU *
AAGGA
XXXXX XXXXX

fU * fG
GAAUUGCUUG
XXXXX XXXX

WV-
fC * fA * fU * fG * fC * fU * fA * fA * fC * mU * mC * mA * mU * mG *
CAUGCUAACUCAUGC
XXXXX XXXXX

10541
mC * mC * mU * mG * mU * mA * mA * fU * fC * fC * fU * fA * fG * fU *
CUGUA
XXXXX XXXXX

fG * fC
AUCCUAGUGC
XXXXX XXXX

WV-
fU * fC * fA * fA * fA * fA * fG * fU * fC * mU * mA * mC * mU * mG *
UCAAAAGUCUACUGG
XXXXX XXXXX

10542
mG * mC * mU * mA * mG * mG * mC * fA * fU * fG * fC * fU * fA * fA *
CUAGG
XXXXX XXXXX

fC * fU
CAUGCUAACU
XXXXX XXXX

WV-
fC * fU * fA * fG * fG * fA * fA * fG * fG * mA * mA * mU * mU * mA *
CUAGGAAGGAAUUA
XXXXX XXXXX

10543
mA * mG * mC * mC * mC * mG * mA * fA * fU * fG * fG * fU * fU * fG *
AGCCCG
XXXXX XXXXX

fA * fC
AAUGGUUGAC
XXXXX XXXX

WV-
fA * fA * fG * fA * fU * fA * fU * fG * fA * mA * mA * mG * mA * mG *
AAGAUAUGAAAGAG
XXXXX XXXXX

10544
mU * mA * mG * mA * mC * mC * mU * fG * fU * fU * fA * fC * fU * fU *
UAGACC
XXXXX XXXXX

fU * fU
UGUUACUUUU
XXXXX XXXX

WV-
fA * fC * fC * fC * fA * fC * fU * fC * fA * mC * mC * mC * mC * mC *
ACCCACUCACCCCCA
XXXXX XXXXX

10545
mA * mU * mU * mU * mC * mU * mU * fG * fA * fU * fC * fC * fA * fG *
UUUCU
XXXXX XXXXX

fG * fG
UGAUCCAGGG
XXXXX XXXX

WV-
fA * fG * fU * fA * fC * fU * fC * fC * fU * mU * mA * mU * mU * mC *
AGUACUCCUUAUUCC
XXXXX XXXXX

10546
mC * mU * mC * mC * mC * mC * mA * fA * fU * fC * fC * fU * fG * fA *
UCCCC
XXXXX XXXXX

fU * fA
AAUCCUGAUA
XXXXX XXXX

WV-
fA * fG * fA * fA * fU * fG * fG * fG * fG * mG * mG * mA * mG * mA *
AGAAUGGGGGGAGA
XXXXX XXXXX

10547
mA * mA * mG * mU * mG * mA * mG * fA * fG * fU * fA * fC * fU * fC *
AAGUGA
XXXXX XXXXX

fC * fU
GAGUACUCCU
XXXXX XXXX

WV-
fA * fU * fU * fU * fG * fA * fG * fG * fA * mA * mA * mU * mU * mU *
AUUUGAGGAAAUUU
XXXXX XXXXX

10548
mC * mA * mG * mA * mG * mG * mA * fA * fA * fG * fA * fG * fA * fA *
CAGAGG
XXXXX XXXXX

fA * fG
AAAGAGAAAG
XXXXX XXXX

WV-
fU * fA * fG * fA * fC * fU * fA * fC * fU * mA * mA * mG * mC * mA *
UAGACUACUAAGCAG
XXXXX XXXXX

10549
mG * mA * mC * mA * mG * mA * mU * fA * fU * fU * fU * fG * fA * fG *
ACAGA
XXXXX XXXXX

fG * fA
UAUUUGAGGA
XXXXX XXXX

WV-
fU * fC * fU * fU * fU * fU * fA * fU * fC * mC * mU * mG * mA * mG *
UCUUUUAUCCUGAGG
XXXXX XXXXX

10550
mG * mA * mA * mU * mU * mA * mU * fA * fG * fA * fC * fU * fA * fC *
AAUUA
XXXXX XXXXX

fU * fA
UAGACUACUA
XXXXX XXXX

WV-
fU * fA * fA * fG * fU * fU * fU * fG * fA * mA * mG * mG * mG * mA *
UAAGUUUGAAGGGA
XXXXX XXXXX

10551
mU * mU * mA * mA * mA * mC * mG * fC * fA * fU * fG * fC * fA * fA *
UUAAAC
XXXXX XXXXX

fA * fG
GCAUGCAAAG
XXXXX XXXX

WV-
fC * fC * fU * fC * fC * fU * fA * fC * fC * mA * mU * mG * mU * mU *
CCUCCUACCAUGUUA
XXXXX XXXXX

10552
mA * mC * mU * mU * mC * mC * mC * fU * fG * fC * fU * fC * fA * fA *
CUUCC
XXXXX XXXXX

fA * fA
CUGCUCAAAA
XXXXX XXXX

WV-
fC * fA * fA * fG * fU * fG * fC * fC * fC * mA * mA * mU * mC * mU *
CAAGUGCCCAAUCUG
XXXXX XXXXX

10553
mG * mA * mU * mC * mA * mA * mC * fC * fU * fC * fC * fU * fA * fC *
AUCAA CCUCCUACCA
XXXXX XXXXX

fC * fA

XXXXX XXXX

WV-
fA * fU * fA * fG * fA * fG * fG * fG * fU * mU * mU * mU * mG * mA *
AUAGAGGGUUUUGA
XXXXX XXXXX

10554
mU * mC * mA * mA * mG * mU * mG * fC * fC * fC * fA * fA * fU * fC *
UCAAGU
XXXXX XXXXX

fU * fG
GCCCAAUCUG
XXXXX XXXX

WV-
fC * fC * fA * fU * fG * fU * fU * fG * fG * mG * mG * mG * mA * mC *
CCAUGUUGGGGGACA
XXXXX XXXXX

10555
mA * mG * mC * mU * mC * mC * mU * fA * fA * fG * fA * fA * fU * fG *
GCUCC
XXXXX XXXXX

fG * fC
UAAGAAUGGC
XXXXX XXXX

WV-
fU * fA * fU * fA * fC * fA * fU * fA * fA * mC * mU * mU * mC * mC *
UAUACAUAAUUUCCA
XXXXX XXXXX

10556
mA * mG * mG * mC * mC * mU * mG * fG * fC * fC * fA * fU * fA * fA *
GGCCU
XXXXX XXXXX

fA * fA
GGCCAUAAAA
XXXXX XXXX

WV-
fU * fG * fG * fC * fU * fA * fU * fG * fA * mC * mA * mG * mA * mG *
UGGCUAUGACAGAGA
XXXXX XXXXX

10557
mA * mU * mU * mG * mG * mC * mU * fA * fA * fA * fA * fG * fC * fU *
UUGGC
XXXXX XXXXX

fC * fA
UAAAAGCUCA
XXXXX XXXX

WV-
fU * fA * fG * fC * fA * fG * fC * fU * fC * mA * mG * mG * mU * mC *
UAGCAGCUCAGGUCC
XXXXX XXXXX

10558
mC * mC * mU * mU * mC * mG * mA * fU * fA * fA * fA * fA * fU * fG *
CUUCG
XXXXX XXXXX

fG * fC
AUAAAAUGGC
XXXXX XXXX

WV-
fA * fG * fA * fU * fU * fC * fU * fA * fU * mA * mU * mA * mU * mU *
AGAUUCUAUAUAUU
XXXXX XXXXX

10559
mA * mC * mA * mU * mA * mG * mU * fC * fA * fG * fA * fC * fC * fA *
ACAUAG
XXXXX XXXXX

fG * fG
UCAGACCAGG
XXXXX XXXX

WV-
fA * fG * fA * fA * fU * fA * fA * fC * fC * mA * mC * mA * mU * mG *
AGAAUAACCACAUGA
XXXXX XXXXX

10560
mA * mU * mU * mC * mU * mA * mU * fA * fU * fU * fU * fU * fA * fC *
UUCUA
XXXXX XXXXX

fA * fU
UAUAUUACAU
XXXXX XXXX

WV-
fC * fU * fA * fU * fC * fA * fC * fU * fG * mU * mA * mU * mG * mC *
CUAUCACUGUAUGCC
XXXXX XXXXX

10561
mC * mU * mC * mU * mC * mA * mU * fC * fU * fC * fU * fC * fC * fU *
UCUCA UCUCUCCUUC
XXXXX XXXXX

fU * fC

XXXXX XXXX

WV-
fC * fU * fA * fC * fC * fA * fG * fA * fG * mU * mC * mC * mU * mC *
CUACCAGAGUCCUCU
XXXXX XXXXX

10562
mU * mU * mG * mC * mC * mC * mU * fA * fG * fU * fC * fA * fA * fA *
UGCCC
XXXXX XXXXX

fU * fC
UAGUCAAAUC
XXXXX XXXX

WV-
fA * fU * fU * fC * fC * fU * fA * fA * fA * mC * mA * mC * mA * mG *
AUUCCUAAACACAGA
XXXXX XXXXX

10563
mA * mG * mC * mA * mC * mA * mA * fA * fC * fA * fA * fA * fA * fA *
GCACA
XXXXX XXXXX

fA * fU
AACAAAAAAU
XXXXX XXXX

WV-
fA * fA * fA * fC * fC * fA * fA * fU * fA * mU * mA * mU * mA * mU *
AAACCAAUAUAUAUA
XXXXX XXXXX

10564
mA * mA * mA * mG * mU * mG * mA * fC * fU * fA * fG * fC * fA * fU *
AAGUG
XXXXX XXXXX

fA * fC
ACUAGCAUAC
XXXXX XXXX

WV-
fC * fA * fA * fA * fG * fA * fG * fU * fG * mU * mU * mU * mU * mU *
CAAAGAGUGUUUUU
XXXXX XXXXX

10565
mG * mA * mA * mA * mG * mG * mA * fU * fG * fA * fA * fA * fU * fA *
GAAAGG
XXXXX XXXXX

fA * fA
AUGAAAUAAA
XXXXX XXXX

WV-
fG * fA * fA * fG * fA * fG * fG * fA * fA * mG * mC * mC * mU * mG *
GAAGAGGAAGCCUGU
XXXXX XXXXX

10566
mU * mG * mA * mG * mG * mU * mC * fA * fU * fC * fU * fA * fC * fA *
GAGGU
XXXXX XXXXX

fA * fG
CAUCUACAAG
XXXXX XXXX

WV-
fA * fG * fA * fC * fA * fA * fU * fU * fG * mG * mA * mA * mG * mA *
AGACAAUUGGAAGA
XXXXX XXXXX

10567
mG * mG * mA * mA * mG * mC * mC * fU * fG * fU * fG * fA * fG * fG *
GGAAGC
XXXXX XXXXX

fU * fC
CUGUGAGGUC
XXXXX XXXX

WV-
fA * fC * fC * fA * fU * fU * fU * fU * fA * mU * mU * mU * mG * mC *
ACCAUUUUAUUUGCU
XXXXX XXXXX

10568
mU * mC * mC * mC * mU * mA * mC * fC * fU * fU * fU * fU * fA * fG *
CCCUA
XXXXX XXXXX

fA * fA
CCUUUUAGAA
XXXXX XXXX

WV-
fC * fG * fG * fA * fG * fC * fA * fA * fG * mG * mG * mG * mG * mU *
CGGAGCAAGGGGGUG
XXXXX XXXXX

10569
mG * mU * mU * mG * mC * mU * mU * fU * fA * fG * fC * fC * fA * fU *
UUGCU
XXXXX XXXXX

fU * fU
UUAGCCAUUU
XXXXX XXXX

WV-
fA * fU * fC * fU * fU * fA * fG * fG * fC * mA * mC * mA * mC * mA *
AUCUUAGGCACACAG
XXXXX XXXXX

10570
mG * mA * mC * mU * mC * mA * mG * fA * fA * fA * fG * fA * fA * fC *
ACUCA
XXXXX XXXXX

fU * fU
GAAAGAACUU
XXXXX XXXX

WV-
fC * fC * fU * fU * fG * fU * fG * fA * fG * mG * mC * mU * mC * mA *
CCUUGUGAGGCUCAC
XXXXX XXXXX

10571
mC * mA * mG * mG * mC * mU * mC * fU * fC * fU * fU * fG * fU * fU *
AGGCU
XXXXX XXXXX

fA * fA
CUCUUGUUAA
XXXXX XXXX

WV-
fA * fA * fU * fC * fA * fC * fA * fG * fC * mU * mC * mU * mC * mC *
AAUCACAGCUCUCCA
XXXXX XXXXX

10572
mA * mA * mG * mG * mC * mU * mG * fU * fA * fG * fA * fC * fA * fU *
AGGCU
XXXXX XXXXX

fA * fG
GUAGACAUAG
XXXXX XXXX

WV-
fG * fA * fG * fG * fU * fG * fC * fU * fG * mC * mA * mA * mA * mG *
GAGGUGCUGCAAAGG
XXXXX XXXXX

10573
mG * mA * mG * mG * mC * mU * mG * fG * fC * fU * fG * fC * fU * fG *
AGGCU
XXXXX XXXXX

fU * fA
GGCUGCUGUA
XXXXX XXXX

WV-
fA * fC * fU * fG * fG * fC * fU * fC * fA * mA * mA * mU * mU * mU *
ACUGGCUCAAAUUUU
XXXXX XXXXX

10574
mC * mA * mA * mG * mA * mG * mU * fU * fA * fU * fA * fA * fC * fA *
AAGAG
XXXXX XXXXX

fG * fU
UUAUAACAGU
XXXXX XXXX

WV-
fU * fA * fA * fA * fU * fG * fU * fC * fA * mG * mA * mC * mC * mA *
UAAAUGUCAGACCAG
XXXXX XXXXX

10575
mG * mC * mA * mA * mG * mG * mA * fC * fA * fU * fA * fA * fA * fG *
CAAGG
XXXXX XXXXX

fA * fU
ACAUAAAGAU
XXXXX XXXX

WV-
fU * fU * fU * fU * fU * fC * fU * fA * fA * mA * mU * mA * mA * mA *
UUUUUCUAAAUAAA
XXXXX XXXXX

10576
mA * mG * mG * mA * mG * mG * mA * fG * fU * fU * fU * fU * fU * fU *
AGGAGG
XXXXX XXXXX

fC * fU
AGUUUUUUCU
XXXXX XXXX

WV-
fA * fG * fC * fC * fA * fC * fC * fG * fC * mG * mC * mC * mC * mG *
AGCCACCGCGCCCGG
XXXXX XXXXX

10577
mG * mC * mC * mU * mC * mA * mC * fC * fA * fU * fU * fC * fU * fU *
CCUCA
XXXXX XXXXX

fU * fU
CCAUUCUUUU
XXXXX XXXX

WV-
fC * fU * fG * fC * fC * fU * fC * fG * fG * mC * mC * mU * mC * mC *
CUGCCUCGGCCUCCC
XXXXX XXXXX

10578
mC * mA * mA * mA * mG * mU * mG * fC * fU * fG * fG * fG * fA * fU *
AAAGU
XXXXX XXXXX

fU * fA
GCUGGGAUUA
XXXXX XXXX

WV-
fC * fG * fU * fG * fA * fU * fC * fU * fG * mC * mC * mU * mG * mC *
CGUGAUCUGCCUGCC
XXXXX XXXXX

10579
mC * mU * mC * mG * mG * mC * mC * fU * fC * fC * fC * fA * fA * fA *
UCGGC
XXXXX XXXXX

fG * fU
CUCCCAAAGU
XXXXX XXXX

WV-
fG * fU * fA * fU * fU * fU * fU * fU * fA * mG * mU * mA * mG * mA *
GUAUUUUUAGUAGA
XXXXX XXXXX

10580
mG * mA * mC * mA * mG * mG * mG * fU * fU * fU * fC * fA * fC * fC *
GACAGG
XXXXX XXXXX

fA * fU
GUUUCACCAU
XXXXX XXXX

WV-
fG * fC * fA * fU * fG * fC * fA * fG * fC * mA * mC * mC * mA * mC *
GCAUGCAGCACCACG
XXXXX XXXXX

10581
mG * mC * mC * mA * mG * mG * mC * fU * fA * fG * fU * fU * fU * fU *
CCAGG
XXXXX XXXXX

fU * fG
CUAGUUUUUG
XXXXX XXXX

WV-
fC * fA * fA * fG * fU * fA * fG * fC * fU * mG * mG * mG * mA * mC *
CAAGUAGCUGGGACU
XXXXX XXXXX

10582
mU * mA * mC * mA * mG * mG * mC * fA * fU * fG * fC * fA * fG * fC *
ACAGG
XXXXX XXXXX

fA * fC
CAUGCAGCAC
XXXXX XXXX

WV-
fC * fC * fU * fC * fA * fG * fC * fC * fU * mC * mC * mC * mA * mA *
CCUCAGCCUCCCAAG
XXXXX XXXXX

10583
mG * mU * mA * mG * mC * mU * mG * fG * fG * fA * fC * fU * fA * fC *
UAGCU
XXXXX XXXXX

fA * fG
GGGACUACAG
XXXXX XXXX

WV-
fU * fU * fU * fG * fG * fG * fA * fG * fA * mG * mA * mC * mA * mG *
UUUGGGAGAGACAG
XXXXX XXXXX

10584
mA * mA * mA * mU * mC * mU * mG * fG * fG * fA * fU * fU * fG * fG *
AAAUCU
XXXXX XXXXX

fC * fC
GGGAUUGGCC
XXXXX XXXX

WV-
fA * fC * fC * fU * fA * fU * fU * fC * fA * mC * mU * mG * mG * mG *
ACCUAUUCACUGGGA
XXXXX XXXXX

10585
mA * mG * mG * mU * mU * mG * mU * fG * fA * fG * fG * fA * fA * fC *
GGUUG
XXXXX XXXXX

fA * fC
UGAGGAACAC
XXXXX XXXX

WV-
fU * fG * fC * fA * fG * fA * fG * fU * fG * mA * mG * mC * mA * mU *
UGCAGAGUGAGCAUG
XXXXX XXXXX

10586
mG * mG * mA * mG * mA * mA * mG * fA * fU * fA * fA * fU * fG * fA *
GAGAA
XXXXX XXXXX

fG * fU
GAUAAUGAGU
XXXXX XXXX

WV-
fG * fG * fU * fU * fU * fA * fG * fG * fU * mG * mC * mC * mU * mG *
GGUUUAGGUGCCUGU
XXXXX XXXXX

10587
mU * mU * mA * mG * mA * mU * mA * fG * fU * fG * fG * fU * fG * fC *
UAGAU
XXXXX XXXXX

fU * fA
AGUGGUGCUA
XXXXX XXXX

WV
fA * fA * fA * fG * fG * fG * fU * fU * fU * mA * mA * mG * mA * mC *
AAAGGGUUUAAGAC
XXXXX XXXXX

10588
mA * mG * mA * mU * mU * mA * mC * fC * fU * fG * fG * fC * fU * fU *
AGAUUA
XXXXX XXXXX

fC * fU
CCUGGCUUCU
XXXXX XXXX

WV-
fC * fU * fA * fU * fC * fC * fC * fU * fC * mU * mG * mU * mG * mC *
CUAUCCCUCUGUGCA
XXXXX XXXXX

10589
mA * mU * mC * mC * mC * mC * mA * fC * fA * fC * fA * fU * fC * fC *
UCCCC ACACAUCCAU
XXXXX XXXXX

fA * fU

XXXXX XXXX

WV-
fU * fU * fA * fU * fA * fG * fG * fC * fU * mA * mG * mA * mG * mA *
UUAUAGGCUAGAGAC
XXXXX XXXXX

10590
mC * mU * mC * mA * mC * mU * mC * fA * fA * fU * fA * fA * fU * fC *
UCACU
XXXXX XXXXX

fC * fA
CAAUAAUCCA
XXXXX XXXX

WV-
fU * fA * fU * fG * fC * fU * fU * fU * fU * mU * mC * mA * mC * mC *
UAUGCUUUUUCACCC
XXXXX XXXXX

10591
mC * mU * mU * mG * mA * mC * mC * fU * fU * fC * fA * fA * fC * fU *
UUGAC
XXXXX XXXXX

fG * fU
CUUCAACUGU
XXXXX XXXX

WV-
fC * fU * fU * fG * fG * fG * fG * fU * fG * mC * mG * mC * mA * mU *
CUUGGGGUGUGCAUC
XXXXX XXXXX

10592
mC * mC * mC * mA * mC * mU * mG * fA * fG * fG *fG * fU * fA * fU *
CCACU
XXXXX XXXXX

fG * fC
GAGGGUAUGC
XXXXX XXXX

WV-
fU * fA * fC * fU * fU * fU * fA * fG * fU * mA * mC * mA * mC * mA *
UACUUUAGUACACAU
XXXXX XXXXX

10593
mU * mA * mC * mU * mU * mG * mG * fG * fA * fC * fU * fU * fU * fU *
ACUUG
XXXXX XXXXX

fU * fC
GGACUUUUUC
XXXXX XXXX

WV-
fC * fA * fA * fC * fU * fU * fA * fU * fC * mA * mU * mA * mG * mC *
CAACUUAUCAUAGCA
XXXXX XXXXX

10594
mA * mG * mG * mC * mU * mA * mC * fU * fU * fU * fA * fG * fU * fA *
GGCUA
XXXXX XXXXX

fC * fA
CUUUAGUACA
XXXXX XXXX

WV-
fA * fU * fU * fC * fC * fA * fA * fU * fU * mA * mC * mA * mA * mA *
AUUCCAAUUACAAAC
XXXXX XXXXX

10595
mC * mC * mC * mU * mU * mU * mU * fU * fC * fA * fA * fC * fU * fU *
CCUUU
XXXXX XXXXX

fA * fU
UUCAACUUAU
XXXXX XXXX

WV-
fA * fA * fA * fA * fU * fA * fU * fA * fG * mU * mC * mC * mC * mC *
AAAAUAUAGUCCCCA
XXXXX XXXXX

10596
mA * mG * mA * mA * mU * mA * mA * fU * fU * fA * fA * fA * fA * fC *
GAAUA
XXXXX XXXXX

fU * fC
AUUAAAACUC
XXXXX XXXX

WV-
fU * fA * fG * fA * fA * fA * fG * fA * fC * mC * mC * mC * mA * mC *
UAGAAAGACCCCACA
XXXXX XXXXX

10597
mA * mA * mA * mA * mC * mU * mA * fG * fU * fG * fA * fU * fU * fG *
AAACU
XXXXX XXXXX

fU * fA
AGUGAUUGUA
XXXXX XXXX

WV-
fC * fU * fC * fC * fA * fG * fC * fC * fU * mG * mG * mG * mU * mG *
CUCCAGCCUGGGUGA
XXXXX XXXXX

10598
mA * mC * mA * mG * mA * mG * mC * fA * fA * fA * fA * fC * fU * fC *
CAGAG
XXXXX XXXXX

fC * fA
CAAAACUCCA
XXXXX XXXX

WV-
fU * fU * fG * fA * fA * fC * fC * fC * fG * mG * mG * mA * mG * mG *
UUGAACCCGGGAGGC
XXXXX XXXXX

10599
mC * mA * mG * mA * mG * mG * mU * fU * fG * fC * fA * fG * fU * fG *
AGAGG
XXXXX XXXXX

fA * fG
UUGCAGUGAG
XXXXX XXXX

WV-
fA * fG * fG * fC * fU * fG * fA * fG * fG * mC * mA * mG * mG * mA *
AGGCUGAGGCAGGAG
XXXXX XXXXX

10600
mG * mA * mA * mU * mC * mA * mC * fU * fU * fG * fA * fA * fC * fC *
AAUCA
XXXXX XXXXX

fC * fG
CUUGAACCCG
XXXXX XXXX

WV-
fG * fC * fU * fA * fC * fU * fC * fA * fG * mG * mA * mG * mG * mC *
GCUACUCAGGAGGCU
XXXXX XXXXX

10601
mU * mG * mA * mG * mG * mC * mA * fG * fG * fA * fG * fA * fA * fU *
GAGGC
XXXXX XXXXX

fC * fA
AGGAGAAUCA
XXXXX XXXX

WV-
fA * fG * fC * fA * fC * fA * fC * fG * fC * mC * mU * mG * mU * mA *
AGCACACGCCUGUAA
XXXXX XXXXX

10602
mA * mU * mC * mC * mC * mA * mG * fC * fU * fA * fC * fU * fC * fA *
UCCCA
XXXXX XXXXX

fG * fG
GCUACUCAGG
XXXXX XXXX

WV-
fA * fG * fC * fC * fU * fG * fA * fC * fC * mG * mA * mC * mA * mU *
AGCCUGACCGACAUG
XXXXX XXXXX

10603
mG * mC * mU * mG * mA * mA * mA * fC * fC * fC * fA * fG * fU * fC *
CUGAA
XXXXX XXXXX

fU * fC
ACCCAGUCUC
XXXXX XXXX

WV-
fG * fU * fU * fC * fG * fA * fG * fA * fC * mC * mA * mG * mC * mC *
GUUCGAGACCAGCCU
XXXXX XXXXX

10604
mU * mG * mA * mC * mC * mG * mA * fC * fA * fU * fG * fC * fU * fG *
GACCG
XXXXX XXXXX

fA * fA
ACAUGCUGAA
XXXXX XXXX

WV-
fG * fG * fU * fC * fU * fC * fU * fG * fG * mG * mA * mG * mG * mC *
GGUCUCUGGGAGGCC
XXXXX XXXXX

10605
mC * mA * mA * mA * mG * mC * mG * fG * fG * fU * fG * fG * fA * fU *
AAAGC
XXXXX XXXXX

fC * fA
GGGUGGAUCA
XXXXX XXXX

WV-
fG * fC * fU * fC * fA * fC * fG * fC * fC * mU * mG * mU * mA * mA *
GCUCACGCCUGUAAU
XXXXX XXXXX

10606
mU * mC * mC * mC * mA * mG * mG * fU * fC * fU * fC * fU * fG * fG *
CCCAG
XXXXX XXXXX

fG * fA
GUCUCUGGGA
XXXXX XXXX

WV-
fG * fG * fU * fG * fG * fC * fU * fC * fA * mC * mG * mC * mC * mU *
GGUGGCUCACGCCUG
XXXXX XXXXX

10607
mG * mU * mA * mA * mU * mC * mC * fC * fA * fG * fG * fU * fC * fU *
UAAUC
XXXXX XXXXX

fC * fU
CCAGGUCUCU
XXXXX XXXX

WV-
fU * fU * fU * fU * fU * fA * fA * fU * fU * mA * mA * mC * mC * mC *
UUUUUAAUUAACCCU
XXXXX XXXXX

10608
mU * mG * mU * mU * mG * mC * mC * fU * fC * fC * fA * fC * fA * fA *
GUUGC
XXXXX XXXXX

fA * fG
CUCCACAAAG
XXXXX XXXX

WV-
fU * fA * fA * fA * fG * fA * fG * fC * fA * mA * mG * mG * mG * mA *
UAAAGAGCAAGGGA
XXXXX XXXXX

10609
mG * mA * mG * mA * mA * mG * mG * fU * fC * fA * fA * fA * fG * fA *
GAGAAG
XXXXX XXXXX

fA * fU
GUCAAAGAAU
XXXXX XXXX

WV-
fU * fG * fA * fU * fG * fA * fC * fA * fG * mA * mG * mG * mU * mC *
UGAUGACAGAGGUCA
XXXXX XXXXX

10610
mA * mG * mC * mC * mU * mC * mC * fC * fA * fG * fA * fA * fU * fA *
GCCUC
XXXXX XXXXX

fA * fA
CCAGAAUAAA
XXXXX XXXX

WV-
fG * fC * fA * fU * fG * fG * fG * fA * fG * mC * mC * mC * mA * mA *
GCAUGGGAGCCCAAU
XXXXX XXXXX

10611
mU * mG * mA * mU * mG * mA * mC * fA * fG * fA * fG * fG * fU * fC *
GAUGA
XXXXX XXXXX

fA * fG
CAGAGGUCAG
XXXXX XXXX

WV-
fG * fA * fA * fG * fC * fC * fA * fA * fA * mG * mG * mG * mC * mA *
GAAGCCAAAGGGCAU
XXXXX XXXXX

10612
mU * mG * mG * mG * mA * mG * mC * fC * fC * fA * fA * fU * fG * fA *
GGGAG
XXXXX XXXXX

fU * fG
CCCAAUGAUG
XXXXX XXXX

WV-
fA * fU * fA * fU * fC * fU * fU * fG * fA * mC * mC * mU * mC * mA *
AUAUCUUGACCUCAC
XXXXX XXXXX

10613
mC * mU * mU * mU * mA * mC * mC * fU * fC * fC * fU * fG * fU * fC *
UUUAC
XXXXX XXXXX

fU * fU
CUCCUGUCUU
XXXXX XXXX

WV-
fA * fA * fC * fC * fU * fC * fA * fA * fA * mG * mG * mG * mA * mG *
AACCUCAAAGGGAGG
XXXXX XXXXX

10614
mG * mG * mA * mA * mU * mU * mA * fG * fG * fA * fG * fA * fA * fU *
GAAUU
XXXXX XXXXX

fA * fA
AGGAGAAUAA
XXXXX XXXX

WV-
fG * fG * fA * fC * fA * fU * fA * fG * fU * mC * mA * mG * mC * mC *
GGACAUAGUCAGCCU
XXXXX XXXXX

10615
mU * mG * mU * mG * mG * mC * mA * fA * fC * fC * fU * fC * fA * fA *
GUGGC
XXXXX XXXXX

fA * fG
AACCUCAAAG
XXXXX XXXX

WV-
fU * fG * fA * fG * fA * fA * fA * fC * fC * mA * mC * mC * mC * mU *
UGAGAAACCACCCUG
XXXXX XXXXX

10616
mG * mA * mG * mA * mA * mG * mA * fG * fC * fA * fA * fU * fA * fA *
AGAAG
XXXXX XXXXX

fC * fC
AGCAAUAACC
XXXXX XXXX

WV-
fA * fU * fG * fA * fG * fG * fG * fG * fA * mG * mG * mG * mA * mA *
AUGAGGGGAGGGAA
XXXXX XXXXX

10617
mA * mA * mG * mU * mG * mG * mC * fC * fA * fA * fA * fA * fG * fC *
AAGUGG
XXXXX XXXXX

fA * fG
CCAAAAGCAG
XXXXX XXXX

WV-
fG * fG * fC * fC * fC * fA * fA * fG * fG * mG * mA * mU * mG * mA *
GGCCCAAGGGAUGAG
XXXXX XXXXX

10618
mG * mG * mG * mG * mA * mG * mG * fG * fA * fA * fA * fA * fG * fU *
GGGAG
XXXXX XXXXX

fG * fG
GGAAAAGUGG
XXXXX XXXX

WV-
fA * fC * fU * fA * fC * fA * fU * fC * fU * mA * mG * mG * mC * mC *
ACUACAUCUAGGCCC
XXXXX XXXXX

10619
mC * mA * mA * mG * mG * mG * mA * fU * fG * fA * fG * fG * fG * fG *
AAGGG
XXXXX XXXXX

fA * fG
AUGAGGGGAG
XXXXX XXXX

WV-
fA * fU * fA * fA * fA * fA * fC * fC * fC * mU * mU * mC * mA * mA *
AUAAAACCCUUCAAU
XXXXX XXXXX

10620
mU * mG * mU * mU * mU * mC * mC * fC * fU * fA * fC * fU * fG * fU *
GUUUC
XXXXX XXXXX

fC * fU
CCUACUGUCU
XXXXX XXXX

WV-
fA * fC * fU * fG * fC * fA * fC * fU * fC * mC * mC * mU * mC * mU *
ACUGCACUCCCUCUU
XXXXX XXXXX

10621
mU * mA * mU * mA * mA * mA * mA * fC * fC * fC * fU * fU * fC * fA *
AUAAA
XXXXX XXXXX

fA * fU
ACCCUUCAAU
XXXXX XXXX

WV-
fU * fG * fU * fA * fA * fA * fU * fU * fC * mU * mA * mC * mC * mC *
UGUAAAUUCUACCCC
XXXXX XXXXX

10622
mC * mA * mA * mU * mU * mA * mA * fA * fG * fA * fU * fU * fA * fA *
AAUUA
XXXXX XXXXX

fA * fA
AAGAUUAAAA
XXXXX XXXX

WV-
fC * fU * fC * fC * fC * fA * fG * fA * fC * mC * mC * mA * mA * mA *
CUCCCAGACCCAAAU
XXXXX XXXXX

10623
mU * mC * mU * mC * mU * mG * mU * fU * fU * fU * fA * fG * fA * fA *
CUCUG
XXXXX XXXXX

fU * fG
UUUUAGAAUG
XXXXX XXXX

WV-
fC * fC * fC * fU * fC * fA * fC * fA * fU * mC * mC * mA * mU * mA *
CCCUCACAUCCAUAA
XXXXX XXXXX

10624
mA * mG * mA * mG * mG * mC * mU * fC * fU * fA * fU * fA * fU * fC *
GAGGC
XXXXX XXXXX

fA * fU
UCUAUAUCAU
XXXXX XXXX

WV-
fC * fA * fU * fU * fU * fU * fU * fU * fG * mC * mC * mC * mU * mC *
CAUUUUUUGCCCUCA
XXXXX XXXXX

10625
mA * mC * mA * mU * mC * mC * mA * fU * fA * fA * fG * fA * fG * fG *
CAUCC
XXXXX XXXXX

fC * fU
AUAAGAGGCU
XXXXX XXXX

WV-
fU * fA * fA * fG * fC * fG * fU * fC * fA * mC * mC * mC * mA * mA *
UAAGCGUCACCCAAC
XXXXX XXXXX

10626
mC * mA * mC * mC * mU * mC * mA * fU * fA * fU * fA * fA * fU * fU *
ACCUC
XXXXX XXXXX

fA * fG
AUAUAAUUAG
XXXXX XXXX

WV-
fC * fU * fA * fC * fU * fU * fU * fA * fU * mC * mC * mC * mU * mU *
CUACUUUAUCCCUUA
XXXXX XXXXX

10627
mA * mA * mG * mC * mA * mU * mG * fA * fA * fA * fC * fC * fU * fG *
AGCAU
XXXXX XXXXX

fA * fU
GAAACCUGAU
XXXXX XXXX

WV-
fC * fC * fA * fA * fG * fA * fG * fG * fG * mA * mG * mG * mU * mA *
CCAAGAGGGAGGUAC
XXXXX XXXXX

10628
mC * mU * mA * mU * mA * mU * mA * fG * fA * fU * fU * fC * fU * fA *
UAUAU
XXXXX XXXXX

fC * fU
AGAUUCUACU
XXXXX XXXX

WV-
fG * fU * fG * fA * fG * fC * fC * fA * fC * mC * mG * mC * mG * mC *
GUGAGCCACCGCGCC
XXXXX XXXXX

10629
mC * mU * mG * mG * mC * mC * mA * fA * fC * fU * fU * fC * fU * fU *
UGGCC
XXXXX XXXXX

fU * fU
AACUUCUUUU
XXXXX XXXX

WV-
fU * fC * fG * fG * fC * fC * fU * fC * fC * mC * mA * mA * mA * mG *
UCGGCCUCCCAAAGU
XXXXX XXXXX

10630
mU * mG * mC * mU * mG * mG * mG * fA * fU * fU * fA * fC * fA * fG *
GCUGG
XXXXX XXXXX

fG * fC
GAUUACAGGC
XXXXX XXXX

WV-
fU * RfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * RmGmGfC
UCAAGGAAGAUGGCA
RSSSSSOSO

10634
* SfA * SfU * RfU * RfU * RfC * SfU
UUUCU
SROOSSRRRS

WV-
fU * SfC * RfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGAUGGCA
SRSSSSOSO

10635
* RFA * SfU * SfU * SfU * SfC * RfU
UUUCU
SSOORSSSSR

WV-
fU * SfC * SfA * RfA * RfG * SfG * SmAfA * RmGmA * SfU * SmGmGfC
UCAAGGAAGAUGGCA
SSRRSSORO

10636
* SfA * RfU * SfU * SfU * SfC * SfU
UUUCU
SSOOSRSSSS

WV-
fU * SfC * SfA * SfA * SfG * RfG * RmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGAUGGCA
SSSSRROSO

10637
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSOOSSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SmU * SmU * SmCmU * SmG * SmA *
CUCCGGUUCUGAAGG
SSSSSSSSO

10670
SmAmG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSOSSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SmU * SmU * SmC * SmU * SmG *
CUCCGGUUCUGAAGG
SSSSSSSSS

10671
SmA * SmAmGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSOOSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SmU * SmU * SmCmU * SmG * SmA *
CUCCGGUUCUGAAGG
SSSSSSSSO

10672
SmAmGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSSOOSSSSS

WV-
fU * RfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGAUGGCA
RSSSSS O S O SS O

10868
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
O SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * RmGmGfC
UCAAGGAAGAUGGCA
SSSSSS O S O SR O

10869
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
O SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC *
UCAAGGAAGAUGGCA
SSSSSS O S O SS O

10870
SfA * SfU * SfU * SfU * RfC * SfU
UUUCU
O SSSSRS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC *
UCAAGGAAGAUGGCA
SSSSSS O S O SS O

10871
SfA * SfU * SfU * RfU * SfC * SfU
UUUCU
O SSSRSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfG * SmGmGfC *
UCAAGGAAGAUGGCA
SSSSSS O S O SS O

10872
SfA * SfU * RfU * SfU * SfC * SfU
UUUCU
O SSRSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC *
UCAAGGAAGAUGGCA
SSSSSS O S O SS O

10873
SfA * SfU * SfU * SfU * SfC * RfU
UUUCU
O SSSSSR

WV-
fG * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC *
UCAAGGAAGAUGGCA
SSSSSS O S O SS O

10874
RfA * SfU * SfU * SfU * SfC * SfU
UUUCU
O RSSSSS

WV-
fU * SfC * RfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGAUGGCA
SRSSSS O S O SS O

10875
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
O SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC *
UCAAGGAAGAUGGCA
SSSSSS O S O SS O

10876
SfA * RfU * SfU * SfU * SfC * SfU
UUUCU
O SRSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * RmGmA * SfU * SmGmGfC
UCAAGGAAGAUGGCA
SSSSSS O R O SS O

10877
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
O SSSSSS

WV-
fU * SfC * SfA * SfA * RfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGAUGGCA
SSSRSS O S O SS O

10878
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
O SSSSSS

WV-
fU * SfC * SfA * RfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGAUGGCA
SSRSSS O S O SS O

10879
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
O SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * RfG * SmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGAUGGCA
SSSSRS O S O SS O

10880
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
O SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * RmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGAUGGCA
SSSSSR O S O SS O

10881
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
O SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * RfU * SmGmGfC
UCAAGGAAGAUGGCA
SSSSSS O S O RS O

10882
* SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
O SSSSSS

WV-
Mod012L001fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU *
UCAAGGAAGAUGGCA
O SSSSSS O S O SS

10883
SmGmGfC * SfA * SfU * SfU * SfU * SfU * SfU
UUUCU
O O SSSSSS

WV-
Mod085L001fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU *
UCAAGGAAGAUGGCA
O SSSSSS O S O SS

10884
SmGmGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
O O SSSSSS

WV-
Mod086L001fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU *
UCAAGGAAGAUGGCA
O SSSSSS O S O SS

10885
SmGmGfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
O O SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC *
UCAAGGAAGAUGGCA
SSSSSS O S O SS O

10886
SfA * SfU * SfU * SfU * SfC * SfUL004Mod012
UUUCU
O SSSSSSO

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC *
UCAAGGAAGAUGGCA
SSSSSS O S O SS O

10887
SfA * SfU * SfU * SfU * SfC * SfUL004Mod085
UUUCU
O SSSSSSO

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC *
UCAAGGAAGAUGGCA
SSSSSS O S O SS O

10888
SfA * SfU * SfU * SfU * SfC * SfUL004Mod086
UUUCU
O SSSSSSO

WV-
fU * SfU * SfA * SfA * SfA * SfA * SmA * SmG * SmU * SmC * SmU *
UUAAAAAGUCUGCUA
SSSSSSSSS

11047
SmG * SmC * SmU * SfA * SfA * SfA * SfA * SfU * SfG
AAAUG
SSSSSSSSSS

WV-
fA * SfA * SfG * SfU * SfC * SfU * SmG * SmC * SmU * SmA * SmA *
AAGUCUGCUAAAAUG
SSSSSSSSS

11048
SmA * SmA * SmU * SfG * SfU * SfU * SfU * SfU * SfC
UUUUC
SSSSSSSSSS

WV-
fU * SfG * SfC * SfU * SfA * SfA * SmA * SmA * SmU * SmG * SmU *
UGCUAAAAUGUUUUC
SSSSSSSSS

11049
SmU * SmU * SmU * SfC * SfA * SfU * SfU * SfC * SfC
AUUCC
SSSSSSSSSS

WV-
fA * SfA * SfA * SfU * SfG * SfU * SmU * SmU * SmU * SmC * SmA *
AAAUGUUUUCAUUCC
SSSSSSSSS

11050
SmU * SmU * SmC * SfC * SfU * SfA * SfU * SfU * SfA
UAUUA
SSSSSSSSSS

WV-
fU * SfU * SfU * SfU * SfC * SfA * SmU * SmU * SmC * SmC * SmU *
UUUUCAUUCCUAUUA
SSSSSSSSS

11051
SmA * SmU * SmU * SfA * SfG * SfA * SfU * SfC * SfU
GAUCU
SSSSSSSSSS

WV-
fA * SfU * SfU * SfC * SfC * SfU * SmA * SmU * SmU * SmA * SmG *
AUUCCUAUUAGAUCU
SSSSSSSSS

11052
SmA * SmU * SmC * SfU * SfG * SfU * SfC * SfG * SfC
GUCGC
SSSSSSSSSS

WV-
fU * SfA * SfU * SfU * SfA * SfG * SmA * SmU * SmC * SmU * SmG *
UAUUAGAUCUGUCGC
SSSSSSSSS

11053
SmU * SmC * SmG * SfC * SfC * SfC * SfU * SfA * SfC
CCUAC
SSSSSSSSSS

WV-
fG * SfA * SfU * SfC * SfU * SfG * SmU * SmC * SmG * SmC * SmC *
GAUCUGUCGCCCUAC
SSSSSSSSS

11054
SmC * SmU * SmA * SfC * SfC * SfU * SfC * SfU * SfU
CUCUU
SSSSSSSSSS

WV-
fG * SfU * SfC * SfG * SfC * SfC * SmC * SmU * SmA * SmC * SmC *
GUCGCCCUACCUCUU
SSSSSSSSS

11055
SmU * SmC * SmU * SfU * SfU * SfU * SfU * SfU * SfC
UUUUC
SSSSSSSSSS

WV-
fC * SfC * SfU * SfA * SfC * SfC * SmU * SmC * SmU * SmU * SmU *
CCUACCUCUUUUUUC
SSSSSSSSS

11056
SmU * SmU * SmU * SfC * SfU * SfG * SfU * SfC * SfU
UGUCU
SSSSSSSSSS

WV-
fC * SfU * SfC * SfU * SfU * SfU * SmU * SmU * SmU * SmC * SmU *
CUCUUUUUUCUGUCU
SSSSSSSSS

11057
SmG * SmU * SmC * SfU * SfG * SfA * SfC * SfA * SfG
GACAG
SSSSSSSSSS

WV-
fU * SfU * SfU * SfU * SfC * SfU * SmG * SmU * SmC * SmU * SmG *
UUUUCUGUCUGACAG
SSSSSSSSS

11058
SmA * SmC * SmA * SfG * SfC * SfU * SfG * SfU * SfU
CUGUU
SSSSSSSSSS

WV-
fU * SfG * SfU * SfC * SfU * SfG * SmA * SmC * SmA * SmG * SmC *
UGUCUGACAGCUGUU
SSSSSSSSS

11059
SmU * SmG * SmU * SfU * SfU * SfG * SfC * SfA * SfG
UGCAG
SSSSSSSSSS

WV-
fG * SfA * SfC * SfA * SfG * SfC * SmU * SmG * SmU * SmU * SmU *
GACAGCUGUUUGCAG
SSSSSSSSS

11060
SmG * SmC * SmA * SfG * SfA * SfC * SfC * SfU * SfC
ACCUC
SSSSSSSSSS

WV-
fU * SfU * SfG * SfU * SfU * SfU * SmG * SmC * SmA * SmG * SmA *
CUGUUUGCAGACCUC
SSSSSSSSS

11061
SmC * SmC * SmU * SfC * SfC * SfU * SfG * SfC * SfC
CUGCC
SSSSSSSSSS

WV-
fU * SfG * SfC * SfA * SfG * SfA * SmC * SmC * SmU * SmC * SmC *
UGCAGACCUCCUGCC
SSSSSSSSS

11062
SmU * SmG * SmC * SfC * SfA * SfC * SfC * SfG * SfC
ACCGC
SSSSSSSSSS

WV-
fA * SfC * SfC * SfU * SfC * SfC * SmU * SmG * SmC * SmC * SmA *
ACCUCCUGCCACCGC
SSSSSSSSS

11063
SmC * SmC * SmG * SfC * SfA * SfG * SfA * SfU * SfU
AGAUU
SSSSSSSSSS

WV-
fC * SfU * SfG * SfC * SfC * SfA * SmC * SmC * SmG * SmC * SmA *
CUGCCACCGCAGAUU
SSSSSSSSS

11064
SmG * SmA * SmU * SfU * SfC * SfA * SfG * SfG * SfC
CAGGC
SSSSSSSSSS

WV-
fA * SfC * SfC * SfG * SfC * SfA * SmG * SmA * SmU * SmU * SmC *
ACCGCAGAUUCAGGC
SSSSSSSSS

11065
SmA * SmG * SmG * SfC * SfU * SfU * SfC * SfC * SfC
UUCCC
SSSSSSSSSS

WV-
fA * SfG * SfA * SfU * SfG * SfC * SmA * SmG * SmG * SmC * SmU *
AGAUUCAGGCUUCCC
SSSSSSSSS

11066
SmU * SmC * SmC * SfC * SfA * SfA * SfU * SfU * SfU
AAUUU
SSSSSSSSSS

WV-
fC * SfA * SfG * SfG * SfC * SfU * SmU * SmC * SmC * SmC * SmA *
CAGGCUUCCCAAUUU
SSSSSSSSS

11067
SmA * SmU * SmU * SfU * SfU * SfU * SfC * SfC * SfU
UUCCU
SSSSSSSSSS

WV-
fU * SfU * SfC * SfC * SfC * SfA * SmA * SmU * SmU * SmU * SmU *
UUCCCAAUUUUUCCU
SSSSSSSSS

11068
SmU * SmC * SmC * SfU * SfG * SfU * SfA * SfG * SfA
GUAGA
SSSSSSSSSS

WV-
fA * SfA * SfU * SfU * SfU * SfU * SmU * SmC * SmC * SmU * SmG *
AAUUUUUCCUGUAGA
SSSSSSSSS

11069
SmU * SmA * SmG * SfA * SfA * SfU * SfA * SfC * SfU
AUACU
SSSSSSSSSS

WV-
fU * SfU * SfC * SfC * SfU * SfG * SmU * SmA * SmG * SmA * SmA *
UUCCUGUAGAAUACU
SSSSSSSSS

11070
SmU * SmA * SmC * SfU * SfG * SfG * SfC * SfA * SfU
GGCAU
SSSSSSSSSS

WV-
fG * SfU * SfA * SfG * SfA * SfA * SmU * SmA * SmC * SmU * SmG *
GUAGAAUACUGGCAU
SSSSSSSSS

11071
SmG * SmC * SmA * SfU * SfC * SfU * SfG * SfU * SfU
CUGUU
SSSSSSSSSS

WV-
fA * SfG * SfA * SfC * SfU * SfG * SmG * SmC * SmA * SmU * SmC *
AUACUGGCAUCUGUU
SSSSSSSSS

11072
SmU * SmG * SmU * SfU * SfU * SfU * SfU * SfG * SfA
UUUGA
SSSSSSSSSS

WV-
fG * SfG * SfC * SfA * SfU * SfC * SmU * SmG * SmU * SmU * SmU *
GGCAUCUGUUUUUGA
SSSSSSSSS

11073
SmU * SmU * SmG * SfA * SfG * SfG * SfA * SfU * SfU
GGAUU
SSSSSSSSSS

WV-
fC * SfU * SfG * SfU * SfU * SfU * SmU * SmU * SmG * SmA * SmG *
CUGUUUUUGAGGAU
SSSSSSSSS

11074
SmG * SmA * SmU * SfU * SfG * SfC * SfU * SfG * SfA
UGCUGA
SSSSSSSSSS

WV-
fU * SfU * SfU * SfG * SfA * SfG * SmG * SmA * SmU * SmU * SmG *
UUUGAGGAUUGCUG
SSSSSSSSS

11075
SmC * SmU * SmG * SfA * SfA * SfU * SfU * SfA * SfU
AAUUAU
SSSSSSSSSS

WV-
fG * SfG * SfA * SfU * SfU * SfG * SmC * SmU * SmG * SmA * SmA *
GGAUUGCUGAAUUA
SSSSSSSSS

11076
SmU * SmU * SmA * SfU * SfU * SfU * SfC * SfU * SfU
UUUCUU
SSSSSSSSSS

WV-
fG * SfC * SfU * SfG * SfA * SfA * SmU * SmU * SmA * SmU * SmU *
GCUGAAUUAUUUCUU
SSSSSSSSS

11077
SmU * SmC * SmU * SfU * SfC * SfC * SfC * SfC * SfA
CCCCA
SSSSSSSSSS

WV-
fA * SfU * SfU * SfA * SfU * SfU * SmU * SmC * SmU * SmU * SmC *
AUUAUUUCUUCCCCA
SSSSSSSSS

11078
SmC * SmC * SmC * SfA * SfG * SfU * SfU * SfG * SfC
GUUGC
SSSSSSSSSS

WV-
fU * SfU * SfC * SfU * SfU * SfC * SmC * SmC * SmC * SmA * SmG *
UUCUUCCCCAGUUGC
SSSSSSSSS

11079
SmU * SmU * SmG * SfC * SfA * SfU * SfU * SfC * SfA
AUUCA
SSSSSSSSSS

WV-
fC * SfC * SfC * SfC * SfA * SfG * SmU * SmU * SmG * SmC * SmA *
CCCCAGUUGCAUUCA
SSSSSSSSS

11080
SmU * SmU * SmC * SfA * SfA * SfU * SfG * SfU * SfU
AUGUU
SSSSSSSSSS

WV-
fG * SfU * SfU * SfG * SfC * SfA * SmU * SmU * SmC * SmA * SmA *
GUUGCAUUCAAUGUU
SSSSSSSSS

11081
SmU * SmG * SmU * SfU * SfU * SfU * SfG * SfA * SfC
CUGAC
SSSSSSSSSS

WV-
fA * SfU * SfU * SfC * SfA * SfA * SmU * SmG * SmU * SmU * SmC *
AUUCAAUGUUCUGAC
SSSSSSSSS

11082
SmU * SmG * SmA * SfC * SfA * SfA * SfC * SfA * SfG
AACAG
SSSSSSSSSS

WV-
fA * SfU * SfG * SfU * SfU * SfC * SmU * SmG * SmA * SmC * SmA *
AUGUUCUGACAACAG
SSSSSSSSS

11083
SmA * SmC * SmA * SfG * SfU * SfU * SfU * SfG * SfC
UUUGC
SSSSSSSSSS

WV-
fC * SfU * SfG * SfA * SfC * SfA * SmA * SmC * SmA * SmG * SmU *
CUGACAACAGUUUGC
SSSSSSSSS

11084
SmU * SmU * SmG * SfC * SfC * SfG * SfC * SfU * SfG
CGCUG
SSSSSSSSSS

WV-
fA * SfA * SfC * SfA * SfG * SfU * SmU * SmU * SmG * SmC * SmC *
AACAGUUUGCCGCUG
SSSSSSSSS

11085
SmG * SmC * SmU * SfG * SfC * SfC * SfC * SfA * SfA
CCCAA
SSSSSSSSSS

WV-
fU * SfU * SfU * SfG * SfC * SfC * SmG * SmC * SmU * SmG * SmC *
UUUGCCGCUGCCCAA
SSSSSSSSS

11086
SmC * SmC * SmA * SfA * SfU * SfG * SfC * SfC * SfA
UGCCA
SSSSSSSSSS

WV-
fC * SfG * SfC * SfU * SfG * SfC * SmC * SmC * SmA * SmA * SmU *
CGCUGCCCAAUGCCA
SSSSSSSSS

11087
SmG * SmC * SmC * SfA * SfU * SfC * SfC * SfU * SfG
UCCUG
SSSSSSSSSS

WV-
fC * SfC * SfC * SfA * SfA * SfU * SmG * SmC * SmC * SmA * SmU *
CCCAAUGCCAUCCUG
SSSSSSSSS

11088
SmC * SmC * SmU * SfG * SfG * SfA * SfG * SfU * SfU
GAGUU
SSSSSSSSSS

WV-
fU * SfG * SfC * SfC * SfA * SfU * SmC * SmC * SmU * SmG * SmG *
UGCCAUCCUGGAGUU
SSSSSSSSS

11089
SmA * SmG * SmU * SfU * SfC * SfC * SfU * SfG * SfU
CCUGU
SSSSSSSSSS

WV-
fU * SfC * SfC * SfU * SfG * SfG * SmA * SmG * SmU * SmU * SmC *
UCCUGGAGUUCCUGU
SSSSSSSSS

11090
SmC * SmU * SmG * SfU * SfA * SfA * SfG * SfA * SfU
AAGAU
SSSSSSSSSS

WV-
fG * SfA * SfG * SfU * SfU * SfC * SmC * SmU * SmG * SmU * SmA *
GAGUUCCUGUAAGAU
SSSSSSSSS

11091
SmA * SmG * SmA * SfU * SfA * SfC * SfC * SfA * SfA
ACCAA
SSSSSSSSSS

WV-
fC * SfC * SfU * SfG * SfU * SfA * SmA * SmG * SmA * SmU * SmA *
CCUGUAAGAUACCAA
SSSSSSSSS

11092
SmC * SmC * SmA * SfA * SfA * SfA * SfA * SfG * SfG
AAAGG
SSSSSSSSSS

WV-
fA * SfA * SfG * SfA * SfU * SfA * SmC * SmC * SmA * SmA * SmA *
AAGAUACCAAAAAGG
SSSSSSSSS

11093
SmA * SmA * SmG * SfG * SfC * SfA * SfA * SfA * SfA
CAAAA
SSSSSSSSSS

WV-
fA * SfC * SfC * SfA * SfA * SfA * SmA * SmA * SmG * SmG * SmC *
ACCAAAAAGGCAAAA
SSSSSSSSS

11094
SmA * SmA * SmA * SfA * SfC * SfA * SfA * SfA * SfA
CAAAA
SSSSSSSSSS

WV-
fA * SfA * SfA * SfG * SfG * SfC * SmA * SmA * SmA * SmA * SmC *
AAAGGCAAAACAAAA
SSSSSSSSS

11095
SmA * SmA * SmA * SfA * SfA * SfU * SfG * SfA * SfA
AUGAA
SSSSSSSSSS

WV-
fC * SfA * SfA * SfA * SfA * SfC * SmA * SmA * SmA * SmA * SmA *
CAAAACAAAAAUGAA
SSSSSSSSS

11096
SmU * SmG * SmA * SfA * SfG * SfC * SfC * SfC * SfC
GCCCC
SSSSSSSSSS

WV-
fC * SfA * SfA * SfA * SfA * SfA * SmU * SmG * SmA * SmA * SmG *
CAAAAAUGAAGCCCC
SSSSSSSSS

11097
SmC * SmC * SmC * SfC * SfA * SfU * SfG * SfU * SfC
AUGUC
SSSSSSSSSS

WV-
fA * SfU * SfG * SfA * SfA * SfG * SmC * SmC * SmC * SmC * SmA *
AUGAAGCCCCAUGUC
SSSSSSSSS

11098
SmU * SmG * SmU * SfC * SfU * SfU * SfU * SfU * SfU
UUUUU
SSSSSSSSSS

WV-
fG * SfC * SfC * SfC * SfC * SfA * SmU * SmG * SmU * SmC * SmU *
GCCCCAUGUCUUUUU
SSSSSSSSS

11099
SmU * SmU * SmU * SfU * SfA * SfU * SfU * SfU * SfG
AUUUG
SSSSSSSSSS

WV-
fA * SfU * SfG * SfU * SfC * SfU * SmU * SmU * SmU * SmU * SmA *
AUGUCUUUUUAUUU
SSSSSSSSS

11100
SmU * SmU * SmU * SfG * SfA * SfG * SfA * SfA * SfA
GAGAAA
SSSSSSSSSS

WV-
fU * SfU * SfU * SfU * SfU * SfA * SmU * SmU * SmU * SmG * SmA *
UUUUUAUUUGAGAA
SSSSSSSSS

11101
SmG * SmA * SmA * SfA * SfA * SfG * SfA * SfU * SfU
AAGAUU
SSSSSSSSSS

WV-
fA * SfU * SfU * SfU * SfG * SfA * SmG * SmA * SmA * SmA * SmA *
AUUUGAGAAAAGAU
SSSSSSSSS

11102
SmG * SmA * SmU * SfU * SfA * SfA * SfA * SfC * SfA
UAAACA
SSSSSSSSSS

WV-
fA * SfG * SfA * SfA * SfA * SfA * SmG * SmA * SmU * SmU * SmA *
AGAAAAGAUUAAAC
SSSSSSSSS

11103
SmA * SmA * SmC * SfA * SfG * SfU * SfG * SfU * SfG
AGUGUG
SSSSSSSSSS

WV-
fA * SfG * SfA * SfU * SfU * SfA * SmA * SmA * SmC * SmA * SmG *
AGAUUAAACAGUGU
SSSSSSSSS

11104
SmU * SmG * SmU * SfG * SfC * SfU * SfA * SfC * SfC
GCUACC
SSSSSSSSSS

WV-
fA * SfA * SfA * SfC * SfA * SfG * SmU * SmG * SmU * SmG * SmC *
AAACAGUGUGCUACC
SSSSSSSSS

11105
SmU * SmA * SmC * SfC * SfA * SfC * SfA * SfU * SfG
ACAUG
SSSSSSSSSS

WV-
fU * fC * fA * fC * fU * fC * mAfG * mAmU * fA * mGmUfU * fG * fA *
UCACUCAGAUAGUUG
XXXXXX O X O

11231
fA * fG * fC * fC
AAGCC
XX O O XXXXXX

WV-
fU * fC * fA * fC * fU * fC * fA * fG * mAmU * fA * mGmUfU * fG * fA *
UCACUCAGAUAGUUG
XXXXXXXX O XX

11232
fA * fG * fC * fC
AAGCC
O O XXXXXX

WV-
fU * fC * fA * fC * fU * fC * mAfG * fA * mU * fA * mGmUfU * fG * fA *
UCACUCAGAUAGUUG
XXXXXX O XXXX

11233
fA * fG * fC * fC
AAGCC
O O XXXXXX

WV-
fU * RfC * RfA * RfC * RfU * RfC * RmAfG * RmAmU * RfA *
UCACUCAGAUAGUUG
RRRRRR O R O RR

11234
RmGmUfU * RfG * RfA * RfA * RfG * RfC * RfC
AAGCC
O O RRRRRR

WV-
fU * RfC * RfA * RfC * RfU * RfC * RfA * RfG * RmAmfU * RfA *
UCACUCAGAUAGUUG
RRRRRRRR O RR

11235
RmGmUfU * RfG * RfA * RfA * RfG * RfC * RfC
AAGCC
O O RRRRRR

WV-
fU * RfC * RfA * RfC * RfU * RfC * RmAfG * RfA * RmU * RfA *
UCACUCAGAUAGUUG
RRRRRR O RRRR

11236
RmGmUfU * RfG * RfA * RfA * RfG * RfC * RfC
AAGCC
O O RRRRRR

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAn001fA * SmGn001mA * SfU *
UCAAGGAAGAUGGCA
SSSSSSn O Sn O

11237
SmGn001mGn001fC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SSn O n O SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAn001SfA * SmGn001SmA * SfU *
UCAAGGAAGAUGGCA
SSSSSSnSSnSS

11238
SmGn001SmGn001SfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SnSnSSSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAn001RfA * SmGn001RmA * SfU *
UCAAGGAAGAUGGCA
SSSSSSnRSnRSSn

11239
SmGn001RmGn001RfC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
RnRSSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001fU * SmG * SfA *
CUCCGGUUCUGAAGG
SSSSSSSSn O SSSn

11340
SmAn001mGn001fG * SfU * SfG * SfU * SfU * SfC
UGUUC
O n O SSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001fU * SmG * SfA *
CUCCGGUUCUGAAGG
SSSSSSSSn O SSSn

11341
SmAn001fG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
O SSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001fU * SmG * SfA *
CUCCGGUUCUGAAGG
SSSSSSSSn O SSSn

11342
SmAn001mG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
O SSSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SmAn001mU * SfA *
UCACUCAGAUAGUUG
SSSSSSn O Sn O

11343
SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
SSn O n O SSSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SfA * SfG * SmAn001mU * SfA *
UCACUCAGAUAGUUG
SSSSSSSSn O SSn O

11344
SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
n O SSSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA* SmU * SfA *
UCACUCAGAUAGUUG
SSSSSSn O SSSSn O

11345
SmGn001mUn001fU SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
n O SSSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SmAn001mU * SfA *
UCACUCAGAUAGUUG
SSSSSSn O Sn O

11346
SfG * SmUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
SSSn O SSSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SmAn001mU * SfA *
UCACUCAGAUAGUUG
SSSSSSn O Sn O

11347
SmGn001fU * SfU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
SSn O SSSSSSS

WV-
BrfUfCfAfCfUfCmAfGfAmU fAmGmUfUfGfAfAfGfCfC
UCACUCAGAUAGUUG
SSSSSSOSSSS

11544

AAGCC
OOSSSSSS

WV-
Acet5fUfCfAfCfUfCmAfGf AmUfAmGmUfUfGfAfAfGfCfC
UCACUCAGAUAGUUG
SSSSSSOSSSS

11545

AAGCC
OOSSSSSS

WV-
BrfUfCfAfCfUfCmAfGfAmU fAmGmUfUfGfAfAfGfCfC
UCACUCAGAUAGUUG
XXXXXXOXXXX

11546

AAGCC
OOXXXXXX

WV-
Acet5fUfCfAfCfUfCmAfGf AmUfAmGmUfUfGfAfAfGfCfC
UCACUCAGAUAGUUG
XXXXXXOXXXX

11547

AAGCC
OOXXXXXX

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001 fUn001 mGn001
CUCCGGUUCUGAAGG
SSSSSSSSnXnX

12123
fAn001 mAn001mG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
nXnXnX SSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001fUn001mG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSnXnX

12124
* SmAn001mG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSnXSSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001fU * SmGn001fA
CUCCGGUUCUGAAGG
SSSSSSSSnXS

12125
* SmAn001mG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
nXSnXSSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001fU * SmG *
CUCCGGUUCUGAAGG
SSSSSSSSnXSS

12126
SfAn001mAn001mG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
nXnXSSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001fU *
CUCCGGUUCUGAAGG
SSSSSSSSnXS

12127
SmGn001fAn001mAn001mG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
nXnXnXSSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001fUn001mG *
CUCCGGUUCUGAAGG
SSSSSSSSnXnX

12128
SfAn001mAn001mG * SfG * SfU * SfG * SfG * SfU * SfC
UGUUC
SnXnXSSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU *
CUCCGGUUCUGAAGG
SSSSSSSSnXnX

12129
SmCn001fUn001mGn001fA * SmAn001mG * SfG * SfU * SfG * SfU * SfU
UGUUC
nXSnXSSSSSS

* SfC

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAn001fAn001mGn001 mAn001
UCAAGGAAGAUGGCA
SSSSSSnXnX

12130
fUn001 mGn001 mGn001fC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
nXnXnX nXnX

SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAn001fAn001mGn001mA * SfU *
UCAAGGAAGAUGGCA
SSSSSSnXnXnXSSn

12131
SmGn001mGn001fC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
XnX SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAn001fA * SmGn001mAn001fU *
UCAAGGAAGAUGGCA
SSSSSSnXSnXnXSn

12132
SmGn001mGn001fC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
XnX SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAn001fA * SmGn001mA *
UCAAGGAAGAUGGCA
SSSSSSnXSnXSnXn

12133
SfUn001mGn001mGn001fC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
XnX SSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAn001fA *
UCAAGGAAGAUGGCA
SSSSSSnXSnXnXn

12134
SmGn001mAn001fUn001 mGn001 mGn001fC * SfA * SfU * SfU * SfU *
UUUCU
XnXnX SSSSSS

SfC * SfU

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAn001fAn001 mGn001mA *
UCAAGGAAGAUGGCA
SSSSSSnXnXnXS

12135
SfUn001mGn001mGn001fC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
nXnXnXSSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAn001fAn001 mGn001mAn001fU *
UCAAGGAAGAUGGCA
SSSSSSnXnXnX

12136
SmGn001mGn001fC * SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
nXSnXnX SSSSSS

WV-
rGrGrCrUrUrCrArArCrUrArU rCrUrGrArGrUrGrA
GGCUUCAACUAUCUG
OOOOOOOOOOOO

12422

AGUGA
O OOOOOO

WV-
rGrArArCrArCrCrUrUrCrArG rArArCrCrGrGrArG
GAACACCUUCAGAAC
OOOOOOOOOO

12423

CGGAG
OOO OOOOOO

WV-
fA * SfU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU *
AUCAAGGAAGAUGGC
SSSSSSSOSOS

12494
SmGmGfC * SfA * SfU * SfU * SfU * SfC * SfU
AUUUCU
SOOSSSS SS

WV-
fU * SfU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU *
UUCAAGGAAGAUGGC
SSSSSSSOSOS

12495
SmGmGfC * SfA * SfU * SfU * SfU * SfC * SfU
AUUUCU
SOOSSSS SS

WV-
fUfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC *
UCAAGGAAGAUGGCA
OSSSS

12496
SfA * SfU * SfU * SfU * SfC * SfU
UUUCU
SOSOSSOOSSSS SS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001fU * SmG * SfA *
CUCCGGUUCUGAAGG
SSSSSSSSnXS

12553
SmAn001mGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSnXOSSSS S

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001RfU * SmG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSnRS

12554
* SmAn001RmGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSnROSSSS S

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001RfU * SmG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSnRS

12555
* SmAn001RfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSnRSSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001RfU * SmG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSnRS

12556
* SmAn001RmG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSnRSSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001SfU * SmG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSnSSS

12557
* SmAn001SmGfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SnSOSSSS S

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001SfU * SmG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSnSS

12558
* SmAn001SfG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSnSSSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001SfU * SmG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSnSS

12559
* SmAn001SmG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSnSSSSSSS

WV-
L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU * SfA *
UCACUCAGAUAGUUG
OSSSS SSOSSSS

12566
SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
OOSSSS SS

WV-
Mod092L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU *
UCACUCAGAUAGUUG
OSSSS SSOSSSS

12567
SfA * SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
OOSSSS SS

WV-
Mod093L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU *
UCACUCAGAUAGUUG
OSSSS SSOSSSS

12568
SfA * SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
OOSSSS SS

WV-
L001TTTfU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU * SfA *
TTTUCACUCAGAUAG
OOOOSSSS

12569
SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
UUGAAGCC
SSOSSSS OOSSSS

SS

WV-
Mod020L001TTTfU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU
TTTUCACUCAGAUAG
OOOOSSSS

12570
* SfA * SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
UUGAAGCC
SSOSSSS OOSSSS

SS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU * SfA *
UCACUCAGAUAGUUG
SSSSSSOSSSS

12571
SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfCTTTL005
AAGCCTTT
OOSSSS SSOOOO

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU * SfA *
UCACUCAGAUAGUUG
SSSSSSOSSSS

12572
SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfCTTTL005Mod020
AAGCCTTT
OOSSSS SSOOOOO

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001RfU * SmG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSnRS

12872
* SmAn001RmGn001RfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSnRnRSSSSS

WV-
fU * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001SfU * SmG * SfA
CUCCGGUUCUGAAGG
SSSSSSSSnSS

12873
* SmAn001SmGn001SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
SSnSnSSSSSS

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCn001fU * SmG *
CUCCGGUUCUGAAGG
SSnXSSnXSSnX

12876
SfA * SmAn001mGn001fG * SfU * SfGn001fU * SfU * SfC
UGUUC
SSSnXnXSSnXSS

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCn001fU * SmG *
CUCCGGUUCUGAAGG
SSnXSSnXSSnXS

12877
SfA * SmAn001fG * SfG * SfU * SfGn001fU * SfU * SfC
UGUUC
SSnXSSSnXSS

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCn001fU * SmG *
CUCCGGUUCUGAAGG
SSnXSSnXSSnXS

12878
SfA * SmAn001mG * SfG * SfU * SfGn001fU * SfU * SfC
UGUUC
SSnXSSSnXSS

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU * SmG * SfA *
CUCCGGUUCUGAAGG
SSnXSSnXSSOS

12879
SmAmGfG * SfU * SfGn001fU * SfU * SfC
UGUUC
SSOOSSnXSS

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU * SmG * SfA *
CUCCGGUUCUGAAGG
SSnXSSnXSSOS

12880
SmAfG * SfG * SfU * SfGn001fU * SfU * SfC
UGUUC
SSOSSSnXSS

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU * SmG * SfA *
CUCCGGUUCUGAAGG
SSnXSSnXSSOS

12881
SmAmG * SfG * SfU * SfGn001fU * SfU * SfC
UGUUC
SSOSSSnXSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SmUn001mU * SmCn001mU *
CUCCGGUUCUGAAGG
SSSSSSnXSnXS

12882
SmGn001mA * SmAn001mG * SfG * SfU * SfG * SfU * SfU * SfC
UGUUC
nXSnXSSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SmUn001mUn001 mCn001mUn001
CUCCGGUUCUGAAGG
SSSSSSnXnXnXnXn

12883
mGn001mAn001 mAn001mGn001fG * SfU * SfG * SfU * SfU * SfC
UGUUC
X nXnXnXSSSSS

WV-
fU * SfC * SfAn001fC * SfU * SfCn001mAn001fG * SfA * SmU * SfA *
UCACUCAGAUAGUUG
SSnXSSnXnXSSS

12884
SmGn001mUn001fU * SfG * SfA * SfAn001fG * SfC * SfC
AAGCC
SnXnXSSSnXSS

WV-
fU * SfC * SfAn001fC * SfU * SfCn001mAfG * SfA * SmU * SfA *
UCACUCAGAUAGUUG
SSnXSSnXOSSSS

12885
SmGmUfU * SfG * SfA * SfAn001fG * SfC * SfC
AAGCC
OOSSSnXSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmA * SmG * SmA * SmU * SmA *
UCACUCAGAUAGUUG
SSSSSSSSSSS

12886
SmG * SmU * SmU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
SSSSSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAn001mG * SmAn001mU *
UCACUCAGAUAGUUG
SSSSSSnXSnX

12887
SmAn001mG * SmUn001mU * SfG * SfA * SfA * SfG * SfC * SfC
AAGCC
SnXSnX SSSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAn001mGn001mAn001 mUn001
UCACUCAGAUAGUUG
SSSSSSnXnXnXnXn

12888
mAn001mGn001 mUn001 mUn001fG * SfA * SfA * SfG * SfC * SfC
AAGCC
X nXnXnXSSSSS

WV-
GCGTGGTACCACGCL012mU * Geom5Ceom5CeomA * G * G * C * T * G
GCGTGGTACCACGCU
OOOOOOOOOO

12904
* G * T * T * A * T * mG * mA * mC * mU * mC
GCCA
OOOOOXOOO

GGCTGGTTATGACUC
XXXXXXXXXXXX

XXX

WV-
GCGTGG * T * A * CCACGCL012mU * Geom5Ceom5CeomA * G * G * C
GCGTGGTACCACGCU
OOOOOXXXOO

12905
* T * G * G * T * T * A * T * mG * mA * mC * mU * mC
GCCA
OOOOOXOOO

GGCTGGTTATGACUC
XXXXXXXXXXXX

XXX

WV-
G * C * G * T * G * G * T * A * C * C * A * C * G * CL012mU *
GCGTGGTACCACGCU
XXXXXXXXXXXX

12906
Geom5Ceom5CeomA * G * G * C * T * G * G * T * T * A * T * mG * mA *
GCCA
XOOXOOOXXX

mC * mU * mC
GGCTGGTTATGACUC
XXXXXXXXXXXX

WV-
GfCGfUGGTACfCAfCGfCL012mU * Geom5Ceom5CeomA * G * G * C * T
GCGUGGTACCACGCU
OOOOOOOOOOO

12907
* G * G * T * T * A * T * mG * mA * mC * mU * mC
GCCA
OOOOXOOO

GGCTGGTTATGACUC
XXXXXXXXXXXX

XXX

WV-
G * fCG * fUG * G * T * A * CfCA * fCG * fCL012mU *
GCGUGGTACCACGCU
XOXOXXXXOOXO

12908
Geom5Ceom5CeomA * G * G * C * T * G * G * T * T * A * T * mG * mA *
GCCA
XOOXOOO

mC * mU * mC
GGCTGGTTATGACUC
XXXXXXXXXXXX

XXX

WV-
G * fC * G * fU * G * G * T * A * C * fC * A * fC * G * fCL012mU *
GCGUGGTACCACGCU
XXXXXXXXXXXX

12909
Geom5Ceom5CeomA * G * G * C * T * G * G * T * T * A * T * mG * mA *
GCCA
XOOXOOO

mC * mU * mC
GGCTGGTTATGACUC
XXXXXXXXXXXX

XXX

WV-
GCGTGGTACCACGCL012BrmU * Geom5Ceom5CeomA * G * G * C * T *
GCGTGGTACCACGCU
OOOOOOOOOOO

12910
G * G * T * T * A * T * mG * mA * mC * mU * mC
GCCA
OOOOXOOO

GGCTGGTTATGACUC
XXXXXXXXXXXX

XXX

WV-
GCGTGG * T * A * CCACGCL012BrmU * Geom5Ceom5CeomA * G * G *
GCGTGGTACCACGCU
OOOOOXXXOOO

12911
C * T * G * G * T * T * A * T * mG * mA * mC * mU * mC
GCCA
OOOOXOOO

GGCTGGTTATGACUC
XXXXXXXXXXXX

XXX

WV-
G * C * G * T * G * G * T * A * C * C * A * C * G * CL012BrmU *
GCGTGGTACCACGCU
XXXXXXXXXXXX

12912
Geom5Ceo m5CeomA * G * G * C * T * G * G * T * T * A * T * mG * mA *
GCCA
XOOXOOO

mC * mU * mC
GGCTGGTTATGACUC
XXXXXXXXXXXX

XXX

WV-
GfCGfUGGTACfCAfCGfCL012BrmU * Geom5Ceom5CeomA * G * G * C
GCGUGGTACCACGCU
OOOOOOOOOOO

12913
* T * G * G * T * T * A * T * mG * mA * mC * mU * mC
GCCA
OOOOXOOO

GGCTGGTTATGACUC
XXXXXXXXXXXX

XXX

WV-
G * fCG * fUG * G * T * A * CfCA * fCG * fCL012BrmU * Geom5Ceo
GCGUGGTACCACGCU
XOXOXXXXOOXO

12914
m5CeomA * G * G * C * T * G * G * T * T * A * T * mG * mA * mC * mU
GCCA
XOOXOOOXXX

* mC
GGCTGGTTATGACUC
XXXXXXXXXXXX

WV-
G * fC * G * fU * G * G * T * A * C * fC * A * fC * G * fCL012BrmU *
GCGUGGTACCACGCU
XXXXXXXXXXXX

12915
Geom5Ceo m5CeomA * G * G * C * T * G * G * T * T * A * T * mG * mA
GCCA
XOOXOOOXXXX

mC * mU * mC
GGCTGGTTATGACUC
XXXXXXXXXXX

WV-
fC * SfU * SfC * SfC * SfU * SfG * SfU * SfU * SmCfU * SmG * SfC *
CUCCUGUUCUG
SSSSSSSSOSS

13319
SmAmGfC * SfU * SfG * SfU * SfU * SfC
CAGCUGUUC
SOOSSSSS

WV-
fC * SfU * SfC * SfC * SfU * SfG * SfU * SfU * SmCfU * SmG * SfC *
CUCCUGUUCUG
SSSSSSSSOSS

13320
SmAfG * SfC * SfU * SfG * SfU * SfU * SfC
CAGCUGUUC
SOSSSSSS

WV-
fC * SfU * SfC * SfC * SfU * SfG * SfU * SfU * SmCfU * SmG * SfC *
CUCCUGUUCUG
SSSSSSSSOSS

13321
SmAmG * SfC * SfU * SfG * SfU * SfU * SfC
CAGCUGUUC
SOSSSSSS

WV-
fC * SfU * SfC * SfC * SfU * SfG * SfU * SfU * SfC * SfU * SmG * SfC *
CUCCUGUUCUG
SSSSSSSSSSS

13322
SmAmGfC * SfU * SfG * SfU * SfU * SfC
CAGCUGUUC
SOOSSSSS

WV-
GTTGCCTCCGGTTCTGA AGGTGTTC +all PMO
GTTGCCTCCGG
OOOOOOOOOOO

13405

TTCTGAAGGTGTTC
OOOOOOOOOOOOO

WV-
CTCCGGTTCTGAAGGTGTTC +all PMO
CTCCGGTTCTG
OOOOOOOOOOO

13406

AAGGTGTTC
OOOOOOOO

WV-
TGCCTCCGGTTCTGA AGGTGTTCTTGTA +all PMO
TGCCTCCGGTT
OOOOOOOOOOO

13407

CTGAAGGTGTT
OOOOOOOOOOO

CTTGTA
OOOOO

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001RfU * SmG * SfA
CUCCGGUUC
SSSSSSSSnRS

13408
* SmAn001RfGn001RfG * SfU * SfG * SfU * SfU * SfC
UGAAGGUGUUC
SSnRnRSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCn001RfU * SmG * SfA
CUCCGGUUC
SSSSSSSSnRSSS

13409
* SmAn001RfGfG * SfU * SfG * SfU * SfU * SfC
UGAAGGUGUUC
nROSSSSS

WV-
fU * fU * fG * fu * fA * fC * fU * mU * mC * mA * mU *
UUGUACUUCAUCCCACUGAUUCUGA
XXXXXXXXXXXXXX

13594
mC * mC * mC * mA * mC * mU * fG * fA *

XXXXXnXnXnXnXnX

fUn001fUn001fCn001fUn001fGn00fA

WV-
fC * fC * fG * fG * fU * fU * fC * mU * mG * mA * mA *
CCGGUUCUGAAGGUGUUCUUGUACU
XXXXXXXXXXXXXX

13595
mG * mG * mU * mG * mU * mU * fC * fU *

XXXXXnXnXnXnXnX

fUn001fGn001fUn001fAn001fCn001fU

WV-
fUn001fUn001fGn001fUn001fAn001fC * fU * mU * mC *
UUGUACUUCAUCCCACUGAUUCUGA
nXnXnXnXnXXXXXXX

13596
mA * mU * mC * mC * mC * mA * mC * mU * fG * fA * fU

XXXXXXX XXXXXX

* fU * fC * fU * fG * fA

WV-
fCn001fCn001fGn001fGn001fUn001fU * fC * mU * mG *
CCGGUUCUGAAGGUGUUCUUGUACU
nXnXnXnXnXXXXXXX

13597
mA * mA * mG * mG * mU * mG * mU * mU * fC * fU *

XXXXXXX XXXXXX

fU * fG * fU * fA * fC * fU

WV-
fU * SfG * SfA * SfC * SfU * SfU * SmG * SmC * SmU *
UGACUUCUCAAGCUUUUCU
SSSSS SSSSS SSSSS

13701
SmC * SmA * SmA * SmG * SmC * SfU * SfU * SfU * SfU

SSSS

* SfC * SfU

WV-
fC * SfA * SfA * SfG * SfC * SfU * SmU * SmU * SmU *
CAAGCUUUUCUUUUAGUUGC
SSSSS SSSSS SSSSS

13702
SmC * SmU * SmU * SmU * SmU * SfA * SfG * SfU * SfU

SSSS

* SfG * SfC

WV-
fC * SfU * SfU * SfU * SfU * SfA * SmG * SmU * SmU *
CUUUUAGUUGCUGCUCUUUU
SSSSS SSSSS SSSSS

13703
SmG * SmC * SmU * SmG * SmC * SfU * SfC * SfU * SfU

SSSS

* SfU * SfU

WV-
fG * SfC * SfU * SfG * SfC * SfU * SmC * SmU * SmU *
GCUGCUCUUUUCCAGGUUCA
SSSSS SSSSS SSSSS

13704
SmU * SmU * SmC * SmC * SmA * SfG * SfG * SfU * SfU

SSSS

* SfC * SfA

WV-
fU * SfU * SfC * SfC * SfA * SfG * SmG * SmU * SmU *
UUCCAGGUUCAAGUGGGAUA
SSSSS SSSSS SSSSS

13705
SmC * SmA * SmA * SmG * SmU * SfG * SfG * SfG * SfA

SSSS

* SfU * SfA

WV-
fC * SfA * SfA * SfG * SfU * SfG * SmG * SmG * SmA *
CAAGUGGGAUACUAGCAAUG
SSSSS SSSSS SSSSS

13706
SmU * SmA * SmC * SmU * SmA * SfG * SfC * SfA * SfA

SSSS

* SfU * SfG

WV-
fU * SfA * SfC * SfU * SfA * SfG * SmC * SmA * SmA *
UACUAGCAAUGUUAUCUGCU
SSSSS SSSSS SSSSS

13707
SmU * SmG * SmU * SmU * SmA * SfU * SfC * SfU * SfG

SSSS

* SfC * SfU

WV-
fU * SfG * SfU * SfU * SfA * SfU * SmC * SmU * SmG *
UGUUAUCUGCUUCCUCCAAC
SSSSS SSSSS SSSSS

13708
SmC * SmU * SmU * SmC * SmC * SfU * SfC * SfC * SfA

SSSS

* SfA * SfC

WV-
fC * SfU * SfU * SfC * SfC * SfU * SmC * SmC * SmA *
CUUCCUCCAACCAUAAAACA
SSSSS SSSSS SSSSS

13709
SmA * SmC * SmC * SmA * SmU * SfA * SfA * SfA * SfA

SSSS

* SfC * SfA

WV-
fC * SfC * SfA * SfU * SfA * SfA * SmA * SmA * SmC *
CCAUAAAACAAAUUCAUUUA
SSSSS SSSSS SSSSS

13710
SmA * SmA * SmA * SmU * SmU * SfC * SfA* SfU * SfU

SSSS

* SfU * SfA

WV-
fA * SfA * SfU * SfU * SfC * SfA * SmU * SmU * SmU *
AAUUCAUUUAAAUCUCUUUG
SSSSS SSSSS SSSSS

13711
SmA * SmA * SmA * SmU * SmC * SfU * SfC * SfU * SfU

SSSS

* SfU * SfG

WV-
fA * SfA * SfU * SfC * SfU * SfC * SmU * SmU * SmU *
AAUCUCUUUGAAAUUCUGAC
SSSSS SSSSS SSSSS

13712
SmG * SmA * SmA * SmA * SmU * SfU * SfC * SfU * SfG

SSSS

* SfA * SfC

WV-
fU * SfG * SfA * SfA * SfA * SfU * SmU * SmC * SmU *
UGAAAUUCUGACAAGAUAUU
SSSSS SSSSS SSSSS

13713
SmG * SmA * SmC * SmA * SmA * SfG * SfA * SfU * SfA

SSSS

* SfU * SfU

WV-
fA * SfC * SfA * SfA * SfG * SfA * SmU * SmA * SmU *
ACAAGAUAUUCUUUUGUUCU
SSSSS SSSSS SSSSS

13714
SmU * SmC * SmU * SmU * SmU * SfU * SfG * SfU * SfU

SSSS

* SfC * SfU

WV-
fU * SfA * SfU * SfU * SfC * SfU * SmU * SmU * SmU *
UAUUCUUUUGUUCUUCUAGC
SSSSS SSSSS SSSSS

13715
SmG * SmU * SmU * SmC * SmU * SfU * SfC * SfU * SfA

SSSS

* SfG * SfC

WV-
fU * SfU * SfC * SfU * SfU * SfU * SmU * SmG * SmU *
UUCUUUUGUUCUUCUAGCCU
SSSSS SSSSS SSSSS

13716
SmU * SmC * SmU * SmU * SmC * SfU * SfA * SfG * SfC

SSSS

* SfC * SfU

WV-
fA * SfU * SfC * SfC * SfA * SfC * SmU * SmG * SmG *
AUCCACUGGAGAUUUGUCUG
SSSSS SSSSS SSSSS

13717
SmA * SmG * SmA * SmU * SmU * SfU * SfG * SfU * SfC

SSSS

* SfU * SfG

WV-
fA * SfG * SfA * SfU * SfU * SfU * SmG * SmU * SmC *
AGAUUUGUCUGCUUGAGCUU
SSSSS SSSSS SSSSS

13718
SmU * SmG * SmC * SmU * SmU * SfG * SfA * SfG * SfC

SSSS

* SfU * SfU

WV-
fU * SfG * SfC * SfU * SfU * SfG * SmA * SmG * SmC *
UGCUUGAGCUUAUUUUCAAG
SSSSS SSSSS SSSSS

13719
SmU * SmU * SmA * SmU * SmU * SfU * SfU * SfC * SfA

SSSS

* SfA * SfG

WV-
fU * SfA * SfU * SfU * SfU * SfU * SmC * SmA * SmA *
UAUUUUCAAGUUUAUCUUGC
SSSSS SSSSS SSSSS

13720
SmG * SmU * SmU * SmU * SmA * SfU * SfC * SfU * SfU

SSSS

* SfG * SfC

WV-
fU * SfU * SfU * SfA * SfU * SfC * SmU * SmU * SmG *
UUUAUCUUGCUCUUCUGGGC
SSSSS SSSSS SSSSS

13721
SmC * SmU * SmC * SmU * SmU * SfC * SfU * SfG * SfG

SSSS

* SfG * SfC

WV-
fU * SfC * SfU * SfU * SfC * SfU * SmG * SmG * SmG *
UCUUCUGGGCUUAUGGGAGC
SSSSS SSSSS SSSSS

13722
SmC * SmU * SmU * SmA * SmU * SfG * SfG * SfG * SfA

SSSS

* SfG * SfC

WV-
fU * SfU * SfA * SfU * SfG * SfG * SmG * SmA * SmG *
UUAUGGGAGCACUUACAAGC
SSSSS SSSSS SSSSS

13723
SmC * SmA * SmC * SmU * SmU * SfA * SfC * SfA * SfA

SSSS

* SfG * SfC

WV-
fG * SfC * SfA * SfC * SfU * SfU * SmA * SmC * SmA *
GCACUUACAAGCACGGGUCC
SSSSS SSSSS SSSSS

13724
SmA * SmG * SmC * SmA * SmC * SfG * SfG * SfG * SfU

SSSS

* SfC * SfC

WV-
fG * SfC * SfA * SfC * SfG * SfG * SmG * SmU * SmC *
GCACGGGUCCUCCAGUUUCA
SSSSS SSSSS SSSSS

13725
SmC * SmU * SmC * SmC * SmA * SfG * SfU * SfU * SfU

SSSS

* SfC * SfA

WV-
fU * SfC * SfC * SfA * SfG * SfU * SmU * SmU * SmC *
UCCAGUUUCAUUUAAUUGUU
SSSSS SSSSS SSSSS

13726
SmA * SmU * SmU * SmU * SmA * SfA * SfU * SfU * SfG

SSSS

* SfU * SfU

WV-
fU * SfU * SfU * SfA * SfA * SfU * SmU * SmG * SmU *
UUUAAUUGUUUGAGAAUUCC
SSSSS SSSSS SSSSS

13727
SmU * SmU * SmG * SmA * SmG * SfA * SfA * SfU * SfU

SSSS

* SfC * SfC

WV-
fG * SfA * SfG * SfA * SfA * SfU * SmU * SmC * SmC *
GAGAAUUCCCUGGCGCAGGG
SSSSS SSSSS SSSSS

13728
SmC * SmU * SmG * SmG * SmC * SfG * SfC * SfA * SfG

SSSS

* SfG * SfG

WV-
fC * SfU * SfG * SfG * SfC * SfG * SmC * SmA * SmG *
CUGGCGCAGGGGCAACUCUU
SSSSS SSSSS SSSSS

13729
SmG * SmG * SmG * SmC * SmA * SfA * SfC * SfU * SfC

SSSS

* SfU * SfU

WV-
fG * SfC * SfA * SfG * SfG * SfG * SmG * SmC * SmA *
GCAGGGGCAACUCUUCCACC
SSSSS SSSSS SSSSS

13730
SmA * SmC * SmU * SmC * SmU * SfU * SfC * SfC * SfA

SSSS

* SfU * SfC

WV-
fG * SfG * SfC * SfA * SfA * SfC * SmU * SmC * SmU *
GGCAACUCUUCCACCAGUAA
SSSSS SSSSS SSSSS

13731
SmU * SmC * SmC * SmA * SmC * SfC * SfA * SfG * SfU

SSSS

* SfA * SfA

WV-
fC * SfU * SfC * SfU * SfU * SfC * SmC * SmA * SmC *
CUCUUCCACCAGUAACUGAA
SSSSS SSSSS SSSSS

13732
SmC * SmA * SmG * SmU * SmA * SfA * SfC * SfU * SfG

SSSS

* SfA * SfA

WV-
fU * SfU * SfC * SfG * SfA * SfU * SmC * SmC * SmG *
UUCGAUCCGUAAUGAUUGUU
SSSSS SSSSS SSSSS

13733
SmU * SmA * SmA * SmU * SmG * SfA * SfU * SfU * SfG

SSSS

* SfU * SfU

WV-
fA * SfA * SfU * SfG * SfA * SfU * SmU * SmG * SmU *
AAUGAUUGUUCUAGCCUCUU
SSSSS SSSSS SSSSS

13734
SmU * SmC * SmU * SmA * SmG * SfC * SfC * SfU * SfC

SSSS

* SfU * SfU

WV-
fC * SfU * SfA * SfG * SfC * SfC * SmU * SmC * SmU *
CUAGCCUCUUGAUUGCUGGU
SSSSS SSSSS SSSSS

13735
SmU * SmG * SmA * SmU * SmU * SfG * SfC * SfU * SfG

SSSS

* SfG * SfU

WV-
fG * SfA * SfU * SfU * SfG * SfC * SmU * SmG * SmG *
GAUUGCUGGUCUUGUUUUUC
SSSSS SSSSS SSSSS

13736
SmU * SmC * SmU * SmU * SmG * SfU * SfU * SfU * SfU

SSSS

* SfU * SfC

WV-
fC * SfU * SfU * SfG * SfU * SfU * SmU * SmU * SmU *
CUUGUUUUUCAAAUUUUGGG
SSSSS SSSSS SSSSS

13737
SmC * SmA * SmA * SmA * SmU * SfU * SfU * SfU * SfG

SSSS

* SfG * SfG

WV-
fA * SfA * SfA * SfU * SfU * SfU * SmU * SmG * SmG *
AAAUUUUGGGCAGCGGUAAU
SSSSS SSSSS SSSSS

13738
SmG * SmC * SmA * SmG * SmC * SfG * SfG * SfU * SfA

SSSS

* SfA * SfU

WV-
fC * SfA * SfG * SfC * SfG * SfG * SmU * SmA * SmA *
CAGCGGUAAUGAGUUCUUCC
SSSSS SSSSS SSSSS

13739
SmU * SmG * SmA * SmG * SmU * SfU * SfC * SfU * SfU

SSSS

* SfC * SfC

WV-
fG * SfA * SfG * SfU * SfU * SfC * SmU * SmU * SmC *
GAGUUCUUCCAACUGGGGAC
SSSSS SSSSS SSSSS

13740
SmC * SmA * SmA * SmC * SmU* SfG * SfG * SfG * SfG

SSSS

* SfA * SfC

WV-
fA * SfA * SfC * SfU * SfG * SfG * SmG * SmG * SmA *
AACUGGGGACGCCUCUGUUC
SSSSS SSSSS SSSSS

13741
SmC * SmG * SmC * SmC * SmU * SfC * SfU * SfG * SfU

SSSS

* SfU * SfC

WV-
fG * SfC * SfC * SfU * SfC * SfU * SmG * SmU * SmU *
GCCUCUGUUCCAAAUCCUGC
SSSSS SSSSS SSSSS

13742
SmC * SmC * SmA * SmA * SmA * SfU * SfC * SfC * SfU

SSSS

* SfG * SfC

WV-
fU * SfG * SfU * SfU * SfC * SfC * SmA * SmA * SmA *
UGUUCAAAUCCUGCAUUGU
SSSSS SSSSS SSSSS

13743
SmU * SmC * SmC * SmU * SmG * SfC * SfA * SfU * SfU

SSSS

* SfG * SfU

WV-
fC * SfA * SfA * SfA * SfU * SfC * SmC * SmU * SmG *
CAAAUCCUGCAUUGUUGCCU
SSSSS SSSSS SSSSS

13744
SmC * SmA * SmU * SmU * SmG * SfU * SfU * SfG * SfC

SSSS

* SfC * SfU

WV-
fC * SfU * SfU * SfU * SfU * SfA * SmU * SmG * SmA *
CUUUUAUGAAUGCUUCUCCA
SSSSS SSSSS SSSSS

13745
SmA * SmU * SmG * SmC * SmU * SfU * SfC * SfU * SfC

SSSS

* SfC * SfA

WV-
fA * SfU * SfG * SfC * SfU * SfU * SmC * SmU * SmC *
AUGCUUCUCCAAGAGGCAUU
SSSSS SSSSS SSSSS

13746
SmC * SmA * SmA * SmG * SmA * SfG * SfG * SfC * SfA

SSSS

* SfU * SfU

WV-
fA * SfA * SfG * SfA * SfG * SfG * SmC * SmA * SmU *
AAGAGGCAUUGAUAUUCUCU
SSSSS SSSSS SSSSS

13747
SmU * SmG * SmA * SmU * SmA * SfU * SfU * SfC * SfU

SSSS

* SfC * SfU

WV-
fG * SfA * SfU * SfA * SfU * SfU * SmC * SmU * SmC *
GAUAUUCUCUGUUAUCAUGU
SSSSS SSSSS SSSSS

13748
SmU * SmG * SmU * SmU * SmA * SfU * SfC * SfA * SfU

SSSS

* SfG * SfU

WV-
fG * SfU * SfU * SfA * SfU * SfC * SmA * SmU * SmG *
GUUAUCAUGUGGACUUUUCU
SSSSS SSSSS SSSSS

13749
SmU * SmG * SmG * SmA * SmC * SfU * SfU * SfU * SfU

SSSS

* SfC * SfU

WV-
fG * SfG * SfA * SfC * SfU * SfU * SmU * SmU * SmC *
GGACUUUUCUGGUAUCAUCU
SSSSS SSSSS SSSSS

13750
SmU * SmG * SmG * SmU * SmA * SfU * SfC * SfA * SfU

SSSS

* SfC * SfU

WV-
fG * SfG * SfU * SfA * SfU * SfC * SmA * SmU * SmC *
GGUAUCAUCUGCAGAAUAAU
SSSSS SSSSS SSSSS

13751
SmU * SmG * SmC * SmA * SmG * SfA * SfA * SfU * SfA

SSSS

* SfA * SfU

WV-
fG * SfC * SfA * SfG * SfA * SfA * SmU * SmA * SmA *
GCAGAAUAAUCCCGGAGAAG
SSSSS SSSSS SSSSS

13752
SmU * SmC * SmC * SmC * SmG * SfG * SfA * SfG * SfA

SSSS

* SfA * SfG

WV-
fC * SfC * SfG * SfG * SfA * SmG * SmA * SmA * SmG *
CCGGAGAAGUUUCAGGGCCA
SSSSS SSSSS SSSSS

13753
SmU * SmU * SmU * SmC * SfA * SfG * SfG * SfG * SfC *

SSSS

SfC * SfA

WV-
fU * SfU * SfU * SfC * SfA * SfG * SmG * SmG * SmC *
UUUCAGGGCCAAGUCAUUUG
SSSSS SSSSS SSSSS

13754
SmC * SmA * SmA * SmG * SmU * SfC * SfA * SfU * SfU

SSSS

* SfU * SfG

WV-
fA * SfA * SfG * SfU * SfC * SfA * SmU * SmU * SmU *
AAGUCAUUUGCCACAUCUAC
SSSSS SSSSS SSSSS

13755
SmG * SmC * SmC * SmA * SmC * SfA * SfU * SfC * SfU

SSSS

* SfA * SfC

WV-
fC * SfC * SfA * SfC * SfA * SfU * SmC * SmU * SmA *
CCACAUCUACAUUUGUCUGC
SSSSS SSSSS SSSSS

13756
SmC * SmA * SmU * SmU * SmU * SfG * SfU * SfC * SfU

SSSS

* SfG * SfC

WV-
fA * SfU * SfU * SfU * SfG * SfU * SmC * SmU * SmG *
AUUUGUCUGCCACUGGCGGA
SSSSS SSSSS SSSSS

13757
SmC * SmC * SmA * SmC * SmU * SfG * SfG * SfC * SfG

SSSS

* SfG * SfA

WV-
fC * SfA * SfC * SfU * SfG * SfG * SmC * SmG * SmG *
CACUGGCGGAGGUCUUUGGC
SSSSS SSSSS SSSSS

13758
SmA * SmG * SmG * SmU * SmC * SfU * SfU * SfU * SfG

SSSS

* SfG * SfC

WV-
fG * SfC * SfG * SfG * SfA * SfG * SmG * SmU * SmC *
GCGGAGGUCUUUGGCCAACU
SSSSS SSSSS SSSSS

13759
SmU * SmU * SmU * SmG * SmG * SfC * SfC * SfA * SfA

SSSS

* SfC * SfU

WV-
fG * SfG * SfU * SfC * SfU * SfU * SmU * SmG * SmG *
GGUCUUUGGCCAACUGCUAU
SSSSS SSSSS SSSSS

13760
SmC * SmC * SmA * SmA * SmC * SfU * SfG * SfC * SfU

SSSS

* SfA * SfU

WV-
fU * SfU * SfG * SfC * SfC * SfA * SmU * SmU * SmG *
UUGCCAUUGUUUCAUCAGCU
SSSSS SSSSS SSSSS

13761
SmU * SmU * SmU * SmC * SmA * SfU * SfC * SfA * SfG

SSSS

* SfC * SfU

WV-
fU * SfU * SfU * SfC * SfA * SfU * SmC * SmA * SmG *
UUUCAUCAGCUCUUUUACUC
SSSSS SSSSS SSSSS

13762
SmC * SmU * SmC * SmU * SmU * SfU * SfU * SfA * SfC

SSSS

* SfU * SfC

WV-
fU * SfC * SfU * SfU * SfU * SfU * SmA * SmC * SmU *
UCUUUUACUCCCUUGGAGUC
SSSSS SSSSS SSSSS

13763
SmC * SmC * SmC * SmU * SmU * SfG * SfG * SfA * SfG

SSSS

* SfU * SfC

WV-
fC * SfC * SfU * SfU * SfG * SfG * SmA * SmG * SmU *
CCUUGGAGUCUUCUAGGAGC
SSSSS SSSSS SSSSS

13764
SmC * SmU * SmU * SmC * SmU * SfA * SfG * SfG * SfA

SSSS

* SfG * SfC

WV-
fU * SfU * SfC * SfU * SfA * SfG * SmG * SmA * SmG *
UUCUAGGAGCCUUUCCUUAC
SSSSS SSSSS SSSSS

13765
SmC * SmC * SmU * SmU * SmU * SfC * SfC * SfU * SfU

SSSS

* SfA * SfC

WV-
fC * SfU * SfU * SfU * SfC * SfC * SmU * SmU * SmA *
CUUUCCUUACGGGUAGCAUC
SSSSS SSSSS SSSSS

13766
SmC * SmG * SmG * SmG * SmU * SfA * SfG * SfC * SfA

SSSS

* SfU * SfC

WV-
fG * SfG * SfG * SfU * SfA * SfG * SmC * SmA * SmU *
GGGUAGCAUCCUGUAGGACA
SSSSS SSSSS SSSSS

13767
SmC * SmC * SmU * SmG * SmU * SfA * SfG * SfG * SfA

SSSS

* SfC * SfA

WV-
fC * SfU * SfG * SfU * SfA * SfG * SmG * SmA * SmC *
CUGUAGGACAUUGGCAGUUG
SSSSS SSSSS SSSSS

13768
SmA * SmU * SmU * SmG * SmG * SfC * SfA * SfG * SfU

SSSS

* SfU * SfG

WV-
fU * SfU * SfG * SfG * SfC * SfA * SmG * SmU * SmU *
UUGGCAGUUGUUUCAGCUUC
SSSSS SSSSS SSSSS

13769
SmG * SmU * SmU * SmU * SmC * SfA * SfG * SfC * SfU

SSSS

* SfU * SfC

WV-
fU * SfU * SfU * SfC * SfA * SfG * SmC * SmU * SmU *
UUUCAGCUUCUGUAAGCCAG
SSSSS SSSSS SSSSS

13770
SmC * SmU * SmG * SmU * SmA * SfA * SfG * SfC * SfC

SSSS

* SfA * SfG

WV-
fU * SfG * SfU * SfA * SfA * SfG * SmC * SmC * SmA *
UGUAAGCCAGGCAAGAAACU
SSSSS SSSSS SSSSS

13771
SmG * SmG * SmC * SmA * SmA * SfG * SfA * SfA * SfA

SSSS

* SfC * SfU

WV-
fG * SfC * SfA * SfA * SfG * SfA * SmA * SmA * SmC *
GCAAGAAACUUUUCCAGGUC
SSSSS SSSSS SSSSS

13772
SmU * SmU * SmU * SmU * SmC * SfC * SfA * SfG * SfG

SSSS

* SfU * SfC

WV-
fU * SfU * SfU * SfC * SfC * SfA * SmG * SmG * SmU *
UUUCCAGGUCCAGGGGGAAC
SSSSS SSSSS SSSSS

13773
SmC * SmC * SmA * SmG * SmG * SfG * SfG * SfG * SfA

SSSS

* SfA * SfC

WV-
fC * SfA * SfG * SfG * SfG * SfG * SmG * SmA * SmA *
CAGGGGGAACUGUUGCAGUA
SSSSS SSSSS SSSSS

13774
SmC * SmU * SmG * SmU * SmU * SfG * SfC * SfA * SfG

SSSS

* SfU * SfA

WV-
fU * SfG * SfU * SfU * SfG * SfC * SmA * SmG * SmU *
UGUUGCAGUAAUCUAUGAGU
SSSSS SSSSS SSSSS

13775
SmA * SmA * SmU * SmC * SmU * SfA * SfU * SfG * SfA

SSSS

* SfG * SfA

WV-
fA * SfU * SfC * SfU * SfA * SfU * SmG * SmA * SmG *
AUCUAUGAGUUUCUUCCAAA
SSSSS SSSSS SSSSS

13776
SmU * SmU * SmU * SmC * SmU * SfU * SfC * SfC * SfA

SSSS

* SfA * SfA

WV-
fU * SfG * SfC * SfU * SfU * SfC * SmC * SmA * SmA *
UUCUUCCAAAGCAGCCUCUC
SSSSS SSSSS SSSSS

13777
SmA * SmG * SmC * SmA * SmG * SfC * SfC * SfU * SfC

SSSS

* SfU * SfC

WV-
fG * SfC * SfA * SfG * SfC * SfC * SmU * SmC * SmU *
GCAGCCUCUCGCUCACUCAC
SSSSS SSSSS SSSSS

13778
SmC * SmG * SmC * SmU * SmC * SfA * SfC * SfU * SfC

SSSS

* SfA * SfC

WV-
fC * SfU * SfC * SfU * SfC * SfG * SmC * SmU * SmC *
CUCUCGCUCACUCACCCUGC
SSSSS SSSSS SSSSS

13779
SmA * SmC * SmU * SmC * SmA * SfC * SfC * SfC * SfU

SSSS

* SfG * SfC

WV-
fA * SfG * SfG * SfU * SfU * SfC * SmA * SmA * SmG *
AGGUUCAAGUGGGAUACUAG
SSSSS SSSSS SSSSS

13780
SmU * SmG * SmG * SmG * SmA * SfU * SfA * SfC * SfU

SSSS

* SfA * SfG

WV-
fU * SfC * SfC * SfA * SfG * SfG * SmU * SmU * SmC *
UCCAGGUUCAAGUGGGAUAC
SSSSS SSSSS SSSSS

13781
SmA * SmA * SmG * SmU * SmG * SfG * SfG * SfA * SfU

SSSS

* SfA * SfC

WV-
fU * SfU * SfG * SfC * SfU * SfG * SmG * SmU * SmC *
UUGCUGGUCUUGUUUUUCAA
SSSSS SSSSS SSSSS

13782
SmU * SmU * SmG * SmU * SmU * SfU * SfU * SfU * SfC

SSSS

* SfA * SfA

WV-
fA * SfC * SfU * SfG * SfG * SfG * SmG * SmA * SmC *
ACUGGGGACGCCUCUGUUCC
SSSSS SSSSS SSSSS

13783
SmG * SmC * SmC * SmU * SmC * SfU * SfG * SfU * SfU

SSSS

* SfC * SfC

WV-
fU * SfA * SfC * SfA * SfU * SfU * SmU * SmG * SmU *
UACAUUUGUCUGCCACUGGC
SSSSS SSSSS SSSSS

13784
SmC * SmU * SmG * SmC * SmC * SfA * SfC * SfU * SfG

SSSS

* SfG * SfC

WV-
fC * SfC * SfC * SfG * SfG * SfA * SmG * SmA * SmA *
CCCGGAGAAGUUUCAGGGCC
SSSSS SSSSS SSSSS

13785
SmG * SmU * SmU * SmU * SmC * SfA * SfG * SfG * SfG

SSSS

* SfC * SfC

WV-
fU * SfC * SfC * SfU * SfG * SfU * SmA * SmG * SmG *
UCCUGUAGGACAUUGGCAGU
SSSSS SSSSS SSSSS

13786
SmA * SmC * SmA * SmU * SmU * SfG * SfG * SfC * SfA

SSSS

* SfG * SfU

WV-
fG * SfA * SfG * SfU * SfC * SfU * SmU * SmC * SmU *
GAGUCUUCUAGGAGCCUUUC
SSSSS SSSSS SSSSS

13787
SmA * SmG * SmG * SmA * SmG * SfC * SfC * SfU * SfU

SSSS

* SfU * SfC

WV-
fC * SfU * SfU * SfG * SfA * SfG * SmC * SmU * SmU *
CUUGAGCUUAUUUUCAAGUU
SSSSS SSSSS SSSSS

13788
SmA * SmU * SmU * SmU * SmU * SfC * SfA * SfA * SfG

SSSS

* SfU * SfU

WV-
fA * SfG * SfC * SfA * SfC * SfU * SmU * SmA * SmC *
AGCACUUACAAGCACGGGUC
SSSSS SSSSS SSSSS

13789
SmA * SmA * SmG * SmC * SmA * SfC * SfG * SfG * SfG

SSSS

* SfU * SfC

WV-
fU * SfU * SfG * SfU * SfA * SfC * SfU * SmU * SmC *
UUGUACUUCAUCCCACUGAUUCUGA
SSSSSSSSSSSSSSS

13790
SmA * SmU * SmC * SmC * SmC * SmA * SmC * SmU *

SSSSSSSSS

SfG * SfA * SfU * SfU * SfC * SfU * SfG * SfA

WV-
fU * SfU * SfU * SfU * SfA * SfC * SfU * SfU * SfC *
UUGUACUUCAUCCCACUGAUUCUGA
SSSSSSSSSOSSSS

13791
SmAfU * SfC * SfC * SfC * SmAfC * SfU * SmGfA * SfU *

OSSOSSSSSS

SfU * SfC * SfU * SfG * SfA

WV-
fU * SfU * SfG * SfU * SfA * SfC * SfU * SmUmCfA *
UUGUACUUCAUCCCACUGAUUCUGA
SSSSSSSOOSOOO

13792
SmUmCmCmCfA * SmCmUfG * SfA * SfU * SfU * SfC *

OSOOSSSSSSS

SfU * SfG * SfA

WV-
fU * SfU * SfG * SfU * SfA * SfC * SfU * SmUfC * SmAfU
UUGUACUUCAUCCCACUGAUUCUGA
SSSSSSSOSOSOSO

13793
* SmCfC * SmCfA * SmCfU * SmGfA * SfU * SfU * SfC *

SOSOSSSSSS

SfU * SfG * SfA

WV-
fU * SfU * SfG * SfU * SfA * SfC * SfU * SfU * SmCfA *
UUGUACUUCAUCCCACUGAUUCUGA
SSSSSSSSOSOSOS

13794
SmUfC * SmCfC * SmAfC * SmUfG * SfA * SfU * SfU *

OSOSSSSSSS

SfC * SfU * SfG * SfA

WV-
fC * SfC * SfG * SfG * SfU * SfG * SfC * SmU * SmG *
CCGGUUCUGAAGGUGUUCUUGUACU
SSSSSSSSSSSSSSS

13795
SmA * SmA * SmG * SmG * SmU * SmG * SmU * SmU *

SSSSSSSSS

SfC * SfU * SfU * SfG * SfU * SfA * SfC * SfC

WV-
fC * SfC * SfG * SfG * SfU * SfU * SfC * SfU *
CCGGUUCUGAAGGUGUUCUUGUACU
SSSSSSSSOOOOO

13796
SmGmAmAmGmGfU * SmGfU * SfU * SfC * SfU * SfU *

SOSSSSSSSSS

SfG * SfU * SfA * SfC * SfU

WV-
fC * SfC * SfG * SfG * SfU * SfU * SfC * SmUfG * SfA *
CCGGUUCUGAAGGUGUUCUUGUACU
SSSSSSSOSSSSSO

13797
SfA * SfG * SfG * SmUfG * SmUmUmCfU * SfU * SfG *

SOOOSSSSSS

SfU * SfA * SfC * SfU

WV-
fC * SfC * SfG * SfG * SfU * SfU * SfC * SmUfG * SmAfA
CCGGUUCUGAAGGUGUUCUUGUACU
SSSSSSSOSOSOS

13798
* SmGfG * SmUfG * SmUfU * SmCfU * SfU * SfG * SfU *

OSOSOSSSSSS

SfA * SfC * SfU

WV-
fC * SfC * SfG * SfG * SfU * SfU * SfC * SfU * SmGfA *
CCGGUUCUGAAGGUGUUCUUGUACU
SSSSSSSSOSOSO

13799
SmAfG * SmGfU * SmGfU * SmU * SfC * SfU * SfU * SfG

SOSSSSSSSSS

SfU * SfA * SfC * SfU

WV-
fU * SfU * SfU * SfG * SfC * SfC * SfG * SfC * SmUfG *
UUUGCCGCUGCCCAAUGCCA
SSSSSSSSOSSS

13810
SmC * SfC * SmCmAfA * SfU * SfG * SfC * SfC * SfA

OOSSSSS

WV-
fU * SfU * SfU * SfG * SfC * SfC * SfG * SfC * SmUfG *
UUUGCCGCUGCCCAAUGCCA
SSSSSSSSOSSS

13811
SmC * SfC * SmCfA * SfA * SfU * SfG * SfC * SfC * SfA

OSSSSSS

WV-
fU * SfU * SfU * SfG * SfC * SfC * SfG * SfC *
UUUGCCGCUGCCCAAUGCCA
SSSSSSSSnXSSS

13812
SmUn001fG * SmC * SfC * SmCn001mAn001fA * SfU *

nXnXSSSSS

SfG * SfC * SfC * SfA

WV-
fU * SfU * SfU * SfG * SfC * SfC * SfG * SfC *
UUUGCCGCUGCCCAAUGCCA
SSSSSSSSnXSSS

13813
SmUn001fG * SmC * SfC * SmCn001fA * SfA * SfU * SfG

nXSSSSSS

* SfC * SfC * SfA

WV-
fU * SfU * SfUn001fG * SfC * SfCn001fG * SfC * SmUfG *
UUUGCCGCUGCCCAAUGCCA
SSnXSSnXSSOSS

13814
SmC * SfC * SmCmAfA * SfU * SfGn001fC * SfC * SfA

SOOSSnXSS

WV-
fU * SfU * SfUn001fG * SfC * SfCn001fG * SfC * SmUfG *
UUUGCCGCUGCCCAAUGCCA
SSnXSSnXSSOSS

13815
SmC * SfC * SmCfA * SfA * SfU * SfGn001fC * SfC * SfA

SOSSSnXSS

WV-
fU * SfU * SfUn001fG * SfC * SfCn001fG * SfC *
UUUGCCGCUGCCCAAUGCCA
SSnXSSnXSSnXSSS

13816
SmUn001fG * SmC * SfC * SmCn001mAn001fA * SfU *

nXnXSSnXSS

SfGn001fC * SfC * SfA

WV-
fU * SfU * SfUn001fG * SfC * SfCn001fG * SfC *
UUUGCCGCUGCCCAAUGCCA
SSnXSSnXSSnXSSS

13817
SmUn001fG * SmC * SfC * SmCn001fA * SfA * SfU
*
nXSSSnXSS

SfGn001fC * SfC * SfA

WV-
fU * SfG * SfC * SfC * SfA * SfU * SfC * SfC * SmUfG *
UGCCAUCCUGGAGUUCCUGU
SSSSSSSSOSSS

13818
SmG * SfA * SmGmUfU * SfC * SfC * SfU * SfG * SfU

OOSSSSS

WV-
fU * SfG * SfC * SfC * SfA * SfU * SfC * SfC * SmUfG *
UGCCAUCCUGGAGUUCCUGU
SSSSSSSSOSSS

13819
SmG * SfA * SmGfU * SfU * SfU * SfC * SfU * SfG * SfU

OSSSSSS

WV
fU * SfG * SfC * SfC * SfA * SfU * SfC * SfC *
UGCCAUCCUGGAGUUCCUGU
SSSSSSSSnXSSS

13820
SmUn001fG * SmG * SfA * SmGn001mUn001fU * SfC *

nXnXSSSSS

SfC * SfU * SfG * SfU

WV-
fU * SfG * SfC * SfC * SfA * SfU * SfC * SfC *
UGCCAUCCUGGAGUUCCUGU
SSSSSSSSnXSSS

13821
SmUn001fG * SmG * SfA * SmGn001fU * SfU * SfC * SfC

nXSSSSSS

* SfU * SfG * SfU

WV-
fU * SfG * SfCn001fC * SfA * SfUn001fC * SfC * SmUfG *
UGCCAUCCUGGAGUUCCUGU
SSnXSSnXSSOSSSO

13822
SmG * SfA * SmGmUfU * SfC * SfCn001fU * SfG * SfU

OSSnXSS

WV-
fU * SfG * SfCn001fC * SfA * SfUn001fC * SfC * SmUfG *
UGCCAUCCUGGAGUUCCUGU
SSnXSSnXSSOSSSO

13823
SmG * SfA * SmGfU * SfU * SfC * SfCn001fU * SfG * SfU

SSSnXSS

WV-
fU * SfG * SfCn001fC * SfA * SfUn001fC * SfC *
UGCCAUCCUGGAGUUCCUGU
SSnXSSnXSSnXSSS

13824
SmUn001fG * SmG * SfA * SmGn001mUn001fU * SfC *

nXnXSSnXSS

SfCn001fU * SfG * Sfu

WV-
fU * SfG * SfCn001fC * SfA * SfUn001fC * SfC *
UGCCAUCCUGGAGUUCCUGU
SSnXSSnXSSnXSSS

13825
SmUn001fG * SmG * SfA * SmGn001fU * SfU * SfC *

nXSSSnXSS

SfCn001fU * SfG * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU * SmG *
UCCGGUUCUGAAGGUGUUC
SSSSSSSOSSS

13826
SfA * SmAmGfG * SfU * SfG * SfU * SfU * SfC

OOSSSSS

WV-
fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUU
SSSSSSSSOSSS

13827
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU

OOSSSS

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU * SmG *
UCCGGUUCUGAAGGUGUU
SSSSSSSOSSS OOSSSS

13828
SfA * SmAmGfG * SfU * SfG * SfU * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU * SmG *
UCCGGUUCUGAAGGUGUUCU
SSSSSSSOSSS

13835
SfA * SmAmGfG * SfU * SfG * SfU * SfU * SfC * SfU

OOSSSSSS

WV-
fC * SfC * SfU * SfC * SfC * SfG * SfG * SfU * SfU *
CCUCCGGUUCUGAAGGUGUU
SSSSSSSSSOSSS

13836
SmCfU * SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU

OOSSSS

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnXSSnXSSOS

13857
SmGn001fA * SmAfG * SfG * SfU * SfGn001fU * SfU *

nXSOSSSnXSS

SfC

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUU
SSnXSSnXSSOSS

13858
SmG * SfA * SmAfG * SfG * SfU * SfGn001fU * SfU

SOSSSnXS

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUU
SSnXSSnXSSOS

13859
SmGn001fA * SmAfG * SfG * SfU * SfGn001fU * SfU

nXSOSSSnXS

WV-
fU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU * SmG
UCCGGUUCUGAAGGUGUUC
SnXSSnXSSOSSSO

13860
* SfA * SmAfG * SfG * SfU * SfGn001fU * SfU * SfC

SSSnXSS

WV-
fU * SfCn001fC * SfG * SfGn001fU * SffU * SmCfU *
UCCGGUUCUGAAGGUGUUC
SnXSSnXSSOSnX

13861
SmGn001fA * SmAfG * SfG * SfU * SfGn001fU * SfU *

SOSSSnXSS

SfC

WV-
fU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU * SmG
UCCGGUUCUGAAGGUGUU
SnXSSnXSSOSSS

13862
* SfA * SmAfG * SfG * SfU * SfGn001fU * SfU

OSSSnXS

WV-
fU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUU
SnXSSnXSSOSnX

13863
SmGn001fA * SmAfG * SfG * SfU * SfGn001fU * SfU

SOSSSnXS

WV-
fC * SfG * SfCn001RfC * SfG * SfGn001RfU * SfU *
CUCCGGUUCUGAAGGUGUUC
SSnRSSnRSSOSS

13864
SmCfU * SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU

SOSSSnRSS

* SfU * SfC

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU *
CUCCGGUUCUGAAGGUGUUC
SSnRSSnRSSOS

13865
SmCfU * SmGn001RfA * SmAfG * SfG * SfU *

nRSOSSSnRSS

SfGn001RfU * SfU * SfC

WV-
fA * SfC * SfA * SfA * SfG * SfU * SmU * SmC * SmU *
ACAAGUUCUCCUUCUGGAAA
SSSSS SSSSS SSSSS

13963
SmC * SmC * SmU * SmU * SmC * SfU * SfG * SfG * SfA

SSSS

* SfA * SfA

WV-
fC * SfU * SfU * SfC * SfU * SfG * SmG * SmA * SmA *
CUUCUGGAAAGGUUCCAACA
SSSSS SSSSS SSSSS

13964
SmA * SmG * SmG * SmU * SmU * SfC * SfC * SfA * SfA

SSSS

* SfC * SfA

WV-
fG * SfG * SfU * SfU * SfC * SfC * SmA * SmA * SmC *
GGUUCCAACAUAAAGCCGAA
SSSSS SSSSS SSSSS

13965
SmA * SmU * SmA * SmA * SmA * SfG * SfC * SfC * SfG

SSSS

* SfA * SfA

WV-
fA * SfA * SfA * SfG * SfC * SfC * SmG * SmA * SmA *
AAAGCCGAAAUACACACUGC
SSSSS SSSSS SSSSS

13966
SmA * SmU * SmA * SmC * SmA * SfC * SfA * SfC * SfU

SSSS

* SfG * SfC

WV-
fA * SfC * SfA * SfC * SfA * SfC * SmU * SmG * SmC *
ACACACUGCCCCAAAGCCAC
SSSSS SSSSS SSSSS

13967
SmC * SmC * SmC * SmA * SmA * SfA * SfG * SfC * SfC

SSSS

* SfA * SfC

WV-
fC * SfA * SfA * SfA * SfG * SfC * SmC * SmA * SmC *
CAAAGCCACAAAACACCUUG
SSSSS SSSSS SSSSS

13968
SmA * SmA * SmA * SmA * SmC * SfA * SfC * SfC * SfU

SSSS

* SfU * SfG

WV-
fA * SfA * SfC * SfA * SfC * SfC * SmU * SmU * SmG *
AACACCUUGCUGUUACGAUG
SSSSS SSSSS SSSSS

13969
SmC * SmU * SmG * SmU * SmU * SfA * SfC * SfG * SfA

SSSS

* SfG * SfG

WV-
fG * SfU * SfU * SfA * SfC * SfG * SmA * SmU * SmG *
GUUACGAUGCUUCCCUCUGU
SSSSS SSSSS SSSSS

13970
SmC * SmU * SmU * SmC * SmC * SfC * SfU * SfC * SfU

SSSS

* SfG * SfU

WV-
fU * SfC * SfC * SfC * SfU * SfC * SmU * SmG * SmU *
UCCCUCUGUCACAGAUUCAA
SSSSS SSSSS SSSSS

13971
SmC * SmA * SmC * SmA * SmG * SfA * SfU * SfU * SfC

SSSS

* SfA * SfA

WV-
fC * SfA * SfG * SfA * SfU * SfU * SmC * SmA * SmA *
CAGAUUCAAUUAUAUUUUGC
SSSSS SSSSS SSSSS

13972
SmU * SmU * SmA * SmU * SmA * SfU * SfU * SfU * SfU

SSSS

* SfA * SfC

WV-
fA * SfU * SfA * SfU * SfU * SfU * SmU * SmG * SmC *
AUAUUUUGCAGUUUAUCAGA
SSSSS SSSSS SSSSS

13973
SmA * SmG * SmU * SmU * SmU * SfA * SfU * SfC * SfA

SSSS

* SfG * SfA

WV-
fU * SfU * SfU * SfA * SfU * SfC * SmA * SmG * SmA *
UUUAUCAGAUAAACCAGCUC
SSSSS SSSSS SSSSS

13974
SmU * SmA * SmA * SmA * SmC * SfC * SfA * SfG * SfC

SSSS

* SfU * SfC

WV-
fA * SfA * SfC * SfC * SfA * SfG * SmC * SmU * SmC *
AACCAGCUCCGUCCAGGCAA
SSSSS SSSSS SSSSS

13975
SmC * SmG * SmU * SmC * SmC * SfA * SfG * SfG * SfC

SSSS

* SfA * SfA

WV-
fU * SfC * SfC * SfA * SfG * SfG * SmC * SmA * SmA *
UCCAGGCAAACUCUCUCAUC
SSSSS SSSSS SSSSS

13976
SmA * SmC * SmU * SmC * SmU * SfC * SfU * SfC * SfA

SSSS

* SfU * SfC

WV-
fU * SfC * SfU * SfC * SfU * SfC * SmA * SmU * SmC *
UCUCUCAUCCUGACACAAAA
SSSSS SSSSS SSSSS

13977
SmC * SmU * SmG * SmA * SmC * SfA * SfC * SfA * SfA

SSSS

* SfA * SfA

WV-
fG * SfA * SfC * SfA * SfC * SfA * SmA * SmA * SmA *
GACACAAAAAGUCCAUAGCA
SSSSS SSSSS SSSSS

13978
SmA * SmG * SmU * SmC * SmC * SfA * SfU * SfA * SfG

SSSS

* SfC * SfA

WV-
fU * SfC * SfC * SfA * SfU * SfA * SmG * SmC * SmA *
UCCAUAGCACCGUGCUCUAA
SSSSS SSSSS SSSSS

13979
SmC * SmC * SmG * SmU * SmG * SfC * SfU * SfC * SfU

SSSS

* SfA * SfA

WV-
fG * SfU * SfG * SfC * SfU * SfC * SmU * SmA * SmA *
GUGCUCUAAUAUUAUCAUUA
SSSSS SSSSS SSSSS

13980
SmU * SmA * SmU * SmU * SmA * SfU * SfC * SfA * SfU

SSSS

* SfU * SfA

WV-
fU * SfU * SfA * SfU * SfC * SfA * SmU * SmU * SmA *
UUAUCAUUAUGAUAAUUUUC
SSSSS SSSSS SSSSS

13981
SmU * SmG * SmA * SmU * SmA * SfA * SfU * SfU * SfU

SSSS

* SfU * SfC

WV-
fA * SfU * SfA * SfA * SfU * SfU * SmU * SmU * SmC *
AUAAUUUUCUUUCUAGUAAU
SSSSS SSSSS SSSSS

13982
SmU * SmU * SmU * SmC * SmU * SfA * SfG * SfU * SfA

SSSS

* SfA * SfU

WV-
fA * SfA * SfU * SfG * SfA * SfU * SmG * SmA * SmC *
AAUGAUGACAACAACAGUCA
SSSSS SSSSS SSSSS

13983
SmA * SmA * SmC * SmA * SmA * SfC * SfA * SfG * SfU

SSSS

* SfC * SfA

WV-
fC * SfA * SfA * SfC * SfA * SfG * SmU * SmC * SmA *
CAACAGUCAAAAGUAAUUUC
SSSSS SSSSS SSSSS

13984
SmA * SmA * SmA * SmG * SmU * SfA * SfA * SfU * SfU

SSSS

* SfU * SfC

WV-
fA * SfG * SfU * SfA * SfA * SfU * SmU * SmU * SmC *
AGUAAUUUCCAUCACCCUUC
SSSSS SSSSS SSSSS

13985
SmC * SmA * SmU * SmC * SmA * SfC * SfC * SfC * SfU

SSSS

* SfU * SfC

WV-
fU * SfC * SfA * SfC * SfC * SfC * SmU * SmU * SmC *
UCACCCUUCAGAACCUGAUC
SSSSS SSSSS SSSSS

13986
SmA * SmG * SmA * SmA * SmC * SfC * SfU * SfG * SfA

SSSS

* SfU * SfC

WV-
fA * SfA * SfC * SfC * SfU * SfG * SmA * SmU * SmC *
AACCUGAUCUUUAAGAAGUU
SSSSS SSSSS SSSSS

13987
SmU * SmU * SmU * SmA * SmA * SfG * SfA * SfA * SfG

SSSS

* SfU * SfU

WV-
fU * SfA * SfA * SfG * SfA * SfA * SmG * SmU * SmU *
UAAGAAGUUAAAGAGUCCAG
SSSSS SSSSS SSSSS

13988
SmA * SmA * SmA * SmG * SmA * SfG * SfU * SfC * SfC

SSSS

* SfA * SfG

WV-
fA * SfG * SfA * SfG * SfU * SfC * SmC * SmA * SmG *
AGAGUCCAGAUGUGCUGAAG
SSSSS SSSSS SSSSS

13989
SmA * SmU * SmG * SmU * SmG * SfC * SfU * SfG * SfA

SSSS

* SfA * SfG

WV-
fG * SfU * SfG * SfC * SfU * SfG * SmA * SmA * SmG *
GUGCUGAAGAUAAAUACAAU
SSSSS SSSSS SSSSS

13990
SmA * SmU * SmA * SmA * SmA * SfU * SfA * SfC * SfA

SSSS

* SfA * SfU

WV-
fU * SfA * SfA * SfA * SfU * SfA * SmC * SmA * SmA *
UAAAUACAAUUUCGAAAAAA
SSSSS SSSSS SSSSS

13991
SmU * SmU * SmU * SmC * SmG * SfA * SfA * SfA * SfA

SSSS

* SfA * SfA

WV-
fA * SfC * SfA * SfA * SfU * SfU * SmU * SmC * SmG *
ACAAUUUCGAAAAAACAAAU
SSSSS SSSSS SSSSS

13992
SmA * SmA * SmA * SmA * SmA * SfA * SfC * SfA * SfA

SSSS

* SfA * SfU

WV-
fU * SfC * SfG * SfA * SfA * SfA * SmA * SmA * SmA *
UCGAAAAAACAAAUCAAAGA
SSSSS SSSSS SSSSS

13993
SmC * SmA * SmA * SmA * SmU * SfC * SfA * SfA * SfA

SSSS

* SfG * SfA

WV-
fA * SfA * SfA * SfC * SfA * SfA * SmA * SmU * SmC *
AAACAAAUCAAAGACUUACC
SSSSS SSSSS SSSSS

13994
SmA * SmA * SmA * SmG * SmA * SfC * SfU * SfU * SfA

SSSS

* SfC * SfC

WV-
fA * SfU * SfC * SfA * SfA * SfA * SmG * SmA * SmC *
AUCAAAGACUUACCUUAAGA
SSSSS SSSSS SSSSS

13995
SmU * SmU * SmA * SmC * SmC * SfU * SfU * SfA * SfA

SSSS

* SfG * SfA

WV-
fG * SfA * SfC * SfU * SfU * SfA * SmC * SmC * SmU *
GACUUACCUUAAGAUACCAU
SSSSS SSSSS SSSSS

13996
SmU * SmA * SmA * SmG * SmA * SfU * SfA * SfC * SfC

SSSS

* SfA * SfU

WV-
fU * SfU * SfA * SfC * SfC * SfU * SmU * SmA * SmA *
UUACCUUAAGAUACCAUUUG
SSSSS SSSSS SSSSS

13997
SmG * SmA * SmU * SmA * SmC * SfC * SfA * SfU * SfU

SSSS

* SfU * SfG

WV-
fU * SfA * SfC * SfC * SfU * SfU * SmA * SmA * SmG *
UACCUUAAGAUACCAUUUGU
SSSSS SSSSS SSSSS

13998
SmA * SmU * SmA * SmC * SmC * SfA * SfU * SfU * SfU

SSSS

* SfG * SfU

WV-
fA * SfC * SfC * SfU * SfU * SfA * SmA * SmG * SmA *
ACCUUAAGAUACCAUUUGUA
SSSSS SSSSS SSSSS

13999
SmU * SmA * SmC * SmC * SmA * SfU * SfU * SfU * SfG

SSSS

* SfU * SfA

WV-
fC * SfC * SfU * SfU * SfA * SfA * SmG * SmA * SmU *
CCUUAAGAUACCAUUUGUAU
SSSSS SSSSS SSSSS

14000
SmA * SmC * SmC * SmA * SmU * SfU * SfU * SfG * SfU

SSSS

* SfA * SfU

WV-
fG * SfA * SfU * SfA * SfC * SfC * SmA * SmU * SmU*
GAUACCAUUUGUAUUUAGCA
SSSSS SSSSS SSSSS

14001
SmU * SmG * SmU * SmA * SmU * SfU * SfU * SfA * SfG

SSSS

* SfC * SfA

WV-
fA * SfU * SfU * SfU * SfG * SfU * SmA * SmU * SmU *
AUUUGUAUUUAGCAUGUUCC
SSSSS SSSSS SSSSS

14002
SmU * SmA * SmG * SmC * SmA * SfU * SfG * SfU * SfU

SSSS

* SfC * SfC

WV-
fA * SfU * SfU * SfU * SfA * SfG * SmC * SmA * SmU *
AUUUAGCAUGUUCCCAAUUC
SSSSS SSSSS SSSSS

14003
SmG * SmU * SmU * SmC * SmC * SfC * SfA * SfA * SfU

SSSS

* SfU * SfC

WV-
fC * SfA * SfU * SfG * SfU * SfU * SmC * SmC * SmC *
CAUGUUCCCAAUUCUCAGGA
SSSSS SSSSS SSSSS

14004
SmA * SmA * SmU * SmU * SmC * SfU * SfC * SfA * SfG

SSSS

* SfG * SfA

WV-
fC * SfC * SfC * SfA * SfA * SfU * SmU * SmC * SmU *
CCCAAUUCUCAGGAAUUUGU
SSSSS SSSSS SSSSS

14005
SmC * SmA * SmG * SmG * SmA * SfA * SfU * SfU * SfU

SSSS

* SfG * SfU

WV-
fU * SfC * SfU * SfC * SfA * SfG * SmG * SmA * SmA *
UCUCAGGAAUUUGUGUCUUU
SSSSS SSSSS SSSSS

14006
SmU * SmU * SmU * SmG * SmU * SfG * SfU * SfC * SfU

SSSS

* SfU * SfU

WV-
fG * SfA * SfA * SfU * SfU * SfU * SmG * SmU * SmG *
GAAUUUGUGUCUUUCUGAGA
SSSSS SSSSS SSSSS

14007
SmU * SmC * SmU * SmU * SmU * SfC * SfU * SfG * SfA

SSSS

* SfG * SfA

WV-
fG * SfU * SfG * SfU * SfC * SfU * SmU * SmU * SmC *
GUGUCUUUCUGAGAAACUGU
SSSSS SSSSS SSSSS

14008
SmU * SmG * SmA * SmG * SmA * SfA * SfA * SfC * SfU

SSSS

* SfG * SfU

WV-
fU * SfU * SfC * SfU * SfG * SfA * SmG * SmA * SmA *
UUCUGAGAAACUGUUCAGCU
SSSSS SSSSS SSSSS

14009
SmA * SmC * SmU * SmG * SmU * SfU * SfC * SfA * SfG

SSSS

* SfC * SfU

WV-
fG * SfA * SfA * SfA * SfC * SfU * SmG * SmU * SmU *
GAAACUGUUCAGCUUCUGUU
SSSSS SSSSS SSSSS

14010
SmC * SmA * SmG * SmC * SmU * SfU * SfC * SfU * SfG

SSSS

* SfU * SfU

WV-
fG * SfU * SfU * SfC * SfA * SfG * SmC * SmU * SmU *
GUUCAGCUUCUGUUAGCCAC
SSSSS SSSSS SSSSS

14011
SmC * SmU * SmG * SmU * SmU * SfA * SfG * SfC * SfC

SSSS

* SfA * SfC

WV-
fC * SfU * SfU * SfC * SfU * SfG * SmU * SmU * SmA *
CUUCUGUUAGCCACUGAUUA
SSSSS SSSSS SSSSS

14012
SmG * SmC * SmC * SmA * SmC * SfU * SfG * SfA * SfU

SSSS

* SfU * SfA

WV-
fU * SfU * SfA * SfG * SfC * SfC * SmA * SmC * SmU *
UUAGCCACUGAUUAAAUAUC
SSSSS SSSSS SSSSS

14013
SmG * SmA * SmU * SmU * SmA * SfA * SfA * SfU * SfA

SSSS

* SfU * SfC

WV-
fA * SfC * SfU * SfG * SfA * SfU * SmU * SmA * SmA *
ACUGAUUAAAUAUCUUUAUA
SSSSS SSSSS SSSSS

14014
SmA * SmU * SmA * SmU * SmC * SfU * SfU * SfU * SfA

SSSS

* SfU * SfA

WV-
fA * SfU * SfC * SfU * SfU * SfU * SmA * SmU * SmA *
AUCUUUAUAUCAUAAUGAAA
SSSSS SSSSS SSSSS

14015
SmU * SmC * SmA * SmU * SmA * SfA * SfU * SfG * SfA

SSSS

* SfA * SfA

WV-
fA * SfU * SfA * SfA * SfU * SfG * SmA * SmA * SmA *
AUAAUGAAAACGCCGCCAUU
SSSSS SSSSS SSSSS

14016
SmA * SmC * SmG * SmC * SmC * SfG * SfC * SfC * SfA

SSSS

* SfU * SfU

WV-
fG * SfC * SfC * SfG * SfC * SfC * SmA * SmU * SmU *
GCCGCCAUUUCUCAACAGAU
SSSSS SSSSS SSSSS

14017
SmU * SmC * SmU * SmC * SmA * SfA * SfC * SfA * SfG

SSSS

* SfA * SfU

WV-
fU * SfC * SfA * SfA * SfC * SfA * SmG * SmA * SmU *
UCAACAGAUCUGUCAAAUCG
SSSSS SSSSS SSSSS

14018
SmC * SmU * SmG * SmU * SmC * SfA * SfA * SfA * SfU

SSSS

* SfC * SfG

WV-
fU * SfG * SfA * SfA * SfG * SfA * SmU * SmA * SmA *
UGAAGAUAAAUACAAUUUCG
SSSSS SSSSS SSSSS

14019
SmA * SmU * SmA * SmC * SmA * SfA * SfU * SfU * SfU

SSSS

* SfC * SfG

WV-
fA * SfU * SfU * SfU * SfC * SfG * SmA * SmA * SmA *
AUUUCGAAAAAACAAAUCAA
SSSSS SSSSS SSSSS

14020
SmA * SmA * SmA * SmC * SmA * SfA * SfA * SfU * SfC

SSSS

* SfA * SfA

WV-
fA * SfA * SfA * SfA * SfA * SfA * SmC * SmA * SmA *
AAAAAACAAAUCAAAGACUU
SSSSS SSSSS SSSSS

14021
SmA * SmU * SmC * SmA * SmA * SfA * SfG * SfA * SfC

SSSS

* SfU * SfU

WV-
fC * SfA * SfA * SfA * SfU * SfC * SmA * SmA * SmA *
CAAAUCAAAGACUUACCUUA
SSSSS SSSSS SSSSS

14022
SmG * SmA * SmC * SmU * SmU * SfA * SfC * SfC * SfU

SSSS

* SfU * SfA

WV-
fA * SfA * SfA * SfG * SfA * SfC * SmU * SmU * SmA *
AAAGACUUACCUUAAGAUAC
SSSSS SSSSS SSSSS

14023
SmC * SmC * SmU * SmU * SmA * SfA * SfG * SfA * SfU

SSSS

* SfA * SfC

WV-
fU * SfA * SfA * SfG * SfA * SfU * SmA * SmC * SmC *
UAAGAUACCAUUUGUAUUUA
SSSSS SSSSS SSSSS

14024
SmA * SmU * SmU * SmU * SmG * SfU * SfA * SfU * SfU

SSSS

* SfU * SfA

WV-
fA * SfC * SfC * SfA * SfU * SfU * SmU * SmG * SmU *
ACCAUUUGUAUUUAGCAUGU
SSSSS SSSSS SSSSS

14025
SmA * SmU * SmU * SmU * SmA * SfG * SfC * SfA * SfU

SSSS

* SfG * SfU

WV-
fU * SfG * SfU * SfA * SfU * SfU * SmU * SmA * SmG *
UGUAUUUAGCAUGUUCCCAA
SSSSS SSSSS SSSSS

14026
SmC * SmA * SmU * SmG * SmU * SfU * SfC * SfC * SfC

SSSS

* SfA * SfA

WV-
fU * SfG * SfC * SfU * SfG * SfA * SmA * SmG * SmA *
UGCUGAAGAUAAAUACAA
SSSSS SSSSS SSSSS SS

14027
SmU * SmA * SmA * SfA * SfU * SfA * SfC * SfA * SfA

WV-
fA * SfA * SfA * SfU * SfA * SfC * SmA * SmA * SmU *
AAAUACAAUUUCGAAAAA
SSSSS SSSSS SSSSS SS

14028
SmU * SmU * SmC * SfG * SfA * SfA * SfA * SfA * SfA

WV-
fC * SfA * SfA * SfU * SfU * SfU * SmC * SmG * SmA *
CAAUUUCGAAAAAACAAA
SSSSS SSSSS SSSSS SS

14029
SmA * SmA * SmA * SfA * SfA * SfC * SfA * SfA * SfA

WV-
fC * SfG * SfA * SfA * SfA * SfA * SmA * SmA * SmC *
CGAAAAAACAAAUCAAAG
SSSSS SSSSS SSSSS SS

14030
SmA * SmA * SmA * SfU * SfC * SfA * SfA * SfA * SfG

WV-
fA * SfA * SfC * SfA * SfA * SfA * SmU * SmC * SmA *
AACAAAUCAAAGACUUAC
SSSSS SSSSS SSSSS SS

14031
SmA * SmA * SmG * SfA * SfC * SfU * SfU * SfA * SfC

WV-
fU * SfC * SfA * SfA * SfA * SfG * SmA * SmC * SmU *
UCAAAGACUUACCUUAAG
SSSSS SSSSS SSSSS SS

14032
SmU * SmA * SmC * SfC * SfU * SfU * SfA * SfA * SfG

WV-
fA * SfC * SfU * SfU * SfA * SfC * SmC * SmU * SmU *
ACUUACCUUAAGAUACCA
SSSSS SSSSS SSSSS SS

14033
SmA * SmA * SmG * SfA * SfU * SfA * SfC * SfC * SfA

WV-
fU * SfA * SfC * SfC * SfU * SfU * SmA * SmA * SmG *
UACCUUAAGAUACCAUUU
SSSSS SSSSS SSSSS SS

14034
SmA * SmU * SmA * SfC * SfC * SfA * SfU * SfU * SfU

WV-
fA * SfC * SfC * SfU * SfU * SfA * SmA * SmG * SmA *
ACCUUAAGAUACCAUUUG
SSSSS SSSSS SSSSS SS

14035
SmU * SmA * SmC * SfC * SfA * SfU * SfU * SfU * SfG

WV-
fC * SfC * SfU * SfU * SfA * SfA * SmG * SmA * SmU *
CCUUAAGAUACCAUUUGU
SSSSS SSSSS SSSSS SS

14036
SmA * SmC * SmC * SfA * SfU * SfU * SfU * SfG * SfU

WV-
fC * SfU * SfU * SfA * SfA * SfG * SmA * SmU * SmA *
CUUAAGAUACCAUUUGUA
SSSSS SSSSS SSSSS SS

14037
SmC * SmC * SmA * SfU * SfU * SfU * SfG * SfU * SfA

WV-
fA * SfU * SfA * SfC * SfC * SfA * SmU * SmU * SmU *
AUACCAUUUGUAUUUAGC
SSSSS SSSSS SSSSS SS

14038
SmG * SmU * SmA * SfU * SfU * SfU * SfA * SfG * SfC

WV-
fU * SfU * SfU * SfG * SfU * SfA * SmU * SmU * SmU *
UUUGUAUUUAGCAUGUUC
SSSSS SSSSS SSSSS SS

14039
SmA * SmG * SmC * SfA * SfU * SfG * SfU * SfU * SfC

WV-
fU * SfU * SfU * SfA * SfG * SfC * SmA * SmU * SmG *
UUUAGCAUGUUCCCAAUU
SSSSS SSSSS SSSSS SS

14040
SmU * SmU * SmC * SfC * SfC * SfA * SfA * SfU * SfU

WV-
fA * SfU * SfG * SfU * SfU * SfC * SmC * SmC * SmA *
AUGUUCCCAAUUCUCAGG
SSSSS SSSSS SSSSS SS

14041
SmA * SmU * SmU * SfC * SfU * SfC * SfA * SfG * SfG

WV-
fC * SfC * SfA * SfA * SfU * SfU * SmC * SmU * SmC *
CCAAUUCUCAGGAAUUUG
SSSSS SSSSS SSSSS SS

14042
SmA * SmG * SmG * SfA * SfA * SfU * SfU * SfU * SfG

WV-
fC * SfU * SfC * SfA * SfG * SfG * SmA * SmA * SmU *
CUCAGGAAUUUGUGUCUU
SSSSS SSSSS SSSSS SS

14043
SmU * SmU * SmG * SfU * SfG * SfU * SfC * SfU * SfU

WV-
fA * SfA * SfU * SfU * SfU * SfG * SmU * SmG * SmU *
AAUUUGUGUCUUUCUGAG
SSSSS SSSSS SSSSS SS

14044
SmC * SmU * SmU * SfU * SfC * SfU * SfG * SfA * SfG

WV-
fU * SfG * SfU * SfC * SfU * SfU * SmU * SmC * SmU *
UGUCUUUCUGAGAAACUG
SSSSS SSSSS SSSSS SS

14045
SmG * SmA * SmG * SfA * SfA * SfA * SfC * SfU * SfG

WV-
fU * SfC * SfU * SfG * SfA * SfG * SmA * SmA * SmA *
UCUGAGAAACUGUUCAGC
SSSSS SSSSS SSSSS SS

14046
SmC * SmU * SmG * SfU * SfU * SfC * SfA * SfG * SfC

WV-
fA * SfA * SfA * SfC * SfU * SfG * SmU * SmU * SmC *
AAACUGUUCAGCUUCUGU
SSSSS SSSSS SSSSS SS

14047
SmA * SmG * SmC * SfU * SfU * SfC * SfU * SfG * SfU

WV-
fU * SfU * SfC * SfA * SfG * SfC * SmU * SmU * SmC *
UUCAGCUUCUGUUAGCCA
SSSSS SSSSS SSSSS SS

14048
SmU * SmG * SmU * SfU * SfA * SfG * SfC * SfC * SfA

WV-
fU * SfU * SfC * SfU * SfG * SfU * SmU * SmA * SmG *
UUCUGUUAGCCACUGAUU
SSSSS SSSSS SSSSS SS

14049
SmC * SmC * SmA * SfC * SfU * SfG * SfA * SfU * SfU

WV-
fU * SfA * SfG * SfC * SfC * SfA * SmC * SmU * SmG *
UAGCCACUGAUUAAAUAU
SSSSS SSSSS SSSSS SS

14050
SmA * SmU * SmU * SfA * SfA * SfA * SfU * SfA * SfU

WV-
fG * SfA * SfA * SfG * SfA * SfU * SmA * SmA * SmA *
GAAGAUAAAUACAAUUUC
SSSSS SSSSS SSSSS SS

14051
SmU * SmA * SmC * SfA * SfA * SfU * SfU * SfU * SfC

WV-
fU * SfU * SfU * SfC * SfG * SfA * SmA * SmA * SmA *
UUUCGAAAAAACAAAUCA
SSSSS SSSSS SSSSS SS

14052
SmA * SmA * SmC * SfA * SfA * SfA * SfU * SfC * SfA

WV-
fA * SfA * SfA * SfA * SfA * SfC * SmA * SmA * SmA *
AAAAACAAAUCAAAGACU
SSSSS SSSSS SSSSS SS

14053
SmU * SmC * SmA * SfA * SfA * SfG * SfA * SfC * SfU

WV-
fA * SfA * SfA * SfU * SfC * SfA * SmA * SmA * SmG *
AAAUCAAAGACUUACCUU
SSSSS SSSSS SSSSS SS

14054
SmA * SmC * SmU * SfU * SfA * SfC * SfC * SfU * SfU

WV-
fA * SfA * SfG * SfA * SfC * SfU * SmU * SmA * SmC *
AAGACUUACCUUAAGAUA
SSSSS SSSSS SSSSS SS

14055
SmC * SmU * SmU * SfA * SfA * SfG * SfA * SfU * SfA

WV-
fA * SfA * SfG * SfA * SfU * SfA * SmC * SmC * SmA *
AAGAUACCAUUUGUAUUU
SSSSS SSSSS SSSSS SS

14056
SmU * SmU * SmU * SfG * SfU * SfA * SfU * SfU * SfU

WV-
fC * SfC * SfA * SfU * SfU * SfU * SmG * SmU * SmA *
CCAUUUGUAUUUAGCAUG
SSSSS SSSSS SSSSS SS

14057
SmU * SmU * SmU * SfA * SfG * SfC * SfA * SfU * SfG

WV-
fG * SfU * SfA * SfU * SfU * SfU * SmA * SmG * SmC *
GUAUUUAGCAUGUUCCCA
SSSSS SSSSS SSSSS SS

14058
SmA * SmU * SmG * SfU * SfU * SfC * SfC * SfC * SfA

WV-
fA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC *
AGGAAGAUGGCAUUUCU
SSSOSOSS OOSSSSSS

14107
SfA * SfU * SfU * SfU * SfC * SfU

WV-
fG * SfG * SmAfA * SmGmA * SfU * SmGmGfC * SfA *
GGAAGAUGGCAUUUCU
SSOSOSS OOSSSSSS

14108
SfU * SfU * SfU * SfC * SfU

WV-
fG * SmAfA * SmGmA * SfU * SmGmGfC * SfA * SfU *
GAAGAUGGCAUUUCU
SOSOSSO OSSSSSS

14109
SfU * SfU * SfC * SfU

WV-
mAfA * SmGmA * SfU * SmGmGfC * SfA * SfU * SfU *
AAGAUGGCAUUUCU
OSOSSOOSSSSSS

14110
SfU * SfC * SfU

WV-
fA * SmGmA * SfU * SmGmGfC * SfA * SfU * SfU * SfU *
AGAUGGCAUUUCU
SOSSOOSSSSSS

14111
SfC * SfG

WV-
mGmA * SfU * SmGmGfC * SfA * SfU * SfU * SfU * SfC *
GAUGGCAUUUCU
OSSOOSSSSSS

14112
SfU

WV-
mA * SfU * SmGmGfC * SfA * SfU * SfU * SfU * SfC *
AUGGCAUUUCU
SSOOSSSSSS

14113
SfU

WV-
fU * SmGmGfC * SfA * SfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
SOOSSSSSS

14114

WV-
mGmGfC * SfA * SfU * SfU * SfU * SfC * SfU
GGCAUUUCU
OOSSSSSS

14115

WV-
mGfC * SfA * SfU * SfU * SfU * SfC * SfU
GCAUUUCU
OSSSSSS

14116

WV-
fC * SfA * SfU * SfU * SfU * SfC * SfU
CAUUUCU
SSSSSS

14117

WV-
fA * SfU * SfU * SfU * SfC * SfU
AUUUCU
SSSSS

14118

WV-
fU * SfU * SfC * SfU
UUCU
SSS

14119

WV-
fU * SfC * SfU
UCU
SS

14120

WV-
fC * RfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU *
CAAGGAAGAUGGCAUUUCU
RSSSSOSOSS

14121
SmGmGfC * SfA * SfU * SfU * SfU * SfC * SfU

OOSSSSSS

WV-
fA * RfA * SfG * SfG * SmAfA * SmGmA * SfU *
AAGGAAGAUGGCAUUUCU
RSSSOSOSS

14122
SmGmGfC * SfA * SfU * SfU * SfU * SfC * SfU

OOSSSSSS

WV-
fA * RfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC *
AGGAAGAUGGCAUUUCU
RSSOSOSS OOSSSSSS

14123
SfA * SfU * SfU * SfU * SfC * SfU

WV-
fG * RfG * SmAfA * SmGmA * SfU * SmGmGfC * SfA *
GGAAGAUGGCAUUUCU
RSOSOSSOOSSSSSS

14124
SfU * SfU * SfU * SfC * SfU

WV-
fG * RmAfA * SmGmA * SfU * SmGmGfC * SfA * SfU *
GAAGAUGGCAUUUCU
ROSOSSOOSSSSSS

14125
SfU * SfU * SfC * SfU

WV-
fA * RmGmA * SfU * SmGmGfC * SfA * SfU * SfU * SfU *
AGAUGGCAUUUCU
ROSSOOSSSSSS

14126
SfC * SfU

WV-
mA * RfU * SmGmGfC * SfA * SfU * SfU * SfU * SfC *
AUGGCAUUUCU
RSOOSSSSSS

14127
SfU

WV-
fU * RmGmGfC * SfA * SfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
ROOSSSSSS

14128

WV-
fC * RfA * SfU * SfU * SfU * SfC * SfU
CAUUUCU
RSSSSS

14129

WV-
fA * RfU * SfU * SfU * SfC * SfU
AUUUCU
RSSSS

14130

WV-
fU * RfU * SfC * SfU
UUCU
RSS

14131

WV-
fU * RfC * SfU
UCU
RS

14132

WV-
Mod097L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG *
UCACUCAGAUAGUUGAAGCC
OSSSSSSOSSSS

14332
SfA * SmU * SfA * SmGmUfU * SfG * SfA * SfA * SfG *

OOSSSSSS

SfC * SfC

WV-
Mod059L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG *
UCACUCAGAUAGUUGAAGCC
OSSSSSSOSSSS

14333
SfA * SmU * SfA * SmGmUfU * SfG * SfA * SfA * SfG *

OOSSSSSS

SfC * SfC

WV-
Mod070L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG *
UCACUCAGAUAGUUGAAGCC
OSSSSSSOSSSS

14334
SfA * SmU * SfA * SmGmUfU * SfG * SfA * SfA * SfG *

OOSSSSSS

SfC * SfC

WV-
Mod057L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG *
UCACUCAGAUAGUUGAAGCC
OSSSSSSOSSSS

14335
SfA * SmU * SfA * SmGmUfU * SfG * SfA * SfA * SfG *

OOSSSSSS

SfC * SfC

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnXSSnXSSOS

14342
SmG * SfA * SmAfGfG * SfU * SfGn001fU * SfU * SfC

SSOOSSnXSS

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnXSSnXSSOS

14343
SmGn001fA * SmAfGfG * SfU * SfGn001fU * SfU * SfC

nXSOOSSnXSS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU *
CUCCGGUUCUGAAGGUGUUC
SSnRSSnRSSOS

14344
SmCfU * SmG * SfA * SmAfGfG * SfU * SfGn001RfU *

SSOOSSnRSS

SfU * SfC

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001fU * SfU *
CUCCGGUUCUGAAGGUGUUC
SSnRSSnRSSOS

14345
SmCfU * SmGn001RfA * SmAfGfG * SfU * SfGn001RfU *

nRSOOSSnRSS

SfU * SfC

WV-
Mod098L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG *
UCACUCAGAUAGUUGAAGCC
OSSSSSSOSSSS

14346
SfA * SmU * SfA * SmGmUfU * SfG * SfA * SfA * SfG *

OOSSSSSS

SfC * SfC

WV-
Mod099L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG *
UCACUCAGAUAGUUGAAGCC
OSSSSSSOSSSS

14347
SfA * SmU * SfA * SmGmUfU * SfG * SfA * SfA * SfG *

OOSSSSSS

SfC * SfC

WV-
Mod100L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG *
UCACUCAGAUAGUUGAAGCC
OSSSSSSOSSSS

14348
SfA * SmU * SfA * SmGmUfU * SfG * SfA * SfA * SfG *

OOSSSSSS

SfC * SfC

WV-
fU * SfC * SfAn001fA * SfG * SfGn001mAfA * SmGmA *
UCAAGGAAGAUGGCAUUUCU
SSnXSSnXOSOS

14522
SfU * SmGmGfC * SfA * SfU * SfUn001fU * SfC * SfU

SOOSSSnXSS

WV-
fU * SfC * SfAn001fA * SfG * SfGn001mAfA * SmGmA *
UCAAGGAAGAUGGCAUUUCU
SSnXSSnXOSOS

14523
SfU * SmGmGfCn001fA * SfU * SfUn001fU * SfC * SfU

SOOnXSSnXSS

WV-
fU * SfU * SfU * SfG * SfC * SfC * SmGfC * SmUmG *
UUUGCCGCUGCCCAAUGCCA
SSSSSSOSOSS

14524
SfC * SmCmCmA * SfA * SfU * SfG * SfC * SfC * SfA

OOSSSSSS

WV-
fU * SfU * SfUn001fG * SfC * SfCn001mGfC * SmUmG *
UUUGCCGCUGCCCAAUGCCA
SSnXSSnXOSOSS

14525
SfC * SmCmCmA * SfA * SfU * SfGn001fC * SfC * SfA

OOSSSnXSS

WV-
fU * SfU * SfUn001fG * SfC * SfCn001mGfC * SmUmG *
UUUGCCGCUGCCCAAUGCCA
SSnXSSnXOSOSS

14526
SfC * SmCmCmAn001fA * SfU * SfGn001fC * SfC * SfA

OOnXSSnXSS

WV-
fU * SfG * SfC * SfC * SfA * SfU * SmCfC * SmUmG
*UGCCAUCCUGGAGUUCCUGU
SSSSSSOSOSS

14527
SfG * SmAmGfU * SfU * SfC * SfC * SfU * SfG * SfU

OOSSSSSS

WV-
fU * SfG * SfCn001fC * SfA * SfUn001mCfC * SmUmG *
UGCCAUCCUGGAGUUCCUGU
SSnXSSnXOSOS

14528
SfG * SmAmGfU * SfU * SfC * SfCn001fU * SfG * SfU

SOOSSSnXSS

WV-
fU * SfG * SfCn001fC * SfA * SfUn001mCfC * SmUmG *
UGCCAUCCUGGAGUUCCUGU
SSnXSSnXOSOS

14529
SfG * SmAmGfUn001fU * SfC * SfCn001fU * SfG * SfU

SOOnXSSnXSS

WV-
fU * SfC * SfAn001fC * SfU * SfCn001mAfG * SfA * SmU
UCACUCAGAUAGUUGAAGCC
SSnXSSnXOSSSS

14530
* SfA * SmGmUfUn001fG * SfA * SfAn001fG * SfC * SfC

OOnXSSnXSS

WV-
fU * SfU * SfU * SfG * SfC * SfC * SmGfC * SmUmG
*UUUGCCGCUGCCCAAUGCCA
SSSSSSOSOSS

14531
SfC * SmCmCfA * SfA * SfU * SfG * SfC * SfC * SfA

OOSSSSSS

WV-
fU * SfU * SfUn001fG * SfC * SfCn001mGfC * SmUmG *
UUUGCCGCUGCCCAAUGCCA
SSnXSSnXOSOSS

14532
SfC * SmCmCfA * SfA * SfU * SfGn001fC * SfC * SfA

OOSSSnXSS

WV-
fU * SfU * SfUn001fG * SfC * SfCn001mGfC * SmUmG *
UUUGCCGCUGCCCAAUGCCA
SSnXSSnXOSOSS

14533
SfC * SmCmCfAn001fA * SfU * SfGn001fC * SfC * SfA

OOnXSSnXSS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU *
CUCCGGUUCUGAAGGUGUU
SSnRSSnRSSOSSS

14565
SmCfU * SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU

OSSSnRS

* SfU

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU *
CUCCGGUUCUGAAGGUGUU
SSnRSSnRSSOSS

14566
SmCfU * SmG * SfA * SmAfGfG * SfU * SfGn001RfU *

SOOSSnRS

SfU

WV-
fU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SnRSSnRSSOS

14773
SmAmGfG * SfU * SfGn001RfU * SfU * SfC * SfU
AGGUGUUCU
SSOOSSnRSSS

WV-
fU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SnRSSnRSSOS

14774
SmAmGfG * SfUn001RfG * SfU * SfUn001RfC * SfU
AGGUGUUCU
SSOOSnRSSnRS

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SSSSSSSOSSS

14775
SmAfGfG * SfU * SfG * SfU * SfU * SfC * SfU
AGGUGUUCU
OOSSSSSS

WV-
fU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SnRSSnRSSOS

14776
SmAfGfG * SfU * SfGn001RfU * SfU * SfC * SfU
AGGUGUUCU
SSOOSSnRSSS

WV-
fU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SnRSSnRSSOS

14777
SmAfGfG * SfUn001RfG * SfU * SfUn001RfC * SfU
AGGUGUUCU
SSOOSnRSSnRS

WV-
fU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SnRSSnRSSOS

14778
SmAmGfG * SfU * SfG * SfUn001RfU * SfC * SfU
AGGUGUUCU
SSOOSSSnRSS

WV-
fU * SfC * SfCn001RfG * SfG * SfUn001RfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SSnRSSnRSO

14779
SmAmGfG * SfU * SfG * SfUn001RfU * SfC * SfU
AGGUGUUCU
SSSOOSSSnRSS

WV-
fU * SfC * SfCn001RfG * SfG * SfUn001RfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SSnRSSnRSO

14790
SmAmGfG * SfU * SfGn001fU * SfU * SfC * SfU
AGGUGUUCU
SSSOOSSnXSSS

WV-
fU * SfC * SfCn001RfG * SfG * SfUn001RfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SSnRSSnRSO

14791
SmAmGfG * SfU * SfGn001RfU * SfU * SfC * SfU
AGGUGUUCU
SSSOOSSnRSSS

WV-
BrfU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA * SmU * SfA *
UCACUCAGAUA
SSSSSSnXSSSS

15052
SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
nXnXSSSSSS

WV-
Acet5fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA * SmU *
UCACUCAGAUA
SSSSSSnXSSSS

15053
SfA * SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
nXnXSSSSSS

WV-
Mod102L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU *
UCACUCAGAUA
OSSSSSSOSSS

15074
SfA * SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SOOSSSSSS

WV-
Mod103L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU *
UCACUCAGAUA
OSSSSSSOSSS

15075
SfA * SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SOOSSSSSS

WV-
Mod104L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU *
UCACUCAGAUA
OSSSSSSOSSS

15076
SfA * SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SOOSSSSSS

WV-
fC * SfU * SfCn001SfC * SfG * SfGn001RfU * SfU * SmCfU * SmG *
CUCCGGUUCUGA
SSnSSSnRSSOS

15143
SfA * SmAfGfG * SfU * SfGn001RfU * SfU * SfC
AGGUGUUC
SSOOSSnRSS

WV-
fC * SfU * SfCn001SfC * SfG * SfGn001SfU * SfU * SmCfU * SmG *
CUCCGGUUCUGA
SSnSSSnSSSOSSS

15322
SfA * SmAfGfG * SfU * SfGn001SfU * SfU * SfC
AGGUGUUC
OOSSnSSS

WV-
fC * fU * fCn001SfC * fG * fGn001SfU * fU * mCfU * mG * fA *
CUCCGGUUCUGA
XXnSXXnSXXO

15323
mAfGfG * fU * fGn001SfU * fU * fC
AGGUGUUC
XXXOOXXnSXX

WV-
fC * fU * fCn001RfC * fG * fGn001RfU * fU * mCfU * mG * fA *
CUCCGGUUCUGA
XXnRXXnRXXO

15324
mAfGfG * fU * fGn001RfU * fU * fC
AGGUGUUC
XXXOOXXnRXX

WV-
fC * fU * fCn001fC * fG * fGn001fU * fU * mCfU * mG * fA * mAfGfG
CUCCGGUUCUGA
XXnXXXnXXXO

15325
* fU * fGn001fU * fU * fC
AGGUGUUC
XXXOOXXnXXX

WV-
fU * SfC * SfCn001SfG * SfG * SfUn001SfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SSnSSSnSSOSSS

15326
SmAmGfG * SfU * SfGn001SfU * SfU * SfC * SfU
AGGUGUUCU
OOSSnSSSS

WV-
fU * fC * fCn001SfG * fG * fUn001SfU * mCfU * mG * fA * mAmGfG
UCCGGUUCUGA
XXnSXXnSX

15327
* fU * fGn001SfU * fU * fC * fU
AGGUGUUCU
OXXXOOXX nSXXX

WV-
fU * fC * fCn001RfG * fG * fUn001RfU * mCfU * mG * fA * mAmGfG
UCCGGUUCUGA
XXnRXXnRX

15328
* fU * fGn001RfU * fU * fC * fU
AGGUGUUCU
OXXXOOXX nRXXX

WV-
fU * fC * fUn001fG * fG * fUn001fU * mCfU * mG * fA * mAmGfU *
UCCGGUUCUGA
XXnXXXnXXO

15329
fU * fGn001fU * fU * fC * fU
AGGUGUUCU
XXXOOXXnXXXX

WV-
fC * SfU * SfCn001SfC * SfG * SfGn001SfU * SfU * SmCfU * SmG *
CUCCGGUUCUGA
SSnSSSnSSSOSSS

15330
SfA * SmAfG * SfG * SfU * SfGn001SfU * SfU * SfC
AGGUGUUC
OSSSnSSS

WV-
fC * fU * fCn001SfC * fG * fGn001SfU * fU * mCfU * mG * fA * mAfG
CUCCGGUUCUGA
XXnSXXnSXXO

15331
* fG * fU * fGn001SfU * fU * fC
AGGUGUUC
XXXOXXXnSXX

WV-
fC * fU * fCn001RfC * fG * fGn001RfU * fU * mCfU * mG * fA *
CUCCGGUUCUGA
XXnRXXnRXXO

15332
mAfG * fG * fU * fGn001RfU * fU * fC
AGGUGUUC
XXXOXXXnRXX

WV-
fC * fU * fCn001fC * fG * fGn001fU * fU * mCfU * mG * fA * mAfG *
CUCCGGUUCUGA
XXnXXXnXXXO

15333
fG * fU * fGn001fU * fU * fC
AGGUGUUC
XXXOXXXnXXX

WV-
fU * SfC * SfCn001RfG * SfG * SfUn001RfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SSnRSSnRSO

15334
SmAmGfG * SfU * SfG * SfUn001fU * SfC * SfU
AGGUGUUCU
SSSOOSSSnXSS

WV-
fU * SfC * SfCn001SfG * SfG * SfUn001SfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SSnSSSnSSOSSS

15335
SmAmGfG * SfU * SfG * SfUn001SfU * SfC * SfU
AGGUGUUCU
OOSSSnSSS

WV-
L001fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA * SmU *
UCACUCAGAUA
OSSSSSSnXSSSS

15336
SfA * SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
nXnXSSSSSS

WV-
Mod059L001fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA *
UCACUCAGAUA
OSSSSSSnXSSSS

15337
SmU * SfA * SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
nXnXSSSSSS

WV-
Mod098L001fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA *
UCACUCAGAUA
OSSSSSSnX SSSS

15338
SmU * SfA * SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
nXnXSSSSSS

WV-
L001L005fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA * SmU
UCACUCAGAUA
OOSSSSSSnX SSSS

15366
* SfA * SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
nXnXSSSSSS

WV-
Mod1051L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU *
UCACUCAGAUA
OSSSSSSOSSS

15367
SfA * SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SOOSSSSSS

WV-
Mod074L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU *
UCACUCAGAUA
OSSSSSSOSSS

15368
SfA * SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SOOSSSSSS

WV-
fU * SfC * SfCn001RfG * SfG * SfUn001RfU * SmCfU * SmG * SfA *
UCCGGUUCUGA
SSnRSSnRSO

15369
SmAmGfG * SfU * SfG * SfU * SfU * SfC * SfU
AGGUGUUCU
SSSOOSSSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SfA * SmGfA * SmU * SfA *
UCACUCAGAUA
SSSSSSSOSSS

15588
SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
OOSSSSSS

WV-
fU * SfU * SfAn001fC * SfU * SfCn001fA * SmGfA * SmU * SfA *
UCACUCAGAUA
SSnXSSnXSOSS

15589
SmGmUfU * SfG * SfA * SfAn001fG * SfC * SfC
GUUGAAGCC
SOOSSSnXSS

WV-
Mod098L001fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
CUCCGGUUCUGA
OSSSSSSSSOSSS

15646
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU * SfC
AGGUGUUC
OOSSSSS

WV-
Mod098L001fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU
CUCCGGUUCUGA
OSSnXSSnXSSOSSS

15647
* SmG * SfA * SmAfG * SfG * SfU * SfGn001fU * SfU * SfC
AGGUGUUC
OSSSnXSS

WV-
Mod106fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA * SmU *
UCACUCAGAUA
SSSSSSnXSSSS

15844
SfA * SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
nXnXSSSSSS

WV-
Mod107fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA * SmU *
UCACUCAGAUA
SSSSSSnXSSSS

15845
SfA * SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
nXnXSSSSSS

WV-
Mod071L001fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA *
UCACUCAGAUA
OSSSSSSnXSSSS

15846
SmU * SfA * SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
nXnXSSSSSS

WV-
L00lfC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU * SmG *
CUCCGGUUCUGA
OSSSSSSSSOSSS

15847
SfA * SmAmGfG * SfU * SfG * SfU * SfU * SfC
AGGUGUUC
OOSSSSS

WV-
Mod071L001fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
CUCCGGUUCUGA
OSSSSSSSSOSSS

15848
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU * SfC
AGGUGUUC
OOSSSSS

WV-
Mod102L001fC * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
CUCCGGUUCUGA
OSSSSSSSSOSSS

15849
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU * SfC
AGGUGUUC
OOSSSSS

WV-
L001fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU * SmG *
CUCCGGUUCUGA
OSSnXSSnXSSOSSS

15850
SfA * SmAfG * SfG * SfU * SfGn001fU * SfU * SfC
AGGUGUUC
OSSSnXSS

WV-
Mod071L001fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU
CUCCGGUUCUGA
OSSnXSSnXSSOSSS

15851
* SmG * SfA * SmAfG * SfG * SfU * SfGn001fU * SfU * SfC
AGGUGUUC
OSSSnXSS

WV-
Mod102L001fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU
CUCCGGUUCUGA
OSSnXSSnXSSOSSS

15852
* SmG * SfA * SmAfG * SfG * SfU * SfGn001fU * SfU * SfC
AGGUGUUC
OSSSnXSS

WV-
fU * SfC * SfAn001fC * SfU * SfC * SfA * SmGfA * SmU * SfA *
UCACUCAGAUA
SSnXSSSS OSSS

15853
SmGmUfUn001fG * SfA * SfAn001fG * SfC * SfC
GUUGAAGCC
OOnXSSnXSS

WV-
fU * SfC * SfAn001fC * SfU * SfCn001fA * SmGfA * SmU * SfA *
UCACUCAGAUA
SSnXSSnXSOSSS

15854
SmGmUfUn001fG * SfA * SfAn001fG * SfC * SfC
GUUGAAGCC
OOnXSSnXSS

WV-
fU * SfC * SfAn001fC * SfU * SfCn001fA * SmGfA * SmU * SfA *
UCACUCAGAUA
SSnXSSnXSOSSS

15855
SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
OOSSSSSS

WV-
fG * SfC * SfA * SfC * SfU * SfC * SfA * SmGfA * SmU * SfA *
UCACUCAGAUA
SSSSSSSOSSS

15856
SmGmUfUn001fG * SfA * SfAn001fG * SfC * SfC
GUUGAAGCC
OOnXSSnXSS

WV-
fU * SfC * SfAn001fC * SfU * SfC * SmAfG * SfA * SmU * SfA *
UCACUCAGAUA
SSnXSSSOSSS

15857
SmGmUfUn001fG * SfA * SfAn001fG * SfC * SfC
GUUGAAGCC
SOOnXSSnXSS

WV-
fU * SfC * SfAn001fC * SfU * SfCn001mAfG * SfA * SmU * SfA *
UCACUCAGAUA
SSnXSSnXOSSSS

15858
SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
OOSSSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU * SfA *
UCACUCAGAUA
SSSSSSOSSS

15859
SmGmUfUn001fG * SfA * SfAn001fG * SfC * SfC
GUUGAAGCC
SOOnXSSnXSS

WV-
fU * SfC * SfAn001fA * SfG * SfG * SmAfA * SmGmA * SfU *
UCAAGGAAGAU
SSnXSSSOSOSSO

15860
SmGmGfCn001fA * SfU * SfUn001fU * SfC * SfU
GGCAUUUCU
OnXSSnXSS

WV-
fU * SfC * SfAn001fA * SfG * SfGn001mAfA * SmGmA * SfU *
UCAAGGAAGAU
SSnXSSnXOSOSS

15861
SmGmGfC * SfA * SfU * SfU * SfU * SfC * SfU
GGCAUUUCU
OOSSSSSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU *
UCAAGGAAGAU
SSSSSSOSOSSOO

15862
SmGmGfCn001fA * SfU * SfUn001fU * SfC * SfU
GGCAUUUCU
nXSSnXSS

WV-
Mod071L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU *
UCACUCAGAUA
O SSSSSSO SSSSOO

15882
SfA * SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SSSSSS

WV-
fC * SfU * SfCn002 RfC * SfG * SfGn002 RfU * SfU * SmCfU * SmG *
CUCCGGUUCUGAAG
SSnR SSnR

15883
SfA * SmAfGfG * SfU * SfGn002 RfG * SfU * SfC
GUGUUC
SSOSSSOOSSnR SS

WV-
mU * SGeon002 m5Ceon002 m5Ceon002 mA * SG * SG * RC * ST *
UGCCAGGCTGG
SnXnXnXSS RSSRSSR

15884
SG * RG * ST * ST * RA * ST * SmG * SmA * SmC * SmU * SmC
TTATGACUC
SSSSSS

WV-
mU * SGeon002 Rm5Ceon002 Rm5Ceon002 RmA * SG * SG * RC * ST
UGCCAGGCTGG
SnRnRnR SSRSSRSSR

15885
* SG * RG * ST * ST * RA * ST * SmG * SmA * SmC * SmU * SmC
TTATGACUC
SSSSSS

WV-
fC * SfU * SfCn002 fC * SfG * SfGn002 fU * SfU * SmCfU * SmG *
CUCCGGUUCUGAAG
SSnXSSnXSSOSSSOOSS

15886
SfA * SmAfGfG * SfU * SfGn002 fU * SfU * SfC
GUGUUC
nXSS

WV-
fCn001 fUn001 fCn001 fCn001 fGn001 fGn001 fUn001 fUn001
CUCCGGUUCUGAAG
nXnXnXnXnX

15912
mCfUn001 mGn001 fAn001 mAfGfGn001 fUn001 fGn001 fUn001
GUGUUC
nXnXnXOnXnXnX

fCn001 fC

OOnXnXnXnXnX

WV-
fCn001 fUn001 fCn001 fCn001 fGn001 fGn001 fUn001 fUn001 mCn001
CUCCGGUUCUGAAG
nXnXnXnXnX nXnX

15913
fUn001 mGn001 fAn001 mAn001 fGn001 fGn001 fUn001 fGn001
GUGUUC
nXnXnX nXnXnXnXnX

fUn001 fUn001 fC

nXnXnXnX

WV-
fA * SfU * SfU * SfU * SfA * SfG * SfC * SfA * SmU * SfG * SmU *
AUUUAGCAUGUU
SSSS SSSS SSSS

15927
SfU * SmC * SfC * SfC * SfA * SfA * SfU * SfU * SfC
CCCAAUUC
SSSSSSS

WV-
fA * SfU * SfUn001 fU * SfA * SfGn001 fC * SfA * SmUn001 fG * SmU
AUUUAGCAUGUU
SSnXSSnXSSnX SSSnX

15928
* SfU * SmCn001 fC * SfC * SfA * SfAn001 fU * SfU * SfC
CCCAAUUC
SSSnXSS

WV-
fA * SfU * SfUn001 fU * SfA * SfGn001 fC * SfA * SmU * SfG * SmU
AUUUAGCAUGUU
SSnXSSnX SSSSSSnX

15929
* SfU * SmCn001 fC * SfC * SfA * SfAn001 fU * SfU * SfC
CCCAAUUC
SSSnXSS

WV-
fA * SfU * SfUn001 fU * SfA * SfGn001 fC * SfA * SmU * SfG * SmU
AUUUAGCAUGUU
SSnXSSnX SSSS

15930
* SfU * SmC * SfC * SfC * SfA * SfAn001 fU * SfU * SfC
CCCAAUUC
SSSSSSnXSS

WV-
fA * SfG * SfU * SfU * SfA * SfUn001 fC * SfA * SmUn001 fG * SmU
AUUUAGCAUGUU
SSSSSnXSSnX SSSnX

15931
* SfU * SmCn001 fC * SfC * SfA * SfA * SfU * SfU * SfC
CCAAUUC
SSSSSS

WV-
fA * SfU * SfUn001 fU * SfA * SfG * SfC * SfA * SmU * SfG * SmU *
AUUUAGCAUGUU
SSnX SSSS SSSSSnX

15932
SfU * SmCn001 fC * SfC * SfA * SfAn001 fU * SfU * SfC
CCCAAUUC
SSSnXSS

WV-
fA * SfU * SfUn001 fU * SfA * SfG * SfC * SfA * SmU * SfG * SmU *
AUUUAGCAUGUU
SSnX SSSS SSSS

15933
SfU * SmC * SfC * SfC * SfA * SfAn001 fU * SfU * SfC
CCCAAUUC
SSSSSnXSS

WV-
fA * SfU * SfUn001 fU * SfA * SfGn001 fC * SfA * SmU * SfG * SmU
AUUUAGCAUGUU
SSnXSSnX SSSS SSSS

15934
* SfU * SmC * SfC * SfC * SfA * SfA * SfU * SfU * SfC
CCCAAUUC
SSSSS

WV-
fA * SfU * SfU * SfU * SfA * SfG * SfC * SfA * SmU * SfG * SmU *
AUUUAGCAUGUU
SSSS SSSS SSSSnX

15935
SfU * SmCn001 fC * SfC * SfA * SfAn001 fU * SfU * SfC
CCCAAUUC
SSSnXSS

WV-
mA * SmU * SmU * SmU * SmA * SmG * SmC * SmA * SmU * SmG *
AUUUAGCAUGUU
SSSS SSSS SSSS

15936
SmU * SmU * SmC * SmC * SmC * SmA * SmA * SmU * SmU * SmC
CCCAAUUC
SSSSSSS

WV-
mA * SmU * SmUn001 mU * SmA * SmGn001 mC * SmA * SmUn001
AUUUAGCAUGUU
SSnXSSnXSSnX SSSnX

15937
mG * SmU * SmU * SmCn001 mC * SmC * SmA * SmAn001 mU *
CCCAAUUC
SSSnXSS

SmU * SmC

WV-
Aeo * STeo * STeo * STeo * SAeo * SGeo * Sm5Ceo * SAeo * STeo *
ATTTAGCATGTT
SSSS SSSS SSSS

15938
SGeo * STeo * STeo * Sm5Ceo * Sm5Ceo * Sm5Ceo * SAeo * SAeo *
CCCAATTC
SSSSSSS

STeo * STeo * Sm5Ceo

WV-
Aeo * STeo * STeon001 Teo * SAeo * SGeon001 m5Ceo * SAeo *
ATTTAGCATGTT
SSnXSSnXSSnX SSSnX

15939
STeon001 Geo * STeo * STeo * Sm5Ceon001 m5Ceo * Sm5Ceo * SAeo
CCCAATTC
SSSnXSS

* SAeon001 Teo * STeo * Sm5Ceo

WV-
fG * SfC * SfAn001 fU * SfG * SfUn001 fU * SfC * SmCn001 fC * SmA
GCAUGUUCCC
SSnXSSnXSSnX SSSnX

15940
* SfA * SmUn001 fU * SfC * SfU * SfCn001 fA * SfG * SfG
AAUUCUCAGG
SSSnXSS

WV-
fA * SfG * SfCn001 fA * SfU * SfGn001 fU * SfU * SmCn001 fC * SmC
AGCAUGUU CC
SSnXSSnXSSnX SSSnX

15941
* SfA * SmAn001 fU * SfU * SfC * SfUn001 fC * SfA * SfG
CAAUUCUCAG
SSSnXSS

WV-
fU * SfA * SfGn001 fC * SfA * SfUn001 fG * SfU * SmUn001 fC * SmC
UAGCAUGUU
SSnXSSnXSSnX SSSnX

15942
* SfC * SmAn001 fA * SfU * SfU * SfCn001 fU * SfC * SfA
CCCAAUUCUCA
SSSnXSS

WV-
fU * SfU * SfAn001 fG * SfC * SfAn001 fU * SfG * SmUn001 fU * SmC
UUAGCAUGUU
SSnXSSnXSSnX SSSnX

15943
* SfC * SmCn001 fA * SfA * SfU * SfUn001 fC * SfU * SfC
CCCAAUUCUC
SSSnXSS

WV-
fU * SfU * SfUn001 fA * SfG * SfCn001 fA * SfU * SmGn001 fU * SmU
UUUAGCAUGUU
SSnXSSnXSSnX SSSnX

15944
* SfC * SmCn001 fC * SfA * SfA * SfUn001 fU * SfC * SfU
CCCAAUUCU
SSSnXSS

WV-
fU * SfA * SfUn001 fU * SfU * SfAn001 fG * SfC * SmAn001 fU * SmG
UAUUUAGCAUGUU
SSnXSSnXSSnX SSSnX

15945
* SfU * SmUn001 fC * SfC * SfC * SfAn001 fA * SfU * SfU
CCCAAUU
SSSnXSS

WV-
fG * SfG * SfAn001 fU * SfU * SfUn001 fA * SfG * SmCn001 fA * SmU
GUAUUUAGCA UGUU
SSnXSSnXSSnX SSSnX

15946
* SfC * SmUn001 fU * SfC * SfC * SfCn001 fA * SfA * SfU
CCCAAU
SSSnXSS

WV-
fU * SfG * SfUn001 fA * SfU * SfUn001 fU * SfA * SmGn001 fC * SmA
UGUAUUUAGCA
SSnXSSnXSSnX SSSnX

15947
* SfU * SmGn001 fU * SfU * SfC * SfCn001 fC * SfA * SfA
UGUU CCCAA
SSSnXSS

WV-
fU * SfU * SfGn001 fU * SfA * SfUn001 fU * SfU * SmAn001 fG * SmC
UUGUAUUUAGCAUGU
SSnXSSnXSSnX SSSnX

15948
* SfA * SmUn001 fG * SfU * SfU * SfCn001 fC * SfC * SfA
U CCCA
SSSnXSS

WV-
fU * SfU * SfUn001 fG * SfU * SfAn001 fU * SfU * SmUn001 fA *
UUUGUAUUU
SSnXSSnXSSnX SSSnX

15949
SmG * SfC * SmAn001 fU * SfG * SfU * SfUn001 fC * SfC * SfC
AGCAUGUU CCC
SSSnXSS

WV-
fG * SfC * SfU * SfG * SfC * SfU * SfC * SfU * SmU * SfU * SmU *
GCUGCUCUUU
SSSS SSSS SSSS

15950
SfC * SmC * SfA * SfG * SfG * SfU * SfU * SfC * SfA
UCCAGGUUCA
SSSSSSS

WV-
fC * SfU * SfU * SfC * SfC * SfU * SfC * SfC * SmA * SfA * SmC *
CUUCCUCCAACCA
SSSS SSSS SSSS

15951
SfC * SmA * SfU * SfA * SfA * SfA * SfA * SfC * SfA
UAAAACA
SSSSSSS

WV-
fA * SfG * SfG * SfU * SfU * SfC * SfA * SfA * SmG * SfU * SmG *
AGGUUCAAGU
SSSS SSSS SSSS

15952
SfG * SmG * SfA * SfU * SfA * SfC * SfU * SfA * SfG
GGGAUACUAG
SSSSSSS

WV-
fG * SfC * SfA * SfC * SfU * SfU * SfA * SfC * SmA * SfA * SmG *
GCACUUACAAG
SSSS SSSS SSSS

15953
SfC * SmA * SfC * SfG * SfG * SfG * SfU * SfC * SfC
CACGGGUCC
SSSSSSS

WV-
fG * SfG * SfC * SfA * SfA * SfC * SfU * SfC * SmU * SfU * SmC *
GGCAACUCUU
SSSS SSSS SSSS

15954
SfC * SmA * SfC * SfC * SfA * SfG * SfU * SfA * SfA
CCACCAGUAA
SSSSSSS

WV-
fG * SfA * SfG * SfU * SfU * SfC * SfU * SfU * SmC * SfC * SmA *
GAGUUCUUCC
SSSS SSSS SSSS

15955
SfA * SmC * SfU * SfG * SfG * SfG * SfG * SfA * SfC
AACUGGGGAC
SSSSSSS

WV-
fG * SfG * SfU * SfA * SfU * SfC * SfA * SfU * SmC * SfU * SmG *
GGUAUCAUCU
SSSS SSSS SSSS

15956
SfC * SmA * SfG * SfA * SfA * SfU * SfA * SfA * SfU
GCAGAAUAAU
SSSSSSS

WV-
fU * SfU * SfU * SfC * SfA * SfG * SfG * SfG * SmC * SfC * SmA *
UUUCAGGGCCA
SSSS SSSS SSSS

15957
SfA * SmG * SfU * SfC * SfA * SfU * SfU * SfU * SfG
AGUCAUUUG
SSSSSSS

WV-
fC * SfC * SfA * SfC * SfA * SfU * SfC * SfU * SmA * SfC * SmA *
CCACAUCUACAU
SSSS SSSS SSSS

15958
SfU * SmU * SfU * SfG * SfU * SfC * SfU * SfG * SfC
UUGUCUGC
SSSSSSS

WV-
fC * SfU * SfU * SfU * SfC * SfC * SfU * SfU * SmA * SfC * SmG *
CUUUCCUUACG
SSSS SSSS SSSS

15959
SfG * SmG * SfU * SfA * SfG * SfC * SfA * SfU * SfC
GGUAGCAUC
SSSSSSS

WV-
fU * SfU * SfC * SfU * SfU * SfC * SfC * SfA * SmA * SfA * SmG *
UUCUUCC
SSSS SSSS SSSS

15960
SfC * SmA * SfG * SfC * SfC * SfU * SfC * SfU * SfC
AAAGCAGCCUCUC
SSSSSSS

WV-
fU * SfC * SfC * SfU * SfG * SfU * SfA * SfG * SmG * SfA * SmC *
UCCUGUAGGA
SSSS SSSS SSSS

15961
SfA * SmU * SfU * SfG * SfG * SfC * SfA * SfG * SfU
CAUUGGCAGU
SSSSSSS

WV-
fG * SfC * SfUn001 fG * SfC * SfUn001 fC * SfU * SmUn001 fU * SmU
GCUGCUCUUU
SSnXSSnXSSnX SSSnX

15962
* SfC * SmCn001 fA * SfG * SfG * SfUn001 fU * SfC * SfA
UCCAGGUUCA
SSSnXSS

WV-
fC * SfU * SfUn001 fC * SfC * SfUn001 fC * SfC * SmAn001 fA * SmC
CUUCCUCCAACCA
SSnXSSnXSSnX SSSnX

15963
* SfC * SmAn001 fU * SfA * SfA * SfAn001 fA * SfC * SfA
UAAAACA
SSSnXSS

WV-
fA * SfG * SfGn001 fU * SfU * SfCn001 fA * SfA * SmGn001 fU * SmG
AGGUUCAAGU
SSnXSSnXSSnX SSSnX

15964
* SfG * SmGn001 fA * SfU * SfA * SfCn001 fU * SfA * SfG
GGGAUACUAG
SSSnXSS

WV-
fG * SfC * SfAn001 fC * SfU * SfUn001 fA * SfC * SmAn001 fA * SmG
GCACUUACAAG
SSnXSSnXSSnX SSSnX

15965
* SfC * SmAn001 fC * SfG * SfG * SfGn001 fU * SfC * SfC
CACGGGUCC
SSSnXSS

WV-
fG * SfG * SfCn001 fA * SfA * SfCn001 fU * SfC * SmUn001 fU * SmC
GGCAACUCUU
SSnXSSnXSSnX SSSnX

15966
* SfC * SmAn001 fC * SfC * SfA * SfGn001 fU * SfA * SfA
CCACCAGUAA
SSSnXSS

WV-
fG * SfA * SfGn001 fU * SfU * SfCn001 fU * SfU * SmCn001 fC * SmA
GAGUUCUUCC
SSnXSSnXSSnX SSSnX

15967
* SfA * SmCn001 fU * SfG * SfG * SfGn001 fG * SfA * SfC
AACUGGGGAC
SSSnXSS

WV-
fG * SfG * SfUn001 fA * SfU * SfCn001 fA * SfU * SmCn001 fU * SmG
GGUAUCAUCU
SSnXSSnXSSnX SSSnX

15968
* SfC * SmAn001 fG * SfA * SfA * SfUn001 fA * SfA * SfU
GCAGAAUAAU
SSSnXSS

WV-
fU * SfU * SfUn001 fC * SfA * SfGn001 fG * SfG * SmCn001 fC * SmA
UUUCAGGGCCA
SSnXSSnXSSnX SSSnX

15969
* SfA * SmGn001 fU * SfC * SfA * SfUn001 fU * SfU * SfG
AGUCAUUUG
SSSnXSS

WV-
fC * SfC * SfAn001 fC * SfA * SfUn001 fC * SfU * SmAn001 fC * SmA
CCACAUCUACAU
SSnXSSnXSSnX SSSnX

15970
* SfU * SmUn001 fU * SfG * SfU * SfCn001 fU * SfG * SfC
UUGUCUGC
SSSnXSS

WV-
fC * SfU * SfUn001 fU * SfC * SfCn001 fU * SfU * SmAn001 fC * SmG
CUUUCCUUACG
SSnXSSnXSSnX SSSnX

15971
* SfG * SmGn001 fU * SfA * SfG * SfCn001 fA * SfU * SfC
GGUAGCAUC
SSSnXSS

WV-
fU * SfU * SfCn001 fU * SfU * SfCn001 fC * SfA * SmAn001 fA * SmG
UUCUUCC
SSnXSSnXSSnX SSSnX

15972
* SfC * SmAn001 fG * SfC * SfC * SfUn001 fC * SfU * SfC
AAAGCAGCCUCUC
SSSnXSS

WV-
fU * SfC * SfCn001 fU * SfG * SfUn001 fA * SfG * SmGn001 fA * SmC
UCCUGUAGGA
SSnXSSnXSSnX SSSnX

15973
* SfA * SmUn001 fU * SfG * SfG * SfCn001 fA * SfG * SfU
CAUUGGCAGU
SSSnXSS

WV-
L00lfC * SfU * SfCn001 RfC * SfG * SfGn001 RfU * SfU * SmCfU *
CUCCGGUUCUGAAG
OSSnR SSnR

16004
SmG * SfA * SmAfGfG * SfU * SfGn001 RfU * SfU * SfC
GUGUUC
SSOSSSOOSSnR SS

WV-
Mod071L001fC * SfU * SfCn001 RfC * SfG * SfGn001 RfU * SfU *
CUCCGGUUCUGAAG
OSSnR SSnR

16005
SmCfU * SmG * SfA * SmAfGfG * SfU * SfCn001 RfU * SfU * SfC
GUGUC
SSOSSSOOSSnR SS

WV-
fC * SfU * SfCn003RfC * SfG * SfGn003RfU * SfU * SmCfU * SmG *
CUCCGGUUCUGAAG
SSnR SSnR

16006
SfA * SmAfGfG * SfU * SfGn003RfU * SfU * SfC
GUGUUC
SSOSSSOOSSnR SS

WV-
fC * SfU * SfCn004RfC * SfG * SfGn004RfU * SfU * SmCfU * SmG *
CUCCGGUUCUGAAG
SSnR SSnR

16007
SfA * SmAfGfG * SfU * SfGn004RfU * SfU * SfC
GUGUUC
SSOSSSOOSSnR SS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAn003fG * SfA * SmU * SfA *
UCACUCAGAUA
SSSSSSnX SSSSnXnX

16008
SmGn003mUn003fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SSSSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAn004fG * SfA * SmU * SfA *
UCACUCAGAUA
SSSSSSnX SSSSnXnX

16009
SmGn004mUn004fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SSSSSS

WV-
L001L005fC * SfU * SfCn001 RfC * SfG * SfGn001 RfU * SfU *
CUCCGGUUCUGAAG
OOSSnR SSnR

16010
SmCfU * SmG * SfA * SmAfGfG * SfU * SfGn001 RfU * SfU * SfC
GUGUUC
SSOSSSOOSSnR SS

WV-
Mod107fC * SfU * SfCn001 RfC * SfG * SfUn001 RfU * SfU * SmCfU *
CUCCGGUUCUGAAG
SSnR SSnR

16011
SmG * SfA * SmAfGfG * SfU * SfGn001 RfU * SfU * SfC
GUGUUC
SSOSSSOOSSnR SS

WV-
Mod108L001fC * SfU * SfCn001 RfC * SfG * SfGn001 RfU * SfU *
CUCCGGUUCUGAAG
OSSnR SSnR

16366
SmCfU * SmG * SfA * SmAfGfG * SfU * SfGn001 RfU * SfU * SfC
GUGUUC
SSOSSSOOSSnR SS

WV-
fC * SfC * SfG * SfG * SfU * SfU * SmCfU * SmG * SfA * SmAmGfG *
CCGGUUCUGAAG
SSSSSSOSSSOO

16367
SfU * SfG * SfU * SfU * SfC * SfU
GUGUUCU
SSSSSS

WV-
fU * SfCn001 RfC * SfG * SfGn001 RfU * SfU * SmCfU * SmG * SfA *
UCCGGUUCUGAAG
SnRSSnR

16368
SmAfG * SfG * SfU * SfGn001 RfU * SfU * SfC
GUGUUC
SSOSSSOSSSnR SS

WV-
fU * SfCn001 RfC * SfG * SfGn001 RfU * SfU * SmCfU * SmG * SfA *
UCCGGUUCUGAAG
SnRSSnR

16369
SmAfGfG * SfU * SfGn001 RfU * SfU * SfC
GUGUUC
SSOSSSOOSSnR SS

WV-
fC * SfC * SfG * SfG * SfU * SfU * SmCfU * SmG * SfA * SmAmGfG *
CCGGUUCUGAAG
SSSSSSOSSSOO SSSSS

16370
SfU * SfG * SfU * SfU * SfC
GUGUUC

WV-
fU * SfCn001 RfC * SfG * SfGn001 RfU * SfU * SmCfU * SmG * SfA *
UCCGGUUCUGAAG
SnRSSnR

16371
SmAfG * SfG * SfU * SfGn001 RfU * SfU
GUGUU
SSOSSSOSSSnRS

WV-
fU * SfCn001 RfC * SfG * SfGn001 RfU * SfU * SmCfU * SmG * SfA *
UCCGGUUCUGAAG
SnRSSnR

16372
SmAfGfG * SfU * SfUn001 RfU * SfU
GUGUU
SSOSSSOOSSnRS

WV-
Mod105L001fC * SfU * SfCn001 RfC * SfG * SfGn001 RfU * SfU *
CUCCGGUUCUGAAG
OSSnR SSnR

16499
SmCfU * SmG * SfA * SmAfGfG * SfU * SfGn001 RfU * SfU * SfC
GUGUUC
SSOSSSOOSSnR SS

WV-
mU * mC * mA * mC * mU * mC * mA * mG * mA * mU * mA * mG *
UCACUCAGAUA
XXXXX XXXXX

16500
mU * mU * mG * mA * mA * mG * mC * mC
GUUGAAGCC
XXXXX XXXX

WV-
fU * fA * fA * fG * fG * mAfA * mGmA * fU * mGmGfC * fA * fU * fU
CAAGGAAGA UGG
XXXXX

16501
* fU * fC * fU
CAUUUCU
OXOXXOOXXXXX X

WV-
fA * fA * fG * fG * mAfA * mGmA * fU * mGmGfC * fA * mU * fU * fU * fU
AAGGAAGA UG
XXXXOXOXXOOXXXX

16502
* fC * fU
GCAUUUCU
X X

WV-
fUfC * fA * fA * fG * fG * mAfA * mGmA * fU * mGmGfC * fA * fU *
UCAAGGAAGA
OXXXXX

16503
fU * fU * fC * fU
UGGCAUUUCU
OXOXXOOXXXXX X

WV-
fU * fU * fC * fA * fA * fG * fG * mAfA * mGmA * fU * mGmGfC * fA
UUCAAGGAAGA
XXXXX

16504
* fU * fU * fU * fC * fU
UGGCAUUUCU
XXOXOXXOOXXXXX

X

WV-
Mod105L001fU * SfC * SfA * SfC * SfU * SfC * SmAn001 fG * SfA *
UCACUCAGAUA
O SSSSSSnX SSSSnXnX

16505
SmU * SfA * SmGn001 mUn001 fU * SfG * SfA * SfA * SfG * SfC *
GUUGAAGCC
SSSSSS

SfC

WV-
Mod108L001fU * SfC * SfA * SfC * SfU * SfC * SmAn001 fG * SfA *
UCACUCAGAUA
O SSSSSSnX SSSSnXnX

16506
SmU * SfA * SmGn001 mUn001 fU * SfG * SfA * SfA * SfG * SfC *
GUUGAAGCC
SSSSSS

SfC

WV-
Mod099L001fU * SfC * SfA * SfC * SfU * SfC * SmAn001 fG * SfA *
UCACUCAGAUA
O SSSSSSnX SSSSnXnX

16507
SmU * SfA * SmGn001 mUn001 fU * SfG * SfA * SfA * SfG * SfC *
GUUGAAGCC
SSSSSS

SfC

WV-
Mod102L001fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA *
UCACUCAGAU
OSSSS SSnXSS

17765
SmU * SfA * SmGn001 mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
AGUUGAAGCC
SSnXnXS SSSSS

WV-
fU * SfC * SfAn001RfC * SfU * SfCn001RmAfG * SfA * SmU * SfA *
UCACUCAGAU
SSnRSS nR OSSSS

17774
SmGmUfU * SfG * SfA * SfAn001RfG * SfC * SfC
AGUUGAAGCC
OOSS SnRSS

WV-
L001fU * SfC * SfAn001RfC * SfU * SfCn001RmAfG * SfA * SmU *
UCACUCAGAU
OSSnRS SnROSS SSOOS

17775
SfA * SmGmUfU * SfG * SfA * SfAn001RfG * SfC * SfC
AGUUGAAGCC
SSnRSS

WV-
fU * SfC * SfAn001SfC * SfU * SfCn001SmAfG * SfA * SmU * SfA *
UCACUCAGAU
SSnSSSnS OSSSS

17801
SmGmUfU * SfG * SfA * SfAn001SfG * SfC * SfC
AGUUGAAGCC
OOSSSnS SS

WV-
fU * SfC * SfAn001RfC * SfU * SfC * SmAn001RfG * SfA * SmU * SfA
UCACUCAGAU
SSnRSS SnRSSS SOOSS

17802
* SmGmUfU * SfG * SfA * SfAn001RfG * SfC * SfC
AGUUGAAGCC
SnRSS

WV-
fU * SfC * SfAn001RfC * SfU * SfCn001RmA * SfG * SfA * SmU * SfA
UCACUCAGAU
SSnRSS nR SSSSS OOSS

17803
* SmGmUfU * SfG * SfA * SfAn001RfG * SfC * SfC
AGUUGAAGCC
SnRSS

WV-
Mod007L001fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA *
UCACUCAGAU
OSSSS SSnXSS

17831
SmU * SfA * SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
AGUUGAAGCC
SSnXnXS SSSSS

WV-
Mod027L001fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA *
UCACUCAGAU
OSSSS SSnXSS

17832
SmU * SfA * SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
AGUUGAAGCC
SSnXnXS SSSSS

WV-
Mod028L001fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA *
UCACUCAGAU
OSSSS SSnXSS

17833
SmU * SfA * SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
AGUUGAAGCC
SSnXnXS SSSSS

WV-
Mod029L001fU * SfC * SfA * SfC * SfU * SfC * SmAn001fG * SfA *
UCACUCAGAU
OSSSS SSnXSS

17834
SmU * SfA * SmGn001mUn001fU * SfG * SfA * SfA * SfG * SfC * SfC
AGUUGAAGCC
SSnXnXS SSSSS

WV-
fG * SfG * SfU * SfU * SmCfU * SmG * SfA * SmAmGfG * SfU * SfG *
GGUUCUGAAG
SSSSO SSSOO SSSSS S

17835
SfU * SfU * SfC * SfU
GUGUUCU

WV-
fUfC * SfC * SfG * SfG * SfU * SfU * SmCfU * SmG * SfA *
UCCGGUUCUG
OSSSS SSOSS SOOSS

17836
SmAmGfG * SfU * SfG * SfU * SfU * SfC * SfU
AAGGUGUUCU
SSSS

WV-
fG * SfU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU * SmG * SfA *
GUCCGGUUCU
SSSSS SSSOS SSOOS

17837
SmAmGfG * SfU * SfG * SfU * SfU * SfC * SfU
GAAGGUGUUCU
SSSSS

WV-
fCn001RfC * SfG * SfGn001RfU * SfU * SmCfU * SmG * SfA *
CCGGUUCUGA
nRSSnRS SOSSS

17838
SmAfGfG * SfU * SfGn001RfU * SfU * SfC
AGGUGUUC
OOSSnRSS

WV-
fCfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU * SmG * SfA
CUCCGGUUCU
OSnRSSnR SSOSS

17839
* SmAfGfG * SfU * SfGn001RfU * SfU * SfC
GAAGGUGUUC
SOOSSnRSS

WV-
fC * SfC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CCUCCGGUUC
SSSnRS SnRSSO SSSOO

17840
SmG * SfA * SmAfGfG * SfU * SfGn001RfU * SfU * SfC
UGAAGGUGUUC
SSnRSS

WV-
fCn001RfC * SfG * SfGn001RfU * SfU * SmCfU * SmG * SfA * SmAfG
CCGGUUCUGA
nRSSnRS SOSSS

17841
* SfG * SfU * SfGn001RfU * SfU * SfC
AGGUGUUC
OSSSnRSS

WV-
fCfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU * SmG * SfA
CUCCGGUUCU
OSnRSSnR SSOSS

17842
* SmAfG * SfG * SfU * SfGn001RfU * SfU * SfC
GAAGGUGUUC
SOSSSnRSS

WV-
fC * SfC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CCUCCGGUUC
SSSnRS

17843
SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU * SfU * SfC
UGAAGGUGUUC
SnRSSOSSSOSSSnRSS

WV-
rC rA rG rA rG rU rA rA rC rA rG rU rC rU rG rA rG rU rA rG rG rU rU
CAGAGUAACA
OOOOO OOOOO

17844
rU rU rA rG rA rG rC rU rA
GUCUGAGUAG
OOOOO OOOOO

GUUUUAGAGC UA
OOOOO OOOOO O

WV-
rG rA rG rU rA rA rC rA rG rU rC rU rG rA rG rU rA rG rG rU rU rU rU
GAGUAACAGU
OOOOO OOOOO

17845
rA rG rA rG rC rU rA
CUGAGUAGGU
OOOOO OOOOO

UUUAGAGCUA
OOOOO OOOO

WV-
rG * rA * rG * rU * rA * rA * rC * rA * rG rU rC rU rG rA rG rU rA
GAGUAACAGU
XXXXX XXXOO

17846
rG rG rU rU rU rU * rA * rG * rA * rG * rC * rU * rA
CUGAGUAGGU
OOOOO OOOOO

UUUAGAGCUA
OOXXXXXXX

WV-
rG * rA * rG * rU * rA * rA * rC * rA * rG * rU * rC * rU * rG *
GAGUAACAGU
XXXXX XXXXX

17847
rA * rG * rU * rA * rG * rG * rU * rU * rU * rU * rA * rG * rA *
CUGAGUAGGU
XXXXX XXXXX

rG * rC * rU * rA
UUUAGAGCUA
XXXXX XXXX

WV-
mGmAmGmUmAmAmCmA rG rU rC rU rG rA rG rU rA rG rG rU rU
GAGUAACAGU
OOOOO OOOOO

17848
rUmUmAmGmAmGmCmUmA
CUGAGUAGGU
OOOOO OOOOO

UUUAGAGCUA
OOOOO OOOO

WV-
mG * mA * mG * mU * mA * mA * mC * mA * rG rU rC rU rG rA rG
GAGUAACAGU
XXXXX XXXOO

17849
rU rA rG rG rU rU rUmU * mA * mG * mA * mG * mC * mU * mA
CUGAGUAGGU
OOOOO OOOOO

UUUAGAGCUA
OOXXXXXXX

WV-
mG * mA * mG * mU * mA * mA * mC * mA * rG * rU * rC * rU *
GAGUAACAGU
XXXXX XXXXX

17850
rG * rA * rG * rU * rA * rG * rG * rU * rU * rU * mU * mA * mG *
CUGAGUAGGU
XXXXX XXXXX

mA * mG * mC * mU * mA
UUUAGAGCUA
XXXXX XXXX

WV-
fGfAfGfUfAfAfCfA rG rU rC rU rG rA rG rU rA rG rG rU rU
GAGUAACAGU
OOOOO OOOOO

17851
rUfUfAfGfAfGfCfUfA
CUGAGUAGGU
OOOOO OOOOO

UUUAGAGCUA
OOOOO OOOO

WV-
fG * fA * fG * fU * fA * fA * fC * fA * rG rU rC rU rG rA rG rU rA rG
GAGUAACAGU
XXXXX XXXOO

17852
rG rU rU rUfU * fA* fG * fA * fG * fC * fU * fA
CUGAGUAGGU
OOOOO OOOOO

UUUAGAGCUA
OOXXXXXXX

WV-
fG * fA * fG * fU * fA * fA * fC * fA * rG * rU * rC * rU * rG * rA *
GAGUAACAGU
XXXXX XXXXX

17853
rG * rU * rA * rG * rG * rU * rU * rU * fU * fA * fG * fA * fG * fC
CUGAGUAGGU
XXXXX XXXXX

* fU * fA
UUUAGAGCUA
XXXXX XXXX

WV-
rG rA rG rU rAn001 rAn001 rCn001 rAn001 rG rU rC rU rG rA rG rU rA
GAGUAACAGU
OOOOnX nXnXnXOO

17854
rG rG rU rU rU rU rA rG rA rGn001 rCn001 rUn001 rA
CUGAGUAGGU
OOOOO OOOOO

UUUAGAGCUA
OOOOO OnXnXnX

WV-
rG rA rG rU rA rA rC rA rG rU rC rU rG rA rG rU rA rG rG rU rU rU rU
GAGUAACAGU
OOOOO OOOOO

17855
rA rG rA rGn001 rCn001 rUn001 rA
CUGAGUAGGU
OOOOO OOOOO

UUUAGAGCUA
OOOOO OnXnXnX

WV-
rG rA rG rU rAn001 rAn001 rCn001 rAn001 rG rU rC rU rG rA rG rU rA
GAGUAACAGU
OOOOnX nXnXnXOO

17856
rG rG rU rU rU rU rA rG rA rG rC rU rA
CUGAGUAGGU
OOOOO OOOOO

UUUAGAGCUA
OOOOO OOOO

WV-
rG rA rG rU rA rAn001 rC rAn001 rG rU rC rU rG rA rG rU rA rG rG rU
GAGUAACAGU
OOOOO nXOnXOO

17857
rU rU rU rA rG rA rGn001 rC rUn001 rA
CUGAGUAGGU
OOOOO OOOOO

UUUAGAGCUA
OOOOO OnXOnX

WV-
rG rA rG rU rAn001 rA rCn001 rA rG rU rC rU rG rA rG rU rA rG rG rU
GAGUAACAGU
OOOOnX OnXOOO

17858
rU rU rU rA rG rA rG rCn001 rUn001 rA
CUGAGUAGGU
OOOOO OOOOO

UUUAGAGCUA
OOOOO OOnXnX

WV-
fU * SfC * SfAn001fA * SfG * SfG * SmA * SfA * SmGmA * SfU *
UCAAGGAAGA
SSnXSS SSSOS SOOnXS

17859
SmGmGfCn001fA * SfU * SfUn001fU * SfC * SfU
UGGCAUUUCU
SnXSS

WV-
fU * SfC * SfAn001fA * SfG * SfG * SmAfA * SmGmA * SfU *
UCAAGGAAGA
SSnXSS SOSOS SOSnXS

17860
SmGmG * SfCn001fA * SfU * SfUn001fU * SfC * SfU
UGGCAUUUCU
SnXSS

WV-
fU * SfC * SfAn001fA * SfG * SfG * SmA * SfA * SmGmA * SfU *
UCAAGGAAGA
SSnXSS SSSOS SOSnXS

17861
SmGmG * SfCn001fA * SfU * SfUn001fU * SfC * SfU
UGGCAUUUCU
SnXSS

WV-
fU * SfC * SfAn001fA * SfG * SfG * SfA * SfA * SmGmA * SfU *
UCAAGGAAGA
SSnXSS SSSOS SOSnXS

17862
SmGfG * SfCn001fA * SfU * SfUn001fU * SfC * SfU
UGGCAUUUCU
SnXSS

WV-
fU * SfC * SfAn001fA * SfG * SfGn001mA * SfA * SmGmA * SfU *
UCAAGGAAGA
SSnXSS nXSSOS SOOSS

17863
SmGmGfC * SfA * SfU * SfUn001fU * SfC * SfU
UGGCAUUUCU
SnXSS

WV-
fU * SfC * SfAn001fA * SfG * SfGn001mAfA * SmGmA * SfU *
UCAAGGAAGA
SSnXSS nXOSOS SOSSS

17864
SmGmG * SfC * SfA * SfU * SfUn001fU * SfC * SfU
UGGCAUUUCU
SnXSS

WV-
fU * SfC * SfAn001fA * SfG * SfGn001mA * SfA * SmGmA * SfU *
UCAAGGAAGA
SSnXSS nXSSOS SOSSS

17865
SmGmG * SfC * SfA * SfU * SfUn001fU * SfC * SfU
UGGCAUUUCU
SnXSS

WV-
fU * SfC * SfAn001fA * SfG * SfGn001fA * SfA * SmGmA * SfU *
UCAAGGAAGA
SSnXSS nXSSOS SOSSS

17866
SmGfG * SfC * SfA * SfU * SfUn001fU * SfC * SfU
UGGCAUUUCU
SnXSS

WV-17881
fG fA fG fUn001 fA fA fCn001 fA rG rU rC rU rG rA rG rU
GAGUAACAGUCUGAGUA
XXXnXX XnXXO OOOOO

rA rG rG rU rU rU fU fA fGn001 fA fG fCn001 fU fA
GGUU UUAGAGCUA
OOOOO OOOXX nXXXnXX

WV-17882
fG fA fG fUn001 fA fA fCn001 fA rG rU rC rU rG rA rG rU
GAGUAACAGUCUGAGUA
XXXnXX XnXXO OOOOO

rA rG rG rU rU rUn001 fU fA fGn001 fA fG fCn001 fU fA
GGUU UUAGAGCUA
OOOOO OOnXXX nXXXnXX

WV-17883
fG fA fG fUn001 fA fA fCn001 fA rG rU rC rU rG rA rG rU
GAGUAACAGUCUGAGUA
XXXnXX XnXXO OOOOO

rA rG rGn001 rU rU rUn001 fU fA fUn001 fA fG fCn001 fU
GGUU UUAGAGCUA
OOOOnX OOnXXX

fA

nXXXnXX

WV-18853
fC fC fUn001 fA fC fCn001 fC fU mA fU mG fU mAn001 fC
CCUACCCUAUGUACAUC
SSnXSS nXSSSS SSnXSS

fA fU fCn001 fG fU fU
GUU
SnXSS

WV-18854
fC fC fUn001 fA fU fGn001 fU fA mC fA mU fC mGn001 fU
CCUAUGUACAUCGUUCU
SSnXSS nXSSSS SSnXSS

fU fC fUn001 fG fC fU
GCU
SnXSS

WV-18855
fG fU fAn001 fC fA fUn001 fC fG mU fU mC fU mGn001 fC
GUACAUCGUUCUGCUUC
SSnXSS nXSSSS SSnXSS

fU fU fCn001 fU fG fA
UGA
SnXSS

WV-18856
fU fC fGn001 fU fU fCn001 fU fG mC fU mU fC mUn001 fG
UCGUUCUGCUUCUGAAC
SSnXSS nXSSSS SSnXSS

fA fA fCn001 fU fG fC
UGC
SnXSS

WV-18857
fU fC fUn001 fG fC fUn001 fU fC mU fG mA fA mCn001 fU
UCUGCUUCUGAACUGCU
SSnXSS nXSSSS SSnXSS

fG fC fUn001 fG fG fA
GGA
SnXSS

WV-18858
fU fU fCn001 fU fG fAn001 fA fC mU fG mC fU mGn001 fG
UUCUGAACUGCUGGAAA
SSnXSS nXSSSS SSnXSS

fA fA fAn001 fG fU fC
GUC
SnXSS

WV-18859
fA fA fCn001 fU fG fCn001 fU fG mG fA mA fA mGn001 fU
AACUGCUGGAAAGUCGC
SSnXSS nXSSSS SSnXSS

fC fG fCn001 fC fU fC
CUC
SnXSS

WV-18860
fA fA fGn001 fU fC fGn001 fC fC mU fC mC fA mAn001 fU
AAGUCGCCUCCAAUAGG
SSnXSS nXSSSS SSnXSS

fA fG fGn001 fU fG fC
UGC
SnXSS

WV-18861
fG fC fCn001 fU fC fCn001 fA fA mU fA mG fG mUn001 fG
GCCUCCAAUAGGUGCCU
SSnXSS nXSSSS SSnXSS

fC fC fUn001 fG fC fC
GCC
SnXSS

WV-18862
fC fA fAn001 fU fA fGn001 fG fU mG fC mC fU mGn001 fC
CAAUAGGUGCCUGCCGG
SSnXSS nXSSSS SSnXSS

fC fG fGn001 fC fU fU
CUU
SnXSS

WV-18863
fU fG fUn001 fG fC fCn001 fU fG mC fC mG fG mCn001 fU
GGUGCCUGCCGGCUUAA
SSnXSS nXSSSS SSnXSS

fU fA fAn001 fU fU fC
UUC
SnXSS

WV-18864
fC fU fGn001 fC fU fGn001 fG fC mU fU mA fA mUn001 fU
CUGCCGGCUUAAUUCAU
SSnXSS nXSSSS SSnXSS

fC fA fUn001 fC fA fU
CAU
SnXSS

WV-18865
fG fG fCn001 fU fU fAn001 fA fU mU fC mA fU mCn001 fA
GGCUUAAUUCAUCAUCU
SSnXSS nXSSSS SSnXSS

fU fC fUn001 fU fU fC
UUC
SnXSS

WV-18866
fA fA fUn001 fU fC fAn001 fU fC mA fU mC fU mUn001 fU
AAUUCAUCAUCUUUCAG
SSnXSS nXSSSS SSnXSS

fC fA fGn001 fC fU fG
CUG
SnXSS

WV-18867
fA fU fCn001 fA fU fCn001 fU fU mU fC mA fG mCn001 fU
AUCAUCUUUCAGCUGUA
SSnXSS nXSSSS SSnXSS

fG fU fAn001 fG fC fC
GCC
SnXSS

WV-18868
fC fU fUn001 fU fC fAn001 fG fC mU fG mU fA mGn001 fC
CUUUCAGCUGUAGCCAC
SSnXSS nXSSSS SSnXSS

fC fA fCn001 fA fC fC
ACC
SnXSS

WV-18869
fA fG fCn001 fU fG fUn001 fA fG mC fC mA fC mAn001 fC
AGCUGUAGCCACACCAG
SSnXSS nXSSSS SSnXSS

fC fA fGn001 fA fA fG
AAG
SnXSS

WV-18870
fU fA fGn001 fC fC fAn001 fC fA mC fC mA fG mAn001 fA
UAGCCACACCAGAAGUU
SSnXSS nXSSSS SSnXSS

fG fU fUn001 fC fC fU
CCU
SnXSS

WV-18871
fA fC fAn001 fC fC fAn001 fG fA mA fG mU fU mCn001 fC
ACACCAGAAGUUCCUGC
SSnXSS nXSSSS SSnXSS

fU fG fCn001 fA fG fA
AGA
SnXSS

WV-18872
fA fG fAn001 fA fG fUn001 fU fC mC fU mG fC mAn001 fG
AGAAGUUCCUGCAGAGA
SSnXSS nXSSSS SSnXSS

fA fG fAn001 fA fA fG
AAG
SnXSS

WV-18873
fU fC fCn001 fU fG fCn001 fA fG mA fG mA fA mAn001 fG
UCCUGCAGAGAAAGGUG
SSnXSS nXSSSS SSnXSS

fG fU fGn001 fC fA fG
CAG
SnXSS

WV-18874
fC fA fGn001 fA fG fAn001 fA fA mG fG mU fG mCn001 fA
CAGAGAAAGGUGCAGAC
SSnXSS nXSSSS SSnXSS

fG fA fCn001 fG fC fU
GCU
SnXSS

WV-18875
fA fA fAn001 fG fG fUn001 fG fC mA fG mA fC mGn001 fC
AAAGGUGCAGACGCUUC
SSnXSS nXSSSS SSnXSS

fU fU fCn001 fC fA fC
CAC
SnXSS

WV-18876
fU fG fCn001 fA fG fAn001 fC fG mC fU mU fC mCn001 fA
UGCAGACGCUUCCACUG
SSnXSS nXSSSS SSnXSS

fC fU fGn001 fG fU fC
GUC
SnXSS

WV-18877
fA fC fGn001 fC fU fUn001 fC fC mA fC mU fG mGn001 fU
ACGCUUCCACUGGUCAG
SSnXSS nXSSSS SSnXSS

fC fA fGn001 fA fA fC
AAC
SnXSS

WV-18878
fU fC fCn001 fA fC fUn001 fG fG mU fC mA fG mAn001 fA
UCCACUGGUCAGAACUG
SSnXSS nXSSSS SSnXSS

fC fU fGn001 fG fC fU
GCU
SnXSS

WV-18879
fU fG fGn001 fU fC fAn001 fG fA mA fC mU fG mGn001 fC
UGGUCAGAACUGGCUUC
SSnXSS nXSSSS SSnXSS

fU fU fCn001 fC fA fA
CAA
SnXSS

WV-18880
fA fG fAn001 fA fC fUn001 fG fG mC fU mU fC mCn001 fA
AGAACUGGCUUCCAAAU
SSnXSS nXSSSS SSnXSS

fA fA fCn001 fG fG fG
GGG
SnXSS

WV-18881
fU fG fGn001 fC fU fUn001 fC fC mA fA mA fU mGn001 fG
UGGCUUCCAAAUGGGAC
SSnXSS nXSSSS SSnXSS

fG fA fCn001 fC fU fG
CUG
SnXSS

WV-18882
fA fG fGn001 fC fA fCn001 fG fA mG fG mC fU mUn001 fA
AGGCACGAGGCUUAAAA
SSnXSS nXSSSS SSnXSS

fA fA fAn001 fA fU fG
AUG
SnXSS

WV-18883
fG fG fCn001 fA fC fGn001 fA fG mG fC mU fU mAn001 fA
GGCACGAGGCUUAAAAA
SSnXSS nXSSSS SSnXSS

fA fA fAn001 fU fG fU
UGU
SnXSS

WV-18884
fG fC fAn001 fC fG fAn001 fG fG mC fU mU fA mAn001 fA
GCACGAGGCUUAAAAAU
SSnXSS nXSSSS SSnXSS

fA fA fUn001 fG fU fC
GUC
SnXSS

WV-18885
fC fA fCn001 fG fA fGn001 fG fC mU fU mA fA mAn001 fA
CACGAGGCUUAAAAAUG
SSnXSS nXSSSS SSnXSS

fA fU fGn001 fU fC fC
UCC
SnXSS

WV-18886
fA fC fGn001 fA fG fGn001 fC fU mU fA mA fA mAn001 fA
ACGAGGCUUAAAAAUGU
SSnXSS nXSSSS SSnXSS

fU fG fUn001 fC fC fU
CCU
SnXSS

WV-18887
fC fG fAn001 fG fG fCn001 fU fU mA fA fA mAn001 fU
CGAGGCUUAAAAAUGUC
SSnXSS nXSSSS SSnXSS

fG fU fCn001 fC fU fA
CUA
SnXSS

WV-18888
fG fA fGn001 fG fC fUn001 fU fA mA fA mA fA mUn001 fG
GAGGCUUAAAAAUGUCC
SSnXSS nXSSSS SSnXSS

fU fC fCn001 fU fA fC
UAC
SnXSS

WV-18889
fA fG fGn001 fC fU fUn001 fA fA mA fA mA fU mGn001 fU
AGGCUUAAAAAUGUCCU
SSnXSS nXSSSS SSnXSS

fC fC fUn001 fA fC fC
ACC
SnXSS

WV-18890
fG fG fCn001 fU fU fAn001 fA fA mA fA mU fG mUn001 fC
GGCUUAAAAAUGUCCUA
SSnXSS nXSSSS SSnXSS

fC fU fAn001 fC fC fC
CCC
SnXSS

WV-18891
fG fC fUn001 fU fA fAn001 fA fA mA fU mG fU mCn001 fC
GCUUAAAAAUGUCCUAC
SSnXSS nXSSSS SSnXSS

fU fA fCn001 fC fC fU
CCU
SnXSS

WV-18892
fC fU fUn001 fA fA fAn001 fA fA mU fG mU fC mCn001 fU
CUUAAAAAUGUCCUACC
SSnXSS nXSSSS SSnXSS

fA fC fCn001 fC fU fA
CUA
SnXSS

WV-18893
fU fU fAn001 fA fA fAn001 fA fU mG fU mC fC mUn001 fA
UUAAAAAUGUCCUACCC
SSnXSS nXSSSS SSnXSS

fC fC fCn001 fU fA fU
UAU
SnXSS

WV-18894
fU fA fAn001 fA fA fAn001 fU fG mU fC mC fU mAn001 fC
UAAAAAUGUCCUACCCU
SSnXSS nXSSSS SSnXSS

fC fC fUn001 fA fU fG
AUG
SnXSS

WV-18895
fA fA fAn001 fA fA fUn001 fG fU mC fC mU fA mCn001 fC
AAAAAUGUCCUACCCUA
SSnXSS nXSSSS SSnXSS

fC fU fAn001 fU fG fU
UGU
SnXSS

WV-18896
fA fA fAn001 fA fU fGn001 fU fC mC fU mA fC mCn001 fC
AAAAUGUCCUACCCUAU
SSnXSS nXSSSS SSnXSS

fU fA fUn001 fG fU fA
GUA
SnXSS

WV-18897
fA fA fAn001 fU fG fUn001 fU fC mU fA mC fC mCn001 fU
AAAUGUCCUACCCUAUG
SSnXSS nXSSSS SSnXSS

fA fU fGn001 fU fA fC
UAC
SnXSS

WV-18898
fA fA fUn001 fG fU fCn001 fC fU mA fC mC fC mUn001 fA
AAUGUCCUACCCUAUGU
SSnXSS nXSSSS SSnXSS

fU fG fUn001 fA fC fA
ACA
SnXSS

WV-18899
fA fU fGn001 fU fC fCn001 fU fA mC fC mC fU mAn001 fU
AUGUCCUACCCUAUGUA
SSnXSS nXSSSS SSnXSS

fG fU fAn001 fC fA fU
CAU
SnXSS

WV-18900
fU fG fUn001 fC fC fUn001 fA fC mC fC mU fA mAn001 fG
UGUCCUACCCUAUGUAC
SSnXSS nXSSSS SSnXSS

fU fA fCn001 fA fU fC
AUC
SnXSS

WV-18901
fG fU fCn001 fC fU fAn001 fC fC mC fU mA fU mGn001 fU
GUCCUACCCUAUGUACA
SSnXSS nXSSSS SSnXSS

fA fC fAn001 fU fC fG
UCG
SnXSS

WV-18902
fU fC fCn001 fU fA fCn001 fC fC mU fA mU fG mUn001 fA
UCCUACCCUAUGUACAU
SSnXSS nXSSSS SSnXSS

fC fA fUn001 fC fG fU
CGU
SnXSS

WV-18903
fC fU fAn001 fC fC fCn001 fU fA mU fG mU fA mCn001 fA
CUACCCUAUGUACAUCG
SSnXSS nXSSSS SSnXSS

fU fC fGn001 fU fU fC
UUC
SnXSS

WV-18904
fU fA fCn001 fC fC fUn001 fA fU mG fU mA fC mAn001 fU
UACCCUAUGUACAUCGU
SSnXSS nXSSSS SSnXSS

fC fG fUn001 fU fC fU
UCU
SnXSS

WV-18905
fU fU fCn001 fG fA fAn001 fA fA mA fA mC fA mAn001 fA
UUCGAAAAAACAAAUCA
SSnXSS nXSSSS SSnXSS

fU fC fAn001 fA fA fG
AAG
SnXSS

WV-18906
fU fC fGn001 fA fA fAn00l fA fA mA fC mA fA mAn001 fU
UCGAAAAAACAAAUCAA
SSnXSS nXSSSS SSnXSS

fC fA fAn001 fA fG fA
AGA
SnXSS

WV-18907
fC fG fAn001 fA fA fAn001 fA fA mC fA mA fA mUn001 fC
CGAAAAAACAAAUCAAA
SSnXSS nXSSSS SSnXSS

fA fA fAn00l fG fA fC
GAC
SnXSS

WV-18908
fG fA fAn001 fA fA fAn001 fA fC mA fA mA fU mCn001 fA
GAAAAAACAAAUCAAAG
SSnXSS nXSSSS SSnXSS

fA fA fGn001 fA fC fU
ACU
SnXSS

WV-18909
fA fA fAn001 fA fA fAn001 fC fA mA fA mU fC mAn001 fA
AAAAAACAAAUCAAAGA
SSnXSS nXSSSS SSnXSS

fA fG fAn001 fC fU fU
CUU
SnXSS

WV-18910
fA fA fAn001 fA fA fCn001 fA fA mA fU mC fA mAn001 fA
AAAAACAAAUCAAAGAC
SSnXSS nXSSSS SSnXSS

fG fA fCn001 fU fU fA
UUA
SnXSS

WV-18911
fA fA fAn001 fA fC fAn001 fA fA mU fC mA fA mAn001 fG
AAAACAAAUCAAAGACU
SSnXSS nXSSSS SSnXSS

fA fC fUn001 fU fA fC
UAC
SnXSS

WV-18912
fA fA fAn001 fC fA fAn001 fA fU mC fA mA fA mGn001 fA
AAACAAAUCAAAGACUU
SSnXSS nXSSSS SSnXSS

fC fU fUn001 fA fC fC
ACC
SnXSS

WV-18913
fA fA fCn001 fA fA fAn001 fU fC mA fA mA fG mAn001 fC
AACAAAUCAAAGACUUA
SSnXSS nXSSSS SSnXSS

fU fU fAn001 fC fC fU
CCU
SnXSS

WV-18914
fA fC fAn001 fA fA fUn001 fC fA mA fA mG fA mCn001 fU
ACAAAUCAAAGACUUAC
SSnXSS nXSSSS SSnXSS

fU fA fCn001 fC fU fU
CUU
SnXSS

WV-18915
fC fA fAn001 fA fU fCn001 fA fA mA fG mA fC mUn001 fU
CAAAUCAAAGACUUACC
SSnXSS nXSSSS SSnXSS

fA fC fCn001 fU fU fA
UUA
SnXSS

WV-18916
fA fA fAn001 fU fC fAn001 fA fA mG fA mC fU mUn001 fA
AAAUCAAAGACUUACCU
SSnXSS nXSSSS SSnXSS

fC fC fUn001 fU fA fA
UAA
SnXSS

WV-18917
fA fA fUn001 fC fA fAn001 fA fG mA fC mU fU mAn001 fC
AAUCAAAGACUUACCUU
SSnXSS nXSSSS SSnXSS

fC fU fUn001 fA fA fG
AAG
SnXSS

WV-18918
fA fU fCn001 fA fA fAn001 fG fA mC fU mU fA mCn001 fC
AUCAAAGACUUACCUUA
SSnXSS nXSSSS SSnXSS

fU fU fAn001 fA fG fA
AGA
SnXSS

WV-18919
fU fC fAn001 fA fA fGn001 fA fC mU fU mA fC mCn001 fU
UCAAAGACUUACCUUAA
SSnXSS nXSSSS SSnXSS

fU fA fAn001 fG fA fU
GAU
SnXSS

WV-18920
fC fA fAn001 fA fG fAn00l fC fU mU fA fC fC mUn001 fU
CAAAGACUUACCUUAAG
SSnXSS nXSSSS SSnXSS

fA fA fGn001 fA fU fA
AUA
SnXSS

WV-18921
fA fA fAn00l fG fA fCn001 fU fU mA fC mC fU mUn001 fA
AAAGACUUACCUUAAGA
SSnXSS nXSSSS SSnXSS

fA fG fAn001 fU fA fC
UAC
SnXSS

WV-18922
fA fA fGn001 fA fC fUn001 fU fA mC fC mU fU mAn001 fA
AAGACUUACCUUAAGAU
SSnXSS nXSSSS SSnXSS

fG fA fUn001 fA fC fC
ACC
SnXSS

WV-18923
fA fG fAn001 fC fU fUn001 fA fC mC fU mU fA mAn001 fG
AGACUUACCUUAAGAUA
SSnXSS nXSSSS SSnXSS

fA fU fAn001 fC fC fA
CCA
SnXSS

WV-18924
fG fA fCn001 fU fU fAn001 fC fC mU fU mA fA mGn001 fA
GACUUACCUUAAGAUAC
SSnXSS nXSSSS SSnXSS

fU fA fCn001 fC fA fU
CAU
SnXSS

WV-18925
fA fC fUn001 fU fA fCn001 fC fU mU fA mA fG mAn001 fU
ACUUACCUUAAGAUACC
SSnXSS nXSSSS SSnXSS

fA fC fCn001 fA fU fU
AUU
SnXSS

WV-18926
fC fU fUn001 fA fC fCn001 fU fU mA fA mG fA mUn001 fA
CUUACCUUAAGAUACCA
SSnXSS nXSSSS SSnXSS

fC fC fAn001 fU fU fU
UUU
SnXSS

WV-18927
fU fU fAn001 fC fC fUn001 fU fA mA fG mA fU mAn001 fC
UUACCUUAAGAUACCAU
SSnXSS nXSSSS SSnXSS

fC fA fUn001 fU fU fG
UUG
SnXSS

WV-18928
fU fA fCn001 fC fU fUn001 fA fA mG fA mU fA mCn001 fC
UACCUUAAGAUACCAUU
SSnXSS nXSSSS SSnXSS

fA fU fUn001 fU fG fU
UGU
SnXSS

WV-18929
fA fG fGn001 fC fA fAn001 fA fA mC fA mA fA mAn001 fA
AGGCAAAACAAAAAUGA
SSnXSS nXSSSS SSnXSS

fU fG fAn001 fA fG fC
AGC
SnXSS

WV-18930
fG fC fAn001 fA fA fAn001 fC fA mA fA mA fA mUn001 fG
GCAAAACAAAAAUGAAG
SSnXSS nXSSSS SSnXSS

fA fA fGn001 fC fC fC
CCC
SnXSS

WV-18931
fA fA fAn001 fA fC fAn001 fA fA mA fA mU fG mAn001 fA
AAAACAAAAAUGAAGCC
SSnXSS nXSSSS SSnXSS

fG fC fCn001 fC fC fA
CCA
SnXSS

WV-18932
fA fA fCn001 fA fA fAn001 fA fA mU fG mA fA mGn001 fC
AACAAAAAUGAAGCCCC
SSnXSS nXSSSS SSnXSS

fC fC fCn001 fA fU fG
AUG
SnXSS

WV-18933
fC fA fAn001 fA fA fAn001 fU fG mA fA mG fC mCn001 fC
CAAAAAUGAAGCCCCAU
SSnXSS nXSSSS SSnXSS

fC fA fUn001 fG fU fC
GUC
SnXSS

WV-18934
fA fA fAn001 fA fU fGn001 fA fA mG fC mC fC mCn001 fA
AAAAUGAAGCCCCAUGU
SSnXSS nXSSSS SSnXSS

fU fG fUn001 fC fU fU
CUU
SnXSS

WV-18935
fA fA fUn001 fG fA fAn001 fG fC mC fC mC fA mUn001 fG
AAUGAAGCCCCAUGUCU
SSnXSS nXSSSS SSnXSS

fU fC fUn001 fU fU fU
UUU
SnXSS

WV-18936
fA fU fGn001 fA fA fGn001 fC fC mC fC mA fU mGn001 fU
AUGAAGCCCCAUGUCUU
SSnXSS nXSSSS SSnXSS

fC fU fUn001 fU fU fU
UUU
SnXSS

WV-18937
fG fA fAn001 fG fC fCn001 fC fC mA fU mG fU mCn001 fU
GAAGCCCCAUGUCUUUU
SSnXSS nXSSSS SSnXSS

fU fU fUn001 fU fA fU
UAU
SnXSS

WV-18938
fA fG fCn001 fC fC fCn001 fA fU mG fU mC fU mUn001 fU
AGCCCCAUGUCUUUUUA
SSnXSS nXSSSS SSnXSS

fU fU fAn001 fU fU fU
UUU
SnXSS

WV-18939
fC fC fCn001 fC fA fUn001 fG fU mC fU mU fU mUn001 fU
CCCCAUGUCUUUUUAUU
SSnXSS nXSSSS SSnXSS

fA fU fUn001 fU fG fA
UGA
SnXSS

WV-18940
fU fG fAn001 fA fG fCn001 fC fC mC fA mU fG mUn001 fC
UGAAGCCCCAUGUCUUU
SSnXSS nXSSSS SSnXSS

fU fU fUn001 fU fU fA
UUA
SnXSS

WV-18941
fA fA fGn001 fC fC fCn001 fC fA mU fG mU fC mUn001 fU
AAGCCCCAUGUCUUUUU
SSnXSS nXSSSS SSnXSS

fU fU fUn001 fA fU fU
AUU
SnXSS

WV-18942
fG fC fCn001 fC fC fAn001 fU fG mU fC mU fU mUn001 fU
GCCCCAUGUCUUUUUAU
SSnXSS nXSSSS SSnXSS

fU fA fUn001 fU fU fG
UUG
SnXSS

WV-18944
fU fC fA fC fU fC mAn001 fG fA mU fA mGn001 mUn001
UCACUCAGAUAGUUGAA
XXXXX XnXXXX XnXnXXX

fU fG fA fA fG fC fC
GCC
XXXX

WV-18945
fU fC fAn001 fC fU fCn001 mA fG fA mU fA mG mU fU fG
UCACUCAGAUAGUUGAA
XXnXXX nXOXXX

fA fAn001 fG fC fC
GCC
XOOXXX nXXX

WV-18983
fC fC fU fA fC fC fC fU mA fU mG fU mA fC fA fU fC fG
CCUACCCUAUGUACAUC
SSSSS SSSSS SSSSS SSSS

fU fU
GUU

WV-18984
fC fC fU fA fU fG fU fA mC fA mU fC mG fU fU fC fU fG
CCUAUGUACAUCGUUCU
SSSSS SSSSS SSSSS SSSS

fC fU
GCU

WV-18985
fG fU fA fC fA fU fC fG mU fU mC fU mG fC fU fU fC fU
GUACAUCGUUCUGCUUC
SSSSS SSSSS SSSSS SSSS

fG fA
UGA

WV-18986
fU fC fG fU fU fC fU fG mC fU mU fC mU fG fA fA fC fU
UCGUUCUGCUUCUGAAC
SSSSS SSSSS SSSSS SSSS

fG fC
UGC

WV-18987
fU fC fU fG fC fU fU fC mU fG mA fA mC fU fG fC fU fG
UCUGCUUCUGAACUGCU
SSSSS SSSSS SSSSS SSSS

fG fA
GGA

WV-18988
fU fU fC fU fG fA fA fC mU fG mC fU mG fG fA fA fA fG
UUCUGAACUGCUGGAAA
SSSSS SSSSS SSSSS SSSS

fU fC
GUC

WV-18989
fA fA fC fU fG fC fU fG mG fA mA fA mG fU fC fG fC fC
AACUGCUGGAAAGUCGC
SSSSS SSSSS SSSSS SSSS

fU fC
CUC

WV-18990
fA fA fG fU fC fG fC fC mU fC mC fA mA fU fA fG fG fU
AAGUCGCCUCCAAUAGG
SSSSS SSSSS SSSSS SSSS

fG fC
UGC

WV-18991
fG fC fC fU fC fC fA fA mU fA mG fG mU fG fC fC fU fG
GCCUCCAAUAGGUGCCU
SSSSS SSSSS SSSSS SSSS

fC fC
GCC

WV-18992
fC fA fA fU fA fG fG fU mG fC mC fU mG fC fC fG fG fC
CAAUAGGUGCCUGCCGG
SSSSS SSSSS SSSSS SSSS

fU fU
CUU

WV-18993
fG fG fU fG fC fC fU fG mC fC mG fG mC fU fU fA fA fU
GGUGCCUGCCGGCUUAA
SSSSS SSSSS SSSSS SSSS

fU fC
UUC

WV-18994
fC fU fG fC fC fG fG fC mU fU mA fA mU fU fC fA fU fC
CUGCCGGCUUAAUUCAU
SSSSS SSSSS SSSSS SSSS

fA fU
CAU

WV-18995
fG fG fC fU fU fA fA fU mU fC mA fU mC fA fU fC fU fU
GGCUUAAUUCAUCAUCU
SSSSS SSSSS SSSSS SSSS

fU fC
UUC

WV-18996
fA fA fU fU fC fA fU fC mA fU mC fU mU fU fC fA fG fC
AAUUCAUCAUCUUUCAG
SSSSS SSSSS SSSSS SSSS

fU fG
CUG

WV-18997
fA fU fC fA fU fC fU fU mU fC mA fG mC fU fG fU fA fG
AUCAUCUUUCAGCUGUA
SSSSS SSSSS SSSSS SSSS

fC fC
GCC

WV-18998
fC fU fU fU fC fA fG fC mU fG mU fA mG fC fC fA fC fA
CUUUCAGCUGUAGCCAC
SSSSS SSSSS SSSSS SSSS

fC fC
ACC

WV-18999
fA fG fC fU fG fU fA fG mC fC mA fC mA fC fC fA fG fA
AGCUGUAGCCACACCAG
SSSSS SSSSS SSSSS SSSS

fA fG
AAG

WV-19000
fU fA fG fC fC fA fC fA mC fC mA fG mA fA fG fU fU fC
UAGCCACACCAGAAGUU
SSSSS SSSSS SSSSS SSSS

fC fU
CCU

WV-19001
fA fC fA fC fC fA fG fA mA fG mU fU mC fC fU fG fC fA
ACACCAGAAGUUCCUGC
SSSSS SSSSS SSSSS SSSS

fG fA
AGA

WV-19002
fA fG fA fA fG fU fU fC mC fU mG fC mA fG fA fG fA fA
AGAAGUUCCUGCAGAGA
SSSSS SSSSS SSSSS SSSS

fA fG
AAG

WV-19003
fU fC fC fU fG fC fA fG mA fG mA fA mA fG fG fU fG fC
UCCUGCAGAGAAAGGUG
SSSSS SSSSS SSSSS SSSS

fA fG
CAG

WV-19004
fC fA fG fA fG fA fA fA mG fG mU fG mC fA fG fA fC fG
CAGAGAAAGGUGCAGAC
SSSSS SSSSS SSSSS SSSS

fC fU
GCU

WV-19005
fA fA fA fG fG fU fG fC mA fG mA fC mG fC fU fU fC fC
AAAGGUGCAGACGCUUC
SSSSS SSSSS SSSSS SSSS

fA fC
CAC

WV-19006
fU fG fC fA fG fA fC fG mC fU mU fC mC fA fC fU fG fG
UGCAGACGCUUCCACUG
SSSSS SSSSS SSSSS SSSS

fU fC
GUC

WV-19007
fA fC fG fC fU fU fC fC mA fC mU fG mG fU fC fA fG fA
ACGCUUCCACUGGUCAG
SSSSS SSSSS SSSSS SSSS

fA fC
AAC

WV-19008
fU fC fC fA fC fU fG fG mU fC mA fG mA fA fC fU fG fG
UCCACUGGUCAGAACUG
SSSSS SSSSS SSSSS SSSS

fC fU
GCU

WV-19009
fU fG fG fU fC fA fG fA mA fC mU fG mG fC fU fU fC fC
UGGUCAGAACUGGCUUC
SSSSS SSSSS SSSSS SSSS

fA fA
CAA

WV-19010
fA fG fA fA fC fU fG fG mC fU mU fC mC fA fA fA fU fG
AGAACUGGCUUCCAAAU
SSSSS SSSSS SSSSS SSSS

fG fG
GGG

WV-19011
fU fG fG fC fU fU fC fC mA fA mA fU mG fG fG fA fC fC
UGGCUUCCAAAUGGGAC
SSSSS SSSSS SSSSS SSSS

fU fG
CUG

WV-19012
fA fG fG fC fA fC fG fA mG fG mC fU mU fA fA fA fA fA
AGGCACGAGGCUUAAAA
SSSSS SSSSS SSSSS SSSS

fU fG
AUG

WV-19013
fG fG fC fA fC fG fA fG mG fC mU fU mA fA fA fA fA fU
GGCACGAGGCUUAAAAA
SSSSS SSSSS SSSSS SSSS

fG fU
UGU

WV-19014
fG fC fA fC fG fA fG fG mC fU mU fA mA fA fA fA fU fG
GCACGAGGCUUAAAAAU
SSSSS SSSSS SSSSS SSSS

fU fC
GUC

WV-19015
fC fA fC fG fA fG fG fC mU fU mA fA mA fA fA fU fG fU
CACGAGGCUUAAAAAUG
SSSSS SSSSS SSSSS SSSS

fC fC
UCC

WV-19016
fA fC fG fA fG fG fC fU mU fA mA fA mA fA fU fG fU fC
ACGAGGCUUAAAAAUGU
SSSSS SSSSS SSSSS SSSS

fC fU
CCU

WV-19017
fC fG fA fG fG fC fU fU mA fA mA fA mA fU fG fU fC fC
CGAGGCUUAAAAAUGUC
SSSSS SSSSS SSSSS SSSS

fU fA
CUA

WV-19018
fG fA fG fG fC fU fU fA mA fA mA fA mU fG fU fC fC fU
GAGGCUUAAAAAUGUCC
SSSSS SSSSS SSSSS SSSS

fA fC
UAC

WV-19019
fA fG fG fC fU fU fA fA mA fA mA fU mG fU fC fC fU fA
AGGCUUAAAAAUGUCCU
SSSSS SSSSS SSSSS SSSS

fC fC
ACC

WV-19020
fG fG fC fU fU fA fA fA mA fA mU fG mU fC fC fU fA fC
GGCUUAAAAAUGUCCUA
SSSSS SSSSS SSSSS SSSS

fC fC
CCC

WV-19021
fG fC fU fU fA fA fA fA mA fU mG fU mC fC fU fA fC fC
GCUUAAAAAUGUCCUAC
SSSSS SSSSS SSSSS SSSS

fC fU
CCU

WV-19022
fC fU fU fA fA fA fA fA mU fG mU fC mC fU fA fC fC fC
CUUAAAAAUGUCCUACC
SSSSS SSSSS SSSSS SSSS

fU fA
CUA

WV-19023
fU fU fA fA fA fA fA fU mG fU mC fC mU fA fC fC fC fU
UUAAAAAUGUCCUACCC
SSSSS SSSSS SSSSS SSSS

fA fU
UAU

WV-19024
fU fA fA fA fA fA fU fG mU fC mC fU mA fC fC fC fU fA
UAAAAAUGUCCUACCCU
SSSSS SSSSS SSSSS SSSS

fU fG
AUG

WV-19025
fA fA fA fA fA fU fG fU mC fC mU fA mC fC fC fU fA fU
AAAAAUGUCCUACCCUA
SSSSS SSSSS SSSSS SSSS

fG fU
UGU

WV-19026
fA fA fA fA fU fG fU fC mC fU mA fC mC fC fU fA fU fG
AAAAUGUCCUACCCUAU
SSSSS SSSSS SSSSS SSSS

fU fA
GUA

WV-19027
fA fA fA fU fG fU fC fC mU fA mC fC mC fU fA fU fG fU
AAAUGUCCUACCCUAUG
SSSSS SSSSS SSSSS SSSS

fA fC
UAC

WV-19028
fA fA fU fG fU fC fC fU mA fC mC fC mU fA fU fG fU fA
AAUGUCCUACCCUAUGU
SSSSS SSSSS SSSSS SSSS

fC fA
ACA

WV-19029
fA fU fG fU fC fC fU fA mC fC mC fU mA fU fG fU fA fC
AUGUCCUACCCUAUGUA
SSSSS SSSSS SSSSS SSSS

fA fU
CAU

WV-19030
fU fG fU fC fC fU fA fC mC fC mU fA mU fG fU fA fC fA
UGUCCUACCCUAUGUAC
SSSSS SSSSS SSSSS SSSS

fU fC
AUC

WV-19031
fG fU fC fC fU fA fC fC mC fU mA fU mG fU fA fC fA fG
GUCCUACCCUAUGUACA
SSSSS SSSSS SSSSS SSSS

fC fG
UCG

WV-19032
fU fC fC fU fA fC fC fC mU fA mU fG mU fA fC fA fU fC
UCCUACCCUAUGUACAU
SSSSS SSSSS SSSSS SSSS

fG fU
CGU

WV-19033
fC fU fA fC fC fC fU fA mU fG mU fA mC fA fU fC fG fU
CUACCCUAUGUACAUCG
SSSSS SSSSS SSSSS SSSS

fU fC
UUC

WV-19034
fU fA fC fC fC fU fA fU mG fU mA fC mA fU fC fG fU fU
UACCCUAUGUACAUCGU
SSSSS SSSSS SSSSS SSSS

fC fU
UCU

WV-19801
fC fC fU fU fC fC mC fU fG mA fA mG mG fU fU fC fC fU
CCUUCCCUGAAGGUUCC
XXXXX XOXXX XOOXX

fC fC
UCC
XXXX

WV-19802
fC fC fU fU fC fC mC fU fG mA fA mG mG fU fU fC fC fU
CCUUCCCUGAAGGUUCC
SSSSS SOSSS SOOSS SSSS

fC fC
UCC

WV-19803
fC fC fU fU fC fC mCn001 fU fG mA fA mGn001 mGn001
CCUUCCCUGAAGGUUCC
XXXXX XnXXXX XnXnXXX

fU fU fC fC fU fC fC
UCC
XXXX

WV-19804
fC fC fU fU fC fC mCn001 fU fG mA fA mGn001 mGn001
CCUUCCCUGAAGGUUCC
SSSSS SnXSSS SnXnXSS

fU fU fC fC fU fC fC
UCC
SSSS

WV-19805
fC fC fUn001 fU fC fCn001 mC fU fG mA fA mG mG fU fU
CCUUCCCUGAAGGUUCC
XXnXXX nXOXXX XOOXX

fC fCn001 fU fC fC
UCC
XnXXX

WV-19806
fC fC fUn001 R fU fC fCn001 R mC fU fG mA fA mG mG fU
CCUUCCCUGAAGGUUCC
SSnRSS nROSSS SOOSS

fU fC fCn001 R fU fC fC
UCC
SnRSS

WV-19886
fC fU fUn001 fC fU fGn001 fC fC mA fA mC fU mU fU fU
CUUCUGCCAACUUUUAU
SSnXSS nXSSSS SSSSS

fA fUn001 fC fA fU
CAU
SnXSS

WV-19887
fU fU fCn001 fU fG fCn001 fC fA mA fC mU fU mU fU fA
UUCUGCCAACUUUUAUC
SSnXSS nXSSSS SSSSS

fU fCn001 fA fU fU
AUU
SnXSS

WV-19888
fU fC fUn001 fG fC fCn001 fA fA mC fU mU fU mU fA fU
UCUGCCAACUUUUAUCA
SSnXSS nXSSSS SSSSS

fC fAn001 fU fU fU
UUU
SnXSS

WV-19889
fC fU fGn001 fC fC fAn001 fA fC mU fU mU fU mA fU fC
CUGCCAACUUUUAUCAU
SSnXSS nXSSSS SSSSS

fA fUn001 fU fU fU
UUU
SnXSS

WV-19890
fU fG fCn001 fC fA fAn001 fC fU mU fU mU fA mU fC fA
UGCCAACUUUUAUCAUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fU fU
UUU
SnXSS

WV-19891
fG fC fCn001 fA fA fCn001 fU fU mU fU mA fU mC fA fU
GCCAACUUUUAUCAUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fU fU
UUU
SnXSS

WV-19892
fC fC fAn001 fA fC fUn001 fU fU mU fA mU fC mA fU fU
CCAACUUUUAUCAUUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fU fC
UUC
SnXSS

WV-19893
fC fA fAn001 fC fU fUn001 fU fU mA fU mC fA mU fU fU
CAACUUUUAUCAUUUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fC fU
UCU
SnXSS

WV-19894
fA fA fCn001 fU fU fUn001 fU fA mU fC mA fU mU fU fU
AACUUUUAUCAUUUUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fC fU fC
CUC
SnXSS

WV-19895
fA fC fUn001 fU fU fUn001 fA fU mC fA mU fU mU fU fU
ACUUUUAUCAUUUUUUC
SSnXSS nXSSSS SSSSS

fU fCn001 fU fC fA
UCA
SnXSS

WV-19896
fC fU fUn001 fU fU fAn001 fU fC mA fU mU fU mU fU fU
CUUUUAUCAUUUUUUCU
SSnXSS nXSSSS SSSSS

fC fUn001 fC fA fU
CAU
SnXSS

WV-19897
fU fU fUn001 fU fA fUn001 fC fA mU fU mU fU mU fU fC
UUUUAUCAUUUUUUCUC
SSnXSS nXSSSS SSSSS

fU fCn001 fA fU fA
AUA
SnXSS

WV-19898
fU fU fUn001 fA fU fCn001 fA fU mU fU mU fU mU fC fU
UUUAUCAUUUUUUCUCA
SSnXSS nXSSSS SSSSS

fC fAn001 fU fA fC
UAC
SnXSS

WV-19899
fU fU fAn001 fU fC fAn001 fU fU mU fU mU fU mC fU fC
UUAUCAUUUUUUCUCAU
SSnXSS nXSSSS SSSSS

fA fUn001 fA fC fC
ACC
SnXSS

WV-19900
fU fA fUn001 fC fA fUn001 fU fU mU fU mU fC mU fC fA
UAUCAUUUUUUCUCAUA
SSnXSS nXSSSS SSSSS

fU fAn001 fC fC fU
CCU
SnXSS

WV-19901
fA fU fCn001 fA fU fUn001 fU fU mU fU mC fU mC fA fU
AUCAUUUUUUCUCAUAC
SSnXSS nXSSSS SSSSS

fA fCn001 fC fU fU
CUU
SnXSS

WV-19902
fU fC fAn001 fU fU fUn001 fU fU mU fC mU fC mA fU fA
UCAUUUUUUCUCAUACC
SSnXSS nXSSSS SSSSS

fC fCn001 fU fU fC
UUC
SnXSS

WV-19903
fC fA fUn001 fU fU fUn001 fU fU mC fU mC fA mU fA fC
CAUUUUUUCUCAUACCU
SSnXSS nXSSSS SSSSS

fC fUn001 fU fC fU
UCU
SnXSS

WV-19904
fA fG fUn001 fU fU fUn001 fU fC mU fC mA fU mA fC fC
AUUUUUUCUCAUACCUU
SSnXSS nXSSSS SSSSS

fU fUn001 fC fU fG
CUG
SnXSS

WV-19905
fU fU fUn001 fU fU fUn001 fC fU mC fA mU fA mC fC fU
UUUUUUCUCAUACCUUC
SSnXSS nXSSSS SSSSS

fU fCn001 fU fG fC
UGC
SnXSS

WV-19906
fU fU fUn001 fU fU fCn001 fU fC mA fU mA fC mC fU fU
UUUUUCUCAUACCUUCU
SSnXSS nXSSSS SSSSS

fC fUn001 fG fC fU
GCU
SnXSS

WV-19907
fU fU fUn001 fU fC fUn001 fC fA mU fA mC fC mU fU fC
UUUUCUCAUACCUUCUG
SSnXSS nXSSSS SSSSS

fU fGn001 fC fU fU
CUU
SnXSS

WV-19908
fU fU fUn001 fC fU fCn001 fA fU mA fC mC fU mU fC fU
UUUCUCAUACCUUCUGC
SSnXSS nXSSSS SSSSS

fG fCn001 fU fU fG
UUG
SnXSS

WV-19909
fU fU fCn001 fU fC fAn001 fU fA mC fC mU fU mC fU fG
UUCUCAUACCUUCUGCU
SSnXSS nXSSSS SSSSS

fC fUn001 fU fG fA
UGA
SnXSS

WV-19910
fU fC fUn001 fC fA fUn001 fA fC mC fU mU fC mU fG fC
UCUCAUACCUUCUGCUU
SSnXSS nXSSSS SSSSS

fU fUn001 fG fA fU
GAU
SnXSS

WV-19911
fC fU fCn001 fA fU fAn001 fC fC mU fU mC fU mG fC fU
CUCAUACCUUCUGCUUG
SSnXSS nXSSSS SSSSS

fU fGn001 fA fU fG
AUG
SnXSS

WV-19912
fU fC fAn001 fU fA fCn001 fC fU mU fC mU fG mC fU fU
UCAUACCUUCUGCUUGA
SSnXSS nXSSSS SSSSS

fG fAn001 fU fG fA
UGA
SnXSS

WV-19913
fC fA fUn001 fA fC fCn001 fU fU mC fU mG fC mU fU fG
CAUACCUUCUGCUUGAU
SSnXSS nXSSSS SSSSS

fA fUn001 fG fA fU
GAU
SnXSS

WV-19914
fA fU fAn001 fC fC fUn001 fU fC mU fG mC fU mU fG fA
AUACCUUCUGCUUGAUG
SSnXSS nXSSSS SSSSS

fU fGn001 fA fU fC
AUC
SnXSS

WV-19915
fU fA fCn001 fc fU fUn001 fC fU mG fC mU fU mG fA fU
UACCUUCUGCUUGAUGA
SSnXSS nXSSSS SSSSS

fG fAn001 fU fC fA
UCA
SnXSS

WV-19916
fA fC fCn001 fU fU fCn001 fU fG mC fU mU fG mA fU fG
ACCUUCUGCUUGAUGAU
SSnXSS nXSSSS SSSSS

fA fUn001 fC fA fU
CAU
SnXSS

WV-19917
fC fC fUn001 fU fC fUn001 fG fC mU fU mG fA mU fG fA
CCUUCUGCUUGAUGAUC
SSnXSS nXSSSS SSSSS

fU fCn001 fA fU fC
AUC
SnXSS

WV-19918
fC fU fUn001 fC fU fGn001 fC fU mU fG mA fU mG fA fU
CUUCUGCUUGAUGAUCA
SSnXSS nXSSSS SSSSS

fC fAn001 fU fC fU
UCU
SnXSS

WV-19919
fU fU fCn001 fU fG fCn001 fU fU mG fA mU fG mA fU fC
UUCUGCUUGAUGAUCAU
SSnXSS nXSSSS SSSSS

fA fUn001 fC fU fC
CUC
SnXSS

WV-19920
fU fC fUn001 fG fC fUn001 fU fG mA fU mG fA mU fC fA
UCUGCUUGAUGAUCAUC
SSnXSS nXSSSS SSSSS

fU fCn001 fU fC fG
UCG
SnXSS

WV-19921
fC fU fGn001 fC fU fUn001 fG fA mU fG mA fU mC fA fU
CUGCUUGAUGAUCAUCU
SSnXSS nXSSSS SSSSS

fC fUn001 fC fG fU
CGU
SnXSS

WV-19922
fU fU fCn001 fU fU fGn001 fA fU mG fA mU fC mA fU fC
UGCUUGAUGAUCAUCUC
SSnXSS nXSSSS SSSSS

fU fCn001 fG fU fU
GUU
SnXSS

WV-19923
fG fC fUn001 fU fG fAn001 fU fG mA fU mC fA mU fC fU
GCUUGAUGAUCAUCUCG
SSnXSS nXSSSS SSSSS

fC fGn001 fU fU fG
UUG
SnXSS

WV-19924
fC mU fUn001 fG fA fU fUn001 fG fA mU fC mA fU mC fU fC
CUUGAUGAUCAUCUCGU
SSnXSS nXSSSS SSSSS

fG fUn001 fU fG fA
UGA
SnXSS

WV-19925
fU fU fGn001 fA fU fGn001 fA fU mC fA mU fC mU fC fG
UUGAUGAUCAUCUCGUU
SSnXSS nXSSSS SSSSS

fU fUn001 fG fA fU
GAU
SnXSS

WV-19926
fU fG fAn001 fU fG fAn001 fU fC mA fU mC fU mC fG fU
UGAUGAUCAUCUCGUUG
SSnXSS nXSSSS SSSSS

fU fGn001 fA fU fA
AUA
SnXSS

WV-19927
fG fA fUn001 fG fA fUn001 fC fA mU fC mU fC mG fU fU
GAUGAUCAUCUCGUUGA
SSnXSS nXSSSS SSSSS

fG fAn001 fU fA fU
UAU
SnXSS

WV-19928
fA fU fGn001 fA fU fCn001 fA fU mC fU mC fG mU fU fG
AUGAUCAUCUCGUUGAU
SSnXSS nXSSSS SSSSS

fA fUn001 fA fU fC
AUC
SnXSS

WV-19929
fU fG fAn001 fU fC fAn001 fU fC mU fC mG fU mU fG fA
UGAUCAUCUCGUUGAUA
SSnXSS nXSSSS SSSSS

fU fAn001 fU fC fC
UCC
SnXSS

WV-19930
fG fA fUn001 fC fA fUn001 fC fU mC fG mU fU mG fA fU
GAUCAUCUCGUUGAUAU
SSnXSS nXSSSS SSSSS

fA fUn001 fC fC fU
CCU
SnXSS

WV-19931
fA fU fCn001 fA fU fCn001 fU fC mG fU mU fG mA fU fA
AUCAUCUCGUUGAUAUC
SSnXSS nXSSSS SSSSS

fU fCn001 fC fU fC
CUC
SnXSS

WV-19932
fU fC fAn001 fU fC fUn001 fC fG mU fU mG fA mU fA fU
UCAUCUCGUUGAUAUCC
SSnXSS nXSSSS SSSSS

fC fCn001 fU fC fA
UCA
SnXSS

WV-19933
fC fA fUn001 fC fu fCn001 fG fU mU fG mA fU mA fU fC
CAUCUCGUUGAUAUCCU
SSnXSS nXSSSS SSSSS

fC fUn001 fC fA fA
CAA
SnXSS

WV-19934
fA fU fCn001 fU fC fGn001 fU fU mG fA mU fA mU fC fC
AUCUCGUUGAUAUCCUC
SSnXSS nXSSSS SSSSS

fU fCn001 fA fA fG
AAG
SnXSS

WV-19935
fU fC fUn001 fC fG fUn001 fU fG mA fU mA fU mC fC fU
UCUCGUUGAUAUCCUCA
SSnXSS nXSSSS SSSSS

fC fAn001 fA fG fG
AGG
SnXSS

WV-19936
fC fU fCn001 fG fU fUn001 fG fA mU fA mU fC mC fU fC
CUCGUUGAUAUCCUCAA
SSnXSS nXSSSS SSSSS

fA fAn001 fG fG fU
GGU
SnXSS

WV-19937
fU fC fGn001 fU fU fGn001 fA fU mA fU mC fC mU fC fA
UCGUUGAUAUCCUCAAG
SSnXSS nXSSSS SSSSS

fA fGn001 fG fU fC
GUC
SnXSS

WV-19938
fC fG fUn001 fU fG fAn001 fU fA mU fC mC fU mC fA fA
CGUUGAUAUCCUCAAGG
SSnXSS nXSSSS SSSSS

fG fGn001 fU fC fA
UCA
SnXSS

WV-19939
fG fU fUn001 fG fA fUn001 fA fU mC fC mU fC mA fA fG
GUUGAUAUCCUCAAGGU
SSnXSS nXSSSS SSSSS

fG fUn001 fC fA fC
CAC
SnXSS

WV-19940
fU fU fGn001 fA fU fAn001 fU fC mC fU mC fA mA fG fG
UUGAUAUCCUCAAGGUC
SSnXSS nXSSSS SSSSS

fU fCn001 fA fC fC
ACC
SnXSS

WV-19941
fU fG fAn001 fU fA fUn001 fC fC mU fC mA fA mG fG fU
UGAUAUCCUCAAGGUCA
SSnXSS nXSSSS SSSSS

fC fAn001 fC fC fC
CCC
SnXSS

WV-19942
fG fA fUn001 fA fU fCn001 fC fU mC fA mA fG mG fU fC
GAUAUCCUCAAGGUCAC
SSnXSS nXSSSS SSSSS

fA fCn001 fC fC fA
CCA
SnXSS

WV-19943
fA fU fAn001 fU fC fCn001 fU fC mA fA mG fG mU fC fA
AUAUCCUCAAGGUCACC
SSnXSS nXSSSS SSSSS

fC fUn001 fC fA fC
CAC
SnXSS

WV-19944
fU fA fUn001 fC fC fUn001 fC fA mA fG mG fU mC fA fC
UAUCCUCAAGGUCACCC
SSnXSS nXSSSS SSSSS

fC fCn001 fA fC fC
ACC
SnXSS

WV-19945
fA fU fCn001 fC fU fCn001 fA fA mG fG mU fC mA fC fC
AUCCUCAAGGUCACCCA
SSnXSS nXSSSS SSSSS

fC fAn001 fC fC fA
CCA
SnXSS

WV-19946
fU fC fCn001 fU fC fAn001 fA fG mG fU mC fA mC fC fC
UCCUCAAGGUCACCCACC
SSnXSS nXSSSS SSSSS

fA fCn001 fC fA fU
AU
SnXSS

WV-19947
fC fC fUn001 fC fA fAn001 fG fG mU fC mA fC mC fC fA
CCUCAAGGUCACCCACCA
SSnXSS nXSSSS SSSSS

fC fCn001 fA fU fC
UC
SnXSS

WV-19948
fC fU fCn001 fA fA fGn001 fG fU mC fA mC fC mC fA fC
CUCAAGGUCACCCACCA
SSnXSS nXSSSS SSSSS

fC fAn001 fU fC fA
UCA
SnXSS

WV-19949
fU fC fAn001 fA fG fGn001 fU fC mA fC mC fC mA fC fC
UCAAGGUCACCCACCAU
SSnXSS nXSSSS SSSSS

fA fUn001 fC fA fC
CAC
SnXSS

WV-19950
fC fA fAn001 fG fG fUn001 fC fA mC fC mC fA mC fC fA
CAAGGUCACCCACCAUC
SSnXSS nXSSSS SSSSS

fU fCn001 fA fC fC
ACC
SnXSS

WV-19951
fA fA fGn001 fG fU fCn001 fA fC mC fC mA fC mC fA fU
AAGGUCACCCACCAUCA
SSnXSS nXSSSS SSSSS

fC fAn001 fC fC fC
CCC
SnXSS

WV-19952
fA fG fGn001 fU fC fAn001 fC fC mC fA mC fC mA fU fC
AGGUCACCCACCAUCACC
SSnXSS nXSSSS SSSSS

fA fCn001 fC fC fU
CU
SnXSS

WV-19953
fG fG fUn001 fC fA fCn001 fC fC mA fC mC fA mU fC fA
GGUCACCCACCAUCACCC
SSnXSS nXSSSS SSSSS

fC fCn001 fC fU fC
UC
SnXSS

WV-19954
fG fU fCn001 fA fC fCn001 fC fA mC fC mA fU mC fA fC
GUCACCCACCAUCACCCU
SSnXSS nXSSSS SSSSS

fC fCn001 fU fC fU
CU
SnXSS

WV-19955
fU fC fAn001 fC fC fCn001 fA fC mC fA mU fC mA fC fC
UCACCCACCAUCACCCUC
SSnXSS nXSSSS SSSSS

fC fUn001 fC fU fG
UG
SnXSS

WV-19956
fC fA fCn001 fC fC fAn001 fC fC mA fU mC fA mC fC fC
CACCCACCAUCACCCUCU
SSnXSS nXSSSS SSSSS

fU fCn001 fU fG fU
GU
SnXSS

WV-19957
fA fC fCn001 fC fA fCn001 fC fA mU fC mA fC mC fC fU
ACCCACCAUCACCCUCUG
SSnXSS nXSSSS SSSSS

fC fUn001 fG fU fG
UG
SnXSS

WV-19958
fC fC fCn001 fA fC fCn001 fA fU mC fA mC fC mC fU fC
CCCACCAUCACCCUCUGU
SSnXSS nXSSSS SSSSS

fU fGn001 fU fG fA
GA
SnXSS

WV-19959
fC fC fAn001 fC fC fAn001 fU fC mA fC mC fC mU fC fU
CCACCAUCACCCUCUGUG
SSnXSS nXSSSS SSSSS

fG fUn001 fG fA fU
AU
SnXSS

WV-19960
fC fA fCn001 fC fA fUn001 fC fA mC fC mC fU mC fU fG
CACCAUCACCCUCUGUG
SSnXSS nXSSSS SSSSS

fU fGn001 fA fU fU
AUU
SnXSS

WV-19961
fA fC fCn001 fA fU fUn001 fA fC mC fC mU fC mU fG fU
ACCAUCACCCUCUGUGA
SSnXSS nXSSSS SSSSS

fG fAn001 fU fU fU
UUU
SnXSS

WV-19962
fC fC fAn001 fU fC fAn001 fC fC mC fU mC fU mG fU fG
CCAUCACCCUCUGUGAU
SSnXSS nXSSSS SSSSS

fA fUn001 fU fU fU
UUU
SnXSS

WV-19963
fC fA fUn001 fC fA fCn001 fC fC mU fC mU fG mU fG fA
CAUCACCCUCUGUGAUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fU fA
UUA
SnXSS

WV-19964
fA fU fCn001 fA fC fCn001 fC fU mC fU mG fU mG fA fU
AUCACCCUCUGUGAUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fA fU
UAU
SnXSS

WV-19965
fU fC fAn001 fC fC fCn001 fU fC mU fG mU fG mA fU fU
UCACCCUCUGUGAUUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fA fU fA
AUA
SnXSS

WV-19966
fC fA fCn001 fC fC fUn001 fC fU mG fU mG fA mU fU fU
CACCCUCUGUGAUUUUA
SSnXSS nXSSSS SSSSS

fU fAn001 fU fA fA
UAA
SnXSS

WV-19967
fA fC fCn001 fC fU fCn001 fU fG mU fG mA fU mU fU fU
ACCCUCUGUGAUUUUAU
SSnXSS nXSSSS SSSSS

fA fUn001 fA fA fC
AAC
SnXSS

WV-19968
fC fC fCn001 fU fC fUn001 fG fU mG fA mU fU mU fU fA
CCCUCUGUGAUUUUAUA
SSnXSS nXSSSS SSSSS

fU fAn001 fA fC fU
ACU
SnXSS

WV-19969
fC fC fUn001 fC fU fGn001 fU fG mA fU mU fU mU fA fU
CCUCUGUGAUUUUAUAA
SSnXSS nXSSSS SSSSS

fA fAn001 fC fU fU
CUU
SnXSS

WV-19970
fC fU fCn001 fU fG fUn001 fG fA mU fU mU fU mA fU fA
CUCUGUGAUUUUAUAAC
SSnXSS nXSSSS SSSSS

fA fCn001 fU fU fG
UUG
SnXSS

WV-19971
fU fC fUn001 fG fU fGn001 fA fU mU fU mU fA mU fA fA
UCUGUGAUUUUAUAACU
SSnXSS nXSSSS SSSSS

fC fUn001 fU fG fA
UGA
SnXSS

WV-19972
fC fU fGn001 fU fG fAn001 fU fU mU fU mA fU mA fA fC
CUGUGAUUUUAUAACUU
SSnXSS nXSSSS SSSSS

fU fUn001 fG fA fU
GAU
SnXSS

WV-19973
fU fG fUn001 fG fA fUn001 fU fU mU fA mU fA mA fC fU
UGUGAUUUUAUAACUUG
SSnXSS nXSSSS SSSSS

fU fGn001 fA fU fC
AUC
SnXSS

WV-19974
fG fU fGn001 fA fU fUn001 fU fU mA fU mA fA mC fU fU
GUGAUUUUAUAACUUGA
SSnXSS nXSSSS SSSSS

fG fAn001 fU fC fA
UCA
SnXSS

WV-19975
fU fG fAn001 fU fU fUn001 fU fA mU fA mA fC mU fU fG
UGAUUUUAUAACUUGAU
SSnXSS nXSSSS SSSSS

fA fUn001 fC fA fA
CAA
SnXSS

WV-19976
fG fA fUn001 fU fU fUn001 fA fU mA fA mC fU mU fG fA
GAUUUUAUAACUUGAUC
SSnXSS nXSSSS SSSSS

fU fCn001 fA fA fG
AAG
SnXSS

WV-19977
fA fU fUn001 fU fU fAn001 fU fA mA fC mU fU mG fA fU
AUUUUAUAACUUGAUCA
SSnXSS nXSSSS SSSSS

fC fAn001 fA fG fC
AGC
SnXSS

WV-19978
fU fU fUn001 fU fA fUn001 fA fA mC fU mU fG mA fU fC
UUUUAUAACUUGAUCAA
SSnXSS nXSSSS SSSSS

fA fAn001 fG fC fA
GCA
SnXSS

WV-19979
fU fU fUn001 fA fU fAn001 fA fC mU fU mG fA mU fC fA
UUUAUAACUUGAUCAAG
SSnXSS nXSSSS SSSSS

fA fGn001 fC fA fG
CAG
SnXSS

WV-19980
fU fU fAn001 fU fA fAn001 fC fU mU fG mA fU mC fA fA
UUAUAACUUGAUCAAGC
SSnXSS nXSSSS SSSSS

fG fCn001 fA fG fA
AGA
SnXSS

WV-19981
fU fA fUn001 fA fA fCn001 fU fU mG fA mU fC mA fA fG
UAUAACUUGAUCAAGCA
SSnXSS nXSSSS SSSSS

fC fAn001 fG fA fG
GAG
SnXSS

WV-19982
fA fU fAn001 fA fC fUn001 fU fG mA fU mC fA mA fG fC
AUAACUUGAUCAAGCAG
SSnXSS nXSSSS SSSSS

fA fGn001 fA fG fA
AGA
SnXSS

WV-19983
fU fA fAn001 fC fU fUn001 fG fA mU fC mA fA mG fC fA
UAACUUGAUCAAGCAGA
SSnXSS nXSSSS SSSSS

fG fAn001 fG fA fA
GAA
SnXSS

WV-19984
fA fA fCn001 fU fU fGn001 fA fU mC fA mA fG mC fA fG
AACUUGAUCAAGCAGAG
SSnXSS nXSSSS SSSSS

fA fGn001 fA fA fA
AAA
SnXSS

WV-19985
fA fC fUn001 fU fG fAn001 fU fC mA fA mG fC mA fG fA
ACUUGAUCAAGCAGAGA
SSnXSS nXSSSS SSSSS

fG fAn001 fA fA fG
AAG
SnXSS

WV-19986
fC fU fUn001 fG fA fUn001 fC fA mA fG mC fA mG fA fG
CUUGAUCAAGCAGAGAA
SSnXSS nXSSSS SSSSS

fA fAn001 fA fG fC
AGC
SnXSS

WV-19987
fU fU fGn001 fA fU fCn001 fA fA mG fC mA fG mA fG fA
UUGAUCAAGCAGAGAAA
SSnXSS nXSSSS SSSSS

fA fAn001 fG fC fC
GCC
SnXSS

WV-19988
fU fG fAn001 fU fC fAn001 fA fG mC fA mG fA mG fA fA
UGAUCAAGCAGAGAAAG
SSnXSS nXSSSS SSSSS

fA fGn001 fC fC fA
CCA
SnXSS

WV-19989
fG fA fUn001 fC fA fAn001 fG fC mA fG mA fG mA fA fA
GAUCAAGCAGAGAAAGC
SSnXSS nXSSSS SSSSS

fG fCn001 fC fA fG
CAG
SnXSS

WV-19990
fA fU fCn001 fA fA fGn001 fC fA mG fA mG fA mA fA fG
AUCAAGCAGAGAAAGCC
SSnXSS nXSSSS SSSSS

fC fCn001 fA fG fU
AGU
SnXSS

WV-19991
fU fC fAn001 fA fG fCn001 fA fG mA fG mA fA mA fG fC
UCAAGCAGAGAAAGCCA
SSnXSS nXSSSS SSSSS

fC fAn001 fG fU fC
GUC
SnXSS

WV-19992
fC fA fAn001 fG fC fAn001 fG fA mG fA mA fA mG fC fC
CAAGCAGAGAAAGCCAG
SSnXSS nXSSSS SSSSS

fA fGn001 fU fC fG
UCG
SnXSS

WV-19993
fA fA fGn001 fC fA fGn001 fA fG mA fA mA fG mC fC fA
AAGCAGAGAAAGCCAGU
SSnXSS nXSSSS SSSSS

fG fUn001 fC fG fG
CGG
SnXSS

WV-19994
fA fG fCn001 fA fG fAn001 fG fA mA fA mG fC mC fA fG
AGCAGAGAAAGCCAGUC
SSnXSS nXSSSS SSSSS

fU fCn001 fG fG fU
GGU
SnXSS

WV-19995
fG fC fAn001 fG fA fGn001 fA fA mA fG mC fC mA fG fU
GCAGAGAAAGCCAGUCG
SSnXSS nXSSSS SSSSS

fC fGn001 fG fU fA
GUA
SnXSS

WV-19996
fC fA fGn001 fA fG fAn001 fA fA mG fC mC fA mG fU fC
CAGAGAAAGCCAGUCGG
SSnXSS nXSSSS SSSSS

fG fGn001 fU fA fA
UAA
SnXSS

WV-19997
fA fG fAn001 fG fA fAn001 fA fG mC fC mA fG mU fC fG
AGAGAAAGCCAGUCGGU
SSnXSS nXSSSS SSSSS

fG fUn001 fA fA fG
AAG
SnXSS

WV-19998
fG fA fGn001 fA fA fAn001 fG fC mC fA mG fU mC fG fG
GAGAAAGCCAGUCGGUA
SSnXSS nXSSSS SSSSS

fU fAn001 fA fG fU
AGU
SnXSS

WV-19999
fA fG fAn001 fA fA fGn001 fC fC mA fG mU fC mG fG fU
AGAAAGCCAGUCGGUAA
SSnXSS nXSSSS SSSSS

fA fAn001 fG fU fU
GUU
SnXSS

WV-20000
fG fA fAn001 fA fG fCn001 fC fA mG fU mC fG mG fU fA
GAAAGCCAGUCGGUAAG
SSnXSS nXSSSS SSSSS

fA fGn001 fU fU fC
UUC
SnXSS

WV-20001
fA fA fAn001 fG fC fCn001 fA fG mU fC mG fG mU fA fA
AAAGCCAGUCGGUAAGU
SSnXSS nXSSSS SSSSS

fG fUn001 fU fC fU
UCU
SnXSS

WV-20002
fA fA fGn001 fC fC fAn001 fG fU mC fG mG fU mA fA fG
AAGCCAGUCGGUAAGUU
SSnXSS nXSSSS SSSSS

fU fUn001 fC fU fG
CUG
SnXSS

WV-20003
fA fG fCn001 fC fA fGn001 fU fC mG fG mU fA mA fG fU
AGCCAGUCGGUAAGUUC
SSnXSS nXSSSS SSSSS

fU fCn001 fU fG fU
UGU
SnXSS

WV-20004
fG fC fCn001 fA fG fUn001 fC fG mG fU mA fA mG fU fU
GCCAGUCGGUAAGUUCU
SSnXSS nXSSSS SSSSS

fC fUn001 fG fU fC
GUC
SnXSS

WV-20005
fC fC fAn001 fG fU fCn001 fG fG mU fA mA fG mU fU fC
CCAGUCGGUAAGUUCUG
SSnXSS nXSSSS SSSSS

fU fGn001 fU fC fC
UCC
SnXSS

WV-20006
fC fA fGn001 fU fC fGn001 fG fU mA fA mG fU mU fC fU
CAGUCGGUAAGUUCUGU
SSnXSS nXSSSS SSSSS

fG fUn001 fC fC fA
CCA
SnXSS

WV-20007
fA fG fUn001 fC fG fGn001 fU fA mA fG mU fU mC fU fG
AGUCGGUAAGUUCUGUC
SSnXSS nXSSSS SSSSS

fU fCn001 fC fA fA
CAA
SnXSS

WV-20008
fG fU fCn001 fG fG fUn001 fA fA mG fU mU fC mU fG fU
GUCGGUAAGUUCUGUCC
SSnXSS nXSSSS SSSSS

fC fCn001 fA fA fG
AAG
SnXSS

WV-20009
fU fC fGn001 fG fU fAn001 fA fG mU fU mC fU mG fU fC
UCGGUAAGUUCUGUCCA
SSnXSS nXSSSS SSSSS

fC fAn001 fA fG fC
AGC
SnXSS

WV-20010
fC fG fGn001 fU fA fAn001 fG fU mU fC mU fG mU fC fC
CGGUAAGUUCUGUCCAA
SSnXSS nXSSSS SSSSS

fA fAn001 fG fC fC
GCC
SnXSS

WV-2001
fG fG fUn001 fA fA fGn001 fU fU mC fU mG fU mC fC fA
GGUAAGUUCUGUCCAAG
SSnXSS nXSSSS SSSSS

fA fGn001 fC fC fC
CCC
SnXSS

WV-20012
fG fU fAn001 fA fG fUn001 fU fC mU fG mU fC mC fA fA
GUAAGUUCUGUCCAAGC
SSnXSS nXSSSS SSSSS

fG fCn001 fC fC fG
CCG
SnXSS

WV-20013
fG fA fAn001 fG fU fUn001 fC fU mG fU mC fC mA fA fG
UAAGUUCUGUCCAAGCC
SSnXSS nXSSSS SSSSS

fC fCn001 fC fG fG
CGG
SnXSS

WV-20014
fA fA fGn001 fU fU fCn001 fU fG mU fC mC fA mA fG fC
AAGUUCUGUCCAAGCCC
SSnXSS nXSSSS SSSSS

fC fCn001 fG fG fU
GGU
SnXSS

WV-20015
fA fG fUn001 fU fC fUn001 fG fU mC fC mA fA mG fC fC
AGUUCUGUCCAAGCCCG
SSnXSS nXSSSS SSSSS

fC fGn001 fG fU fU
GUU
SnXSS

WV-20016
fG fU fUn001 fC fU fGn001 fU fC mC fA mA fG mC fC fC
GUUCUGUCCAAGCCCGG
SSnXSS nXSSSS SSSSS

fG fGn001 fU fU fG
UUG
SnXSS

WV-20017
fU fU fCn001 fU fG fUn001 fC fC mA fA mG fC mC fC fG
UUCUGUCCAAGCCCGGU
SSnXSS nXSSSS SSSSS

fG fUn001 fU fG fA
UGA
SnXSS

WV-20018
fU fC fUn001 fG fU fCn001 fC fA mA fG mC fC mC fG fG
UCUGUCCAAGCCCGGUU
SSnXSS nXSSSS SSSSS

fU fUn001 fG fA fA
GAA
SnXSS

WV-20019
fC fU fGn001 fU fC fCn001 fA fA mG fC mC fC mG fU fU
CUGUCCAAGCCCGGUUG
SSnXSS nXSSSS SSSSS

fU fGn001 fA fA fA
AAA
SnXSS

WV-20020
fU fG fUn001 fC fC fAn001 fA fG mC fC mC fG mG fU fU
UGUCCAAGCCCGGUUGA
SSnXSS nXSSSS SSSSS

fG fAn001 fA fA fU
AAU
SnXSS

WV-20021
fG fU fCn001 fC fA fAn001 fG fC mC fC mG fG mU fU fG
GUCCAAGCCCGGUUGAA
SSnXSS nXSSSS SSSSS

fA fAn001 fA fU fC
AUC
SnXSS

WV-20022
fU fC fCn001 fA fA fGn001 fC fC mC fG mG fU mU fG fA
UCCAAGCCCGGUUGAAA
SSnXSS nXSSSS SSSSS

fA fAn001 fU fC fU
UCU
SnXSS

WV-20023
fC fC fAn001 fA fG fCn001 fC fC mG fG mU fU mG fA fA
CCAAGCCCGGUUGAAAU
SSnXSS nXSSSS SSSSS

fA fUn001 fC fU fG
CUG
SnXSS

WV-20024
fC fA fAn001 fG fC fCn001 fC fG mG fU mU fG mA fA fA
CAAGCCCGGUUGAAAUC
SSnXSS nXSSSS SSSSS

fU fCn001 fU fG fC
UGC
SnXSS

WV-20025
fA fA fGn001 fC fC fCn001 fG fG mU fU mG fA mA fA fU
AAGCCCGGUUGAAAUCU
SSnXSS nXSSSS SSSSS

fC fUn001 fG fC fC
GCC
SnXSS

WV-20026
fA fG fCn001 fC fC fGn001 fG fU mU fG mA fA mA fU fC
AGCCCGGUUGAAAUCUG
SSnXSS nXSSSS SSSSS

fU fGn001 fC fC fA
CCA
SnXSS

WV-20027
fG fC fCn001 fC fG fGn001 fU fU mG fA mA fA mU fC fU
GCCCGGUUGAAAUCUGC
SSnXSS nXSSSS SSSSS

fG fCn001 fC fA fG
CAG
SnXSS

WV-20028
fC fC fCn001 fG fG fUn001 fU fG mA fA mA fU mC fU fG
CCCGGUUGAAAUCUGCC
SSnXSS nXSSSS SSSSS

fC fCn001 fA fG fA
AGA
SnXSS

WV-20029
fC fC fGn001 fG fU fUn001 fG fA mA fA mU fC mU fG fC
CCGGUUGAAAUCUGCCA
SSnXSS nXSSSS SSSSS

fC fAn001 fG fA fG
GAG
SnXSS

WV-20030
fC fG fGn001 fU fU fGn001 fA fA mA fU mC fU mG fC fC
CGGUUGAAAUCUGCCAG
SSnXSS nXSSSS SSSSS

fA fGn001 fA fG fC
AGC
SnXSS

WV-20031
fG fG fUn001 fU fG fAn001 fA fA mU fC mU fG mC fC fA
GGUUGAAAUCUGCCAGA
SSnXSS nXSSSS SSSSS

fG fAn001 fG fC fA
GCA
SnXSS

WV-20032
fG fU fUn001 fG fA fAn001 fA fU mC fU mG fC mC fA fG
GUUGAAAUCUGCCAGAG
SSnXSS nXSSSS SSSSS

fA fGn001 fC fA fG
CAG
SnXSS

WV-20033
fU fU fGn001 fA fA fAn001 fU fC mU fG mC fC mA fG fA
UUGAAAUCUGCCAGAGC
SSnXSS nXSSSS SSSSS

fG fCn001 fA fG fG
AGG
SnXSS

WV-20034
fU fG fAn001 fA fA fUn001 fC fU mG fC mC fA mG fA fG
UGAAAUCUGCCAGAGCA
SSnXSS nXSSSS SSSSS

fC fAn001 fG fG fU
GGU
SnXSS

WV-20035
fG fA fAn001 fA fU fCn001 fU fG mC fC mA fG mA fG fC
GAAAUCUGCCAGAGCAG
SSnXSS nXSSSS SSSSS

fA fGn001 fG fU fA
GUA
SnXSS

WV-20036
fA fA fAn001 fU fC fUn001 fG fC mC fA mG fA mG fC fA
AAAUCUGCCAGAGCAGG
SSnXSS nXSSSS SSSSS

fG fGn001 fU fA fC
UAC
SnXSS

WV-20037
fA fA fUn001 fC fU fGn001 fC fC mA fG mA fG mC fA fG
AAUCUGCCAGAGCAGGU
SSnXSS nXSSSS SSSSS

fG fUn001 fA fC fC
ACC
SnXSS

WV-20038
fA fU fCn001 fU fG fCn001 fC fA mG fA mG fC mA fG fG
AUCUGCCAGAGCAGGUA
SSnXSS nXSSSS SSSSS

fU fAn001 fC fC fU
CCU
SnXSS

WV-20039
fU fC fUn001 fG fC fCn001 fA fG mA fG mC fA mG fG fU
UCUGCCAGAGCAGGUAC
SSnXSS nXSSSS SSSSS

fA fCn001 fC fU fC
CUC
SnXSS

WV-20040
fC fU fGn001 fC fC fAn001 fG fA mG fC mA fG mG fU fA
CUGCCAGAGCAGGUACC
SSnXSS nXSSSS SSSSS

fC fCn001 fU fC fC
UCC
SnXSS

WV-20041
fU fG fCn001 fC fA fGn001 fA fG mC fA mG fG mU fA fC
UGCCAGAGCAGGUACCU
SSnXSS nXSSSS SSSSS

fC fUn001 fC fC fA
CCA
SnXSS

WV-20042
fG fC fCn001 fA fG fAn001 fG fC mA fG mG fU mA fC fC
GCCAGAGCAGGUACCUC
SSnXSS nXSSSS SSSSS

fU fCn001 fC fA fA
CAA
SnXSS

WV-20043
fC fC fAn001 fG fA fGn001 fC fA mG fG mU fA mC fC fU
CCAGAGCAGGUACCUCC
SSnXSS nXSSSS SSSSS

fC fCn001 fA fA fC
AAC
SnXSS

WV-20044
fC fA fGn001 fA fG fCn001 fA fG mG fU mA fC mC fU fC
CAGAGCAGGUACCUCCA
SSnXSS nXSSSS SSSSS

fC fAn001 fA fC fA
ACA
SnXSS

WV-20045
fA fG fAn001 fG fC fAn001 fG fG mU fA mC fC mU fC fC
AGAGCAGGUACCUCCAA
SSnXSS nXSSSS SSSSS

fA fAn001 fC fA fU
CAU
SnXSS

WV-20046
fG fA fGn001 fC fA fGn001 fG fU mA fC mC fU mC fC fA
GAGCAGGUACCUCCAAC
SSnXSS nXSSSS SSSSS

fA fCn001 fA fU fC
AUC
SnXSS

WV-20047
fA fG fCn001 fA fG fGn001 fU fA mC fC mU fC mC fA fA
AGCAGGUACCUCCAACA
SSnXSS nXSSSS SSSSS

fC fAn001 fU fC fA
UCA
SnXSS

WV-20048
fG fC fAn001 fG fG fUn001 fA fC mC fU mC fC mA fA fC
GCAGGUACCUCCAACAU
SSnXSS nXSSSS SSSSS

fA fUn001 fC fA fA
CAA
SnXSS

WV-20049
fC fA fGn001 fG fU fAn001 fC fC mU fC mC fA mA fC fA
CAGGUACCUCCAACAUC
SSnXSS nXSSSS SSSSS

fU fCn001 fA fA fG
AAG
SnXSS

WV-20050
fA fG fGn001 fU fA fCn001 fC fU mC fC mA fA mC fA fU
AGGUACCUCCAACAUCA
SSnXSS nXSSSS SSSSS

fC fAn001 fA fG fG
AGG
SnXSS

WV-20051
fG fG fUn001 fA fC fCn001 fU fC mC fA mA fC mA fU fC
GGUACCUCCAACAUCAA
SSnXSS nXSSSS SSSSS

fA fAn001 fG fG fA
GGA
SnXSS

WV-20052
fG fU fAn001 fC fC fUn001 fC fC mA fA mC fA mU fC fA
GUACCUCCAACAUCAAG
SSnXSS nXSSSS SSSSS

fA fGn001 fG fA fA
GAA
SnXSS

WV-20053
fU fA fCn001 fC fU fCn001 fC fA mA fC mA fU mC fA fA
UACCUCCAACAUCAAGG
SSnXSS nXSSSS SSSSS

fG fGn001 fA fA fG
AAG
SnXSS

WV-20054
fA fC fCn001 fU fC fCn001 fA fA mC fA mU fC mA fA fG
ACCUCCAACAUCAAGGA
SSnXSS nXSSSS SSSSS

fG fAn001 fA fG fA
AGA
SnXSS

WV-20055
fC fC fUn001 fC fC fAn001 fA fC mA fU mC fA mA fG fG
CCUCCAACAUCAAGGAA
SSnXSS nXSSSS SSSSS

fA fAn001 fG fA fU
GAU
SnXSS

WV-20056
fC fU fCn001 fC fA fAn001 fC fA mU fC mA fA mG fG fA
CUCCAACAUCAAGGAAG
SSnXSS nXSSSS SSSSS

fA fGn001 fA fU fG
AUG
SnXSS

WV-20057
fU fC fCn001 fA fA fCn001 fA fU mC fA mA fG mG fA fA
UCCAACAUCAAGGAAGA
SSnXSS nXSSSS SSSSS

fG fAn001 fU fG fG
UGG
SnXSS

WV-20058
fC fC fAn001 fA fC fAn001 fU fC mA fA mG fG mA fA fG
CCAACAUCAAGGAAGAU
SSnXSS nXSSSS SSSSS

fA fUn001 fG fG fC
GGC
SnXSS

WV-20059
fC fA fAn001 fC fA fUn001 fC fA mA fG mG fA mA fG fA
CAACAUCAAGGAAGAUG
SSnXSS nXSSSS SSSSS

fU fGn001 fG fC fA
GCA
SnXSS

WV-20060
fA fA fCn001 fA fU fCn001 fA fA mG fG mA fA mG fA fU
AACAUCAAGGAAGAUGG
SSnXSS nXSSSS SSSSS

fG fGn001 fC fA fU
CAU
SnXSS

WV-20061
fA fC fAn001 fU fC fAn001 fA fG mG fA mA fG mA fU fG
ACAUCAAGGAAGAUGGC
SSnXSS nXSSSS SSSSS

fG fCn001 fA fU fU
AUU
SnXSS

WV-20062
fC fA fUn001 fC fA fAn001 fG fG mA fA mG fA mU fG fG
CAUCAAGGAAGAUGGCA
SSnXSS nXSSSS SSSSS

fC fAn001 fU fU fU
UUU
SnXSS

WV-20063
fA fU fCn001 fA fA fGn001 fG fA mA fG mA fU mG fG fC
AUCAAGGAAGAUGGCAU
SSnXSS nXSSSS SSSSS

fA fUn001 fU fU fC
UUC
SnXSS

WV-20064
fU fC fAn001 fA fG fGn001 fA fA mG fA mU fG mG fC fA
UCAAGGAAGAUGGCAUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fC fU
UCU
SnXSS

WV-20065
fC fA fAn001 fG fG fAn001 fA fG mA fU mG fG mC fA fU
CAAGGAAGAUGGCAUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fC fU fA
CUA
SnXSS

WV-20066
fA fA fGn001 fG fA fAn001 fG fA mU fG mG fC mA fU fU
AAGGAAGAUGGCAUUUC
SSnXSS nXSSSS SSSSS

fU fCn001 fU fA fG
UAG
SnXSS

WV-20067
fA fG fGn001 fA fA fGn001 fA fU mG fG mC fA mU fU fU
AGGAAGAUGGCAUUUCU
SSnXSS nXSSSS SSSSS

fC fUn001 fA fG fU
AGU
SnXSS

WV-20068
fG fG fAn001 fA fG fAn001 fU fG mG fC mA fU mU fU fC
GGAAGAUGGCAUUUCUA
SSnXSS nXSSSS SSSSS

fU fAn001 fG fU fU
GUU
SnXSS

WV-20069
fG fA fAn001 fG fA fUn001 fG fG mC fA mU fU mU fC fU
GAAGAUGGCAUUUCUAG
SSnXSS nXSSSS SSSSS

fA fGn001 fU fU fU
UUU
SnXSS

WV-20070
fA fA fGn001 fA fU fGn001 fG fC mA fU mU fU mC fU fA
AAGAUGGCAUUUCUAGU
SSnXSS nXSSSS SSSSS

fG fUn001 fU fU fG
UUG
SnXSS

WV-20071
fA fG fAn001 fU fG fGn001 fC fA mU fU mU fC mU fA fG
AGAUGGCAUUUCUAGUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fG fG
UGG
SnXSS

WV-20072
fG fA fUn001 fG fG fCn001 fA fU mU fU mC fU mA fG fU
GAUGGCAUUUCUAGUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fG fG fA
GGA
SnXSS

WV-20073
fA fU fGn001 fG fC fAn001 fU fU mU fC mU fA mG fU fU
AUGGCAUUUCUAGUUUG
SSnXSS nXSSSS SSSSS

fU fGn001 fG fA fG
GAG
SnXSS

WV-20074
fU fG fGn001 fC fA fUn001 fU fU mC fU mA fG mU fU fU
UGGCAUUUCUAGUUUGG
SSnXSS nXSSSS SSSSS

fG fGn001 fA fG fA
AGA
SnXSS

WV-20075
fG fG fCn001 fA fU fUn001 fU fC mU fA mG fU mU fU fG
GGCAUUUCUAGUUUGGA
SSnXSS nXSSSS SSSSS

fG fAn001 fG fA fU
GAU
SnXSS

WV-20076
fG fC fAn001 fU fU fUn001 fC fU mA fG mU fU mU fG fG
GCAUUUCUAGUUUGGAG
SSnXSS nXSSSS SSSSS

fA fGn001 fA fU fG
AUG
SnXSS

WV-20077
fC fA fUn001 fU fU fCn001 fU fA mG fU mU fU mG fG fA
CAUUUCUAGUUUGGAGA
SSnXSS nXSSSS SSSSS

fG fAn001 fU fG fG
UGG
SnXSS

WV-20078
fA fU fUn001 fU fC fUn001 fA fG mU fU mU fG mG fA fG
AUUUCUAGUUUGGAGAU
SSnXSS nXSSSS SSSSS

fA fUn001 fG fG fC
GGC
SnXSS

WV-20079
fU fU fUn001 fC fU fAn001 fG fU mU fU mG fG mA fG fA
UUUCUAGUUUGGAGAUG
SSnXSS nXSSSS SSSSS

fU fGn001 fG fC fA
GCA
SnXSS

WV-20080
fU fU fCn001 fU fA fGn001 fU fU mU fG mG fA mG fA fU
UUCUAGUUUGGAGAUGG
SSnXSS nXSSSS SSSSS

fG fGn001 fC fA fG
CAG
SnXSS

WV-20081
fU fC fUn001 fA fG fUn001 fU fU mG fG mA fG mA fU fG
UCUAGUUUGGAGAUGGC
SSnXSS nXSSSS SSSSS

fG fCn001 fA fG fU
AGU
SnXSS

WV-20082
fC fU fAn001 fG fU fUn001 fU fG mG fA mG fA mU fG fG
CUAGUUUGGAGAUGGCA
SSnXSS nXSSSS SSSSS

fC fAn001 fG fU fU
GUU
SnXSS

WV-20083
fU fA fGn001 fU fU fUn001 fG fG mA fG mA fU mG fG fC
UAGUUUGGAGAUGGCAG
SSnXSS nXSSSS SSSSS

fA fGn001 fU fU fU
UUU
SnXSS

WV-20084
fA fG fUn001 fU fU fGn001 fG fA mG fA mU fG mG fC fA
AGUUUGGAGAUGGCAGU
SSnXSS nXSSSS SSSSS

fG fUn001 fU fU fC
UUC
SnXSS

WV-20085
fG fU fUn001 fU fG fGn001 fA fG mA fU mG fG mC fA fG
GUUUGGAGAUGGCAGUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fC fC
UCC
SnXSS

WV-20086
fU fU fUn001 fG fG fAn001 fG fA mU fG mG fC mA fG fU
UUUGGAGAUGGCAGUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fC fC fU
CCU
SnXSS

WV-20087
fU fU fGn001 fG fA fGn001 fA fU mG fG mC fA mG fU fU
UUGGAGAUGGCAGUUUC
SSnXSS nXSSSS SSSSS

fU fCn001 fC fU fU
CUU
SnXSS

WV-20088
fU fG fGn001 fA fG fAn001 fU fG mG fC mA fG mU fU fU
UGGAGAUGGCAGUUUCC
SSnXSS nXSSSS SSSSS

fC fCn001 fU fU fA
UUA
SnXSS

WV-20089
fG fG fAn001 fG fA fUn001 fG fG mC fA mG fU mU fU fC
GGAGAUGGCAGUUUCCU
SSnXSS nXSSSS SSSSS

fC fUn001 fU fA fG
UAG
SnXSS

WV-20090
fG fA fGn001 fA fU fGn001 fG fC mA fG mU fU mU fC fC
GAGAUGGCAGUUUCCUU
SSnXSS nXSSSS SSSSS

fU fUn001 fA fG fU
AGU
SnXSS

WV-20091
fA fG fAn001 fU fG fGn001 fC fA mG fU mU fU mC fC fU
AGAUGGCAGUUUCCUUA
SSnXSS nXSSSS SSSSS

fU fAn001 fG fU fA
GUA
SnXSS

WV-20092
fG fA fUn001 fG fG fCn001 fA fG mU fU mU fC mC fU fU
GAUGGCAGUUUCCUUAG
SSnXSS nXSSSS SSSSS

fA fGn001 fU fA fA
UAA
SnXSS

WV-20093
fA fU fGn001 fG fC fAn001 fG fU mU fU mC fC mU fU fA
AUGGCAGUUUCCUUAGU
SSnXSS nXSSSS SSSSS

fG fUn001 fA fA fC
AAC
SnXSS

WV-20094
fU fG fGn001 fC fA fGn001 fU fU mU fC mC fU mU fA fG
UGGCAGUUUCCUUAGUA
SSnXSS nXSSSS SSSSS

fU fAn001 fA fC fC
ACC
SnXSS

WV-20095
fG fG fCn001 fA fG fUn001 fU fU mC fC mU fU mA fG fU
GGCAGUUUCCUUAGUAA
SSnXSS nXSSSS SSSSS

fA fAn001 fC fC fA
CCA
SnXSS

WV-20096
fG fC fAn001 fG fU fUn001 fU fC mC fU mU fA mG fU fA
GCAGUUUCCUUAGUAAC
SSnXSS nXSSSS SSSSS

fA fCn001 fC fA fC
CAC
SnXSS

WV-20097
fC fA fGn001 fU fU fUn001 fC fC mU fU mA fG mU fA fA
CAGUUUCCUUAGUAACC
SSnXSS nXSSSS SSSSS

fC fCn001 fA fC fA
ACA
SnXSS

WV-20098
fA fG fUn001 fU fU fCn001 fC fU mU fA mG fU mA fA fC
AGUUUCCUUAGUAACCA
SSnXSS nXSSSS SSSSS

fC fAn001 fC fA fG
CAG
SnXSS

WV-20099
fG fU fUn001 fU fC fCn001 fU fU mA fG mU fA mA fC fC
GUUUCCUUAGUAACCAC
SSnXSS nXSSSS SSSSS

fA fCn001 fA fG fG
AGG
SnXSS

WV-20100
fU fU fUn001 fC fC fUn001 fU fA mG fU mA fA mC fC fA
UUUCCUUAGUAACCACA
SSnXSS nXSSSS SSSSS

fC fAn001 fG fG fU
GGU
SnXSS

WV-20101
fU fU fCn001 fC fU fUn001 fA fG mU fA mA fC mC fA fC
UUCCUUAGUAACCACAG
SSnXSS nXSSSS SSSSS

fA fGn001 fG fU fU
GUU
SnXSS

WV-20102
fU fC fCn001 fU fU fAn001 fG fU mA fA mC fC mA fC fA
UCCUUAGUAACCACAGG
SSnXSS nXSSSS SSSSS

fG fGn001 fU fU fG
UUG
SnXSS

WV-20103
fC fC fUn001 fU fA fGn001 fU fA mA fC mC fA mC fA fG
CCUUAGUAACCACAGGU
SSnXSS nXSSSS SSSSS

fG fUn001 fU fG fU
UGU
SnXSS

WV-20104
fC fU fUn001 fA fG fUn001 fA fA mC fC mA fC mA fG fG
CUUAGUAACCACAGGUU
SSnXSS nXSSSS SSSSS

fU fUn001 fG fU fG
GUG
SnXSS

WV-20105
fU fU fAn001 fG fU fAn001 fA fC mC fA mC fA mG fG fU
UUAGUAACCACAGGUUG
SSnXSS nXSSSS SSSSS

fU fGn001 fU fG fU
UGU
SnXSS

WV-20106
fU fA fGn001 fU fA fAn001 fC fC mA fC mA fG mG fU fU
UAGUAACCACAGGUUGU
SSnXSS nXSSSS SSSSS

fG fUn001 fG fU fC
GUC
SnXSS

WV-20107
fA fG fUn001 fA fA fCn001 fC fA mC fA mG fG mU fU fG
AGUAACCACAGGUUGUG
SSnXSS nXSSSS SSSSS

fU fGn001 fU fC fA
UCA
SnXSS

WV-20108
fG fU fAn001 fA fC fCn001 fA fC mA fG mG fU mU fG fU
GUAACCACAGGUUGUGU
SSnXSS nXSSSS SSSSS

fG fUn001 fC fA fC
CAC
SnXSS

WV-20109
fU fA fAn001 fC fC fAn001 fC fA mG fG mU fU mG fU fG
UAACCACAGGUUGUGUC
SSnXSS nXSSSS SSSSS

fU fCn001 fA fC fC
ACC
SnXSS

WV-20110
fA fA fCn001 fC fA fCn001 fA fG mG fU mU fG mU fG fU
AACCACAGGUUGUGUCA
SSnXSS nXSSSS SSSSS

fC fAn001 fC fC fA
CCA
SnXSS

WV-20111
fA fC fCn001 fA fC fAn001 fG fG mU fU mG fU mG fU fC
ACCACAGGUUGUGUCAC
SSnXSS nXSSSS SSSSS

fA fCn001 fC fA fG
CAG
SnXSS

WV-20112
fC fC fAn001 fC fA fGn001 fG fU mU fG mU fG mU fC fA
CCACAGGUUGUGUCACC
SSnXSS nXSSSS SSSSS

fC fCn001 fA fG fA
AGA
SnXSS

WV-20113
fC fA fCn001 fA fG fGn001 fU fU mG fU mG fU mC fA fC
CACAGGUUGUGUCACCA
SSnXSS nXSSSS SSSSS

fC fAn001 fG fA fG
GAG
SnXSS

WV-20114
fA fC fAn001 fG fG fUn001 fU fG mU fG mU fC mA fC fC
ACAGGUUGUGUCACCAG
SSnXSS nXSSSS SSSSS

fA fGn001 fA fG fU
AGU
SnXSS

WV-20115
fC fA fGn001 fG fU fUn001 fG fU mG fU mC fA mC fC fA
CAGGUUGUGUCACCAGA
SSnXSS nXSSSS SSSSS

fG fAn001 fG fU fA
GUA
SnXSS

WV-20116
fA fG fGn001 fU fU fGn001 fU fG mU fC mA fC mC fA fG
AGGUUGUGUCACCAGAG
SSnXSS nXSSSS SSSSS

fA fGn001 fU fA fA
UAA
SnXSS

WV-20117
fG fG fUn001 fU fG fUn001 fG fU mC fA mC fC mA fG fA
GGUUGUGUCACCAGAGU
SSnXSS nXSSSS SSSSS

fG fUn001 fA fA fC
AAC
SnXSS

WV-20118
fG fU fUn001 fG fU fUn001 fU fC mA fC mC fA mG fA fG
GUUGUGUCACCAGAGUA
SSnXSS nXSSSS SSSSS

fU fAn001 fA fC fA
ACA
SnXSS

WV-20119
fU fU fGn001 fU fG fUn001 fC fA mC fC mA fG mA fG fU
UUGUGUCACCAGAGUAA
SSnXSS nXSSSS SSSSS

fA fAn001 fC fA fG
CAG
SnXSS

WV-20120
fU fG fUn001 fG fU fCn001 fA fC mC fA mG fA mG fU fA
UGUGUCACCAGAGUAAC
SSnXSS nXSSSS SSSSS

fA fCn001 fA fG fU
AGU
SnXSS

WV-20121
fG fU fUn001 fU fC fAn001 fC fC mA fG mA fG mU fA fA
GUGUCACCAGAGUAACA
SSnXSS nXSSSS SSSSS

fC fAn001 fG fU fC
GUC
SnXSS

WV-20122
fU fG fUn001 fC fA fCn001 fC fA mG fA mG fU mA fA fC
UGUCACCAGAGUAACAG
SSnXSS nXSSSS SSSSS

fA fGn001 fU fC fU
UCU
SnXSS

WV-20123
fG fU fCn001 fA fC fCn001 fA fG mA fG mU fA mA fC fA
GUCACCAGAGUAACAGU
SSnXSS nXSSSS SSSSS

fG fUn001 fC fU fG
CUG
SnXSS

WV-20124
fU fC fAn001 fC fC fAn001 fG fA mG fU mA fA mC fA fG
UCACCAGAGUAACAGUC
SSnXSS nXSSSS SSSSS

fU fCn001 fU fG fA
UGA
SnXSS

WV-20125
fC fA fCn001 fC fA fGn001 fA fG mU fA mA fC mA fG fU
CACCAGAGUAACAGUCU
SSnXSS nXSSSS SSSSS

fC fUn001 fG fA fG
GAG
SnXSS

WV-20126
fA fC fCn001 fA fG fAn001 fG fU mA fA mC fA mG fU fC
ACCAGAGUAACAGUCUG
SSnXSS nXSSSS SSSSS

fU fGn001 fA fG fU
AGU
SnXSS

WV-20127
fC fC fAn001 fG fA fGn001 fU fA mA fC mA fG mU fC fU
CCAGAGUAACAGUCUGA
SSnXSS nXSSSS SSSSS

fG fAn001 fG fU fA
GUA
SnXSS

WV-20128
fC fA fGn001 fA fG fUn001 fA fA mC fA mG fU mC fU fG
CAGAGUAACAGUCUGAG
SSnXSS nXSSSS SSSSS

fA fGn001 fU fA fG
UAG
SnXSS

WV-20129
fA fG fAn001 fG fU fAn001 fA fC mA fG mU fC mU fG fA
AGAGUAACAGUCUGAGU
SSnXSS nXSSSS SSSSS

fG fUn001 fA fG fG
AGG
SnXSS

WV-20130
fG fA fGn001 fU fA fAn001 fC fA mG fU mC fU mG fA fG
GAGUAACAGUCUGAGUA
SSnXSS nXSSSS SSSSS

fU fAn001 fG fG fA
GGA
SnXSS

WV-20131
fA fG fUn001 fA fA fCn001 fA fG mU fC mU fG mA fG fU
AGUAACAGUCUGAGUAG
SSnXSS nXSSSS SSSSS

fA fGn001 fG fA fG
GAG
SnXSS

WV-20132
fG fU fAn001 fA fC fAn001 fG fU mC fU mG fA mG fU fA
GUAACAGUCUGAGUAGG
SSnXSS nXSSSS SSSSS

fG fGn001 fA fG fC
AGC
SnXSS

WV-20133
fU fA fAn001 fC fA fGn001 fU fC mU fG mA fG mU fA fG
UAACAGUCUGAGUAGGA
SSnXSS nXSSSS SSSSS

fG fAn001 fG fC fU
GCU
SnXSS

WV-20134
fA fA fCn001 fA fG fUn001 fC fU mG fA mG fU mA fG fG
AACAGUCUGAGUAGGAG
SSnXSS nXSSSS SSSSS

fA fGn001 fC fU fA
CUA
SnXSS

WV-20135
fA fC fAn001 fG fU fCn001 fU fG mA fG mU fA mG fG fA
ACAGUCUGAGUAGGAGC
SSnXSS nXSSSS SSSSS

fG fCn001 fU fA fA
UAA
SnXSS

WV-20136
fC fA fGn001 fU fC fUn001 fG fA mG fU mA fG mG fA fG
CAGUCUGAGUAGGAGCU
SSnXSS nXSSSS SSSSS

fC fUn001 fA fA fA
AAA
SnXSS

WV-20137
fA fG fUn001 fC fG fGn001 fA fG mU fA mG fG mA fG fC
AGUCUGAGUAGGAGCUA
SSnXSS nXSSSS SSSSS

fU fAn001 fA fA fA
AAA
SnXSS

WV-20138
fG fU fCn001 fU fG fAn001 fG fU mA fG mG fA mG fC fU
GUCUGAGUAGGAGCUAA
SSnXSS nXSSSS SSSSS

fA fAn001 fA fA fU
AAU
SnXSS

WV-20139
fU fC fUn001 fG fA fGn001 fU fA mG fG mA fG mC fU fA
UCUGAGUAGGAGCUAAA
SSnXSS nXSSSS SSSSS

fA fAn001 fA fU fA
AUA
SnXSS

WV-20140
fC fU fGn001 fA fG fUn001 fA fG mG fA mG fC mU fA fA
CUGAGUAGGAGCUAAAA
SSnXSS nXSSSS SSSSS

fA fAn001 fU fA fU
UAU
SnXSS

WV-20141
fU fG fAn001 fG fU fAn001 fG fG mA fG mC fU mA fA fA
UGAGUAGGAGCUAAAAU
SSnXSS nXSSSS SSSSS

fA fUn001 fA fU fU
AUU
SnXSS

WV-20142
fG fA fGn001 fU fA fGn001 fG fA mG fC mU fA mA fA fA
GAGUAGGAGCUAAAAUA
SSnXSS nXSSSS SSSSS

fU fAn001 fU fU fU
UUU
SnXSS

WV-20143
fA fG fUn001 fA fG fGn001 fA fG mC fU mA fA mA fA fU
AGUAGGAGCUAAAAUAU
SSnXSS nXSSSS SSSSS

fA fUn001 fU fU fU
UUU
SnXSS

WV-20144
fG fU fAn001 fG fG fAn001 fG fC mU fA mA fA mA fU fA
GUAGGAGCUAAAAUAUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fU fG
UUG
SnXSS

WV-20145
fU fA fGn001 fG fA fGn001 fC fU mA fA mA fA mU fA fU
UAGGAGCUAAAAUAUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fG fG
UGG
SnXSS

WV-20146
fA fG fGn001 fA fG fCn001 fU fA mA fA mA fU mA fU fU
AGGAGCUAAAAUAUUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fG fG fG
GGG
SnXSS

WV-20147
fG fG fAn001 fG fC fUn001 fA fA mA fA mU fA mU fU fU
GGAGCUAAAAUAUUUUG
SSnXSS nXSSSS SSSSS

fU fGn001 fG fG fU
GGU
SnXSS

WV-20148
fG fA fGn001 fC fU fAn001 fA fA mA fU mA fU mU fU fU
GAGCUAAAAUAUUUUGG
SSnXSS nXSSSS SSSSS

fG fGn001 fG fU fU
GUU
SnXSS

WV-20149
fA fG fCn001 fU fA fAn001 fA fA mU fA mU fU mU fU fG
AGCUAAAAUAUUUUGGG
SSnXSS nXSSSS SSSSS

fG fGn001 fU fU fU
UUU
SnXSS

WV-20150
fG fC fUn001 fA fA fAn001 fA fU mA fU mU fU mU fG fG
GCUAAAAUAUUUUGGGU
SSnXSS nXSSSS SSSSS

fG fUn001 fU fU fU
UUU
SnXSS

WV-20151
fC fU fAn001 fA fA fAn001 fU fA mU fU mU fU mG fG fG
CUAAAAUAUUUUGGGUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fU fU
UUU
SnXSS

WV-20152
fU fA fAn001 fA fA fUn001 fA fU mU fU mU fG mG fG fU
UAAAAUAUUUUGGGUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fU fG
UUG
SnXSS

WV-20153
fA fA fAn001 fA fU fAn001 fU fU mU fU mG fG mG fU fU
AAAAUAUUUUGGGUUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fU fG fC
UGC
SnXSS

WV-20154
fA fA fAn001 fU fA fUn001 fU fU mU fG mG fG mU fU fU
AAAUAUUUUGGGUUUUU
SSnXSS nXSSSS SSSSS

fU fUn001 fG fC fA
GCA
SnXSS

WV-20155
fA fA fUn001 fA fU fUn001 fU fU mG fG mG fU mU fU fU
AAUAUUUUGGGUUUUUG
SSnXSS nXSSSS SSSSS

fU fGn001 fC fA fA
CAA
SnXSS

WV-20156
fA fU fAn001 fU fU fUn001 fU fG mG fG mU fU mU fU fU
AUAUUUUGGGUUUUUGC
SSnXSS nXSSSS SSSSS

fG fCn001 fA fA fA
AAA
SnXSS

WV-20157
fU fA fUn001 fU fU fUn001 fG fG mG fU mU fU mU fU fG
UAUUUUGGGUUUUUGCA
SSnXSS nXSSSS SSSSS

fC fAn001 fA fA fA
AAA
SnXSS

WV-20158
fA fU fUn001 fU fU fGn001 fG fG mU fU mU fU mU fG fC
AUUUUGGGUUUUUGCAA
SSnXSS nXSSSS SSSSS

fA fAn001 fA fA fA
AAA
SnXSS

WV-20159
fU fU fUn001 fU fG fGn001 fG fU mU fU mU fU mG fC fA
UUUUGGGUUUUUGCAAA
SSnXSS nXSSSS SSSSS

fA fAn001 fA fA fG
AAG
SnXSS

WV-20160
fU fU fUn001 fG fG fGn001 fU fU mU fU mU fG mC fA fA
UUUGGGUUUUUGCAAAA
SSnXSS nXSSSS SSSSS

fA fAn001 fA fG fG
AGG
SnXSS

WV-20314
fU fU fC fG fA fA fA fA mA fA mC fA mA fA fU fC fA fA
UUCGAAAAAACAAAUCA
SSSSS SSSSS SSSSS SSSS

fA fG
AAG

WV-20315
fU fC fG fA fA fA fA fA mA fC mA fA mA fU fC fA fA fA
UCGAAAAAACAAAUCAA
SSSSS SSSSS SSSSS SSSS

fG fA
AGA

WV-20316
fC fG fA fA fA fA fA fA mC fA mA fA mU fC fA fA fA fG
CGAAAAAACAAAUCAAA
SSSSS SSSSS SSSSS SSSS

fA fC
GAC

WV-20317
fG fA fA fA fA fA fA fC mA fA mA fU mC fA fA fA fG fA
GAAAAAACAAAUCAAAG
SSSSS SSSSS SSSSS SSSS

fC fU
ACU

WV-20318
fA fA fA fA fA fA fC fA mA fA mU fC mA fA fA fG fA fC
AAAAAACAAAUCAAAGA
SSSSS SSSSS SSSSS SSSS

fU fU
CUU

WV-20319
fA fA fA fA fA fC fA fA mA fU mC fA mA fA fG fA fC fU
AAAAACAAAUCAAAGAC
SSSSS SSSSS SSSSS SSSS

fU fA
UUA

WV-20320
fA fA fA fA fC fA fA fA mU fC mA fA mA fG fA fC fU fU
AAAACAAAUCAAAGACU
SSSSS SSSSS SSSSS SSSS

fA fC
UAC

WV-20321
fA fA fA fC fA fA fA fU mC fA mA fA mG fA fC fU fU fA
AAACAAAUCAAAGACUU
SSSSS SSSSS SSSSS SSSS

fC fC
ACC

WV-20322
fA fA fC fA fA fA fU fC mA fA mA fG mA fC fU fU fA fC
AACAAAUCAAAGACUUA
SSSSS SSSSS SSSSS SSSS

fC fU
CCU

WV-20323
fA fC fA fA fA fU fC fA mA fA mG fA mC fU fU fA fC fC
ACAAAUCAAAGACUUAC
SSSSS SSSSS SSSSS SSSS

fU fU
CUU

WV-20324
fC fA fA fA fU fC fA fA mA fG mA fC mU fU fA fC fC fU
CAAAUCAAAGACUUACC
SSSSS SSSSS SSSSS SSSS

fU fA
UUA

WV-20325
fA fA fA fU fC fA fA fA mG fA mC fU mU fA fC fC fU fU
AAAUCAAAGACUUACCU
SSSSS SSSSS SSSSS SSSS

fA fA
UAA

WV-20326
fA fA fU fC fA fA fA fG mA fC mU fU mA fC fC fU fU fA
AAUCAAAGACUUACCUU
SSSSS SSSSS SSSSS SSSS

fA fG
AAG

WV-20327
fA fU fC fA fA fA fG fA mC fU mU fA mC fC fU fU fA fA
AUCAAAGACUUACCUUA
SSSSS SSSSS SSSSS SSSS

fG fA
AGA

WV-20328
fU fC fA fA fA fG fA fC mU fU mA fC mC fU fU fA fA fG
UCAAAGACUUACCUUAA
SSSSS SSSSS SSSSS SSSS

fA fU
GAU

WV-20329
fC fA fA fA fG fA fC fU mU fA mC fC mU fU fA fA fG fA
CAAAGACUUACCUUAAG
SSSSS SSSSS SSSSS SSSS

fU fA
AUA

WV-20330
fA fA fA fG fA fC fU fU mA fC mC fU mU fA fA fG fA fU
AAAGACUUACCUUAAGA
SSSSS SSSSS SSSSS SSSS

fA fC
UAC

WV-20331
fA fA fG fA fC fU fU fA mC fC mU fU mA fA fG fA fU fA
AAGACUUACCUUAAGAU
SSSSS SSSSS SSSSS SSSS

fC fC
ACC

WV-20332
fA fG fA fC fU fU fA fC mC fU mU fA mA fG fA fU fA fC
AGACUUACCUUAAGAUA
SSSSS SSSSS SSSSS SSSS

fC fA
CCA

WV-20333
fG fA fC fU fU fA fC fC mU fU mA fA mG fA fU fA fC fC
GACUUACCUUAAGAUAC
SSSSS SSSSS SSSSS SSSS

fA fU
CAU

WV-20334
fA fC fU fU fA fC fC fU mU fA mA fG mA fU fA fC fC fA
ACUUACCUUAAGAUACC
SSSSS SSSSS SSSSS SSSS

fU fU
AUU

WV-20335
fC fU fU fA fC fC fU fU mA fA mG fA mU fA fC fC fA fU
CUUACCUUAAGAUACCA
SSSSS SSSSS SSSSS SSSS

fU fU
UUU

WV-20336
fU fU fA fC fC fU fU fA mA fG mA fU mA fC fC fA fU fU
UUACCUUAAGAUACCAU
SSSSS SSSSS SSSSS SSSS

fU fG
UUG

WV-20337
fU fA fC fC fU fU fA fA mG fA mU fA mC fC fA fU fU fU
UACCUUAAGAUACCAUU
SSSSS SSSSS SSSSS SSSS

fG fU
UGU

WV-20338
fA fG fG fC fA fA fA fA mC fA mA fA mA fA fU fG fA fA
AGGCAAAACAAAAAUGA
SSSSS SSSSS SSSSS SSSS

fG fC
AGC

WV-20339
fG fC fA fA fA fA fC fA mA fA mA fA mU fG fA fA fG fC
GCAAAACAAAAAUGAAG
SSSSS SSSSS SSSSS SSSS

fC fC
CCC

WV-20340
fA fA fA fA fC fA fA fA mA fA mU fG mA fA fG fC fC fC
AAAACAAAAAUGAAGCC
SSSSS SSSSS SSSSS SSSS

fC fA
CCA

WV-20341
fA fA fC fA fA fA fA fA mU fG mA fA mG fC fC fC fC fA
AACAAAAAUGAAGCCCC
SSSSS SSSSS SSSSS SSSS

fU fG
AUG

WV-20342
fC fA fA fA fA fA fU fG mA fA mG fC mC fC fC fA fU fG
CAAAAAUGAAGCCCCAU
SSSSS SSSSS SSSSS SSSS

fU fC
GUC

WV-20343
fA fA fA fA fU fG fA fA mG fC mC fC mC fA fU fG fU fC
AAAAUGAAGCCCCAUGU
SSSSS SSSSS SSSSS SSSS

fU fU
CUU

WV-20344
fA fA fU fG fA fA fG fC mC fC mC fA mU fG fU fC fU fU
AAUGAAGCCCCAUGUCU
SSSSS SSSSS SSSSS SSSS

fU fU
UUU

WV-20345
fA fU fG fA fA fG fC fC mC fC mA fU mG fU fC fU fU fU
AUGAAGCCCCAUGUCUU
SSSSS SSSSS SSSSS SSSS

fU fU
UUU

WV-20346
fG fA fA fG fC fC fC fC mA fU mG fU mC fU fU fU fU fU
GAAGCCCCAUGUCUUUU
SSSSS SSSSS SSSSS SSSS

fA fU
UAU

WV-20347
fA fG fC fC fC fC fA fU mG fU mC fU mU fU fU fU fA fU
AGCCCCAUGUCUUUUUA
SSSSS SSSSS SSSSS SSSS

fU fU
UUU

WV-20348
fC fC fC fC fA fU fG fU mC fU mU fU mU fU fA fU fU fU
CCCCAUGUCUUUUUAUU
SSSSS SSSSS SSSSS SSSS

fG fA
UGA

WV-20349
fU fG fA fA fG fC fC fC mC fA mU fG mU fC fU fU fU fU
UGAAGCCCCAUGUCUUU
SSSSS SSSSS SSSSS SSSS

fU fA
UUA

WV-20350
fA fA fG fC fC fC fC fA mU fG mU fC mU fU fU fU fU fA
AAGCCCCAUGUCUUUUU
SSSSS SSSSS SSSSS SSSS

fU fU
AUU

WV-20351
fG fC fC fC fC fA fU fG mU fC mU fU mU fU fU fA fU fU
GCCCCAUGUCUUUUUAU
SSSSS SSSSS SSSSS SSSS

fU fG
UUG

WV-20352
fC fU fG fC fA fU mA mU mU mC mA mA mA mG fG fA fC
CUGCAUAUUCAAAGGAC
SSSSS SSSSS SSSSS SSSS

fA fC fC
ACC

WV-20353
fC fU fG fC fA fU mU mG mU mU mU mU mG mG fC fC fU
CUGCAUUGUUUUGGCCU
SSSSS SSSSS SSSSS SSSS

fC fU fG
CUG

WV-20354
fA fU fA fA fA fG mC mC mG mA mA mA mU mA fC fA fC
AUAAAGCCGAAAUACAC
SSSSS SSSSS SSSSS SSSS

fA fC fU
ACU

WV-20355
fG fC fU fG fU fU mA mC mG mA mU mG mC mU fU fC fC
GCUGUUACGAUGCUUCC
SSSSS SSSSS SSSSS SSSS

fC fU fC
CUC

WV-20356
fC fU fU fC fC fC mU mC mU mG mU mC mA mC fA fG fA
CUUCCCUCUGUCACAGA
SSSSS SSSSS SSSSS SSSS

fU fU fC
UUC

WV-20357
fC fA fG fA fU fA mA mA mC mC mA mG mC mU fC fC fG
CAGAUAAACCAGCUCCG
SSSSS SSSSS SSSSS SSSS

fU fC fC
UCC

WV-20358
fC fU fC fC fG fU mC mC mA mG mG mC mA mA fA fC fU
CUCCGUCCAGGCAAACU
SSSSS SSSSS SSSSS SSSS

fC fU fC
CUC

WV-20359
fG fG fC fA fA fA mC mU mC mU mC mU mC mA fU fC fC
GGCAAACUCUCUCAUCC
SSSSS SSSSS SSSSS SSSS

fU fG fA
UGA

WV-20360
fC fU fC fU fC fU mC mA mU mC mC mU mG mA fC fA fC
CUCUCUCAUCCUGACAC
SSSSS SSSSS SSSSS SSSS

fA fA fA
AAA

WV-20361
fC fA fA fA fC fU mC mU mC mU mC mA mU mC fC fU fG
CAAACUCUCUCAUCCUG
SSSSS SSSSS SSSSS SSSS

fA fC fA
ACA

WV-20362
fG fC fU fC fU fA mA mU mA mU mU mA mU mC fA fU fU
GCUCUAAUAUUAUCAUU
SSSSS SSSSS SSSSS SSSS

fA fU fG
AUG

WV-20363
fA fU fA fG fC fA mC mC mG mU mG mC mU mC fU fA fA
AUAGCACCGUGCUCUAA
SSSSS SSSSS SSSSS SSSS

fU fA fU
UAU

WV-20364
fC fC fG fU fG fC mU mC mU mA mA mU mA mU fU fA fU
CCGUGCUCUAAUAUUAU
SSSSS SSSSS SSSSS SSSS

fC fA fU
CAU

WV-20365
fU fA fU fG fA fU mA mA mU mU mU mU mC mU fU fU
UAUGAUAAUUUUCUUUC
SSSSS SSSSS SSSSS SSSS

fC fU fA fG
UAG

WV-20366
fC fU fU fU fC fU mA mG mU mA mA mU mA mU fA fA
CUUUCUAGUAAUAUAAU
SSSSS SSSSS SSSSS SSSS

fU fG fA fU
GAU

WV-20367
fU fA fA fU fU fU mU mC mU mU mU mC mU mA fG fU
UAAUUUUCUUUCUAGUA
SSSSS SSSSS SSSSS SSSS

fA fA fU fA
AUA

WV-20368
fA fC fA fA fC fA mA mC mA mG mU mC mA mA fA fA fG
ACAACAACAGUCAAAAG
SSSSS SSSSS SSSSS SSSS

fU fA fA
UAA

WV-20369
fA fA fU fA fU fA mA mU mG mA mU mG mA mC fA fA
AAUAUAAUGAUGACAAC
SSSSS SSSSS SSSSS SSSS

fC fA fA fC
AAC

WV-20370
fU fG fA fU fG fA mC mA mA mC mA mA mC mA fG fU fC
UGAUGACAACAACAGUC
SSSSS SSSSS SSSSS SSSS

fA fA fA
AAA

WV-20371
fU fA fA fU fU fU mC mC mA mU mC mA mC mC fC fU fU
UAAUUUCCAUCACCCUU
SSSSS SSSSS SSSSS SSSS

fC fA fG
CAG

WV-20372
fC fA fC fC fC fU mU mC mA mG mA mA mC mC fU fG fA
CACCCUUCAGAACCUGA
SSSSS SSSSS SSSSS SSSS

fU fC fU
UCU

WV-20373
fU fC fC fA fU fC mA mC mC mC mU mU mC mA fG fA fA
UCCAUCACCCUUCAGAA
SSSSS SSSSS SSSSS SSSS

fC fC fU
CCU

WV-20374
fA fC fC fU fG fA mU mC mU mU mU mA mA mG fA fA fG
ACCUGAUCUUUAAGAAG
SSSSS SSSSS SSSSS SSSS

fU fU fA
UUA

WV-20375
fC fA fC fC fC fU mU mC mA mG mA mA mC mC fU fG fA
CACCCUUCAGAACCUGA
SSSSS SSSSS SSSSS SSS

fU fC
UC

WV-20376
fC fA fG fA fA fC mC mU mG mA mU mC mU mU fU fA fA
CAGAACCUGAUCUUUAA
SSSSS SSSSS SSSSS SSSS

fG fA fA
GAA

WV-20377
fA fG fA fG fU fC mC mA mG mA mU mG mU mG fC fU fG
AGAGUCCAGAUGUGCUG
SSSSS SSSSS SSSSS SSS

fA fA
AA

WV-20378
fC fU fG fA fA fG mA mU mA mA mA mU mA mC fA fA
CUGAAGAUAAAUACAAU
SSSSS SSSSS SSSSS SSSS

fU fu fU fC
UUC

WV-20379
fU fG fU fG fC fU mG mA mA mG mA mU mA mA fA fU
UGUGCUGAAGAUAAAUA
SSSSS SSSSS SSSSS SSSS

fA fC fA fA
CAA

WV-20380
fA fC fA fA fU fU mU mC mG mA mA mA mA mA fA fC fA
ACAAUUUCGAAAAAACA
SSSSS SSSSS SSSSS SSS

fA fA
AA

WV-20381
fC fU fG fA fA fG mA mU mA mA mA mU mA mC fA fA
CUGAAGAUAAAUACAAU
SSSSS SSSSS SSSSS SSS

fU fU fU
UU

WV-20382
fU fA fA fA fU fA mC mA mA mU mU mU mC mG fA fA
UAAAUACAAUUUCGAAA
SSSSS SSSSS SSSSS SSS

fA fA fA
AA

WV-20383
fA fC fU fU fA fC mC mU mU mA mA mG mA mU fA fC fC
ACUUACCUUAAGAUACC
SSSSS SSSSS SSSSS SSSS

fA fU fU
AUU

WV-20384
fA fA fU fC fA fA mA mG mA mC mU mU mA mC fC fU fU
AAUCAAAGACUUACCUU
SSSSS SSSSS SSSSS SSSS

fA fA fG
AAG

WV-20385
fA fA fG fA fC fU mU mA mC mC mU mU mA mA fG fA fU
AAGACUUACCUUAAGAU
SSSSS SSSSS SSSSS SSSS

fA fC fC
ACC

WV-20386
fA fU fU fC fU fC mA mG mG mA mA mU mU mU fG fU
AUUCUCAGGAAUUUGUG
SSSSS SSSSS SSSSS SSSS

fG fU fC fU
UCU

WV-20387
fC fA fU fG fU fU mC mC mC mA mA mU mU mC fU fC fA
CAUGUUCCCAAUUCUCA
SSSSS SSSSS SSSSS SSS

fG fG
GG

WV-20388
fC fC fC fA fA fU mU mC mU mC mA mG mG mA fA fU fU
CCCAAUUCUCAGGAAUU
SSSSS SSSSS SSSSS SSS

fU fG
UG

WV-20389
fC fU fU fU fC fU mG mA mG mA mA mA mC mU fG fU fU
CUUUCUGAGAAACUGUU
SSSSS SSSSS SSSSS SSSS

fC fA fG
CAG

WV-20390
fA fG fG fA fA fU mU mU mG mU mG mU mC mU fU fU
AGGAAUUUGUGUCUUUC
SSSSS SSSSS SSSSS SSSS

fC fU fG fA
UGA

WV-20391
fU fG fU fG fU fC mU mU mU mC mU mG mA mG fA fA
UGUGUCUUUCUGAGAAA
SSSSS SSSSS SSSSS SSSS

fA fC fU fG
CUG

WV-20392
fC fU fU fU fA fU mA mU mC mA mU mA mA mU fG fA
CUUUAUAUCAUAAUGAA
SSSSS SSSSS SSSSS SSSS

fA fA fA fC
AAC

WV-20393
fC fA fC fU fG fA mU mU mA mA mA mU mA mU fC fU fU
CACUGAUUAAAUAUCUU
SSSSS SSSSS SSSSS SSSS

fU fA fU
UAU

WV-20789
L001 fU fC fA fA fG fG mA fA mG fA mU fG mG fC fA fU
UCAAGGAAGAUGGCAUU
ORRRR RRORO ROROR

fU fU fC fU
UCU
RRRRR

WV-20790
Mod012L001 fU fC fA fA fG fG mA fA mG fA mU fG mG
UCAAGGAAGAUGGCAUU
ORRRR RRORO ROROR

fC fA fU fU fU fC fU
UCU
RRRRR

WV-21210
Mod118L001 fU fC fA fC fU fC mAn001 fG fA mU fA
UCACUCAGAUAGUUGAA
OSSSS SSnXSS SSnXnXS

mGn001 mUn001 fU fG fA fA fG fC fC
GCC
SSSSS

WV-21211
Mod119L001 fU fC fA fC fU fC mAn001 fG fA mU fA
UCACUCAGAUAGUUGAA
OSSSS SSnXSS SSnXnXS

mGn001 mUn001 fU fG fA fA fG fC fC
GCC
SSSSS

WV-21212
Mod120L001 fU fC fA fC fU fC mAn001 fG fA mU fA
UCACUCAGAUAGUUGAA
OSSSS SSnXSS SSnXnXS

mGn001 mUn001 fU fG fA fA fG fC fC
GCC
SSSSS

WV-21217
fC fU fCn001 R fC fG fGn001 R fU fU mC
CUCCGGUUC
SSnRSS nRSS

WV-21218
fU fC fAn001 R fC fU fCn001 R mA fG fA mU fA mG mU
UCACUCAGAUAGUUGAA
SSnRSS nROSSS SOSSS

fU fG fA fAn001 R fG fC fC
GCC
SnRSS

WV-21245
fU fC fAn001 R fC fU fCn001 R mA fG fA mU fA mG mU
UCACUCAGAUAGUUGAA
SSnRSS nROSSS SSOSS

fU fG fA fAn001 R fG fC fC
GCC
SnRSS

WV-21257
fC fG fGn001 R fU fU mC fU mG fA mA fG fG fU fGn001 R
CGGUUCUGAAGGUGUUC
SSnRSS OSSSO SSSnRS S

fU fU fC

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA
UCAAGGAAGAUGGCAUUUCG
SSSSSSOSOSSOOSSSSSS

24310
* SfU * SmGmGfC * SfA * SfU * SfU * SfU * SfC *

SmG

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA
UCAAGGAAGAUGGCACCCCG
SSSSSSOSOSSOOSSSSSS

24311
* SfU * SmGmGfC * SfA * SfC * SfC * SfC * SfC *

SfG

WV-
fU * SfC * SfG * SfA * SfG * SfA * SmAfA * SmGmA
UCGAGAAAGAUGGCAUUUCU
SSSSSSOSOSSOOSSSSSS

24463
* SfU * SmGmGfC * SfA * SfU * SfU * SfU * SfC *

SfU

WV-
fU * SfU * SfA * SfA * SfG * SfG * SmAfA * SmGmA
UUAAGGAAGAUGGCAUUCCU
SSSSSSOSOSSOOSSSSSS

24464
* SfU * SmGmGfC * SfA * SfU * SfU * SfC * SfC *

SfU

WV-
fU * RfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
RSSSSSSOSSSOOSSSSSS

25439
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU *

SfC * SfU

WV-
fU * SfC * RfC * SfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SRSSSSSOSSSOOSSSSSS

25440
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU *

SfC * SfU

WV-
fU * SfC * SfC * RfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSRSSSSOSSSOOSSSSSS

25441
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU *

SfC * SfU

WV-
fU * SfC * SfC * SfG * RfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSRSSSOSSSOOSSSSSS

25442
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU *

SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * RfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSSRSSOSSSOOSSSSSS

25443
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU *

SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * RfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSSSRSOSSSOOSSSSSS

25444
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU *

SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * RmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSSSSROSSSOOSSSSSS

25445
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU *

SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSSSSSORSSOOSSSSSS

25446
RmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU *

SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSSSSSOSRSOOSSSSSS

25447
SmG * RfA * SmAmGfG * SfU * SfG * SfU * SfU *

SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSSSSSOSSROOSSSSSS

25448
SmG * SfA * RmAmGfG * SfU * SfG * SfU * SfU *

SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSSSSSOSSSOORSSSSS

25449
SmG * SfA * SmAmGfG * RfU * SfG * SfU * SfU *

SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSSSSSOSSSOOSRSSSS

25450
SmG * SfA * SmAmGfG * SfU * RfG * SfU * SfU *

SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSSSSSOSSSOOSSRSSS

25451
SmG * SfA * SmAmGfG * SfU * SfG * RfU * SfU *

SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSSSSSOSSSOOSSSRSS

25452
SmG * SfA * SmAmGfG * SfU * SfG * SfU * RfU *

SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSSSSSOSSSOOSSSSRS

25453
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU *

RfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUCU
SSSSSSSOSSSOOSSSSSR

25454
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU *

SfC * RfU

WV-
fC * SfG * SfG * SfU * SfU * SmCfU * SmG * SfA *
CGGUUCUGAAGGUGUUCU
SSSSSOSSSOOSSSSSS

25455
SmAmGfG * SfU * SfG * SfU * SfU * SfC * SfU

WV-
fU * SfU * SfC * SfC * SfG * SfG * SfU * SfU *
UUCCGGUUCUGAAGGUGUUCU
SSSSSSSSOSSSOOSSSSSS

25456
SmCfU * SmG * SfA * SmAmGfG * SfU * SfG * SfU *

SfU * SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SfU *
UCCGGUUUCUGAAGGUGUUCU
SSSSSSSSOSSSOOSSSSSS

25457
SmCfU * SmG * SfA * SmAmGfG * SfU * SfG * SfU *

SfU * SfC * SfU

WV-
fU * SfC * SfC * SfG * SfG * SfU * SfU * SmCfU *
UCCGGUUCUGAAGGUGUUUCU
SSSSSSSOSSSOOSSSSSSS

25458
SmG * SfA * SmAmGfG * SfU * SfG * SfU * SfU *

SfU * SfC * SfU

WV
fU * SfC * SfC * SfG * SfG * SfU * SmCfU * SmG *
UCCGGUCUGAAGGUGUUCU
SSSSSSOSSSOOSSSSSS

25459
SfA * SmAmGfG * SfU * SfG * SfU * SfU * SfC * SfU

WV-
lT * SfC * SlA * SfC * SfU * SfC * SmAfG * SfA *
TCACUCAGAUAGUUGAAGCC
SSSSSSOSSSSOOSSSSSS

25536
SmU * SfA * SmGmUfU * SfG * SfA * SfA * SfG *

SfC * SfC

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA *
UCACUCAGAUAGUUGAAGCC
SSSSSSOSSSSOOSSSSSS

25537
SmU * SfA * SmGmUfU * SfG * SfA * SfA * SlG * SfC

* SfC

WV-
lT * SfC * SlA * SfC * SfU * SfC * SmAfG * SfA *
TCACUCAGAUAGUUGAAGCC
SSSSSSOSSSSOOSSSSSS

25538
SmU * SfA * SmGmUfU * SfG * SfA * SfA * SlG * SfC

* SfC

WV-
fU * SfC * SfA * SfC * SfU * SfC * SlAfG * SfA * SmU
UCACUCAGAUAGTUGAAGCC
SSSSSSOSSSSOOSSSSSS

25539
* SfA * SfGlTfU * SfG * SfA * SfA * SfG * SfC * SfC

WV-
fU * SfC * SfA * SfC * SfU * SfC * SlAfG * SfA * SmU
UCACUCAGAUAGTTGAAGCC
SSSSSSOSSSSOOSSSSSS

25540
* SfA * SlGlTlT * SfG * SfA * SfA * SfG * SfC * SfC

WV-
fU * SfC * SfA * SfC * SfU * SfC * S1An001RfG * SfA
UCACUCAGAUAGTTGAAGCC
SSSSSSnRSSSSnRnRSSSSSS

25541
* SmU * SfA * SlGn001RlTn001RlT * SfG * SfA * SfA

* SfG * SfC * SfC

WV-
lT * SfC * SlA * SfC * SfU * SfC * SmAn001RfG * SfA
TCACUCAGAUAGUUGAAGCC
SSSSSSnRSSSSnRnRSSSSSS

25542
* SmU * SfA * SmGn001RmUn001RfU * SfG * SfA *

SfA * SfG * SfC * SfC

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAn001RfG *
UCACUCAGAUAGUUGAAGCC
SSSSSSnRSSSSnRnRSSSSSS

25543
SfA * SmU * SfA * SmGn001RmUn001RfU * SfG *

SfA * SfA * SlG * SfC * SfC

WV-
lT * SfC * SlA * SfC * SfU * SfC * SmAn001RfG * SfA
TCACUCAGAUAGUUGAAGCC
SSSSSSnRSSSSnRnRSSSSSS

25544
* SmU * SfA * SmGn001RmUn001RfU * SfG * SfA *

SfA * SlG * SfC * SfC

WV-
L001fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA
UCACUCAGAUAGUUGAAGCC
OSSSSSSOSSSSOSSSSSSS

27163
* SmU * SfA * SmGmU * SfU * SfG * SfA * SfA * SfG

* SfC * SfC

WV-
L001fU * SfC * SfAn001RfC * SfU * SfCn001RmAfG *
UCACUCAGAUAGUUGAAGCC
OSSnRSSnROSSSSOSSSSnRSS

27164
SfA * SmU * SfA * SmGmU * SfU * SfG * SfA *

SfAn001RfG * SfC * SfC

WV-19790
Mod020L001 fU fC fA fC fU fC mAn001 fG fA mU fA
UCACUCAGAUAGUUGAA
OSSSS SSnXSS SSnXnXS

mGn001 mUn001 fU fG fA fA fG fC fC
GCC
SSSSS

WV-19791
Mod015L001 fU fC fA fC fU fC mAn001 fG fA mU fA
UCACUCAGAUAGUUGAA
OSSSS SSnXSS SSnXnXS

mGn001 mUn001 fU fG fA fA fG fC fC
GCC
SSSSS

WV-19792
Mod109L001 fU fC fA fC fU fC mAn00l fG fA mU fA
UCACUCAGAUAGUUGAA
OSSSS SSnXSS SSnXnXS

mGn001 mUn001 fU fG fA fA fG fC fC
GCC
SSSSS

WV-19793
Mod110L001 fU fC fA fC fU fC mAn001 fG fA mU fA
UCACUCAGAUAGUUGAA
OSSSS SSnXSS SSnXnXS

mGn001 mUn001 fU fG fA fA fG fC fC
GCC
SSSSS

WV-19794
Mod111L001 fU fC fA fC fU fC mAn001 fG fA mU fA
UCACUCAGAUAGUUGAA
OSSSS SSnXSS SSnXnXS

mGn001 mUn001 fU fG fA fA fG fC fC
GCC
SSSSS

WV-19795
Mod112L001 fU fC fA fC fU fC mAn00l fG fA mU fA
UCACUCAGAUAGUUGAA
OSSSS SSnXSS SSnXnXS

mGn001 mUn001 fU fG fA fA fG fC fC
GCC
SSSSS

WV-19796
Mod113L001 fU fC fA fC fU fC mAn001 fG fA mU fA
UCACUCAGAUAGUUGAA
OSSSS SSnXSS SSnXnXS

mGn001 mUn001 fU fG fA fA fG fC fC
GCC
SSSSS

WV-19797
Mod114L001 fU fC fA fC fU fC mAn001 fG fA mU fA
UCACUCAGAUAGUUGAA
OSSSS SSnXSS SSnXnXS

mGn001 mUn001 fU fG fA fA fG fC fC
GCC
SSSSS

WV-19798
Mod115L001 fU fC fA fC fU fC mAn001 fG fA mU fA
UCACUCAGAUAGUUGAA
OSSSS SSnXSS SSnXnXS

mGn001 mUn001 fU fG fA fA fG fC fC
GCC
SSSSS

WV-15883
fC * SfU * SfCn002RfC * SfG * SfGn002RfU * SfU * SmCfU
CUCCGGUUCUGAAGGUG
SSnR SSnR SSOSSS OOSSnR

* SmC * SfA * SmAfGfG * SfU * SfGn002RfU * SfU * SfC
UUC
SS

WV-15884
mU * SGeon002m5Ceon002m5Ceon002mA * SG * SG * RC
UGCCAGGCTGGTTATGAC
SnX nX nX SSRSSR

* ST * SG * RG * ST * ST * RA * ST * SmG * SmA * SmC *
UC
SSRSSSSSS

SmU * SmC

WV-15885
mU * SGeon002Rm5Ceon002Rm5Ceon002RmA * SG * SG *
UGCCAGGCTGGTTATGAC
SnR nR nR SSRSSR

RC * ST * SG * RG * ST * ST * RA * ST * SmG * SmA *
UC
SSRSSSSSS

SmC * SmU * SmC

WV-15886
fC * SfU * SfCn002fC * SfG * SfUn002fU * SfU * SmCfU *
CUCCGGUUCUGAAGGUG
SSnX SSnX SSOSSS OOSSnX

SmG * SfA * SmAfGfG * SfU * SfUn002fU * SfU * SfC
UUC
SS

WV-15887
mU * SGeon002Sm5Ceon002Sm5Ceon002SmA * SG * SG *
UGCCAGGCTGGTTATGAC
SnS nS nS SSRSSR

RC * ST * SG * RG * ST * ST * RA * ST * SmG * SmA *
UC
SSRSSSSSS

SmC * SmU * SmC

WV-16006
fCfUfCn003RfCfGfGn003RfUfUmCfUmGfAmAfGfGfUfGn0
CUCCGGUUCUGAAGGUG
SSnR SSnR SSOSSS

03RfUfUfC
UUC
OOSSnR SS

WV-16008
fUfCfAfCfUfCmAn003fGfAmUfAmGn003mUn003fUfGfAfA
UCACUCAGAUAGUUGAA
SSSSSSnX SSSSnX

fGfCfC
GCC
nX SSSSSS

WV-16007
fCfUfCn004RfCfGfGn004RfUfUmCfU
CUCCGGUUCUGAAGGUG
SSnR SSnR SSOSSS

mGfAmAfGfGfUGn004RfUfUfC
UUC
OOSSnR SS

WV-16009
fUfCfAfCfUfCmAn004fGfAmUfAmG
UCACUCAGAUAGUUGAA
SSSSSS nX SSSSnX

n004mUn004fUfGfAfAfGfCfC
GCC
nX SSSSSS

WV-24088
fU * SfC * SfA * SfC * SfU * SfC * SmAn005fG * SfA *
UCACUCAGAUAGUUGAA
SSSSS S nX SSSS

SmU * SfA * SmGn005mUn005fU * SfG * SfA * SfA * SfG *
GCC
nX nX

SfC * SfC

SSSSS S

WV-24089
fU * SfC * SfA * SfC * SfU * SfC * SmAn005RfG * SfA *
UCACUCAGAUAGUUGAA
SSSSS S nR SSSS

SmU * SfA * SmGn005RmUn005RfU * SfG * SfA * SfA *
GCC
nR nR

SfG * SfC * SfC

SSSSS S

WV-24090
fU * SfU * SfA * SfC * SfU * SfC * SmAn005SfG * SfA *
UCACUCAGAUAGUUGAA
SSSSS S nS SSSS

SmU * SfA * SmGn005SmUn005SfU * SfG * SfA * SfA *
GCC
nS nS

SfG * SfC * SfC

SSSSS S

WV-24100
mU * SGeon005m5Ceon005m5Ceon005mA * SG * SG * RC
UGCCAGGCTGGTTATGAC
S nX nX nX SSRSS

* ST * SG * RG * ST * ST * RA * ST * SmG * SmA * SmC *
UC
RSSRSS

SmU * SmC

SSSS

WV-24101
mU * SGeon005Rm5Ceon005Rm5Ceon005RmA * SG * SG *
UGCCAGGCTGGTTATGAC
S nR nR nR SSRSS

RC * ST * SG * RG * ST * ST * RA * ST * SmG * SmA *
UC
RSSRSS

SmC * SmU * SmC

SSSS

WV-24102
mU * SGeon005Sm5Ceon005Sm5Ceon005SmA * SG * SG *
UGCCAGGCTGGTTATGAC
S nS nS nS SSRSS

RC * ST * SG * RG * ST * ST * RA * ST * SmG * SmA *
UC
RSSRSS

SmC * SmU * SmC

SSSS

WV-24091
fU * SfC * SfA * SfC * SfU * SfC * SmAn006fG * SfA *
UCACUCAGAUAGUUGAA
SSSSS S nX SSSS

SmU * SfA * SmGn006mUn006fU * SfG * SfA * SfA * SfG *
GCC
nX nX

SfC * SfC

SSSSS S

WV-24092
fU * SfC * SfA * SfC * SfU * SfC * SmAn006RfG * SfA *
UCACUCAGAUAGUUGAA
SSSSS S nR SSSS

SmU * SfA * SmGn006RmUn006RfU * SfG * SfA * SfA *
GCC
nR nR

SfG * SfC * SfC

SSSSS S

WV-24093
fU * SfC * SfA * SfC * SfU * SfC * SmAn006SfG * SfA *
UCACUCAGAUAGUUGAA
SSSSS S nS SSSS

SmU * SfA * SmGn006SmUn006SfU * SfG * SfA * SfA *
GCC
nS nS

SfG * SfC * SfC

SSSSS S

WV-24103
mU * SGeon006m5Ceon006m5Ceon006mA * SG * SG * RC
UGCCAGGCTGGTTATGAC
S nX nX nX SSRSS

* ST * SG * RG * ST * ST * RA * ST * SmG * SmA * SmC *
UC
RSSRSS

SmU * SmC

SSSS

WV-24104
mU * SGeon006Rm5Ceon006Rm5Ceon006RmA * SG * SG *
UGCCAGGCTGGTTATGAC
S nR nR nR SSRSS

RC * ST * SG * RG * ST * ST * RA * ST * SmG * SmA *
UC
RSSRSS

SmC * SmU * SmC

SSSS

WV-24105
mU * SGeon006Sm5Ceon006Sm5Ceon006SmA * SG * SG *
UGCCAGGCTGGTTATGAC
S nS nS nS SSRSS

RC * ST * SG * RG * ST * ST * RA * ST * SmG * SmA *
UC
RSSRSS

SmC * SmU * SmC

SSSS

WV-24094
fU * SfC * SfA * SfC * SfU * SfC * SmAn007fG * SfA *
UCACUCAGAUAGUUGAA
SSSSS S nX SSSS

SmU * SfA * SmGn007mUn007fU * SfG * SfA * SfA * SfG *
GCC
nX nX

SfC * SfC

SSSSS S

WV-24095
fU * SfC * SfA * SfC * SfU * SfC * SmAn007RfG * SfA *
UCACUCAGAUAGUUGAA
SSSSS S nR SSSS

SmU * SfA * SmGn007RmUn0071RfU * SfG * SfA * SfA *
GCC
nR nR

SfG * SfC * SfC

SSSSS S

WV-24096
fU * SfC * SfA * SfC * SfU * SfC * SmAn007SfG * SfA *
UCACUCAGAUAGUUGAA
SSSSS S nS SSSS

SmU * SfA * SmGn007SmUn007SfU * SfG * SfA * SfA *
GCC
nS nS

SfG * SfU * SfC

SSSSS S

WV-24106
mU * SGeon007Rm5Ceon007Rm5Ceon007RmA * SG * SG *
UGCCAGGCTGGTTATGAC
S nR nR nR SSRSS

RC * ST * SG * RG * ST * ST * RA * ST * SmG * SmA *
UC
RSSRSS

SmC * SmU * SmC

SSSS

WV-24107
mU * SGeon007Sm5Ceon007Sm5Ceon007SmA * SG * SG *
UGCCAGGCTGGTTATGAC
S nS nS nS SSRSS

RC * ST * SG * RG * ST * ST * RA * ST * SmG * SmA *
UC
RSSRSS

SmC * SmU * SmC

SSSS

WV-24097
fU * SfC * SfA * SfC * SfU * SfC * SmAn008fG * SfA *
UCACUCAGAUAGUUGAA
SSSSS S nX SSSS

SmU * SfA * SmGn008mUn008fU * SfG * SfA * SfA * SfG *
GCC
nX nX

SfC * SfC

SSSSS S

WV-24098
fU * SfC * SfA * SfC * SfU * SfC * SmAn008RfG * SfA *
UCACUCAGAUAGUUGAA
SSSSS S nR SSSS

SmU * SfA * SmGn008RmUn008RfU * SfG * SfA * SfA *
GCC
nR nR

SfG * SfC * SfC

SSSSS S

WV-24099
fU * SfC * SfA * SfC * SfU * SfC * SmAn008SfG * SfA *
UCACUCAGAUAGUUGAA
SSSSS S nS SSSS

SmU * SfA * SmGn008SmUn008SfU * SfG * SfA * SfA *
GCC
nS nS

SfG * SfC * SfC

SSSSS S

WV-24108
mU * SGeon008m5Ceon008m5Ceon008mA * SG * SG * RC
UGCCAGGCTGGTTATGAC
S nX nX nX SSRSS

* ST * SG * RG * ST * ST * RA * ST * SmG * SmA * SmC *
UC
RSSRSS

SmU * SmC

SSSS

WV-24109
mU * SGeon008Rm5Ceon008Rm5Ceon008RmA * SG * SG *
UGCCAGGCTGGTTATGAC
S nR nR nR SSRSS

RC * ST * SG * RG * ST * ST * RA * ST * SmG * SmA *
UC
RSSRSS

SmC * SmU * SmC

SSSS

WV-24110
mU * SGeon008Sm5Ceon008Sm5Ceon008SmA * SG * SG *
UGCCAGGCTGGTTATGAC
S nS nS nS SSRSS

RC * ST * SG * RG * ST * ST * RA * ST * SmG * SmA *
UC
RSSRSS

SmC * SmU * SmC

SSSS

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU * SmG
CUCCGGUUCUGAAGGUGUUC
SSnX SSnX SSOSS

12880
* SfA * SmAfG * SfG * SfU * SfGn001fU * SfU * SfC

SOSSSnX SS

WV-
fC * SfU * SfCn001fC * SfG * SfGn001fU * SfU * SmCfU * SmG
CUCCGGUUCUGAAGGUGUUC
SSnX SSnX SSOSS

12880
* SfA * SmAfG * SfG * SfU * SfGn001fU * SfU * SfC

SOSSSnX SS

WV-
fGn001RfU
GU
nR

21219

WV-
fCn001RfC
CC
nR

21226

WV-
fGn001SfU
GU
nS

21252

WV-
fCn001SfC
CC
nS

21253

WV-
fGn001RmA
GA
nR

21258

WV-
fC * RfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
RSnR SSnR SSOSS

21374
SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU * SfU * SfC

SOSSSnR SS

WV-
fC * SfU * RfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SRnR SSnR SSOSS

21375
SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU * SfU * SfC

SOSSSnR SS

WV-
fC * SfU * SfCn001SfC * SfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnS SSnR SSOSS

21376
SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU * SfU * SfC

SOSSSnR SS

WV-
fC * SfU * SfCn001RfC * RfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR RSnR SSOSS

21377
SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU * SfU * SfC

SOSSSnR SS

WV-
fC * SfU * SfCn001RfC * SfG * RfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSRnR SSOSS

21378
SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU * SfU * SfC

SOSSSnR SS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001SfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSnS SSOSS

21379
SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU * SfU * SfC

SOSSSnR SS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * RfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSnR

21380
SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU * SfU * SfC

RSOSSSO SS SnR

SS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU * RmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSnR

21381
SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU * SfU * SfC

SROSSSO SS SnR

SS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSnR

21382
RmG * SfA * SmAfG * SfG * SfU * SfGn001RfU * SfU * SfC

SSORSSOSS SnR

SS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSnR

21383
SmG * RfA * SmAfG * SfG * SfU * SfGn001RfU * SfU * SfC

SSOSRSOSSSnR SS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSnR SSOSS

21384
SmG * SfA * RmAfG * SfG * SfU * SfGn001RfU * SfU * SfC

ROSSSnR SS

WV
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSnR SSOSS

21385
SmG * SfA * SmAfG * RfG * SfU * SfGn001RfU * SfU * SfC

SORSSnR SS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSnR SSOSS

21386
SmG * SfA * SmAfG * SfG * RfU * SfGn001RfU * SfU * SfC

SOSRSnR SS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSnR SSOSS

21387
SmG * SfA * SmAfG * SfG * SfU * RfGn001RfU * SfU * SfC

SOSSRnR SS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSnR SSOSS

21388
SmG * SfA * SmAfG * SfG * SfU * SfGn001SfU * SfU * SfC

SOSSSnS SS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSnR SSOSS

21389
SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU * RFU * SfC

SOSSSnR RS

WV-
fC * SfU * SfCn001RfC * SfG * SfGn001RfU * SfU * SmCfU *
CUCCGGUUCUGAAGGUGUUC
SSnR SSnR SSOSS

21390
SmG * SfA * SmAfG * SfG * SfU * SfGn001RfU * SfU * RfC

SOSSSnR SR

WV-
fC * SfU * SfUn001fA * SfA * SfGn001fA * SfU * SmA * SfC *
CUUAAGAUACCAUUUGUAUU
SSnX SSnX SSSSS

21578
SmC * SfA * SmU * SfU * SfU * SfG * SfUn001fA * SfU * SfU

SSSSS nX SS

WV-
fU * SfU * SfAn001fA * SfG * SfAn001fU * SfA * SmC * SfC *
UUAAGAUACCAUUUGUAUUU
SSnX SSnX SSSSS

21579
SmA * SfU * SmU * SfU * SfG * SfU * SfAn001fU * SfU * SfU

SSSSS nX SS

WV-
fU * SfA * SfAn001fG * SfA * SfUn001fA * SfC * SmC * SfA *
UAAGAUACCAUUUGUAUUUA
SSnX SSnX SSSSS

21580
SmU * SfU * SmU * SfG * SfU * SfA * SfUn001fU * SfU * SfA

SSSSS nX SS

WV-
fA * SfA * SfGn001fA * SfU * SfAn001fC * SfC * SmA * SfU *
AAGAUACCAUUUGUAUUUAG
SSnX SSnX SSSSS

21581
SmU * SfU * SmG * SfU * SfA * SfU * SfUn001fU * SfA * SfG

SSSSS nX SS

WV-
fA * SfG * SfAn001fU * SfA * SfCn001fC * SfA * SmU * SfU *
AGAUACCAUUUGUAUUUAGC
SSnX SSnX SSSSS

21582
SmU * SfG * SmU * SfA * SfU * SfU * SfUn001fA * SfG * SfC

SSSSS nX SS

WV-
fG * SfA * SfUn001fA * SfC * SfCn001fA * SfU * SmU * SfU *
GAUACCAUUUGUAUUUAGCA
SSnX SSnX SSSSS

21583
SmG * SfU * SmA * SfU * SfU * SfU * SfAn001fG * SfC * SfA

SSSSS nX SS

WV-
fA * SfU * SfAn001fC * SfC * SfAn001fU * SfU * SmU * SfG *
AUACCAUUUGUAUUUAGCAU
SSnX SSnX SSSSS

21584
SmU * SfA * SmU * SfU * SfU * SfA * SfGn001fC * SfA * SfU

SSSSS nX SS

WV-
fU * SfA * SfCn001fC * SfA * SfUn001fU * SfU * SmG * SfU *
UACCAUUUGUAUUUAGCAUG
SSnX SSnX SSSSS

21585
SmA * SfU * SmU * SfU * SfA * SfG * SfCn001fA * SfU * SfG

SSSSS nX SS

WV-
fA * SfC * SfCn001fA * SfU * SfUn001fU * SfG * SmU * SfA *
ACCAUUUGUAUUUAGCAUGU
SSnX SSnX SSSSS

21586
SmU * SfU * SmU * SfA * SfG * SfC * SfAn001fU * SfG * SfU

SSSSS nX SS

WV-
fC * SfC * SfAn001fU * SfU * SfUn001fG * SfU * SmA * SfU *
CCAUUUGUAUUUAGCAUGUU
SSnX SSnX SSSSS

21587
SmU * SfU * SmA * SfG * SfC * SfA * SfUn001fG * SfU * SfU

SSSSS nX SS

WV-
fC * SfA * SfUn001fU * SfU * SfGn001fU * SfA * SmU * SfU *
CAUUUGUAUUUAGCAUGUUC
SSnX SSnX SSSSS

21588
SmU * SfA * SmG * SfC * SfA * SfU * SfGn001fU * SfU * SfC

SSSSS nX SS

WV-
fA * SfU * SfUn001fU * SfG * SfUn001fA * SfU * SmU * SfU *
AUUUGUAUUUAGCAUGUUCC
SSnX SSnX SSSSS

21589
SmA * SfG * SmC * SfA * SfU * SfG * SfUn001fU * SfC * SfC

SSSSS nX SS

WV-
fU * SfU * SfUn001fG * SfU * SfAn001fU * SfU * SmU * SfA *
UUUGUAUUUAGCAUGUUCCC
SSnX SSnX SSSSS

21590
SmG * SfC * SmA * SfU * SfG * SfU * SfUn001fC * SfC * SfC

SSSSS nX SS

WV-
fU * SfU * SfGn001fU * SfA * SfUn001fU * SfU * SmA * SfG *
UUGUAUUUAGCAUGUUCCCA
SSnX SSnX SSSSS

21591
SmC * SfA * SmU * SfG * SfU * SfU * SfCn001fC * SfC * SfA

SSSSS nX SS

WV-
fU * SfG * SfUn001fA * SfU * SfUn001fU * SfA * SmG * SfC *
UGUAUUUAGCAUGUUCCCAA
SSnX SSnX SSSSS

21592
SmA * SfU * SmG * SfU * SfU * SfC * SfCn001fC * SfA * SfA

SSSSS nX SS

WV-
fG * SfU * SfAn001fU * SfU * SfUn001fA * SfG * SmC * SfA *
GUAUUUAGCAUGUUCCCAAU
SSnX SSnX SSSSS

21593
SmU * SfG * SmU * SfU * SfC * SfC * SfCn001fA * SfA * SfU

SSSSS nX SS

WV-
fU * SfA * SfUn001fU * SfU * SfAn001fG * SfC * SmA * SfU *
UAUUUAGCAUGUUCCCAAUU
SSnX SSnX SSSSS

21594
SmG * SfU * SmU * SfC * SfC * SfC * SfAn001fA * SfU * SfU

SSSSS nX SS

WV-
fU * SfU * SfUn001fA * SfG * SfCn001fA * SfU * SmG * SfU *
UUUAGCAUGUUCCCAAUUCU
SSnX SSnX SSSSS

21595
SmU * SfC * SmC * SfC * SfA * SfA * SfUn001fU * SfC * SfU

SSSSS nX SS

WV-
fU * SfU * SfAn001fG * SfC * SfAn001fU * SfG * SmU * SfU *
UUAGCAUGUUCCCAAUUCUC
SSnX SSnX SSSSS

21596
SmC * SfC * SmC * SfA * SfA * SfU * SfUn001fC * SfU * SfC

SSSSS nX SS

WV-
fU * SfA * SfGn001fC * SfA * SfUn001fG * SfU * SmU * SfC *
UAGCAUGUUCCCAAUUCUCA
SSnX SSnX SSSSS

21597
SmC * SfC * SmA * SfA * SfU * SfU * SfCn001fU * SfU * SfA

SSSSS nX SS

WV-
fA * SfG * SfCn001fA * SfU * SfGn001fU * SfG * SmC * SfC *
AGCAUGUUCCCAAUUCUCAG
SSnX SSnX SSSSS

71598
SmC * SfA * SmA * SfU * SfU * SfC * SfUn001fC * SfA * SfG

SSSSS nX SS

WV-
fG * SfC * SfAn001fU * SfG * SfUn001fU * SfC * SmC * SfC *
GCAUGUUCCCAAUUCUCAGG
SSnX SSnX SSSSS

21599
SmA * SfA * SmU * SfU * SfC * SfU * SfCn001fA * SfG * SfG

SSSSS nX SS

WV-
fC * SfA * SfUn001fG * SfU * SfUn001fC * SfC * SmC * SfA *
CAUGUUCCCAAUUCUCAGGA
SSnX SSnX SSSSS

21600
SmA * SfU * SmU * SfC * SfU * SfC * SfAn001fG * SfG * SfA

SSSSS nX SS

WV-
fA * SfU * SfGn001fU * SfU * SfCn001fC * SfC * SmA * SfA *
AUGUUCCCAAUUCUCAGGAA
SSnX SSnX SSSSS

21601
SmU * SfU * SmC * SfU * SfC * SfA * SfGn001fG * SfA * SfA

SSSSS nX SS

WV-
fU * SfG * SfUn001fU * SfC * SfCn001fC * SfA * SmA * SfU *
UGUUCCCAAUUCUCAGGAAU
SSnX SSnX SSSSS

21602
SmU * SfC * SmU * SfC * SfA * SfG * SfGn001fA * SfA * SfU

SSSSS nX SS

WV-
fG * SfU * SfUn001fC * SfC * SfCn001fA * SfA * SmU * SfU *
GUUCCCAAUUCUCAGGAAUU
SSnX SSnX SSSSS

21603
SmC * SfU * SmC * SfA * SfG * SfG * SfAn001fA * SfU * SfU

SSSSS nX SS

WV-
fU * SfU * SfCn001fC * SfC * SfAn001fA * SfU * SmU * SfC *
UUCCCAAUUCUCAGGAAUUU
SSnX SSnX SSSSS

21604
SmU * SfC * SmA * SfG * SfG * SfA * SfAn001fU * SfU * SfU

SSSSS nX SS

WV-
fU * SfC * SfCn001fC * SfA * SfAn001fU * SfU * SmC * SfU *
UCCCAAUUCUCAGGAAUUUG
SSnX SSnX SSSSS

21605
SmC * SfA * SmG * SfG * SfA * SfA * SfUn001fU * SfU * SfG

SSSSS nX SS

WV-
fC * SfC * SfCn001fA * SfA * SfUn001fU * SfC * SmU * SfC *
CCCAAUUCUCAGGAAUUUGU
SSnX SSnX SSSSS

21606
SmA * SfG * SmG * SfA * SfA * SfU * SfUn001fU * SfG * SfU

SSSSS nX SS

WV-
fC * SfC * SfAn001fA * SfU * SfUn001fC * SfU * SmC * SfA *
CCAAUUCUCAGGAAUUUGUG
SSnX SSnX SSSSS

21607
SmG * SfG * SmA * SfA * SfU * SfU * SfUn001fG * SfU * SfG

SSSSS nX SS

WV-
fC * SfA * SfAn001fU * SfU * SfCn001fU * SfC * SmA * SfG *
CAAUUCUCAGGAAUUUGUGU
SSnX SSnX SSSSS

21608
SmG * SfA * SmA * SfU * SfU * SfU * SfGn001fU * SfG * SfU

SSSSS nX SS

WV-
fA * SfA * SfUn001fU * SfC * SfUn001fC * SfA * SmG * SfG *
AAUUCUCAGGAAUUUGUGUC
SSnX SSnX SSSSS

21609
SmA * SfA * SmU * SfU * SfU * SfG * SfUn001fG * SfU * SfC

SSSSS nX SS

WV-
fA * SfU * SfUn001fC * SfU * SfCn001fA * SfG * SmG * SfA *
AUUCUCAGGAAUUUGUGUCU
SSnX SSnX SSSSS

21610
SmA * SfU * SmU * SfU * SfG * SfU * SfGn001fU * SfC * SfU

SSSSS nX SS

WV-
fU * SfU * SfCn001fU * SfC * SfAn001fG * SfG * SmA * SfA *
UUCUCAGGAAUUUGUGUCUU
SSnX SSnX SSSSS

21611
SmU * SfU * SmU * SfG * SfU * SfG * SfUn001fC * SfU * SfU

SSSSS nX SS

WV-
fU * SfC * SfUn001fC * SfA * SfGn001fG * SfA * SmA * SfU *
UCUCAGGAAUUUGUGUCUUU
SSnX SSnX SSSSS

21612
SmU * SfU * SmG * SfU * SfG * SfU * SfCn001fU * SfU * SfU

SSSSS nX SS

WV-
fC * SfU * SfCn001fA * SfG * SfGn001fA * SfA * SmU * SfU *
CUCAGGAAUUUGUGUCUUUC
SSnX SSnX SSSSS

21613
SmU * SfG * SmU * SfG * SfU * SfC * SfUn001fU * SfU * SfC

SSSSS nX SS

WV-
fU * SfC * SfAn001fG * SfG * SfAn001fA * SfU * SmU * SfU *
UCAGGAAUUUGUGUCUUUCU
SSnX SSnX SSSSS

21614
SmG * SfU * SmG * SfU * SfC * SfU * SfUn001fU * SfC * SfU

SSSSS nX SS

WV-
fC * SfA * SfGn001fG * SfA * SfAn001fU * SfU * SmU * SfG *
CAGGAAUUUGUGUCUUUCUG
SSnX SSnX SSSSS

21615
SmU * SfG * SmU * SfC * SfU * SfU * SfUn001fC * SfU * SfG

SSSSS nX SS

WV-
fA * SfG * SfGn001fA * SfA * SfUn001fU * SfU * SmG * SfU *
AGGAAUUUGUGUCUUUCUGA
SSnX SSnX SSSSS

21616
SmG * SfU * SmC * SfU * SfU * SfU * SfCn001fU * SfG * SfA

SSSSS nX SS

WV-
fG * SfG * SfAn001fA * SfU * SfUn001fU * SfG * SmU * SfG *
GGAAUUUGUGUCUUUCUGAG
SSnX SSnX SSSSS

21617
SmU * SfC * SmU * SfU * SfU * SfC * SfUn001fG * SfA * SfG

SSSSS nX SS

WV-
fG * SfA * SfAn001fU * SfU * SfUn001fG * SfU * SmG * SfU *
GAAUUUGUGUCUUUCUGAGA
SSnX SSnX SSSSS

21618
SmC * SfU * SmU * SfU * SfC * SfU * SfGn001fA * SfG * SfA

SSSSS nX SS

WV-
fA * SfA * SfUn001fU * SfU * SfGn001fU * SfG * SmU * SfC *
AAUUUGUGUCUUUCUGAGAA
SSnX SSnX SSSSS

21619
SmU * SfU * SmU * SfC * SfU * SfG * SfAn001fG * SfA * SfA

SSSSS nX SS

WV-
fA * SfU * SfUn001fU * SfG * SfU001fG * SfU * SmC * SfU *
AUUUGUGUCUUUCUGAGAAA
SSnX SSnX SSSSS

21620
SmU * SfU * SmC * SfU * SfG * SfA * SfGn001fA * SfA * SfA

SSSSS nX SS

WV-
fU * SfU * SfUn001fG * SfU * SfGn001fU * SfC * SmU * SfU *
UUUGUGUCUUUCUGAGAAAC
SSnX SSnX SSSSS

21621
SmU * SfC * SmU * SfG * SfA * SfG * SfAn001fA * SfA * SfC

SSSSS nX SS

WV-
fU * SfU * SfGn001fU * SfG * SfUn001fC * SfU * SmU * SfU *
UUGUGUCUUUCUGAGAAACU
SSnX SSnX SSSSS

21622
SmC * SfU * SmG * SfA * SfG * SfA * SfAn001fA * SfC * SfU

SSSSS nX SS

WV-
fU * SfG * SfUn001fG * SfU * SfCn001fU * SfU * SmU * SfC *
UGUGUCUUUCUGAGAAACUG
SSnX SSnX SSSSS

21623
SmU * SfG * SmA * SfG * SfA * SfA * SfAn001fC * SfU * SfG

SSSSS nX SS

WV-
fG * SfU * SfGn001fU * SfC * SfUn001fU * SfU * SmC * SfU *
GUGUCUUUCUGAGAAACUGU
SSnX SSnX SSSSS

21624
SmG * SfA * SmG * SfA * SfA * SfA * SfCn001fU * SfG * SfU

SSSSS nX SS

WV-
fU * SfG * SfUn001fC * SfU * SfUn001fU * SfC * SmU * SfG *
UGUCUUUCUGAGAAACUGUU
SSnX SSnX SSSSS

21625
SmA * SfG * SmA * SfA * SfA * SfC * SfUn001fG * SfU * SfU

SSSSS nX SS

WV-
fG * SfU * SfCn001fU * SfU * SfUn001fC * SfU * SmG * SfA *
GUCUUUCUGAGAAACUGUUC
SSnX SSnX SSSSS

21626
SmG * SfA * SmA * SfA * SfC * SfU * SfGn001fU * SfU * SfC

SSSSS nX SS

WV-
fU * SfC * SfUn001fU * SfU * SfCn001fU * SfG * SmA * SfG *
UCUUUCUGAGAAACUGUUCA
SSnX SSnX SSSSS

21627
SmA * SfA * SmA * SfC * SfU * SfG * SfUn001fU * SfC * SfA

SSSSS nX SS

WV-
fC * SfU * SfUn001fU * SfC * SfUn001fG * SfA * SmG * SfA *
CUUUCUGAGAAACUGUUCAG
SSnX SSnX SSSSS

21628
SmA * SfA * SmC * SfU * SfG * SfU * SfUn001fC * SfA * SfG

SSSSS nX SS

WV-
fU * SfU * SfUn001fC * SfU * SfGn001fA * SfG * SmA * SfA *
UUUCUGAGAAACUGUUCAGC
SSnX SSnX SSSSS

21629
SmA * SfC * SmU * SfG * SfU * SfU * SfCn001A * SfG * SfC

SSSSS nX SS

WV-
fU * SfU * SfCn001fU * SfG * SfAn001fG * SfA * SmA * SfA *
UUCUGAGAAACUGUUCAGCU
SSnX SSnX SSSSS

21630
SmC * SfU * SmG * SfU * SfU * SfC * SfAn001fG * SfC * SfU

SSSSS nX SS

WV-
fU * SfC * SfUn001fG * SfA * SfGn001fA * SfA * SmA * SfC *
UCUGAGAAACUGUUCAGCUU
SSnX SSnX SSSSS

21631
SmU * SfG * SmU * SfU * SfC * SfA * SfGn001fC * SfU * SfU

SSSSS nX SS

WV-
fC * SfU * SfGn001fA * SfG * SfAn001fA * SfA * SmC * SfU *
CUGAGAAACUGUUCAGCUUC
SSnX SSnX SSSSS

21632
SmG * SfU * SmU * SfC * SfA * SfG * SfCn001fU * SfU * SfC

SSSSS nX SS

WV-
fU * SfG * SfAn001fG * SfA * SfAn001fA * SfC * SmU * SfG *
UGAGAAACUGUUCAGCUUCU
SSnX SSnX SSSSS

21633
SmU * SfU * SmC * SfA * SfG * SfC * SfUn001fU * SfC * SfU

SSSSS nX SS

WV-
fG * SfA * SfGn001fA * SfA * SfAn001fC * SfU * SmG * SfU *
GAGAAACUGUUCAGCUUCUG
SSnX SSnX SSSSS

21634
SmU * SfC * SmA * SfG * SfC * SfU * SfUn001fC * SfU * SfG

SSSSS nX SS

WV-
fA * SfG * SfAn001fA * SfA * SfCn001fU * SfG * SmU * SfU *
AGAAACUGUUCAGCUUCUGU
SSnX SSnX SSSSS

21635
SmC * SfA * SmG * SfC * SfU * SfU * SfCn001fU * SfG * SfU

SSSSS nX SS

WV-
fG * SfA * SfAn001fA * SfC * SfUn001fG * SfU * SmU * SfC *
GAAACUGUUCAGCUUCUGUU
SSnX SSnX SSSSS

21636
SmA * SfG * SmC * SfU * SfU * SfC * SfUn001fG * SfU * SfU

SSSSS nX SS

WV-
fA * SfA * SfAn001fC * SfU * SfGn001fU * SfU * SmC * SfA *
AAACUGUUCAGCUUCUGUUA
SSnX SSnX SSSSS

21637
SmG * SfC * SmU * SfU * SfC * SfU * SfGn001fU * SfU * SfA

SSSSS nX SS

WV-
fA * SfA * SfCn001fU * SfG * SfUn001fU * SfC * SmA * SfG *
AACUGUUCAGCUUCUGUUAG
SSnX SSnX SSSSS

21638
SmC * SfU * SmU * SfC * SfU * SfG * SfUn001fU * SfA * SfG

SSSSS nX SS

WV-
fA * SfC * SfUn001fG * SfU * SfUn001fC * SfA * SmG * SfC *
ACUGUUCAGCUUCUGUUAGC
SSnX SSnX SSSSS

21639
SmU * SfU * SmC * SfU * SfG * SfU * SfUn001fA * SfG * SfC

SSSSS nX SS

WV-
fC * SfU * SfGn001fU * SfU * SfCn001fA * SfG * SmC * SfU *
CUGUUCAGCUUCUGUUAGCC
SSnX SSnX SSSSS

21640
SmU * SfC * SmU * SfG * SfU * SfU * SfAn001fG * SfC * SfC

SSSSS nX SS

WV-
fU * SfG * SfUn001fU * SfC * SfAn001fG * SfC * SmU * SfU *
UGUUCAGCUUCUGUUAGCCA
SSnX SSnX SSSSS

21641
SmC * SfU * SmG * SfU * SfU * SfA * SfGn001fC * SfC * SfA

SSSSS nX SS

WV-
fG * SfU * SfUn001fC * SfA * SfGn001fC * SfU * SmU * SfC *
GUUCAGCUUCUGUUAGCCAC
SSnX SSnX SSSSS

21642
SmU * SfG * SmU * SfU * SfA * SfG * SfCn001fC * SfA * SfC

SSSSS nX SS

WV-
fU * SfU * SfCn001fA * SfG * SfCn001fU * SfU * SmC * SfU *
UUCAGCUUCUGUUAGCCACU
SSnX SSnX SSSSS

21643
SmG * SfU * SmU * SfA * SfG * SfC * SfCn001A * SfC * SfU

SSSSS nX SS

WV-
fU * SfC * SfAn001fG * SfC * SfUn001fU * SfC * SmU * SfG *
UCAGCUUCUGUUAGCCACUG
SSnX SSnX SSSSS

21644
SmU * SfU * SmA * SfG * SfC * SfC * SfAn001fC * SfG * SfG

SSSSS nX SS

WV-
fC * SfA * SfGn001fC * SfU * SfUn001fC * SfU * SmG * SfU *
CAGCUUCUGUUAGCCACUGA
SSnX SSnX SSSSS

21645
SmU * SfA * SmG * SfC * SfC * SfA * SfCn001fU * SfG * SfA

SSSSS nX SS

WV-
fA * SfG * SfCn001fU * SfU * SfCn001fU * SfG * SmU * SfU *
AGCUUCUGUUAGCCACUGAU
SSnX SSnX SSSSS

21646
SmA * SfG * SmC * SfC * SfA * SfC * SfUn001fG * SfA * SfU

SSSSS nX SS

WV-
fG * SfC * SfUn001fU * SfC * SfUn001fG * SfU * SmU * SfA *
GCUUCUGUUAGCCACUGAUU
SSnX SSnX SSSSS

21647
SmG * SfC * SmC * SfA * SfC * SfU * SfGn001fA * SfU * SfU

SSSSS nX SS

WV-
fC * SfU * SfUn001fC * SfU * SfGn001fU * SfU * SmA * SfG *
CUUCUGUUAGCCACUGAUUA
SSnX SSnX SSSSS

21648
SmC * SfC * SmA * SfC * SfU * SfG * SfAn001fU * SfU * SfA

SSSSS nX SS

WV-
fU * SfU * SfCn001fU * SfG * SfUn001fU * SfA * SmG * SfC *
UUCUGUUAGCCACUGAUUAA
SSnX SSnX SSSSS

21649
SmC * SfA * SmC * SfU * SfG * SfA * SfUn001fU * SfA * SfA

SSSSS nX SS

WV-
fU * SfC * SfUn001fG * SfU * SfUn001fA * SfG * SmC * SfC *
UCUGUUAGCCACUGAUUAAA
SSnX SSnX SSSSS

21650
SmA * SfC * SmU * SfG * SfA * SfU * SfUn001fA * SfA * SfA

SSSSS nX SS

WV-
fC * SfU * SfGn001fU * SfU * SfAn001fG * SfC * SmC * SfA *
CUGUUAGCCACUGAUUAAAU
SSnX SSnX SSSSS

21651
SmC * SfU * SmG * SfA * SfU * SfU * SfAn001fA * SfA * SfU

SSSSS nX SS

WV-
fU * SfG * SfUn001fU * SfA * SfGn001fC * SfC * SmA * SfC *
UGUUAGCCACUGAUUAAAUA
SSnX SSnX SSSSS

21652
SmU * SfG * SmA * SfU * SfU * SfA * SfAn001fA * SfU * SfA

SSSSS nX SS

WV-
fG * SfU * SfUn001fA * SfG * SfCn001fC * SfA * SmC * SfU *
GUUAGCCACUGAUUAAAUAU
SSnX SSnX SSSSS

21653
SmG * SfA * SmU * SfU * SfA * SfA * SfAn001fU * SfA * SfU

SSSSS nX SS

WV-
fU * SfU * SfAn001fG * SfC * SfCn001fA * SfC * SmU * SfG *
UUAGCCACUGAUUAAAUAUC
SSnX SSnX SSSSS

21654
SmA * SfU * SmU * SfA * SfA * SfA * SfUn001fA * SfU * SfC

SSSSS nX SS

WV-
fU * SfA * SfGn001fC * SfC * SfAn001fC * SfU * SmG * SfA *
UAGCCACUGAUUAAAUAUCU
SSnX SSnX SSSSS

21655
SmU * SfU * SmA * SfA * SfA * SfU * SfAn001fU * SfC * SfU

SSSSS nX SS

WV-
fA * SfG * SfCn001fC * SfA * SfCn001fU * SfG * SmA * SfU *
AGCCACUGAUUAAAUAUCUU
SSnX SSnX SSSSS

21656
SmU * SfA * SmA * SfA * SfU * SfA * SfUn001fC * SfU * SfU

SSSSS nX SS

WV-
fG * SfC * SfCn001fA * SfC * SfUn001fG * SfA * SmU * SfU *
GCCACUGAUUAAAUAUCUUU
SSnX SSnX SSSSS

21657
SmA * SfA * SmA * SfU * SfA * SfU * SfCn001fU * SfU * SfU

SSSSS nX SS

WV-
fC * SfC * SfAn001fC * SfU * SfGn001fA * SfU * SmU * SfA *
CCACUGAUUAAAUAUCUUUA
SSnX SSnX SSSSS

21658
SmA * SfA * SmU * SfA * SfU * SfC * SfUn001fU * SfU * SfA

SSSSS nX SS

WV-
fC * SfA * SfCn001fU * SfG * SfAn001fU * SfU * SmA * SfA *
CACUGAUUAAAUAUCUUUAU
SSnX SSnX SSSSS

21659
SmA * SfU * SmA * SfU * SfC * SfU * SfUn001fU * SfA * SfU

SSSSS nX SS

WV-
fA * SfC * SfUn001fG * SfA * SfUn001fU * SfA * SmA * SfA *
ACUGAUUAAAUAUCUUUAUA
SSnX SSnX SSSSS

21660
SmU * SfA * SmU * SfC * SfU * SfU * SfUn001fA * SfU * SfA

SSSSS nX SS

WV-
fC * SfU * SfGn001fA * SfU * SfUn001fA * SfA * SmA * SfU *
CUGAUUAAAUAUCUUUAUAU
SSnX SSnX SSSSS

21661
SmA * SfU * SmC * SfU * SfU * SfU * SfAn001fU * SfA * SfU

SSSSS nX SS

WV-
fU * SfG * SfAn001fU * SfU * SfAn001fA * SfA * SmU * SfA *
UGAUUAAAUAUCUUUAUAUC
SSnX SSnX SSSSS

21662
SmU * SfC * SmU * SfU * SfU * SfA * SfUn001fA * SfU * SfC

SSSSS nX SS

WV-
fG * SfA * SfUn001fU * SfA * SfAn001fA * SfU * SmA * SfU *
GAUUAAAUAUCUUUAUAUCA
SSnX SSnX SSSSS

21663
SmC * SfU * SmU * SfU * SfA * SfU * SfAn001fU * SfC * SfA

SSSSS nX SS

WV-
fA * SfU * SfUn001fA * SfA * SfAn001fU * SfA * SmU * SfC *
AUUAAAUAUCUUUAUAUCAU
SSnX SSnX SSSSS

21664
SmU * SfU * SmU * SfA * SfU * SfA * SfUn001fC * SfA * SfU

SSSSS nX SS

WV-
fU * SfU * SfAn001fA * SfA * SfUn001fA * SfU * SmC * SfU *
UUAAAUAUCUUUAUAUCAUA
SSnX SSnX SSSSS

21665
SmU * SfU * SmA * SfU * SfA * SfU * SfCn001fA * SfU * SfA

SSSSS nX SS

WV-
fU * SfA * SfAn001fA * SfU * SfAn001fU * SfC * SmU * SfU *
UAAAUAUCUUUAUAUCAUAA
SSnX SSnX SSSSS

21666
SmU * SfA * SmU * SfA * SfU * SfC * SfAn001fU * SfA * SfA

SSSSS nX SS

WV-
fA * SfA * SfAn001fU * SfA * SfUn001fC * SfU * SmU * SfU *
AAAUAUCUUUAUAUCAUAAU
SSnX SSnX SSSSS

21667
SmA * SfU * SmA * SfU * SfC * SfA * SfUn001fA * SfA * SfU

SSSSS nX SS

WV-
fA * SfA * SfUn001fA * SfU * SfCn001fU * SfU * SmU * SfA *
AAUAUCUUUAUAUCAUAAUG
SSnX SSnX SSSSS

21668
SmU * SfA * SmU * SfC * SfA * SfU * SfAn001fA * SfU * SfG

SSSSS nX SS

WV-
fA * SfU * SfAn001fU * SfC * SfUn001fU * SfU * SmA * SfU *
AUAUCUUUAUAUCAUAAUGA
SSnX SSnX SSSSS

21669
SmA * SfU * SmC * SfA * SfU * SfA * SfAn001fU * SfG * SfA

SSSSS nX SS

WV-
fU * SfA * SfUn001fC * SfU * SfUn001fU * SfA * SmU * SfA *
UAUCUUUAUAUCAUAAUGAA
SSnX SSnX SSSSS

21670
SmU * SfC * SmA * SfU * SfA * SfA * SfUn001fG * SfA * SfA

SSSSS nX SS

WV-
fA * SfU * SfCn001fU * SfU * SfUn001fA * SfU * SmA * SfU *
AUCUUUAUAUCAUAAUGAAA
SSnX SSnX SSSSS

21671
SmC * SfA * SmU * SfA * SfA * SfU * SfUn001fA * SfA * SfA

SSSSS nX SS

WV-
fU * SfC * SfUn001fU * SfU * SfAn001fU * SfA * SmU * SfC *
UCUUUAUAUCAUAAUGAAAA
SSnX SSnX SSSSS

21672
SmA * SfU * SmA * SfA * SfU * SfG * SfAn001fA * SfA * SfA

SSSSS nX SS

WV-
fC * SfU * SfUn001fU * SfA * SfUn001fA * SfU * SmC * SfA *
CUUUAUAUCAUAAUGAAAAC
SSnX SSnX SSSSS

21673
SmU * SfA * SmA * SfU * SfG * SfA * SfAn001fA * SfA * SfC

SSSSS nX SS

WV-
fC * SfU * SfGn001fA * SfA * SfUn001fU * SfA * SmU * SfU *
CUGAAUUAUUUCUUCCCCAG
SSnX SSnX SSSSS

21723
SmU * SfC * SmU * SfU * SfC * SfC * SfCn001fC * SfA * SfG

SSSSS nX SS

WV-
fU * SfG * SfAn001fA * SfU * SfUn001fA * SfU * SmU * SfU *
UGAAUUAUUUCUUCCCCAGU
SSnX SSnX SSSSS

21724
SmC * SfU * SmU * SfC * SfC * SfC * SfCn001fA * SfG * SfU

SSSSS nX SS

WV-
fG * SfA * SfAn001fU * SfU * SfAn001fU * SfU * SmU * SfC *
GAAUUAUUUCUUCCCCAGUU
SSnX SSnX SSSSS

21725
SmU * SfU * SmC * SfC * SfC * SfC * SfAn001fG * SfU * SfU

SSSSS nX SS

WV-
fA * SfA * SfUn001fU * SfA * SfUn001fU * SfU * SmC * SfU *
AAUUAUUUCUUCCCCAGUUG
SSnX SSnX SSSSS

21726
SmU * SfC * SmC * SfU * SfC * SfA * SfGn001fU * SfU * SfG

SSSSS nX SS

WV-
fA * SfU * SfUn001fA * SfU * SfUn001fU * SfC * SmU * SfU *
AUUAUUUCUUCCCCAGUUGC
SSnX SSnX SSSSS

21727
SmC * SfC * SmC * SfC * SfA * SfG * SfUn001fU * SfG * SfC

SSSSS nX SS

WV-
fU * SfU * SfAn001fU * SfU * SfUn001fC * SfU * SmU * SfC *
UUAUUUCUUCCCCAGUUGCA
SSnX SSnX SSSSS

21728
SmC * SfC * SmC * SfA * SfG * SfU * SfUn001fG * SfC * SfA

SSSSS nX SS

WV-
fU * SfA * SfUn001fU * SfU * SfCn001fU * SfU * SmC * SfC *
UAUUUCUUCCCCAGUUGCAU
SSnX SSnX SSSSS

21729
SmC * SfC * SmA * SfG * SfU * SfU * SfGn001fC * SfA * SfU

SSSSS nX SS

WV-
fA * SfU * SfUn001fU * SfC * SfUn001fU * SfC * SmC * SfC *
AUUUCUUCCCCAGUUGCAUU
SSnX SSnX SSSSS

21730
SmC * SfA * SmG * SfU * SfU * SfG * SfCn001fA * SfU * SfU

SSSSS nX SS

WV-
fU * SfU * SfUn001fC * SfU * SfUn001fC * SfC * SmC * SfC *
UUUCUUCCCCAGUUGCAUUC
SSnX SSnX SSSSS

21731
SmA * SfG * SmU * SfU * SfG * SfC * SfAn001fU * SfU * SfC

SSSSS nX SS

WV-
fU * SfU * SfCn001fU * SfU * SfCn001fC * SfU * SmC * SfA *
UUCUUCCCCAGUUGCAUUCA
SSnX SSnX SSSSS

21732
SmG * SfU * SmU * SfG * SfC * SfA * SfUn001fU * SfC * SfA

SSSSS nX SS

WV-
fU * SfC * SfUn001fU * SfC * SfCn001fC * SfC * SmA * SfG *
UCUUCCCCAGUUGCAUUCAA
SSnX SSnX SSSSS

21733
SmU * SfU * SmG * SfC * SfA * SfU * SfUn001fC * SfA * SfA

SSSSS nX SS

WV-
fC * SfU * SfUn001fC * SfC * SfCn001fC * SfA * SmG * SfU *
CUUCCCCAGUUGCAUUCAAU
SSnX SSnX SSSSS

21734
SmU * SfG * SmC * SfA * SfU * SfU * SfCn001fA * SfA * SfU

SSSSS nX SS

WV-
fU * SfU * SfCn001fC * SfC * SfCn001fA * SfG * SmU * SfU *
UUCCCCAGUUGCAUUCAAUG
SSnX SSnX SSSSS

21735
SmG * SfC * SmA * SfU * SfU * SfC * SfAn001fA * SfU * SfG

SSSSS nX SS

WV-
fU * SfC * SfCn001fC * SfC * SfAn001fG * SfU * SmU * SfG *
UCCCCAGUUGCAUUCAAUGU
SSnX SSnX SSSSS

21736
SmC * SfA * SmU * SfU * SfC * SfA * SfAn001fU * SfG * SfU

SSSSS nX SS

WV-
fC * SfC * SfCn001fC * SfA * SfGn001fU * SfU * SmG * SfC *
CCCCAGUUGCAUUCAAUGUU
SSnX SSnX SSSSS

21737
SmA * SfU * SmU * SfC * SfA * SfA * SfUn001fG * SfU * SfU

SSSSS nX SS

WV-
fC * SfC * SfCn001fA * SfG * SfUn001fU * SfG * SmC * SfA *
CCCAGUUGCAUUCAAUGUUC
SSnX SSnX SSSSS

21738
SmU * SfU * SmC * SfA * SfA * SfU * SfUn001fU * SfU * SfC

SSSSS nX SS

WV-
fC * SfC * SfAn001fG * SfU * SfUn001fG * SfC * SmA * SfU *
CCAGUUGCAUUCAAUGUUCU
SSnX SSnX SSSSS

21739
SmU * SfC * SmA * SfA * SfU * SfG * SfUn001fU * SfC * SfU

SSSSS nX SS

WV-
fC * SfA * SfGn001fU * SfU * SfGn001fC * SfA * SmU * SfU *
CAGUUGCAUUCAAUGUUCUG
SSnX SSnX SSSSS

21740
SmC * SfA * SmA * SfU * SfG * SfU * SfUn001fC * SfU * SfG

SSSSS nX SS

WV-
fA * SfG * SfUn001fU * SfG * SfCn001fA * SfU * SmU * SfC *
AGUUGCAUUCAAUGUUCUGA
SSnX SSnX SSSSS

21741
SmA * SfA * SmU * SfG * SfU * SfU * SfCn001fU * SfG * SfA

SSSSS nX SS

WV-
fG * SfU * SfUn001fG * SfC * SfAn001fU * SfU * SmC * SfA *
GUUGCAUUCAAUGUUCUGAC
SSnX SSnX SSSSS

21742
SmA * SfU * SmG * SfU * SfU * SfC * SfUn001fG * SfA * SfC

SSSSS nX SS

WV-
fU * SfU * SfUn001fC * SfA * SfUn001fU * SfC * SmA * SfA *
UUGCAUUCAAUGUUCUGACA
SSnX SSnX SSSSS

21743
SmU * SfG * SmU * SfU * SfC * SfU * SfGn001fA * SfC * SfA

SSSSS nX SS

WV-
fU * SfG * SfCn001fA * SfU * SfUn001fC * SfA * SmA * SfU *
UGCAUUCAAUGUUCUGACAA
SSnX SSnX SSSSS

21744
SmG * SfU * SmU * SfC * SfU * SfG * SfAn001fC * SfA * SfA

SSSSS nX SS

WV-
fG * SfC * SfAn001fU * SfU * SfCn001fA * SfA * SmU * SfG *
GCAUUCAAUGUUCUGACAAC
SSnX SSnX SSSSS

21745
SmU * SfU * SmC * SfU * SfG * SfA * SfCn001fA * SfA * SfC

SSSSS nX SS

WV-
fC * SfA * SfUn001fU * SfC * SfAn001fA * SfU * SmG * SfU *
CAUUCAAUGUUCUGACAACA
SSnX SSnX SSSSS

21746
SmU * SfC * SmU * SfG * SfA * SfC * SfAn001fA * SfC * SfA

SSSSS nX SS

WV-
fA * SfU * SfUn001fC * SfA * SfAn001fU * SfG * SmU * SfU *
AUUCAAUGUUCUGACAACAG
SSnX SSnX SSSSS

21747
SmC * SfU * SmG * SfA * SfA * SfA * SfAn001fC * SfA * SfG

SSSSS nX SS

WV-
fU * SfU * SfCn001fA * SfA * SfUn001fG * SfU * SmU * SfC *
UUCAAUGUUCUGACAACAGU
SSnX SSnX SSSSS

21748
SmU * SfG * SmA * SfC * SfA * SfA * SfCn001fA * SfG * SfU

SSSSS nX SS

WV-
fU * SfC * SfAn001fA * SfU * SfGn001fU * SfU * SmC * SfU *
UCAAUGUUCUGACAACAGUU
SSnX SSnX SSSSS

21749
SmG * SfA * SmC * SfA * SfA * SfC * SfAn001fG * SfU * SfU

SSSSS nX SS

WV-
fC * SfA * SfAn001fU * SfG * SfUn001fU * SfC * SmU * SfG *
CAAUGUUCUGACAACAGUUU
SSnX SSnX SSSSS

21750
SmA * SfC * SmA * SfA * SfC * SfA * SfGn001fU * SfU * SfU

SSSSS nX SS

WV-
fA * SfA * SfUn001fG * SfU * SfUn001fC * SfU * SmG * SfA *
AAUGUUCUGACAACAGUUUG
SSnX SSnX SSSSS

21751
SmC * SfA * SmA * SfC * SfA * SfG * SfUn001fU * SfU * SfG

SSSSS nX SS

WV-
fA * SfU * SfGn001fU * SfU * SfCn001fU * SfG * SmA * SfC *
AUGUUCUGACAACAGUUUGC
SSnX SSnX SSSSS

21752
SmA * SfA * SmC * SfA * SfG * SfU * SfUn001fU * SfG * SfC

SSSSS nX SS

WV-
fU * SfG * SfUn001fU * SfC * SfUn001fG * SfA * SmC * SfA *
UGUUCUGACAACAGUUUGCC
SSnX SSnX SSSSS

21753
SmA * SfC * SmA * SfG * SfU * SfU * SfUn001fG * SfC * SfC

SSSSS nX SS

WV-
fG * SfU * SfUn001fC * SfU * SfGn001fA * SfC * SmA * SfA *
GUUCUGACAACAGUUUGCCG
SSnX SSnX SSSSS

21754
SmC * SfA * SmG * SfU * SfU * SfU * SfGn001fC * SfC * SfG

SSSSS nX SS

WV-
fU * SfU * SfCn001fU * SfG * SfAn001fC * SfA * SmA * SfC *
UUCUGACAACAGUUUGCCGC
SSnX SSnX SSSSS

21755
SmA * SfG * SmU * SfU * SfU * SfG * SfCn001fC * SfG * SfC

SSSSS nX SS

WV-
fU * SfC * SfUn001fG * SfA * SfCn0001fA * SfA * SmC * SfA *
UCUGACAACAGUUUGCCGCU
SSnX SSnX SSSSS

21756
SmG * SfU * SmU * SfU * SfG * SfC * SfCn001fG * SfU * SfU

SSSSS nX SS

WV-
fC * SfU * SfGn001fA * SfC * SfAn001fA * SfC * SmA * SfG *
CUGACAACAGUUUGCCGCUG
SSnX SSnX SSSSS

21757
SmU * SfU * SmU * SfG * SfC * SfC * SfGn001fC * SfU * SfG

SSSSS nX SS

WV-
fU * SfG * SfAn001fC * SfA * SfAn001fC * SfA * SmG * SfU *
UGACAACAGUUUGCCGCUGC
SSnX SSnX SSSSS

21758
SmU * SfU * SmG * SfC * SfC * SfG * SfCn00lfU * SfG * SfC

SSSSS nX SS

WV-
fG * SfA * SfCn001fA * SfA * SfCn001fA * SfG * SmU * SfU *
GACAACAGUUUGCCGCUGCC
SSnX SSnX SSSSS

21759
SmU * SfG * SmC * SfC * SfG * SfC * SfUn001fG * SfC * SfC

SSSSS nX SS

WV-
fA * SfC * SfAn001fA * SfC * SfAn001fG * SfU * SmU * SfU *
ACAACAGUUUGCCGCUGCCC
SSnX SSnX SSSSS

21760
SmG * SfC * SmC * SfG * SfC * SfU * SfGn001fC * SfC * SfC

SSSSS nX SS

WV-
fC * SfA * SfAn001fC * SfA * SfGn001fU * SfU * SmU * SfG *
CAACAGUUUGCCGCUGCCCA
SSnX SSnX SSSSS

21761
SmC * SfC * SmG * SfC * SfU * SfG * SfCn001fC * SfC * SfA

SSSSS nX SS

WV-
fA * SfA * SfCn001fA * SfG * SfUn001fU * SfU * SmG * SfC *
AACAGUUUGCCGCUGCCCAA
SSnX SSnX SSSSS

21762
SmC * SfG * SmC * SfU * SfG * SfC * SfUn001fC * SfA * SfA

SSSSS nX SS

WV-
fA * SfC * SfAn001fG * SfU * SfUn001fU * SfG * SmC * SfC *
ACAGUUUGCCGCUGCCCAAU
SSnX SSnX SSSSS

21763
SmG * SfC * SmU * SfG * SfC * SfC * SfCn001fA * SfA * SfU

SSSSS nX SS

WV-
fC * SfA * SfGn001fU * SfU * SfUn001fG * SfC * SmC * SfG *
CAGUUUGCCGCUGCCCAAUG
SSnX SSnX SSSSS

21764
SmC * SfU * SmG * SfC * SfC * SfC * SfAn001fA * SfU * SfG

SSSSS nX SS

WV-
fA * SfG * SfUn001fU * SfU * SfGn001fC * SfC * SmG * SfC *
AGUUUGCCGCUGCCCAAUGC
SSnX SSnX SSSSS

21765
SmU * SfG * SmC * SfC * SfC * SfA * SfAn001fU * SfG * SfC

SSSSS nX SS

WV-
fG * SfU * SfUn001fU * SfG * SfCn001fC * SfG * SmC * SfU *
GUUUGCCGCUGCCCAAUGCC
SSnX SSnX SSSSS

21766
SmG * SfC * SmC * SfC * SfA * SfA * SfUn001fG * SfC * SfC

SSSSS nX SS

WV-
fU * SfU * SfUn001fG * SfC * SfCn001fG * SfC * SmU * SfG *
UUUGCCGCUGCCCAAUGCCA
SSnX SSnX SSSSS

21767
SmC * SfC * SmC * SfA * SfA * SfU * SfGn001fC * SfC * SfA

SSSSS nX SS

WV-
fU * SfU * SfGn001fC * SfC * SfGn001fC * SfU * SmG * SfC *
UUGCCGCUGCCCAAUGCCAU
SSnX SSnX SSSSS

21768
SmC * SfC * SmA * SfA * SfU * SfG * SfCn001fC * SfA * SfU

SSSSS nX SS

WV-
fU * SfG * SfCn001fC * SfG * SfCn001fU * SfG * SmC * SfC *
UGCCGCUGCCCAAUGCCAUC
SSnX SSnX SSSSS

21769
SmC * SfA * SmA * SfU * SfG * SfC * SfCn001fA * SfU * SfC

SSSSS nX SS

WV-
fG * SfC * SfCn001fG * SfC * SfUn001fG * SfC * SmC * SfC *
GCCGCUGCCCAAUGCCAUCC
SSnX SSnX SSSSS

21770
SmA * SfA * SmU * SfG * SfC * SfC * SfAn001fU * SfC * SfC

SSSSS nX SS

WV-
fC * SfC * SfGn001fC * SfU * SfGn001fC * SfC * SmC * SfA *
CCGCUGCCCAAUGCCAUCCU
SSnX SSnX SSSSS

21771
SmA * SfU * SmG * SfC * SfC * SfA * SfUn001fC * SfC * SfU

SSSSS nX SS

WV-
fA * SfU * SfUn001fU * SfU * SfGn001fG * SfG * SmC * SfA *
AUUUUGGGCAGCGGUAAUGA
SSnX SSnX SSSSS

21772
SmG * SfC * SmG * SfG * SfU * SfA * SfAn001fU * SfG * SfA

SSSSS nX SS

WV-
fU * SfU * SfUn001fU * SfG * SfGn001fG * SfC * SmA * SfG *
UUUUGGGCAGCGGUAAUGAG
SSnX SSnX SSSSS

21773
SmC * SfG * SmG * SfU * SfA * SfA * SfUn001fG * SfA * SfG

SSSSS nX SS

WV-
fU * SfU * SfUn001fG * SfG * SfGn001fC * SfA * SmG * SfC *
UUUGGGCAGCGGUAAUGAGU
SSnX SSnX SSSSS

21774
SmG * SfG * SmU * SfA * SfA * SfU * SfGn001fA * SfG * SfU

SSSSS nX SS

WV-
fU * SfU * SfGn001fG * SfG * SfCn001fA * SfG * SmC * SfG *
UUGGGCAGCGGUAAUGAGUU
SSnX SSnX SSSSS

21775
SmG * SfU * SmA * SfA * SfU * SfG * SfAn001fG * SfU * SfU

SSSSS nX SS

WV-
fU * SfG * SfGn001fG * SfC * SfAn001fG * SfC * SmG * SfG *
UGGGCAGCGGUAAUGAGUUC
SSnX SSnX SSSSS

21776
SmU * SfA * SmA * SfU * SfG * SfA * SfGn00fU * SfU * SfC

SSSSS nX SS

WV-
fG * SfG * SfGn001fC * SfA * SfGn001fC * SfG * SmG * SfU *
GGGCAGCGGUAAUGAGUUCU
SSnX SSnX SSSSS

21777
SmA * SfA * SmU * SfG * SfA * SfG * SfUn001fU * SfC * SfU

SSSSS nX SS

WV-
fG * SfG * SfCn001fA * SfG * SfCn001fG * SfG * SmU * SfA *
GGCAGCGGUAAUGAGUUCUU
SSnX SSnX SSSSS

21778
SmA * SfU * SmG * SfA * SfG * SfU * SfUn001fC * SfU * SfU

SSSSS nX SS

WV-
fG * SfC * SfAn001fG * SfC * SfGn001fG * SfU * SmA * SfA *
GCAGCGGUAAUGAGUUCUUC
SSnX SSnX SSSSS

21779
SmU * SfG * SmA * SfG * SfU * SfU * SfCn001fU * SfU * SfC

SSSSS nX SS

WV-
fC * SfA * SfGn001fC * SfG * SfGn001fU * SfA * SmA * SfU *
CAGCGGUAAUGAGUUCUUCC
SSnX SSnX SSSSS

21780
SmG * SfA * SmG * SfU * SfU * SfC * SfUn001fU * SfC * SfC

SSSSS nX SS

WV-
fA * SfG * SfCn001fG * SfG * SfUn001fA * SfA * SmU * SfG *
AGCGGUAAUGAGUUCUUCCA
SSnX SSnX SSSSS

21781
SmA * SfG * SmU * SfU * SfC * SfU * SfUn001fC * SfC * SfA

SSSSS nX SS

WV-
fG * SfC * SfGn001fG * SfU * SfAn001fA * SfU * SmG * SfA *
GCGGUAAUGAGUUCUUCCAA
SSnX SSnX SSSSS

21782
SmG * SfU * SmU * SfC * SfU * SfU * SfCn001fC * SfA * SfA

SSSSS nX SS

WV-
fC * SfG * SfGn001fU * SfA * SfAn001fU * SfG * SmA * SfG *
CGGUAAUGAGUUCUUCCAAC
SSnX SSnX SSSSS

21783
SmU * SfU * SmC * SfU * SfU * SfC * SfCn001fA * SfA * SfC

SSSSS nX SS

WV-
fG * SfG * SfUn001fA * SfA * SfUn001fG * SfA * SmG * SfU *
GGUAAUGAGUUCUUCCAACU
SSnX SSnX SSSSS

21784
SmU * SfC * SmU * SfU * SfC * SfC * SfAn001fA * SfC * SfU

SSSSS nX SS

WV-
fG * SfU * SfAn001fA * SfU * SfGn001fA * SfG * SmU * SfU *
GUAAUGAGUUCUUCCAACUG
SSnX SSnX SSSSS

21785
SmC * SfU * SmU * SfC * SfC * SfA * SfAn001fC * SfU * SfG

SSSSS nX SS

WV-
fU * SfA * SfAn001fU * SfG * SfAn001fG * SfU * SmU * SfC *
UAAUGAGUUCUUCCAACUGG
SSnX SSnX SSSSS

21786
SmU * SfU * SmC * SfC * SfA * SfA * SfCn001fU * SfG * SfG

SSSSS nX SS

WV-
fA * SfA * SfUn001fG * SfA * SfGn001fU * SfU * SmC * SfU *
AAUGAGUUCUUCCAACUGGG
SSnX SSnX SSSSS

21787
SmU * SfC * SmC * SfA * SfA * SfC * SfUn001fG * SfG * SfG

SSSSS nX SS

WV-
fA * SfU * SfGn001fA * SfG * SfUn001fU * SfC * SmU * SfU *
AUGAGUUCUUCCAACUGGGG
SSnX SSnX SSSSS

21788
SmC * SfC * SmA * SfA * SfC * SfU * SfGn001fG * SfG * SfG

SSSSS nX SS

WV-
fU * SfG * SfAn001fG * SfU * SfUn001fC * SfU * SmU * SfC *
UGAGUUCUUCCAACUGGGGA
SSnX SSnX SSSSS

21789
SmC * SfA * SmA * SfC * SfU * SfG * SfGn001fG * SfG * SfA

SSSSS nX SS

WV-
fG * SfA * SfGn001fU * SfU * SfCn001fU * SfU * SmC * SfC *
GAGUUCUUCCAACUGGGGAC
SSnX SSnX SSSSS

21790
SmA * SfA * SmC * SfU * SfG * SfG * SfGn001fG * SfA * SfC

SSSSS nX SS

WV-
fA * SfG * SfUn001fU * SfC * SfUn001fU * SfC * SmC * SfA *
AGUUCUUCCAACUGGGGACG
SSnX SSnX SSSSS

21791
SmA * SfC * SmU * SfG * SfG * SfG * SfGn001fA * SfG * SfG

SSSSS nX SS

WV-
fG * SfU * SfUn001fC * SfU * SfUn001fC * SfC * SmA * SfA *
GUUCUUCCAACUGGGGACGC
SSnX SSnX SSSSS

21792
SmC * SfU * SmG * SfG * SfG * SfG * SfAn001fC * SfG * SfC

SSSSS nX SS

WV-
fU * SfU * SfCn001fU * SfU * SfCn001fC * SfA * SmA * SfC *
UUCUUCCAACUGGGGACGCC
SSnX SSnX SSSSS

21793
SmU * SfG * SmG * SfG * SfG * SfA * SfCn001fG * SfC * SfC

SSSSS nX SS

WV-
fU * SfC * SfUn001fU * SfC * SfCn001fA * SfA * SmC * SfU *
UCUUCCAACUGGGGACGCCU
SSnX SSnX SSSSS

21794
SmG * SfG * SmG * SfG * SfA * SfC * SfGn001fC * SfC * SfU

SSSSS nX SS

WV-
fC * SfU * SfUn001fC * SfC * SfAn001fA * SfC * SmU * SfG *
CUUCCAACUGGGGACGCCUC
SSnX SSnX SSSSS

21795
SmG * SfG * SmG * SfA * SfC * SfG * SfCn001fC * SfU * SfC

SSSSS nX SS

WV-
fU * SfU * SfCn001fC * SfA * SfAn001fC * SfU * SmG * SfG *
UUCCAACUGGGGACGCCUCU
SSnX SSnX SSSSS

21796
SmG * SfG * SmA * SfC * SfG * SfC * SfCn001fU * SfC * SfU

SSSSS nX SS

WV-
fU * SfC * SfCn001fA * SfA * SfCn001fU * SfG * SmG * SfG *
UCCAACUGGGGACGCCUCUG
SSnX SSnX SSSSS

21797
SmG * SfA * SmC * SfG * SfC * SfC * SfUn001fC * SfU * SfG

SSSSS nX SS

WV-
fC * SfC * SfAn001fA * SfC * SfUn001fG * SfG * SmG * SfG *
CCAACUGGGGACGCCUCUGU
SSnX SSnX SSSSS

21798
SmA * SfC * SmG * SfC * SfC * SfU * SfCn001fU * SfG * SfU

SSSSS nX SS

WV-
fC * SfA * SfAn001fC * SfU * SfGn001fG * SfG * SmG * SfA *
CAACUGGGGACGCCUCUGUU
SSnX SSnX SSSSS

21799
SmC * SfG * SmC * SfC * SfU * SfC * SfUn001fG * SfU * SfU

SSSSS nX SS

WV-
fA * SfA * SfCn001fU * SfG * SfGn001fG * SfG * SmA * SfC *
AACUGGGGACGCCUCUGUUC
SSnX SSnX SSSSS

21800
SmG * SfC * SmC * SfU * SfC * SfU * SfGn001fU * SfU * SfC

SSSSS nX SS

WV-
fA * SfC * SfUn001fG * SfG * SfGn001fG * SfA * SmC * SfG *
ACUGGGGACGCCUCUGUUCC
SSnX SSnX SSSSS

21801
SmC * SfC * SmU * SfC * SfU * SfG * SfUn001fU * SfC * SfC

SSSSS nX SS

WV-
fC * SfU * SfGn001fG * SfG * SfGn001fA * SfC * SmG * SfC *
CUGGGGACGCCUCUGUUCCA
SSnX SSnX SSSSS

21802
SmC * SfU * SmC * SfU * SfG * SfU * SfUn001fC * SfC * SfA

SSSSS nX SS

WV-
fU * SfG * SfGn001fG * SfG * SfAn001fC * SfG * SmC * SfC *
UGGGGACGCCUCUGUUCCAA
SSnX SSnX SSSSS

21803
SmU * SfC * SmU * SfG * SfU * SfU * SfCn001fC * SfA * SfA

SSSSS nX SS

WV-
fG * SfG * SfGn001fG * SfA * SfCn001fG * SfC * SmC * SfU *
GGGGACGCCUCUGUUCCAAA
SSnX SSnX SSSSS

21804
SmC * SfU * SmG * SfU * SfU * SfC * SfCn001fA * SfA * SfA

SSSSS nX SS

WV-
fG * SfG * SfGn001fA * SfC * SfGn001fC * SfC * SmU * SfC *
GGGACGCCUCUGUUCCAAAU
SSnX SSnX SSSSS

21805
SmU * SfG * SmU * SfU * SfC * SfC * SfAn001fA * SfA * SfU

SSSSS nX SS

WV-
fG * SfG * SfAn001fC * SfG * SfCn001fC * SfU * SmC * SfU *
GGACGCCUCUGUUCCAAAUC
SSnX SSnX SSSSS

21806
SmG * SfU * SmU * SfC * SfC * SfA * SfAn001fA * SfU * SfC

SSSSS nX SS

WV-
fG * SfA * SfCn001fG * SfC * SfCn001fU * SfC * SmU * SfG *
GACGCCUCUGUUCCAAAUCC
SSnX SSnX SSSSS

21807
SmU * SfU * SmC * SfC * SfA * SfA * SfAn001fU * SfC * SfC

SSSSS nX SS

WV-
fA * SfC * SfGn001fC * SfC * SfUn001fC * SfU * SmG * SfU *
ACGCCUCUGUUCCAAAUCCU
SSnX SSnX SSSSS

21808
SmU * SfC * SmC * SfA * SfA * SfA * SfUn001fC * SfC * SfU

SSSSS nX SS

WV-
fC * SfG * SfCn001fC * SfU * SfCn001fU * SfG * SmU * SfU *
CGCCUCUGUUCCAAAUCCUG
SSnX SSnX SSSSS

21809
SmC * SfC * SmA * SfA * SfA * SfU * SfCn001fC * SfU * SfG

SSSSS nX SS

WV-
fG * SfC * SfCn001fU * SfC * SfUn001fG * SfU * SmU * SfC *
GCCUCUGUUCCAAAUCCUGC
SSnX SSnX SSSSS

21810
SmC * SfA * SmA * SfA * SfU * SfC * SfCn001fU * SfG * SfC

SSSSS nX SS

WV-
fC * SfC * SfUn001fC * SfU * SfGn001fU * SfU * SmC * SfC *
CCUCUGUUCCAAAUCCUGCA
SSnX SSnX SSSSS

21811
SmA * SfA * SmA * SfU * SfC * SfC * SfUn001fG * SfC * SfA

SSSSS nX SS

WV-
fC * SfU * SfCn001fU * SfG * SfUn001fU * SfC * SmC * SfA *
CUCUGUUCCAAAUCCUGCAU
SSnX SSnX SSSSS

21812
SmA * SfA * SmU * SfC * SfC * SfU * SfGn001fC * SfA * SfU

SSSSS nX SS

WV-
fU * SfC * SfUn001fG * SfU * SfUn001fC * SfC * SmA * SfA *
UCUGUUCCAAAUCCUGCAUU
SSnX SSnX SSSSS

21813
SmA * SfU * SmC * SfC * SfU * SfG * SfCn001fA * SfU * SfU

SSSSS nX SS

WV-
fC * SfU * SfGn001fU * SfU * SfCn001fC * SfA * SmA * SfA *
CUGUUCCAAAUCCUGCAUUG
SSnX SSnX SSSSS

21814
SmU * SfC * SmC * SfU * SfG * SfC * SfAn001fU * SfU * SfG

SSSSS nX SS

WV-
fU * SfG * SfUn001fU * SfC * SfCn001fA * SfA * SmA * SfU *
UGUUCCAAAUCCUGCAUUGU
SSnX SSnX SSSSS

21815
SmC * SfC * SmU * SfG * SfC * SfA * SfUn001fU * SfG * SfU

SSSSS nX SS

WV-
fG * SfU * SfUn001fC * SfC * SfAn001fA * SfA * SmU * SfC *
GUUCCAAAUCCUGCAUUGUU
SSnX SSnX SSSSS

21816
SmC * SfU * SmG * SfC * SfA * SfU * SfUn001fG * SfU * SfU

SSSSS nX SS

WV-
fU * SfU * SfCn001fC * SfA * SfAn001fA * SfU * SmC * SfC *
UUCCAAAUCCUGCAUUGUUG
SSnX SSnX SSSSS

21817
SmU * SfG * SmC * SfA * SfU * SfU * SfUn001fU * SfU * SfG

SSSSS nX SS

WV-
fU * SfC * SfCn001fA * SfA * SfAn001fU * SfC * SmC * SfU *
UCCAAAUCCUGCAUUGUUGC
SSnX SSnX SSSSS

21818
SmG * SfC * SmA * SfU * SfU * SfG * SfUn001fU * SfG * SfC

SSSSS nX SS

WV-
fU * SfC * SfAn001RfC * SfU * SfCn001RmA * SfG * SfA *
UCACUCAGAUAGUUGAAGCC
SSnR SSnR SSSSS

22753
SmU * SfA * SmG * SmU * SfU * SfG * SfA * SfAn001RfG *

SSSSS nR SS

SfC * SfC

WV-
L009n001L009n001L009n001L009fU * SfC * SfA * SfC * SfU *
UCACUCAGAUAGUUGAAGCC
nX nX nX OSSSSS

23576
SfC * SmAfG * SfA * SmU * SfA * SmGmUfU * SfG * SfA *

SOSS SSOOSSSSS

SfA * SfG * SfC * SfC

S

WV-
L009n001L009n001L009n001fU * SfC * SfA * SfC * SfU * SfC *
UCACUCAGAUAGUUGAAGCC
nX nX nX SSSSS

23577
SmAfG * SfA * SmU * SfA * SmGmUfU * SfG * SfA * SfA *

SOSS SSOOSSSSS

SfG * SfC * SfC

S

WV-
L009n001L009n001L009n001L009fU * SfC * SfAn001fC * SfU *
UCACUCAGAUAGUUGAAGCC
nX nX nX OSSnX

23578
SfCn001mAfG * SfA * SmU * SfA * SmGmUfU * SfG * SfA *

SSnX

SfAn001fG * SfC * SfC

OSSSSOOSSSnX SS

WV-
L009n001L009n001L009n001fU * SfC * SfAn001fC * SfU *
UCACUCAGAUAGUUGAAGCC
nX nX nX SSnX

23579
SfCn001mAfG * SfA * SmU * SfA * SmGmUfU * SfG * SfA *

SSnX

SfAn001fG * SfC * SfC

OSSSSOOSSSnX SS

WV-
L010n001L010n001L010n001L009fU * SfC * SfA * SfC * SfU *
UCACUCAGAUAGUUGAAGCC
nX nX nX OSSSSS

23936
SfC * SmAfG * SfA * SmU * SfA * SmGmUfU * SfG * SfA *

SOSS SSOOSSSSS

SfA * SfG * SfC * SfC

S

WV-
L010n001L010n001L010n001fU * SfC * SfA * SfC * SfU * SfC *
UCACUCAGAUAGUUGAAGCC
nX nX nX SSSSS

23937
SmAfG * SfA * SmU * SfA * SmGmUfU * SfG * SfA * SfA *

SOSS SSOOSSSSS

SfG * SfC * SfC

S

WV-
L010n001L010n001L010n001L009fU * SfC * SfAn001fC * SfU *
UCACUCAGAUAGUUGAAGCC
nX nX nX OSSnX

23938
SfCn001mAfG * SfA * SmU * SfA * SmGmUfU * SfG * SfA *

SSnX

SfAn001fG * SfC * SfC

OSSSSOOSSSnX SS

WV-
L010n001L010n001L010n001fU * SfC * SfAn001fC * SfU *
UCACUCAGAUAGUUGAAGCC
nX nX nX SSnX

23939
SfCn001mAfG * SfA * SmU * SfA * SmGmUfU * SfG * SfA *

SSnX OSSSSO

SfAn001fG * SfC * SfC

OSSSnX SS

WV-
mU * SGeon009m5Ceon009m5Ceon009mA * SG * SG * RC * ST
UGCCAGGCTGGTTATGACUC
S nX nX nX SSRSS

XBD108
* SG * RG * ST * ST * RA * ST * SmG * SmA * SmC * SmU *

RSSRSS SSSS

SmC

WV-XBD
mU * SGeon009Rm5Ceon009Rm5Ceon009RmA * SG * SG * RC
UGCCAGGCTGGTTATGACUC
S nR nR nR SSRSS

109
* ST * SG * RG * ST * ST * RA * ST * SmG * SmA * SmC *

RSSRSS SSSS

SmU * SmC

WV-XBD
mU * SGeon009Sm5Ceon009Sm5Ceon009SmA * SG * SG * RC *
UGCCAGGCTGGTTATGACUC
S nS nS nS SSRSS

110
ST * SG * RG * ST * ST * RA * ST * SmG * SmA * SmC * SmU

RSSRSS SSSS

* SmC

WV-
mU * SGeon010m5Ceon010m5Ceon010mA * SG * SG * RC * ST
UGCCAGGCTGGTTATGACUC
S nX nX nX SSRSS

XKCD108
* SG * RG * ST * ST * RA * ST * SmG * SmA * SmC * SmU *

RSSRSS SSSS

SmC

WV-
mU * SGeon010Rm5Ceon010Rm5Ceon010RmA * SG * SG * RC
UGCCAGGCTGGTTATGACUC
S nR nR nR SSRSS

XKCD
* ST * SG * RG * ST * ST * RA * ST * SmG * SmA * SmC *

RSSRSS SSSS

109
SmU * SmC

WV-
mU * SGeon010Sm5Ceon010Sm5Ceon010SmA * SG * SG * RC *
UGCCAGGCTGGTTATGACUC
S nS nS nS SSRSS

XKCD
ST * SG * RG * ST * ST * RA * ST * SmG * SmA * SmC * SmU

RSSRSS SSSS

110
* SmC

WV-3519
Mod032fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA
UCAAGGAAGA
O XXXXX XOXOX

* fU * fU * fU * fC * fU
UGGCAUUUCU
OXO XXXXX X

WV-3518
Mod031fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA
UCAAGGAAGA
O XXXXX XOXOX

* fu * fU * fU * fC * fU
UGGCAUUUCU
OXO XXXXX X

WV-3517
Mod030fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA
UCAAGGAAGA
O XXXXX XOXOX

* fU * fU * fU * fC * fU
UGGCAUUUCU
OXO XXXXX X

WV-3516
fU * fC * fA * fA * fG * fG * mAfA * mGfA * mUfG * mGfC * fA * fU *
UCAAGGAAGA
XXXXX XOXOX

fU * fU * fC * fU
UGGCAUUUCU
OXO XXXXX X

WV-3515
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmAfU * SmGmGfC *
UCAAGGAAGA
SSSSS SOSOO

SfAfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
SOOSOSSSS

WV-3514
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGfAfU * SmGmGfC *
UCAAGGAAGA
SSSSS SOSOO

SfAfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
SOOSOSSSS

WV-3513
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmAfU * SmGmGfC *
UCAAGGAAGA
SSSSS SOSOO

SmAfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
SOOSOSSSS

WV-3512
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGfAU * SmGmGfC *
UCAAGGAAGA
SSSSS SOSOO

SmAfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
SOOSOSSSS

WV-3511
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmAfU * SmGmGfC *
UCAAGGAAGA
SSSSS SOSOO SOO

SmA * SfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
SSSSS S

WV-3510
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGfAfU * SmGmGfC *
UCAAGGAAGA
SSSSS SOSOO SOO

SmA * SfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
SSSSS S

WV-3509
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGA
SSSSS SOSOS

* SfAfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
SOOSOSSSS

WV-3508
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGfA * SfU * SmGmGfC *
UCAAGGAAGA
SSSSS SOSOS

SfAfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
SOOSOSSSS

WV-3507
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmAfU * SmGmGfC *
UCAAGGAAGA
SSSSS SOSOO SOO

SfA * SfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
SSSSS S

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAn011fG * SfA * SmU * SfA *
UCACUCAGAUA
SSSSS SnXSSSS

27250
SmGn011mUn011fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
nXnX SSSSS S

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAn010fG * SfA * SmU * SfA *
UCACUCAGAUA
SSSSS

27249
SmGn010mUn010fU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SnXSSSSnXnX SSSSS

S

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGA
SSSSS SOSOS SOO

24086
* SfA * SfU * SfU * SfU * SfC * SfG
UGGCAUUUCG
SSSSS S

WV-
fG * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC
GCAAGGAAGAU
SSSSS SOSOS SOO

24085
* SfA * SfU * SfU * SfU * SfC * SfU
GGCAUUUCU
SSSSS S

WV-
fU * SfG * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmG *
UCAAGGAAGA
SSSSS SOSOS SO

22919
SfC * SfA * SfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
SSSSS SS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmG *
UCAAGGAAGA
SSSSS SOSOS SSO

22918
SmGfC * SfA * SfU * SfU * SfU * SfC * SfU
UGGCAUUUCU
SSSSS S

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmG
UCAAGGAAGA UG
SSSSS SOSOS S

22765

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGA
SSSSS SOSOS SOOS

22764
* SfA
UGGCA

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGA
SSSSS SOSOS

22763
* SfA * SfU
UGGCAU
SOOSS

WV-
fU * SfC * SfA * SfA * SfG * SfG * SmAfA * SmGmA * SfU * SmGmGfC
UCAAGGAAGA
SSSSS SOSOS

22762
* SfA * SfU * SfU
UGGCAUU
SOOSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmA * SfG * SfA * SmU * SfA * SmG
UCACUCAGAUA
SSSSS SSSSS

22752
* SmU * SfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SSSSS SSSS

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmA * SfG * SfA * SmU * SfA *
UCACUCAGAUA
SSSSS SSSSS SOO

22751
SmGmUfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SSSSS S

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU * SfA * SmG *
UCACUCAGAUA
SSSSS SO SSSSS O

22750
SmUfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SSSSS S

WV-
fU * SfC * SfA * SfC * SfU * SfC * SmAfG * SfA * SmU * SfA * SmGmU
UCACUCAGAUA
SSSSS SOSSSSO

22749
* SfU * SfG * SfA * SfA * SfG * SfC * SfC
GUUGAAGCC
SSSSS SS

WV-
fA * SfU * SfC * SfA * SfU * SfU * SfU * SfU * SmU * SfU * SmC * SfU *
AUCAUUUUUU
SSSSS SSSSS

21502
SmC * SfA * SfU * SfA * SfC * SfC * SfU * SfU
CUCAUACCUU
SSSSS SSSS

WV-
fU * SfA * SfU * SfC * SfA * SfU * SfU * SfU * SmU * SfU * SmU * SfC *
UAUCAUUUUU
SSSSS SSSSS

21501
SmU * SfC * SfA * SfU * SfA * SfC * SfC * SfU
UCUCAUACCU
SSSSS SSSS

WV-
fU * SfU * SfA * SfU * SfC * SfA * SfU * SfU * SmU * SfU * SmU * SfU *
UUAUCAUUUUU
SSSSS SSSSS

21500
SmC * SfU * SfC * SfA * SfU * SfA * SfC * SfC
UCUCAUACC
SSSSS SSSS

WV-
fU * SfU * SfU * SfA * SfU * SfC * SfA * SfU * SmU * SfU * SmU * SfU *
UUUAUCAUUUU
SSSSS SSSSS

21499
SmU * SfC * SfU * SfC * SfA * SfU * SfA * SfC
UUCUCAUAC
SSSSS SSSS

WV-
fU * SfU * SfU * SfU * SfA * SfU * SfC * SfA * SmU * SfU * SmU * SfU *
UUUUAUCAUUUU
SSSSS SSSSS

21498
SmU * SfU * SfC * SfU * SfC * SfA * SfU * SfA
UUCUCAUA
SSSSS SSSS

WV-
fC * SfU * SfU * SfU * SfU * SfA * SfU * SfC * SmA * SfU * SmU * SfU *
CUUUUAUCAUUU
SSSSS SSSSS

21497
SmU * SfU * SfU * SfC * SfU * SfC * SfA * SfU
UUUCUCAU
SSSSS SSSS

WV-
fA * SfC * SfU * SfU * SfU * SfU * SfA * SfU * SmC * SfA * SmU * SfU *
ACUUUUAUCAUU
SSSSS SSSSS

21496
SmU * SfU * SfU * SfU * SfC * SfU * SfC * SfA
UUUUCUCA
SSSSS SSSS

WV-
fA * SfA * SfC * SfU * SfU * SfU * SfU * SfA * SmU * SfC * SmA * SfU *
AACUUUUAUCAU
SSSSS SSSSS

21495
SmU * SfU * SfU * SfU * SfU * SfC * SfU * SfC
UUUUUCUC
SSSSS SSSS

WV-
fC * SfA * SfA * SfC * SfU * SfU * SfU * SfU * SmA * SfU * SmC * SfA *
CAACUUUUAUCAU
SSSSS SSSSS

21494
SmU * SfU * SfU * SfU * SfU * SfU * SfC * SfU
UUUUUCU
SSSSS SSSS

WV-
fC * SfC * SfA * SfA * SfC * SfU * SfU * SfU * SmU * SfA * SmU * SfC *
CCAACUUUUAU
SSSSS SSSSS

21493
SmA * SfU * SfU * SfU * SfU * SfU * SfU * SfU
CAUUUUUUC
SSSSS SSSS

WV-
fG * SfC * SfC * SfA * SfA * SfC * SfU * SfU * SmU * SfU * SmA * SfU *
GCCAACUUUUA
SSSSS SSSSS

21492
SmC * SfA * SfU * SfU * SfU * SfU * SfU * SfU
UCAUUUUUU
SSSSS SSSS

WV-
fU * SfG * SfC * SfC * SfA * SfA * SfC * SfU * SmU * SfU * SmU * SfA *
UGCCAACUUUU
SSSSS SSSSS

21491
SmU * SfC * SfA * SfU * SfU * SfU * SfU * SfU
AUCAUUUUU
SSSSS SSSS

WV-
fC * SfU * SfG * SfC * SfC * SfA * SfA * SfC * SmU * SfU * SmU * SfU *
CUGCCAACUUUU
SSSSS SSSSS

21490
SmA * SfU * SfC * SfA * SfU * SfU * SfU * SfU
AUCAUUUU
SSSSS SSSS

WV-
fU * SfC * SfU * SfG * SfC * SfC * SfA * SfA * SmC * SfU * SmU * SfU *
UCUGCCAACUUU
SSSSS SSSSS

21489
SmU * SfA * SfU * SfC * SfA * SfU * SfU * SfU
UAUCAUUU
SSSSS SSSS

WV-
fU * SfU * SfC * SfU * SfG * SfC * SfC * SfA * SmA * SfC * SmU * SfU *
UUCUGCCAACUU
SSSSS SSSSS

21488
SmU * SfU * SfA * SfU * SfC * SfA * SfU * SfU
UUAUCAUU
SSSSS SSSS

WV-
fC * SfU * SfU * SfC * SfU * SfG * SfC * SfC * SmA * SfA * SmC * SfU *
CUUCUGCCAACU
SSSSS SSSSS

21487
SmU * SfU * SfU * SfA * SfU * SfC * SfA * SfU
UUUAUCAU
SSSSS SSSS

WV-
fC * SfU * SfCfC * SfG * SfGfU * SfU * SmCfU * SmG * SfA * SmAfG *
CUCCGGUUCUGA
SSOSS OSSOS SSOSS

21373
SfG * SfU * SfGfU * SfU * SfC
AGGUGUUC
SOSS

In Table A1 (including Table A1.1., Table A1.2, Table A1.3, etc.):

Spaces in Table A1 are utilized for formatting and readability, e.g., OXXXXX XXXXX XXXXX XXXX illustrates the same stereochemistry as OXXXXXXXXXXXXXXXXXXX *S and *S both indicate phosphorothioate internucleotidic linkage wherein the linkage phosphorus has Sp configuration; etc.

All oligonucleotides listed in Tables A1 are single-stranded. As described in the present application, they may be used as a single strand, or as a strand to form complexes with one or more other strands.

Some sequences, due to their length, are divided into multiple lines.

ID: Identification number for an oligonucleotide.

WV-8806, WV-13405, WV-13406 and WV-13407 are fully PMO (morpholino oligonucleotides; [all PMO] in Table).

Abbreviations in Tables:

m5Ceo:5-Methyl 2′-Methoxyethyl C

embedded image

5MS: 5′-(S)—CH₃modification of sugar moieties;

5MSfC: 2′-F-5′-(S)-methyl C (in oligonucleotides

embedded image

wherein in BA is nucleobase C and R^2sis —F, and the 5′ and 3′ positions independently connect to —OH, internucleotidic linkages, linkers/linkages-H, linkers/linkages-Mod, etc. Nucleoside form is

embedded image

wherein in BA is nucleobase C and R^2sis —F);

C6:C6 amino linker (L001, —NH—(CH₂)₆— wherein —NH— is connected to Mod (e.g., through —C(O)— in Mod) or —H, and —(CH₂)₆— is connected to the 5′-end (or 3′-end if indicated) of oligonucleotide chain through, e.g., phosphodiester (—O—P(O)(OH)—O—. May exist as a salt form. May be illustrated in the Tables as O or PO), phosphorothioate (—O—P(O)(SH)—O—. May exist as a salt form. May be illustrated in the Tables as * if the phosphorothioate not chirally controlled; *S, S or Sp, if chirally controlled and has an Sp configuration, and *R, R, or Rp, if chirally controlled and has an Rp configuration), or phosphorodithioate (—O—P(S)(SH)—O—. May exist as a salt form. May be illustrated in the Tables as PS2 or : or D) linkage. May also be referred to as C6 linker or C6 amine linker); or D: Phosphodithioate (Phosphorodithioate), represented by D or a colon(:);

n001: non-negatively charged linkage

embedded image

(which is stereorandom unless otherwise indicated (e.g., as n001R, or n001S));

n002: non-negatively charged linkage

embedded image

(which is stereorandom unless otherwise indicated (e.g., as n002R, or n002S));

n003: non-negatively charged linkage

embedded image

(which is stereorandom unless otherwise indicated (e.g., as n003R. or n003S));

n004: non-negatively charged linkage

embedded image

(which is stereorandom unless otherwise indicated (e.g., as n004R, or n004S));

n005: non-negatively charged linkage

embedded image

(which is stereorandom unless otherwise indicated (e.g., as n005R, or n005S));

n006: non-negatively charged linkage

embedded image

(which is stereorandom unless otherwise indicated (e.g., as n006R, or n006S):

n007: non-negatively charged linkage

embedded image

(which is stereorandom at linkage phosphorus unless otherwise indicated (e.g., as n007R or n007S));

n008: non-negatively charged linkage

embedded image

(which is stereorandom unless otherwise indicated (e.g., as n008R, or n008S));

n009: non-negatively charged linkage

embedded image

(which is stereorandom unless otherwise indicated (e.g., as n009R, or n009S));

n010: non-negatively charged linkage

embedded image

(which is stereorandom unless otherwise indicated (e.g., as n010R, or n010S));

n001R: n001 being chirally controlled and having the Rp configuration;

n002R: n002 being chirally controlled and having the Rp configuration;

n003R: n003 being chirally controlled and having the Rp configuration;

n004R: n004 being chirally controlled and having the Rp configuration;

n005R: n005 being chirally controlled and having the Rp configuration;

n006R: n006 being chirally controlled and having the Rp configuration:

n007R: n007 being chirally controlled and having the Rp configuration;

n008R: n008 being chirally controlled and having the Rp configuration;

n009R: n009 being chirally controlled and having the Rp configuration;

n010R: n010 being chirally controlled and having the Rp configuration;

n001S: n001 being chirally controlled and having the Sp configuration:

n002S: n002 being chirally controlled and having the Sp configuration;

n003S: n003 being chirally controlled and having the Sp configuration:

n004S: n004 being chirally controlled and having the Sp configuration;

n005S: n005 being chirally controlled and having the Sp configuration;

n006S: n006 being chirally controlled and having the Sp configuration;

n007S: n007 being chirally controlled and having the Sp configuration;

n008S: n008 being chirally controlled and having the Sp configuration;

n009S: n009 being chirally controlled and having the Sp configuration:

n010S: n010 being chirally controlled and having the Sp configuration; nO, nX: in Linkage/Stereochemistry, nO or nX indicates a stereorandom n001; nR: in Linkage/Stereochemistry, nR indicates a linkage, e.g., n001, n002, n003, n004, n005, n006, n007, n008, n009, etc., being chirally controlled and having the Rp configuration (e.g., for n001, n001R in Description);

nS: in Linkage/Stereochemistry, nS indicates a linkage, e.g., n001, n002, n003, n004, n005, n006, n007, n008, n009, etc., being chirally controlled and having the Sp configuration (e.g., for n001, n001R in Description):

BrfU: a nucleoside unit wherein the nucleobase is BrU

embedded image

and wherein the sugar has a 2′-F (f) modification

embedded image

BrmU: a nucleoside unit wherein the nucleobase is BrU

embedded image

and wherein the sugar has a 2′-OMe (m) modification

embedded image

BrdU: a nucleoside unit wherein the nucleobase is BrU

embedded image

and wherein the sugar is 2-deoxyribose (as widely found in natural DNA; 2′-deoxy (d))

embedded image

L004: linker having the structure of —NH(CH₂)₄CH(CH₂OH)CH₂—, wherein —NH— is connected to Mod (e.g., through —C(O)— in Mod) or —H, and the —CH₂— connecting site is connected to a linkage, e.g., phosphodiester (—O—P(O)(OH)—O—. May exist as a salt form. May be illustrated in the Tables as O or PO), phosphorothioate (—O—P(O)(SH)—O—. May exist as a salt form. May be illustrated in the Tables as * if the phosphorothioate not chirally controlled; *S, S, or Sp, if chirally controlled and has an Sp configuration, and *R. R, or Rp, if chirally controlled and has an Rp configuration), or phosphorodithioate (—O—P(S)(SH)—O—. May exist as a salt form. May be illustrated in the Tables as PS2 or : or D) linkage, at the 5′- or 3′-end of an oligonucleotide chain as indicated. For example, an asterisk immediately preceding a L004 (e.g., *L004) indicates that the linkage is a phosphorothioate linkage, and the absence of the indication of any other linkage immediately preceding L004 indicates that the linkage is a phosphodiester linkage. For example, in WV-9858, which terminates in fUL004, the linker L004 is connected (via the —CH₂— site) to the phosphodiester linkage at the 3′ position at the 3′-terminal sugar (which is 2′-F and connected to the nucleobase U), and the L004 linker is connected via —NH— to —H; similarly, in WV-10886, WV-10887, and WV-10888, the L004 linker is connected (via the —CH₂— site) to the phosphodiester linkage at the 3′ position of the 3′-terminal sugar, and the L004 is connected via —NH— to Mod012 (WV-10886), Mod085 (WV-10887) or Mod086 (WV-10888);

L005: linker having the structure of —NH(CH₂)₅C(O)N(CH₂CH₂OH)CH₂CH₂—, wherein —NH— is connected to Mod (e.g., through —C(O)— in Mod) or —H, and the —CH₂— connecting site is connected to a linkage, e.g., phosphodiester (—O—P(O)(OH)—O—. May exist as a salt form. May be illustrated in the Tables as O or PO), phosphorothioate (—O—P(O)(SH)—O—. May exist as a salt form. May be illustrated in the Tables as * if the phosphorothioate not chirally controlled; *S, S, or Sp, if chirally controlled and has an Sp configuration, and *R, R, or Rp, if chirally controlled and has an Rp configuration), or phosphorodithioate (—O—P(S)(SH)—O—. May exist as a salt form. May be illustrated in the Tables as PS2 or : or D) linkage, at the 5′- or 3′-end of an oligonucleotide chain as indicated. For example, an asterisk immediately preceding a L005 (e.g., *L005) indicates that the linkage is a phosphorothioate linkage, and the absence of the indication of any other linkage immediately preceding L005 indicates that the linkage is a phosphodiester linkage. For example, in WV-12571, L005 is connected to —H (no Mod following L005; via the —NH— site) and the phosphodiester linkage at the 3′ position of the 3′-terminal sugar (via the —CH₂— site); and in WV-12572, L005 is connected to Mod020 (via the —NH— site) and the phosphodiester linkage at the 3′ position of the 3′-terminal sugar (via the —CH₂— site); L001L005: linker having the structure of —NH(CH₂), C(O)N(CH₂CH₂—, —P(O)(OH)—O—(CH₂)₆NH—)CH₂CH₂—, wherein each of the two —NH— is independently connected to Mod (e.g., through —C(O)—) or —H, and the —CH₂— connecting site is connected to a linkage, e.g., phosphodiester (—O—P(O)(OH)—O—. May exist as a salt form. May be illustrated in the Tables as O or PO), phosphorothioate (—O—P(O)(SH)—O—. May exist as a salt form. May be illustrated in the Tables as * if the phosphorothioate not chirally controlled: *S, S. or Sp, if chirally controlled and has an Sp configuration, and *R. R, or Rp, if chirally controlled and has an Rp configuration), or phosphorodithioate (—O—P(S)(SH)—O—. May exist as a salt form. May be illustrated in the Tables as PS2 or: or D) linkage at the 5′- or 3′-end of an oligonucleotide chain as indicated.

eo: 2′-MOE (2′-OCH₂CH₂OCH₃) modification on the preceding nucleoside (e.g., Aeo(

embedded image

wherein BA is nucleobase A));

F, f: 2′-F modification on the following nucleoside (e.g., fA

embedded image

wherein BA is nucleobase A));

m: 2′-OMe modification on the following nucleoside (e.g., m A

embedded image

wherein BA is nucleobase A));

r: 2′-OH on the following nucleoside (e.g., rA

embedded image

wherein BA is nucleobase A, as existed in natural RNA));

L012: internucleotidic linkage having the structure of —O—P(O)[O(CH₂)₂O(CH₂)₂O(CH₂)₂OH]—O—. May be illustrated as OO in the Tables;

*, PS: Phosphorothioate:

PS2, : D: phosphorodithioate (e.g., WV-3078, wherein a colon (:) indicates a phosphorodithioate);

*R, R, Rp: Phosphorothioate in Rp conformation;

*S, S, Sp: Phosphorothioate in Sp conformation;

X: Phosphorothioate stereorandom;

embedded image

NA: Not Applicable;

O, PO: phosphodiester (phosphate). When no internucleotidic linkage is specified between two nucleoside units, the internucleotidic linkage is a phosphodiester linkage (natural phosphate linkage). When used to indicate linkage between Mod and a linker, e.g., L001, O may indicate —C(O)— (connecting Mod and L001, for example:

Mod013L001fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC*SfA*SfU*SfU*SfU*SfC *SfU (Description), OOSSSSSSOSOSSOOSSSSSS (Linkage/Stereochemistry). Note the second 0 in OOSSSSSSOSOSSOOSSSSSS (Linkage/Stereochemistry) represents phosphodiester linkage connecting L001 and the 5′-O— of the 5′-terminal sugar of the oligonucleotide chain (see illustrations below. Alternatively, the 5′-O— may be considered part of the phosphodiester linkage (or another type of linkage such as a phosphorothioate linkage), in which case the phosphodiester linkage (or another type of linkage such as phosphorothioate linkage) is connected to the 5′ position of the 5′-terminal sugar of the oligonucleotide chain). In some instances, “O” for —C(O)— (connecting Mod and L001) is omitted (e.g., for Mod013L001fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC*SfA*SfU*SfU*SfU*SfC*SfU, “Linkage/Stereochemistry” OSSSSSSOSOSSOOSSSSSS);

Various Mods:

Mod001 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Lauric (in Mod013). Myristic (in Mod014). Palmitic (in Mod005), Stearic (in Mod015), Oleic (in Mod016). Linoleic (in Mod017), alpha-Linoleinc (in Mod018), gamma-Linolenic (in Mod019), DHA (in Mod006), Turbinaric (in Mod020), Dilinoleic (in Mod021), TriG1cNAc (in Mod024). TrialphaMannose (in Mod026), MonoSulfonamide (in Mod 027), TriSulfonamide (in Mod029), Lauric (in Mod030), Myristic (in Mod031). Palmitic (in Mod032), and Stearic (in Mod033): Lauric acid (for Mod013), Myristic acid (for Mod014), Palmitic acid (for Mod005), Stearic acid (for Mod015), Oleic acid (for Mod016). Linoleic acid (for Mod017), alpha-Linolenic acid (for Mod018), gamma-Linolenic acid (for Mod019), docosahexaenoic acid (for Mod006), Turbinaric acid (for Mod020), alcohol for Dilinoleyl (for Mod021), acid for TriG1cNAc (for Mod024), acid for TrialphaMannose (for Mod026), acid for MonoSulfonamide (for Mod 027), acid for TriSulfonamide (for Mod029), Lauryl alcohol (for Mod030). Myristyl alcohol (for Mod031). Palmityl alcohol (for Mod032), and Stearyl alcohol (for Mod033), respectively, conjugated to oligonucleotide chains, e.g., through an amide group, a linker (e.g., C6 amino linker, (L001)), and/or a linkage group (e.g., phosphodiester linkage (PO), phosphorothioate linkage (PS), etc.): e.g., Mod013 (Lauric acid with C6 amino linker and PO or PS), Mod014 (Myristic acid with C6 amino linker and PO or PS), Mod005 (Palmitic acid with C6 amino linker and PO or PS), Mod015 (Stearic acid with C6 amino linker and PO or PS), Mod016 (Oleic acid with C6 amino linker and PO or PS), Mod017 (Linoleic acid with C6 amino linker and PO or PS), Mod018 (alpha-Linolenic acid with C6 amino linker and PO or PS), Mod019 (gamma-Linolenic acid with C6 amino linker and PO or PS), Mod006 (DHA with C6 amino linker and PO or PS), Mod020 (Turbinaric acid with C6 amino linker and PO or PS), Mod021 (alcohol (see below) with PO or PS), Mod024 (acid (see below) with C6 amino linker and PO or PS), Mod026 (acid (see below) with C6 amino linker and PO or PS), Mod027 (acid (see below) with C6 amino linker and PO or PS), Mod029 (acid (see below) with C6 amino linker and PO or PS), Mod030 (Lauryl alcohol with PO or PS), Mod031 (Myristyl alcohol with PO or PS), Mod032 (Palmityl alcohol with PO or PS), and Mod033 (Stearyl alcohol with PO or PS), with PO or PS for each oligonucleotide indicated in Table A1. For example, WV-3557 Steary alcohol conjugated to oligonucleotide chain of WV-3473 via PS: Mod033*fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC*SfA*SfU*SfU*SfU*SfC*Sf U (Description), XSSSSSSOSOSSOOSSSSSS (Stereochemistry); and

WV-4106 Stearic acid conjugated to oligonucleotide chain of WV-3473 via amide group, C6, and PS: Mod015L001*fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC*SfA*SfU*SfU*SfU*Sf C*SfU (Description), XSSSSSSOSOSSOOSSSSSS (Stereochemistry). Certain moieties for conjugation, and example reagents (many of which were previously known and are commercially available or can be readily prepared using known technologies in accordance with the present disclosure, e.g., Laurie acid (for Mod013), Myristic acid (for Mod014), Palmitic acid (for Mod005), Stearic acid (for Mod015), Oleic acid (for Mod016). Linoleic acid (for Mod017), alpha-Linolenic acid (for Mod018), gamma-Linolenic acid (for Mod019), docosahexaenoic acid (for Mod006), Turbinaric acid (for Mod2), alcohol for Dilinoleyl (for Mod021), Lauryl alcohol (for Mod030), Myristyl alcohol (for Mod031), Palmityl alcohol (for Mod032). Stearyl alcohol (for Mod033), etc.) are listed below. Certain example moieties (e.g., lipid moieties, targeting moiety, etc.) and/or example preparation reagents (e.g., acids, alcohols, etc.) for conjugation to oligonucleotide chains include the below with a non-limiting example of a linker; Mod005 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001) and Palmitic acid:

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Mod005L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod006 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001) and DHA:

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Mod006L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod009 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod012 (with —C(O)— connecting to e.g. —NH— of a linker such as L001:

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Mod013 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001) and Lauric acid:

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Mod013L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod014 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001) and Myristic acid:

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Mod014L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod015 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001) and Stearic acid:

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Mod015L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod016 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001) and Oleic acid:

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Mod016L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod017 (with —C(O)— connecting to e.g., —NH— of a linker such as L001) and Linoleic acid:

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Mod 017L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod018 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001) and alpha-Linolenic acid:

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Mod018L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod019 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001) and gamma-Linolenic acid:

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Mod019L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod020 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001) and Turbinaric acid:

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Mod020L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod021 (with PO or PS connecting to 5′-O— of an oligonucleotide chain) and alcohol:

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Mod024 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001) and acid:

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Mod024L001(with PO or PS connecting to 5′-O—of an oligonucleotide chain):

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Mod026 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001) and acid:

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Mod026L001(with PO or PS connecting to 5′-O—of an oligonucleotide chain):

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Mod027 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001) and acid:

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Mod027L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod028 (with —C(O)— connecting to, e.g., —NH— of a linker such a L001):

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Mod029 (with —C(O)— connecting to, e.g. —NH— of a linker such as L00) and acid:

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Mod029L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod030 (with PO or PS connecting to 5′-O— of an oligonucleotide chain) and Lauryl alcohol:

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Mod031 (with PO or PS connecting to 5′-O— of an oligonucleotide chain) and Myristyl alcohol:

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Mod032 (with PO or PS connecting to 5′-O— of an oligonucleotide chain) and Palmityl alcohol:

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Mod033 (with PO or PS connecting to 5′-O— of an oligonucleotide chain) and Stearyl alcohol:

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Mod053 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod 070 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod071 (with —C(O)— connecting to e.g., —NH— of a linker such as L001):

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Mod086 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod092 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod093 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod007 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod050 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod043 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod057 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod058(with—C(O)-connecting to, e.g., —NH— of a linker such as L001):

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Mod059 (with —C(O)— connecting to, e.g., —NH— of a linker such as(L001):

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Mod066 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod074 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod085 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod091L001 (with PO PS connecting to 5′-O— of a oligonucleotide chain):

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(e.g., in WV-11114, X=O (PO) and connecting to 5′-O— of the oligonucleotide chain)

Mod097 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod098 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod099 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod100 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod102 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod103 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod104 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod105 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod106 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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(e.g., in WV-15844, X=O (PO) and connecting to 5′-O— of the oligonucleotide chain)

Mod107 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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(e.g., in WV-15845 and WV-16011, X=O(PO) and connecting to 5′-O— of the oligonucleotide chain)

Mod108 (with —C(O)— connecting to, e.g., —NH— of a linker such as L001):

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Mod109:

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Mod109L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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(e.g., in WV-19792, X=O)
Mod110:

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Mod110L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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(e.g., in WV-19793, X=O)
Mod111:

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Mod 111L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod 112:

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Mod112L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod113:

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Mod 113L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod 114:

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Mod114L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod115:

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Mod115L001(with PO or PS connecting to 5-O— of an oligonucleotide chain):

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Mod118:

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Mod118L001 with PO or PS connecting to 5′-O— of an oligonucleotide chain:

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Mod 119L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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Mod120:

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Mod120L001 (with PO or PS connecting to 5′-O— of an oligonucleotide chain):

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L009n001009n001L009n001L009: connected to the 5-position of the 5′ terminal sugar of an oligonucleotide chain (e.g., for WV-23576 and WV-23578, sugar of fU) through a phosphodiester:

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L009n001L009n001L009n001: connected to the 5-position of the 5′ terminal sugar of an oligonucleotide chain (e.g., for WV-23577 and WV-23579, sugar of fU) through n001:

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L010n001L010n001L010n001L009: connected to the 5′-position of the 5′ terminal sugar of an oligonucleotide chain (e.g., for WV-23936 and WV-23938, sugar of fU) through a phosphodiester:

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L010n001L10n001L10n001: connected to the 5′-position of the 5′ terminal sugar of an oligonucleotide chain (e.g., for WV-23937 and WV-23939, sugar of fU) through n001:

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In some embodiments, some functional groups are optionally protected, e.g., for Mod024 and/or Mod 026, the hydroxyl groups are optionally protected as AcO—, before and/or during conjugation to oligonucleotide chains, and the functional groups, e.g., hydroxyl groups, can be deprotected, for example, during oligonucleotide cleavage and/or deprotection:

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Applicant notes that presented in Table A1 are example ways of presenting structures of provided oligonucleotides, for example, WV-3546 (Mod020L001fU*SfC*SfA*SfA*Sf*Sf*SmAfA*SmGmA*SfU*SmGmGfC*SfA*SfU*SfU*SfU*Sf C*SfU) can be presented as a lipid moiety (Mod020,

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connected via —C(O)-(OOSSSSSSOSOSSOOSSSSSS, which “O” may be omitted as in Table A1) to the —NH— of —NH—(CH₂)₆—, wherein the —(CH₂)₆— is connected to the 5′-end of the oligonucleotide chain via a phosphodiester linkage (OOSSSSSSOSOSSOOSSSSSS). One having ordinary skill in the art understands that a provided oligonucleotide can be presented as combinations of lipid, linker and oligonucleotide chain units in many different ways, wherein in each way the combination of the units provides the same oligonucleotide. For example, WV-3546, can be considered to have a structure of A^c-[-L^LD-(R^LD)_a]_b, wherein a is 1, b is 1, and have a lipid moiety R^LDof

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connected to its oligonucleotide chain (A^c) unit through a linker L^LDhaving the structure of —C(O)—NH—(CH₂)₆—OP(═O)(OH)—O—, wherein —C(O)— is connected to R^LD, and —O— is connected to A^c(as 5′-O— of the oligonucleotide chain); one of the many alternative ways is that R^LDis

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and L^LDis —NH—(CH₂)₆—OP(═O)(OH)—O—, wherein —NH— is connected to R^LD, and —O— is connected to A^c(as 5′-O— of the oligonucleotide chain).

In some embodiments, each phosphorothioate internucleotidic linkage of an oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, a provided oligonucleotide composition is a chirally controlled oligonucleotide composition of an oligonucleotide type listed in Table A1, wherein each phosphorothioate internucleotidic linkage of the oligonucleotide is independently a chirally controlled internucleotidic linkage.

In some embodiments, the present disclosure provides compositions comprising or consisting of a plurality of provided oligonucleotides (e.g., chirally controlled oligonucleotide compositions). In some embodiments, all oligonucleotides of the plurality are of the same type, i.e., all have the same base sequence, pattern of backbone linkages, pattern of backbone chiral centers, and pattern of backbone phosphorus modifications. In some embodiments, all oligonucleotides of the same type are structural identical. In some embodiments, provided compositions comprise oligonucleotides of a plurality of oligonucleotides types, typically in controlled amounts. In some embodiments, a provided chirally controlled oligonucleotide composition comprises a combination of two or more provided oligonucleotide types.

In some embodiments, an oligonucleotide composition of the present disclosure is a chirally controlled oligonucleotide composition, wherein the sequence of the oligonucleotides of its plurality comprises or consists of a base sequence listed in Table A1.

In some experiments, provided oligonucleotides can provide surprisingly high activities, e.g., when compared to those of Drisapersen and/or Eteplirsen. For example, chirally controlled oligonucleotide compositions of WV-887, WV-892, WV-896, WV-1714, WV-2444, WV-2445, WV-2526, WV-2527, WV-2528, and WV-2530, and many others, each showed a superior capability, in some embodiments many fold higher, to mediate skipping of an exon in dystrophin, compared to Drisapersen and/or Eteplirsen. Certain data are provided in the present disclosure as examples.

In some embodiments, the present disclosure pertains to a composition comprising a chirally controlled oligonucleotide selected from any DMD oligonucleotide listed herein, or any DMD oligonucleotide having a base sequence comprising at least 15 consecutive bases of any DMD oligonucleotide listed herein.

In some embodiments, a provided oligonucleotide is no more than 25 bases long. In some embodiments, a provided oligonucleotide is no more than 25 to 60 bases long. In some embodiments, a U can be replaced with T, or vice versa.

In some embodiments, when assaying example oligonucleotides in mice, oligonucleotides (e.g., WV-3473, WV-3545, WV-3546, WV-942, etc.) are intravenous injected via tail vein in male C57BL/10ScSndmdmdx mice (4-5 weeks old), at tested amounts, e.g., 10 mg/kg, 30 mg/kg, etc. In some embodiments, tissues are harvested at tested times, e.g., on Day, e.g., 2, 7 and/or 14, etc., after injection, in some embodiments, fresh-frozen in liquid nitrogen and stored in −80° C. until analysis.

Various assays can be used to assess oligonucleotide levels in accordance with the present disclosure. In some embodiments, hybrid-ELISA is used to quantify oligonucleotide levels in tissues using test article serial dilution as standard curve: for example, in an example procedure, maleic anhydride activated 96-well plate (Pierce 15110) was coated with 50 μl of capture probe at 500 nM in 2.5% NaHCO3 (Gibco, 25080-094) for 2 hours at 37° C. The plate was then washed 3 times with PBST (PBS+0.1% Tween-20), and blocked with 5% fat free milk-PBST at 37° C. for 1 hour. Test article oligonucleotide was serial diluted into matrix. This standard together with original samples were diluted with lysis buffer (4 M Guanidine; 0.33% N-Lauryl Sarcosine; 25 mM Sodium Citrate; 10 mM DTT) so that oligonucleotide amount in all samples is less than 100 ng/mL. 20 μl of diluted samples were mixed with 180 μl of 333 nM detection probe diluted in PBST, then denatured in PCR machine (65° C., 10 min, 95° C. 15 min, 4° C. ∞). 50 μl of denatured samples were distributed in blocked ELISA plate in triplicates, and incubated overnight at 4° C. After 3 washes of PBST, 1:2000 streptavidin-AP in PBST was added, 50 μl per well and incubated at room temperature for 1 hour. After extensive wash with PBST, 100 μl of AttoPhos (Promega S1000) was added, incubated at room temperature in dark for 10 min and read on plate reader (Molecular Device, M5) fluorescence channel: Ex435 nm, Em555 nm. Oligonucleotides in samples were calculated according to standard curve by 4-parameter regression.

In some embodiments, provided oligonucleotides are stable in both plasma and tissue homogenates.

Additional Embodiments and Examples of Oligonucleotides and Compositions, Including Dystrophin (DMD) Oligonucleotides and Compositions

Among other things, the present disclosure provides oligonucleotides, compositions, and methods for, modulating splicing, reducing target levels, treating various conditions, disorders, diseases, etc. For example, in some embodiments, the present disclosure provides dystrophin (DMD) oligonucleotides and/or DMD oligonucleotide compositions that are useful for various purposes. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 23 in the mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 44 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 46 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 47 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 51 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 52 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 53 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 54 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 55 in the human or mouse DMD gene.

In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of multiple exons in the human or mouse DMD gene.

In some embodiments, a provided oligonucleotide, e.g., a DMD oligonucleotide, comprises a modification. In some embodiments, a DMD oligonucleotide comprises a sugar modification. In some embodiments, a DMD oligonucleotide comprises a sugar modification at the 2′ position. In some embodiments, a DMD oligonucleotide comprises a sugar modification at the 2′ position selected from 2′-F, 2′-OMe and 2′-MOE.

In some embodiments, a DMD oligonucleotide comprises a 2′-F, 2′-OMe and/or 2′-MOE. In some embodiments, a DMD oligonucleotide comprises a 2′-F. In some embodiments, in a DMD oligonucleotide, each sugar comprises a 2′-F.

In some embodiments, a DMD oligonucleotide comprises a 2′-OMe. In some embodiments, in a DMD oligonucleotide, each sugar comprises a 2′-OMe. In some embodiments, a DMD oligonucleotide comprises a 2′-MOE. In some embodiments, in a DMD oligonucleotide, each sugar comprises a 2′-MOE.

In some embodiments, a provided oligonucleotide, e.g., a DMD oligonucleotide comprises a 2′-OMe and a 2′-F. In some embodiments, a provided oligonucleotide, e.g., a DMD oligonucleotide, comprises a pattern of 2′ sugar modifications, wherein the pattern comprises a sequence selected from: fm, mf, ffm, fffm, ffffm, fffffm, ffffffm, fffffffm, ffffffffm, fffffffffim, mf, mff, mff, mffff, mfffff, mffffff, mfffffff, mffffff, fmf, fmmf, fmmmf, fmmmmf, fmmmmmf, fmmmmmmf, fmmmmmmmf, fmmmmmmmmf, fmmmmmmmmmf, ffffffmmmmmmmmffffff, fffffmmmmmmmmmmmfffff, ffffmmmmmmmmmmmmmffff, fffmmmmmmmmmmmmfff, ffmmmmmmmmmmmmmmmmff, fmmmmmmmmmmmmmmmmmmf, ffffffffffmmmmmmmmmm, fffffmmmmmmmmffffff, ffffmmmmmmmmmmfffff, fffmmmmmmmmmmmmffff, ffmmmmmmmmmmmmmmfff, fmmmmmmmmmmmmmmmmff, mmmmmmmmmmmmmmmmmmf, fffffffffmmmmmmmmmm, ffffmmmmmmmmffffff, fffmmmmmmmmmmfffff, ffmmmmmmmmmmmmffff, fmmmmmmmmmmmmmmfff, mmmmmmmmmmmmmmmmff, mmmmmmmmmmmmmmmmmf, ffffffffmmmmmmmmmm, fffmmmmmmmmffffff, ffmmmmmmmmmmfffff, fmmmmmmmmmmmmffff, mmmmmmmmmmmmmmfff, mmmmmmmmmmmmmmmff, mmmmmmmmmmmmmmmmf, fffffffmmmmmmmmmm, ffmmmmmmmmffffff, fmmmmmmmmmmfffff, mmmmmmmmmmmmffff, mmmmmmmmmmmmmfff, mmmmmmmmmmmmmmff, mmmmmmmmmmmmmmmf, ffffffmmmmmmmmmm, fmmmmmmmmffffff, mmmmmmmmmmfffff, mmmmmmmmmmmffff, mmmmmmmmmmmmfff, mmmmmmmmmmmmmff, mmmmmmmmmmmmmmf, fffffmmmmmmmmmm, mmmmmmmmffffff, mmmmmmmmmfffff, mmmmmmmmmmffff, mmmmmmmmmmmfff, mmmmmmmmmmmmff, mmmmmmmmmmmmmf, ffffmmmmmmmmmm, ffffffmmmmmmmmfffff, fffffmmmmmmmmmmffff, ffffmmmmmmmmmmmmfff, fffmmmmmmmmmmmmmmff, ffmmmmmmmmmmmmmmmmf, fmmmmmmmmmmmmmmmmmm, ffffffffffmmmmmmmmm, ffffffmmmmmmmmffff, fffffmmmmmmmmmmmfff, ffffmmmmmmmmmmmmff, fffmmmmmmmmmmmmmmf, ffmmmmmmmmmmmmmmmm, fmmmmmmmmmmmmmmmmm, ffffffffffmmmmmmmm, ffffffmmmmmmmmfff, fffffmmmmmmmmmmff, ffffmmmmmmmmmmmmf, fffmmmmmmmmmmmmmm, ffmmmmmmmmmmmmmmm, fmmmmmmmmmmmmmmmm, ffffffffffmmmmmmm, ffffffmmmmmmmmff, fffffmmmmmmmmmmf, ffffmmmmmmmmmmmm, fffmmmmmmmmmmmmm, ffmmmmmmmmmmmmm, fmmmmmmmmmmmmmmm, ffffffffffmmmmmm, ffffffmmmmmmmmf, fffffmmmmmmmmmm, ffffmmmmmmmmmmm, fffmmmmmmmmmmmm, ffmmmmmmmmmmmmm, fmmmmmmmmmmmmm, ffffffffffmmmmm, ffffffmmmmmmm, fffffmmmmmmmmm, ffffmmmmmmmmmm, fffmmmmmmmmmm, ffmmmmmmmmmmmm, fmmmmmmmmmmmmm, ffffffffffmmmm, ffffffmmmmmmm, fffffmmmmmmmm, ffffmmmmmmmmm, fffmmmmmmmmmm, ffmmmmmmmmmmm, fmmmmmmmmmmmm, ffffffffffmmm, ffffffmmmmmm, fffffmmmmmmm, ffffmmmmmmmm, fffmmmmmmmmm, ffmmmmmmmmmm, fmmmmmmmmmmm, ffffffffffmm, ffffffmmmmm, fffffmmmmmm, ffffmmmmmmm, fffmmmmmmmm, ffmmmmmmmmm, fmmmmmmmmmm, ffffffffffm, mmmmmmmmmmfffffffff, ffffffmmmmmmmmmmmmmm, mmmmmmmmmmmmmmffffff, ffmmmmmmmfmmfmfffff, mmffffffffmffmfmmmmm, mfmfmfmfmfmfinfmfmfmf, mmmmmmffffffffmmmmmm, ffffffmmmmmmmmffffff, mfmmffmfnmfffmmmmfn, fmffmmffmffmmmffffmf, fmff, mffm, fmffm, mfmmf, fmmf, fmffmm, mfnmff, mmff, fmmff, mmffm, fmffmmf, mfmmffm, mfmm, mfmmf, mfnmff, fmffmmf, mfmmffm, mmffm, ffmmf, fmfff, mfffm, fmfffm, fmfffmm, mfmmfff, mmfff, fmmfff, mmfffm, fmfffmmf, mfmmfffm, mfmm, mfmmf, mfmmfff, fmfffmmf, mfmmfffm, mmfffm, fffmmf, mfmmmf, fmmmf, fmffmmm, mfmmmff, mmmff, fmmff, mmmffm, fmfmmmf, mfmmmffm, mfmmm, mfmmmf, mfmmmff, fmffmmmf, mfmmmffm, mmmffm, ffmmmf, or any portion thereof comprising at least five consecutive modifications, wherein f is 2′-F and m is 2′-OMe.

In some embodiments, a provided oligonucleotide, e.g., a DMD oligonucleotide, comprises a pattern which comprises any of O, OO, OOO, OOOO, OOOOO, OOOOOO, OOOOOOO, OOOOOOOO, OOOOOOOOO, OOOOOOOOOO, OOOOOOOOOOO, S, SS, SSS, SSSS, SSSSS, SSSSSS, SSSSSSS, SSSSSSSS, SSSSSSSSS, SSSSSSSSSS, SSSSSSSSSSS, X, XX, XXX, XXXX, XXXXX, XXXXXX, XXXXXXX, XXXXXXXX, XXXXXXXXX, XXXXXXXXXX, XXXXXXXXXXX, R, RR, RRR. RRRR, RRRRR, RRRRRR, RRRRRRR, RRRRRRRR, RRRRRRRRR, RRRRRRRRRR, RRRRRRRRRRR, OSOOO, OSOO, OSO, SOOO, OXOOO, OXOO, OXO, XOO, ROOOR, ROROR, ROROR, ROORR, RROOR, ROOR, OOR, RRROR, RRRO, RROR, ROR, SOOOR, ROOOS, ROOO, ROO, RO, OOOS, SOOOS, SOOO, SOOSS, SOSOS, SOSO, OSOS, SOS, SSOOS, SSOO, SSO, SOO, SSSOS, SSSO, SOS, XOOOX, XOOO, XOO, XO, OOOX, OOX, OX SOOOS, SOOO, SOO, SO, OOOS, OOS, XXXXXXXXXXXXX, XXXXXXXXXXXX, XXXXXXXXXXX, XXXXXXXXXX, XXXXXXXXX, XXXXXXXX, XXXXXXX, XXXXXX, XXXXX, XXXX, SSSSRSSRSS, SSSSRSSRS, SSSSRSSR, SSSSRSS, SSSSRS, SSSS, SSS, SSSRSSRSS, SSRSSRSS, SRSSRSS, RSSRSS, SSRSS, SSRS, SSSRSSRSSS, SSRSSRSSS, SSSRSSRSS, SSRSSRSSSS, SRSSRSSSS, SSRSSRSSS, SSRSSSSSSS, SRSSSSSSS, SSRSSSSSS, SSSSSSRSSS, SSSSSRSSS, SSSSSSRSS, SSO, SOS, OSO, OSSO, SSOS, SSOSS, SSOSSO, SSOSSOS, SSOSSOSS, XO, XXO, XOX, XXOX, XXOXX, XXXOXX, XXXOX, XXOXX, XXXOXXX, XXOXXO, XXOXX, XXOXXOX, or XXOXXOXX, or any portion thereof comprising at least 5 consecutive internucleotidic linkages, wherein X is a stereorandom phosphorothioate linkage, S is a phosphorothioate linkage of the Sp configuration, and R is a phosphorothioate linkage of the Rp configuration.

Various oligonucleotides, including DMD oligonucleotides, having these modifications and patterns thereof, or portions thereof, are described in the present disclosure, including those listed in Table A1.

In some embodiments, a DMD oligonucleotide comprises a non-negatively charged internucleotidic linkage. Non-limiting examples of such an oligonucleotide include, inter alia: WV-11237, WV-11238, WV-11239, WV-11340, WV-11341, WV-11342, WV-11343, WV-11344, WV-11345, WV-11346, WV-11347, WV-12123, WV-12124, WV-12125, WV-12126, WV-12127, WV-12128, WV-12129, WV-12130, WV-12131, WV-12132, WV-12133, WV-12134, WV-12135, WV-12136, WV-12553, WV-12554, WV-12555, WV-12556, WV-12557, WV-12558, WV-12559, WV-12872, WV-12873, WV-12876, WV-12877, WV-12878, WV-12879, WV-12880, WV-12881, WV-12882, WV-12883, WV-12884, WV-12885, WV-12887, WV-12888, WV-13408, WV-13409, WV-13594, WV-13595, WV-13596, WV-13597, WV-13812, WV-13813, WV-13814, WV-13815, WV-13816, WV-13817, WV-13820, WV-13821, WV-13822, WV-13823, WV-13824, WV-13825, WV-13857, WV-13858, WV-13859, WV-13860, WV-13861, WV-13862, WV-13863, WV-13864, WV-13865, WV-14342, WV-14343, WV-14344, WV-14345, WV-14522, WV-14523, WV-14525, WV-14526, WV-14528, WV-14529, WV-14530, WV-14532, WV-14533, WV-14565, WV-14566, WV-14773, WV-14774, WV-14776, WV-14777, WV-14778, WV-14779, WV-14790, WV-14791, WV-15052, WV-15053, WV-15143, WV-15322, WV-15323, WV-15324, WV-15325, WV-15326, WV-15327, WV-15328, WV-15329, WV-15330, WV-15331, WV-15332, WV-15333, WV-15334, WV-15335, WV-15336, WV-15337, WV-15338, WV-15366, WV-15369, WV-15589, WV-15647, WV-15844, WV-15845, WV-15846, WV-15850, WV-15851, WV-15852, WV-15853, WV-15854, WV-15855, WV-15856, WV-15857, WV-15858, WV-15859, WV-15860, WV-15861, WV-15862, WV-15912, WV-15913, WV-15928, WV-15929, WV-15930, WV-15931, WV-15932, WV-15933, WV-15934, WV-15935, WV-15937, WV-15939, WV-15940, WV-15941, WV-15942, WV-15943, WV-15944, WV-15945, WV-15946, WV-15947, WV-15948, WV-15949, WV-15962, WV-15963, WV-15964, WV-15965, WV-15966, WV-15967, WV-15968, WV-15969, WV-15970, WV-15971, WV-15972, WV-15973, WV-16004, WV-16005, WV-16010, WV-16011, WV-16366, WV-16368, WV-16369, WV-16371, WV-16372, WV-16499, WV-16505, WV-16506, WV-16507, WV-17765, WV-17774, WV-17775, WV-17801, WV-17802, WV-17803, WV-17831, WV-17832, WV-17833, WV-17834, WV-17838, WV-17839, WV-17840, WV-17841, WV-17842, WV-17843, WV-17854, WV-17855, WV-17856, WV-17857, WV-17858, WV-17859, WV-17860, WV-17861, WV-17862, WV-17863, WV-17864, WV-17865, WV-17866, WV-17881, WV-17882, WV-17883, WV-18853, WV-18854, WV-18855, WV-18856, WV-18857, WV-18858, WV-18859, WV-18860, WV-18861, WV-18862, WV-18863, WV-18864, WV-18865, WV-18866, WV-18867, WV-18868, WV-18869, WV-18870, WV-18871, WV-18872, WV-18873, WV-18874, WV-18875, WV-18876, WV-18877, WV-18878, WV-18879, WV-18880, WV-18881, WV-18882, WV-18883, WV-18884, WV-18885, WV-18886, WV-18887, WV-18888, WV-18889, WV-18890, WV-18891, WV-18892, WV-18893, WV-18894, WV-18895, WV-18896, WV-18897, WV-18898, WV-18899, WV-18900, WV-18901, WV-18902, WV-18903, WV-18904, WV-18905, WV-18906, WV-18907, WV-18908, WV-18909, WV-18910, WV-18911, WV-18912, WV-18913, WV-18914, WV-18915, WV-18916, WV-18917, WV-18918, WV-18919, WV-18920, WV-18921, WV-18922, WV-18923, WV-18924, WV-18925, WV-18926, WV-18927, WV-18928, WV-18929, WV-18930, WV-18931, WV-18932, WV-18933, WV-18934, WV-18935, WV-18936, WV-18937, WV-18938, WV-18939, WV-18940, WV-18941, WV-18942, WV-18944, WV-18945, WV-19790, WV-19791, WV-19792, WV-19793, WV-19794, WV-19795, WV-19796, WV-19797, WV-19798, WV-19803, WV-19804, WV-19805, WV-19806, WV-19886, WV-19887, WV-19888, WV-19889, WV-19890, WV-19891, WV-19892, WV-19893, WV-19894, WV-19895, WV-19896, WV-19897, WV-19898, WV-19899, WV-19900, WV-19901, WV-19902, WV-19903, WV-19904, WV-19905, WV-19906, WV-19907, WV-19908, WV-19909, WV-19910, WV-19911, WV-19912, WV-19913, WV-19914, WV-19915, WV-19916, WV-19917, WV-19918, WV-19919, WV-19920, WV-19921, WV-19922, WV-19923, WV-19924, WV-19925, WV-19926, WV-19927, WV-19928, WV-19929, WV-19930, WV-19931, WV-19932, WV-19933, WV-19934, WV-19935, WV-19936, WV-19937, WV-19938, WV-19939, WV-19940, WV-19941, WV-19942, WV-19943, WV-19944, WV-19945, WV-19946, WV-19947, WV-19948, WV-19949, WV-19950, WV-19951, WV-19952, WV-19953, WV-19954, WV-19955, WV-19956, WV-19957, WV-19958, WV-19959, WV-19960, WV-19961, WV-19962, WV-19963, WV-19964, WV-19965, WV-19966, WV-19967, WV-19968, WV-19969, WV-19970, WV-19971, WV-19972, WV-19973, WV-19974, WV-19975, WV-19976, WV-19977, WV-19978, WV-19979, WV-19980, WV-19981, WV-19982, WV-19983, WV-19984, WV-19985, WV-19986, WV-19987, WV-19988, WV-19989, WV-19990, WV-19991, WV-19992, WV-19993, WV-19994, WV-19995, WV-19996, WV-19997, WV-19998, WV-19999, WV-20000, WV-20001, WV-20002, WV-20003, WV-20004, WV-20005, WV-20006, WV-20007, WV-20008, WV-20009, WV-20010, WV-20011, WV-20012, WV-20013, WV-20014, WV-20015, WV-20016, WV-20017, WV-20018, WV-20019, WV-20020, WV-20021, WV-20022, WV-20023, WV-20024, WV-20025, WV-20026, WV-20027, WV-20028, WV-20029, WV-20030, WV-20031, WV-20032, WV-20033, WV-20034, WV-20035, WV-20036, WV-20037, WV-20038, WV-20039, WV-20040, WV-20041, WV-20042, WV-20043, WV-20044, WV-20045, WV-20046, WV-20047, WV-20048, WV-20049, WV-20050, WV-20051, WV-20052, WV-20053, WV-20054, WV-20055, WV-20056, WV-20057, WV-20058, WV-20059, WV-20060, WV-20061, WV-20062, WV-20063, WV-20064, WV-20065, WV-20066, WV-20067, WV-20068, WV-20069, WV-20070, WV-20071, WV-20072, WV-20073, WV-20074, WV-20075, WV-20076, WV-20077, WV-20078, WV-20079, WV-20080, WV-20081, WV-20082, WV-20083, WV-20084, WV-20085, WV-20086, WV-20087, WV-20088, WV-20089, WV-20090, WV-20091, WV-20092, WV-20093, WV-20094, WV-20095, WV-20096, WV-20097, WV-20098, WV-20099, WV-20100, WV-20101, WV-20102, WV-20103, WV-20104, WV-20105, WV-20106, WV-20107, WV-20108, WV-20109, WV-20110, WV-20111, WV-20112, WV-20113, WV-20114, WV-20115, WV-20116, WV-20117, WV-20118, WV-20119, WV-20120, WV-20121, WV-20122, WV-20123, WV-20124, WV-20125, WV-20126, WV-20127, WV-20128, WV-20129, WV-20130, WV-20131, WV-20132, WV-20133, WV-20134, WV-20135, WV-20136, WV-20137, WV-20138, WV-20139, WV-20140, WV-20141, WV-20142, WV-20143, WV-20144, WV-20145, WV-20146, WV-20147, WV-20148, WV-20149, WV-20150, WV-20151, WV-20152, WV-20153, WV-20154, WV-20155, WV-20156, WV-20157, WV-20158, WV-20159, WV-20160, WV-21210, WV-21211, WV-21212, WV-21217, WV-21218, WV-21219, WV-21226, WV-21245, WV-21252, WV-21253, WV-21257, WV-21258, WV-21374, WV-21375, WV-21376, WV-21377, WV-21378, WV-21379, WV-21380, WV-21381, WV-21382, WV-21383, WV-21384, WV-21385, WV-21386, WV-21387, WV-21388, WV-21389, WV-21390, WV-21578, WV-21579, WV-21580, WV-21581, WV-21582, WV-21583, WV-21584, WV-21585, WV-21586, WV-21587, WV-21588, WV-21589, WV-21590, WV-21591, WV-21592, WV-21593, WV-21594, WV-21595, WV-21596, WV-21597, WV-21598, WV-21599, WV-21600, WV-21601, WV-21602, WV-21603, WV-21604, WV-21605, WV-21606, WV-21607, WV-21608, WV-21609, WV-21610, WV-21611, WV-21612, WV-21613, WV-21614, WV-21615, WV-21616, WV-21617, WV-21618, WV-21619, WV-21620, WV-21621, WV-21622, WV-21623, WV-21624, WV-21625, WV-21626, WV-21627, WV-21628, WV-21629, WV-21630, WV-21631, WV-21632, WV-21633, WV-21634, WV-21635, WV-21636, WV-21637, WV-21638, WV-21639, WV-21640, WV-21641, WV-21642, WV-21643, WV-21644, WV-21645, WV-21646, WV-21647, WV-21648, WV-21649, WV-21650, WV-21651, WV-21652, WV-21653, WV-21654, WV-21655, WV-21656, WV-21657, WV-21658, WV-21659, WV-21660, WV-21661, WV-21662, WV-21663, WV-21664, WV-21665, WV-21666, WV-21667, WV-21668, WV-21669, WV-21670, WV-21671, WV-21672, WV-21673, WV-21723, WV-21724, WV-21725, WV-21726, WV-21727, WV-21728, WV-21729, WV-21730, WV-21731, WV-21732, WV-21733, WV-21734, WV-21735, WV-21736, WV-21737, WV-21738, WV-21739, WV-21740, WV-21741, WV-21742, WV-21743, WV-21744, WV-21745, WV-21746, WV-21747, WV-21748, WV-21749, WV-21750, WV-21751, WV-21752, WV-21753, WV-21754, WV-21755, WV-21756, WV-21757, WV-21758, WV-21759, WV-21760, WV-21761, WV-21762, WV-21763, WV-21764, WV-21765, WV-21766, WV-21767, WV-21768, WV-21769, WV-21770, WV-21771, WV-21772, WV-21773, WV-21774, WV-21775, WV-21776, WV-21777, WV-21778, WV-21779, WV-21780, WV-21781, WV-21782, WV-21783, WV-21784, WV-21785, WV-21786, WV-21787, WV-21788, WV-21789, WV-21790, WV-21791, WV-21792, WV-21793, WV-21794, WV-21795, WV-21796, WV-21797, WV-21798, WV-21799, WV-21800, WV-21801, WV-21802, WV-21803, WV-21804, WV-21805, WV-21806, WV-21807, WV-21808, WV-21809, WV-21810, WV-21811, WV-21812, WV-21813, WV-21814, WV-21815, WV-21816, WV-21817, WV-21818, WV-22753, WV-23576, WV-23577, WV-23578, WV-23579, WV-23936, WV-23937, WV-23938, and WV-23939.

Example Dystrophin Oligonucleotides and Compositions for Exon Skipping of Exon 23

In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for mediating skipping of exon 23 in mouse DMD. Non-limiting examples include oligonucleotides and compositions of WV-10256, WV-10257, WV-10258, WV-10259, WV-10260, WV-1093, WV-1094, WV-1095, WV-1096, WV-1097. WV-1098, WV-1099, WV-1100, WV-1101, WV-1102, WV-1103, WV-1104, WV-1105, WV-1106, WV-1121, WV-1122, WV-1123, WV-11231, WV-11232, WV-11233, WV-11234, WV-11235, WV-11236, WV-1124, WV-1125, WV-1126, WV-1127, WV-1128, WV-1129, WV-1130, WV-11343, WV-11344, WV-11345, WV-11346, WV-11347, WV-1141, WV-1142, WV-1143, WV-1144, WV-1145, WV-1146, WV-1147, WV-1148, WV-1149, WV-1150, WV-1678. WV-1679, WV-1680, WV-1681, WV-1682, WV-1683, WV-1684, WV-1685, WV-2733, WV-2734, WV-4610, WV-4611, WV-4614, WV-4615, WV-4616, WV-4617, WV-4618, WV-4619, WV-4620, WV-4621, WV-4622, WV-4623, WV-4624, WV-4625, WV-4626, WV-4627, WV-4628, WV-4629, WV-4630, WV-4631, WV-4632, WV-4633, WV-4634, WV-4635, WV-4636, WV-4637, WV-4638, WV-4639, WV-4640, WV-4641, WV-4642. WV-4643, WV-4644, WV-4645, WV-4646, WV-4647, WV-4648, WV-4649, WV-4650, WV-4651, WV-4652, WV-4653, WV-4654, WV-4655, WV-4656, WV-4657, WV-4658, WV-4659, WV-4660, WV-4661, WV-4662, WV-4663, WV-4664, WV-4665, WV-4666, WV-4667, WV-4668, WV-4669, WV-4670, WV-4671, WV-4672. WV-4673, WV-4674, WV-4675, WV-4676, WV-4677, WV-4678, WV-4679, WV-4680, WV-4681, WV-4682, WV-4683, WV-4684, WV-4685, WV-4686, WV-4687, WV-4688, WV-4689, WV-4690, WV-4691, WV-4692, WV-4693, WV-4694, WV-4695, WV-4696, WV-4697, WV-6010, WV-7677, WV-7678, WV-7679, WV-7680, WV-7681, WV-7682, WV-7683, WV-7684, WV-7685, WV-7686, WV-7687, WV-7688, WV-7689, WV-7690, WV-7691, WV-7692. WV-7693. WV-7694, WV-7695, WV-7696, WV-7697, WV-7698, WV-7699, WV-7700, WV-7701, WV-7702, WV-7703, WV-7704, WV-7705, WV-7706, WV-7707, WV-7708, WV-7709, WV-7710, WV-7711, WV-7712, WV-7713, WV-7714, WV-7715, WV-7716, WV-7717, WV-7718, WV-7719, WV-7720, WV-7721, WV-7722. WV-7723, WV-7724, WV-7725, WV-7726, WV-7727, WV-7728, WV-7729, WV-7730, WV-7731, WV-7732, WV-7733, WV-7734, WV-7735, WV-7736, WV-7737. WV-7738. WV-7739, WV-7740, WV-7741, WV-7742, WV-7743, WV-7744, WV-7745, WV-7746, WV-7747, WV-7748, WV-7749, WV-7750, WV-7751, WV-7752, WV-7753, WV-7754, WV-7755, WV-7756, WV-7757, WV-7758, WV-7759, WV-7760, WV-7761, WV-7762, WV-7763, WV-7764, WV-7765, WV-7766, WV-7767. WV-7768, WV-7769, WV-7770, WV-7771, WV-9163, WV-9164, WV-9165, WV-9166, WV-9167, WV-9168, WV-9169, WV-9170, WV-9171, WV-9172, WV-9173, WV-9174, WV-9175, WV-9176, WV-9177, WV-9178, WV-9179, WV-9180, WV-9181, WV-9182, WV-9183, WV-9184, WV-9185, WV-9186, WV-9187, WV-9188, WV-9189, WV-9190, WV-9191, WV-9192, WV-9193, WV-9194, WV-9195, WV-9196, WV-9197, WV-9198, WV-9199, WV-9200. WV-9201. WV-9202, WV-9203, WV-9204, WV-9205, WV-9206, WV-9207, WV-9208, WV-9209, WV-9210, WV-9408, WV-9409, WV-9410, WV-9411, WV-9412, WV-9413, WV-9414, WV-9415, WV-9416, WV-9417, WV-9418, WV-9419, WV-9420, WV-943, WV-9875, WV-9876, WV-9877, WV-9878, and WV-9879, and other oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

In some embodiments, a DMD oligonucleotide is capable of mediating skipping of exon 23. Non-limiting examples of such DMD oligonucleotides include: WV-12566, WV-12567, WV-12568, WV-12884, WV-12885, WV-12886, WV-12887, WV-12888, WV-12571, and WV-12572, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

Exon skipping of DMD exon 23 and other exons may be assayed in patient-derived cell lines and in cells from the mdx mouse model (which carries a nonsense point mutation in the in-frame exon 23 (Sicinski et al. 1989 Science 244: 1578-1580). By skipping exon 23 the nonsense mutation is bypassed while the reading frame is maintained). Additional strains of mdx mice, including the mdx^2cv, mdx^4cvand mdx^5cvalleles were reported by Wha Bin Im et al. 1996 Hum. Mol. Gen. 5: 1149-1153.

Data showing the capability of various DMD oligonucleotides to mediate skipping of exon 23 is shown herein, inter alia, in Table 1A.1, Table 1A.2, Table 1A.3, and Table 25C.1 to Table 25C.5.

Example Dystrophin Oligonucleotides and Compositions Targeting Exon 44 and Adjoining Intronic Region 3′ to Exon 44

In some embodiments, a DMD oligonucleotide targets DMD exon 44 or the adjoining intronic region 3′ to DMD exon 44.

In some embodiments, a DMD oligonucleotide targets DMD exon 44 or the adjoining intronic region 3′ to DMD exon 44, and the oligonucleotide is capable of mediating multiple exon skipping (e.g., of exons 45 to 55, or 45 to 57).

Reportedly, a phenomenon known as back-splicing can occur, in which, for example, a portion of the 3′ end of exon 55 interacts with a portion of the 5′ end of exon 45, forming a circular RNA (circRNA), which can thus skip multiple exons, e.g., all exons from exon 45 to 55, inclusive. The phenomenon can also reportedly occur between exon 57 and exon 45, skipping multiple exons, e.g., all exons from exon 45 to 57, inclusive. Back-splicing is described in the literature, e.g., in Suzuki et al. 2016 Int. J. Mol. Sci. 17.

Without wishing to be bound by any particular theory, the present disclosure suggests that it may be possible for a DMD oligonucleotide targeting DMD exon 44 or the adjoining intronic region 3′ to exon 44 may be able to mediate splicing of exons 45 to 55, or of exons 45 to 57, which exons are excised as a single piece of circular RNA (circRNA) designated 45-55 (or 55-45) or 45-57 (or 5745), respectively.

Several oligonucleotides were designed to target exon 44 or intron 44, or which straddle exon 44 and intron 44. In some embodiments, oligonucleotides designed to target exon 44 or intron 44, or which straddle exon 44 and intron 44 are tested to determine if they can increase the amount of backslicing and/or multiple-exon skipping.

In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for mediating exon skipping in human DMD, wherein the base sequence of the oligonucleotide is a sequence of exon 44 or intron 44, or a portion of both exon 44 and intron 44. Non-limiting examples include oligonucleotides and compositions of WV-13963, WV-13964, WV-13965, WV-13966, WV-13967, WV-13968, WV-13969, WV-13970, WV-13971, WV-13972, WV-13973, WV-13974, WV-13975, WV-13976, WV-13977, WV-13978, WV-13979, WV-13980, WV-13981, WV-13982, WV-13983, WV-13984, WV-13985, WV-13986, WV-13987, WV-13988, WV-13989, WV-13990, WV-13991, WV-13992, WV-13993, WV-13994, WV-13995, WV-13996, WV-13997, WV-13998, WV-13999, WV-14000, WV-14001, WV-14002, WV-14003, WV-14004, WV-14005, WV-14006, WV-14007, WV-14008, WV-14009, WV-14010, WV-14011, WV-14012, WV-14013, WV-14014, WV-14015, WV-14016, WV-14017, WV-14018, WV-14019, WV-14020, WV-14021, WV-14022, WV-14023, WV-14024, WV-14025, WV-14026, WV-14027, WV-14028, WV-14029, WV-14030, WV-14031, WV-14032, WV-14033, WV-14034, WV-14035, WV-14036, WV-14037, WV-14038, WV-14039, WV-14040, WV-14041, WV-14042, WV-14043, WV-14044, WV-14045, WV-14046, WV-14047, WV-14048, WV-14049, WV-14050, WV-14051, WV-14052, WV-14053, WV-14054, WV-14055, WV-14056, WV-14057, and WV-14058, and other oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

Data showing the capability of various DMD oligonucleotides targeting exon 44 or the adjacent intron 3′ to exon 44 are shown in Table 22A.2 and Table 22A.3.

TABLE 1A.1

Example data of certain oligonucleotides

Oligo-

nucleotide
10
3.33
1.11
0.37
0.12

WV-7684
4.2
2.1
1
0.2
0.1

4.1
2.1
0.9
0.2
0.1

5.2
3.2
1.5
0
0

5.1
3.3
1.1
0
0

WV-12886
27.7
17.5
10
5
2.4

28
17.6
9.8
5
2.3

29.8
22.8
13.1

3.7

32.7
21.5
11.9

3.5

WV-11231
3.8
2.1
1.4
0.4
0.3

3.8
2.1
1.3
0.5
0.3

5.3
2.7
1.4
0.7
0.2

5.1
2.4
1.6
0.8
0.2

WV-10258
24.5
19.9
9.5
4.8
2.8

25.3
20.1
9.1
4.8
2.7

24.4
19.4
13.2
6.2
3.4

24.2
19.7
13.6
6.3
3.5

WV-11345
29.2
24.9
15.9
12.1
5

30.2
24.9
15.5
11.9
5.1

30.8
25.8
17.8

32.3
25.3
17.6

WV-12885
26.8
23.3
16.5
8
2.8

27.5
23
17.2
8.2
3.8

32.3
25.8
16.3

6.1

30.7
27.1
16.3

6.3

WV-15589
22.2
14.8
11.2
4.6
2.2

21.7
15
12.3
4.4
2.3

24.1
11.3
11.4

23.5
8.6
10.8

Oligonucleotides to DMD exon 23 were tested in vitro for their ability to induce skipping of exon 23.

H2K cells were dosed with oligonucleotide in differentiation media for 4 days. RNA was extracted with Trizol, pre-amp then treated with TaqMan with multiplexed reading of skipped and total DMD transcript; absolute quantification was via standard curve g-Blocks. In these and various other studies, numbers indicate amount of skipping (i.e., skipping efficiency; or the percentage of skipping as a percentage of total mRNA transcript).

Oligonucleotides were tested at 10, 3.33, 1.11, 0.37, or 0.12 uM.

TABLE 1A.2

Activity of certain oligonucleotides

PBS
WV-11345
WV-17774
WV-18945

Quadriceps

0.01
0.01
28.61
30.25
3.93
3.92
2.1
1.53

0.01
0.12
26.34
24.53
10.82
10.73
1.16
0.91

0.15
0.06
40.29
36.57
14.79
13.47
2.04
0.92

30
30.05
10.13
6.19
5.05
3.97

23.24
25.18
13.92
14.36
2.4
1.77

Gastrocnemius

0.02
0.02
22.27
13.18
36.41
33.55
2.46
1.95

0.02
0.01
14.74
8.03
18.02
19.55
0.6
0.27

0.09
0.11
11.12
3.68
16.17
15.44
0.36
0.41

22.82
28.29
11.22
10.94
0.72
0.75

18.09
15.66
28.85
27.9
0.61
3.14

Diaphram

0.04
0.03
27.05
24
7.11
4.07
0.72
0.82

0.01
1.13
16.22
16.2
18.1
18.6
0.81
0.68

0.04
0.09
15.16
13.23
9.66
10.02
0.33
0.32

33.66
36.52
4.55
4.86
0.63
0.21

20.03
20.55
8.38
9.46
0.56
0.91

Tibialis

0.01
0.01
34.34
35.04
16.2
15.77
0
0

0
0
28.7
23.07
42.94
42.97

0.04
0.02
7.87
9.87
12.1
14.51

17.01
14.68
15.16
13.91

45.6
41.54

In this study, in vivo skipping activity was measured in MDX mouse model after single IV dose.

MDX mice received single IV dose of 150 mg/kg. Necropsied flash frozen tissues (Quadriceps, Diaphragm, etc.) were pulverized and RNA extracted with Trizol. Skipping efficiency was determined by multiplex TaqMan assay for ‘total’ and ‘exon-23 skipped’ DMD transcripts, normalized to gBlock standard curves.

Numbers indicate amount of skipping DMD exon 23 (as a percentage of total mRNA, where 100 would represent 100% skipped).

TABLE 1A.3

Activity of certain oligonucleotides

10 uM
3.3 uM
1.1 uM
0.3 uM
0.1 uM

WV-
32.1
17.7
11.1
3.9
1.9

10258
33.2
19.4
13
4.6
2.1

29
18.5
11.5
11.1
6.4

29
18.6
12.4
11.3
6

WV-
6.8
7.6
0.7
1.6
0.1

11233
6.9
7.8
0.5
1.3
0

11.1
1.3
1.6
0.6
0.7

11
1.3
1.6
0.4
0.7

WV-

11345

42
29.3
16.6
8.1
5

40
27.4
17.4
8.2
4.7

WV-

18944

7.7
4
1.4
1
0.7

8
4
1.7
1
0.8

WV-
44.5
38.2
26.7
11.9
6.6

17774
45.2
37.5
26.3
12.5
6.6

44
37.2
26.7
14.7
4.8

44.7
35.6
27.2
13.2
4.5

WV-
14.1
11.6
5
1.9
1.5

18945
14.3
11.2
4.8
2
1.5

21.4
11.4
4.7
2.4
2.6

21.3
11.1
4.7
2.3
3

Mock
0.2

0.6
0

0.3

0.8
0

2.5
0
0.3
2.5
1.2

2
0
0.4
2.5
1.1

Oligonucleotides were tested in vitro for ability to skip DMD exon 23.

Oligonucleotides were tested at 10, 3.3., 1.1, 0.3, and 0.1 uM.

Numbers indicate amount of skipping DMD exon 23 (as a percentage of total mRNA, where 100 would represent 100% skipped).

Example Dystrophin Oligonucleotides and Compositions for Exon Skipping of Exon 45

In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for mediating skipping of exon 45 in DMD (e.g., of mouse, human, etc.).

In some embodiments, a provided DMD oligonucleotide and/or composition is capable of mediating skipping of exon 45. Non-limiting examples of such DMD oligonucleotides and compositions include those of: WV-11047, WV-11048, WV-11049, WV-11050, WV-11051, WV-11052, WV-11053, WV-11054, WV-11055, WV-11056, WV-11057, W4V-11058, WV-11059, WV-11060, WV-11061, WV-11062, WV-11063, WV-11064, WV-11065, WV-11066, WV-11067, WV-11068, WV-11069, WV-11070, WV-11071, WV-11072, WV-11073, WV-11074, WV-11075, WV-11076, WV-11077, WV-11078, WV-11079, WV-11080, WV-11081, WV-11082, WV-11083, WV-11084, WV-11085, WV-11086, WV-11087, WV-11088, WV-11089, WV-11090, WV-11091, WV-11092, WV-11093, WV-11094, WV-11095, WV-11096, WV-11097, WV-11098, WV-11099, WV-11100, WV-11101, WV-11102, WV-11103, WV-11104, WV-11105, WV-9594, WV-9595, WV-9596, WV-9597, WV-9598, WV-9599, WV-9600, WV-9601, WV-9602, WV-9603, WV-9604, WV-9605, WV-9606, WV-9607, WV-9608. WV-9609, WV-9610, WV-9611, WV-9612, WV-9613, WV-9614, WV-9615, WV-9616, WV-9617, WV-9618, WV-9619, WV-9620, WV-9621, WV-9622, WV-9623, WV-9624, WV-9625, WV-9626, WV-9627, WV-9628, WV-9629, WV-9630, WV-9631, WV-9632, WV-9633, WV-9634, WV-9635, WV-9636, WV-9637, WV-9638, WV-9639, WV-9640, WV-9641, WV-9642, WV-9643, WV-9644, WV-9645, WV-9646, WV-9647, WV-9648, WV-9649, WV-9650. WV-9651. WV-9652, WV-9653, WV-9654, WV-9655, WV-9656, WV-9657, WV-9658. WV-9659. WV-9762. WV-9763, WV-9764, WV-9765, WV-9766, WV-9767, WV-9768, WV-9769, WV-9770, WV-9771, WV-9772, WV-9773, WV-9774, WV-9775, WV-9776, WV-9777, WV-9778, WV-9779, WV-9780, WV-9781, WV-9782, WV-9783, WV-9784, WV-9785, WV-9786, WV-9787, WV-9788, WV-9789, WV-9790, WV-9791. WV-9792, WV-9793, WV-9794, WV-9795, WV-9796, WV-9797, WV-9798, WV-9799, WV-9800, WV-9801, WV-9802, WV-9803, WV-9804, WV-9805, WV-9806, WV-9807, WV-9808, WV-9809, WV-9810, WV-9811, WV-9812, WV-9813, WV-9814, WV-9815, WV-9816, WV-9817, WV-9818, WV-9819, WV-9820, WV-9821, WV-9822, WV-9823, WV-9824, WV-9825, and WV-9826, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

As shown in various tables from Table 1 to Table 22 (and parts thereof), various DMD oligonucleotides comprising various patterns of modifications were testing for skipping of various exons. The Tables show test results of certain DMD oligonucleotides. To assay exon skipping of DMD, certain DMD oligonucleotides were tested in vitro in Δ52 human patient-derived myoblast cells (also designated DEL52) and/or Δ45-52 human patient-derived myoblast cells (human cells wherein the exon 52 or exons 45-52 were already deleted, also designated DEL45-52). Unless noted otherwise, in various experiments, oligonucleotides were delivered gymnotically. In the tables, generally, 100.00 would represent 100⁰% skipping and 0.0 would represent 0% skipping. Various DMD oligonucleotides are described in detail in Table A1.

Table 1A.4, below, shows example data of some DMD oligonucleotides in skipping exon 45. Procedure: A48-50 (De148-50 or D48-50) myoblasts were treated with 10 uM oligonucleotides for 4 days in differentiation media.

TABLE 1A.4

Example data of certain oligonucleotides.

WV-11047
0.024
0.009
0.012
0.016

WV-11051
0.022
0.024
0.046
0.014

WV-11052
0.024
0.032
0.014
0.026

WV-11053
0.027
0.009
0.017
0.023

WV-11054
0.029
0.038
0.035
0.028

WV-11055
0.030
0.025
0.016
0.033

WV-11056
0.029
0.043
0.018
0.031

WV-11057
0.000
0.015
0.000
0.032

WV-11058
0.044
0.029
0.049
0.024

WV-11059
0.025
0.041
0.049
0.024

WV-11062
0.218
0.175
0.151
0.231

WV-11063
0.472
0.730
0.456
0.594

WV-11064
0.297
0.307
0.334
0.345

WV-11065
0.651
0.630
0.675
0.544

WV-11066
0.124
0.087
0.137
0.153

WV-11067
0.183
0.210
0.238
0.224

WV-11068
0.212
0.266
0.244
0.406

WV-11069
0.389
0.715
0.407
0.744

WV-11070
1.677
1.473
1.483
1.677

WV-11071
0.385
0.362
0.413
0.310

WV-11072
0.146
0.250
0.142
0.268

WV-11073
0.709
0.876
0.721
0.835

WV-11074
2.015
2.207
1.992
2.527

WV-11075
0.254
0.238
0.157
0.220

WV-11076
0.000
2.715
0.000
2.315

WV-11077
1.568
1.414
1.388
1.308

WV-11078
3.915
3.122
4.175
3.076

WV-11079
7.178
8.083
8.257
6.955

WV-11080
1.467
1.202
1.726
1.155

WV-11081
9.279
4.780
10.244
4.512

WV-11082
3.377
2.646
3.242
2.256

WV-11083
3.964
2.631
4.001
2.419

WV-11084
11.336
7.481
13.752
8.270

WV-11085
1.818
0.679
1.787
2.003

WV-11086
16.017
15.215
17.207
15.191

WV-11087
1.104
0.766
1.728
1.030

WV-11088
14.320
12.940
14.287
10.746

WV-11089
16.126
13.507
15.515
15.389

WV-11090
1.148
0.596
1.405
0.647

WV-11091
0.105
0.069
0.311
0.049

WV-11092
0.094
0.066
0.111
0.066

WV-11093
0.123
0.060
0.087
0.037

WV-11094
0.054
0.062
0.060
0.038

WV-11095
0.317
0.064
0.241
0.109

WV-11096
0.062
0.061
0.096
0.059

WV-11098
0.026
0.033
0.032
0.024

WV-11100
0.015
0.012
0.014
0.011

WV-11101
0.000
0.021
0.000
0.011

WV-11102
0.019
0.030
0.025
0.017

WV-11103
0.017
0.023
0.014
0.029

WV-11104
0.053
0.050
0.067
0.035

WV-11105
0.017
0.033
0.034
0.051

Mock
0.050
0.018
0.010
0.037

Mock
0.019
0.023
0.009
0.023

Numbers represent level of skipping, wherein 100 would represent 100% skipping and 0 would represent 0% skipping. For various data described herein, “Mock” is a negative control, in which water was used instead of an oligonucleotide.

Table 1B.1, and 1B.2 Example data of certain oligonucleotides.

The Tables below show example data of some DMD oligonucleotides in skipping exon 45. Procedure: Δ48-50 (De148-50 or DEL48-50 or D48-50) myoblasts were treated with 10 or 3 uM oligonucleotides for 4 days in differentiation media.

Oligonucleotides were dosed at 10 μM and 3 μM for 4 days in DEL48-50 Myoblasts. Certain oligonucleotides comprise a non-negatively charged internucleotidic linkage, as detailed in Table A1.

TABLE 1B.1

Example data of certain oligonucleotides.

10 um
3 um

WV-13810
7.0
6.5
7.1
6.5
2.7
2.8
2.5
2.3

WV-13811
8.4
8.0
9.1
9.5
3.3
3.2
2.4
2.8

WV-13812
22.8
21.1
22.9
23.7
9.2
9.2
10.0
9.7

WV-13813
19.4
19.9
20.1
20.2
7.6
8.1
7.5
7.4

WV-13814
13.6
13.6
13.5
13.3
5.1
4.3
4.9
4.9

WV-13815
26.9
25.6
23.9
24.3
9.0
8.9
8.2
8.6

WV-13816
37.0
35.0
31.8
33.8
14.0
14.5
14.6
12.0

WV-13817
52.7
55.4
54.3
54.2
24.9
26.1
21.9
21.7

WV-14531
2.9
2.7
2.8
2.9
0.7
0.9
1.0
1.2

WV-14532
4.3
4.3
3.8
4.1
1.4
1.3
1.1
1.0

WV-14533
7.9
7.6
7.3
7.9
1.9
2.1
2.4
2.1

WV-11086
18.3
20.1
18.4
18.4
7.9
7.7
7.6
8.1

TABLE 1B.2

Example data of certain oligonucleotides.

10 uM
3 uM

WV-13818
3.2
2.8
3.2
2.9
0.9
0.8
1.1
1.2

WV-13819
3.8
3.8
3.0
2.9
1.0
0.9
0.9
1.0

WV-13820
6.6
6.7
6.4
6.3
3.2
3.0
2.9
3.0

WV-13821
7.4
6.5
7.4
6.9
2.2
1.9
2.5
1.9

WV-13822
9.5
9.5
8.1
8.6
3.4
3.5
3.4
3.9

WV-13823
10.4
10.9
11.2
10.5
4.2
5.0
4.1
4.4

WV-13824
17.1
16.3
16.1
15.6
8.1
7.6
7.1
7.0

WV-13825
20.1
19.3
22.5
20.6
9.9
9.8
9.0
9.6

WV-14527
2.2
1.9
1.4
2.0
0.7
0.7
0.9
0.7

WV-14528
2.3
2.2
2.5
2.4
1.0
0.9
1.0
1.0

WV-14529
5.2
1.8
2.0
2.0
0.7
0.7
0.8
0.8

WV-11089
2.6
2.7
2.9
2.5
0.9
0.9
1.4
1.3

Additional data related to multiple exon skipping mediated by DMD oligonucleotides which target DMD exon 45 are shown in Table 22A.1.

Example Dystrophin Oligonucleotides and Compositions which Target Exon 46

In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for targeting exon 46 and/or mediating skipping of exon 46 in human DMD. Non-limiting examples include oligonucleotides and compositions of WV-13701, WV-13702, WV-13703, WV-13704, WV-13705, WV-13706, WV-13707, WV-13708, WV-13709, WV-13710, WV-13711, WV-13712, WV-13713, WV-13714, WV-13715, WV-13716, WV-13780, and WV-13781, and other oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

In some embodiments, DMD oligonucleotides are first tested for single exon skipping to select suitable oligonucleotides, then tested combinatorially (in combination with another DMD oligonucleotide) for multi-exon skipping.

In some embodiments, DMD oligonucleotides targeting DMD exon 46, 47, 52, 54 or 55 are first tested for single exon skipping to select suitable oligonucleotides, then tested combinatorially (in combination with another DMD oligonucleotide) for multi-exon skipping.

TABLE 2A

Example data of certain oligonucleotides. Numbers

indicate percentage of exon 46 skipping.

WV-13701
0.3
0.3
0.5
0.4

WV-13702
0.3
0.4
0.5
0.3

WV-13703
0.9
0.9
1.1
0.8

WV-13704

9.7
5.4

WV-13705
4.9
5.1
5.9
3.4

WV-13706

4.6
4.8

WV-13707
8.5
7.4
5.2
5.1

WV-13708
9.4
10.8
6.0
5.6

WV-13709
8.8
12.1
8.1
4.9

WV-13710
0.1
0.1
0.1
0.1

WV-13711
0.1
0.1
0.0
0.1

WV-13712
3.4
4.7
2.4
2.4

WV-13713
0.5

0.7
0.5

WV-13714
0.6

0.5
0.4

WV-13715
0.9

0.6
0.7

WV-13716
1.5
3.9
1.1
2.8

WV-13780
10.1
5.2

6.1

WV-13781
7.7
6.4

5.0

Mock
0.0
0.0
0.0
0.0

Mock
0.0

0.0

Example Dystrophin Oligonucleotides and Compositions which Target Exon 47

In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for targeting exon 47 and/or mediating skipping of exon 47 in human DMD. Non-limiting examples include oligonucleotides and compositions of exon 47 oligos include: WV-13717, WV-13718, WV-13719, WV-13720, WV-13721, WV-13722, WV-13723, WV-13724, WV-13725, WV-13726, WV-13727, WV-13728, WV-13729, WV-13730, WV-13731, WV-13732, WV-13788, and WV-13789, and other oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides

TABLE 3A

Example data of certain oligonucleotides. Numbers

represent percentage of exon 47 skipping.

WV-13717
0.0
0.0

WV-13718
0.0
0.0

WV-13719
0.0
0.0

WV-13720
0.0
0.0

WV-13721
0.0
0.0

WV-13722
0.0
0.0

WV-13723
0.5
0.5

WV-13724
1.4
1.8

WV-13725
0.6
0.4

WV-13726
0.0
0.0

WV-13727
1.1
1.1

WV-13728
1.1
1.1

WV-13729
0.2
0.2

WV-13730
0.5
0.6

WV-13731
1.6
1.8

WV-13732
0.1
0.6

Example Dystrophin Oligonucleotides and Compositions for Exon Skipping of Exon 51

In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for mediating skipping of exon 51 in DMD (e.g., of mouse, human, etc.).

In some embodiments, a provided DMD oligonucleotide and/or composition is capable of mediating skipping of exon 51. Non-limiting examples of such DMD oligonucleotides and compositions include those of: ONT-395, WV-10255, WV-10261, WV-10262, WV-10634, WV-10635, WV-10636, WV-10637, WV-10868, WV-10869, WV-10870, WV-10871, WV-10872, WV-10873, WV-10874, WV-10875, WV-10876, WV-10877, WV-10878, WV-10879, WV-10880, WV-10881, WV-10882, WV-10883, WV-10884, WV-10885, WV-10886, WV-10887, WV-10888, WV-1107, W4V-1108, WV-1109, WV-1110, WV-1111, WV-1112, WV-1113, WV-1114, WV-1115, WV-1116, WV-1117, WV-1118, WV-1119, WV-1120, WV-11237, WV-11238, WV-11239, WV-1131, WV-1132, WV-1133, WV-1134, WV-1135, WV-1136, WV-1137, WV-1138, WV-1139, WV-1140, WV-1151, WV-1152, WV-1153, WV-1154, WV-1155, WV-1156, WV-1157, WV-1158, WV-1159, WV-1160, WV-1709, WV-1710, WV-1711, WV-1712, WV-1713, WV-1714, WV-1715, WV-1716, WV-2095, WV-2096, WV-2097, WV-2098, WV-2099, WV-2100, WV-2101, WV-2102, WV-2103, WV-2104. WV-2105. WV-2106, WV-2107, WV-2108, WV-2109, WV-2165, WV-2179, WV-2180, WV-2181, WV-2182, WV-2183, WV-2184, WV-2185, WV-2186, WV-2187, WV-2188, WV-2189, WV-2190, WV-2191, WV-2192, WV-2193, WV-2194, WV-2195, WV-2196, WV-2197, WV-2198, WV-2199, WV-2200, WV-2201, WV-2202. WV-2203, WV-2204, WV-2205, WV-2206, WV-2207, WV-2208, WV-2209, WV-2210, WV-2211, WV-2212, WV-2213, WV-2214, WV-2215, WV-2216, WV-2217, WV-2218, WV-2219, WV-2220, WV-2221, WV-2222, WV-2223, WV-2224, WV-2225, WV-2226, WV-2227, WV-2228, WV-2229, WV-2230, WV-2231, WV-2232, WV-2233, WV-2234, WV-2235, WV-2236, WV-2237, WV-2238, WV-2239, WV-2240, WV-2241, WV-2242, WV-2243, WV-2244. WV-2245. WV-2246, WV-2247, WV-2248, WV-2249, WV-2250, WV-2251, WV-2252, WV-2253, WV-2254, WV-2255, WV-2256, WV-2257, WV-2258, WV-2259, WV-2260, WV-2261, WV-2262, WV-2263, WV-2264, WV-2265, WV-2266, WV-2267, WV-2268, WV-2273, WV-2274, WV-2275, WV-2276, WV-2277, WV-2278. WV-2279, WV-2280, WV-2281, WV-2282, WV-2283, WV-2284, WV-2285, WV-2286, WV-2287, WV-2288, WV-2289, WV-2290, WV-2291, WV-2292, WV-2293, WV-2294, WV-2295, WV-2296, WV-2297, WV-2298, WV-2299, WV-2300, WV-2301, WV-2302, WV-2303, WV-2304, WV-2305, WV-2306, WV-2307, WV-2308, WV-2309, WV-2310, WV-2311, WV-2312, WV-2313, WV-2314, WV-2315, WV-2316, WV-2317, WV-2318, WV-2319, WV-2320, WV-2321, WV-2322, WV-2323, WV-2324, WV-2325, WV-2326, WV-2327, WV-2328, WV-2329. WV-2330. WV-2331, WV-2332, WV-2333, WV-2334, WV-2335, WV-2336, WV-2337, WV-2338, WV-2339, WV-2340, WV-2341, WV-2342, WV-2343, WV-2344, WV-2345, WV-2346, WV-2347, WV-2348, WV-2349, WV-2350, WV-2351, WV-2352, WV-2353, WV-2354, WV-2361, WV-2362, WV-2363, WV-2364, WV-2365. WV-2366, WV-2367, WV-2368, WV-2369, WV-2370, WV-2381, WV-2382, WV-2383, WV-2384, WV-2385, WV-2432, WV-2433, WV-2434, WV-2435, WV-2436, WV-2437, WV-2438, WV-2439, WV-2440, WV-2441, WV-2442, WV-2443, WV-2444, WV-2445, WV-2446, WV-2447, WV-2448, WV-2449, WV-2526, WV-2527, WV-2528, WV-2529, WV-2530, WV-2531, WV-2532, WV-2533, WV-2534, WV-2535, WV-2536, WV-2537, WV-2538, WV-2578. WV-2579. WV-2580, WV-2581, WV-2582, WV-2583, WV-2584, WV-2585, WV-2586, WV-2587, WV-2588, WV-2625, WV-2627, WV-2628, WV-2660, WV-2661, WV-2662, WV-2663, WV-2664, WV-2665, WV-2666, WV-2667, WV-2668, WV-2669, WV-2670, WV-2737, WV-2738, WV-2739, WV-2740, WV-2741, WV-2742. WV-2743, WV-2744, WV-2745, WV-2746, WV-2747, WV-2748, WV-2749, WV-2750, WV-2752, WV-2783, WV-2784, WV-2785, WV-2786, WV-2787, WV-2788, WV-2789, WV-2790, WV-2791, WV-2792, WV-2793, WV-2794, WV-2795, WV-2796, WV-2797, WV-2798, WV-2799, WV-2800, WV-2801, WV-2802, WV-2803, WV-2804, WV-2805, WV-2806, WV-2807, WV-2808, WV-2812, WV-2813, WV-2814, WV-3017, WV-3018, WV-3019, WV-3020, WV-3022, WV-3023, WV-3024, WV-3025, WV-3026, WV-3027, WV-3028, WV-3029, WV-3030. WV-3031. WV-3032, WV-3033, WV-3034, WV-3035, WV-3036, WV-3037, WV-3038, WV-3039, WV-3040, WV-3041, WV-3042, WV-3043, WV-3044, WV-3045, WV-3046, WV-3047, WV-3048, WV-3049, WV-3050, WV-3051, WV-3052, WV-3053, WV-3054, WV-3055, WV-3056, WV-3057, WV-3058, WV-3059, WV-3060. WV-3061, WV-3070, WV-3071, WV-3072, WV-3073, WV-3074, WV-3075, WV-3076, WV-3077, WV-3078, WV-3079, WV-3080, WV-3081, WV-3082, WV-3083, WV-3084, WV-3085, WV-3086, WV-3087, WV-3088, WV-3089, WV-3113, WV-3114, WV-3115, WV-3116, WV-3117, WV-3118, WV-3120, WV-3121, WV-3152, WV-3153, WV-3357, WV-3358, WV-3359, WV-3360, WV-3361, WV-3362, WV-3363, WV-3364, WV-3365, WV-3366, WV-3463. WV-3464. WV-3465, WV-3466, WV-3467, WV-3468, WV-3469, WV-3470, WV-3471, WV-3472, WV-3473, WV-3506, WV-3507, WV-3508, WV-3509, WV-3510, WV-3511, WV-3512, WV-3513, WV-3514, WV-3515, WV-3516, WV-3517, WV-3518, WV-3519, WV-3520, WV-3543, WV-3544, WV-3545, WV-3546, WV-3547. WV-3548, WV-3549, WV-3550, WV-3551, WV-3552, WV-3553, WV-3554, WV-3555, WV-3556, WV-3557, WV-3558, WV-3559, WV-3560, WV-3753, WV-3754, WV-3820, WV-3821, WV-3855, WV-3856, WV-3971, WV-4106, WV-4107, WV-4191, WV-4231, WV-4232, WV-4233, WV-4890, WV-6137, WV-6409, WV-6410, WV-6560, WV-6826, WV-6827, WV-6828, WV-7109, WV-7110, WV-7333, WV-7334, WV-7335, WV-7336, WV-7337, WV-7338, WV-7339, WV-7340, WV-7341, WV-7342, WV-7343, WV-7344, WV-7345, WV-7346, WV-7347. WV-7348. WV-7349, WV-7350, WV-7351, WV-7352, WV-7353, WV-7354, WV-7355, WV-7356, WV-7357, WV-7358, WV-7359, WV-7360, WV-7361, WV-7362, WV-7363, WV-7364, WV-7365, WV-7366, WV-7367, WV-7368, WV-7369, WV-7370, WV-7371, WV-7372, WV-7373, WV-7374, WV-7375, WV-7376, WV-7377. WV-7378, WV-7379, WV-7380, WV-7381, WV-7382, WV-7383, WV-7384, WV-7385, WV-7386, WV-7387, WV-7388, WV-7389, WV-7390, WV-7391, WV-7392, WV-7393, WV-7394, WV-7395, WV-7396, WV-7397, WV-7398, WV-7399, WV-7400, WV-7401, WV-7402, WV-7410, WV-7411, WV-7412, WV-7413, WV-7414, WV-7415, WV-7457, WV-7458, WV-7459, WV-7460, WV-7461, WV-7506, WV-7596, WV-8130, WV-8131, WV-8230, WV-8231. WV-8232. WV-8449, WV-8478, WV-8479, WV-8480, WV-8481, WV-8482, WV-8483, WV-8484, WV-8485, WV-8486, WV-8487, WV-8488, WV-8489, WV-8490, WV-8491, WV-8492, WV-8493, WV-8494, WV-8495, WV-8496, WV-8497, WV-8498, WV-8499, WV-8500, WV-8501, WV-8502, WV-8503, WV-8504, WV-8505. WV-8506, WV-8806, WV-884, WV-885, WV-886, WV-887, WV-888, WV-889, WV-890, WV-891, WV-892, WV-893, WV-894, WV-895, WV-896, WV-897, WV-9222, WV-9223, WV-9224, WV-9225, WV-9226, WV-9227, WV-942, WV-9540, WV-9541, WV-9737, WV-9738, WV-9739, WV-9740, WV-9741, WV-9742, WV-9827, WV-9828, WV-9829, WV-9830, WV-9831, WV-9832, WV-9833, WV-9834, WV-9835, WV-9836, WV-9837, WV-9838, WV-9839, WV-9840, WV-9841, WV-9842, WV-9843, WV-9844, WV-9845, WV-9846, WV-9847, WV-9848, WV-9849. WV-9850. WV-9851, WV-9852, WV-9858, and WV-8937, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

Additional non-limiting examples of such DMD oligonucleotides and compositions include those of: WV-2444, WV-2528, WV-2531, WV-2578, WV-2579, WV-2580, WV-2581, WV-2669, WV-2745, WV-3032, WV-3152, WV-3153, WV-3360, WV-3363, WV-3364, WV-3465, WV-3466, WV-3470, WV-3472, WV-3473, WV-3507, WV-3545, WV-3546, WV-3552, WV-4106, WV-4231, WV-4232, WV-4233, WV-887, WV-896, WV-942, and other DMD oligonucleotides having abase sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

Additional non-limiting examples of such DMD oligonucleotides and compositions include those of: WV-12494, WV-12130, WV-12131, WV-12132, WV-12133, WV-12134, WV-12135, WV-12136, WV-12496, WV-12495, WV-12123, WV-12124, WV-12125, WV-12126, WV-12127, WV-12128, WV-12129, WV-12553, WV-12554, WV-12555, WV-12556, WV-12557, WV-12558, WV-12559, WV-12872, WV-12873, WV-12876, WV-12877, WV-12878, WV-12879, WV-12880, WV-12881, WV-12882, and WV-12883, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

In some embodiments, the sequence of the region of interest for exon 51 skipping differs between the mouse and human.

Various assays can be utilized to assess oligonucleotides for exon skipping in accordance with the present disclosure. In some embodiments, in order to test the efficacy of a particular combination of chemistry and stereochemistry of an oligonucleotide intended for exon 51 skipping in human, a corresponding oligonucleotide can be prepared which has the mouse sequence, and then tested in mouse. The present disclosure recognizes that in the human and mouse homologs of exon 51, a few differences exist (underlined below):

M GTGGTTACTAAGGAAACTGTCATCTCCAAACTAGAAATGCCATCTTC

TTTGCTGTTGGAGH GTGGTTACTAAGGAAACTGCCATCTCCAAACTAG

AAATGCCATCTTCCTTGATGTTGGAG.

where M is Mouse, nt 7571-7630; and H is Human, nt 7665-7724.

Because of these differences, slightly different DMD oligonucleotides for skipping exon 51 can be prepared for testing in mouse and human. As a non-limiting example, the following DMD oligonucleotide sequences can be used for testing in human and mouse:

HUMAN DMD oligonucleotide sequence:

UCAAGGAAGAUGGCAUUCU

MOUSE DMD oligonucleotide sequence:

GCAAAGAAGAUGGCAUUUCU

Mismatches between human and mouse are underlined.

A DMD oligonucleotide intended for treating a human subject can be constructed with a particular combination of base sequence (e.g., UCAAGGAAGAUGGCAUUUCU), and a particular pattern of chemistry, internucleotidic linkages, stereochemistry, and additional chemical moieties (if any). Such a DMD oligonucleotide can be tested in vitro in human cells or in vivo in human subjects, but may have limited suitability for testing in mouse, for example, because base sequences of the two have mismatches.

A corresponding DMD oligonucleotide can be constructed with the corresponding mouse base sequence (GCAAAGAAGAUGGCAUUUCU) and the same pattern of chemistry, internucleotidic linkages, stereochemistry, and additional chemical moieties (if any). Such an oligonucleotide can be tested in vivo in mouse. Several DMD oligonucleotides comprising the mouse base sequence were constructed and tested.

In some embodiments, a human DMD exon skipping oligonucleotide can be tested in a mouse which has been modified to comprise a DMD gene comprising the human sequence.

Various DMD oligonucleotides comprising various patterns of modifications are described herein. The Tables below show test results of certain DMD oligonucleotides. To assay exon skipping of DMD, DMD oligonucleotides were tested in vitro in Δ52 human patient-derived myoblast cells and/or Δ45-52 human patient-derived myoblast cells (human cells wherein the exon 52 or exons 45-52 were already deleted). Unless noted otherwise, in various experiments, oligonucleotides were delivered gymnotically.

TABLE 4A

Example data of certain oligonucleotides.

10 uM
3 uM

WV-942
1.0
2.2
1.5
0.2
0.5
0.2

WV-1709
8.5
12.9
7.7
3.3
5.8
3.7

WV-1710
4.1
6.1
4.7
1.1
2.5
1.3

WV-1711
4.4
5.8
3.7
1.1
2.4
1.4

WV-1712
2.6
4.4
3.1
0.9
2.0
1.7

WV-1713
2.1
3.5
2.3
0.6
1.6
0.3

WV-1714
7.8
10.5
10.2
2.3
4.1
2.3

WV-1715
2.2
3.8
3.3
0.8
1.8
1.1

WV-1716
2.1
3.5
2.4
0.9
1.8
0.9

DMD oligonucleotides were tested in vitro at 10 uM and 3 uM, in triplicates. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown. Full descriptions of the oligonucleotides tested in this Table (and other Tables) are provided in Table A1.

In Table 4B, below, additional data of DMD oligonucleotides for skipping exon 51 were presented.

TABLE 4B

Example data of certain oligonucleotides.

10 uM
3 uM

WV-942
1.0
2.2
1.5
0.2
0.5
0.2

WV-1714
7.8
10.5
10.2
2.3
4.1
2.3

WV-2444
22.2
26.7
28.6
9.1
12.6
11.9

WV-2445
17.1
20.7
18.7
7.0
9.7
9.1

WV-2528
32.4
34.6
39.3
16.9
19.9
22.3

WV-2529
3.2
5.8
6.1
2.2
4.5
3.0

WV-2530
18.6
21.1
25.4
7.6
11.5
11.4

DMD oligonucleotides were tested at 10 uM and 3 uM, in triplicates. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

In Table 4C, below, additional data of DMD oligonucleotides for skipping exon 51 were presented.

TABLE 4C

Example data of certain oligonucleotides.

WV-942
WV-887
WV-1714
WV-2438

10
uM
1.1
0.7
5.1
3.9
3.6
3.7
9.3
9.3

3
uM
0.5
0.3
1.0
2.2
1.6
1.5
3.9
3.1

1
uM
0.2
0.2
0.6
0.7
0.6
0.3
1.4
1.1

WV-2439
WV-2444
WV-2445
Mock

10
uM
3.2
2.1
12.9
14.3
9.7
8.9
0.4
0.1

3
uM
0.8
0.7
4.7
4.1
3.3
3.5
0.1
0.1

1
uM
0.4
0.3
1.4
1.0
1.1
1.0
0.1

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

In Table 4D, below, additional data of DMD oligonucleotides for skipping exon 51 were presented.

Table 4D. Example data of certain oligonucleotides.

TABLE 4D

Example data of certain oligonucleotides.

10 uM

WV-942
0.6
0.6
0.6
0.6

WV-2660
0.2
0.3
0.1
0.1

WV-2661
0.4
0.4

WV-2662
0.2
0.2
0.1
0.1

WV-2663
0.5
0.5
0.4
0.5

WV-2670
5.1
5.2
6.2
7.3

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

In Table 5, below, additional data of DMD oligonucleotides for skipping exon 51 were presented.

TABLE 5

Example data of certain oligonucleotides.

10 uM
3 uM
1 uM

Mock
0.0
0.1
0.0

WV-2531
21.7
8.7
3.2

WV-3152
26.1
15.3
5.7

WV-2745
24.0
10.7
4.8

WV-3463
6.6
3.0
0.8

WV-3464
16.1
6.2
2.4

WV-3465
16.4
6.0
1.8

WV-3466
13.0
5.7
2.0

WV-3467
12.6
5.8
2.6

WV-3469
14.2
6.0
1.5

WV-3470
24.9
11.9
6.4

WV-3471
4.9
1.6
1.0

WV-3472
20.1
12.4
7.2

WV-3473
24.9
11.4
7.6

WV-942
3.3
2.1
0.7

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

TABLE 6

Example data of certain oligonucleotides.

5 uM
1 uM

WV-942
.2

PMO
.1

WV-6137
1
.9

WV-7333
.3
.2

WV-7334
.7
.4

WV-7335
1.7
.4

WV-7336
2.2
.6

WV-7337
1.7
.4

WV-7343
1.4
.5

WV-7344
2.8
.7

WV-7345
2.9
1

WV-7346
1.9
.7

WV-7347
1.2
.5

WV-7348
2.5
1

WV-7349
3
.6

WV-7350
3.1
1

WV-7351
1.7
.6

WV-7352
2.7
.8

WV-7353
2.8
.2

WV-7354
2.2
.3

WV-7355
2.7
1.6

WV-7356
3.3
1.2

WV-7357
2.7
1.1

WV-7358
2.2
.6

WV-7359
.7
.3

WV-7360
.6
.5

WV-7361
2.8
.8

WV-7362
4.1
.8

WV-7363
2
.7

Numbers represent skipping efficiency wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown. Numbers are approximate. Oligonucleotides were delivered gymnotically to Δ48-50 patient-derived myoblasts (4 days post-differentiation). The oligonucleotide designated as “PMO” in this table and other tables related to skipping of DMD exon 51 is WV-8806 CTCCAACATCAAGGAAGATGGCATTTCTAG, which is fully PMO (Morpholino).

In Table 7, below, additional data of DMD oligonucleotides for skipping exon 51 were presented.

TABLE 7

Example data of certain oligonucleotides.

Mock
.1

WV-942
.2

PMO
.1

WV-7364
2
.5

WV-7365
1.8
.5

WV-7366
1.1
5.7

WV-7367
.2
.3

WV-7368
.4
.4

WV-7369
.4
.2

WV-7370
.2
.3

WV-7371
.3
.2

WV-7372
.3

WV-7373
.5
1.3

WV-7374
.3
.4

WV-7375
.2
.8

WV-7376
.2
.5

WV-7377
.3
.5

WV-7378
.4

WV-7379
7.8
1

WV-7380
2.8
.3

WV-7381
4.1
.2

WV-7382
1.3
.1

WV-7383
1.7
.3

WV-7384
2.8
.4

WV-7385

1.8

WV-7386
4
1.6

WV-7387
3
1.8

WV-7388
1.2
.7

WV-7389
.5
.4

WV-7390
1
.5

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown. Numbers are approximate.

In some embodiments, the present disclosure pertains to metabolites of any oligonucleotide, e.g., DMD oligonucleotide, disclosed herein, or any combination thereof. In some embodiments, a metabolite of an oligonucleotide, e.g., a DMD oligonucleotide is the result of an oligonucleotide, e.g., a DMD oligonucleotide being acted upon by a nuclease (e.g., an exonuclease or endonuclease or other enzymes, including those may chemically process one or more modifications of an oligonucleotide). In some embodiments, a “metabolite” of an oligonucleotide, e.g., a DMD oligonucleotide is not the physical product of such an oligonucleotide being metabolized or physically treated with a nuclease, but rather a compound which corresponds chemically to a product of an oligonucleotide being metabolized or treated with an enzyme. e.g., a nuclease. In some embodiments, metabolite of an oligonucleotide, e.g., a DMD oligonucleotide, is chemically synthesized, without any metabolic process, and optionally administered to a subject.

In some embodiments, a metabolite is a truncation of an oligonucleotide on the 5′ end and/or 3′ end by one or two nucleotides or nucleosides. In some embodiments, the present disclosure provides an oligonucleotide, e.g., DMD oligonucleotide which corresponds to an oligonucleotide, e.g., DMD oligonucleotide listed herein, but is truncated at the 5′ end by one or two nucleotides. In some embodiments, the present disclosure provides an oligonucleotide, e.g., a DMD oligonucleotide which corresponds to an oligonucleotide, e.g., a DMD oligonucleotide listed herein, but is truncated at the 3′ end by one or two nucleotides. In some embodiments, the present disclosure provides an oligonucleotide, e.g., a DMD oligonucleotide which corresponds to an oligonucleotide, e.g., a DMD oligonucleotide listed herein, but is truncated at the 3′ end and 5′ end by one or two nucleotides. Among other things, such oligonucleotides may perform various of biological functions, e.g., such DMD oligonucleotides can mediate skipping of exon 23, 45, 51, 53, or any other DMD exon.

In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has the base sequence of a DMD oligonucleotide listed herein, except that the base sequence is shorter on the 5′ end by one or two bases. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has the base sequence of a DMD oligonucleotide listed herein, except that the base sequence is shorter on the 3′ end by one or two bases. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has the base sequence of a DMD oligonucleotide disclosed herein, except that the base sequence is shorter on the 3′ end and the 5′ end by one or two bases. Such DMD oligonucleotides, among other things, can mediate skipping of exon 23, 45, 51, 53, or any other DMD exon.

In some embodiments, a metabolite of a DMD oligonucleotide has removed from the oligonucleotide an additional moiety (e.g., a lipid or other conjugated moiety).

In some embodiments, an oligonucleotide of the present disclosure may be a metabolite of another oligonucleotide. For example, several oligonucleotides may be metabolite of WV-3473, for example, WV-4231 (3′n-1, truncated at the 3′ end by one nucleotide), WV-4232 (3′ n-2), WV-4233 (5′ n-1), etc. Example data of such “metabolite” oligonucleotides were presented in Table 9 below (at 1, 3 and 10 uM, in replicates). Generally, an oligonucleotide can be used independently whether or not it can be a metabolite of another oligonucleotide.

TABLE 9

Example data of certain oligonucleotides.

Oligonucleotide
10 uM
3 uM
1 uM

PMO
2.4
1.6
0.4
1.1
0.4
0.6

WV-3473
78.8
73.5
62.5
59.8
38.8
38.8

WV-4231 (3′ n-1)
83.8
71.4
65.0
67.2
44.4
43.0

WV-4232 (3′ n-2)
48.5
66.5
42.2
57.5

30.0

WV-4233 (5′ n-1)
54.2
45.9
37.1
31.6
18.6
14.5

Results of replicate experiments are shown. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown. In this and other tables PMO is a Morpholino oligonucleotide control.

In some embodiments, the present disclosure pertains to DMD oligonucleotides corresponding to any DMD oligonucleotide to exon 51 or any other exon listed herein (e.g., in Table A1), but which are truncated by one, two or more nucleotides on the 5′ end and/or 3′ end.

In some embodiments, the length of a provided oligonucleotide, e.g., a DMD oligonucleotide, is 15 to 45 bases. In some embodiments, the length of a provided oligonucleotide, e.g., a DMD oligonucleotide, is 20 to 45 bases. In some embodiments, the length of a provided oligonucleotide, e.g., a DMD oligonucleotide, is 20 to 40 bases. In some embodiments, the length of a provided oligonucleotide, e.g., a DMD oligonucleotide, is 35 bases. In some embodiments, the length of a provided oligonucleotide, e.g., a DMD oligonucleotide, is 20 to 25 bases.

In some experiments, lengths of DMD oligonucleotides for skipping exon 51 are 20 or 25 bases.

Tables 10A and 10B. Example data of certain oligonucleotides.

Table 10A shows data of 20-mers for skipping DMD exon 51: Table 10B shows data of 25-mers for skipping DMD exon 51. Sequences are provided in Table A1. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

TABLE 10A

20-mers

untreated
WV-2313
WV-2314
WV-2315
WV-2316

0.1
0.1
1.0
1.4
1.7
1.6
2.0
2.0
4.6
2.5

WV-2317
WV-2318
WV-2319
WV-2320
WV-942

1.7
1.1
4.3
4.3
5.0
6.5
2.9
3.7
3.9
3.4

TABLE 10B

25-mers

WV-2223
WV-2224

WV-2225

WV-2226

15.7
14.8
6.6
7.3
13.4
16.1
7.7
7.7

WV-2227
WV-2228

WV-2229

WV-2230

9.8
9.7
15.7
15.6
8.5
8.9
12.9
13.4

Additional data are provided.

TABLE 10C

Example data of certain oligonucleotides.

10 uM

3 uM

1 uM

WV-2531
21.7
25.1
8.7
10.6
3.2
4.6

WV-3152
26.1
21.7
15.3
10.7
5.7
4.1

WV-3472
20.1
16.3
12.4
8.5
7.2
3.8

WV-3473
24.9
38.4
11.4
11.2
7.6
6.5

WV-942
3.3
0.2
2.1

0.7
0.1

Oligonucleotides were tested in vitro at 10, 3 and 1 μM. Results of replicate experiments are shown. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

TABLE 10D

Example data of certain oligonucleotides.

10 uM
3 uM
1 uM

WV-1714
5.8
6.2
8.1
2.4
3.0
2.7
0.7
0.7
2.0

WV-3030
29.9
27.2
35.2
6.2
5.6
5.6
0.6
0.6
1.6

WV-3032
31.7
29.3
37.9
7.8
6.4
7.7
1.2
1.1
1.1

WV-2669
3.1
3.1
4.1
1.4
1.7
1.7
0.6
0.7
0.8

WV-3035
13.2
16.4
17.6
1.9
2.5
2.8
1.0
1.1
0.8

TABLE 10E

Example data of certain oligonucleotides.

10 uM

3 uM

1 uM

WV-2531
24.7
21.7
11.0
8.7
4.8
3.2

WV-3360

25.1
12.9
10.1

3.3

WV-3363

24.0

7.7

3.4

WV-3364
72.8
45.5
17.2
9.8

4.0

TABLE 10F

Example data of certain oligonucleotides.

10 uM
3 uM
1 uM

Mock
0.0
0.1
0.0

WV-2531
21.7
8.7
3.2

WV-3360
25.1
10.1
3.3

WV-3363
24.0
7.7
3.4

WV-3364
45.5
9.8
4.0

Oligonucleotides were tested in vitro at 10, 3 and 1 μM. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

TABLE 10G

Example data of certain oligonucleotides.

10 uM
3 uM
1 uM

WV-1714
5.8
6.2
8.1
2.4
3.0
2.7
0.7
0.7
2.0

WV-3030
29.9
27.2
35.2
6.2
5.6
5.6
0.6
0.6
1.6

WV-3032
31.7
29.3
37.9
7.8
6.4
7.7
1.2
1.1
1.1

WV-2669
3.1
3.1
4.1
1.4
1.7
1.7
0.6
0.7
0.8

WV-3035
13.2
16.4
17.6
1.9
2.5
2.8
1.0
1.1
0.8

Oligonucleotides were tested in vitro at 10, 3 and 1 M. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency, results from replicate experiments are shown.

TABLE 10H

Example data of certain oligonucleotides.

10 uM, 15% serum
10 uM 5% serum

Mock
0.0
0.1

0.0
0.1

WV-942
1.0
1.0
0.2
0.2
0.7
0.5
0.4
0.4

WV-2578
3.2

2.2
2.4
2.3
2.2
0.9

WV-2579

3.1
2.9

2.5
2.5

WV-2580
2.5
2.9
2.4
3.1
6.8
6.4
2.8
3.2

WV-2581
3.3
3.6
3.9
3.7
4.4
5.8
5.8
5.4

10 uM 5% serum

10 uM 5% serum

20 mg/ml BSA

4 mg/ml BSA

Mock
0.1
0.1

0.1
0.1

WV-942
0.7
0.6
1.4
1.3
0.2
0.3
0.6
0.5

WV-2578
0.9
0.5
0.5
0.6
0.6
0.6
0.5
0.7

WV-2579
0.1
0.1
0.5
0.3
0.1
0.1
0.5
0.4

WV-2580
0.4
0.3
0.2
0.2

0.2
0.1

WV-2581
0.2
0.2
0.4
0.4
0.2
0.2
0.1
0.1

3 uM 15% serum

3 uM 5% serum

Mock
0.0
0.0

0.0
0.0

WV-942
0.1
0.0
0.3
0.3
0.1
0.1
0.2
0.2

WV-2578
0.5
0.3
0.3
0.4
0.3
0.5
0.6
0.2

WV-2579
0.6
0.5
1.8
1.5
0.5
0.4
0.3
0.3

WV-2580
1.0
1.0
0.5
0.6
1.2
1.0
0.5
0.7

WV-2581
0.0
0.0
0.6
0.6
0.4
0.5
0.8
0.7

3 uM 5% serum

3 uM 5% serum

20 mg/ml BSA

4 mg/ml BSA

Mock
0.0
0.0

0.0
0.0

WV-942
0.1
0.1
0.1
0.1
0.1
0.1
0.4
0.3

WV-2578
0.2
0.2
0.2
0.3
0.2
0.1
0.1

WV-2579
0.4
0.4
0.2
0.2
0.1
0.1
0.2
0.2

WV-2580
0.2
0.2
0.2
0.3
0.0
0.0
0.3
0.3

WV-2581
0.0
0.0
0.3
0.3
0.1
0.1
0.1
0.1

10 uM, 15% serum
10 uM 5% serum

Mock
0.0
0.1

0.0
0.1

WV-942
1.0
1.0
0.2
0.2
0.7
0.5
0.4
0.4

WV-2578
3.2

2.2
2.4
2.3
2.2
0.9

WV-2579

3.1
2.9

2.5
2.5

WV-2580
2.5
2.9
2.4
3.1
6.8
6.4
2.8
3.2

WV-2581
3.3
3.6
3.9
3.7
4.4
5.8
5.8
5.4

10 uM 5% serum

10 uM 5% serum

20 mg/ml BSA

4 mg/ml BSA

Mock
0.1
0.1

0.1
0.1

WV-942
0.7
0.6
1.4
1.3
0.2
0.3
0.6
0.5

WV-2578
0.9
0.5
0.5
0.6
0.6
0.6
0.5
0.7

WV-2579
0.1
0.1
0.5
0.3
0.1
0.1
0.5
0.4

WV-2580
0.4
0.3
0.2
0.2

0.2
0.1

WV-2581
0.2
0.2
0.4
0.4
0.2
0.2
0.1
0.1

3 uM 15% serum

3 uM 5% serum

Mock
0.0
0.0

0.0
0.0

WV-942
0.1
0.0
0.3
0.3
0.1
0.1
0.2
0.2

WV-2578
0.5
0.3
0.3
0.4
0.3
0.5
0.6
0.2

WV-2579
0.6
0.5
1.8
1.5
0.5
0.4
0.3
0.3

WV-2580
1.0
1.0
0.5
0.6
1.2
1.0
0.5
0.7

WV-2581
0.0
0.0
0.6
0.6
0.4
0.5
0.8
0.7

3 uM 5% serum

3 uM 5% serum

20 mg/ml BSA

4 mg/ml BSA

Mock
0.0
0.0

0.0
0.0

WV-942
0.1
0.1
0.1
0.1
0.1
0.1
0.4
0.3

WV-2578
0.2
0.2
0.2
0.3
0.2
0.1
0.1

WV-2579
0.4
0.4
0.2
0.2
0.1
0.1
0.2
0.2

WV-2580
0.2
0.2
0.2
0.3
0.0
0.0
0.3
0.3

WV-2581
0.0
0.0
0.3
0.3
0.1
0.1
0.1
0.1

Oligonucleotides were tested in vitro at 10 and 3 □M. In this table, in some cases, serum and/or BSA were added to test the effect on exon skipping. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

TABLE 10I

Example data of certain oligonucleotides.

10 uM
3 uM
1 uM

Mock
0.0
0.1
0.0

WV-2531
21.7
8.7
3.2

WV-3152
26.1
15.3
5.7

WV-2745
24.0
10.7
4.8

WV-3463
6.6
3.0
0.8

WV-3464
16.1
6.2
2.4

WV-3465
16.4
6.0
1.8

WV-3466
13.0
5.7
2.0

WV-3467
12.6
5.8
2.6

WV-3469
14.2
6.0
1.5

WV-3470
24.9
11.9
6.4

WV-3471
4.9
1.6
1.0

WV-3472
20.1
12.4
7.2

WV-3473
24.9
11.4
7.6

WV-942
3.3
2.1
0.7

Oligonucleotides were tested in vitro at 10.3 and 1 M. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency, results from replicate experiments are shown.

TABLE 10J

Example data of certain oligonucleotides.

10 uM

3 uM

1 uM

WV-2531
32.9
32.0
16.9
16.7
6.2
6.2

WV-3360
27.2
26.5
13.4
14.2
6.0
5.9

WV-3361
28.9
28.0
16.7
16.1
6.3
6.0

WV-3362
34.3
32.9
16.2
15.5
6.1
5.8

WV-3363
33.2
33.6
16.4
16.0
6.7
6.4

WV-3364
47.9
47.6
14.2
14.0
6.4
6.5

WV-3365
25.6
24.2
14.7
14.2
6.9
6.4

WV-3366
34.6
34.0
21.1
19.8
8.0
7.4

WV-942
0.6
0.6
0.3
0.3
0.1
0.1

Mock
0.0
0.0
0.1
0.1
0.1
0.0

TABLE 10K

Example data of certain oligonucleotides.

Activity relative to WV-942

WV-942
1.1
0.9

Mock
0.1
0.0

WV-2526
18.4
15.3

WV-2527
17.0
16.3

WV-2528
34.6
27.2

WV-2529
3.7
2.8

WV-2530
17.0
16.9

WV-2533
4.1
3.6

WV-2534
2.0
1.2

WV-2535
0.4
0.2

WV-2536
0.2
0.1

WV-2537
1.1
1.0

Olignucleotides were tested in vitro at 10 μM. Is table, numbers represent skipping efficiency relative to WV-942 (ave): results from replicate experiments are shown.

TABLE 10L

Example data of certain oligonucleotides.

Activity relative to WV-942 at 10 uM

WV-942
0.8
1.8
1.2

WV-1709
7.1
10.7
6.5

WV-1710
3.4
5.1
3.9

WV-1711
3.6
4.9
3.1

WV-1712
2.1
3.7
2.6

WV-1713
1.8
2.9
1.9

WV-1714
6.5
8.8
8.5

WV-1715
1.8
3.1
2.7

WV-1716
1.7
2.9
2.0

WV-2444
18.5
22.2
23.8

WV-2445
14.2
17.2
15.6

WV-2528
27.0
28.8
32.7

WV-2529
2.7
4.8
5.1

WV-2530
15.5
17.6
21.2

Activity relative to WV-942 at 3 uM

WV-942
0.7
1.7
0.6

WV-1709
10.9
19.5
12.2

WV-1710
3.6
8.3
4.3

WV-1711
3.6
8.1
4.6

WV-1712
3.0
6.7
5.8

WV-1713
2.0
5.3
0.9

WV-1714
7.5
13.8
7.8

WV-1715
2.6
5.8
3.6

WV-1716
3.2
6.1
3.1

WV-2444
30.3
41.9
39.7

WV-2445
23.4
32.3
30.2

WV-2528
56.3
66.3
74.4

WV-2529
7.5
15.0
10.0

WV-2530
25.2
38.4
37.8

Oligonucleotides were tested in vitro at 10 and 3 μM. In this table, numbers represent skipping efficiency relative to WV-942 (ave): results from replicate experiments are shown.

In some embodiments, an oligonucleotide, e.g., a DMD)oligonucleotide, can be tested in vivo for capability to skip an exon in a tissue in alive animal; in some embodiments, a tissue is gastrocnemius, triceps, quadriceps, diaphragm, and/or heart. In some embodiments, alive animal is a mouse, rat, monkey, dog, or non-human primate. In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide, is capable of mediating skipping e.g., of exon 23, 45, 51, 53, or any other DMD exon. Various DMD oligonucleotides were shown to mediate skipping of DMD exon 51 in a tissue in anon-human primate (NHP), wherein the tissue was gastrocnemius, triceps, quadriceps, diaphragm, or heart.

In some embodiments, the present disclosure pertains to methods of administering oligonucleotides. e.g., DMD oligonucleotides, wherein the timeline of pre-differentiation (of myoblast cells to myotubules) and treatment with the oligonucleotide are suitably altered. In some embodiments, in a test in vitro, an oligonucleotide, e.g., a DMD oligonucleotide to exon 51, was tested with treatment of day or 4 day.

TABLE 11A

Example data of certain oligonucleotides.

Oligonucleotide
Group A
Group B
Group C

PMO
1.3
0.6
3.3

WV-3473
29.3
23.1
81.6

Numbers represent skipping efficiency wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency. PMO is a Morpholino having the sequence of CTCCAACATCAAGGAAGATGGCGTTTCTAG.

Group A
Group B
Group C

Pre-differentiation
1 day
2 day
0 day

ASO treatment
1 day
1 day
4 days

Wash-out

2 days

2 days
—

Example 19 describes various timelines for experiments suitable for testing oligonucleotides, e.g., DMD oligonucleotides e.g. in patient-derived myoblasts in vitro.

TABLE 11B

Example data of certain oligonucleotides.

Conc.

(uM)
WV-942
PMO

0.3
0.2
0.0
0.1
0.1

0.5
0.4
0.1
0.0

1
0.6
0.1
0.2
0.1

0.1
0.1
0.1
0.3

3
0.1
0.1
0.1
0.2
0.2
0.5
0.3
0.7
0.2

10
0.5
0.3
0.1
0.8
0.7
1.3
0.8
1.6
0.4

30
0.0
1.0
0.5
2.0
3.4
5.5
2.3
0.9
1.7

Conc.

(uM)
WV-3473
WV-3545

0.3
5.1
4.7
1.9
8.7
1.4
3.9
6.4
3.0
4.2
0.9
1.1
2.9

1
15.6
8.5
13.8
5.7
6.2
12.9
13.9
11.7
2.8
5.6
5.2
12.0

3
24.4
25.1
7.7
14.7
18.5
27.3
22.6
21.3
16.9
16.9

23.5

10
36.8
38.1
17.3
31.9
33.8
46.9
49.0
51.7
42.9
34.1
31.0
42.1

30
67.7

49.0
47.6
51.6
69.4
91.2
88.9
89.9
83.7
79.8
84.7

Conc.

(uM)
WV-3546

0.3
6.0
0.7
1.1
0.7
1.6
7.1

1
8.2
12.2
14.2
4.7
5.4
11.1

3
31.5

15.9
29.6

10
62.1
59.1
74.0
49.9
43.6
65.1

30
98.9
98.8
97.4
97.4
95.6
98.1

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency. PMO is a control oligonucleotide which is a Morpholino corresponding to Eteplirsen. WV-942 is an oligonucleotide corresponding to Drisapersen. Oligonucleotides were delivered gymnotically.

TABLE 11C

Example data of certain oligonucleotides.

Conc.

(uM)
WV-942
PMO
WV-3473

0.3
0.2
0.0
0.1
0.4
0.1
0.0
5.1
4.7
1.9

1
0.6
0.1
0.2
0.1
0.1
0.3
15.6
8.5
13.8

3
0.1
0.1
0.1
0.3
0.7
0.2
24.4
25.1
7.7

10
0.5
0.3
0.1
0.8
1.6
0.4
36.8
38.1
17.3

30
0.0
1.0
0.5
2.3
0.9
1.7
67.7

49.0

Conc.

(uM)
WV-3545
WV-3546
WV-3543

0.3
6.4
3.0
4.2
6.0
0.7
1.1
5.1
2.1
4.6

1
13.9
11.7
2.8
8.2
12.2
14.2
8.2
2.8
9.2

3
22.6
21.3
16.9
31.5

17.9
21.6
18.8

10
49.0
51.7
42.9
62.1
59.1
74.0
26.7
28.9
31.2

30
91.2
88.9
89.9
98.9
98.8
97.4
83.2
82.5
75.5

Conc.

(uM)
WV-3544
WV-3554
WV-4107

0.3
5.6
3.0
3.1
2.2
2.0
4.0
1.1
1.0
0.8

1
12.4
9.8
12.0
12.6
4.5
8.4
3.9
2.3
4.0

3
22.7
23.9
15.7
18.6
15.7
18.3
15.7
14.1
13.5

10
37.8
32.0
35.1
42.3
36.8
33.0
70.0
53.6
64.3

30
80.4
81.3
79.1
86.4
91.1
84.3
93.6
92.0
93.0

In some embodiments, an oligonucleotide comprises a derivative of U. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD comprises a derivative of U. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD and comprises a derivative of U and at least one chirally controlled internucleotidic linkage. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD and comprises a derivative of U and at least one chirally controlled phosphorothioate internucleotidic linkage. In some embodiments, a derivative of U is BrU or Acet5

embedded image

In some embodiments, an oligonucleotide comprises BrU. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD comprises BrU. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD and comprises BrU and at least one chirally controlled internucleotidic linkage. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD and comprises BrU and at least one chirally controlled phosphorothioate internucleotidic linkage.

In some embodiments, an oligonucleotide comprises Acct5U. In some embodiments, Acet5U is also designated AcetU or acetU. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD comprises Acet5U. In some embodiments, in an oligonucleotide, e.g., DMD oligonucleotide, any U or T can be optionally replaced by Acet5U (e.g., in a first wing, a core, a second wing, or anywhere in the oligonucleotide). In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD comprises an Acet5mU nucleoside unit, wherein the base is Acet5U and the sugar is the common natural RNA sugar wherein the 2′-OH is replaced with 2′-OMe. In some embodiments, an oligonucleotide comprises an Acet5fU nucleoside unit, wherein the base is Acet5U and the sugar is the common natural RNA sugar wherein the 2′-OH is replaced with 2′-F. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD and comprises Acet5U and at least one chirally controlled internucleotidic linkage. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD and comprises Acet5U and at least one chirally controlled phosphorothioate internucleotidic linkage.

As shown in Table 11D, Table 11E, and Table A1, certain oligonucleotides, e.g., DMD oligonucleotides, were designed and constructed comprising BrU or acet5U. In some oligonucleotides, the nucleoside at the 5′ end comprises BrU or acet5U. In some embodiments, oligonucleotides comprise a BrfU nucleoside unit, wherein the base is BrU and the sugar is the common natural RNA sugar wherein the 2′-OH is replaced with 2′-F. In some oligonucleotides, the oligonucleotide comprises a BrdU nucleoside unit, wherein the base is BrU and the sugar is 2-deoxyribose (common natural DNA sugar). In some embodiments, any U or T can be replaced by BrU (e.g., in a first wing, a core, a second wing, or anywhere within an oligonucleotide). In some embodiments, in an oligonucleotide, e.g., a DMD oligonucleotide, any number of U or T can be replaced by BrU and/or Acet5U.

In some embodiments, an oligonucleotide comprises an acet5fU nucleoside unit, wherein the base is acet5U and the sugar is the common natural RNA sugar wherein the 2′-OH is replaced with 2′-F.

Table 11D shows data of various DMD oligonucleotides which mediate skipping of exon 51, including oligonucleotide WV-7410, which comprises BrfU, and WV-7413, which comprises acet5fU. Percentage was measured using RT-qPCR. Gymnotic delivery of 10 μM and 3 μM oligonucleotides in Δ48-50 patient derived myoblasts (4 days post-differentiation). The experiment was done in technical replicates.

TABLE 11D

Example data of certain oligonucleotides.

WV-3152
WV-3516
WV-7410
WV-7413

10 μM
39
10
49
11

3 μM
20
6
34
6

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency. Approximate numbers are provided.

In some embodiments, the present disclosure provides oligonucleotides, e.g., various DMD oligonucleotides, that comprise BrdU at or near the center of the oligonucleotides (e.g., in a core region, middle region, etc.). In some embodiments, example such oligonucleotides include WV-2812, WV-2813, and WV-2814. Certain exon skipping data of these oligonucleotides were presented below.

TABLE 11E

Example data of certain oligonucleotides.

10 uM

3 uM

WV-1714
0.035
0.034
0.012
0.013

WV-2812
0.094
0.095
0.023
0.024

WV-942
0.004
0.004
0.001
0.001

WV-2814
0.004
0.005
0.002
0.002

WV-2813
0.041
0.042
0.017
0.017

Numbers represent skipping efficiency, wherein 1.000 would represent 100% skipping and 0.0 represents 0% efficiency. Approximate numbers are provided.

TABLE 11F

Example data of certain oligonucleotides.

10 uM
3 uM

WV-9738
44.7
44.0
46.1
45.4
26.6
25.9
25.6
24.4

WV-9739
51.8
49.9
53.2
50.9
32.3
35.4
31.0
33.2

WV-9740
49.9
48.8
47.8
46.1
32.5
30.3
29.0
29.6

WV-9741
36.1
37.8
35.0
35.6
23.5
22.3
21.4
24.6

WV-9742
53.4
54.8
59.1
56.8
41.7
40.4
37.6
40.3

WV-7410
64.8
63.9
65.4
67.0
45.1
43.5
43.9
40.6

WV-7410
66.0
67.2
64.7
64.5
44.9
40.3
33.7
31.7

WV-3152
47.0
45.7
47.1
45.0
28.3
30.2
25.3
22.6

WV-3516
12.5
12.5
9.7
10.4
5.0
4.9
5.2
4.6

MOCK
0.5
0.3
0.5
0.3
0.5
0.6
0.8
0.4

MOCK
0.6
0.4
0.5
0.5
0.6
0.6
0.3
0.4

MOCK
0.3
0.3
0.6
0.2
0.4
0.4
0.2
0.6

Additional DMD oligonucleotides for skipping Exon 51 were constructed. Various DMD oligonucleotides comprise BrU. In some cases, a BrU is attached to a sugar which is 2′-F modified (BrfU). D48-50 myoblasts were dosed at 10 uM and 3 uM in differentiation media for 4 days. Percentage of skipping is shown, wherein 100 would represent 100% skipping and 0 would represent 0% skipping.

TABLE 11G

Activity of certain oligonucleotides

10
3.3
1.1

10
3.3
1.1

WV-
20.8
9
4.1
WV-
36.9
10.4
4.7

3152
22
10
4.9
14522
27.4
10.4
4.2

17.3
9.3
3.2

21
12.6
5.6

21.3
7.2
4.4

26.5
10.4
5.7

WV-
27.4
13.2
12.7
WV-
27.2
8.1
6.2

15860
30.4
15.4
9
14523
28.3
8.5
4.9

33
14.2
6

18.4
9.1
3.6

33.4
16.9
5.9

18.7
9.6
4.4

WV-
26.6
9.2
5.6
Mock
0.21

15861
28.5
6.1
5.4

0.35

34.1
8.2
5.2

0.48

29.9
11.1
4

0.24

WV-
30.7

7.8

15862
33.3

7.2

21.9
15.1
6.8

26.4
13.2
7.2

Activity of various DMD exon 51 oligonucleotides was tested in vitro.

Numbers indicate amount of skipping DMD exon 23 (as a percentage of total mRNA, where 100 would represent 100% skipped).

Amounts tested were: 10, 3.3 and 1.1 uM.

TABLE 11H

Activity of certain oligonucleotides

10
3.3
1.1

10
3.3
1.1

uM
uM
uM

uM
uM
uM

Mock
0.2
0.3
0.2
WV-
37.6
22.6
9

0.3
0.2
0.3
17861
38.8
22.5
8.9

0.2
0
0.2

40.7
24.4
13.2

0.2
0.6
0.2

41.7
25.4
11.6

WV-
3.1
1.6
0.7
WV-
38.4
18.9
8.1

7336
8.9
1.8
0.1
17862
34.1
19.6
9

5.4
1.4
0.9

34.8
26
10

4.9
1.5
0.7

36.1
21.4
9.5

WV-
32.4
26.5
7.5
WV-
32.7
18.2
9.2

3152
27.2
22.2
8.4
17863
35.1
18.9
9.3

28
14.5
7.6

34.8
18.2
8.6

26.8
14.8
7.3

30.7
17
9

WV-
43.3
25.7
10.2
WV-
37.3
23.6
11.7

15860
37.9
23.8
9.6
17864
41.4
23.3
10.6

38.4
24.5
11.2

39.9
20.6
17.5

42.4
21.9
11

38.8
21.7
10.2

WV-
42.3
26.7
16.3
WV-
35.9
16.5
9.3

17859
41.3
26
16.8
17865
34
16.7
7.5

39.9
22.9
15.5

34.4
17.5
11.9

48.6
23.6
14.9

34.1
17.8
9.8

WV-
38.1
19.3
11.7
WV-
48.7
28.4
17.7

17860
35.3
19.2
12
17866
43.3
28.6
13.1

41
28.2
16.4

44.5
24.8
15.4

40.4
21.9
11.1

45.1
30.5
16.3

Oligonucleotides for skipping DMD exon 51 were tested in vitro.

Numbers indicate amount of skipping DMD exon 23 (as a percentage of total mRNA, where 100 would represent 100% skipped).

Concentrations of oligonucleotides used: 10, 3.3 and 1.1 uM.

TABLE 11I

Activity of certain oligonucleotides

10 uM
3.3 uM

Mock
0
0

0
0

0
0

0
0

WV-
15.9
7

20034
17.1
8.4

16.1
7.3

15.3
7.2

WV-
29.7
18.3

20037
27.2
17.5

26.6
19.4

29.2
18.4

WV-
9.6
4.9

20040
9.1
5.2

11.4
3.5

10.9
2.9

WV-
20.2
9.6

20043
20.4
9.8

18.9
9.8

21
10.4

WV-
28.5
14.7

20046
29.8
14.2

29.2
15.8

26.6
14.5

WV-
20.9
11.6

20049
18.6
12.2

18.4
11.7

WV-
28.8
18.8

20052
30.1
18.6

29.6
20.1

WV-
26.8
17

20055
25.3
16.6

24.1
17

WV-
14.6
4.8

20058
12
3.7

12.6
3.5

WV-
35.8
26.5

20061
39.3
24.2

39.9
22.8

WV-
26.5
17.6

20064
24.5
16.4

27.5
17.1

WV-
15.7
8.3

20067
16.8
9.3

17.3
8.6

16.3
8.7

WV-
41.3
26.4

20070
31.7
22.3

39.7
27.2

38.4
26.9

WV-
30.9
21.1

20073
26.9
17.9

31.1
20.2

30.7
22.2

WV-
23.2
16.8

20076
18.9
11.4

21.8
16.9

22.8
15.8

WV-
35.7
24.8

3152
33.5
24.9

32.1
25.3

WV-
41.9
27.5

15860
43.6
30.7

42.4
30

TABLE 11J

Activity of certain oligonucleotides

WV-3152
19
20
12
14

WV-15860
29
31
26
23

WV-20140
1
1
1
1

WV-20139
3
3
2
2

WV-20138

2
3

WV-20137
4
5

WV-20136

WV-20135
5
5
5
5

WV-20134
5
6
5
4

WV-20133
17
17
13
13

WV-20132
8
8
6
6

WV-20131
14
16
12
12

WV-20130
10
9
8
8

WV-20129
12
14
11
11

WV-20128
9
9
8
8

WV-20127

8
8

WV-20126
7
8
8
7

WV-20125
8
8
8
8

WV-20124
22
21
21
21

WV-20123
13
13
14
12

WV-20122
11
12
12
11

WV-20121
21
22
22
21

WV-20120
28
30
32
33

WV-20119
52
50

WV-20118
39
37
27
26

WV-20117
18
17
15
18

WV-20116
20
20
17
17

WV-20115
8
8
8
6

WV-20114
19
20
15
14

WV-20113
20
18
17
15

WV-20112
16
15
12
12

WV-20111
31
30
33
31

WV-20110
14
14
14
12

WV-20109
20
21
25
24

WV-20108
27
25
22
22

WV-20107
20
19
16
14

WV-20106
44
42
34
37

WV-20105
23
22
18
18

WV-20104
41
40
33
28

WV-20103
48
52
53
53

WV-20102
54
52
55
59

WV-20101
38
39
38
43

WV-20100
52
51
48
50

WV-20099
53
51
47
48

WV-20098
46
44
45
46

WV-20097
47
46
51
48

WV-20096
45
41
42
43

WV-20095
43
41
50
47

WV-20094
55
50
57
55

WV-20093
35
34
35
38

WV-20092
25
26
25
25

WV-20091
28
27
30
32

WV-20090
21
19
22
22

WV-20089
8
7
8
9

WV-20088
22
21
26
25

WV-20087
28
28
33
32

WV-20086
25
25
27
26

WV-20085
33
31
30
31

WV-20084
21
22
21
21

WV-20083
21
21
19
17

WV-20082
42
37
32
30

WV-20081
41
41
30
30

WV-20080
49
44
26
25

WV-20079
42
38
53
51

WV-20078
27
28
36
35

WV-20077
10
10
10
10

WV-20076
45
45
45
41

WV-20075
40
31
37
42

WV-20074
55
57
53
56

WV-20073
51
55
51
50

WV-20072
41
36
37
36

WV-20071
42
40
44
46

WV-20070
18
18
25
25

WV-20069
11
11
10
9

WV-20068
20
17
20
18

WV-20067
12
9
11
11

WV-20066
12
11
13
12

WV-20065
16
15
16
14

WV-20064
37
35
37
36

WV-20063
19

24
22

WV-20062
6
6
7
7

WV-20061
24
23
26
24

WV-20060
16
17
16
17

WV-20059
55
42
62
67

WV-20058
28
30
33
33

WV-20057
37
38
37
34

WV-20056
35
34
33
35

WV-20055

40
40

WV-20054
25
25
35
36

WV-20053
43
45
46
46

WV-20052
47
47
53
46

WV-20051
30
33
30
30

WV-20050
29
28
28
26

WV-20049
41
41
38
38

WV-20049
24
23
22
21

Oligonucleotides for skipping DMD exon 51 were tested in vitro.

Oligonucleotides were dosed 4d at 10 uM.

Numbers indicate amount of skipping DMD exon 51 (as a percentage of total mRNA, where 100 would represent 100% skipped).

Example Dystrophin Oligonucleotides and Compositions Which Target Exon 52

In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for targeting exon 52 and/or mediating skipping of exon 52 in human DMD. Non-limiting examples include oligonucleotides and compositions of Exon 52 oligos include: WV-13733, WV-13734, WV-13735, WV-13736, WV-13737, WV-13738, WV-13739, WV-13740, WV-13741, WV-13742, WV-13743, and WV-13744, WV-13782, and WV-13783, and other oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

TABLE 12A

Example data of certain oligonucleotides.

WV-13733
0.3
0.2

WV-13734
0.0
0.0

WV-13735
1.6
0.3

WV-13736
3.9
1.3

WV-13737
0.7
0.4

WV-13738
0.0
0.0

WV-13739
28.3
29.3

WV-13740
29.9
33.3

WV-13741
1.6
1.6

WV-13742
12.9
14.1

WV-13743
0.9
1.0

WV-13744
0.6
0.7

WV-13782
0.1
0.1

WV-13783
0.8
0.0

Mock
0.0
0.0

Mock
0.1
0.1

Skipping efficiency of various DMD olignucleotides, tested for skipping of DMD exon 52.

Example Dystrophin Oligonucleotides and Compositions for Exon Skipping of Exon 53

In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for mediating skipping of exon 53 in DMD (e.g., of mouse, human, etc.).

In some embodiments, an oligonucleotide, e.g., a human DMD exon 53 skipping oligonucleotide can be tested in a mouse which has been modified to comprise a DMD gene comprising the human exon 53 sequence.

In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide, is capable of mediating skipping of exon 53. Non-limiting examples of such oligonucleotides include: WV-10439, WV-10440, WV-10441, WV-10442, WV-10443, WV-10444, WV-10445, WV-10446, WV-10447, WV-10448, WV-10449, WV-10450, WV-10451, WV-10452, WV-10453, WV-10454, WV-10455, WV-10456, WV-10457, WV-10458, WV-10459, WV-10460, WV-10461, WV-10462, WV-10463, WV-10464, WV-10465, WV-10466, WV-10467, WV-10468, WV-10469, WV-10470, WV-10487, WV-10488, WV-10489, WV-10490, WV-10491, WV-10492, WV-10493, WV-10494, WV-10495, WV-10496, WV-10497, WV-10498, WV-10499, WV-10500, WV-10501, WV-10502, WV-10503, WV-10504, WV-10505, WV-10506, WV-10507, WV-10508, WV-10509, WV-10510, WV-10511, WV-10512, WV-10513, WV-10514, WV-10515, WV-10516, WV-10517, WV-10518, WV-10519, WV-10520, WV-10521, WV-10522, WV-10523, WV-10524, WV-10525, WV-10526, WV-10527, WV-10528, WV-10529, WV-10530, WV-10531, WV-10532, WV-10533, WV-10534, WV-10535, WV-10536, WV-10537, WV-10538, WV-10539, WV-10540, WV-10541, WV-10542, WV-10543, WV-10544, WV-10545, WV-10546, WV-10547, WV-10548, WV-10549, WV-10550, WV-10551, WV-10552, WV-10553, WV-10554, WV-10555, WV-10556, WV-10557, WV-10558, WV-10559, WV-10560, WV-10561, WV-10562, WV-10563, WV-10564, WV-10565, WV-10566, WV-10567, WV-10568, WV-10569, WV-10570, WV-10571, WV-10572, WV-10573, WV-10574, WV-10575, WV-10576, WV-10577, WV-10578, WV-10579, WV-10580, WV-10581, WV-10582, WV-10583, WV-10584, WV-10585, WV-10586, WV-10587, WV-10588, WV-10589, WV-10590, WV-10591, WV-10592, WV-10593, WV-10594, WV-10595, WV-10596, WV-10597, WV-10598, WV-10599, WV-10600, WV-10601, WV-10602, WV-10603, WV-10604, WV-10605, WV-10606, WV-10607, WV-10608, WV-10609, WV-10610, WV-10611, WV-10612, WV-10613, WV-10614, WV-10615, WV-10616, WV-10617, WV-10618, WV-10619, WV-10620, WV-10621, WV-10622, WV-10623, WV-10624, WV-10625, WV-10626, WV-10627, WV-10628, WV-10629, WV-10630, WV-10670, WV-10671, WV-10672, WV-11340, WV-11341, WV-11342, WV-11544, WV-11545, WV-11546, WV-11547, WV-13835, WV-13864, WV-14344, WV-4698, WV-4699, WV-4700, WV-4701, WV-4702, WV-4703, WV-4704, WV-4705, WV-4706, WV-4707, WV-4708, WV-4709, WV-4710, WV-4711, WV-4712, WV-4713, WV-4714, WV-4715, WV-4716, WV-4717, WV-4718, WV-4719, WV-4720, WV-4721, WV-4722, WV-4723, WV-4724, WV-4725, WV-4726, WV-4727, WV-4728, WV-4729, WV-4730, WV-4731, WV-4732, WV-4733, WV-4734, WV-4735, WV-4736, WV-4737, WV-4738, WV-4739, WV-4740, WV-4741, WV-4742, WV-4743, WV-4744, WV-4745, WV-4746, WV-4747, WV-4748, WV-4749, WV-4750, WV-4751, WV-4752, WV-4753, WV-4754, WV-4755, WV-4756, WV-4757, WV-4758, WV-4759, WV-4760, WV-4761, WV-4762, WV-4763, WV-4764, WV-4765, WV-4766, WV-4767, WV-4768, WV-4769, WV-4770, WV-4771, WV-4772, WV-4773, WV-4774, WV-4775, WV-4776, WV-4777, WV-4778, WV-4779, WV-4780, WV-4781, WV-4782, WV-4783, WV-4784. WV-4785, WV-4786, WV-4787, WV-4788, WV-4789, WV-4790, WV-4791, WV-4792, WV-4793, WV-9067, WV-9068, WV-9069, WV-9070, WV-9071, WV-9072, WV-9073, WV-9074, WV-9075, WV-9076, WV-9077, WV-9078, WV-9079, WV-9080, WV-9081, WV-9082, WV-9083, WV-9084, WV-9085, WV-9086, WV-9087, WV-9088, WV-9089, WV-9090, WV-9091, WV-9092, WV-9093, WV-9094, WV-9095, WV-9096, WV-9097, WV-9098, WV-9099, WV-9100, WV-9101, WV-9102, WV-9103, WV-9104, WV-9105, WV-9106, WV-9107, WV-9108, WV-9109, WV-9110, WV-9111, WV-9112, WV-9113, WV-9114, WV-9115, WV-9116, WV-9117, WV-9118, WV-9119, WV-9120, WV-9121, WV-9122, WV-9123, WV-9124, WV-9125, WV-9126, WV-9127, WV-9128, WV-9129. WV-9130, WV-9131, WV-9132, WV-9133, WV-9134, WV-9135, WV-9136, WV-9137, WV-9138, WV-9139, WV-9140, WV-9141, WV-9142, WV-9143, WV-9144, WV-9145, WV-9146, WV-9147, WV-9148, WV-9149, WV-9150, WV-9151, WV-9152, WV-9153, WV-9154, WV-9155, WV-9156, WV-9157, WV-9158, WV-9159, WV-9160, WV-9161, WV-9162, WV-9422, WV-9423, WV-9424, WV-9425, WV-9426, WV-9427, WV-9428, WV-9429, WV-9511, WV-9512, WV-9513, WV-9514, WV-9515, WV-9516, WV-9517, WV-9518, WV-9519, WV-9520. WV-9521, WV-9522, WV-9523, WV-9524, WV-9525, WV-9534, WV-9535, WV-9536, WV-9537, WV-9538, WV-9539, WV-9680, WV-9681, WV-9682, WV-9683, WV-9684, WV-9685, WV-9686, WV-9687, WV-9688, WV-9689, WV-9690, WV-9691, WV-9699, WV-9700, WV-9701, WV-9702, WV-9703, WV-9704, WV-9709, WV-9710, WV-9711, WV-9712, WV-9713, WV-9714, WV-9715, WV-9743, WV-9744, WV-9745, WV-9746, WV-9747, WV-9748, WV-9749, WV-9750, WV-9751, WV-9752, WV-9753, WV-9754, WV-9755, WV-9756, WV-9757, WV-9758, WV-9759, WV-9760, WV-9761, WV-9897, WV-9898, WV-9899, WV-9900, WV-9901, WV-9902, WV-9903, WV-9904, WV-9905, WV-9906, WV-9907, WV-9908, WV-9909, WV-9910, WV-9911, WV-9912, WV-9913. WV-9914. WV-7436, WV-7437, WV-7438, WV-7439, WV-7440, WV-7441, WV-7442, WV-7443, WV-7444, WV-7445, WV-7446, WV-7447, WV-7448, WV-7449, WV-7450, WV-7451, WV-7452, WV-7453, WV-7454, WV-7455, and WV-7456, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

Additional examples of such DMD oligonucleotides include: WV-9422, WV-9425, WV-9426, WV-9517, WV-9519, WV-9521, WV-9522, WV-9524, WV-9710, WV-9714, WV-9715, WV-9743, WV-9744, WV-9745, WV-9746, WV-9747, WV-9748, WV-9749, WV-9750, WV-9751, WV-9756. WV-9757, WV-9758, WV-9759, WV-9760, WV-9761, WV-9897, WV-9898, WV-9899, WV-9900, WV-9906, and WV-9912, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

Non-limiting examples of such DMD oligonucleotides also include: WV-12123, WV-12124, WV-12125, WV-12126, WV-12127 WV-12128, WV-12129, WV-12553, WV-12554, WV-12555, WV-12556, WV-12557, WV-12558, WV-12559, WV-12872, WV-12873, WV-12876, WV-12877, WV-12878, WV-12879, WV-12880, WV-12881, WV-12882 and WV-12883 and other DMD oligonuclotides having abase sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

Results of various experiments for skipping Dystrophin exon 53 are described in the present disclosure. For example, data from a sequence identification screen are shown below, in Table

TABLE 13A

Example data of certain oligonucleotides.

Oligonucleotide
Replicate 1
Replicate 2

WV-4698
1.9
2.1

WV-4699
2.0
2.2

WV-4700
2.8
3.0

WV-4701
3.7
2.9

WV-4702
2.9
2.7

WV-4703
1.8
2.4

WV-4704
3.2
3.4

WV-4705
3.7
4.3

WV-4706
2.6
2.6

WV-4707
3.2
3.6

WV-4708
4.8
6.0

WV-4709
6.6
5.2

WV-4710
3.9
4.6

WV-4711
5.4
6.7

WV-4712
5.3
6.4

WV-4713
5.8
8.0

WV-4714
2.9
3.6

WV-4715
3.3
4.3

WV-4716
3.8
4.3

WV-4717
6.8
7.0

WV-4718
4.3
5.0

WV-4719
5.5
6.0

WV-4720
7.7
8.6

WV-4721
2.7
3.8

WV-4722
3.8
4.6

WV-4723
3.4
5.6

WV-4724
3.5
4.7

WV-4725
4.9
6.3

WV-4726
4.2
4.4

WV-4727
2.7
4.9

WV-4728
2.6
5.6

WV-4729
3.9
4.1

WV-4730
2.4
3.3

WV-4731
1.8
2.5

WV-4732
1.8
2.3

WV-4733
2.3
2.1

WV-4734
2.0
2.0

WV-4735
2.5
2.7

WV-4736
2.7
3.0

WV-4737
3.2
3.1

WV-4738
3.1
3.5

WV-4739
2.6
2.4

WV-4740
4.4
3.6

WV-4741
3.7
4.1

WV-4742
4.5
4.9

WV-4743
5.0
5.2

WV-4744
3.6
4.7

WV-4745
4.1
0.0

WV-4746
2.9
2.0

WV-4747
2.5
3.5

WV-4748
2.1
1.7

WV-4749
2.4
2.4

WV-4750
2.3
2.9

WV-4751
1.9
2.5

WV-4752
2.2
1.6

WV-4753
1.6
2.0

WV-4754
1.7
2.0

WV-4755
1.7
1.9

WV-4756
1.7
1.5

WV-4757
1.6
1.9

WV-4758
1.6
2.0

WV-4759
1.6
1.6

WV-4760
1.8
1.8

WV-4761
1.9
1.6

WV-4762
1.2
1.3

WV-4763
0.9
2.0

WV-4764
3.0
2.7

WV-4765
3.4
3.2

WV-4766
2.5
2.3

WV-4767
2.5
2.7

WV-4768
2.3
2.7

WV-4769
2.4
2.4

WV-4770
2.8
2.8

WV-4771
2.3
2.9

WV-4772
4.0
2.5

WV-4773
3.2
1.8

WV-4774
3.0
2.3

WV-4775
4.4
3.3

WV-4776
3.1
3.8

WV-4777
4.5
2.1

WV-4778
0.0
2.0

WV-4779
2.8
3.4

WV-4780
3.2
3.5

WV-4781
2.9
3.2

WV-4782
1.8
2.9

WV-4783
2.1
2.6

WV-4784
2.4
2.4

WV-4785
3.4
3.6

WV-4786
1.8
1.6

WV-4787
2.9
2.7

WV-4788
2.8
3.1

WV-4789
4.3
4.0

WV-4790
3.9
2.6

WV-4791
2.2
2.2

WV-4792
2.5
3.2

WV-4793
2.4
2.6

Mock
1.3
1.6

Skipping efficiency of various DMD oligonucleotides, tested for skipping of DMD exon 53 in vitro in Delta 52 human myoblast cells. Oligonucleotides tested were 6-8-6 gapmers (2′-F-2-OMe-2′-F), wherein each internucleotidic linkage is a stereorandom phosphorothioate. Numbers represent skipping efficiency wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency, results from replicate experiments are shown.

A number of oligonucleotides were generated and tested for efficacy in skipping DMD Exon 53 in vitro in human patient-derived myoblast cells; certain results are shown below in Tables 13B to 21 (A and B). Oligonucleotides were used at concentrations of 3 and 10 uM, in two replicates (R1 and R2). Numbers indicate the percentage of skipping of DMD exon 53, wherein 0.0 would indicate no skipping, and 100.0 would indicate 1001% skipping. Several base sequences were tested in combination with a variety of chemical formats. For example, in some embodiments, abase sequence is GUACUUCAUCCCACUGAUUC, GUGUUCTTGTACTTCAUCCC, UUCUGAAGGTGTFCUUGUAC, or CUCCGTCTGAAGGUGUUC, wherein U is optionally substituted with T and vice versa. Various chemical formats were utilized, including, e.g. gapmers (for example, 6-8-6 wing-core-wing gapmers). In some embodiments, both wings are 2-F, while the core was all 2′-MOE, alternating 2′-MOE/2-OMe, alternating 2-OMe/2′-MOE, alternating 2-MOE/2′-F, alternating 2-F/2′-MOE, alternating 2′-Me/2′-F. and alternating 2-F/2′-Me, etc. In some embodiments, the first wing was 2′-MOE or 2′-M and the second wing was 2′-F (a type of asymmetrical gapmers). In some embodiments, each internucleotidic linkage is a stereorandom phosphorothioate. In some embodiments, some alternating phosphorothioate linkages are replaced by phosphodiester linkages. In some embodiments, 5′-methyl 2-MOE Cis used. Descriptions of certain oligonucleotides tested are provided in Table A1.

TABLE 13B

Example data of certain oligonucleotides.

Replicate 1

Replicate 2

Oligonucleotide
10 uM
3 uM
10 uM
3 uM

WV-9067
6.6

1.9
1.8

WV-9068
6.5

1.5
1.6

WV-9069
6.9
1.8
1.7
1.5

WV-9070
2.9
3.2
2.6
1.9

WV-9071
2.9
1.9
2.0
1.4

WV-9072
9.6
2.4
2.4
1.5

WV-9073
8.6
3.3
2.7
2.1

WV-9074
8.3
2.4
2.5
1.9

WV-9075
7.0
2.1
2.1
2.0

WV-9076
9.6
3.0
3.1
2.0

WV-9077
6.3
1.7
2.0
1.5

WV-9078
6.1
2.3
2.2
1.9

WV-9079
10.0
3.9
3.6
2.3

WV-9080
7.6
3.1
2.8
2.6

WV-9081
5.7
2.2
1.9
1.6

WV-9082
11.2
6.1
6.4
3.2

WV-9083
6.0
1.9
2.1
1.6

WV-9084
6.6
2.4
2.9
2.1

WV-9085
0.0
7.5
7.6
3.4

WV-9086
7.5
3.4
3.1
2.0

WV-9087
7.1
2.4
2.1
1.7

WV-9088
9.0
3.0
2.6
1.6

WV-9089
8.2
2.5
2.3
1.9

WV-9090
0.0
2.3
2.2
1.6

WV-9091
9.9
4.7
3.7
3.2

WV-9092
9.0
3.4
3.4
2.0

WV-9093
8.7
2.9
3.2
2.0

WV-9094
11.9
6.0
5.2
3.1

WV-9095
7.5
3.4
2.6
2.5

WV-9096
10.1
4.0
4.0
2.9

WV-9097
10.7
5.7
4.5
2.8

WV-9098
8.5
3.6
2.9
2.3

WV-9099
8.1
2.9
2.4
2.4

WV-9100
12.7
6.0
4.7
2.9

WV-9101
7.6
2.9
3.1
2.0

WV-9102
9.9
4.0
3.6
2.5

WV-9103
12.6
6.9
6.1
3.0

WV-9104
11.3
3.7
4.3
2.1

WV-9105
6.5
2.9
2.3
2.4

WV-9106
15.1
7.7
5.5
4.3

WV-9107
7.8
2.5
2.2
2.6

WV-9108
11.3
3.3
3.5
2.2

WV-9109
16.1
10.6
8.9
4.1

WV-9110
8.8
3.5
3.4
1.7

WV-9111
7.3
3.4
2.5
1.7

WV-9112
11.5
4.6
3.4
2.2

WV-9113
10.6
4.2
3.1
2.3

WV-9114
10.8
4.9
4.1
2.6

WV-9115
8.4
0.0
2.5
2.1

WV-9116
7.5
0.0
1.6
1.8

WV-9117
6.8
0.0
2.0
1.5

WV-9118
9.3
0.0
2.7
2.1

WV-9119
7.2
0.6
2.0
2.0

WV-9120
8.5
6.1
2.5
2.0

WV-9121
11.8
5.7
3.9
2.5

WV-9122
8.6
4.0
2.4
2.4

WV-9123
10.7
5.2
2.0
2.0

WV-9124
11.0
5.3
3.6
3.2

WV-9125
8.7
3.5
2.3
2.2

WV-9126
10.5
3.4
3.4
2.4

WV-9127
8.5
3.4
2.7
2.5

WV-9128
8.2
2.9
2.0
2.2

WV-9129
7.5
2.6
1.6
1.7

WV-9130
12.6
0.0
5.4
2.7

WV-9131
7.6
2.3
2.2
1.8

WV-9132
8.4
0.7
3.4
2.3

WV-9133
16.2
7.0
6.9
3.2

WV-9134
8.5
3.9
3.0
1.9

WV-9135
12.5
2.8
2.9
1.7

WV-9136
8.7
4.1
3.1
2.2

WV-9137
7.5
2.5
1.7
1.6

WV-9138
7.2
2.7
2.1
1.7

WV-9139
9.3
5.3
5.1
2.8

WV-9140
8.0
3.1
2.5
2.1

WV-9141
7.7
3.3
2.9
1.8

WV-9142
11.9
6.4
6.0
3.2

WV-9143
7.0
3.2
3.9
1.8

WV-9144
9.8
4.0
3.6
2.7

WV-9145
13.0
6.6
5.3
2.6

WV-9146
7.9
3.7
3.4
1.9

WV-9147
8.2
3.9
3.1
2.0

WV-9148
15.0
8.8
6.4
3.3

WV-9149
6.9
2.9
2.3
3.1

WV-9150
10.8
6.9
5.6
1.9

WV-9151
12.9
7.2
5.1
2.7

WV-9152
8.4
3.4
2.6
1.5

WV-9153
7.2
3.9
2.9
1.7

WV-9154
21.5
14.1
12.4
4.3

WV-9155
6.9
3.3
2.5
1.6

WV-9156
11.0
6.4
4.9
2.4

WV-9157
16.7
10.5
9.7
3.9

WV-9158
7.7
3.7
2.3
1.7

WV-9159
7.7
3.1
3.3
1.5

WV-9160
8.0
3.1
2.8
1.8

WV-9161
8.4
4.5
3.2
2.2

WV-9162
8.9
4.5
4.7
2.2

Mock
2.4

Mock
2.1

WV-9746
2.5
2.5
4.6
3.4

WV-9747
3.0
3.1
5.5
4.8

WV-9748
4.9
2.5
4.3
4.0

WV-9749
2.9
2.7
4.5
4.1

WV-9750
3.2
2.5
4.4
3.8

WV-9751
3.5
2.7
4.7
4.8

WV-9758
1.7
1.9
2.1
3.5

WV-9759
2.6
3.6
2.8
6.1

WV-9760
3.1
3.9
3.4
4.8

WV-9761
3.0
4.8
4.6
7.2

WV-9756
3.9
4.4
5.3
8.4

WV-9757
3.7
4.3
6.8
8.1

WV-9517
3.3
2.7
7.1
5.3

WV-9519
2.4
2.1
5.1
4.6

WV-9521
2.4
2.5
6.3
4.9

WV-9522
2.6
2.3
5.8
4.3

WV-9715
4.6
5.7
10.5
4.2

WV-9714
4.5
3.4
9.0
8.5

WV-9422
2.1
2.0
6.2
4.3

WV-9743
4.1
2.4
7.3
6.2

WV-9744
3.4
1.9
4.4
5.1

WV-9745
2.7
2.4
5.6
6.2

Mock
2.4
1.8
1.7
2.5

Efficacy of DMD Exon 53 skipping of various DMD oligonucleotides in vitro. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency. Results from replicate experiments are shown.

TABLE 14

Example data of certain oligonucleotides.

3 uM-R1
3 uM-R2
10 uM-R1
10 uM-R2

WV-9746
2.5
2.5
4.6
3.4

WV-9747
3.0
3.1
5.5
4.8

WV-9748
4.9
2.5
4.3
4.0

WV-9749
2.9
2.7
4.5
4.1

WV-9750
3.2
2.5
4.4
3.8

WV-9751
3.5
2.7
4.7
4.8

WV-9758
1.7
1.9
2.1
3.5

WV-9759
2.6
3.6
2.8
6.1

WV-9760
3.1
3.9
3.4
4.8

WV-9761
3.0
4.8
4.6
7.2

WV-9756
3.9
4.4
5.3
8.4

WV-9757
3.7
4.3
6.8
8.1

WV-9517
3.3
2.7
7.1
5.3

WV-9519
2.4
2.1
5.1
4.6

WV-9521
2.4
2.5
6.3
4.9

WV-9522
2.6
2.3
5.8
4.3

WV-9715
4.6
5.7
10.5
4.2

WV-9714
4.5
3.4
9.0
8.5

WV-9422
2.1
2.0
6.2
4.3

WV-9743
4.1
2.4
7.3
6.2

WV-9744
3.4
1.9
4.4
5.1

WV-9745
2.7
2.4
5.6
6.2

Mock
2.4
1.8
1.7
2.5

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments (RI and 1R2) are shown.

TABLE 15

Example data of certain oligonucleotides.

10 uM
3 uM

WV-9897
7.4
4.8

WV-9898
11.8
4.6

WV-9899
10.1
4.1

WV-9900
10.3
4.7

WV-9901
5.7
2.5

WV-9902
8.8
3.5

WV-9903
7.3
3.4

WV-9904
6.9
3.0

WV-9905
6.7
3.1

WV-9906
12.1
5.0

WV-9907
11.1
3.8

WV-9908
12.6
5.1

WV-9909
11.3
3.9

WV-9910
9.8
4.3

WV-9911
3.5
4.0

WV-9912
11.3
4.7

WV-9913
10.3
3.9

WV-9914
9.4
2.8

WV-9747
7.6
3.4

WV-9749
6.4
3.6

WV-9750
6.0
3.5

WV-9758
3.5
2.5

WV-9517
9.6
4.1

Mock
2.5
2.6

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency.

Additional oligonucleotides were generated and tested for skipping DMD exon 53 in vitro in cells. Certain data are shown below in Table 16. Oligonucleotides were used at concentrations of 3 and 10 uM, in two replicates. Numbers indicate the percentage of skipping of DMD exon 53. As shown, oligonucleotides can have different base sequences in combination with a variety of chemical formats. In some embodiments, oligonucleotides tested were 20-mers, each having a gapmer format of wing-core-wing, wherein each wing was 2′-F, and the core was 2′-OMe or a mixture of 2′-OMe and 2′-F. In some embodiments, each internucleotidic linkage was a chirally controlled phosphorothioate internucleotidic linkage in Sp configuration. In some embodiments, oligonucleotides comprise one or more natural phosphate linkages. In some embodiments, oligonucleotides of the present disclosure comprise one or more 5′-methyl 2′-F C (5MSfC,

embedded image

nucleoside is

embedded image

wherein BA is nucleobase C, R^2sis —F).

TABLE 16

Example data of certain oligonucleotides.

Group A (3 uM)

Group B (10 uM)

WV-9746
8.0
7.5
13.7
7.5

WV-9747
10.2
9.3
17.4
9.3

WV-9748
8.8
8.2
14.1
8.2

WV-9749
9.9
8.7
15.8
8.7

WV-9750
10.0
9.3
17.3
9.3

WV-9751
9.3
8.4
14.5
8.4

WV-9758
6.9
6.1
8.8
6.1

WV-9759
7.5
7.7
11.3
7.7

WV-9760
8.1
7.3
10.2
7.3

WV-9761
7.3
8.2
12.7
8.2

WV-9756
10.9
10.3
20.2
10.3

WV-9757
22.7
10.1
32.1
10.1

WV-9517
10.3
9.2
20.1
9.2

WV-9519
8.8
8.1
16.2
8.1

WV-9521
9.2
8.0
16.0
8.0

WV-9522
9.5
8.8
17.7
8.8

WV-9715
14.3
12.3
26.9
12.3

WV-9714
13.2
11.3
23.7
11.3

WV-9422
8.3
7.3
16.6
7.3

WV-9743
9.8
7.8
20.1
7.8

WV-9744
7.6
6.7
12.9
6.7

WV-9745
9.6
7.4
17.0
7.4

Mock
4.7
4.9
5.2

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

A number of DMD oligonucleotides were also designed, constructed and tested for efficacy in skipping DMD Exon 53 in vitro in differentiated myoblast cells. Certain data are shown

below in Table 17. Oligonucleotides were delivered gymnotically at concentrations of 3 and 10 μM, in two biological replicates (R1 and R2). Numbers indicate the percentage of skipping of DMD exon 53, as determined by RT-qPCR.

TABLE 17

Example data of certain oligonucleotides.

3 uM-R1
3 uM-R2
10 uM-R1
10 uM-R2

WV-9422
2.1
2.0
6.2
4.3

WV-9743
4.1
2.4
7.3
6.2

WV-9744
3.4
1.9
4.4
5.1

WV-9745
2.7
2.4
5.6
6.2

Mock
2.4
1.8
1.7
2.5

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments (R1 and R2) are shown.

A number of oligonucleotides were designed, constructed and tested for efficacy in skipping DMD Exon 53 in vitro in Δ52 differentiated myoblast cells. Certain data were shown below in Table 18. In an example procedure, cells were pre-differentiated for 4 days and oligonucleotides were delivered gymnotically for 4 days. Differentiation medium was DMEM, 2% horse serum and 10 μg/ml insulin. In some embodiments, with certain oligonucleotides, without pre-differentiating these cells, skipping efficiency was relatively low. Oligonucleotides were delivered gymnotically at concentrations of 1, 3 and 10 μM, in biological replicates (R1 and R2). Numbers indicate the percentage of skipping of DMD exon 53, as determined by RT-qPCR. PMO53 is an oligonucleotide also designated as WV-13405, HumDMDEx53, or PMO (in DMD exon 53 experiments), or PMO SR which has abase sequence of GTTGCCTCCGGTTCTGAAGGTGTC and is fully PMO (Morpholino). “-” indicates that no data were available for that particular sample.

TABLE 18

Example data of certain oligonucleotides.

30 uM-
30 uM-
10 uM-
10 uM-
3 uM-
3 uM-
1 uM-
1 uM-

R1
R2
R1
R2
R1
R2
R1
R2

WV-9714
—
—
52.1
31.0
25.0
21.7
7.9
9.2

WV-9715
—
—
—
—
12.6
7.3
11.1
8.7

WV-9517
—
—
—
—
20.5
20.4
7.3
6.9

WV-9519
—
—
39.0
30.5
15.1
13.3
5.3
6.6

WV-9521
—
—
43.2
10.2
16.9
15.1
5.1
5.2

WV-9747
83.0
87.5
50.7
46.6
17.0
19.5
6.4
6.2

WV-9748
66.4
68.2
42.9
33.2
14.5
10.2
4.8
3.9

WV-9749
76.8
80.2
39.2
35.4
18.5
13.0
5.7
23.5

WV-9897
—
—
—
—
26.0
25.3
8.3
8.4

WV-9898
—
—
—
—
22.8
23.6
8.5
7.9

WV-9900
—
—
46.7
45.7
25.5
21.8
7.4
7.9

WV-9899
—
—
28.7
—
27.2
26.1
8.8
8.8

WV-9906
—
—
—
—
37.9
—
9.7
9.8

WV-9912
—
—
—
—
22.5
—
8.8
9.7

WV-9524
—
14.6
—
32.9
15.2
14.5
5.4
6.9

PMO53
112.8
105.4
53.7
49.3
20.4
19.9
6.9
10.4

Mock
2.2
1.7
2.2
1.5
1.6
1.8
2.0
2.0

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping relative to control and 0.0 would represent 0% efficiency; results from replicate experiments (R1 and R2) are shown.

A number of DMD oligonucleotides were designed, constructed and tested for efficacy in skipping DMD Exon 53 in vitro in Δ45-52 differentiated myoblast cell. Certain results, normalized to SFSR9 are shown below in Table 19. Oligonucleotides were delivered gymnotically at concentrations of 13 and 10 μM, in biological replicates (R1 and R2). Numbers indicate the percentage of skipping of DMD exon 53, as determined by RT-qPCR.

TABLE 19

Example data of certain oligonucleotides.

10 uM-
10 uM-
3 uM-
3 uM-
1 uM-
1 uM-

R1
R2
R1
R2
R1
R2

MOCK
0.8
0.8
0.8
0.8
0.9
0.9

MOCK
0.7
0.7
0.8
0.8
0.8
0.8

PMO
18.0
18.0
5.6
5.7
3.8
4.0

PMO
19.3
17.9
9.6
9.4
3.1
3.1

WV-9517
39.4
42.3
16.0
16.1
5.3
5.2

WV-9517
43.8
42.9
18.5
17.5
5.5
5.7

WV-9519
33.7
28.5
14.3
13.3
4.5
4.5

WV-9519
27.6
27.9
12.4
11.3
4.1
4.1

WV-9897
30.8
31.1
11.7
12.5
3.9
3.8

WV-9897
32.3
30.7
12.0
11.9
4.6
4.7

WV-9714
46.8
42.8
21.5
20.6
4.5
4.1

WV-9714
46.5
48.1
25.4
25.6
4.2
2.9

WV-9747
31.1
31.8
12.0
12.5
4.7
4.7

WV-9747
27.6
28.0
10.5
11.1
3.5
3.7

WV-9748
21.7
21.7
7.9
8.0
3.3
3.2

WV-9748
21.1
20.9
8.5
8.1
3.1
3.1

WV-9749
23.2
24.2
10.1
9.4
3.7
3.7

WV-9749
25.3
24.6
10.7
10.5
3.7
3.9

WV-9897
53.2
53.1
24.5
24.4
5.4
5.5

WV-9897
48.3
48.7
22.8
22.8
4.8
4.8

WV-9898
46.5
46.8
21.1
21.1
5.2
5.4

WV-9898
46.3
46.4
23.4
23.8
5.0
4.6

WV-9899
45.4
44.1
19.5
19.5
4.8
5.0

WV-9899
44.9
44.0
21.4
21.2
5.5
5.6

WV-9900
34.9
35.0
19.5
19.6
5.0
5.3

WV-9900
30.2
31.5
17.6
17.6
4.4
4.4

WV-9906
42.9
44.6
18.0
19.0
2.9
3.1

WV-9906
37.5
36.3
17.5
18.2
2.8
3.2

WV-9912
39.8
41.6
19.6
17.7
5.0
4.4

WV-9912
41.6
40.8
21.3
19.9
4.2
4.2

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments (R1 and R2) are shown.

Additional testing of oligonucleotides was performed, and the results were shown below in Tables 20 and 21.

TABLE 20

Example data of certain oligonucleotides.

10 uM
10 uM
3 uM
3 uM
1 uM
1 uM

WV-9517
34.6
35.6
17.0
19.4
6.7
7.8

WV-9897
43.8

26.8
27.3
9.7
9.8

WV-9898
42.7
30.3
22.8
26.7
8.5
9.3

WV-9899
45.0

16.4
26.8
10.0
8.6

WV-10670
32.4
32.9
15.2
18.2
7.2
8.0

WV-10671
28.7
30.9
14.7
16.1
6.7
8.0

WV-10672
25.6
28.1
11.8
12.2
5.0
5.0

PMO
40.8
36.0
19.1
18.6
10.7
11.7

Mock
1.1
1.9
1.8
1.9
1.7
2.5

Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency, results from replicate experiments are shown.

TABLE 21

Example data of certain oligonucleotides.

A.

WV-
WV-
WV-
WV-
WV-
WV-
WV-
WV-
WV-

9422
9425
9426
9517
9519
9521
9522
9524
9536

a) 8,
a) 8
a) 3
a) 10,
a) 9,
a) 8,
a) 8,
a) 9
a) 7

c) 4

c) 6
c) 4
c) 5
c) 5

WV-
WV-
WV-
WV-
WV-
WV-
WV-
WV-
WV-

9700
9701
9702
9703
9704
9709
9710
9711
9713

a) 4
a) 4
a) 6
a) 8
a) 7
a) 4
a) 6
a) 6
a) 4

WV-
WV-
WV-
WV-
WV-
WV-
WV-
WV-
WV-

9714
9715
9746
9747
9748
9749
9750
9751
9756

a) 13,
a) 15,
c) 4
c) 4
c) 4
c) 4
c) 4
c) 4
c) 7

c) 9
c) 9

WV-
WV-
WV-
WV-
WV-
WV-
WV-
WV-

9757
9758
9759
9760
9761
9743
9744
9745

c) 7
c) 2
c) 4
c) 4
c) 6
c) 6
c) 4
c) 6

B.

WV-
WV-
WV-
WV-
WV-

9422
9425
9426
9429
9517

b) 4
b) 2
b) 2
b) 1
b) 5

Oligonucleotides were tested in vitro in delta 52 cells. A, Exon skipping at 10 uM is shown. B, protein restoration. Different replicates or experiments are designated as a), b), and c).

Additional DMD oligonucleotides were tested for their ability to mediate skipping of a DMD exon as shown below. Full PMO (Morpholino)oligonucleotides have the following sequences:

PMO SR
WV-13405
GTTGCCTCCGGTTCTGAAGGTGTTC

PMO WV
WV-13406
CTCCGGTTCTGAAGGTGTTC

PMO
WV-13407
TGCCTCCGGTTCTGAAGGTGTTCTTGTA

WV-13407 is also designated PMO NS.

TABLE 21C

Example data of certain oligonucleotides.

10 uM
3 uM

Mock
0.1
0.2
0.1
0.1
0.1
0.1
0.1
0.1

PMO SR
1.8
1.6
1.1
0.9
0.5
0.5
0.5
0.4

PMO WV
0.8
1.0
1.0
1.1
0.4
0.4
0.5
0.3

PMO
2.3
2.5
1.8
1.8
1.0
0.9
0.6
0.6

WV-10454
5.5
6.1
4.5
3.9
1.3
1.3
0.9
0.7

WV-10455
10.5
13.8
7.3
7.8
2.1
2.8
2.0
2.5

WV-10456
7.2
7.4
5.6
5.0
1.4
1.5
1.7
1.3

WV-10457
9.8
14.2
8.4
9.0
3.8
2.9
3.2
2.9

WV-10458
6.6
5.4
5.6
5.2
1.2
1.1
1.1
1.2

WV-10459
2.4
2.8
2.7
2.5
1.0
1.0
0.5
0.5

WV-10460
7.9
6.0
7.6
7.5
1.9
1.8
1.4
1.4

WV-10461
14.9
11.3

5.7
6.0
2.4
3.7

WV-10462
1.6
2.4
3.4
3.1
0.8
0.8
0.7
0.9

WV-10463
2.6
3.2
2.9
2.7
0.7
0.7
0.7
0.7

WV-10464
1.2
1.1
0.2
0.1
0.4
0.3
0.2
0.3

WV-10465
2.3
1.8

0.6
0.7
0.7
0.7

WV-10466
8.6
9.1
3.9
2.6
1.8
1.6
1.9
1.6

WV-10467
3.2
0.8
1.4
1.1
4.1
4.3
3.3
2.9

WV-10468

2.1
2.0

WV-10469
3.2
3.1
4.8
4.2
0.6
0.6
1.0
0.0

WV-9699
4.6
3.2
2.8
2.4
0.8
0.9
0.7
0.5

WV-9898
19.4
19.0
17.6
18.2
5.4
6.2
5.9
5.4

Numbers represent skipping efficiency, wherein 100 would represent 100% skipping and 0 would represent 0% skipping. Replicate data is shown.

In some embodiments, oligonucleotides, e.g., DMD oligonucleotides, are designed to target Intronic Splice Enhancer elements, e.g., for DMD oligonucleotides for exon 53 skipping, elements within 4kb of Exon53. In some embodiments, provided oligonucleotides are 30-mers. Example data for certain such oligonucleotides are presented in Table 21D.

TABLE 21D

Example data of certain oligonucleotides.

WV-10490
1.6
1.6
1.8
1.9

WV-10491
1.6
1.7
1.7
1.5

WV-10492
1.4
1.5
1.6
1.4

WV-10493

0.9
0.6

WV-10494
1.4
1.5
1.3
1.6

WV-10495

WV-10496
1.8
1.5
1.8
1.7

WV-10497
1.6
1.6
1.5
1.7

WV-10498
0.7
0.7
2.0
1.8

WV-10499
1.5
1.4
1.7
1.6

WV-10500
0.8
1.3
0.9
0.6

WV-10501
1.2
1.7
1.3
1.4

WV-10502
1.4
1.4
1.5
1.4

WV-10503

1.5
1.0
1.7

WV-10504

1.6
1.8

WV-10505
1.5
1.2
1.9
1.5

WV-10506
0.8
0.8
1.4
1.3

WV-10507
1.4
1.1
0.9
1.4

WV-10508
1.5
1.4
1.8
1.7

WV-10509
1.2
1.5
1.4
1.6

WV-10510
1.3
1.7
1.0
1.6

WV-10511
0.5
0.9
0.8
1.2

WV-10512
1.3
1.5
1.7
1.7

WV-10513
1.5
1.6
1.6
1.7

WV-10514
1.1

1.7
1.8

WV-10515
2.0
1.9
1.9
1.9

WV-10516
8.3
8.7
9.1
8.0

WV-10517
0.5
0.5
1.7
1.5

WV-10518
1.7
1.5
1.5
1.7

WV-10519
1.8
1.6
1.8
1.8

WV-10520
2.1
1.8
1.8
1.7

WV-10521
3.3
3.1
2.6
3.4

WV-10522
1.9
2.0
1.7
2.1

WV-10523
2.3
2.1
1.9
1.9

WV-10524
1.8
1.9
2.1
2.0

WV-10525
2.0
2.1
1.1
1.6

WV-10526
1.7
1.9
1.8
1.7

WV-10527
1.1
1.3
1.4
1.5

WV-10528
1.6
1.6
1.7
1.4

WV-10529
1.6
1.1

WV-10530
0.9
1.7
1.7
1.6

WV-10531
1.2
1.5
1.0
1.3

WV-10532
1.4
1.6
1.6
1.5

WV-10533
1.4
0.5
1.5
1.5

WV-10534
1.3
1.4
1.7
1.6

WV-10535
0.9
0.6
1.7
1.6

WV-10536
1.5
1.0
1.4
1.3

WV-10537
1.4
1.6
1.6
1.4

WV-9517
44.5
42.5
41.6
43.2

WV-9699
13.0
12.7
9.8
9.3

Mock
1.6
1.7
1.4
1.3

Results: Gymnotic delivery of 1 μM Intron ASO's in Δ45-52 patient derived myoblasts (4 days post-differentiation). Done in biological replicates. Numbers represent percentage of exon skipping, as determined by RT-qPCR.

TABLE 21E

Example data of certain oligonucleotides.

Conc.
10
3.33
1.11
0.3704
0.1235
0

WV-13405
35.2
23.1
9.0
4.0
2.2
1.0

(PMO)
36.3
23.1
8.7
4.0
2.3
1.2

33.1
20.6
8.3
3.3
2.1
1.0

33.7
20.7
8.3
3.2
2.2
1.2

WV-9898
31.2
22.2
8.6
1.7
1.3
1.1

30.4
22.5
10.3
1.5
1.2
0.9

49.6
23.3
6.2
1.7
1.4
1.2

48.3
22.3
5.5
1.5
1.6
1.5

WV-12880
73.1
53.5
38.4
10.3
4.5
1.0

72.1
54.3
37.6
10.3
4.8
1.1

69.3
51.5
24.4
5.5
3.5
1.2

69.6
52.6
23.7
6.2
3.2
1.0

WV-9517
40.4
28.1
3.5
2.1
1.4
1.0

39.8
28.2
1.2
2.1
1.3
1.0

29.3
18.1
5.5
1.8
1.3
1.6

28.9
17.4
4.9
1.7
1.3
1.4

WV-9897
21.2
20.0
3.9
1.6
2.1
1.3

23.6
18.5
3.7
1.9
2.1
1.2

39.5
18.7
5.1
1.7
2.0
1.5

40.9
18.5
5.2
1.6
1.8
1.0

WV-12887
79.7
59.4
44.2
9.6
5.5
0.9

78.7
58.8
44.1
9.6
5.6
0.9

76.1
61.0
38.1
12.3
6.7
1.1

75.0
61.3
31.9
9.8
5.1
1.1

Δ45-52 DMD patient derived myoblasts, with 7d of pre-differentiation, were treated with oligonucleotides in muscle differentiation medium at indicated concentrations under free uptake condition before being collected and analyzed for RNA skipping efficiency (4d dosing) by qPCR. Relative (SRSF9 normalization) quantification. Oligonucleotides were tested at a concentration of 0 to 10 μM. Results of replicate experiments are shown. Some of the oligonucleotides tested comprise anon-negatively charged internucleotidic linkage (WV-12887 and WV-12880).

TABLE 21F

Example data of certain oligonucleotides.

10 uM
3.3 uM

Mock
0.3
0.3
0.3
0.4
0.3
0.3
0.3
0.3

WV-13405
4.3
4.5
4.2
4.7
1.2
1.1
1.8
1.9

(PMO)

WV-9517
15.0
14.2

5.6
5.8
8.7
9.3

WV-11340
32.4
33.7
35.9
36.9
15.4
13.0
15.9
15.0

WV-12873
38.7
37.5
39.6
39.2
13.6
11.7
17.0
14.5

WV-12872
44.9
41.9
44.1
46.5
15.7
17.5
15.7
19.5

WV-13408
49.0
48.7
50.2
50.3
21.6
22.0
23.0
24.5

WV-12553
18.3
20.7
18.7
24.1
7.4
7.6
9.7
8.4

WV-12557
40.0
39.2
33.8
35.9
15.3
15.5
23.6
23.9

WV-12554
38.8
39.0
43.5
44.9
15.1
14.0
20.5
20.3

WV-13409
34.6
38.4
39.1
40.3
14.7
12.9
18.9
16.5

WV-9898
24.1
22.0

7.9
7.7
9.9
8.5

WV-11342
30.4
34.5
31.3
31.9
14.3
14.4
14.1
13.3

WV-12559
44.3
41.8

16.6
16.5
17.4
19.4

WV-12556
42.5
43.0
39.7
43.3
16.1
17.1
18.8
17.1

WV-9897
20.8
17.9

6.0
5.4
6.8
4.8

WV-11341
36.6
39.4

17.8
16.8
18.2
19.3

WV-12558
41.5
39.4
36.0

18.2
15.1
18.5
16.7

WV-12555
44.3
43.6

20.5
19.0
20.2
22.1

WV12880
41.1
43.2
46.1
45.1
27.4
24.6
25.9
29.1

WV-12877
51.5
53.3

26.2
27.1
30.2
30.7

WV-12125
47.3
49.4
37.8
35.1
21.3
20.6
24.0
23.5

WV-12127
40.0
40.6
41.2
39.7
19.9
15.5
18.3
18.0

WV-12129
33.5
35.0
24.4
24.4
13.9
10.7
14.4
13.7

Δ45-52 DMD patient derived myoblasts were treated with oligos in muscle differentiation medium at indicated concentrations for 4d under free uptake conditions and analyzed for RNA skipping efficiency by qPCR.

TABLE 21G

Example data of certain oligonucleotides.

Oligo Conc

[uM]
10 uM
3.3 uM

Mock
0.6
0.6
0.6
0.8
0.7
0.6
1.0
0.8

WV-13405
6.9
7.4
10.1
10.9
2.2
1.9
4.1
4.4

(PMO)

WV-9517
24.2
22.0
11.5
33.7
9.3
9.8
19.8
20.6

WV-11340
50.8
54.1
61.6
63.9
30.1
22.0
33.2
30.6

WV-12872
70.6
66.4
71.0
74.6
24.7
29.2
27.9
38.9

WV-12873
60.8
59.5
62.9
62.8
20.4
15.3
33.5
24.5

WV-13408
73.5
72.3
75.8
75.6
35.6
35.7
42.2
46.3

WV-12553
32.7
39.1
38.0
51.3
13.7
14.6
22.7
18.9

WV-12557
65.2
64.4
76.7
80.4
26.3
27.1
45.3
45.6

WV-12554
61.0
61.5
69.5
71.7
27.0
22.9
38.5
37.6

WV-13409
57.2
63.6
66.2
69.3
23.6
18.9
34.4
28.4

WV-9898
45.1
40.3
16.3
14.4
13.2
12.1
20.8
16.1

WV-11342
49.9
58.1
57.9
60.0
27.4
27.8
30.3
27.4

WV-12559
72.4
68.4
50.8
56.1
33.3
32.8
35.5
42.5

WV-12556
70.5
71.0
68.4
73.5
31.0
33.5
42.0
37.0

WV-9897
42.0
34.9

41.2
10.2
8.0
17.9
9.4

WV-11341
61.6
67.2
74.1
74.4
37.0
33.8
40.8
42.9

WV-12558
71.6
68.0
66.3

35.6
27.1
40.5
35.5

WV-12555
70.2
68.9
56.0
61.7
35.2
32.4
40.1
45.0

WV12880
58.8
63.0
68.5
66.5
44.4
36.6
44.8
52.1

WV-12877
77.9
80.2
69.5
75.6
46.3
48.2
55.8
58.4

WV-12125
71.1
74.1
83.6
80.4
36.5
34.8
45.6
44.3

WV-12127
61.9
64.0
67.8
66.2
35.0
23.3
35.5
34.7

WV-12129
52.7
55.8
63.1
63.6
23.8
14.7
26.5
24.1

Δ45-52 DMD patient derived myoblasts, with 7 differentiation, were treated with oligos in muscle differentiation medium at indicated concentrations for 4d under free uptake conditions and analyzed for RNA skipping efficiency by qPCR.

TABLE 21H

Example data of certain oligonucleotides.

WV-
27.2
WV-
74.4
WV-
45.0

12553
30.1
12124
67.6
12127
42.3

32.1

67.7

43.2

WV-
63.6
WV-
65.8
WV-
50.2

11341
55.0
12125
74.2
12129
53.3

55.7

92.6

51.2

WV-
51.7
WV-
65.8
WV-
60.6

11342
54.0
12126
57.9
12882
66.9

50.8

55.8

68.6

WV-
81.1
WV-
65.2
WV-
76.0

12555

12880
63.9
12878
75.1

76.2

60.9

78.1

WV-
73.4
WV-
61.9
WV-
67.0

12556
75.1
12881
60.3
12876
62.0

66.9

57.7

66.4

WV-
59.9
WV-
59.5

12558
78.8
12123
55.1

66.0

49.9

WV-
68.3
WV-
78.9

12559
76.3
12877
78.0

73.3

83.1

WV-
59.9

9897
59.6

58.6

WV-
44.7

9898
39.1

46.3

Full length oligonucleotide stability at 5 day timepoint in Human Liver homogenate was tested. Numbers are replicates and represent percentage of full-length oligonucleotide remaining, wherein 100 would represent 100% oligonucleotide remaining (complete stability) and 0 would represent 0% oligonucleotide remaining (complete instability). Some nucleotides tested comprise anon-negatively charged internucleotidic linkage.

TABLE 21I

Example data of certain oligonucleotides.

Oligo Conc
WV-
WV-
WV-
WV-

[uM]
9517
13826
13827
13835
Mock

10 uM
45.7
46.5
23.1
40.5
1.2

46.3
45.8
22.9
58.8
1.1

49.3
46.8
26.8
54.5
1.3

48.5
50.3
28.1
55.2
1.2

3.3 uM
18.1
20.3
7.9
24.6
1

17
19.5
8.3
25.3
1.1

22.6
19.7
8.8
26.6
1.1

22.8
20.2
8.3
27.2
1.1

1.1 uM
6
7
2.9
7.9
1

6
6.2
2.7
7.4
1.2

6.9
7.3
0.7
9.6
0.9

6.6
6.8
0.9
9.1
0.7

WV-
WV-
WV-
WV-

9517
12880
13864
14344
MOCK

10 uM
36.1
60.2
66.8
47.9
0.9

38.3
62.0
67.0
46.8
1.0

44.5
60.9
68.7
56.8
1.2

43.9
59.2
69.6
56.3
1.0

3.3 uM
15.4
38.3
45.3
25.1
0.9

15.8
37.3
45.6
27.0
0.9

18.8
37.9
50.5
39.2
1.0

18.8
39.6
49.3
38.9
1.0

1.1 uM
4.7
15.8
21.5
12.2
0.6

4.9
14.4
22.6
12.4
0.9

6.4
18.5
24.9
17.2
1.1

6.2
16.2
13.2
17.1
0.9

0.3 uM
2.2
5.0
6.6
5.7
0.8

1.8
5.0
5.9
5.7
0.9

2.7
7.4
8.2
7.2
1.0

2.7
7.5
8.2
6.9
1.0

Numbers indicate amount of skipping relative to control.

TABLE 21I.1

Example data of certain oligonucleotides.

10 uM
3.3 uM
1.1 uM
0.3 uM
0.1 uM

Mock
1.1
1.2

0.8
1.0

1.0
1.1
2.0
0.9
1.0

1.1
0.7
1.1
1.0
1.1

1.2
0.7
1.1
0.9
1.0

Wv-
44.8
28.6
18.1
9.5
4.0

13405
44.8
23.4
17.4
8.7
4.0

(PMO)
51.2
26.5
11.4
5.1
3.7

50.8
25.6
11.2
5.5
3.6

WV-
35.9
18.3
6.5
2.2
1.9

9517
36.6
17.3
6.4
2.1
1.9

40.2
23.4
5.5
2.7
1.7

38.7
25.6
5.9
2.2
1.8

Wv-
57.3
36.3
16.4
4.8
7.5

12880
55.8
37.0
18.1
2.8
4.7

57.5
35.9
16.6
8.0
7.4

58.9
33.0
16.5
7.2
6.8

WV-
68.1
45.1
22.6
10.5
7.4

13864
68.0
44.5
23.0
12.0
5.6

67.5
43.1
24.3
8.4
6.0

64.8
44.5
19.9
3.3
6.1

WV-
40.2
21.5
6.3
2.8
2.0

13835
39.4
20.3
9.7
2.5
2.0

50.0
21.0
5.5
3.2
2.0

47.7
20.6
6.0
3.3
2.2

WV-
41.4
25.9
7.4
4.7
0.7

14791
40.3
24.8
5.8
4.0
0.5

40.1
24.9
9.1
4.3
3.9

41.3
27.2
8.9
4.6
3.5

WV-
50.1
28.6
13.6
6.4
3.8

14344
47.4
28.6
8.8
5.8
4.7

54.9
46.1
18.0
11.4
6.6

55.7
38.3
18.7
11.8
6.0

Skipping efficiency of various DMD oligonucleotides, tested for skipping of DMD exon 53. Numbers represent skipping of exon 53.

Δ45-52 patient myoblasts were differentiated for 7 days, then treated with oligonucleotide for 4d under gymnotic conditions in differentiation media. RNA was harvested by Trizol extraction and skipping analyzed by TaqMan.

TABLE 21I.2

Example data of certain oligonucleotides.

10 uM
3.3 uM
1.1 uM
0.3 uM
0.1 uM

Mock
0.7
0.6
0.6
0.6
0.7

0.7
0.7
0.6
0.6
0.7

0.6
0.6
0.6
0.7
0.7

0.5
0.5
0.7
0.6
0.7

Wv-
9.4
1.5
3.4
1.1
0.8

13405
9.3
1.4
3.1
1.1
0.8

(PMO)
6.6
2.8
1.5
0.9
0.8

6.3
2.6
1.5
1.0
0.8

WV-
29.3
8.4
2.6
1.0
0.7

9517
28.7
9.2
3.0
1.1
0.8

16.6
6.6
2.3
1.1
0.7

16.9
6.8
2.2
1.1
0.9

WV-
37.9
17.7
9.6
3.4
1.3

12880
38.8
19.9
9.1
3.3
1.4

31.4
16.1
7.9
3.3
1.6

31.6
16.8
8.0
3.0
1.5

WV-
55.9
28.6
11.7
4.3
2.0

13864
54.3
27.8
11.6
4.6
2.0

43.4
22.2
10.7
4.2
2.0

43.0
22.7
9.8
3.8
2.1

WV-
38.7
11.6
2.9
1.3
0.9

13835
37.2
11.0
2.9
1.3
0.8

42.3
13.1
3.5
1.2
0.9

41.5
10.0
3.1
1.3
0.9

WV-
26.3
12.1
5.2
1.9
1.3

14791
24.8
11.2
4.7
2.1
1.1

28.0
13.0
5.2
2.2
1.2

27.6
12.4
4.9
2.1
1.4

WV-
36.2
17.8
8.0
2.7
1.7

14344
37.4
17.0
7.1
2.7
1.8

37.4
22.3
9.8
3.7
1.7

36.6
22.6
9.9
3.7
1.5

Skipping efficiency of various DMD oligonucleotides, tested for skipping of DMD exon 53. Numbers represent skipping of exon 53.

Δ45-52 patient myoblasts were treated with oligonucleotide for 4d(4 days) under gymnotic conditions in differentiation media. RNA was harvested by Trizol extraction and ski ping analyzed by TaqMan.

Several oligonucleotides (including WV-9517, WV-13864, WV-13835, and WV-14791) were tested at various concentrations up to 30 uM for TLR9 activation in vitro in HEK-blue-TLR9 cells (16 hour gymnotic uptake). WV-13864 and WV-14791 comprise a chirally controlled non-negatively charged internucleotidic linkage in the Rp configuration. WV-9517, WV-13864, WV-13835, and WV-14791 did not exhibit significant TLR9 activation (less than 2-fold TLR9 induction; data not shown). WV-13864 and WV-14791 also exhibited negligible signal up to 30 uM in PBMC cytokine release assay compared to water (data not shown).

Example Dystrophin Oligonucleotides and Compositions which Target Exon 54

In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for targeting exon 54 and/or mediating skipping of exon 54 in human DMD. Non-limiting examples include oligonucleotides and compositions of Exon 54 oligos include: WV-13745, WV-13746, WV-13747, WV-13748, WV-13749, WV-13750, WV-13751, WV-13752, WV-13753, WV-13754, WV-13755, WV-13756, WV-13757, WV-13758, WV-13759, WV-13760, WV-13784, and WV-13785, and other oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

TABLE 21J

Example data of certain oligonucleotides.

WV-13745
0.2
0.3
0.2
0.0

WV-13746
0.6
0.6
0.4
0.4

WV-13747
0.4
0.5
0.4
0.4

WV-13748
1.1
1.2
0.7
0.9

WV-13749
2.5
2.1
1.7
1.8

WV-13750
1.9
2.1
1.4
1.4

WV-13751
4.3
5.1
4.4
5.7

WV-13752
0.0
0.0
3.1
3.9

WV-13753
0.0
0.0
0.0
0.0

WV-13754
6.0

1.4
1.7

WV-13755
1.1
1.2
0.5
0.5

WV-13756
4.7
5.0
2.3
2.4

WV-13757
1.9
2.1
1.1
1.4

WV-13758
2.0
2.2
0.9
1.2

WV-13759
0.7
0.7
0.4
0.2

WV-13760
0.7
0.6
0.3
0.5

WV-13784
0.0
0.0
0.0
0.0

WV-13785
0.0
0.0
0.0
0.0

Mock
0.0

0.0

Mock
0.0

0.0

Skipping efficiency of various DMD oligonucleotides, tested for skipping of DMD exon 54.

Example Dystrophin Oligonucleotides and Compositions which Target Exon 55

In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for targeting exon 55 and/or mediating skipping of exon 55 in human DMD. Non-limiting examples include oligonucleotides and compositions of Exon 55 oligos include: WV-13761, WV-13762, WV-13763, WV-13764, WV-13765, WV-13766, WV-13767, WV-13768, WV-13769, WV-13770, WV-13771, WV-13772, WV-13773, WV-13774, WV-13775, WV-13776, WV-13777, WV-13778, WV-13779, WV-13786, and WV-13787, and other oligonucleotides having a base sequence (naked sequence) which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

In some embodiments, two or more oligonucleotides capable of skipping or targeting exon 44, 46, 47, 51, 52, 53, 54 and/or 55 can be used in any combination to mediate multiple exon skipping.

TABLE 21K

Example data of certain oligonucleotides.

WV-13761
0.5
0.5
0.3
0.4

WV-13762
0.3
0.2
0.1
0.1

WV-13763
0.2
0.2
0.2
0.2

WV-13764
0.1
0.1
0.1
0.1

WV-13765
1.0
1.0
0.4
0.4

WV-13766
2.6
2.7
1.7
1.8

WV-13767
0.2
0.0
1.4
1.6

WV-13768
1.1
1.1
0.7
0.7

WV-13769
1.6
1.8
1.1
1.1

WV-13770
1.4
1.4
0.8
0.9

WV-13771
0.3
0.4
0.2
0.2

WV-13772
1.8
1.7
0.9
0.9

WV-13773
0.0
0.0
0.1
0.1

WV-13774
0.0
0.0
0.0
0.0

WV-13775
1.0
0.8
0.3
0.4

WV-13776
0.7
0.6
0.3
0.7

WV-13777
2.8
2.2
0.4
1.1

WV-13778
0.3
0.3
0.2
0.3

WV-13779
0.0
0.0
0.4
0.4

WV-13786
0.0
0.0
2.0
2.3

WV-13787
0.0
0.0
0.2
0.1

Mock
0.0
0.0
0.0
0.0

Mock
0.0
0.0
0.0
0.0

Skipping efficiency of various DMD oligonucleotides, tested for skipping of DMD exon 55.

Example Dystrophin Oligonucleotides and Compositions which Target Exon 57

In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for targeting exon 57 and/or mediating skipping of exon 57 in human DMD. Non-limiting examples include oligonucleotides and compositions of Exon 57 oligos include: WV-18853, WV-18854, WV-18855, WV-18856, WV-18857, WV-18858, WV-18859, WV-18860, WV-18861, WV-18862, WV-18863, WV-18864, WV-18865, WV-18866, WV-18867, WV-18868, WV-18869, WV-18870, WV-18871, WV-18872, WV-18873, WV-18874, WV-18875, WV-18876, WV-18877, WV-18878, WV-18879, WV-18880, WV-18881, WV-18882, WV-18883, WV-18884, WV-18885, WV-18886, WV-18887, WV-18888, WV-18889, WV-18890, WV-18891, WV-18892, WV-18893, WV-18894, WV-18895, WV-18896, WV-18897, WV-18898, WV-18899, WV-18900, WV-18901, WV-18902, WV-18903, WV-18904, and other oligonucleotides having a base sequence (naked sequence) which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

Example Dystrophin Oligonucleotides and Compositions for Exon Skipping of Multiple Exons (Multi-Exon Skipping)

In some embodiments, the present disclosure provides oligonucleotides, compositions, and methods for splicing modulation, including skipping of multiple exons. In some embodiments, a DMD oligonucleotide or composition thereof is capable of mediating skipping of multiple exons in the human or mouse Dystrophin gene.

In some embodiments, in a patient with muscular dystrophy, the symptoms of muscular dystrophy can at least be partially relieved and/or the disorder at least partially treated by administration of a DMD oligonucleotide capable of skipping one exon or multiple exons. Without wishing to be bound by any particular theory, the present disclosure notes that BMD patients with a deletion of exons 45 to 55 of DMD showed a milder or asymptomatic phenotype.

A non-limiting example of a scheme for multiple exon skipping is shown in FIG. 1. In this Figure, various numbers (43 to 57) indicate exons; and the shapes of the exons (e.g., <, > or |) indicate which reading frame is represented at the 5′ and 3′ end of each exon. Normally exon 44 is joined to exon 45. In a non-limiting example of multiple exon skipping, exons 45 to 55 are skipped, allowing exon 44 to join to exon 56. The 3′ end of exon 44 is represented by the same reading frame (<) as the 5′ end of exon 56: thus skipping exons 45 to 55 maintains or restores the correct reading frame. In some embodiments, skipping multiple exons restores the reading frame if one of the skipped exons comprises a mutation which alters the reading frame (in many cases, for example, producing a missense or prematurely truncated protein).

Among other things, the present disclosure notes that various exons represent at their 5′ and/or 3′ ends different reading frames; thus, some combinations of skipping adjacent reading frames but not other combinations are capable of maintaining or restoring the reading frame. In some embodiments, provided compositions and methods for multiple exon skipping skip, as non-limiting examples, exons 45-46, 4547, 4548, 4549, 45-51, 45-53, 45-55, 47-48, 47-49, 47-51, 47-53, 47-55, 48-49, 48-51, 48-53, 46-55, 50-51, 50-53, 50-55, 49-51, 49-53, 49-55, 52-53, 52-55, 44-45, 44-54, or 44-56, wherein in each case multiple exon skipping maintains or restores the correct reading frame. In some embodiments, skipping of non-overlapping sets of exons is capable of maintaining or restoring reading frame, e.g., skipping of exons 45-46 and exons 49-55; skipping of exons 45-47 and 49-55; skipping of exons 4549 and 52-55; etc.

Without wishing to be bound by any particular theory, the present disclosure notes that some DMD exons may be spliced transcriptionally, while others are spliced post-transcriptionally. For example, each of exons 45 to 55 are reportedly not simultaneously spliced, but rather first as three groups: exons 45 to 49, 50 to 52, and 53 to 55, the individual exons within each group being spliced transcriptionally. Reportedly, the remaining introns (between exons 44/45, 49/50, 52/53, and 55/56) are later spliced post-transcriptionally. Without wishing to be bound by any particular theory, the present disclosure notes that this lag in the timing of splicing may be exploited by oligonucleotides capable of increasing the splicing between exons whose adjacent introns are spliced post-transcriptionally, such as exon 44 and 56. It is reported that in nature, such multi-exon skipping joining exon 44 to exon 56 occurs at a low but detectable frequency (approximately 1/600). Without wishing to be bound by any particular theory, the present disclosure pertains in part to DMD oligonucleotides capable of skipping multiple exons at a therapeutically and clinically significant level.

In some embodiments, a composition capable of mediating multiple exon skipping comprises a DMD oligonucleotide. In some embodiments, a composition capable of mediating multiple exon skipping comprises a combination of (e.g., two or more different) DMD oligonucleotides. In some embodiments, a composition capable of mediating multiple exon skipping comprises a combination of (e.g., two or more different) DMD oligonucleotides, wherein at least one oligonucleotide recognizes a target associated with skipping the 5′ exon to be skipped, and at least one oligonucleotide recognizes a target associated with skipping the 3′ exon to be skipped. In some embodiments, a composition capable of mediating multiple exon skipping comprises a oligonucleotide capable of recognizes both (1) a target associated with skipping the 5′ exon to be skipped and (2) a target associated with skipping the 3′ exon to be skipped.

In some embodiments, an advantage of a composition capable of multiple exon skipping is that it is useful for treatment of dystrophy associated with a mutation in any individual exon included in the group of exons which is skipped. As a non-limiting example, a DMD oligonucleotide capable of mediating skipping of exon 48 is only capable of treating mutations within that exon (or, in some cases, an adjacent or nearby exon) but not mutations within other exons. However, a composition capable of mediating skipping of exons 45 to 55 is capable of treating mutations in any of exons 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55. Thus, both a patient with a mutation in exon 48 and a patient with a mutation in exon 54 can be treated with a composition capable of skipping exons 45 to 55. In some embodiments, a composition capable of mediating skipping of exons 45 to 55 is capable of treating up to about 63% of DMD patients.

In some embodiments, a composition comprises one or more DMD oligonucleotides, wherein the composition is capable of mediating skipping of multiple (two or more) DMD exons.

In some embodiments, a MESO (a composition comprising one or more oligonucleotides, which composition is capable of mediating multiple exon skipping) has an advantage over a DMD oligonucleotide capable of skipping only one exon. In some embodiments, a composition which is capable of mediating skipping of a single exon, is only useful for treating patients treatable by skipping that exon (e.g., patients having a genetic lesion in that exon). In some embodiments, a MESO is useful for treating patients treatable by skipping any of the exons which the MESO is able to skip, which is likely a larger percentage of the patient population. In some embodiments, double or multiple exon skipping can potentially be applicable to 90% of patients.

In addition, in some embodiments, because the 5′ and 3′ ends of an exon are sometimes not in the same frame, deletion of such an exon would cause a frameshift. Skipping of multiple exons, in various such cases, can restore the reading frame.

In some embodiments, multiple exon skipping is useful to treat DMD patients with deletion, duplication, and nonsense mutations.

In addition, in some embodiments, skipping of multiple exons can mimic the genetics of the milder Becker muscular dystrophy. In some embodiments, the more severe Duchenne muscular dystrophy, mediated by a genetic lesion in one exon, can be converted into a milder Becker muscular dystrophy, mediated by an in-frame deletion of multiple exons. It is reported that some BMD patients and an asymptomatic person have in-frame deletions of exons 48 to 51 or 45 to 51. Singh et al. 1997 Hum. Genet. 99: 206-208; Melacini et al. 1993 J. Am. Col., Cardiol. 22: 1927-1934; Melis et al. 1998 Eur. J. Paediatr. Neurol. 2: 255-261; and Aartsma-Rus et al. 2003 Hum. Mol. Genet. 8: 907-914.

In some embodiments, certain exons may be more challenging than others to skip. In some embodiments, the present disclosure provides technologies to skip such exons, e.g., through chemical modifications, linkage phosphorus stereochemistry, and combinations thereof. In some embodiments, the present disclosure encompasses the recognition that multiple exon skipping can be useful for skipping such challenging exons. In some embodiments, the present disclosure provides multiple exon skipping technologies for skipping such challenging exons.

In some embodiments, exon skipping, e.g., DMD exon skipping, can be used to treat patients, e.g., DMD patients, with circular or circularized RNA transcripts (e.g., those of DMD). Circular DMD transcripts are reported in, as a non-limiting example: Gualandi et al. 2003 J. Med. Gen. 40:e100.

In some embodiments, a composition capable of mediating multiple exon skipping (MESO) comprises one DMD oligonucleotide capable of mediating skipping of multiple exons. In some embodiments, a composition capable of mediating multiple exon skipping (MESO) comprises two DMD oligonucleotides which are together (e.g., when used in combination) capable of mediating skipping of multiple exons. In some embodiments, a composition capable of mediating multiple exon skipping (MESO) comprises a cocktail of (e.g., a mixture of three or more) DMD oligonucleotides which are together (e.g., when used in combination as a cocktail) capable of mediating skipping of multiple exons. Combinations or cocktails of oligonucleotides capable of mediating skipple of multiple exons have been reported by, for example, Yokota et al. 2009 Arch. Neurol. 66: 32: Yokota et al. 2012 Nucl. Acid Ther. 22: 306; Adkin et al. 2012 Neur. Dis. 22: 297-305; Echigoya et al. 2013 Nul. Acid. Ther.; and Echigoya et al. 2015 Molecular Therapy-Nucleic Acids 4: e225. Among other things, the present disclosure provides more effective combinations, through, e.g., selected sequences, chemical modifications, and/or linkage phosphorus chemistry, etc.

In some embodiments, the present disclosure provides oligonucleotides that, when combined with other oligonucleotides, can provide dramatically increased activities compared to either oligonucleotides individually prior to combination. For example, in some embodiments, the present disclosure provides DMD oligonucleotides which are individually incapable of mediating efficient skipping of a particular exon; when combined with other oligonucleotides, such oligonucleotides are capable of mediating skipping of multiple exons. Among other things, the present disclosure provides combination therapy, wherein two or more oligonucleotides are used together to provide desired and/or enhanced properties and/or activities. When used in combination therapy, the two or more agents, e.g., oligonucleotides, may be administered concurrently, or separately in suitable ways for them to achieve their combination effects. In some embodiments, two or more oligonucleotides in a combination are all (primarily) for skipping of the same exon, and their combination provides enhanced skipping of such exon, in some embodiments, significantly more than the addition of their separate effects. In some embodiments, two or more oligonucleotide in a combination are for skipping of difference exons, and their combination provides effective skipping, sometimes more than the oligonucleotides individually can achieve, of two or more exons. In some embodiments, the present disclosure provide combinations of oligonucleotides with synergies between two or more different oligonucleotides. In some embodiments, the present disclosure provides combinations of different oligonucleotides wherein one or more, or each oligonucleotide by itself is not effective for exon skipping. Certain combinations are described in Adams et al. 2007 BMC Mol. Biol. 8:57. Among other things, the present disclosure provides more effective combinations, through, e.g., designed control of one or more or all structural elements of oligonucleotides. In some embodiments, a provided combination provides exon skipping of DMD exon 45. In some embodiments, a provided combination provides exon skipping of another DMD exon, including those described herein or otherwise desirable for skipping (e.g., for prevention or treatment of one or more conditions, diseases or disorders etc.) as known in the art.

In some embodiments, cocktails, combinations and mixtures of oligonucleotides, e.g., for multiple exon skipping may have disadvantages compared to single oligonucleotides which can perform the same or comparable functions, such as higher costs of goods, complications in manufacturing and delivery, increased regulatory burden, etc. In accordance with FDA regulations, each component in a combination may need to be separately tested for toxicity, as well as the entire combination. In some embodiments, the present disclosure provides single oligonucleotides that can achieve the same or comparable functions of oligonucleotide combinations, and may be utilized to replace oligonucleotide combinations, through precise and designed control of one or more structural elements of oligonucleotides, e.g., chemical modifications, stereochemistry, and combinations thereof.

Various technologies are suitable for assessing multiple exon skipping in accordance with the present disclosure. Non-limiting examples are described in Example 20 and FIG. 2.

In some embodiments, a composition for skipping multiple DMD exons comprises a DMD oligonucleotide capable of skipping DMD exon 45. Various DMD oligonucleotides were tested for their capability to skip exon 45, as shown in Table A. Various DMD oligonucleotides for skipping exon 45 were also tested for their ability to skip multiple exons, as shown in Table 22A. Among other things, the present disclosure demonstrates that several oligonucleotides, including WV-11088 and WV-11089, can provide low levels of skipping of exons 45-55 (creating a junction between exon 44 and exon 56 or 44-56).

In another experiment, oligonucleotides WV-11047, WV-11051 to WV-11059 did not demonstrate significant skipping under the specific tested condition, and oligonucleotides WV-11062 to WV-11069 each exhibited detectable levels of skipping which were <1% under the specific tested condition. Oligonucleotides WV-11091 to WV-11096, WV-11098, and WV-11100 to WV-11105 exhibited <0.5% skipping of exon 45 under the specific tested condition.

TABLE 22A

Example data of certain oligonucleotides.

WV-11070
1.6

WV-11071
.3

WV-11072
.2

WV-11073
.7

WV-11074
2.2

WV-11075
.2

WV-11076
1.2

WV-11077
1.3

WV-11078
3.3

WV-11079
7.5

WV-11080
1.3

WV-11081
7.2

WV-11082
2.8

WV-11083
3.1

WV-11084
10.1

WV-11085
1.5

WV-11086
15.8

WV-11087
1.1

WV-11088
13

WV-11089
15.1

WV-11090
.9

Oligonucleotides were tested for their ability to skip DMD exon 45 in Δ48-50 cells.

Numbers indicate skipping level, wherein 100 would represent 100% skipping and 0 would represent 0% skipping.

Several oligonucleotides, including WV-11088 and WV-11089, showed detectable levels of multiple exon skipping (specifically exons 45-55) (approximately 0.1% skipping).

In another experiment, various DMD oligonucleotides targeting exon 45 were tested in Δ48-50 for an ability to skip multiple exons (specifically 45 to 53, creating a junction between exon 44 and exon 54 or 44-54). Oligonucleotides tested were: WV-11047, WV-11051, WV-11052, WV-11053, WV-11054, WV-11055, WV-11056, WV-11057, WV-11058, WV-11059, WV-11062, WV-11063, WV-11064, WV-11065, WV-11066, WV-11067, WV-11068, WV-11069, WV-11070, WV-11071, WV-11072, WV-11073, WV-11074, WV-11075, WV-11076, WV-11077, WV-11078, WV-11079, WV-11080, WV-11081, WV-11082, WV-11083, WV-11084, WV-11085, WV-11086, WV-11087, WV-11088, WV-11089, WV-11090, WV-11091, WV-11092, WV-11093, WV-11094, WV-11095, WV-11096, WV-11098, WV-11100, WV-11101. All these oligonucleotides, in one experiment, demonstrated on average about 0.05% or less skipping of exons 44-54 (data not shown).

Oligonucleotides targeting exon 45 were also tested for skipping of exons 45 to 57, as shown in Table 22A.1.

TABLE 22A.1

Example data of certain oligonucleotides.

WV-11047
0.064
0.118
0.048
0.099

WV-11051
0.044
0.101
0.034
0.079

WV-11052
0.076
0.089
0.078
0.090

WV-11053
0.082
0.076
0.078
0.072

WV-11054
0.126
0.083
0.110
0.100

WV-11055
0.037
0.071
0.048
0.073

WV-11056
0.133
0.102
0.116
0.092

WV-11057
0.000
0.001
0.000
0.097

WV-11058
0.102
0.030
0.071
0.042

WV-11059
0.171
0.100
0.157
0.075

WV-11062
0.070
0.112
0.081
0.088

WV-11063
0.088
0.078
0.051
0.081

WV-11064
0.085
0.071
0.071
0.075

WV-11065
0.073
0.114
0.077
0.143

WV-11066
0.083
0.100
0.004
0.143

WV-11067
0.115
0.069
0.094
0.068

WV-11068
0.112
0.071
0.125
0.053

WV-11069
0.075
0.075
0.083
0.053

WV-11070
0.062
0.107
0.067
0.101

WV-11071
0.085
0.116
0.073
0.118

WV-11072
0.080
0.097
0.052
0.084

WV-11073
0.052
0.148
0.047
0.118

WV-11074
0.155
0.098
0.116
0.101

WV-11075
0.145
0.079
0.126
0.113

WV-11076
0.000
0.105
0.000
0.111

WV-11077
0.050
0.087
0.080
0.058

WV-11078
0.087
0.095
0.077
0.103

WV-11079
0.076
0.063
0.079
0.062

WV-11080
0.059
0.058
0.052
0.070

WV-11081
0.077
0.086
0.058
0.055

WV-11082
0.117
0.071
0.112
0.080

WV-11083
0.077
0.108
0.091
0.091

WV-11084
0.080
0.102
0.053
0.069

WV-11085
0.047
0.143
0.041
0.140

WV-11086
0.085
0.087
0.084
0.074

WV-11087
0.114
0.034
0.000
0.056

WV-11088
0.134
0.112
0.057
0.063

WV-11089
0.074
0.113
0.109
0.082

WV-11090
0.119
0.076
0.074
0.081

WV-11091
0.000
0.055
0.031
0.054

WV-11092
0.039
0.057
0.068
0.058

WV-11093
0.147
0.061
0.138
0.061

WV-11094
0.108
0.078
0.061
0.080

WV-11095
0.062
0.061
0.056
0.072

WV-11096
0.104
0.071
0.072
0.101

WV-11098
0.072
0.095
0.081
0.065

WV-11100
0.068
0.079
0.078
0.068

WV-11101
0.000
0.058
0.000
0.048

Oligonucleotides were tested in Δ48-50 for their ability to skip DMD exons 45 to 57, creating a junction between exon 44 and exon 58 or 44-58. Numbers indicate skipping level, wherein 100 would represent 100% skipping and 0 would represent 0% skipping. Replicate data in this and other tables are shown.

In some embodiments, a DMD oligonucleotide targets DMD exon 44 or the adjoining intronic region 3′ to DMD exon 44 and is capable of mediating multiple exon skipping.

Several oligonucleotides were designed to target exon 44 or intron 44, or which straddle exon 44 and intron 44. In some embodiments, oligonucleotides designed to target exon 44 or intron 44, or which straddle exon 44 and intron 44 arc tested to determine if they can increase the amount of backslicing and/or multiple-exon skipping.

As shown in Table 22A.2 and Table 22A.3, below, DMD oligonucleotides targeting Exon44 were tested for the ability to increase circRNA 55-45 (e.g., mediate multiple exon skipping of exons 45 to 55); or for the ability to increase circRNA 57-45 (e.g., mediate multiple exon skipping of exons 45 to 57). Various DMD oligonucleotides comprise various difference including, inter alia, base sequence and length (18 or 20 bases). Numbers indicate relative amount of circRNA 55-45 (Table 22A.2) or circRNA 57-45 (Table 22A.3). In this and various other tables, Rep indicates Replicate.

TABLE 22A.2

Example data of certain oligonucleotides.

WV-13964
0.9
1

WV-13965
1.1
1.1

WV-13966
1.1
0.6

WV-13967
1.3
1.2

WV-13969
1
0.8

WV-13971
0.3
0.9

WV-13972
1.1
1.3

WV-13973
1.1
1.3

WV-13976
1.2
1.2

WV-13979
0.5
0.5

WV-13980
1.3
0.4

WV-13981
0.9
0.7

WV-13982
1
1

WV-13983
0.9
0.6

WV-13984
1.1

WV-13985
1.3
0.8

WV-13987
1.2
1

WV-13988
1.4
0.9

WV-13989
1.6
1

WV-13990
1.7
1

WV-13991
1.4
1

WV-13992
1.6
1

WV-13993
1.2
1

WV-13994
1.2
0.6

WV-13995
1.1
0.9

WV-13996
1.4
1

WV-13997
1.2
1.3

WV-13998
1.2
0.8

WV-13999
1.2
1.3

WV-14000
0.9
0.9

WV-14001
1.1
1.5

WV-14002
1
1.1

WV-14003
2
2.1

WV-14004
1.9
1.2

WV-14005
1.1
1

WV-14006
1.2
1.4

WV-14007
1.3
1.7

WV-14008
1.4
1.1

WV-14009
1.3
1.3

WV-14010
1
1.1

WV-14011
3.2
3.7

WV-14012
1.8
2

WV-14013
1.4
1.8

WV-14014
1.1
1.3

WV-14015
1.1
1.3

WV-14016
1.2
1.5

WV-14017
1.5
1.5

WV-14018
0.8
1

WV-14019
1.2
1.4

WV-14020
1
1

WV-14021
1
1.3

WV-14022
1.3
1.5

WV-14023
1.3
1.7

WV-14024
1.2
1.2

WV-14025
1.5
1.6

WV-14026
2.4
0.6

WV-14027
1.2
1.2

WV-14028
1.1
1.2

WV-14029
1.2
1.4

WV-14030
1.3
1.6

WV-14031
1.3
1.6

WV-14032
1.2
1.5

WV-14033
1.3

WV-14034
1.1
1.2

WV-14035
1.2
1.4

WV-14036
1.1
1.1

WV-14037
1.1
1.2

WV-14038
1.4
1.4

WV-14039
1.2
1.2

WV-14040
2.2
3

WV-14041
2.3
2.4

WV-14042
1.3
1.3

WV-14043
1.1
1.4

WV-14044
1.3
1.5

WV-14045
1.8
2.1

WV-14046
1.3
1.6

WV-14047
1.2
1.6

WV-14048
3.8
4.9

WV-14049
2.1
2.6

WV-14050
1.4
1.5

WV-14051
1.5
1.7

WV-14052
1.4
2.2

WV-14053
1.5
1.4

WV-14054
1.4
1.8

WV-14055
1.3
1.6

WV-14056
1.3
1.4

WV-14057
1.7
2.1

WV-14058
1.8
1.4

TABLE 22A.3

Example data of certain oligonucleotides.

Biological
Biological

Rep1
Rep2

mock

0.9

mock
0.8
1

mock
1
1.4

mock
1
0.5

mock
1.9
1.2

mock
0.7
0.7

mock
0.9
0.6

mock
0.3
1.6

WV-13964
0.8
1

WV-13965
0.8
0.7

WV-13966
1
0.7

WV-13967
1.2
0.9

WV-13969
1.2
1.3

WV-13971

0.5

WV-13972
0.9
1.3

WV-13973
0.6
1.4

WV-13976
1.3
1.6

WV-13979
0.5
0.3

WV-13980
1.4
0.6

WV-13981
0.8
1.3

WV-13982
1.1
1

WV-13983
1
0.8

WV-13984
0.8
0.4

WV-13985
1.3
1.6

WV-13987
1.4
1.1

WV-13988
1.4
1

WV-13989
1.5
0.7

WV-13990
1.3
0.6

WV-13991
1.3
0.8

WV-13992
1.6
2.4

WV-13993
0.9
0.9

WV-13994
0.6
1

WV-13995
0.9
1.6

WV-13996
1.2
0.8

WV-13997
1.4
0.7

WV-13998
1.2
0.8

WV-13999
0.9
0.9

WV-14000
0.6
0.3

WV-14001
0.8
0.9

WV-14002
0.6
1.3

WV-14003
2.1
2

WV-14004
2.1
0.7

WV-14005
0.9
0.8

WV-14006
1.3
1.1

WV-14007
0.9
1.6

WV-14008
1.3
1.1

WV-14009
0.9
1

WV-14010
1
0.6

WV-14011
3.1
4.7

WV-14010
1
0.6

WV-14011
3.1
4.7

WV-14012
1.3
1.7

WV-14013
0.9
1

WV-14014
0.9
1.1

WV-14015
0.4
1.2

WV-14016
0.4
2.1

WV-14017
1.4
1.3

WV-14018
0.8
0.7

WV-14019
1.3
1.5

WV-14020
0.6
1.2

WV-14021
1.2
1.4

WV-14022
1.6
1.6

WV-14023
1.2
1.3

WV-14024
1.4
1.1

WV-14025
0.5
1.6

WV-14026
1.9

WV-14027
1.1
0.9

WV-14028
0.8
1

WV-14029
1.1
1.3

WV-14030
1.2
1.4

WV-14031
1.2
1.5

WV-14032
0.9
1.7

WV-14033
0.9

WV-14034
0.8
1.1

WV-14035
1.3
1.1

WV-14036
0.7
0.9

WV-14037
1.2
1

WV-14038
1.4
1.6

WV-14039
1.1
0.5

WV-14040
2.5
4.4

WV-14041
2
2.8

WV-14042
1.4
1.2

WV-14043
1.4
1.4

WV-14044
1.7
1.2

WV-14045
1.7
2

WV-14046
1.1
1.9

WV-14047
1.3
0

WV-14048
3.1
7.1

WV-14049
1.9
2.5

WV-14050
1.6
1.4

WV-14051
1.8
1.7

WV-14052
0.9
2.6

WV-14053
1.1
1.8

WV-14054
1.2
2

WV-14055
1.2
2

WV-14056
1.4
0.9

WV-14057
1.5
1.9

WV-14058
1.3
1

In some embodiments, a composition capable of mediating exon skipping of a particular DMD exon comprises two or more oligonucleotides targeting a particular exon. In some embodiments, a combination of two or more oligonucleotides provides skipping levels significantly higher than the addition of the skipping level of each oligonucleotide individually. In some embodiments, a combination of two or more oligonucleotides provides significant (1%, 5%, 10%, or more) and/or detectable levels of skipping while each oligonucleotide individually does not provide detectable levels of skipping. Combinations of traditional oligonucleotides (e.g., stereorandom oligonucleotide and/or oligonucleotides without non-negatively charged internucleotidic linkages described in the present disclosure) has been reported to provide certain improved effects, e.g., in Wilton et al. 2007 Mol. Ther. 7: 1288-1296 (exons 10, 20, 34, 65, etc.). Among other things, provided combinations comprise at least one oligonucleotide comprising one or more chirally controlled internucleotidic linkages and/or one or more non-negatively charged internucleotidic linkages, and can provide significantly increased levels of exon skipping.

Among other things, the present disclosure recognizes that certain exons are particularly challenging for skipping. For example, in one report, for exons 47 and 57, individual DMD oligonucleotides were not capable of mediating exon skipping, but pairs of oligonucleotides were capable of mediating exon skipping. In one report, effective skipping of exon 45 was mediated by combining two DMD oligonucleotides which were individually not effective in skipping of this exon. Aartsma-Rus et al. 2006 Mol. Ther. 14: 401. Aartsma-Rus et al. 2006 Mol. Ther. 14: 401. In some embodiments, the present disclosure provides oligonucleotides (e.g., chirally controlled oligonucleotides), and compositions and methods of use thereof, for exon skipping of such challenging exons. With chemistry modifications and/or stereochemistry technologies described herein, the present disclosure provides technologies with greatly improved exon skipping efficiency. In some embodiments, the present disclosure provides single oligonucleotide (e.g., a chirally controlled oligonucleotide) and compositions thereof (e.g., a chirally controlled oligonucleotide composition) for exon skipping of one or more exons that are challenging to skip. In some embodiments, the present disclosure provides combinations of oligonucleotides (e.g., chirally controlled oligonucleotides) and compositions thereof (e.g., chirally controlled oligonucleotide compositions) for exon skipping of one or more exons that are challenging to skip. In some embodiments, combinations of DMD oligonucleotides targeting the same exon mediate increased exon skipping levels relative to individual DMD oligonucleotides.

In some embodiments, a composition comprises two or more DMD oligonucleotides, wherein each individual DMD oligonucleotide mediates low levels of exon skipping, while the combination mediates a higher level of skipping (higher than the addition of levels achieved by each oligonucleotide individually).

In some embodiments, a composition comprises two or more DMD oligonucleotides, wherein the oligonucleotides target different exons.

In some embodiments, a combination of multiple DMD oligonucleotides targeting different exons is capable of mediating skipping of two or more (e.g., multiple) exons.

In some embodiments, a composition comprises two or more DMD oligonucleotides. In some embodiments, a composition comprises two or more DMD oligonucleotides, at least one of which is described herein or has a base sequence, stereochemistry or other chemical characteristic described herein.

Oligonucleotides Comprising Non-Negatively Charged Internucleotidic Linkages can Provide Significantly Improved Activities.

In some embodiments, the present disclosure provides oligonucleotides comprising one or more non-negatively charged internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, the present disclosure provides oligonucleotides comprising one or more neutral internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of formula 1-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

In some embodiments, a non-negatively charged internucleotidic linkage comprises a triazole moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

embedded image

In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

embedded image

In some embodiments, a non-negatively charged internucleotidic linkage comprises a substituted triazolyl group. In some embodiments, a non-negatively charged internucleotidic linkage has the structure

embedded image

wherein W is O or S. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted alkynyl group. In some embodiments, a non-negatively charged internucleotidic linkage has the structure

embedded image

wherein W is O or S.

In some embodiments, the present disclosure provides oligonucleotides comprising an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, which comprises a cyclic guanidine moiety. In some embodiments, an internucleotidic linkage comprises a cyclic guanidine and has the structure of:

embedded image

In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, comprising a cyclic guanidine is stereochemically controlled.

In some embodiments, a non-negatively charged internucleotidic linkage, or a neutral internucleotidic linkage, is or comprising a structure selected from

embedded image

wherein W is O or S. In some embodiments, a non-negatively charged internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, a neutral internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, a nucleic acid or an oligonucleotide comprising a modified internucleotidic linkage comprising a cyclic guanidine moiety is a siRNA, double-straned siRNA, single-stranded siRNA, gapmer, skipmer, blockmer, antisense oligonucleotide, antagomir, microRNA, pre-microRNs, antimir, supermir, ribozyme, U1 adaptor, RNA activator, RNAi agent, decoy oligonucleotide, triplex forming oligonucleotide, aptamer or adjuvant.

In some embodiments, an oligonucleotide comprises a neutral internucleotidic linkage and a chirally controlled internucleotidic linkage. In some embodiments, an oligonucleotide comprises a neutral internucleotidic linkage and a chirally controlled internucleotidic linkage which is a phosphorothioate in the Rp or Sp configuration. In some embodiments, the present disclosure provides an oligonucleotide comprising one or more non-negatively charged internucleotidic linkages and one or more phosphorothioate internucleotidic linkage, wherein each phosphorothioate internucleotidic linkage in the oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, the present disclosure provides an oligonucleotide comprising one or more neutral internucleotidic linkages and one or more phosphorothioate internucleotidic linkage, wherein each phosphorothioate internucleotidic linkage in the oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, a provided oligonucleotide comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more chirally controlled phosphorothioate internucleotidic linkages.

Without wishing to be bound by any particular theory, the present disclosure notes that a neutral internucleotidic linkage is more hydrophobic than a phosphorothioate internucleotidic linkage (PS), which is more hydrophobic than a phosphodiester linkage (natural phosphate linkage, PO). Typically, unlike a PS or PO, a neutral internucleotidic linkage bears less charge. Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more neutral internucleotidic linkages into an oligonucleotide may increase oligonucleotides' ability to be taken up by a cell and/or to escape from endosomes. Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more neutral internucleotidic linkages can be utilized to modulate melting temperature between an oligonucleotide and its target nucleic acid.

Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more non-negatively charged internucleotidic linkages, e.g., neutral internucleotidic linkages, into an oligonucleotide may be able to increase the oligonucleotide's ability to mediate a function such as exon skipping or gene knockdown. In some embodiments, an oligonucleotide capable of altering skipping of one or more exons in a target gene comprises one or more neutral internucleotidic linkages. In some embodiments, an oligonucleotide capable of mediating skipping of an exon(s) in a target gene comprises one or more neutral internucleotidic linkages. In some embodiments, an oligonucleotide capable of mediating skipping of one or more DMD exon(s) comprises one or more neutral internucleotidic linkages.

In some embodiments, an oligonucleotide capable of mediating knockdown of level of a nucleic acid or a product encoded thereby comprises one or more non-negatively charged internucleotidic linkages. In some embodiments, an oligonucleotide capable of mediating knockdown of expression of a target gene comprises one or more non-negatively charged internucleotidic linkages. In some embodiments, an oligonucleotide capable of mediating knockdown of expression of a target gene comprises one or more neutral internucleotidic linkages.

In some embodiments, a non-negatively charged internucleotidic linkage is not chirally controlled. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled and its linkage phosphorus is Rp. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled and its linkage phosphorus is Sp.

In some embodiments, a provided oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more non-negatively charged internucleotidic linkages. In some embodiments, a provided oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more neutral internucleotidic linkages. In some embodiments, each of non-negatively charged internucleotidic linkage and/or neutral internucleotidic linkages is optionally and independently chirally controlled. In some embodiments, each non-negatively charged internucleotidic linkage in an oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, each neutral internucleotidic linkage in an oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

embedded image

wherein W is O or S. In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

embedded image

In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

embedded image

In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure

embedded image

herein W is O or S. In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

embedded image

In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

embedded image

In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

embedded image

wherein W is O or S. In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

embedded image

In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

embedded image

In some embodiments, a provided oligonucleotide comprises at least one non-negatively charged internucleotidic linkage wherein its linkage phosphorus is in Rp configuration, and at least one non-negatively charged internucleotidic linkage wherein its linkage phosphorus is in Sp configuration.

In some embodiments, an oligonucleotide capable of increasing the frequency of skipping of an exon of a target gene comprises a non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide capable of increasing the frequency of skipping of an exon of a target gene comprises a non-negatively charged internucleotidic linkage and is useful for treatment of a disease wherein the exon comprises a deleterious or disease-associated mutation. A non-limiting example is the DMD gene, wherein the skipping of an exon comprising a mutation contributes to muscular dystrophy.

Various oligonucleotides, including DMD oligonucleotides, that comprise one or more non-negatively charged internucleotidic linkages/neutral internucleotidic linkages were designed and/or constructed and/or tested, for example, WV-1343, WV-1344, WV-1345, WV-1346, WV-1347, WV-11237, WV-11238, WV-11239, WV-12130, WV-12131, WV-12132, WV-12133, WV-12134, WV-12135, WV-12136, WV-11340, WV-11341, WV-11342, WV-12123, WV-12124, WV-12125, WV-12126, WV-12127, WV-12128, WV-12129, WV-12553, WV-12554, WV-12555, WV-12556, WV-12557, WV-12558, WV-12559, WV-12872, WV-12873, etc. Example DMD oligonucleotides for skipping exon 23 and comprising a non-negatively charged internucleotidic linkage (e.g., a neutral internucleotidic linkage) include: WV-11343, WV-11344, WV-11345, WV-11346, and WV-1347. Example DMD oligonucleotides for skipping exon 51 and comprising a non-negatively charged internucleotidic linkage (e.g., a neutral internucleotidic linkage) include: WV-11237, WV-11238, WV-11239, WV-12130, WV-12131, WV-12132, WV-12133, WV-12134, WV-12135, and WV-12136. Example DMD oligonucleotides for skipping exon 53 and comprising a non-negatively charged internucleotidic linkage (e.g., a neutral internucleotidic linkage) include: WV-11340, WV-1341, WV-11342, WV-12123, WV-12124, WV-12125, WV-12126, WV-12127, WV-12128, WV-12129, WV-12553, WV-12554, WV-12555, WV-12556, WV-12557, WV-12558, WV-12559, WV-12872, and WV-12873. Certain oligonucleotides are in Table A1.

Additional DMD oligonucleotides comprising a non-negatively charged internucleotidic linkage were designed and/or constructed. These include DMD oligonucleotides for skipping DMD exon 45, WV-14528, WV-14529, WV-14532, and WV-14533.

The efficacy of various DMD oligonucleotides comprising a non-negatively charged internucleotidic linkage in skipping DMD exon 45 is shown in Table 1B.1 and Table 1B.2 herein.

The efficacy of various DMD oligonucleotides comprising a non-negatively charged internucleotidic linkage in skipping DMD exon 53 is shown in Table 21E, Table 21F, Table 21G, and Table 21H herein.

In some embodiments, a non-negatively charged internucleotidic linkage may be designated as nX if stereorandom, or nS chirally controlled and linkage phosphorus in the Sp configuration, or nR if chirally controlled and the linkage phosphorus in the Rp configuration.

In some embodiments, a non-negatively charged internucleotidic linkage may be designated as n001 if stereorandom, or n001S chirally controlled and linkage phosphorus in the Sp configuration, or n001R if chirally controlled and the linkage phosphorus in the Rp configuration (e.g., in Table A1).

Various DMD oligonucleotides comprising a non-negatively charged internucleotidic linkage in the Rp configuration were constructed, including WV-12872, WV-13408, WV-12554, WV-13409, WV-12555, and WV-12556.

Various DMD oligonucleotides comprising a non-negatively charged internucleotidic linkage in the Sp configuration were constructed, including WV-12557, WV-12558, and WV-12559.

Data showing activity and stability of various oligonucleotides comprising a non-negatively charged internucleotidic linkage in the Rp or Sp configuration are shown in Table 21H Table 211, Table 211.1, and Table 211.2

Several oligonucleotides (including WV-9517, WV-13864, WV-13835, and WV-14791) were tested at various concentrations up to 30 uM for TLR9 activation in HEK-blue-TLR9 cells (16 hour gymnotic uptake). WV-13864 and WV-14791 comprise a chirally controlled non-negatively charged internucleotidic linkage in the Rp configuration. WV-9517, WV-13864, WV-13835, and WV-14791 did not exhibit significant TLR9 activation (data not shown).

Several oligonucleotides which target a gene other than DMD were designed and/or constructed which comprise a non-negatively charged internucleotidic linkage.

Below are presented oligonucleotides comprising a cyclic guanidine moiety which target DMD or Malat-1 (Malat1). The DMD oligonucleotides are designed to mediate skipping of exon 23 (in mouse) or exon 51 or exon 53 (in human). The Malat-1 oligonucleotides are designed to for Malat1 mRNA knockdown, e.g., mediated through RNase H.

TABLE 22B

Example Malat-1 oligonucleotides comprising a neutral backbone.

Oligonucleotide
Description
Stereochemistry

WV-11533
mU * SGeon001m5Ceon001 m5Ceo n001mA * SG * SG *
SnXnXnXSSRSSR

RC * ST * SG * RG * ST * ST * RA * ST * SmG * SmA *
SSRSSSSSS

SmC * SmU * SmC

WV-12504
Mod001L00mU * SGeon001 m5Ceon001 m5Ceon001mA *
OSnXnXnXSSRSS

SG * SG * RC * ST * SG * RG * ST * ST * RA * ST * SmG
RSSRSSSSSS

* SmA * SmC * SmU * SmC

WV-12505
L001mU * SGeon001m5Ceon001 m5Ceon001mA * SG * SG
OSnXnXnXSSRSS

* RC * ST * SG * RG * ST * ST * RA * ST * SmG * SmA *
RSSRSSSSSS

SmC * SmU * SmC

All of these oligonucleotides have the base sequence of UGCCAGGCTGGTTATGACUC.

Oligonucleotides comprising non-negatively charged internucleotidic linkages and targeting other gene targets were also designed, constructed and/or tested for their properties and activities, including activities for reducing levels of target mRNAs and/or proteins, e.g., via RNaseH-mediated knockdown. Such oligonucleotides are active in reducing target levels.

TABLE 22C

Malat1 oligonucleotides

Oligonucleotide
Sequence
Stereochemistry

WV-8587
mU * SGeo m5Ceo m5Ceo mA * SG * SG * RC * ST * SG * RG
SOOOSSRSSR

* ST * ST * RA * ST * S mG * S mA * S mC * S mU * S mC
SSRSSSSSS

WV-14733
mU * SGeo m5Ceo m5Ceo mA * SG * SG * SC * ST * SG * SG
SOOOSSSSSS

* ST * ST * SA * ST * S mG * S mA * S mC * S mU * S mC
SSSSSSSSS

WV-15351
mU * SGeo m5Ceo m5Ceo mA * SG * SGn001C * ST *
SOOOSSIASS

SGn001G* ST * STn001A * ST * S mG * S mA * S mC * S mU
nXSSnXSSSSSS

* S mC

WV-15352
mU * SGeo m5Ceo m5Ceo mA * SG * SGn001C * ST * SG *
SOOOSSnXSS

RG * ST * ST * RA * ST * S mG * S mA * S mC * S mU * S mC
RSSRSSSSSS

WV-15353
mU * SGeo m5Ceo m5Ceo mA * SG * SG * RC * ST *
SOOOSSRSSnX

SGn001G * ST* ST * RA * ST* S mG* S mA * S mC * S mU *
SSRSSSSSS

S mC

WV-15354
mU * SGeo m5Ceo m5Ceo mA * SG * SG * RC * ST * SG * RG
SOOOSSRSSRSS

* ST * STn001A * ST * S mG * S mA * S mC * S mU * S mC
nXSSSSSS

WV-15356
mU * SGeo m5Ceo m5Ceo mA * SG * SG * RCn001Tn001G *
SOOOSSRnXnX

RG * ST * ST * RA * ST * S mG * S mA * S mC * S mU * S mC
RSSRSSSSSS

WV-15357
mU * SGeo m5Ceo m5Ceo mA * SG * SG * RC * ST * SG *
SOOOSSRSSR

RGn001Tn001T * RA * ST * S mG * S mA * S mC * S mU * S
nXnXRSSSSSS

mC

WV-15358
mU * SGeo m5Ceo m5Ceo mA * SG * SG * RC * ST * SG * RG
SOOOSSRSSRS

* ST * ST * RAn001Tn001 mG * S mA * S mC * S mU * S mC
SRnXnXSSSS

WV-8582
mU * SGeo m5Ceo m5Ceo mA * SG * SG * SC * ST * SG * SG
SOOOSSSSSSS

* ST * ST * RA * ST * S mG * S mA * S mC * S mU * S mC
SRSSSSSS

WV-15359
mU * SGeo m5Ceo m5Ceo mA * SG * SG * SC * ST * SG * SG
SOOOSSSSSSS

* ST * STn001An001Tn001 mG * S mA * S mC * S mU * S mC
SnXnXnXSSSS

WV-15360
mU * SGeo m5Ceo m5Ceo mA * SG * SG * SC * ST * SG * SG
SOOOSSSSSSS

* ST * STn001A * ST * S mG * S mA * S mC * S mU * S mC
SnXSSSSSS

WV-15361
mU * SGeo m5Ceo m5Ceo mA * SG * SG * SC * ST * SG * SG
SOOOSSSSSSS

* ST * ST * RA * STn001 mGn001 mA * S mC * S mU * S mC
SRSnXnXSSS

WV-15362
mU * SGeo m5Ceo m5Ceo mA * SG * SG * SC * ST * SG * SG
SOOOSSSSSSS

* ST * ST * RAn001T * S mG * S mA * S mC * S mU * S mC
SRnXSSSSS

WV-15363
mU * SGeo m5Ceo m5Ceo mA * SG * SG * SC * ST * SG* SG
SOOOSSSSSSS

* ST * ST * RA * STn001 mG * S mA * S mC * S mU * S mC
SRSnXSSSS

WV-14556
mUn001Geon001 m5Ceon001 m5Ceo mA * SG * SG * RC * ST
nXnXnXOSSRS

* SG * RG * ST * ST * RA * ST * S mG * S mA * S mC * S mU
SRSSRSSSSSS

* S mC

WV-14557
mUn001Geon001 m5Ceo m5Ceon001 mA * SG * SG * RC * ST
nXnXOnXSSRS

* SG * RG * ST * ST * RA * ST * S mG * S mA * S mC * S mU
SRSSRSSSSSS

* S mC

WV-14558
mUn001Geon001 m5Ceo m5Ceo mAn001G * SG * RC * ST *
nXnXOOnXSRS

SG * RG * ST * ST * RA * ST * S mG * S mA * S mC * S mU *
SRSSRSSSSSS

S mC

WV-14559
mUn001Geo m5Ceon001 m5Ceon001 mA * SG * SG * RC * ST
nXOnXnXSSRSS

* SG * RG * ST * ST * RA * ST * S mG * S mA * S mC * S mU
RSSRSSSSSS

* S mC

WV-14560
mUn001Geo m5Ceon001 m5Ceo mAn001G * SG * RC * ST *
nXOnXOnXSRSS

SG * RG * ST * ST * RA * ST * S mG * S mA * S mC * S mU *
RSSRSSSSSS

S mC

WV-14561
mUn001Geo m5Ceo m5Ceon001 mAn001G * SG * RC * ST *
nXOnXOnXSRSS

SG * RG * ST * ST * RA * ST * S mG * S mA * S mC * S mU *
RSSRSSSSSS

S mC

WV-11533
mU * SGeon001 m5Ceon001 m5Ceon001 mA * SG * SG * RC *
SnXnXnXSSRSS

ST * SG * RG * ST * ST * RA * ST * S mG * S mA * S mC * S
RSSRSSSSSS

mU * S mC

WV-14562
mU * SGeon001 m5Ceon001 m5Ceo mAn001G * SG * RC * ST
SnXnXOnXSRSS

* SG * RG * ST * ST * RA * ST * S mG * S mA * S mC * S mU
RSSRSSSSSS

* S mC

WV-14563
mU * SGeon001 m5Ceo m5Ceon001 mAn001G * SG * RC * ST
SnXOnXnXSRSS

* SG * RG * ST * ST * RA * ST * S mG * S mA * S mC * S mU
RSSRSSSSSS

* S mC

WV-14564
mU * SGeo m5Ceon001 m5Ceon001 mAn001G * SG * RC * ST
SOnXnXnXSRSS

* SG * RG * ST * ST * RA * ST * S mG * S mA * S mC * S mU
RSSRSSSSSS

* S mC

WV-14349
Mod098L001 mU * SGeo m5Ceo m5Ceo mA * SG * SG * RC *
OSOOOSSRSSRS

ST * SG * RG * ST * ST * RA * ST * S mG * S mA * S mC * S
SRSSSSSS

mU * S mC

All of the oligonucleotides in this table have the base sequence of UGCCAGGCTGGTTATGACUC.

TABLE 22D

Data of Malat1 oligonucleotides

0.004 uM
0.02 uM
0.1 uM

WV-8587
1.23
1.21
0.94
0.95
0.84
0.81
0.54
0.53
0.61

WV-14733
1.81
1.06
1.36
1.47
1.12
1.17
0.98
0.97
0.72

WV-15351
1.27
0.92
1.00
0.89
0.95
0.92
0.74
0.66
0.71

WV-15352
1.49
1.78
1.52
0.88
0.83
0.91
0.50
0.52
0.73

WV-15353
0.85
0.91
1.10
0.65
0.59
0.68
0.44
0.42
0.40

WV-15354
1.31
1.00
0.90
0.69
0.94
0.79
0.56
0.87
0.74

WV-15356
0.77
0.87
0.68
0.49
0.67
0.63
0.30
0.35
0.31

WV-15357
0.91
1.02
1.13
0.66
0.75
0.79
0.37
0.32
0.36

WV-15358
0.80
0.82
0.90
0.83
0.85
0.85
0.36
0.45
0.43

WV-8582
1.11
1.06
1.15
1.30
1.15
1.14
0.67
0.85
1.06

WV-15359
1.16
1.26
1.02
0.92
0.83
0.83
0.85

0.90

WV-15360
1.57
1.38
1.31
1.05
0.99
0.83
1.03
0.91
0.80

WV-15361
0.92
1.11
1.00
0.71
0.63
0.68
0.74
1.09
0.73

WV-15362
1.23
1.22
1.07
0.90
0.83
0.82
0.99
0.97
0.80

WV-15363
1.16
1.03
0.85
0.89
0.87
0.90
1.10
1.18
1.01

WV-14556
0.81
0.84
0.91
0.46
0.42
0.58
0.15
0.23
0.17

WV-14557
0.75
1.10
0.96
0.46
0.40
0.54
0.19
0.19
0.21

WV-14558
0.96
1.11
0.90
0.77
1.08
0.78
1.27
0.40
0.45

WV-14559
0.80
0.62
0.75
0.35
0.36
0.37
0.12
0.17
0.13

WV-14560
1.11
0.99
1.03
0.44
0.48
0.60
0.29
0.31
0.15

WV-14561
0.71
0.73
1.04
0.47
0.41
0.48
0.22
0.24
0.16

WV-11533
0.74
0.75
0.87
0.40
0.37
0.41
0.14
0.14
0.09

WV-14562
0.79
0.60
0.60
0.53
0.45
0.64
0.22
0.33
0.24

WV-14563
0.76
0.96
0.79
0.57
0.51
0.53
0.23
0.23
0.24

WV-14564
0.72
0.65
0.70
0.58
0.47
0.50
0.17
0.20
0.21

WV-9491
1.02
0.96
1.28
0.82
0.93
1.27
0.88
0.91
1.06

WV-14349
1.07
1.34
1.03
0.86
0.77
1.11
0.63
0.60
0.79

Various Malat1 oligonucleotides were designed, constructed and tested which comprise one or more non-negatively charged internucleotidic linkages in a core. In various embodiments of a Malat1 oligonucleotide, a phosphorothioate in the Rp configuration is replaced by anon-negatively charged internucleotidic linkage.

TABLE 22E

Data of Malat1 oligonucleotides

WV-
WV-
WV-
WV-
WV-
WV-

8587
15351
15352
15353
15354
9491

0.004 uM
1.23
1.27
1.49
0.85
1.31
1.02

1.21
0.92
1.78
0.91
1.00
0.96

0.94
1.00
1.52
1.10
0.90
1.28

0.02 uM
0.95
0.89
0.88
0.65
0.69
0.82

0.84
0.95
0.83
0.59
0.94
0.93

0.81
0.92
0.91
0.68
0.79
1.27

0.1 uM
0.54
0.74
0.50
0.44
0.56
0.88

0.53
0.66
0.52
0.42
0.87
0.91

0.61
0.71
0.73
0.40
0.74
1.06

Numbers represent knockdown of Malat1 mRNA relative to HPRT1, wherein 1.000 would represent no (0.0%) knockdown and 0.000 represents 100.0% knockdown; results from replicate experiments are shown.

TABLE 22F

Data of certain oligonucleotides.

WV-
WV-
WV-
WV-
WV-

8587
15356
15357
15358
9491

0.004
uM
1.23
0.77
0.91
0.80
1.02

1.21
0.87
1.02
0.82
0.96

0.94
0.68
1.13
0.90
1.28

0.02
uM
0.95
0.49
0.66
0.83
0.82

0.84
0.67
0.75
0.85
0.93

0.81
0.63
0.79
0.85
1.27

0.1
uM
0.54
0.30
0.37
0.36
0.88

0.53
0.35
0.32
0.45
0.91

0.61
0.31
0.36
0.43
1.06

Numbers represent knockdown of Malat1 mRNA relative to HPRT1, wherein 1.000 would represent no (0.0%) knockdown and 0.000 represents 100.0% knockdown: results from replicate experiments are shown.

Various Malat1 oligonucleotides were designed, constructed and tested which comprise a non-negatively charged internucleotidic linkage. Various Malat1 oligonucleotides comprise 1 or more non-negatively charged internucleotidic linkages. In various tables and throughout the text herein, the presence or absence of a hyphen in the designation of an oligonucleotide is irrelevant. For example, WV8582 is equivalent to WV-8582.

TABLE 22G

Data of certain oligonucleotides.

WV-
WV-
WV-
WV-
WV-
WV-
WV-

8582
15359
15360
15361
15362
15363
9491

0.004 uM
1.11
1.16
1.57
0.92
1.23
1.16
1.02

1.06
1.26
1.38
1.11
1.22
1.03
0.96

1.15
1.02
1.31
1.00
1.07
0.85
1.28

0.02 uM
1.30
0.92
1.05
0.71
0.90
0.89
0.82

1.15
0.83
0.99
0.63
0.83
0.87
0.93

1.14
0.83
0.83
0.68
0.82
0.90
1.27

0.1 uM
0.67
0.85
1.03
0.74
0.99
1.10
0.88

0.85

0.91
1.09
0.97
1.18
0.91

1.06
0.90
0.80
0.73
0.80
1.01
1.06

Numbers represent knockdown of Malat1 mRNA relative to HPRT1, wherein 1.000 would represent no (0.0%) knockdown and 0.000 represents 100.0% knockdown; results from replicate experiments are shown.

Various Malat1 oligonucleotides were designed, constructed and tested which comprise a non-negatively charged internucleotidic link-age. Various Malat1 oligonucleotides comprise 1 or more non-negatively charged internucleotidic linkages.

TABLE 22H

Data of certain oligonucleotides.

0.004 uM
0.02 uM

WV-11533
0.74
0.75
0.87
0.40
0.37
0.41

WV-14556
0.81
0.84
0.91
0.46
0.42
0.58

WV-14557
0.75
1.10
0.96
0.46
0.40
0.54

WV-14558
0.96
1.11
0.90
0.77
1.08
0.78

WV-14559
0.80
0.62
0.75
0.35
0.36
0.37

WV-14560
1.11
0.99
1.03
0.44
0.48
0.60

WV-14561
0.71
0.73
1.04
0.47
0.41
0.48

WV-14562
0.79
0.60
0.60
0.53
0.45
0.64

WV-14563
0.76
0.96
0.79
0.57
0.51
0.53

WV-14564
0.72
0.65
0.70
0.58
0.47
0.50

WV-9491
1.02
0.96
1.28
0.82
0.93
1.27

0.1 uM

WV-11533
0.14
0.14
0.09

WV-14556
0.15
0.23
0.17

WV-14557
0.19
0.19
0.21

WV-14558
1.27
0.40
0.45

WV-14559
0.12
0.17
0.13

WV-14560
0.29
0.31
0.15

WV-14561
0.22
0.24
0.16

WV-14562
0.22
0.33
0.24

WV-14563
0.23
0.23
0.24

WV-14564
0.17
0.20
0.21

WV-9491
0.88
0.91
1.06

Numbers represent knockdown of Malat1 mRNA relative to HPRT1, wherein 1.000 would represent no (0.0%) knockdown and 0.000 represents 100.0% knockdown; results from replicate experiments are shown.

In some embodiments, oligonucleotides were designed, constructed and tested in vitro against suitable reference oligonucleotides which do not comprise any non-negatively charged internucleotidic linkages, e.g., in iCell Astrocytes, at several suitable doses (e.g., 0, 0.014, 0.041, 0.123, 0.37, 1.11, 3.33, 10 uM) gymnotic for suitable period of time e.g., 2 days.

Tables 23, 24 and 25 present experimental results.

TABLE 23

Data of certain oligonucleotides.

Oliogomscleotide tested

Dose
(Relative fold change Malat1/HPRT1)

(uM)
WV-8587
WV-9696

0
0.924
0.970
1.106
1.162
1.040
0.799

0.013717
0.833
0.930
0.730
0.997
0.844
0.918

0.041152
1.186
0.868
0.874
1.076
0.957
0.844

0.123457
0.772
0.827
0.658
0.970
0.756
0.821

0.37037
0.610
0.610
0.553
0.821
0.520
0.681

1.111111
0.394
0.360
0.425
0.431
0.419
0.402

3.333333
0.157
0.136
0.162
0.225
0.214
0.220

10
0.051
0.052
0.065
0.090
0.086
0.091

Oliogonudeotide tested

Dose
(Relative fold change Malat1/HPRT1)

(uM)
WV-11114
WV-11533

0
0.761
0.881
1.212
0.958
0.985
1.056

0.013717
1.048
1.027
1.187
0.900
0.932
1.020

0.041152
0.912
0.958
1.108
0.453
0.503
0.479

0.123457
0.971
1.063
1.238
0.356
0.387
0.332

0.37037
0.706
0.846
0.692
0.105
0.107
0.096

1.111111
0.429
0.486
0.574
0.048
0.051
0.049

3.333333
0.181
0.196
0.203
0.033
0.032
0.030

10
0.080
0.075
0.087
0.026
0.034
0.031

Numbers represent knockdown of Malat1 mRNA, wherein 1.000 would represent no (0.0%) knockdown and 0.000 re resents 100.0% knockdown; results from replicate experiments are shown.

TABLE 24

IC50 of certain Malat1 oligonucleotides.

Oligonucleotide
IC50

WV-8587
757
nM

WV-9696
806
nM

WV-11114
894
nM

WV-11533
49
nM

Among other things, the present disclosure demonstrates that oligonucleotides comprising one or more non-negatively charged internucleotidic linkages can provide dramatically improved activities—as illustrated in Table 24, more than 15-fold improvement can be achieved in terms of IC50.

In another experiment, several Malat1 oligonucleotides including WV-11533, which comprises three neutral internucleotidic linkages, were assessed for knockdown of Malat1, measured by a decrease in the abundance of a Malat1 RNA WV-7772, which is complementary to the tested oligonucleotides, in the presence of RNaseH.

Linkage/

Oligonucleotide
Description
Naked Sequence
Stereochemistry

WV-11533
mU * SGeon001m5Ceo n001m5Ceo n001mA
UGCCAGGCTG
SnXnXnXSSRSSRS

* SG * SG * RC * ST * SG * RG * ST * ST *
GTTATGACUC
SRSSSSSS

RA * ST * SmG * SmA * SmC * SmU * SmC

WV-8556
mU * Geom5Ceom5CeomA * G * G * C * T
UGCCAGGCTGG
XOOOXXXXXX

* G *G * T * T * A * T * mG * mA * mC *
TTATGACUC
XXXXXXXXX

mU * mC

WV-8587
mU * SGeom5Ceom5CeomA * SG * SG *
UGCCAGGCTGG
SOOOSSRSSRSS

RC * ST * SG * RG * ST * ST * RA * ST *
TTATGACUC
RSSSSSS

SmG * SmA * SmC * SmU * SmC

WV-7772
rC rU rG rA rG rU rC rA rU rA rA rC rC rA
CUGAGUCAUAAC
OOOOOOOOOOOO

rG rC rC rU rG rG rC rA
CAGCCUGGCA
OOOOOOOOO

WV -9696
L001mU * SGeom5Ceom5CeomA * SG * SG
UGCCAGGCT
OSOOOSSRSSRS

* RC * ST * SG * RG * ST * ST * RA * ST *
GGTTATGACUC
SRSSSSSS

SmG * SmA * SmC * SmU * SmC

WV-11114
Mod091L001mU * SGeom5Ceom5CeomA *
UGCCAGGCT
OSOOOSSRSSRS

SG * SG * RC * sT * SG * RG * ST * ST *
GGTTATGACUC
SRSSSSSS

RA * ST * SmG * SmA * SmC * SmU * SmC

At a time point of 45 minutes, less than 20% of the Malat1 RNA remained in the presence of RNase H and WV-11533 or WV-8587, indicating greater than 80% knockdown; and about 60% of the Malat1 RNA remained in the presence of RNase H and WV-8556, which is stereorandom and does not comprise a neutral backbone. Among other things, the present disclosure demonstrates that oligonucleotides comprising non-negatively charged internucleotidic linkages and/or chirally controlled internucleotidic linkages showed significantly improved activities in reducing levels of target nucleic acids, e.g., through RNase H-mediated knockdown.

Certain oligonucleotides were also tested for stability in rat liver homogenate at 0, 1 and 2 days. For both WV-11533 and WV-8587, over 80% of the full-length oligonucleotide remained at 2 days; about 40% of the stereorandom WV-8556 remained.

Oligonucleotides were also tested for Tm with the Malat1 RNA, WV-7772. One example set of test conditions: 1 μM Duplex in 1×PBS (pH 7.2); Temperature Range: 15° C.-90° C.; Temperature Rate: 0.5° C./min; Measurement Interval: 0.5° C. The results showed the following duplex Tm (° C.) with WV-7772; WV-8556, 73.52; WV-8587, 69.57; and WV-11533, 68.67.

In some embodiments, oligonucleotides comprising non-negatively charged internucleotidic linkages provide improved splicing modulation activities. Various oligonucleotides for mediating skipping of an exon in DMD were prepared and/or tested, wherein the oligonucleotides comprise non-negatively charged internucleotidic linkages. Certain oligonucleotides comprising non-negatively charged internucleotidic linkages are listed in Table A1.

TABLE 25 A

Example data of certain oligonucleotides.

Oligonucleotide
10 uM

3 uM

WV-9898
27.13
13.38
11.27
9.69

WV-9897
33.61
31.46
11.82
9.52

WV-9517
20.21
12.08
6.72
6.89

WV-11342
44.84
41.17
19.22
18.43

WV-11341
38.85
44.85
18.95
20.63

WV-11340
41.51
43.08
17.79
16.4

PMO
3.89
4.05
2.08
1.52

Mock
0.49
0.53
0.45
0.52

Numbers indicate the level of exon skipping; e.g., 27.13 in column 2, row 2, represents 27.13% skipping of a DMD exon. Oligonucleotides were tested in vitro on cells at 10 or 3 uM.

TABLE 25B

Example data of certain oligonucleotides.

Mock
WV-11237
WV-3152
WV-3516
PMO

10
um
1
49
35
7
3

3
uM
1
22
16
3
2

Numbers indicate the level of exon skipping relative to control; numbers are approximate. Oligonucleotides were tested in vitro on cells at 10 or 3 uM.

PMO indicates an all-PMO oligonucleotide.

Various DMD oligonucleotides for skipping exon 23 in mouse were constructed, several of which comprise anon-negatively charged internucleotidic linkage, including WV-11343 WV-11344 WV-11345, WV-11346, and WV-11347. These oligonucleotides were tested and demonstrated skipping of exon 23, as shown in the table below.

TABLE 25C.1

Example data of certain oligonucleotides.

10 uM
3.3 uM

WV-7684
5
2

WV-10256
25
13

WV-11343
44
33

WV-10257
16
10

WV-11344
42
29

WV-10258
22
20

WV-11345
48
39

WV-10259
24
10

WV-11346
43
32

WV-10260
23
14

WV-11347
43
32

In some experiments de145-52 cells (patient derived myoblasts) were treated with various oligonucleotides, including WV-13405 (PMO), WV-9517 and WV-9898, in muscle differentiation medium at 15, 10, 3.3, 1.1, 0.3, 0.1 and 0 uM under free uptake conditions for 6 days before being collected and analyzed for dystrophin protein restoration by Western blot. WV-9517 and WV-9898 demonstrated significant DMD production at concentrations of 3.3 uM and higher; WV-13405 did not show significant DMD product at a concentration of 3.3 uM, but did show DMD production at concentrations of 10 and 15 uM. Control was Vinculin.

As shown in Table 25D, additional oligonucleotides were constructed which were capable of mediating skipping of exon 53 and which comprise at least one neutral internucleotidic linkage.

Various additional DMD oligonucleotides for skipping exon 23 in mouse were constructed. These oligonucleotides were tested and demonstrated skipping of exon 23, as shown in the table below.

TABLE 25C.2

Example data of certain oligonucleotides.

WV-11345
WV-24092
WV-24098
Mock

10 uM
37.8
39.8
30.2
32.4
41.5
40.2
0
0

3.3 uM
22.4
22.9
13.4
14.5
24.3
23.5
0
0

1.1 uM
9.2
8.1
3
3.1
10.5
9.9
0
0

DMD oligonucleotides were tested in vitro for their ability to skip DMD exon 23 in H2K murine cells. Oligonucleotide delivery was gymnotic, and 4 day treatment was used.

Numbers represent exon 23 skipping level relative to control. 100.0 would represent 100% of transcripts skipped; 0 would represent 0% of transcripts skipped. Data from replicates are shown.

TABLE 25C.4

Example data of certain oligonucleotides.

10 uM
3.3 uM
1.1 uM

WV-10258
22.9
11.6
3.8

WV-12885
34.2
17.8
6.1

32.4
18.6
6.9

WV-23576
23.7
10.6
3.8

25.6
11.5
3.3

WV-23577
23.3
13.9
6.6

WV-23578
22
11.8
4.9

16.1
13.9
7.1

WV-23579
19.2
8.3
6.7

20.7
29.8
5.5

WV-23937
18.8
9.2
3.5

6.3
4.2
1.3

WV-23938
26.4
16
6.9

30.3
16.7
7.3

WV-23939
35.2
23.3
11.8

33.6
22
12.9

Mock
0
0
0

0
0
0

TABLE 25C.4

Example data of certain oligonucleotides.

WV-
WV-
WV-
WV-

10258
25536
25537
25539
Mock

10 uM
22.9
2.3
10.7
11.8
15.1
12.5
8.1
0
0

3.3 uM
11.6
1.5
3.6
7.3
9.9
5.6
3.8
0
0

1.1 uM
3.8
1.1
1.3
2.7
4.2
1.8
2.3
0
0

DMD oligonucleotides were tested in vitro for their ability to skip DMD exon 23 in H2K murine cells. Oligonucleotide delivery was gymnotic, and 4 day treatment was used. Some of the tested oligonucleotides comprise one or more LNA.

Numbers represent exon 23 skipping level relative to control. 100.0 would represent 100% of transcripts skipped; 0 would represent 0% of transcripts skipped. Data from replicates are shown.

TABLE 25C.5

Example data of certain oligonucleotides.

10 uM
3.3 uM
1.1 uM

WV-
22.9
11.6
3.8

10258

WV-
37.8
22.4
9.2

11345
39.8
22.9
8.1

WV-
34.2
17.8
6.1

12885
32.4
18.6
6.9

WV-
23.7
10.6
3.8

23576
25.6
11.5
3.3

WV-
23.3
13.9
6.6

23577

WV-
22
11.8
4.9

23578
16.1
13.9
7.1

WV-
19.2
8.3
6.7

23579
20.7
29.8
5.5

WV-
18.8
9.2
3.5

23937
6.3
4.2
1.3

WV-
26.4
16
6.9

23938
30.3
16.7
7.3

WV-
35.2
23.3
11.8

23939
33.6
22
12.9

WV-
30.2
13.4
3

24092
32.4
14.5
3.1

WV-
41.5
24.3
10.5

24098
40.2
23.5
9.9

WV-
2.3
1.5
1.1

25536
10.7
3.6
1.3

WV-
11.8
7.3
2.7

25537
15.1
9.9
4.2

WV-
12.5
5.6
1.8

25539
8.1
3.8
2.3

Mock
0
0
0

0
0
0

DMD oligonucleotides were tested in vitro for their ability to skip DMD exon 23 in H2K murine cells. Oligonucleotide delivery was gymnotic, and 4 day treatment was used. Some of the tested oligonucleotides comprise one or more non-negatively charged internucleotidic link-age.

Numbers represent exon 23 skipping level relative to control. 100.0 would represent 100% of transcripts skipped, 0 would represent 0%10 of transcripts skipped. Data from replicates are shown.

TABLE 25C.6

Example data of certain oligonucleotides.

Conc.
WV-24104

WV-24109

−4.70927
0.891
0.837
0.814
1.059

−4.40824
0.942
1.052
0.765
1.208

−4.10721
0.948
1.030
0.754
1.104

−3.80618
0.855
1.143
0.792
1.059

−3.50515
1.067
1.234
0.831
0.891

−3.20412
0.797
0.968
0.760
1.045

−2.90309
0.968
0.825
0.675
1.067

−2.60206
0.825
1.016
0.765
1.135

−2.30103
1.059
0.872
0.648
0.613

−2
0.988
1.067
0.413
0.548

−1.70927
0.754
0.955
0.357
0.362

−1.69897
0.922
0.797
0.313
0.340

−1.40824
0.666
0.739
0.220
0.227

−1.10721
0.548
0.604
0.162
0.170

−0.80618
0.404
0.427
0.096
0.098

−0.50515
0.352
0.427
0.062
0.053

−0.20412
0.272
0.206
0.027
0.027

0.09691
0.132
0.103
0.013
0.014

0.39794
0.061
0.058
0.008
0.011

0.69897
0.028
0.032
0.007
0.008

1
0.018
0.019
0.008
0.009

1.30103
0.016
0.015
0.009
0.010

Oligonucleotides targeting Malat-1, wherein the oligonucleotides comprise a non-negatively charged internucleotidic linkage, were tested for their ability to knock down Malat-1 in GABA neurons in vitro, with 4 day treatment. Numbers represent Malat-1 level relative to HPRT1 control and water, wherein 1.0 would represent 100% Malat-1 level (0% knockdown) and 0 would represent 0% Malat-1 level (100% knockdown). Concentrations (Conc.) tested are provided as [Log (dose uM)].

Data from replicates are shown.

IC50 of WV-24104 was 132 nM; and IC50 of WV-24109 was 12 nM.

TABLE 25D

Example data of certain oligonucleotides.

10 uM
3 uM

mock
0.9
1.0
0.5
0.8
0.9
0.9
1.0
1.0

WV-9517
20.1
18.9
18.3
19.3
9.0
8.9
7.7
7.6

WV-11340
28.9
29.4
26.7
26.7
12.8
12.6
11.5
11.4

WV-11342
18.7
17.9
20.4
20.0
8.3
8.3
7.6
7.7

WV-12553
17.0
19.2
20.0
18.6
8.1
8.1
7.8
8.3

WV-12123
21.7
22.7
21.6
22.4
9.5
9.6
9.9
9.6

WV-12124
17.6
17.5
16.5
17.6
6.7
6.9
7.2
7.0

WV-12125
39.5
38.6
40.6
39.4
18.5
16.8
17.9
17.6

WV-12126
31.2
31.1
32.3
32.2
14.7
14.3
14.1
14.7

WV-12127
36.8
38.0
37.0
38.3
17.4
16.9
17.0
16.9

WV-12128
27.0
26.3
26.3
26.8
10.1
10.8
10.1
10.0

WV-12129
32.9
33.5
35.1
35.3
14.8
14.9
16.0
16.0

Mock
1.6
1.5
1.8
1.8
1.7
1.6
1.5
1.7

WV-9517
30.3
31.1
32.4
29.2
14.1
13.9
13.5
14.5

WV-11340
48.7
50.3
45.1
44.6
24.0
25.8
23.8
23.3

WV-12553
28.7
27.8
27.5
27.0
13.5
13.6
13.1
13.8

WV-9897
39.7
38.5
37.3
35.6
18.8
19.1
18.0
17.7

WV-11341
47.1
47.4

21.8
22.5
22.5
23.1

WV-12555
55.7
54.7
55.7
54.6
27.1
27.7
26.0
26.0

WV-12558
36.0
35.8
49.9
47.3
21.2
19.8
22.1
22.1

WV-9898
43.6
41.7
38.0
38.8
21.1
20.6

WV-11342
43.7
44.3
42.1
41.8
22.5
20.9
19.0
20.1

WV-12556
46.1
46.4
45.6
44.0
24.2
23.1
21.3
21.0

WV-12559
47.4
45.1
45.6
47.2
21.0
21.7
24.5
22.6

Mock
1.7
1.6
1.8
1.7
1.7
1.7
1.6
1.5

WV-9517
29.8
29.8
28.7
29.2
15.6
15.4
16.0
16.2

WV-11340
45.7
44.5
46.1
47.3
25.7
24.0
23.8
24.4

WV-11342
44.6
46.6
45.3
44.2
21.5
21.0
19.8
20.3

WV-12876
42.4
43.3
41.2
41.0
26.2
26.3
24.5
26.0

WV-12877
53.7
53.8
52.4
52.3
37.8
36.5
34.3
32.9

WV-12878
48.5
48.3
45.1
46.2
31.4
30.9
29.3
30.0

WV-12879
34.1
34.9
33.2
34.0
19.7
19.8
21.4
21.1

WV-12880
50.4
50.1
51.4
52.1
33.0
32.5
32.9
32.0

WV-12881
41.6
42.9
38.8
39.4
26.1
25.6
24.3
22.7

WV-12882
29.6
29.7
32.3
31.3
15.3
15.1
15.5
15.2

WV-12129
57.8
57.0
55.5
55.6
33.1
32.2

D45-52 myoblasts were treated for 4 days with 10 and 3 uM oligonucleotide.

Numbers in this and various other tables indicate amount of skipping relative to control.

Various DMD oligonucleotides comprising a chirally, controlled neutral backbone were constructed, including WV-12555, which comprises neutral internucleotidic linkage in the Rp configuration, and WV-12558, which comprises a neutral internucleotidic linkage in the Sp configuration. These were also tested for skipping a DMD exon, as shown in Table 25E.

TABLE 25E

Example data of certain oligonucleotides.

WV-
WV-
WV-
WV-
WV-
WV-

MOCK
9517
11340
9897
11341
12555
12558

10 uM
1.6
30.3
48.7
39.7
47.1
55.7
36.0

1.5
31.1
50.3
38.5
47.4
54.7
35.8

1.8
32.4
45.1
37.3

55.7
49.9

1.8
29.2
44.6
35.6

54.6
47.3

3 uM
1.7
14.1
24.0
18.8
21.8
27.1
21.2

1.6
13.9
25.8
19.1
22.5
27.7
19.8

1.5
13.5
23.8
18.0
22.5
26.0
22.1

1.7
14.5
23.3
17.7
23.1
26.0
22.1

D45-52 myoblasts were treated for 4 days with 10 and 3 uM oligonucleotide. Oligonucleotides were delivered gymnotically. Numbers represent amount of skipping relative to control.

In some embodiments, >2 fold increase in exon skipping efficiency was achieved.

TABLE 25F

Example data of certain oligonucleotides.

MDX mouse
Human
Human
Human

Muscle
Liver
Muscle
Kidney

WV-9517
82.4
77.8
84
73.7

3.08
7.9
2.01
3.59

WV-9897
88.3
82
96.1
75.2

9.12
4.2
5.5
3.8

WV-9898
74
75.8
96.8
81.5

5.07
6.4
8.9
5

WV-3473
69.8
69.8
ND
24

5.91
5.91
ND
0.15

Various DMD oligonucleotides for skipping exon 53 or 51 were incuted in tissue lysate for 5-days; full length oligonucleotides detected by LC-MS. Numbers represent percentage of full-length oligonucleotide remaining. Greater than 75% oligonucleotide remains inhuman and MDX muscle lysates at 5d incubation. Data was from a previous experiment performed for WV-3473, with 2d incubation in MDX muscle lysate. ND; Not determined; WV-3473 stability in human muscle lysate was not performed.

In some embodiments, an oligonucleotide comprising a neutral internucleotidic linkage (e.g., acyclic guanidine type) demonstrated a higher level of exon skipping than a corresponding oligonucleotide which did not comprise such a neutral internucleotidic linkage.

In some embodiments, the present disclosure pertains to an oligonucleotide or an oligonucleotide composition which is capable of mediating single-stranded RNA interference, wherein the oligonucleotide or oligonucleotide composition comprises a non-negatively charged internucleotidic linkage.

As described herein, various oligonucleotides comprising a non-negatively charged internucleotidic linkage and targeting any of several different genes, with different base sequences, patterns of sugar modifications, backbone chemistry, and patterns of stereochemistry of backbone internucleotidic linkages were constructed, including but not limited to various oligonucleotides which target C9orf72 (a different gene than DMD, or Malat).

Described herein are various non-limiting examples of oligonucleotides which target C9orf72 (which is a gene different from the other genes mentioned herein) and which comprise a non-negatively charged internucleotidic linkage.

A hexanucleotide repeat expansion in the C9orf72 gene (Chromosome 9, open reading frame 72) is reportedly the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). C9orf72 gene variants comprising the repeat expansion and/or products thereof are also associated with other C9orf72-related disorders, such as corticobasal degeneration syndrome (CBD), atypical Parkinsonian syndrome, olivopontocerebellar degeneration (OPCD), primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), Huntington's disease (HD) phenocopy, Alzheimer's disease (AD), bipolar disorder, schizophrenia, and other non-motor disorders. Various oligonucleotides were designed and constructed which comprise a neutral internucleotidic linkage and which target a C9orf72 target (e.g., a C9orf72 oligonucleotide) and are capable of knocking down or decreasing expression, level and/or activity of the C9orf72 target gene and/or a gene product thereof (a transcript, particularly a repeat expansion containing transcript, a protein, etc.).

Various oligonucleotides designed to target C9orf72 and comprising a non-negatively charged internucleotidic linkage include, but are not limited to: WV-11532, WV-13305, WV-13307, WV-13309, WV-13311, WV-13312, WV-13313, WV-13803, WV-13804, WV-13805, WV-13806, WV-13807, WV-13808, WV-14553, and WV-14555. These are described below in Table 25G.

TABLE 25G

Oligonucleotides targeting C9orf72 comprising a neutrai intemucleotidic linkage.

Oligo-

nucleo-

tide
Sequence
Naked Sequence
Stereochemistry

WV-
mC * Sm5Ceon001 Teon001 m5Ceon001
CCTCACTCACCC
SnXnXnXSSSRSSR

11532
mA * SC * ST * SC * RA * SC * SC * RC
ACTCGCCA
SSSSSSSS

* SA * Se * ST * SmC * SmG * SmC *

SmC * SmA

WV-
m5Ceo * Rm5Ceon001 Teon001
CCTCACTCACCC
RnXnXnXRSSRSSR

13305
m5Ceon001 Aeo * RC * ST * sC * RA *
ACTCGCCA
SSSSSSSS

SC * SC * RC * SA * SC * ST * SmC*

SmG * SmC * SmC * SmA

WV_
m5Ceo * Sm5Ceon001 Teon001
CCTCACTCACCC
SnXnXnXRSSRSSR

13307
m5Ceon001 Aeo * RC * ST * SC * RA *
ACTCGCCA
SSSSSSSS

SC * SC * RC * SA * Sc * ST * SmC *

SmG * SmC * SmC * SmA

WV_
m5Ceo * Rm5Ceon001 Teon001
CCTCACTCACCC
RnXnXnXRSSRSSS

13309
m5Ceon001 Aeo * RC * ST * SC * RA *
ACTCGCCA
RSSSSSSS

SC * Sc * SC * RA * SC * ST * SmC *

SmG * SmC * SmC * SmA

WV-
m5Ceo * Sm5Ceon001 Teon001
CCTCACTCACCC
SnXnXnX.RSSRSSS

13311
m5Ceon001 Aeo * RC * ST * SC * RA *
ACTCGCCA
RSSSSSSS

SC * SC * SC * RA * SC * ST * SmC *

SmG * SmC * SmC * SmA

WV-
mC * Sm5Ceon001 Teon001 m5Ceon001
CCTCACTCACCC
SnXnXnXSSSR

13312
mA * SC * ST * SC * RA * SC * SC * SC
ACTCGCCA
SSSSSSSSSSS

* SA * SC * ST * SmC * SmG * SmC *

SmC * SmA

WV-
m5Ceo * Rm5Ceon001 Teon001
CCTCACTCACCC
RnXnXnXRSSR

13313
m5Ceon001 Aeo * RC * ST * SC * RA *
ACTCGCCA
SSSSSSSSSSS

Sc * SC * SC * SA * SC * ST * SmC *

SmG
* SmC * SmC *
SmA

WV-
Teo * Geon001 m5Ceon001 m5Ceon001
TGCCGCCTCCT
XnXnXnXXXXXXX

13803
Geo*C*C*T*C*C*I*C*A*
CACTCACCC
XXXXXXXXX

T * mC * mA * mC * mC * mC

WV-
Teo * Geom5Ccom 5CcoGeo * C * C * T
TGCCGCCTCCT
XOOOXXXXXXXXX

13804
* C * C * T * C * A * C * T *mCn001
CACTCACCC
XXnXnXnXX

mAn001 mCn001 mC * mC

WV-
Teo * Geon001 m5Ceon001 m5Ceon001
TGCCGCCTCCT
XnXnXnXXXXXXXX

13805
Geo * C * C * T * C * C * T * C * A * C *
CACTCACCC
XXXXnXnXnXX

T * mCn001 mAn001 mCn001 mC * mC

WV-
Geo * m5Ceon001 Geon001 m5Ceon001
GCGCGACTCCT
XnXnXnXXXXXXXX

13806
Geo * A * C * T * C * C * T * G* A * G
GAGTTCCAG
XXXXOOOX

* T * Teom5Ceom5CeoAeo * Geo

WV-
Geo * m5CeoGeom5CeoGeo * A * C * T
GCGCGACTCCT
XOOOXXXXXXXXXX

13807
* C * C * T * G * A * G * T * Teon001
GAGTTCCAG
XnXnXnXX

m5Ceon001 m5Ceon001 Aeo * Geo

WV-
Geo * m5Ceon001 Geon001 m5Ceon001
GCGCGACTCCT
XnXnXnXXXXXXXXX

13808
Geo * A * C * T * C * C * T * G * A * G
GAGTTCCAG
XXXnXnXnXX

* T * Teon001 m5Ceon001 m5Ceon001

Aeo * Geo

WV-
m5Ceo* Rm5Ceon001 Teon001
CCTCACTCACCC
RnXnXnXRSSRSSR

14553
m5Ceon001 Aeo * RC * ST * SC * RA *
ACTCGCCA
SSSRSSSS

SC * SC * RC * SA * SC* ST * Rm5Ceo

* SmG * SmC * SmC * SmA

WV-
m5Ceo* Rm5Ceon001 Teon001
CCTCACTCACCC
RnXnXnXRSSRSSS

14555
m5Ceon001 Aeo * RC * ST * SC * RA *
ACTCGCCA
RSSRSSSS

SC * SC * SC * RA * SC * ST * Rm5Ceo

* SmG * SmC * SmC * SmA

Several variants of a C9orf72 mRNA are produced from the C9orf72 gene: V2 (which does not comprise the deleterious hexanucleotide repeat and which comprises about 90% of all transcripts); V3 (which comprises the hexanucleotide repeat and comprises about 9% of all transcripts); and V I (which comprises the hexanucleotide repeat and comprises about 1% of all transcripts).

Hexanucleotide repeats reportedly elicit gain of function toxicities, at least partially mediated by the dipeptide repeat proteins and foci formation by, for example, repeat-expansion containing transcripts and/or spliced-out repeat-expansion containing introns and/or antisense transcription of the repeat-expansion containing region and various nucleic-acid binding proteins.

Both WV-8008 and WV-11532 have the same base sequence (or naked sequence). CCTCACTCACCCACTCGCCA. They differ, inter alia, in that the latter comprises 3 contiguous neutral internucleotidic linkages (Xn), but the former does not comprise any neutral internucleotic linkages. The structures of these oligonucleotides is provided below, in Table 25H.

TABLE 25H

C9orf72 oligonucleotides.

Oligo-

nucleotide
Sequence
Stereochemistry

WV-8008
m5Ceo * Rm5CeoTeom5CeoAeo * RC * ST * SC * RA * SC * SC
ROOORSSRSSRS

* RC * SA * SC * ST * SmC * SmG * SmC * SmC * SmA SSSSSSS

WV-11532
mC * Sm5Ceon001Teon001m5Ceon001mA * SC * ST * SC * RA
SnXnXnXSSSRSS

* SC * SC * RC * SA * SC * ST * SmC * SmG * SmC * SmC *
RSSSSSSSS

SmA

,

WV-8008 and WV-11532 were tested for their ability to knock down expression of hexanucleotide-comprising (i.e., disease-associated) transcript V3 compared to total transcripts (all V), as shown below in Table 25I.

Table 25I and J. Activity of various c9orf72 oligonucleotides.

In Tables 25I to 25J, various c9orf72 oligonucleotides were tested in motor neurons, with oligonucleotides delivered gymnotically at concentrations from 0.003 to 10 μM (Concentrations are provided as exp10). Tested c9orf72 oligonucleotide WV-11532 comprises three neutral internucleotidic linkages. In Tables 14A and 14B, shown are residual levels of c9orf72 transcriptions [e.g., all transcripts (all V) or only V3] relative to HPRT1, after treatment with c9orf72 oligonucleotides, wherein 1.000 would represent 100% relative transcript level (no knockdown) and 0.000 would represent 0% relative transcript level (e.g., 100% knockdown). Results from replicate experiments are shown.

TABLE 25I

Activity of various c9orf72 oligonucleotides

(residual level of all V C9orf72 transcripts)

Conc.
WV-8008
WV-11532

−2.495
0.999
0.958
0.913
1.006
0.894
0.900

−1.796
0.965
0.864
0.882
0.972
0.829
0.858

−1.097
1.006
0.900
0.932
0.907
0.888
0.858

−0.398
0.800
0.742
0.806
0.795
0.747
0.742

0.301
0.624
0.611
0.687
0.562
0.554
0.554

1
0.524
0.500
0.521
0.409
0.411
0.387

TABLE 25J

Activity of various c9orf72 oligonucleotides

(residual level of V3 C9orf72 transcripts)

Conc.
WV-8008
WV-11532

−2.495
0.947
0.871
1.014
0.927
0.853
0.908

−1.796
0.877
0.841
0.908
0.836
0.769
0.841

−1.097
0.665
0.743
0.871
0.620
0.633
0.717

−0.398
0.555
0.427
0.707
0.421
0.415
0.427

0.301
0.210
0.178
0.304
0.096
0.105
0.094

1
0.056
0.071
0.083
0.012
0.015
0.015

As described herein and in data not shown, various oligonucleotides comprising a non-negatively charged internucleotidic linkage and targeting different genes, with different base sequences, patterns of sugar modifications, backbone chemistries, and patterns of stereochemistry of backbone internucleotidic linkages were constructed, including but not limited to various oligonucleotides which target DMD, Malat1, or C9orf72.

Oligonucleotides comprising a non-negatively charged internucleotidic linkage were also constructed to target six other genes not described herein (wherein the six genes were not DMD, Malat1, or C9orf72); these oligonucleotides include oligonucleotides designed to target these genes and reduce the expression, level and/or activity of the gene or its gene product. These and various oligonucleotides comprising a neutral internucleotidic linkage described herein are capable of performing various functions, including reducing the level, expression and/or activity of a gene or its gene product (e.g., via a RNaseH- or steric-hindrance-mediated mechanism, or via a single-stranded RNA interference-mediated mechanism) and inducing skipping of an exon (e.g., skipping modulation).

Without wishing to be bound by any particular theory, Applicant notes that a non-negatively charged and/or neutral internucleotidic linkage can improve an oligonucleotide's entry into a cell and/or escape from an endosome.

Oligonucleotides which Comprise a Non-Negatively Charged Internucleotidic Linkage can Provide Desired Levels of TLR9 Activation

Among other things, oligonucleotides comprising non-negatively charged internucleotidic linkages can provide desired levels of properties and/or activities, e.g., TLR9 antagonist or agonist activities. In some embodiments, oligonucleotides comprising non-negatively charged internucleotidic linkages demonstrate lower levels of TLR9 activation in human and/or an animal model (e.g., a mouse) compared to certain comparable oligonucleotides of the same base sequences but having no non-negatively charged internucleotidic linkages. In some embodiments, oligonucleotides comprising non-negatively charged internucleotidic linkages have lower toxicity compared to certain oligonucleotides of the same base sequences but having no non-negatively charged internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic linkage is within a CpG motif and is the internucleotidic linkage between the C and G.

In an experiment, several oligonucleotides to target gene C were constructed. Gene C is a different gene than DMD, or SMalat-1. The sequence of these oligonucleotides comprises a CpG, a motif known to activate TLR9.

Table 25K.

This experiment represents a test of induction of human TLR9 or mouse TLR9 in HEK293 cells. Numbers represent relative inductive relative to negative control, water. Concentrations tested: 0.93 uM, 2.77 uM, 8.33 uM, 25 uM, 75 uM. Positive control: WV-BZ21. The experiment was performed in biological duplicates.

TABLE 25K

Oligonucleotides used in this study

Oligo-

nucleotide
Sequence
Stereochemistry

WV-HZ12
mN * Sm5NeoNeom5NeomN * SN * SN * SN * RN * SN * SN *
SOOOS SSRSS

RN * SN * SN * SN * SmC * SmG * SmN * SmN * SmN
RSSSSSSSS

WV-BZ761
mN * Sm5NeoNeom5NeomN * SN * SN * SN * RN * SN * SN *
SOOOS SSRSS

RN * SN * SN * SN * SmCmG * SmN * SmN * SmN
RSSSSOSSS

WV-BZ762
mN * Sm5NeoNeom5NeomN * SN * SN * SN * RN * SN * SN *
SOOOS SSRSS

RN * SN * SN * SN * Sm5CeomG * SmN * SmN * SmN
RSSSSOSSS

WV-BZ763
mN * Sm5NeoNeom5NeomN * SN * SN * SN * RN * SN * SN *
SOOOS SSRSS

RN * SN * SN * SN * Sm5Ceo * SmG * SmN * SmN * SmN
RSSSSSSSS

WV-BZ764
mN * Sm5NeoNeom5NeomN * SN * SN * SN * RN * SN * SN *
SOOOS SSRSS

RN * SN * SN * SN * Rm5CeomG * SmN * SmN * SmN
RSSSROSSS

WV-BZ765
mN * Sm5NeoNeom5NeomN * SN * SN * SN * RN * SN * SN *
SOOOS SSRSS

RN * SN * SN * SN * Rm5Ceo * SmG * SmN * SmN * SmN
RSSSRSSSS

WV-BZ766
mN * Sm5NeoNeom5NeomN * SN * SN * SN * RN * SN * SN *
SOOOS SSRSS

RN * SN * SN * SN * Sm5mC * StnG * SmN * SmN * SmN
RSSSSSSSS

WV-BA207
mN * Sm5NeoNeom5NeomN * SN * SN * SN * RN * SN * SN *
SOOOS SSRSS

SN * RN * SN * SN * SmCn001mG * SmN * SmN * SmN
SRSSSnXSSS

WV-BA208
m5Neo * Rm5NeoNeom5NeoNeo * RN * SN * SN * RN * SN *
ROOOR SSRSS

SN * RN * SN * SN * SN * SmCn001mG * SmN * SmN * SmN
RSSSSnXSSS

WV-BA209
m5Neo * Rm5NeoNeom5NeoNeo * RN * SN * SN * RN * SN *
ROOOR SSRSS

SN * SN * RN * SN * SN * SmCn001mG * SmN * SmN * SmN
SRSSSnXSSS

WV-BZ21
T * C * G * T * C * G * T * T * T * T * G * T * C * G * T * T * T
XXXXX XXXXX

* T * G * T * C * G * T * T
XXXXX XXXXX

XXX

TABLE 25L

Activity of certain oligonucleotides.

0.93 uM
2.77 uM
8.33 uM
25 uM
75 uM

WV-HZ12
1.0
1.0
1.0
1.0
0.9

1.1
1.0
1.1
1.0
1.0

WV-BZ761
1.0
1.0
1.0
1.0
1.0

1.1
1.0
1.1
1.0
0.9

WV-BZ762
1.0
1.0
1.0
1.1
1.0

1.0
1.1
1.0
1.0
1.0

WV-BZ763
1.0
1.0
1.1
1.1
1.1

1.1
1.1
1.1
1.1
1.0

WV-BZ764
1.0
1.0
1.0
0.9
1.0

1.0
1.0
1.0
1.0
1.0

WV-BZ765
1.0
0.9
1.1
1.0
1.0

1.0
1.1
1.0
0.9
0.9

WV-BZ766
1.1
1.3
1.5
1.5
1.5

1.2
1.3
1.3
1.4
1.4

WV-BA207
1.0
1.0
1.0
1.0
1.0

1.1
1.1
1.0
1.0
1.0

WV-BA208
1.0
1.0
1.0
1.0
1.0

1.0
1.1
1.0
0.9
1.0

WV-BA209
1.0
1.0
1.0
0.9
1.0

1.1
1.0
0.9
1.0
1.0

WV-BZ21
10.0
12.0
12.0
11.4
11.0

(positive
9.4
10.4
11.4
11.5
11.1

control)

All the tested oligonuclotides (WV-HZ12, WV-BZ761, WV-BZ762, WV-BZ763, WV-BZ764, WV-BZ765, WV-BZ766 WV-BA207, WV-BA208, and WV-BA209) target gene C and all have the same base sequence, wherein each base is indicated generically by N, except that the single CpG motif is indicated. WV-BZ21, positive control, has abase sequence of TCGTCGTTTTGTCGTTTTGTCGTT, which comprises several CpG motifs, and is not designed to target gene C. Numbers indicate relative induction of hTLR9 activity relative to water.

TABLE 25M

Activity of certain oligonucleotides.

0.93 uM
2.77 uM
8.33 uM
25 uM
75 uM

WV-HZ12
2.9
4.4
4.7
5.0
4.9

3.0
4.1
4.8
5.1
5.2

WV-BZ761
1.2
1.5
1.8
2.1
2.1

1.2
1.4
1.8
2.1
2.2

WV-BZ762
1.0
1.0
1.0
1.0
1.0

1.0
1.1
1.1
0.9
1.0

WV-BZ763
1.0
1.1
1.1
1.1
1.0

1.1
1.0
1.1
1.1
1.1

WV-BZ764
1.0
1.1
1.1
1.1
1.1

1.0
1.1
1.1
1.1
1.1

WV-BZ765
1.0
1.2
1.3
1.3
1.2

1.1
1.2
1.3
1.3
1.3

WV-BZ766
1.1
1.3
1.4
1.6
1.6

1.1
1.2
1.4
1.6
1.6

WV-BA207
1.1
1.1
1.1
1.1
1.1

1.0
1.0
1.1
1.1
1.2

WV-BA208
1.0
1.1
1.1
1.2
1.1

1.0
1.0
1.1
1.2
1.2

WV-BA209
1.0
1.2
1.1
1.2
1.1

1.0
1.1
1.2
1.2
1.3

WV-BZ21
21.4
22.4
22.9
21.2
18.1

(positive
22.9
24.0
23.8
22.3
18.9

control)

These oligonucleotides were also tested for induction of mouse TLR9.

Numbers indicate relative induction of mTLR9 activity relative to water.

In some embodiments, it was observed that in some instances certain oligonucleotides that did not induce appreciable TLR9 activation, or induced very low level of TLR9 activation above mock against human or mouse TLR9.

Example Oligonucleotides Comprising Additional Moieties

In some embodiments, the present disclosure provides oligonucleotides comprising one or more additional moieties, e.g., targeting moieties, carbohydrate moieties, etc. In some embodiments, the present disclosure provides oligonucleotides comprising one or more sulfonamide moieties. In some embodiments, a provided oligonucleotide comprise one or two or more sulfonamide moieties. In some embodiments, the present disclosure provides oligonucleotides that can modulate splicing, e.g., DMD oligonucleotides that can modulate exon skipping, wherein the oligonucleotides comprise one or more sulfonamide moieties. In some embodiments, the present disclosure provides oligonucleotides that mediate skipping of DMD exon 23, 45, 51 or 53, or multiple DMD exons, wherein the oligonucleotides comprise one or more sulfonamide moieties.

In some embodiments, a sulfonamide moiety has or comprises the structure of -L-SO₂N(R′)₂. In some embodiments, a sulfonamide moiety has or comprises the structure of —SO₂N(R′)₂. In some embodiments, a sulfonamide moiety has or comprises the structure of -Cy-SO₂N(R′)₂. In some embodiments, -Cy- is aromatic. In some embodiments, -Cy- is an optionally substituted phenyl ring. In some embodiments, -Cy- is

embedded image

In some embodiments, -Cy- is an optionally substituted heteroaryl ring. In some embodiments, -Cy- is an optionally substituted 5-6 membered heteroaryl ring having 1-4 heteroatoms. In some embodiments, -Cy- is

embedded image

In some embodiments, each R¹is —H.

A sulfonamide moiety can be connected to an oligonucleotide chain via various suitable linkers in accordance with the present disclosure, such as those described herein and/or in WO/2017/062862, linkers of which is incorporated herein by reference. Example sulfonamides moieties,

embedded image

In some embodiments, an oligonucleotide comprise a modified internucleotidic linkage and a sulfonamide moiety optionally through a linker. In some embodiments, an oligonucleotide comprising a modified internucleotidic linkage and a sulfonamide moiety is a siRNA, double-straned siRNA, single-stranded siRNA, gapmer, skipmer, blockmer, antisense oligonucleotide, antagomir, microRNA, pre-microRNs, antimir, supermir, ribozyme, U1 adaptor, RNA activator, RNAi agent, decoy oligonucleotide, triplex forming oligonucleotide, aptamer or adjuvant. In some embodiments, the present disclosure provides an oligonucleotide which comprises a modified internucleotidic linkage which comprises a sulfonamide. In some embodiments, an oligonucleotide comprises a sulfonamide and a chirally controlled internucleotidic linkage. In some embodiments, an oligonucleotide comprises a sulfonamide and a chirally controlled internucleotidic linkage which is a phosphorothioate internucleotidic linkage.

In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a sulfonamide moiety or a derivative or variant thereof. In some embodiments, the present disclosure pertains to an oligonucleotide composition, wherein the oligonucleotide comprises a sulfonamide moiety or a derivative or variant thereof and the oligonucleotide comprises at least one chirally controlled internucleotidic linkage.

In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a sulfonamide moiety or a derivative or variant thereof, wherein the oligonucleotide is capable of mediating modulation of exon skipping of a target gene. In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a sulfonamide moiety or a derivative or variant thereof, wherein the oligonucleotide is capable of increasing skipping of an exon of a target gene.

Example oligonucleotides that can be utilized for splicing modulation, e.g., exon skipping, that comprise a sulfonamide moiety include WV-3548. WV-3366, etc. Other oligonucleotides comprising a sulfonamide moiety were designed, constructed and/or tested for various activities. For example, oligonucleotides comprising a “mono-sulfonamide” moiety, such as WV-2836, WV-7419 WV-7421, WV-7422, WV-7408, WV-7409, WV-7427, WV-7863, and WV-7864; oligonucleotide comprising a “bi-sulfonamide”, WV-7423; and oligonucleotide comprising a “tri-sulfonamide”, WV-7417.

TABLE 26A

Certain Malat1 oligonucleotides.

Oligo-

Linkage/

nucleotide
Description
Naked Sequence
Stereochemistry

WV-2735
Geo * Geo * Geo * Teo * m5Ceo * A *
GGGTCAGCTG
XXXXXXXXXXX

G*C*T*G*C*C*A*A*T* Geo
CCAATGCTAG
XXXXXXXX

* m5Ceo * Teo * Aeo * Geo

WV-2835
Mod027L001 * Geo * Geo * Geo * Teo *
GGGTCAGCTGC
XXXXXXXXXXX

m5Ceo *A*G*C*T*G*C*C*A
CAATGCTAG
XXXXXXXXX

* A * T * Geo * m5Ceo * Teo * Aeo *

Geo

WV-2836
Mod028L001 * Geo * Geo * Geo * Teo *
GGGTCAGCTGC
XXXXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A
C AATGCTAG
XXXXXXXXX

* A * T * Geo * m5Ceo * Teo * Aeo *

Geo

WV-3174
mU * mG * mC * mC * mA * G * G * C
UGCCAGGCTGG
XXXXXXXXXXX

* T * G * G * T * T * A * T * mG * mA
T TATGACUC
XXXXXXXX

* mC * mU * mC

WV-7301
Teo * Geo * m5Ceo * m5Ceo * Aeo * G
TGCCAGGCTGG
XXXXXXXXXXX

* G * C * T * G * G * T * T * A * T *
T TATGACTC
XXXXXXXX

Geo * Aeo * m5Ceo * Teo * m5Ceo

WV-7408
Mod027L00lGeo * Geo * Geo * Teo *
GGGTCAGCTGC
OXXXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A
CAATGCTAG
X XXXXXXXX

* A * T * Geo * m5Ceo * Teo * Aeo *

Geo

WV-7409
Mod028L001Geo * Geo * Geo * Teo *
GGGTCAGCTGC
OXXXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A
C AATGCTAG
X XXXXXXXX

* A * T * Geo * m5Ceo * Teo * Aeo *

Geo

WV-7417
Mod029L001 * Geo * Geo * Geo * Teo *
GGGTCAGCTGC
XXXXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A
CAATGCTAG
XXXXXXXXX

* A * T * Geo * m5Ceo * Teo * Aeo *

Geo

WV-7419
Mod045L001 * Geo * Geo * Geo * Teo *
GGGTCAGCTGC
XXXXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A
CAATGCTAG
XXXXXXXXX

A * T * Geo * m5Ceo * Teo * Aeo *

Geo

WV-7421
Mod047L001 * Geo * Geo * Geo * Teo *
GGGTCAGCTGC
XXXXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A
CAATGCTAG
XXXXXXXXX

* A * T * Geo * m5Ceo * Teo * Aeo *

Geo

WV-7422
Mod048L001 * Geo * Geo * Geo * Teo *
GGGTCAGCTG
XXXXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A
CCAATGCTAG
XXXXXXXXX

* A * T * Geo * m5Ceo * Teo * Aeo *

Geo

WV-7423
Mod049L001 * Geo * Geo * Geo * Teo *
GGGTCAGCTG
XXXXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A
CCAATGCTAG
XXXXXXXXX

* A * T * Geo * m5Ceo * Teo * Aeo *

Geo

WV-7427
Mod045L001Geo * Geo * Geo * Teo *
GGGTCAGCTG
OXXXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A
CCAATGCTAG
XXXXXXXXX

* A * T * Geo * m5Ceo * Teo * Aeo *

Geo

WV-7863
Mod046L001Geo * Geo * Geo * Teo *
GGGTCAGCTG
OXXXXXXXXXX

m5Ceo *A * G * C * T * G * C * C A
CCAATGCTAG
XXXXXXXXX

A * T * Geo * m5Ceo * Teo * Aeo *

Geo

WV-7864
Mod054L001Geo * Geo * Geo * Teo *
GGGTCAGCTG
OXXXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A
CCAATGCTAG
X XXXXXXXX

* A * T * Geo * m5Ceo * Teo * Aeo *

Geo

WV-9430
Mod029L001mU * mG * mC * mC *
UGCCAGGCTG
OXXXXXXXXX

mA * G * G * C * T * G * G * T * T * A
GTTATGACUC
XXXXXXXXXX

* T * mG * mA * mC * mU * mC

WV-7420
Mod046L001 * Geo * Geo * Geo * Teo *
GGGTCAGCTG
XXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A
CCAATGCTAG
XXXXXXXXXXX

* A * T * Geo * m5Ceo * Teo * Aeo *

Geo

For this Table, descriptions match those of Table A1, and

embedded image

In these Mods, —C(O)— connects to —NH— of a linker (e.g., L001).

Oligonucleotides comprising a sulfonamide moiety were tested for their ability to knockdown Malat1. Tested oligonucleotides were gymnotically delivered to Δ48-50 patient derived myotubes, which were dosed at 3.1, 0.3 and 0.1 μM concentrations. Cells were allowed to differentiate for 4 days (e.g., this experiment was 4 days post-differentiation). qPCR was used to evaluate knockdown of Malat-1. The results are shown in Table 26B.

TABLE 26B

Example data of Malat1 oligonucleotides.

WV-
WV-
WV-
WV-
WV-
WV-
WV-
WV-

3174
8927
8929
8930
8931
8934
9385
9390
Mock

3 μM
10

11
10
11
9
8
33
95

1 μM
18

2.8
24
22
19
20
49
100

0.3 μM
39
56
50
67
46
42
43
67
95

0.1 μM
63
73
68
81
68
69
56
81
100

Numbers represent relative Malat-1 mRNA level.

Various Malat1 oligonucleotides, many comprising a sulfonamide moiety, were tested for their ability to knockdown Malat1 in pre-differentiated myotubes. Certain data are shown in Table 26C. A48-50 patient derived myoblasts were differentiated for 4 days prior to dosing with at 1 and 0.1 M concentrations. RNA was harvested 48 hours post-treatment for measurement.

TABLE 26C

Example data of Malat1 oligonucleotides.

WV-
WV-
WV-
WV-
WV-
WV-
WV-
WV-

3174
8927
8929
8930
8931
8934
9385
9390

1 μM

31
25
25
36
24
18
45

0.1 μM
62
70
79
72
78
55
59
66

WV-
WV-
WV-
WV-

8448
7558
7559
7560
MOCK

1 μM
33
34
22
23
98

0.1 μM
68
72
69
82
98

Numbers represent relative Malat-1 mRNA level. Numbers are approximate.

In some experiments, animals were dosed with oligonucleotides, including some which comprise a sulfonamide moiety, and the animals were later sacrificed and their tissues tested for the level of the oligonucleotides.

In some experiments, the following protocol was used: Animals: 32 male Mdx mice and 32 male C57BL/6 mice (all 8-10 week-old). Test animals were acclimated to the facility for at least 3 days upon arrival. Dosing: S. C. (subcutaneous) dosing on days 1, 3 and 5 (5 mL/kg). Necropsy: animals were euthanized 72 hours after the last SC injection. All animals were perfused with PBS. The following tissues were collected: brain, sciatic nerves, spinal cord, eyes, liver, kidney, spleen, heart, diaphragm, gastrocnemius, quadriceps and triceps, white fat, brown fat. Fresh tissues will be rinsed briefly with PBS, gently blotted dry, weighed and snap frozen in Liquid Nitrogen in 2-mL tubes and stored at −80C (on dry ice). Histology: Quadricep and Kidney postfixed in 10% Formalin and processed to slides (paraffin embedded sections). In some experiments, suitable variants of this protocol were used.

Certain results are shown in Tables 27, 28 and 29.

TABLE 27

Knock-down and oligonucleotide presence in various tissues.

Heart pK

Malat1
Quadriceps pD
Triceps pD
Gastro pD
Diaphragm pD
Heart pD
Mean ± SD

Sequence
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD
(ug/g)

PBS
1.000 ±
1.000 ±
1.000 ±
1.000 ±
1.000 ±
0.000 ±

0.142
0.265
0.042
0.276
0.074
0.000

WV-2735
0.776 ±
0.699 ±
0.731 ±
0.879 ±
0.707 ±
1.631 ±

0.122
0.150
0.107
0.158
0.173
0.692

WV-2835
0.639 ±
0.588 ±
0.417 ±
0.895 ±
0.510 ±
1.987 ±

0.119
0.036
0.065
0.116
0.066
0.203

WV-2836
0.621 ±
0.834 ±
0.616 ±
0.769 ±
0.619 ±
7.001 ±

0.124
0.206
0.169
0.229
0.389
1.331

Numbers indicate Malat1 mRNA levels relative to mHprt (mHPRT or mHPRT1), and presence of oligonucleotide (ug/g). Experimental procedure: Study Species: 5-6 wks MDX mice: Route: Subcutaneous; # Doses: QD for 3 days; Time Point Post Last Dose: 2 days: Daily Dose Level (ug): 12.5 mg/kg.

TABLE 28

Knock-down and oligonucleotide presence in various tissues.

Oligo-
Quadriceps pD
Triceps pD
Gastro pD
Diaphragm pD
Heart pD

nucleotide
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD

PBS
1.000 ± 0.266
1.000 ± 0.207
1.000 ± 0.138
1.000 ± 0.191
1.000 ± 0.221

WV-2735
0.952 ± 0.232
0.876 ± 0.180
0.998 ± 0.072
0.651 ± 0.046
1.032 ± 0.541

WV-2835
0.593 ± 0.167
0.877 ± 0.180
0.645 ± 0.124
0.563 ± 0.091
1.032 ± 0.240

WV-2836
0.556 ± 0.172
0.739 ± 0.047
0.695 ± 0.102
0.614 ± 0.120
0.544 ± 0.109

WV-3174
0.610 ± 0.109
1.009 ± 0.047
0.809 ± 0.137
0.698 ± 0.069
0.588 ± 0.258

WV-7301
0.624 ± 0.074
0.846 ± 0.172
0.837 ± 0.141
0.453 ± 0.031
0.887 ± 0.142

Quadriceps pK
Diaphragm pK
Heart pK

Oligo-
Mean ± SD
Mean ± SD
Mean ± SD

nucleotide
(ug/g)
(ug/g)
(ug/g)

PBS
0.000 ± 0.000
0.096 ± 0.015
0.000 ± 0.000

WV-2735
5.616 ± 2.724
3.207 ± 1.465
0.342 ± 0.169

WV-2835
8.421 ± 3.374
5.734 ± 1.465
0.777 ± 0.203

WV-2836
11.221 ± 7.877
6.142 ± 1.006
0.664 ± 0.441

WV-3174
9.792 ± 8.339
4.609 ± 1.006
0.619 ± 0.122

WV-7301
6.659 ± 3.858
5.728 ± 2.092
0.707 ± 0.191

Numbers indicate Malat1 mRNA levels relative to mHprt, and presence of oligonucleotide (ug/g). Experimental procedure: Study Species: 10-12 wks MDX mice; Route: Subcutaneous; # Doses: QD for 3 days; Time Point Post Last Dose: 3 days; and Daily Dose Level (ug): 12 mg/kg.

TABLE 29

Knock-down and oligonucleotide presence in various tissues.

Oligo-
Quadriceps pD
Triceps pD
Gastro pD
Diaphragm pD
Heart pD

nucleotide
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD

PBS
1.000 ± 0.266
1.000 ± 0.191
1.000 ± 0.249
1.000 ± 0.191
1.000 ± 0.147

WV-2735
0.753 ± 0.230
0.667 ± 0.132
0.756 ± 0.136
0.651 ± 0.046
0.596 ± 0.140

WV-2835
0.611 ± 0.165
0.549 ± 0.077
0.656 ± 0.101
0.563 ± 0.091
0.546 ± 0.092

WV-2836
0.640 ± 0.186
0.596 ± 0.114
0.812 ± 0.216
0.614 ± 0.120
0.774 ± 0.168

WV-3174
0.796 ± 0.142
0.610 ± 0.111
0.870 ± 0.081
0.698 ± 0.069
0.703 ± 0.099

WV-7301
0.456 ± 0.116
0.498 ± 0.097
0.753 ± 0.113
0.453 ± 0.031
0.368 ± 0.031

Quadriceps pK
Diaphragm pK
Heart pK

Oligo-
Mean ± SD
Mean ± SD
Mean ± SD

nucleotide
(ug/g)
(ug/g)
(ug/g)

PBS
0.000 ± 0.000
0.108 ± 0.016
0.000 ± 0.000

WV-2735
2.787 ± 0.734
9.219 ± 3.234
0.428 ± 0.084

WV-2835
2.700 ± 0.891
9.895 ± 2.466
0.726 ± 0.207

WV-2836
2.273 ± 0.621
9.751 ± 6.912
0.670 ± 0.242

WV-3174
2.142 ± 0.778
7.568 ± 1.807
0.612 ± 0.172

WV-7301
2.868 ± 0.334
6.174 ± 2.456
0.975 ± 0.216

Numbers indicate Malat1 mRNA levels relative to mHprt, and presence of oligonucleotide (ug/g). Experimental procedure: Study Species: 10-12 wks wt mice; Route: Subcutaneous; # Doses: QD for 3 days; Time Point Post Last Dose: 3 days; and Daily Dose Level(ug): 12 mg/kg.

TABLE 30

Knock-down and oligonucleotide presence in various tissues.

Malat1
Quadriceps pD
Gastro pD
Diaphragm pD
Heart pD

Sequence
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD

PBS
1.000 ± 0.256
1.000 ± 0.309
1.000 ± 0.345
1.000 ± 0.432

WV-3174
0.752 ± 0.118
0.833 ± 0.160
0.647 ± 0.058
0.599 ± 0.120

WV-3174
0.603 ± 0.118
0.678 ± 0.145
0.421 ± 0.092
0.582 ± 0.185

WV-3174
0.454 ± 0.112
0.523 ± 0.104
0.380 ± 0.081
0.415 ± 0.062

WV-3174
0.342 ± 0.033
0.505 ± 0.119
0.322 ± 0.077
0.340 ± 0.055

Quadriceps pK
Gastro pK
Diaphragm pK
Heart pK

Malat1
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD

Sequence
(ug/g)
(ug/g)
(ug/g)
(ug/g)

PBS
0.011 ± 0.025
0.000 ± 0.000
0.000 ± 0.000
0.000 ± 0.000

WV-3174
1.388 ± 0.677
1.704 ± 0.524
2.502 ± 0.919
1.781 ± 0.668

WV-3174
6.651 ± 5.930
4.563 ± 1.705
7.366 ± 3.939
2.532 ± 0.487

WV-3174
12.374 ± 4.081
14.574 ± 8.235
12.075 ± 3.739
4.611 ± 1.050

WV-3174
15.227 ± 4.925
14.124 ± 2.285
22.734 ± 4.484
12.660 ± 2.437

Numbers indicate Malat1 mRNA levels relative to mHprt, and presence of oligonucleotide (ug/g). Experimental procedure: Study Species: 5-6 wks wt mice; Route: Subcutaneous # Doses: QD for 1 days, Time Point Post Last Dose: 3 days; and Daily Dose Level (ug): 200 mg/kg.

Example Methods for Preparing Oligonucleotides and Compositions

Among other things, the present disclosure provides technologies (methods, reagents, conditions, purification processes, etc.) for preparing oligonucleotides and oligonucleotide compositions, including chirally controlled oligonucleotides and chirally controlled oligonucleotide nucleotides. Various technologies (methods, reagents, conditions, purification processes, etc.), as described herein, can be utilized to prepare provided oligonucleotides and compositions thereof in accordance with the present disclosure, including but not limited to those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the preparation technologies of each of which are incorporated herein by reference.

In some embodiments, the present disclosure provides chirally controlled oligonucleotides. In some embodiments, a provided chirally controlled oligonucleotide is over 50% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 55% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 60% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 65% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 70% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 75% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 80% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 85% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 90% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 91% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 92% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 93% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 94% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 95% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 96% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 97% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 98% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 99% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 99.5% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 99.6% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 99.7% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 99.8% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 99.9% pure. In some embodiments, a provided chirally controlled oligonucleotide is over at least about 99% pure.

In some embodiments, a chirally controlled oligonucleotide composition is a composition designed to comprise a single oligonucleotide type. In certain embodiments, such compositions are about 50% diastereomerically pure. In some embodiments, such compositions are about 50% diastereomerically pure. In some embodiments, such compositions are about 50% diastereomerically pure. In some embodiments, such compositions are about 55% diastereomerically pure. In some embodiments, such compositions are about 60% diastereomerically pure. In some embodiments, such compositions are about 65% diastereomerically pure. In some embodiments, such compositions are about 70% diastereomerically pure. In some embodiments, such compositions are about 75% diastereomerically pure. In some embodiments, such compositions are about 80% diastereomerically pure. In some embodiments, such compositions are about 85% diastereomerically pure. In some embodiments, such compositions are about 90% diasteromerically pure. In some embodiments, such compositions are about 91% diastereomerically pure. In some embodiments, such compositions are about 92% diastereomerically pure. In some embodiments, such compositions are about 93% diastereomerically pure. In some embodiments, such compositions are about 94% diastereomerically pure. In some embodiments, such compositions are about 95% diastereomerically pure. In some embodiments, such compositions are about 96% diastereomerically pure. In some embodiments, such compositions are about 97% diastereomerically pure. In some embodiments, such compositions are about 98% diastereomerically pure. In some embodiments, such compositions are about 99% diastereomerically pure. In some embodiments, such compositions are about 99.5% diastereomerically pure. In some embodiments, such compositions are about 99.6% diastereomerically pure. In some embodiments, such compositions are about 99.7% diastereomerically pure. In some embodiments, such compositions are about 99.8% diastereomerically pure. In some embodiments, such compositions are about 99.9% diastereomerically pure. In some embodiments, such compositions are at least about 99% diastereomerically pure.

Among other things, the present disclosure recognizes the challenge of stereoselective (rather than stereorandom or racemic) preparation of oligonucleotides. Among other things, the present disclosure provides methods and reagents for stereoselective preparation of oligonucleotides comprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) internucleotidic linkages, and particularly for oligonucleotides comprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) chiral internucleotidic linkages. In some embodiments, in a stereorandom or racemic preparation of oligonucleotides, at least one chiral internucleotidic linkage is formed with less than 90:10, 95:5, 96:4, 97:3, or 98:2 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 90:10, 95:5, 96:4, 97:3, or 98:2 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 95:5 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 96:4 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 97:3 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 98:2 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 99:1 diastereoselectivity. In some embodiments, diastereoselectivity of a chiral internucleotidic linkage in an oligonucleotide may be measured through a model reaction, e.g. formation of a dimer under essentially the same or comparable conditions wherein the dimer has the same internucleotidic linkage as the chiral internucleotidic linkage, the 5′-nucleoside of the dimer is the same as the nucleoside to the 5′-end of the chiral internucleotidic linkage, and the 3′-nucleoside of the dimer is the same as the nucleoside to the 3-end of the chiral internucleotidic linkage.

In some embodiments, a chirally controlled oligonucleotide composition is a composition designed to comprise multiple oligonucleotide types. In some embodiments, methods of the present disclosure allow for the generation of a library of chirally controlled oligonucleotides such that a pre-selected amount of any one or more chirally controlled oligonucleotide types can be mixed with any one or more other chirally controlled oligonucleotide types to create a chirally controlled oligonucleotide composition. In some embodiments, the pre-selected amount of an oligonucleotide type is a composition having any one of the above-described diastereomeric purities.

In some embodiments, the present disclosure provides methods for making a chirally controlled oligonucleotide comprising steps of:

(1) coupling:

(2) capping:

(3) optionally modifying;

(4) deblocking; and

(5) repeating steps (1)-(4) until a desired length is achieved.

In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising one or more cycles, each of which independently comprises:

(1) a coupling step;

(2) optionally a pre-modification capping step:

(3) a modification step;

(4) optionally a post-modification capping step; and

(5) optionally a de-blocking step.

In some embodiments, a cycle comprises one or more pre-modification capping steps. In some embodiments, a cycle comprises one or more post-modification capping steps. In some embodiments, a cycle comprises one or more pre- and post-modification capping steps. In some embodiments, a cycle comprises one or more de-blocking steps. In some embodiments, a cycle comprises a coupling step, a pre-modification capping step, a modification step, a post-modification capping step, and a de-blocking step. In some embodiments, a cycle comprises a coupling step, a pre-modification capping step, a modification step, and a de-blocking step. In some embodiments, a cycle comprises a coupling step, a modification step, a post-modification capping step and a de-blocking step. In some embodiments, comprise a coupling step, a pre-modification capping step, a modification step, a post-modification capping step, and a de-blocking step. In some embodiments, one or more cycles comprise a coupling step, a pre-modification capping step, a modification step, and a de-blocking step. In some embodiments, one or more cycles comprise a coupling step, a modification step, a post-modification capping step and a de-blocking step.

When describing the provided methods, the word “cycle” has its ordinary meaning as understood by a person of ordinary skill in the art. In some embodiments, one round of steps (1)-(4) is referred to as a cycle. In some embodiments, some cycles comprise modifying. In some embodiments, some cycles do not comprise modifying. In some embodiments, some cycles comprise and some cycles do not comprise modifying. In some embodiments, each cycle independently comprises a modifying step. In some embodiments, each cycle does not comprise a cycling step.

In some embodiments, to form a chirally controlled internucleotidic linkage, a chirally pure phosphoramidite comprising a chiral auxiliary is utilized to stereoselectively form the chirally controlled internucleotidic linkage. Various phosphoramidite and chiral auxiliaries, e.g., those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the phosphoramidite and chiral auxiliaries of each of which are incorporated herein by reference, may be utilized in accordance with the present disclosure.

In some embodiments, a coupling step provides an oligonucleotide comprises an internucleotidic linkage of formula I, I-a, I-b. I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1. II-a-2. II-b-1, II-b-2, l-c-1, I-c-2, II-d-1, I-d-2, etc., or a salt form thereof, wherein PL is P. In some embodiments, such an internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, such an internucleotidic linkage comprises a chiral auxiliary moiety.

In some embodiments, a modifying step provides an oligonucleotide comprises an internucleotidic linkage of formula I, I-a, 1-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, etc., or a salt form thereof, wherein P^Lis P=W. In some embodiments, a modifying step provides an oligonucleotide comprises an internucleotidic linkage of formula I, I-a. I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof, wherein P^Lis P=W. In some embodiments, W is S. In some embodiments, W is O. In some embodiments, such an internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, such an internucleotidic linkage comprises a chiral auxiliary moiety. In some embodiments, a modifying step provides a non-negatively charged internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof. In some embodiments, such an internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, such an internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, such an internucleotidic linkage comprises a chiral auxiliary moiety. In some embodiments, such an internucleotidic linkage comprises no chiral auxiliary moiety. In some embodiments, a chiral auxiliary moiety falls off during modification.

Provided technologies provide various advantages. Among other things, as demonstrated herein, provided technologies can greatly improve oligonucleotide synthesis crude purity and yield, particularly for modified and/or chirally pure oligonucleotides that provide a number of properties and activities that are critical for therapeutic purposes. With the capability to provide unexpectedly high crude purity and yield for therapeutically important oligonucleotides, provided technologies can significantly reduce manufacturing costs (through, e.g., simplified purification, greatly improved overall yields, etc.). In some embodiments, provided technologies can be readily scaled up to produce oligonucleotides in sufficient quantities and qualities for clinical purposes. In some embodiments, provided technologies comprising chiral auxiliaries that comprise electron-withdrawing groups in G²(e.g., PSM chiral auxiliaries) are particularly useful for preparing chirally controlled internucleotidic linkages comprising P-N bonds (e.g., non-negatively charged internucleotidic linkages such as n001, n002, n003, n004, n005, n006, n007, n008, n009, n010, etc.) and can significantly simplify manufacture operations, reduce cost, and/or facilitate downstream formation.

In some embodiments, provided technologies provides improved reagents compatibility. For example, as demonstrated in the present disclosure, provided technologies provide flexibility to use different reagent systems for oxidation, sulfurization and/or azide reactions, particularly for chirally controlled oligonucleotide synthesis.

Among other things, the present disclosure provides oligonucleotide compositions of high crude purity. In some embodiments, the present disclosure provides chirally controlled oligonucleotide composition of high crude purity. In some embodiments, the present disclosure provides chirally controlled oligonucleotide of high crude purity. In some embodiments, the present disclosure provides oligonucleotide of high crude purity and/or high stereopurity.

Support and Linkers

In some embodiments, oligonucleotides can be prepared in solution. In some embodiments, oligonucleotides can be prepared using a support. In some embodiments, oligonucleotides are prepared using a solid support. Suitable support that can be utilized in accordance with the present disclosure include, e.g., solid support described in U.S. Pat. Nos. 9,695,211, 9,605,019, U.S. Pat. No. 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the solid support of each of which is incorporated herein by reference.

In some embodiments, a linker moiety is utilized to connect an oligonucleotide chain to a support during synthesis. Suitable linkers are widely utilized in the art, and include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the linker of each of which is incorporated herein by reference.

In some embodiments, the linking moiety is a succinamic acid linker, or a succinate linker (—CO—CH₂—CH₂—CO—), or an oxalyl linker (—CO—CO—). In some embodiments, the linking moiety and the nucleoside are bonded together through an ester bond. In some embodiments, a linking moiety and a nucleoside are bonded together through an amide bond. In some embodiments, a linking moiety connects a nucleoside to another nucleotide or nucleic acid. Suitable linkers are disclosed in, for example, Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991, Chapter 1 and Solid-Phase Supports for Oligonucleotide Synthesis, Pon, R. T., Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28. In some embodiments, a universal linker (UnyLinker) is used to attached the oligonucleotide to the solid support (Ravikumar et al., Org. Process Res. Dev., 2008, 12 (3), 399-410). In some embodiments, other universal linkers are used (Pon, R. T., Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28). In some embodiments, various orthogonal linkers (such as disulfide linkers) are used (Pon, R. T., Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28).

Among other things, the present disclosure recognizes that a linker can be chosen or designed to be compatible with a set of reaction conditions employed in oligonucleotide synthesis. In some embodiments, to avoid degradation of oligonucleotides and to avoid desulfurization, auxiliary groups are selectively removed before de-protection. In some embodiments, DPSE group can selectively be removed by F ions. In some embodiments, the present disclosure provides linkers that are stable under a DPSE de-protection condition, e.g., 0.1M TBAF in MeCN, 0.5M HF-Et₃N in THF or MeCN, etc. In some embodiments, a provided linker is a linker as exemplified below:

embedded image

Solvents

Syntheses of provided oligonucleotides are generally performed in aprotic organic solvents. In some embodiments, a solvent is a nitrile solvent such as, e.g., acetonitrile. In some embodiments, a solvent is a basic amine solvent such as, e.g., pyridine. In some embodiments, a solvent is an ethereal solvent such as, e.g., tetrahydrofuran. In some embodiments, a solvent is a halogenated hydrocarbon such as, e.g., dichloromethane. In some embodiments, a mixture of solvents is used. In certain embodiments a solvent is a mixture of any one or more of the above-described classes of solvents.

In some embodiments, when an aprotic organic solvent is not basic, a base is present in the reacting step. In some embodiments where a base is present, the base is an amine base such as, e.g., pyridine, quinoline, or N,N-dimethylaniline. Example other amine bases include pyrrolidine, piperidine, N-methyl pyrrolidine, pyridine, quinoline, N,N-dimethylaminopyridine (DMAP), or N,N-dimethylaniline.

In some embodiments, a base is other than an amine base.

In some embodiments, an aprotic organic solvent is anhydrous. In some embodiments, an anhydrous aprotic organic solvent is freshly distilled. In some embodiments, a freshly distilled anhydrous aprotic organic solvent is a basic amine solvent such as, e.g., pyridine. In some embodiments, a freshly distilled anhydrous aprotic organic solvent is an ethereal solvent such as, e.g., tetrahydrofuran. In some embodiments, a freshly distilled anhydrous aprotic organic solvent is a nitrile solvent such as, e.g., acetonitrile.

Chiral Reagents/Chiral Auxiliaries

In some embodiments, chiral reagents (may also be referred to as chiral auxiliaries) are used to confer stereoselectivity in the production of chirally controlled oligonucleotides. Many chiral reagents, also referred to by those of skill in the art and herein as chiral auxiliaries, may be used in accordance with methods of the present disclosure. Examples of such chiral reagents are described herein and in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/098264, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the chiral auxiliaries of each of which is incorporated by reference.

In some embodiments, a chiral reagent for use in accordance with the methods of the present disclosure is of Formula 3-I, below:

embedded image

wherein:

W¹and W²are any of —O—, —S—, -NG⁵-, or -NG⁵-O—;

U₁and U₃are carbon atoms which are bonded to U₂if present, or to each other if r is 0, via a single, double or triple bond:

U₂is —C—, -CG⁸-, -CG⁸G⁸-. -NG⁸-, —N—, —O—, or —S— where r is an integer of 0 to 5; and

each of G¹, G², G³, G⁴, G⁵, and G⁸is independently R¹as described in the present disclosure.

In some embodiments, W¹and W²are any of —O—, —S—, or -NG⁵-, U₁and U₃are carbon atoms which are bonded to U₂if present, or to each other if r is 0, via a single, double or triple bond. U₂is —C—, -CG⁸-, -CG⁸G⁸-, -NG⁸-, —N—, —O—, or —S— where r is an integer of 0 to 5 and no more than two heteroatoms are adjacent. When any one of U₂is C, a triple bond must be formed between a second instance of U₂, which is C, or to one of U₁or U₃. Similarly, when any one of U₂is CG⁸, a double bond is formed between a second instance of U₂which is -CG⁸- or —N—, or to one of U₁or U₃.

In some embodiments, -U₁G³G⁴-(U₂)_r-U₃G¹G²- is -CG³G⁴-CG¹G²-. In some embodiments, -U₁-(U₂),-U₃- is -CG³=CG¹-. In some embodiments, -U₁-(U₂)_r-U₃- is —C≡C—. In some embodiments, -U₁-(U₂)_r-U₃- is -CG³=CG⁸-CG¹G²-. In some embodiments, U₁(U₂)_r-U₃- is -CG³G⁴-O-CG¹G²-. In some embodiments, -U₁-(U₂)-U₃is -CG³G⁴-NG⁸-CG¹G²-. In some embodiments, -U₁-(U₂)_r-U₃- is -CG³G⁴-N-CG²-. In some embodiments, -U₁-(U₂),-U₃- is -CG³G⁴-N═CG⁸-CG¹G²-.

In some embodiments, G¹, G², G³, G⁴, G⁵, and G⁸are independently R¹as described in the present disclosure. In some embodiments, G¹, G², G³, G⁴, G⁵, and G⁸are independently R as described in the present disclosure. In some embodiments, G¹, G², G³, G⁴, G⁵, and G⁸are independently hydrogen, or an optionally substituted group selected from aliphatic, alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaliphatic, heterocyclyl, heteroaryl, and aryl; or two of G¹, G², G³, G⁴, and G⁵are G⁶(taken together to form an optionally substituted, saturated, partially unsaturated or unsaturated carbocyclic or heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, and is fused or unfused). In some embodiments, a ring so formed is substituted by oxo, thioxo, alkyl, alkenyl, alkynyl, heteroaryl, or aryl moieties. In some embodiments, when a ring formed by taking two G⁶together is substituted, it is substituted by a moiety which is bulky enough to confer stereoselectivity during the reaction.

In some embodiments, a ring formed by taking two of G⁶together is optionally substituted cyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl, cyclopentenyl, tetrahydropyranyl, or piperazinyl. In some embodiments, a ring formed by taking two of G together is optionally substituted cyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl, cyclopentenyl, tetrahydropyranyl, pyrrolidinyl, or piperazinyl.

In some embodiments, G¹is optionally substituted phenyl. In some embodiments, G¹is phenyl. In some embodiments, G²is methyl or hydrogen. In some embodiments, G²is hydrogen. In some embodiments, G¹is optionally substituted phenyl and G²is methyl. In some embodiments, G¹is phenyl and G²is methyl. In some embodiments, G¹is —CH₂Si(R)z, wherein one R is optionally substituted C_1-6aliphatic, and the other two R are each independently an optionally substituted 3-20 membered, monocyclic or polycyclic, saturated, partially unsaturated or aromatic ring having 0-5 heteroatoms. In some embodiments, the other two R are each independently optionally substituted phenyl. In some embodiments, G¹is —CH₂SiMePh₂.

In some embodiments, r is 0.

In some embodiments, W¹is -NG⁵-O—. In some embodiments, W¹is -NG⁵-O—, wherein the —O— is bonded to —H. In some embodiments, W¹is -NG¹-. In some embodiments, one of G³and G⁴is taken together with G⁵to form an optionally substituted 3-10 membered ring. In some embodiments, one of G³and G⁴is taken together with G⁵to form an optionally substituted pyrrolidinyl ring. In some embodiments, one of G³and G⁴is taken together with G⁵to form a pyrrolidinyl ring. In some embodiments, G⁵is optionally substituted C_1-6aliphatic. In some embodiments, G⁵is methyl. In some embodiments, one of G¹and G²and one of G³and G⁴are taken together with their intervening atoms to form an optionally substituted 3-10 membered ring having 0-3 heteroatoms. In some embodiments, a formed ring 3-membered. In some embodiments, a formed ring 4-membered. In some embodiments, a formed ring 5-membered. In some embodiments, a formed ring 6-membered. In some embodiments, a formed ring 7-membered. In some embodiments, a formed ring is substituted. In some embodiments, a formed ring is unsubstituted. In some embodiments, a formed ring has no heteroatom. In some embodiments, a formed ring is saturated. For example compounds, see WV-CA-293 and WV-CA-294.

In some embodiments, W²is —O—.

In some embodiments, a chiral reagent is a compound of Formula 3-AA:

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wherein each variable is independently as defined above and described herein.

In some embodiments of Formula 3AA, W¹and W²are independently -NG⁵-, —O—, or —S—; G¹, G², G³, G⁴, and G⁵are independently hydrogen, or an optionally substituted group selected from alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaliphatic, heterocyclyl, heteroaryl, or aryl; or two of G¹, G², G³, G⁴, and G⁵are G⁶(taken together to form an optionally substituted saturated, partially unsaturated or unsaturated carbocyclic or heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, fused or unfused), and no more than four of G¹, G², G³, G⁴, and G⁵are G⁶. Similarly to the compounds of Formula 3-1, any of G¹, G², G³, G⁴, or G⁵are optionally substituted by oxo, thioxo, alkyl, alkenyl, alkynyl, heteroaryl, or aryl moieties. In some embodiments, such substitution induces stereoselectivity in chirally controlled oligonucleotide production. In some embodiments, a heteroatom-containing moiety, e.g., heteroaliphatic, heterocyclyl, heteroaryl, etc., has 1-5 heteroatoms. In some embodiments, the heteroatoms are selected from nitrogen, oxygen, sulfure and silicon. In some embodiments, at least one heteroatom is nitrogen.

In some embodiments, W¹is -NG⁵-O—. In some embodiments, W¹is -NG⁵-O—, wherein the —O— is bonded to —H. In some embodiments, W¹is -NG⁵-. In some embodiments, G⁵and one of G³and G⁴are taken together to form an optionally substituted 3-10 membered ring having 0-3 heteroatoms in addition to the nitrogen atom of W¹. In some embodiments, G⁵and G³are taken together to form an optionally substituted 3-10 membered ring having 0-3 heteroatoms in addition to the nitrogen atom of W¹. In some embodiments, G⁵and G⁴are taken together to form an optionally substituted 3-10 membered ring having 0-3 heteroatoms in addition to the nitrogen atom of W¹. In some embodiments, a formed ring is an optionally substituted 4, 5, 6, 7, or 8 membered ring. In some embodiments, a formed ring is an optionally substituted 4-membered ring. In some embodiments, a formed ring is an optionally substituted 5-membered ring. In some embodiments, a formed ring is an optionally substituted 6-membered ring. In some embodiments, a formed ring is an optionally substituted 7-membered ring.

In some embodiments, a provided chiral reagent has the structure of

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In some embodiments, a provided chiral reagent has the structure of

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In some embodiments, a provided chiral reagent has the structure of

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In some embodiments, a provided chiral reagent has the structure of

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In some embodiments, a provided chiral reagent has the structure of

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In some embodiments, a provided chiral reagent has the structure of

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In some embodiments, a provided chiral reagent has the structure of

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In some embodiments, a provided chiral reagent has the structure of

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In some embodiments, W¹is -NG⁵, W²is O, each of G¹an G³is independently hydrogen or an optionally substituted group selected from C₁(aliphatic, heterocyclyl, heteroaryl and aryl, G²is —C(R)₂Si(R)₃, and G⁴and G⁵are taken together to form an optionally substituted saturated, partially unsaturated or unsaturated heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, fused or unfused. In some embodiments, each R is independently hydrogen, or an optionally substituted group selected from C₁-C₆aliphatic, carbocyclyl, aryl, heteroaryl, and heterocyclyl. In some embodiments, G²is —C(R)₂Si(R)₃, wherein —C(R)₂- is optionally substituted —CH₂—, and each R of —Si(R) is independently an optionally substituted group selected from C_1-10aliphatic, heterocyclyl, heteroaryl and aryl. In some embodiments, at least one R of —Si(R)₃is independently optionally substituted C_1-10alkyl. In some embodiments, at least one R of —Si(R)₃is independently optionally substituted phenyl. In some embodiments, one R of —Si(R)₃is independently optionally substituted phenyl, and each of the other two R is independently optionally substituted C_1-10alkyl. In some embodiments, one R of —Si(R)₃is independently optionally substituted C_1-10alkyl, and each of the other two R is independently optionally substituted phenyl. In some embodiments, G²is optionally substituted —CH₂Si(Ph)(Me)₂. In some embodiments, G²is optionally substituted —CH₂Si(Me)(Ph)₂. In some embodiments, G²is —CH₂Si(Me)(Ph)₂. In some embodiments, G⁴and G⁵are taken together to form an optionally substituted saturated 5-6 membered ring containing one nitrogen atom (to which G⁵is attached). In some embodiments, G⁴and G⁵are taken together to form an optionally substituted saturated 5-membered ring containing one nitrogen atom. In some embodiments, G¹is hydrogen. In some embodiments, G³is hydrogen. In some embodiments, both G¹and G³are hydrogen.

In some embodiments, W¹is -NG⁵-, W²is O, each of G¹and G³is independently R¹, G²is —R¹, and G⁴and G⁵are taken together to form an optionally substituted saturated, partially unsaturated or unsaturated heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, fused or unfused. In some embodiments, each of G¹and G³is independently R. In some embodiments, each of G¹and G³is independently —H. In some embodiments, G²is connected to the rest of the molecule through a carbon atom, and the carbon atom is substituted with one or more electron-withdrawing groups. In some embodiments, G²is methyl substituted with one or more electron-withdrawing groups. In some embodiments, G²is methyl substituted with one and no more than one electron-withdrawing group. In some embodiments, G²is methyl substituted with two or more electron-withdrawing groups. Among other things, a chiral auxiliary having G²comprising an electron-withdrawing group can be readily removed by a base (base-labile, e.g., under an anhydrous condition substantially free of water; in many instances, preferably before oligonucleotides comprising internucleotidic linkages comprising such chiral auxiliaries are exposed to conditions/reagent systems comprising a substantial amount of water, particular in the presence of a base(e.g., cleavage conditions/reagent systems using NH₄OH)) and provides various advantages as described herein, e.g., high crude purity, high yield, high stereoselectivity, more simplified operation, fewer steps, further reduced manufacture cost, and/or more simplified downstream formulation (e.g., low amount of salt(s) after cleavage), etc. In some embodiments, as described in the Examples, such auxiliaries may provide alternative or additional chemical compatibility with other functional and/or protection groups. In some embodiments, as demonstrated in the Examples, base-labile chiral auxiliaries are particularly useful for construction of chirally controlled non-negatively charged internucleotidic linkages (e.g., neutral internucleotidic linkages such as n001); in some instances, as demonstrated in the Examples, they can provide significantly improved yield and/or crude purity with high stereoselectivity, e.g., when utilized with removal using a base under an anhydrous condition. In some embodiments, such a chiral auxiliary is bonded to a linkage phosphorus via an oxygen atom (e.g., which corresponds to a —OH group in a corresponding chiral auxiliary compound, e.g., a compound of formula I), the carbon atom in the chiral auxiliary to which the oxygen is bonded (the alpha carbon) also bonds to —H (in addition to other groups; in some embodiments, a secondary carbon), and the next carbon atom (the beta carbon) in the chiral auxiliary is boned to one or two electron-withdrawing groups. In some embodiments, —W²—H is —OH. In some embodiments, G¹is —H. In some embodiments, G²comprises one or two electron-withdrawing groups or can otherwise facilitate remove of the chiral auxiliary by a base. In some embodiments, G¹is —H, G²comprises one or two electron-withdrawing groups, -W²—H is —OH. In some embodiments, G¹is —H, G²comprises one or two electron-withdrawing groups, —W²—H is —OH, -W¹—H is -NG⁵-H, and one of G³and G⁴is taken together with G⁵to form with their intervening atoms a ring as described herein (e.g., an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having in addition to the nitrogen atom to which G⁵is on, 0-5 heteroatoms (e.g., an optionally substituted 3, 4, 5, or 6-membered monocyclic saturated ring having in addition to the nitrogen atom to which G⁵is on no other heteroatoms)).

As appreciated by those skilled in the art, various electron-withdrawing groups are known in the art and can be utilized in accordance with the present disclosure. In some embodiments, an electronic-withdrawing group comprises and/or is connected to the carbon atom through, e.g., —S(O)—, —S(O)₂—, —P(O)(R¹)—, —P(S)R¹—, or —C(O)—. In some embodiments, an electron-withdrawing group is —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂. In some embodiments, an electron-withdrawing group is aryl or heteroaryl, e.g., phenyl, substituted with one or more of —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R′)₂, —P(O)(OR′)₂, or—P(S)(R′)₂.

In some embodiments, G²is -L-R′. In some embodiments, G²is -L′-L″-R′, wherein L′ is —C(R)₂— or optionally substituted —CH₂—, and L″ is —P(O)(R′)—, —P(O)(R′)O—, —P(O)(OR′)—, —P(O)(OR′)O—, —P(O)[N(R′)]—, —P(O)[N(R′)]O—, —P(O)[N(R′)][N(R′)]—, —P(S)(R′)—, —S(O)₂—, —S(O)₂—, —S(O)₂O—, —S(O)—, —C(O)—, —C(O)N(R′)—, or —S—. In some embodiments, L′ is —C(R)₂—. In some embodiments, L′ is optionally substituted —CH₂—.

In some embodiments, L′ is —C(R)₂—. In some embodiments, each R is independently hydrogen, or an optionally substituted group selected from C₁-C₆aliphatic, carbocyclyl, aryl, heteroaryl, and heterocyclyl. In some embodiments, L′ is —CH₂—. In some embodiments, L″ is —P(O)(R′)—, —P(S)(R′)—, —S(O)₂—. In some embodiments, G²is -L′-C(O)N(R′)₂. In some embodiments, G²is -L′-P(O)(R′)₂. In some embodiments, G²is -L′-P(S)(R′)₂. In some embodiments, each R′ is independently optionally substituted aliphatic, heteroaliphatic, aryl, or heteroaryl as described in the present disclosure (e.g., those embodiments described for R). In some embodiments, each R′ is independently optionally substituted phenyl. In some embodiments, each R′ is independently optionally substituted phenyl wherein one or more substituents are independently selected from —CN, -OMe, —Cl, —Br, and —F. In some embodiments, each R′ is independently substituted phenyl wherein one or more substituents are independently selected from —CN, -OMe, —Cl, —Br, and —F. In some embodiments, each R′ is independently substituted phenyl wherein the substituents are independently selected from —CN, -OMe, —Cl, —Br, and —F. In some embodiments, each R′ is independently mono-substituted phenyl, wherein the substituent is independently selected from —CN, -OMe, —Cl, —Br, and —F. In some embodiments, two R′ are the same. In some embodiments, two R′ are different. In some embodiments, G²is -L′-S(O)R′. In some embodiments, G²is -L′-C(O)N(R′)₂. In some embodiments, G²is -L′-S(O)₂R′. In some embodiments, R′ is optionally substituted aliphatic, heteroaliphatic, aryl, or heteroaryl as described in the present disclosure (e.g., those embodiments described for R). In some embodiments, R′ is optionally substituted phenyl. In some embodiments, R′ is optionally substituted phenyl wherein one or more substituents are independently selected from —CN, -OMe, —Cl, —Br, and —F. In some embodiments, R′ is substituted phenyl wherein one or more substituents are independently selected from —CN, -OMe, —Cl, —Br, and —F. In some embodiments, R′ is substituted phenyl wherein each substituent is independently selected from —CN, -OMe, —Cl, —Br, and —F. In some embodiments, R′ is mono-substituted phenyl. In some embodiments, R′ is mono-substituted phenyl, wherein the substituent is independently selected from —CN, -OMe, —Cl, —Br, and —F. In some embodiments, a substituent is an electron-withdrawing group. In some embodiments, an electron-withdrawing group is —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂.

In some embodiments, G²is optionally substituted —CH₂-L″-R, wherein each of L″ and R is independently as described in the present disclosure. In some embodiments, G²is optionally substituted —CH(-L″-R)₂, wherein each of L″ and R is independently as described in the present disclosure. In some embodiments, G²is optionally substituted —CH(—S—R)₂. In some embodiments, G²is optionally substituted —CH₂—S—R. In some embodiments, the two R groups are taken together with their intervening atoms to form a ring. In some embodiments, a formed ring is an optionally substituted 5, 6, 7-membered ring having 0-2 heteroatoms in addition to the intervening heteroatoms. In some embodiments, G²is optionally substituted

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In some embodiments, G²is

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In some embodiments, —S— may be converted to —S(O)— or —S(O)₂—, e.g., by oxidation, e.g., to facilitate removal by a base.

In some embodiments, G²is -L′-R′, wherein each variable is as described in the present disclosure. In some embodiments, G²is —CH₂—R′. In some embodiments, G²is —CH(R′)₂. In some embodiments, G²is —C(R′)₃. In some embodiments, R′ is optionally substituted aryl or heteroaryl. In some embodiments, R′ is substituted aryl or heteroaryl wherein one or more substituents are independently an electron-withdrawing group. In some embodiments, -L′- is optionally substituted —CH₂—, and R′ is R, wherein R is optionally substituted aryl or heteroaryl. In some embodiments, R is substituted aryl or heteroaryl wherein one or more substituents are independently an electron-withdrawing group. In some embodiments, R is substituted aryl or heteroaryl wherein each substituent is independently an electron-withdrawing group. In some embodiments, R is aryl or heteroaryl substituted with two or more substituents, wherein each substituent is independently an electron-withdrawing group. In some embodiments, an electron-withdrawing group is —CN, —NO₂, halogen, —C(O)R, —C(O)OR¹, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂. In some embodiments, R′ is

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In some embodiments, R′ is p-NO₂Ph-. In some embodiments, R′ is

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In some embodiments, R′ is

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In some embodiments, R′ is

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In some embodiments, R′ is

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In some embodiments, R′ is

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In some embodiments, G²is

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In some embodiments, R′ is

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In some embodiments, R′ is

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In some embodiments, R′ is 2,4,6-trichlorophenyl. In some embodiments, R′ is 2,4,6-trifluorophenyl. In some embodiments, G²is —CH(4-chlorophenyl)₂. In some embodiments, G²is —CH(R′)₂, wherein each R′ is

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In some embodiments, G²is —CH(R′)₂, wherein each R′ is

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In some embodiments, R′ is —C(O)R. In some embodiments, R′ is CH₃C(O)—.

In some embodiments, G²is -L′-S(O)₂R′, wherein each variable is as described in the present disclosure. In some embodiments, G²is —CH₂—S(O)₂R′. In some embodiments, G²is -L′-S(O)R′, wherein each variable is as described in the present disclosure. In some embodiments, G²is —CH₂—S(O)R′. In some embodiments, G²is -L′-C(O)₂R′, wherein each variable is as described in the present disclosure. In some embodiments, G²is —CH₂—C(O)₂R′. In some embodiments, G²is -L′-C(O)R′, wherein each variable is as described in the present disclosure. In some embodiments, G²is —CH₂—C(O)R′. In some embodiments, -L′- is optionally substituted —CH₂—, and R′ is R. In some embodiments, R is optionally substituted aryl or heteroaryl. In some embodiments, R is optionally substituted aliphatic. In some embodiments, R is optionally substituted heteroaliphatic. In some embodiments, R is optionally substituted heteroaryl. In some embodiments, R is optionally substituted aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is not phenyl, or mono-, di- or tri-substituted phenyl, wherein each substituent is selected from —NO₂, halogen, —CN, —C_1-3alkyl, and C_1-3alkyloxy. In some embodiments, R is substituted aryl or heteroaryl wherein one or more substituents are independently an electron-withdrawing group. In some embodiments, R is substituted aryl or heteroaryl wherein each substituent is independently an electron-withdrawing group. In some embodiments, R is aryl or heteroaryl substituted with two or more substituents, wherein each substituent is independently an electron-withdrawing group. In some embodiments, an electron-withdrawing group is —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂. In some embodiments, R′ is phenyl. In some embodiments, R′ is substituted phenyl. In some embodiments, R′ is

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In some embodiments, R′ is

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In some embodiments, R′ is

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In some embodiments, R′ is optionally substituted C_1-6aliphatic. In some embodiments, R′ is t-butyl. In some embodiments, R′ is isopropyl. In some embodiments, R′ is methyl. In some embodiments, G²is —CH₂C(O)OMe. In some embodiments, G²is —CH₂C(O)Ph. In some embodiments, G²is —CH₂C(O)—tBu.

In some embodiments, G²is -L′-NO₂. In some embodiments, G²is —CH₂—NO₂. In some embodiments, G²is -L′-S(O)₂N(R′)₂. In some embodiments, G²is —CH₂—S(O)₂N(R′)₂. In some embodiments, G²is -L′-S(O)₂NHR′. In some embodiments, G²is —CH₂—S(O)₂NHR′. In some embodiments, R′ is methyl. In some embodiments, G²is —CH₂—S(O)₂NH(CH₃). In some embodiments. R′ is —CH₂Ph. In some embodiments, G²is —CH₂—S(O)₂NH(CH₂Ph). In some embodiments, G²is —CH₂—S(O)₂N(CH₂Ph)₂. In some embodiments, R′ is phenyl. In some embodiments, G²is —CH₂—S(O)₂NHPh. In some embodiments, G²is —CH₂—S(O)₂N(CH₃)Ph. In some embodiments, G²is —CH₂—S(O)₂N(CH₃)₂. In some embodiments, G²is —CH₂—S(O)₂NH(CH₂Ph). In some embodiments, G²is —CH₂—S(O)₂NHPh. In some embodiments, G²is —CH₂—S(O)₂NH(CH₂Ph). In some embodiments, G²is —CH₂—S(O)₂N(CH₃)₂. In some embodiments, G²is —CH₂—S(O)₂N(CH₃)Ph. In some embodiments, G²is -L′-S(O)₂N(R′)(OR′). In some embodiments, G²is —CH₂—S(O)₂N(R′)(OR′). In some embodiments, each R′ is methyl. In some embodiments, G²is —CH₂—S(O)₂N(CH₃)(OCH₃). In some embodiments, G²is —CH₂—S(O)₂N(Ph)(OCH₃). In some embodiments, G²is —CH₂—S(O)₂N(CH₂Ph)(OCH₃). In some embodiments, G²is —CH₂—S(O)₂N(CH₂Ph)(OCH₃). In some embodiments, G²is -L′-S(O)₂OR′. In some embodiments, G²is —CH₂—S(O)₂OR′. In some embodiments, G²is —CH₂—S(O)₂OPh. In some embodiments, G²is —CH₂—S(O)₂OCH₃. In some embodiments, G²is —CH₂—S(O)₂OCH₂Ph.

In some embodiments, G²is -L′-P(O)(R′)₂. In some embodiments, G²is —CH₂—P(O)(R′)₂. In some embodiments, G²is -L′-P(O)[N(R′)₂]₂. In some embodiments, G²is —CH₂—P(O)[N(R′)₂]₂. In some embodiments, G²is -L′-P(O)[O(R′)₂]₂. In some embodiments, G²is —CH₂—P(O)[O(R′)₂]₂. In some embodiments, G²is -L′-P(O)(R′)[N(R′)₂]₂. In some embodiments, G²is —CH₂—P(O)(R′)[N(R′)₂]. In some embodiments, G²is -L′-P(O)(R′)[O(R′)]. In some embodiments, G²is —CH₂—P(O)(R′)[O(R′)]. In some embodiments, G²is -L′-P(O)(OR′)[N(R′)₂]. In some embodiments. G²is —CH₂—P(O)(OR′)[N(R′)₂]. In some embodiments, G²is -L′-C(O)N(R′)₂, wherein each variable is as described in the present disclosure. In some embodiments, G²is —CH₂—C(O)N(R′)₂. In some embodiments, each R′ is independently R. In some embodiments, one R′ is optionally substituted aliphatic, and one R is optionally substituted aryl. In some embodiments, one R′ is optionally substituted C_1-6aliphatic, and one R is optionally substituted phenyl. In some embodiments, each R′ is independently optionally substituted C_1-6aliphatic. In some embodiments, G²is —CH₂—P(O)(CH₃)Ph. In some embodiments, G²is —CH₂—P(O)(CH₃)₂. In some embodiments, G²is —CH₂—P(O)(Ph)₂. In some embodiments, G²is —CH₂—P(O)(OCH₃)₂. In some embodiments, G²is —CH₂—P(O)(CH₂Ph)₂. In some embodiments, G²is —CH₂—P(O)[N(CH₃)Ph]₂. In some embodiments, G²is —CH₂—P(O)[N(CH₃)₂]₂. In some embodiments, G²is —CH₂—P(O)[N(CH₂Ph)₂]₂. In some embodiments, G²is —CH₂—P(O)(OCH₃)₂. In some embodiments, G²is —CH₂—P(O)(OPh)₂.

In some embodiments, G²is -L′-SR′. In some embodiments, G²is —CH₂—SR′. In some embodiments, R′ is optionally substituted phenyl. In some embodiments, R′ is phenyl.

In some embodiments, a provided chiral reagent has the structure of

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wherein each R¹is independently as described in the present disclosure. In some embodiments, a provided chiral reagent has the structure of

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wherein each R¹is independently as described in the present disclosure. In some embodiments, each R¹is independently R as described in the present disclosure. In some embodiments, each R¹is independently R, wherein R is optionally substituted aliphatic, aryl, heteroaliphatic, or heteroaryl as described in the present disclosure. In some embodiments, each R¹is phenyl. In some embodiments, R¹is -L-R′. In some embodiments, R¹is -L-R′, wherein L is —O—, —S—, or —N(R′). In some embodiments, a provided chiral reagent has the structure of

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wherein each X¹is independently —H, an electron-withdrawing group, —NO₂, —CN, —OR, —Cl, —Br, or —F, and W is O or S. In some embodiments, a provided chiral reagent has the structure of

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wherein each X¹is independently —H, an electron-withdrawing group, —NO₂, —CN, —OR, —Cl, —Br, or —F, and W is O or S. In some embodiments, each X¹is independently —CN, —OR, —Cl, —Br, or —F, wherein R is not —H. In some embodiments, R is optionally substituted C_1-6aliphatic. In some embodiments, R is optionally substituted C_1-6alkyl. In some embodiments, R is —CH₃. In some embodiments, one or more X¹are independently electron-withdrawing groups (e.g., —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, —P(S)(R¹)₂, etc.).

In some embodiments, a provided chiral reagent has the structure of

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wherein R¹is as described in the present disclosure. In some embodiments, a provided chiral reagent has the structure of

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wherein R¹is as described in the present disclosure. In some embodiments, R¹is R as described in the present disclosure. In some embodiments, R¹is R, wherein R is optionally substituted aliphatic, aryl, heteroaliphatic, or heteroaryl as described in the present disclosure. In some embodiments, R¹is -L-R′. In some embodiments, R¹is -L-R′, wherein L is —O—, —S—, or —N(R′). In some embodiments, a provided chiral reagent has the structure of

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wherein X¹is —H, an electron-withdrawing group, —NO₂, —CN, —OR, —Cl, —Br, or —F, and W is O or S. In some embodiments, a provided chiral reagent has the structure of

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wherein X¹is —H, an electron-withdrawing group, —NO₂, —CN, —OR, —Cl, —Br, or —F, and W is O or S. In some embodiments, X¹is —CN, —OR, —Cl, —Br, or —F, wherein R is not —H. In some embodiments, R is optionally substituted C_1-6aliphatic. In some embodiments, R is optionally substituted C_1-6alkyl. In some embodiments, R is —CH₃. In some embodiments, X¹is an electron-withdrawing group (e.g., —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R′)₂, —P(O)(R¹)₂, —P(O)OR′)₂, —P(S)(R¹)₂, etc.). In some embodiments, X¹is an electron-withdrawing group that is not —CN, —NO₂, or halogen. In some embodiments, X¹is not —H, —CN, —NO₂, halogen, or C_1-3alkyloxy.

In some embodiments, G²is —CH(R²¹)—CH(R²²)═C(R²³)(R²⁴), wherein each of R²¹, R²², R²³, and R²⁴is independently R. In some embodiments, R²²and R²³are both R, and the two R groups are taken together with their intervening atoms to form an optionally substituted aryl or heteroaryl ring as described herein. In some embodiments, one or more substituents are independently electron-withdrawing groups. In some embodiments, R²¹and R²⁴are both R, and the two R groups are taken together with their intervening atoms to form an optionally substituted ring as described herein. In some embodiments, R²¹and R²⁴are both R. and the two R groups are taken together with their intervening atoms to form an optionally substituted saturated or partially saturated ring as described herein. In some embodiments, R²²and R²³are both R, and the two R groups are taken together with their intervening atoms to form an optionally substituted aryl or heteroaryl ring as described herein, and R²¹and R²⁴are both R, and the two R groups are taken together with their intervening atoms to form an optionally substituted partially saturated ring as described herein. In some embodiments, R²¹is —H. In some embodiments, R²⁴is —H. In some embodiments, G²is optionally substituted

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In some embodiments, G²is optionally substituted

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wherein each Ring A²is independently a 3-15 membered monocyclic, bicyclic or polycyclic ring as described herein. In some embodiments, Ring A²is an optionally substituted 5-10 membered monocyclic aryl or heteroaryl ring having 1-5 heteroatoms as described herein. In some embodiments, Ring A²is an optionally substituted phenyl ring as described herein. In some embodiments, In some embodiments, G²is optionally substituted

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In some embodiments, G²is

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In some embodiments, G²is

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In some embodiments, G²is

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Certain useful example compounds for chiral auxiliaries are presented in, e.g., Tables CA-1 to CA-13. In some embodiments, a useful compound is an enantiomer of a compound in, e.g., Tables CA-1 to CA-13. In some embodiments, a useful compound is a diastereomer of a compound in, e.g., Tables CA-1 to CA-13. In some embodiments, a compound useful for chiral auxiliaries for removal under basic conditions (e.g., by a base under an anhydrous condition) is a compound of Tables CA-1 to CA-13, or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-1 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-2 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-3 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-4 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-5 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-6 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-7 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-8 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-9 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-10 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-11 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-12 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-13 or an enantiomer or a diastereomer thereof.

In some embodiments, when contacted with a base, a chiral auxiliary moiety. e.g., of an internucleotidic linkage, whose corresponding compound is a compound of Formula 3-I or 3-AA may be released as an alkene, which has the same structure as a product formed by elimination of a water molecule from the corresponding compound (elimination of -W²—H═—OH and an alpha-H of G²). In some embodiments, such an alkene has the structure of (electron-withdrawing group)₂═C(R¹)-L-N(R⁵)(R⁶), (electron-withdrawing group)H═C(R¹)-L-N(R⁵)(R⁶), CH(-L″-R′)═C(R¹)-L-N(R⁵)(R⁶) wherein the CH group is optionally substituted, or C^x═C(R¹)-L-N(R⁵)(R⁶), wherein C^xis optionally substituted

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and may be optionally fused with one or more optionally substituted rings, and each other variable is independently as described herein. In some embodiments, C^xis optionally substituted

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In some embodiments, C^xis

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In some embodiments, such an alkene is

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In some embodiments such an alkene is

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In some embodiments, such an alkene is

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In some embodiments, a chiral reagent is an aminoalcohol. In some embodiments, a chiral reagent is an aminothiol. In some embodiments, a chiral reagent is an aminophenol. In some embodiments, a chiral reagent is (S)- and (R)-2-methylamino-1-phenylethanol, (1R,2S)-ephedrine, or (IR, 2S)-2-methylamino-1,2-diphenylethanol.

In some embodiments of the disclosure, a chiral reagent is a compound of one of the following formulae:

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In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer (e.g., WV-CA-237 is a related stereoisomer of WV-CA-236 (a related diastereomer, having the same constitution, the same configuration at one chiral center but not the other); WV-CA-108 is a related enantiomer of WV-CA-236 (mirror image of each other)): Table CA-1. Example chiral auxiliaries.

TABLE CA-1

Example chiral auxiliaries.

WV-CA-231

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WV-CA-232

WV-CA-233

WV-CA-234

WV-CA-235

WV-CA-236

WV-CA-237

WV-CA-238

WV-CA-239

WV-CA-240

WV-CA-241

WV-CA-242

WV-CA-243

WV-CA-244

WV-CA-245

WV-CA-246

WV-CA-247

WV-CA-248

WV-CA-249

WV-CA-250

WV-CA-251

WV-CA-252

WV-CA-253

WV-CA-254

WV-CA-255

WV-CA-256

WV-CA-257

WV-CA-258

WV-CA-259

WV-CA-260

WV-CA-261

WV-CA-262

WV-CA-263

WV-CA-264

WV-CA-265

WV-CA-266

WV-CA-267

WV-CA-268

WV-CA-269

WV-CA-270

WV-CA-271

WV-CA-272

WV-CA-273

WV-CA-274

WV-CA-275

WV-CA-276

WV-CA-277

WV-CA-278

WV-CA-279

WV-CA-280

WV-CA-281

WV-CA-282

WV-CA-283

WV-CA-284

WV-CA-285

WV-CA-286

WV-CA-287

WV-CA-288

WV-CA-289

WV-CA-290

WV-CA-291

WV-CA-293

WV-CA-294

In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-1 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-i or a salt thereof.

In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

TABLE CA-2

Example chiral auxiliaries.

WV-CA-231

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WV-CA-239

WV-CA-249

WV-CA-272

WV-CA-273

WV-CA-274

WV-CA-275

WV-CA-276

WV-CA-277

WV-CA-278

WV-CA-279

WV-CA-280

WV-CA-281

WV-CA-282

WV-CA-283

WV-CA-284

WV-CA-285

In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-2 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-2 or a salt thereof.

In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

TABLE CA-3

Example chiral auxiliaries.

WV-CA-236

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WV-CA-237

WV-CA-238

WV-CA-240

WV-CA-241

WV-CA-242

WV-CA-243

WV-CA-252

WV-CA-290

WV-CA-291

WV-CA-108

WV-CA-183

In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-3 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-3 or a salt thereof.

In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

TABLE CA-4

Example chiral auxiliaries.

WV-CA-251

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WV-CA-253

WV-CA-255

WV-CA-257

WV-CA-258

WV-CA-263

In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-4 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-4 or a salt thereof.

In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

TABLE CA-5

Example chiral auxiliaries.

WV-CA-254

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WV-CA-256

WV-CA-259

In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-5 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-5 or a salt thereof.

In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

TABLE CA-6

Example chiral auxiliaries.

WV-CA-260

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WV-CA-261

WV-CA-262

In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-6 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-6 or a salt thereof.

In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

TABLE CA-7

Example chiral auxiliaries.

WV-CA-245

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WV-CA-264

WV-CA-265

WV-CA-266

In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-7 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-7 or a salt thereof.

In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

TABLE CA-8

Example chiral auxiliaries.

WV-CA-267

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WV-CA-269

WV-CA-271

In some embodiments, a provided compound is an enantiomer of a compound from Table CA-8 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-8 or a salt thereof.

In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

TABLE CA-9

Example chiral auxiliaries.

WV-CA-268

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WV-CA-270

In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-9 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-9 or a salt thereof.

In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer particularly enantiomer:

TABLE CA-10

Example chiral auxiliaries.

WV-CA-244

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WV-CA-246

In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-10 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-10 or salt thereof.

In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

TABLE CA-11

Example chiral auxiliaries.

WV-CA-247

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WV-CA-248

In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-11 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-11 or a salt thereof.

In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

TABLE CA-12

Example chiral auxiliaries.

WV-CA-250

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WV-CA-286

WV-CA-287

WV-CA-288

WV-CA-289

In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-12 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-12 or a salt thereof.

In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

TABLE CA-13

Example chiral auxiliaries.

WV-CA-110

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WV-CA-315

WV-CA-110b

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WV-CA-324

In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-13 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-13 or a salt thereof.

As appreciated by those skilled in the art, chiral reagents are typically stereopure or substantially stereopure, and are typically utilized as a single stereoisomer substantially free of other stereoisomers. In some embodiments, compounds of the present disclosure are stereopure or substantially stereopure.

As demonstrated herein, when used for preparing a chiral internucleotidic linkage, to obtain stereoselectivity generally stereochemically pure chiral reagents are utilized. Among other things, the present disclosure provides stereochemically pure chiral reagents, including those having structures described.

The choice of chiral reagent, for example, the isomer represented by Formula Q or its stereoisomer, Formula R, permits specific control of chirality at a linkage phosphorus. Thus, either an Rp or Sp configuration can be selected in each synthetic cycle, permitting control of the overall three dimensional structure of a chirally controlled oligonucleotide. In some embodiments, a chirally controlled oligonucleotide has all Rp stereocenters. In some embodiments of the disclosure, a chirally controlled oligonucleotide has all Sp stereocenters. In some embodiments of the disclosure, each linkage phosphorus in the chirally controlled oligonucleotide is independently Rp or Sp. In some embodiments of the disclosure, each linkage phosphorus in the chirally controlled oligonucleotide is independently Rp or Sp, and at least one is Rp and at least one is Sp. In some embodiments, the selection of Rp and Sp centers is made to confer a specific three dimensional superstructure to a chirally controlled oligonucleotide. Examples of such selections are described in further detail herein.

In some embodiments, a provided oligonucleotide comprise a chiral auxiliary moiety, e.g., in an internucleotidic linkage. In some embodiments, a chiral auxiliary is connected to a linkage phosphorus. In some embodiments, a chiral auxiliary is connected to a linkage phosphorus through W². In some embodiments, a chiral auxiliary is connected to a linkage phosphorus through W², wherein W²is O. Optionally, W¹, e.g., when W¹is -NG⁵-, is capped during oligonucleotide synthesis. In some embodiments, W¹in a chiral auxiliary in an oligonucleotide is capped, e.g., by a capping reagent during oligonucleotide synthesis. In some embodiments, W¹may be purposeful capped to modulate oligonucleotide property. In some embodiments, W¹is capped with —R¹. In some embodiments, R¹is —C(O)R′. In some embodiments, R′ is optionally substituted C_1-6aliphatic. In some embodiments, R′ is methyl.

In some embodiments, a chiral reagent for use in accordance with the present disclosure is selected for its ability to be removed at a particular step in the above-depicted cycle. For example, in some embodiments it is desirable to remove a chiral reagent during the step of modifying the linkage phosphorus. In some embodiments, it is desirable to remove a chiral reagent before the step of modifying the linkage phosphorus. In some embodiments, it is desirable to remove a chiral reagent after the step of modifying the linkage phosphorus. In some embodiments, it is desirable to remove a chiral reagent after a first coupling step has occurred but before a second coupling step has occurred, such that a chiral reagent is not present on the growing oligonucleotide during the second coupling (and likewise for additional subsequent coupling steps). In some embodiments, a chiral reagent is removed during the “deblock” reaction that occurs after modification of the linkage phosphorus but before a subsequent cycle begins. Example methods and reagents for removal are described herein.

In some embodiments, removal of chiral auxiliary is achieved when performing the modification and/or deblocking step, as illustrated in Scheme I. It can be beneficial to combine chiral auxiliary removal together with other transformations, such as modification and deblocking. A person of ordinary skill in the art would appreciate that the saved steps/transformation could improve the overall efficiency of synthesis, for instance, with respect to yield and product purity, especially for longer oligonucleotides. One example wherein the chiral auxiliary is removed during modification and/or deblocking is illustrated in Scheme 1.

In some embodiments, a chiral reagent for use in accordance with methods of the present disclosure is characterized in that it is removable under certain conditions. For instance, in some embodiments, a chiral reagent is selected for its ability to be removed under acidic conditions. In certain embodiments, a chiral reagent is selected for its ability to be removed under mildly acidic conditions. In certain embodiments, a chiral reagent is selected for its ability to be removed by way of an E1 elimination reaction (e.g., removal occurs due to the formation of a cation intermediate on the chiral reagent under acidic conditions, causing the chiral reagent to cleave from the oligonucleotide). In some embodiments, a chiral reagent is characterized in that it has a structure recognized as being able to accommodate or facilitate an E1 elimination reaction. One of skill in the relevant arts will appreciate which structures would be envisaged as being prone toward undergoing such elimination reactions.

In some embodiments, a chiral reagent is selected for its ability to be removed with a nucleophile. In some embodiments, a chiral reagent is selected for its ability to be removed with an amine nucleophile. In some embodiments, a chiral reagent is selected for its ability to be removed with a nucleophile other than an amine.

In some embodiments, a chiral reagent is selected for its ability to be removed with a base. In some embodiments, a chiral reagent is selected for its ability to be removed with an amine. In some embodiments, a chiral reagent is selected for its ability to be removed with a base other than an amine.

In some embodiments, chirally pure phosphoramidites comprising chiral auxiliaries may be isolated before use. In some embodiments, chirally pure phosphoramidites comprising chiral auxiliaries may be used without isolation—in some embodiments, they may be used directly after formation.

Activation

As appreciated by those skilled in the art, oligonucleotide preparation may use various conditions, reagents, etc. to active a reaction component, e.g., during phosphoramidite preparation, during one or more steps during in the cycles, during post-cycle cleavage/deprotection, etc. Various technologies for activation can be utilized in accordance with the present disclosure, including but not limited to those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the activation technologies of each of which are incorporated by reference. Certain activation technologies. e.g., reagents, conditions, methods, etc. are illustrated in the Examples.

Coupling

In some embodiments, cycles of the present disclosure comprise stereoselective condensation/coupling steps to form chirally controlled internucleotidic linkages. For condensation, often an activating reagent is used, such as 4,5-dicyanoimidazole (DCI), 4,5-dichloroimidazole, 1-phenylimidazolium triflate (PhIMT), benzimidazolium triflate (BIT), benztriazole, 3-nitro-4,2,4-triazole (NT), tetrazole, 5-ethylthiotetrazole (ETT), 5-benzylthiotetrazole (BTT), 5-(4-nitrophenyl)tetrazole, N-cyanomethylpyrrolidinium triflate (CMPT), N-cyanomethylpiperidinium triflate, N-cyanomethyldimethylammonium triflate, etc. Suitable conditions and reagents, including chiral phosphoramidites, include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, U.S. Pat. No. 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the condensation reagents, conditions and methods of each of which are incorporated by reference. Certain coupling technologies, e.g., reagents, conditions, methods, etc. are illustrated in the Examples.

In some embodiments, a phosphoramidite for coupling has the structure of

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wherein each variable is independently as described in the present disclosure. In some embodiments, each R is independently optionally substituted C_1-6aliphatic. A person skill in the art will appreciate that two R groups in any structure or formula can either be the same or different. In some embodiments, each R is independently optionally substituted C_1-6alkyl. In some embodiments, each R is independently optionally substituted C_1-6alkenyl. In some embodiments, each R is independently optionally substituted C_1-6alkynyl. In some embodiments, each R is indenpendtly isopropyl. In some embodiments, -X-L-R¹comprises an optionally substituted triazole group. In some embodiments, X is a covalent bond. In some embodiments, L is a covalent bond. In some embodiments, -X-L-R¹is R¹. In some embodiments, R¹comprise an optionally substituted ring. In some embodiments, R¹is R as described herein. In some embodiments, R¹is optionally substituted

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In some embodiments, R¹is

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In some embodiments, R¹is

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In some embodiments, R¹is

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In some embodiments, -L- comprises C_1-6alkylene. In some embodiments, -L- comprises C_1-6alkenylene. In some embodiments, -L- comprises

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In some embodiments, R¹is R as described herein. In some embodiments, -L- is

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and R¹is H. In some embodiments, -L-R is

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In some embodiments, -X-L-R¹is

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In some embodiments, -X-L-R¹is —OCH₂CH₂CN.

In some embodiments, a chiral phosphoramidite for coupling has the structure of

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wherein each variable is independently as described in the present disclosure. In some embodiments, a chiral phosphoramidite for coupling has the structure of

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In some embodiments, a chiral phosphoramidite for coupling has the structure of

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wherein each variable is independently as described in the present disclosure. In some embodiments, G¹or G²comprises an electron-withdrawing group as described in the present disclosure. In some embodiments, a chiral phosphoramidite for coupling has the structure of

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wherein each variable is independently as described in the present disclosure. In some embodiments, R¹is R²as described in the present disclosure. In some embodiments, R¹is R as described in the present disclosure. In some embodiments, R is optionally substituted phenyl as described in the present disclosure. In some embodiments, R is phenyl. In some embodiments, R is 4-methyl phenyl. In some embodiments, R is 4-methoxy phenyl. In some embodiments, R is optionally substituted C_1-6aliphatic as described in the present disclosure. In some embodiments, R is optionally substituted C_1-6alkyl as described in the present disclosure. For example, in some embodiments, R is methyl; in some embodiments, R is isopropyl; in some embodiments, R is t-butyl; etc.

In some embodiments, R^5s-L^s- is R′O—. In some embodiments, R′O— is DMTrO-. In some embodiments, R^4sis —H. In some embodiments, R^4sand R^2sare taken together to form a bridge -L-O- as described in the present disclosure. In some embodiments, the —O— is connected to the carbon at the 2′ position. In some embodiments, L is —CH₂—. In some embodiments, L is —CH(Me)-. In some embodiments, L is —(R)—CH(Me)-. In some embodiments, L is —(S)—CH(Me)-. In some embodiments. R^2sis —H. In some embodiments, R^2sis —F. In some embodiments, R^2sis —OR′. In some embodiments, R^2sis -OMe. In some embodiments, R^2sis -MOE. As appreciated by those skilled in the art, BA may be suitably protected during synthesis.

In some embodiments, an internucleotidic linkage formed in a coupling step has the structure of formula I or a salt form thereof. In some embodiments, P^Lis P. In some embodiments, -X-L-R is

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wherein each variable is independently in accordance with the present disclosure. In some embodiments, -X-L-R¹is —CH₂CH₂CN.

In some embodiments, a coupling forms an internucleotidic linkage with a stereoselectivity of 80%, 85%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the stereoselectivity is 85% or more. In some embodiments, the stereoselectivity is 85% or more. In some embodiments, the stereoselectivity is 90% or more. In some embodiments, the stereoselectivity is 91% or more. In some embodiments, the stereoselectivity is 92% or more. In some embodiments, the stereoselectivity is 93% or more. In some embodiments, the stereoselectivity is 94% or more. In some embodiments, the stereoselectivity is 95% or more. In some embodiments, the stereoselectivity is 96% or more. In some embodiments, the stereoselectivity is 97% or more. In some embodiments, the stereoselectivity is 98% or more. In some embodiments, the stereoselectivity is 99% or more.

Capping

If the final nucleic acid is larger than a dimer, the unreacted —OH moiety is generally capped with a blocking/capping group. Chiral auxiliaries in oligonucleotides may also be capped with a blocking group to form a capped condensed intermediate. Suitable capping technologies (e.g., reagents, conditions, etc.) include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the capping technologies of each of which are incorporated by reference. In some embodiments, a capping reagent is a carboxylic acid or a derivate thereof. In some embodiments, a capping reagent is R′COOH. In some embodiments, a capping step introduces R′COO— to unreacted 5′-OH group and/or amino groups in chiral auxiliaries. In some embodiments, a cycle may comprise two or more capping steps. In some embodiments, a cycle comprises a first capping before modification of a coupling product (e.g., converting P(III) to P(V)), and another capping after modification of a coupling product. In some embodiments, a first capping is performed under an amidation condition, e.g., which comprises an acylating reagent (e.g., an anhydride having the structure of (RC(O))₂O, (e.g., Ac₂O)) and a base (e.g., 2,6-lutidine). In some embodiments, a first capping caps an amino group, e.g., that of a chiral auxiliary in an internucleotidic linkage. In some embodiments, an internucleotidic linkage formed in a coupling step has the structure of formula I or a salt form thereof. In some embodiments, P^Lis P. In some embodiments, -X-L-R¹is

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wherein each variable is independently in accordance with the present disclosure. In some embodiments, R¹is R—C(O)—. In some embodiments, R is CH₃—. In some embodiments, each chirally controlled coupling (e.g., using a chiral auxiliary) is followed with a first capping. Typically, cycles for non-chirally controlled coupling using traditional phosphoramidite to construct natural phosphate linkages do not contain a first capping. In some embodiments, a second capping is performed, e.g., under an esterification condition (e.g., capping conditions of traditional phosphoramidite oligonucleotide synthesis) wherein free 5′-OH are capped.

Certain capping technologies, e.g., reagents, conditions, methods, etc. are illustrated in the Examples.

Modifying

In some embodiments, an internucleotidic linkage wherein its linkage phosphorus exists as P(II) is modified to form another modified internucleotidic linkage (e.g., one of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof) or a natural phosphate linkage. In many embodiments, P(III) is modified by reaction with an electrophile. Various types of reactions suitable for P(III) may be utilized in accordance with the present disclosure. Suitable modifying technologies (e.g., reagents (e.g., sulfurization reagent, oxidation reagent, etc.), conditions, etc.) include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the modifying technologies of each of which are incorporated by reference.

In some embodiments, as illustrated in the Examples, the present disclosure provides modifying reagents for introducing non-negatively charged internucleotidic linkages including neutral internucleotidic linkages.

In some embodiments, modifying is within a cycle. In some embodiments, modifying can be outside of a cycle. For example, in some embodiments, one or more modifying steps can be performed after the oligonucleotide chain has been reached to introduce modifications simultaneously at one or more internucleotidic linkages and/or other locations.

In some embodiments, modifying comprises use of click chemistry. e.g., wherein an alkyne group of an oligonucleotide, e.g., of an internucleotidic linkage, is reacted with an azide. Various reagents and conditions for click chemistry can be utilized in accordance with the present disclosure. In some embodiments, an azide has the structure of R¹-Na₃, wherein R¹is as described in the present disclosure. In some embodiments, R¹is optionally substituted C_1-6alkyl. In some embodiments, R¹is isopropyl.

In some embodiments, as demonstrated in the examples, a P(III) linkage can be converted into a non-negatively charged internucleotidic linkage by reacting the P(III) linkage with an azide or an azido imidazolinium salt (e.g., a compound comprising

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in some embodiments, referred to as an azide reaction) under suitable conditions. In some embodiments, an azido imidazolinium salt is a salt of PF₆⁻. In some embodiments, an azido imidazolinium salt is a salt of

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In some embodiments, a useful reagent, e.g., an azido imidazolinium salt, is a salt of

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In some embodiments, a useful reagent is a salt of

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In some embodiments, a useful reagent is a salt of

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In some embodiments, a useful reagent is a salt of

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Such reagents comprising nitrogen cations also contain counter anions (e.g., Q as described in the present disclosure), which are widely known in the art and are contained in various chemical reagents. In some embodiments, a useful reagent is Q⁺Q⁻, wherein Q⁺ is

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and Q⁺ is a counter anion. In some embodiments, Q⁺ is

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In some embodiments, Q⁺is

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In some embodiments, Q⁺is

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In some embodiments, Q⁻is

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In some embodiments, Q⁺ is

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As appreciated by those skilled in the art, in a compound having the structure of Q⁺Q⁻, typically the number of positive charges in Q⁺ equals the number of negative charges in Q⁻. In some embodiments, Q⁺is a monovalent cation and Q⁻ is a monovalent anion. In some embodiments, Q⁻ is F⁻, Cl⁻, Br⁻, BF₄⁻, PF₆⁻, TfO⁻, Tf₂N⁻, AsF₆⁻, ClO₄⁻, or SbF₆⁻. In some embodiments, Q⁻ is PF₆⁻. Those skilled in the art readily appreciate that many other types of counter anions are available and can be utilized in accordance with the present disclosure. In some embodiments, an azido imidazolinium salt is 2-azido-1,3-dimethylimidazolinium hexafluorophosphate. In some embodiments, an azide is

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In some embodiments, an azido imidazolinium salt is

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In some embodiments, an azido imidazolinium salt is

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In some embodiments, an azide is

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In some embodiments, an azide is

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In some embodiments, an azide is

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In some embodiments, an azido imidazolinium salt is

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In some embodiments, an azido imidazolinium salt is

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In some embodiments, an azido imidazolinium salt is

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In some embodiments, an azido imidazolinium salt is

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In some embodiments, a P(III) linkage is reacted with an electrophile having the structure of R-G^Z, wherein R is as described in the present disclosure, and G^Zis a leaving group, e.g., —Cl, —Br, —I, -OTf, -Oms, -OTosyl, etc. In some embodiments, R is —CH₃. In some embodiments, R is —CH₂CH₃. In some embodiments, R is —CH₂CH₂CH₃. In some embodiments, R is —CH₂OCH₃. In some embodiments, R is CH₃CH₂OCH₂—. In some embodiments, R is PhCH₂OCH₂—. In some embodiments, R is HC≡C—CH₂—. In some embodiments, R is H₃C—C≡C—CH₂—. In some embodiments, R is CH₂═CHCH₂—. In some embodiments, R is CH₃SCH₂—. In some embodiments, R is —CH₂COOCH₃. In some embodiments, R is —CH₂COOCH₂CH₃. In some embodiments, R is —CH₂CONHCH₃.

In some embodiments, after a modifying step, a P(III) linkage phosphorus is converted into a P(V) internucleotidic linkage. In some embodiments, a P(III) linkage phosphorus is converted into a P(V) internucleotidic linkage, and all groups bounded to the linkage phosphorus remain unchanged. In some embodiments, a linkage phosphorus is converted from P into P(═O). In some embodiments, a linkage phosphorus is converted from P into P(═S). In some embodiments, a linkage phosphorus is converted from P into P(═N-L-R). In some embodiments, a linkage phosphorus is converted from P into

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wherein each variable is independently as described in the present disclosure. In some embodiments, P is converted into

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In some embodiments, P is converted into

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In some embodiments, P is converted into

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In some embodiments, P is converted into

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In some embodiments, P is converted into

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As appreciated by those skilled in the art, for each cation there typically exists a counter anion so that the total number of positive charges equals the total number of negative charges in a system (e.g., compound, composition, etc.). In some embodiments, a counter anion is Q⁻ as described in the present disclosure (e.g., F⁻, Cl⁻, Br⁻, BF₄⁻, PF₆⁻, TfO⁻, Tf₂N⁻, AsF₆⁻, ClO₄⁻, SbF₆⁻, etc.). In some embodiments, an internucleotidic linkage having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof, wherein PL is P is converted into an internucleotidic linkage having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof, wherein P^Lis P(═W) or P→B(R′)₃or P^N. In some embodiments, an internucleotidic linkage having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, I-a-1, I-a-2, II-b-1, II-b-2, I-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof, wherein P^Lis P, is converted into an internucleotidic linkage having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, I-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof, wherein PL is P(═W) or P→B(R′). In some embodiments, a linkage phosphorus P, which is P^Lin an internucleotidic linkage having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, I-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof is converted into P^Lwhich is P(═W) or P→B(R′)₃. In some embodiments, a linkage phosphorus P, which is PL in an internucleotidic linkage having the structure of formula I or a salt form thereof is converted into P^Lwhich is P(═W) or P→B(R′)₃. In some embodiments, W is O (e.g., for an oxidation reaction). In some embodiments, W is S (e.g., for a sulfurization reaction). In some embodiments, W is ═N-L-R (e.g., for an azide reaction). In some embodiments, an internucleotidic linkage having the structure of formula I or a salt form thereof (e.g., wherein P^Lis P) is converted into an internucleotidic linkage having the structure of formula III or a salt form thereof:

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wherein:

P^Nis P(═N-L-R⁵),

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Q⁻ is an anion, and

each other variables is independently as described in the present disclosure.

In some embodiments, P^Nis P(═N-L-R⁵). In some embodiments, P^Nis

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In some embodiments, P^Nis

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In some embodiments, P^Nis

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In some embodiments, P^Nis

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In some embodiments, P^Nis

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In some embodiments, internucleotidic linkages of the present disclosure may exist in a salt form. In some embodiments, internucleotidic linkages of formula III may exist in a salt form. In some embodiments, in a salt form of an internucleotidic linkage of formula III P^Nis

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In some embodiments, P^Nis P=W^N, wherein W^Nis as described herein.

In some embodiments, Y, Z, and -X-L-R¹remains the same during the conversion. In some embodiments, each of X, Y and Z is independently —O—. In some embodiments, as described herein, -X-L-R¹is of such a structure that H-X-L-R¹is a chiral reagent described herein, or a capped chiral reagent described herein wherein an amino group of the chiral reagent (typically of -W¹—H or —W²—H, which comprises an amino group -NHG⁴-) is capped, e.g., with —C(O)R′ (replacing a —H, e.g., —N[—C(O)R′]G⁵-). In some embodiments, -X-L-R¹is

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wherein each variable is independently in accordance with the present disclosure. In some embodiments, wherein R¹is —C(O)R. In some embodiments, R¹is CH₃C(O)—. In some embodiments, as described herein, G²comprises an electron-withdrawing group. In some embodiments, G²is —CH₂SO₂Ph.

In some embodiments, an internucleotidic linkage (e.g., a modified internucleotidic linkage, a chiral internucleotidic linkage, a chirally controlled internucleotidic linkage, a non-negatively charged internucleotidic linkage, a neutral internucleotidic linkage, etc.) has the structure of formula I, I-a. I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof, wherein P^Lis P(═N-L-R), or of formula HI or a salt form thereof. In some embodiments, such an internucleotidic linkage is chirally controlled. In some embodiments, all such internucleotidic linkages are chirally controlled. In some embodiments, linkage phosphorus of at least one of such internucleotidic linkages is Rp. In some embodiments, linkage phosphorus of at least one of such internucleotidic linkages is Sp. In some embodiments, linkage phosphorus of at least one of such internucleotidic linkages is Rp, and linkage phosphorus of at least one of such internucleotidic linkages is Sp. In some embodiments, oligonucleotides of the present disclosure comprises one or more (e.g., 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-40, 1-50, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.) such internucleotidic linkages. In some embodiments, such oligonucleotide further comprise one or more other types of internucleotidic linkages, e.g., one or more natural phosphate linkages, and/or one or more phosphorothioate internucleotidic linkages (e.g., in some embodiments, one or more of which are independently chirally controlled; in some embodiments, each of which is independently chirally controlled; in some embodiments, at least one is Rp; in some embodiments, at least one is Sp; in some embodiments, at least one is Rp and at least one is Sp: etc.) In some embodiments, such oligonucleotides are stereopure (substantially free of other stereoisomers). In some embodiments, the present disclosure provides chirally controlled oligonucleotide compositions of such oligonucleotides. In some embodiments, the present disclosure provides chirally pure oligonucleotide compositions of such oligonucleotides.

In some embodiments, modifying proceeds with a stereoselectivity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the stereoselectivity is 85% or more. In some embodiments, the stereoselectivity is 85% or more. In some embodiments, the stereoselectivity is 90% or more. In some embodiments, the stereoselectivity is 91% or more. In some embodiments, the stereoselectivity is 92% or more. In some embodiments, the stereoselectivity is 93% or more. In some embodiments, the stereoselectivity is 94% or more. In some embodiments, the stereoselectivity is 95% or more. In some embodiments, the stereoselectivity is 96% or more. In some embodiments, the stereoselectivity is 97% or more. In some embodiments, the stereoselectivity is 98% or more. In some embodiments, the stereoselectivity is 99% or more. In some embodiments, modifying is stereospecific.

Deblocking

In some embodiments, a cycle comprises a cycle step. In some embodiments, the 5′ hydroxyl group of the growing oligonucleotide is blocked (i.e., protected) and must be deblocked in order to subsequently react with a nucleoside coupling partner.

In some embodiments, acidification is used to remove a blocking group. Suitable deblocking technologies (e.g., reagents, conditions, etc.) include those described in U.S. Pat. No. 9,695,211, U.S. Pat. No. 9,605,019, U.S. Pat. No. 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555. WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the deblocking technologies of each of which are incorporated by reference. Certain deblocking technologies, e.g., reagents, conditions, methods, etc. are illustrated in the Examples.

Cleavage and Deprotection

At certain stage, e.g., after the desired oligonucleotide lengths have been achieved, cleavage and/or deprotection are performed to deprotect blocked nucleobases etc. and cleave the oligonucleotide products from support. In some embodiments, cleavage and deprotection are performed separately. In some embodiments, cleavage and deprotection are performed in one step, or in two or more steps but without separation of products in between. In some embodiments, cleavage and/or deprotection utilizes basic conditions and elevated temperature. In some embodiments, for certain chiral auxiliaries, a fluoride condition is required (e.g., TBAF, HF-ET₃N, etc., optionally with additional base). Suitable cleavage and deprotection technologies (e.g., reagents, conditions, etc.) include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the cleavage and deprotection technologies of each of which are incorporated by reference. Certain cleavage and deprotection technologies, e.g., reagents, conditions, methods, etc. are illustrated in the Examples.

In some embodiments, certain chiral auxiliaries are removed under basic conditions. In some embodiments, oligonucleotides are contacted with a base, e.g., an amine having the structure of N(R)₃, to remove certain chiral auxiliaries (e.g., those comprising an electronic-withdrawing group in G²as described in the present disclosure). In some embodiments, a base is NHR₂. In some embodiments, each R is independently optionally substituted C_1-6aliphatic. In some embodiments, each R is independently optionally substituted C_1-6alkyl. In some embodiments, an amine is DEA. In some embodiments, an amine is TEA. In some embodiments, an amine is provided as a solution, e.g., an acetonitrile solution. In some embodiments, such contact is performed under anhydrous conditions. In some embodiments, such a contact is performed immediately after desired oligonucleotide lengths are achieved (e.g., first step post synthesis cycles). In some embodiments, such a contact is performed before removal of chiral auxiliaries and/or protection groups and/or cleavage of oligonucleotides from a solid support. In some embodiments, contact with a base may remove cyanoethyl groups utilized in standard oligonucleotide synthesis, providing an natural phosphate linkage which may exist in a salt form (with the cation being, e.g., an ammonium salt). In some embodiments, contact with a base provides an internucleotidic linkage of formula I-n-1, I-n-2. I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1. II-b-2, II-c-1, II-c-2,11-d-1, or II-d-2, or a salt form thereof. In some embodiments, contact with a base removes a chiral auxiliary from an internucleotidic linkage of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2, or a salt form thereof. In some embodiments, contact with a base removes a chiral auxiliary (e.g., -X-L-R¹) from an internucleotidic linkage of formula I or a salt form thereof (e.g., wherein P^Lis P(═N-L-R⁵)). In some embodiments, contact with a base removes a chiral auxiliary (e.g., -X-L-R¹) from an internucleotidic linkage of formula III or a salt form thereof. In some embodiments, In some embodiments, contact with a base converts an internucleotidic linkage of formula I or a salt form thereof (e.g., wherein P^Lis P(═N-L-R⁵)), or of formula III or a salt form thereof, into an internucleotidic linkage of formula II-n-1, 1-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2, or a salt form thereof.

Cycles

Suitable cycles for preparing oligonucleotides of the present disclosure include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647 (e.g., Schemes I, I-b, I-c, I-d, I-e, I-f, etc.), WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the cycles of each of which are incorporated by reference. For example, in some embodiments, an example cycle is Scheme 1-f. Certain cycles are illustrated in the Examples (e.g., for preparation of natural phosphate linkages, utilizing other chiral auxiliaries, etc.).

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In some embodiments, R^2sis H or —OR¹, wherein R¹is not hydrogen. In some embodiments, R^2sis H or —OR¹wherein R¹is optionally substituted C_1-6alkyl. In some embodiments, R^2sis H. In some embodiments, R^2sis -OMe. In some embodiments, R^2sis —OCH₂CH₂OCH₃. In some embodiments, R^2sis —F. In some embodiments, R^4sis —H. In some embodiments, R^4sand R^2sare taken together to form abridge -L-O- as described in the present disclosure. In some embodiments, the —O—is connected to the carbon at the 2′ position. In some embodiments, L is —CH₂—. In some embodiments, L is —CH(Me)-. In some embodiments, L is -(R)-CH(Me)-. In some embodiments, L is -(S)-CH(Me)-.

Purification and Characterization

Various purification and/or characterization technologies (methods, instruments, protocols, etc.) can be utilized to purify and/or characterize oligonuclotides and oligonucleotide compositions in accordance with the present disclosure. In some embodiments, purification is performed using various types of HPLC/UPLC technologies. In some embodiments, characterization comprises MS, NMR, UV, etc. In some embodiments, purification and characterization may be performed together, e.g., HPLC-MS, UPLC-MS, etc. Example purification and characterization technologies include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the purification and characterization technologies of each of which are incorporated by reference.

In some embodiments, the present disclosure provides methods for preparing provided oligonucleotide and oligonucleotide compositions. In some embodiments, a provided method comprises providing a provided chiral reagent having the structure of formula 3-I or 3-AA. In some embodiments, a provided method comprises providing a provided chiral reagent having the structure of

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wherein W¹is -NG⁵, W²is O, each of G¹and G³is independently hydrogen or an optionally substituted group selected from C_1-10aliphatic, heterocyclyl, heteroaryl and aryl, G²is —C(R)₂Si(R)₃, and G⁴and G⁵are taken together to form an optionally substituted saturated, partially unsaturated or unsaturated heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, fused or unfused, wherein each R is independently hydrogen, or an optionally substituted group selected from C₁-C₆aliphatic, carbocyclyl, aryl, heteroaryl, and heterocyclyl. In some embodiments, a provided chiral reagent has the structure of

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wherein each variable is independently as described in the present disclosure. In some embodiments, a provided methods comprises providing a phosphoramidite comprising a moiety from a chiral reagent having the structure of

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wherein -W¹H and —W²H, or the hydroxyl and amino groups, form bonds with the phosphorus atom of the phosphoramidite. In some embodiments, -W¹H and —W²H, or the hydroxyl and amino groups, form bonds with the phosphorus atom of the phosphoramidite, e.g., in

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In some embodiments, a phosphoramidite has the structure of

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or wherein B^PROis BA as described in the present disclosure, and each other variable is as described in the present disclosure. In some embodiments, B^PROis a protected nucleobase. In some embodiments, B^PROis protected A, T, G, C, U or a tautomers thereof. In some embodiments, R is a protection group. In some embodiments, R is DMTr.

In some embodiments, G²is —C(R)₂Si(R)₃, wherein —C(R)₂— is optionally substituted —CH₂—, and each R of —Si(R)₃is independently an optionally substituted group selected from Co aliphatic, heterocyclyl, heteroaryl and aryl. In some embodiments, at least one R of —Si(R)₃is independently optionally substituted Co alkyl. In some embodiments, at least one R of —Si(R)₃is independently optionally substituted phenyl. In some embodiments, one R of —Si(R)₃is independently optionally substituted phenyl, and each of the other two R is independently optionally substituted C_1-10alkyl. In some embodiments, one R of —Si(R)₃is independently optionally substituted C_1-10alkyl, and each of the other two R is independently optionally substituted phenyl. In some embodiments, G²is optionally substituted —CH₂Si(Ph)(Me)₂. In some embodiments, G²is optionally substituted —CH₂Si(Me)(Ph)₂. In some embodiments, G²is —CH₂Si(Me)(Ph)₂. In some embodiments, G²is —CH₂SiMe₃. In some embodiments, G²is —CH₂Si(iPr)₃. In some embodiments, G⁴and G⁵are taken together to form an optionally substituted saturated 5-6 membered ring containing one nitrogen atom (to which G⁵is attached). In some embodiments, G⁴and G⁵are taken together to form an optionally substituted saturated 5-membered ring containing one nitrogen atom. In some embodiments, G¹is hydrogen. In some embodiments, G³is hydrogen. In some embodiments, both G¹and G³are hydrogen. In some embodiments, both G¹and G³are hydrogen, G²is —C(R)₂Si(R)₃, wherein —C(R)₂— is optionally substituted —CH₂—, and each R of —Si(R)₃is independently an optionally substituted group selected from C_1-10aliphatic, heterocyclyl, heteroaryl and aryl, and G⁴and G⁵are taken together to form an optionally substituted saturated 5-membered ring containing one nitrogen atom. In some embodiments, a provided method further comprises providing a fluoro-containing reagent. In some embodiments, a provided fluoro-containing reagent removes a chiral reagent, or a product formed from a chiral reagent, from oligonucleotides after synthesis. Various known fluoro-containing reagents, including those F sources for removing —SiR₃groups, can be utilized in accordance with the present disclosure, for example, TBAF, HF₃-Et₃N etc. In some embodiments, a fluoro-containing reagent provides better results, for example, shorter treatment time, lower temperature, less de-sulfurization, etc, compared to traditional methods, such as concentrated ammonia. In some embodiments, for certain fluoro-containing reagent, the present disclosure provides linkers for improved results, for example, less cleavage of oligonucleotides from support during removal of chiral reagent (or product formed therefrom during oligonucleotide synthesis). In some embodiments, a provided linker is an SP linker. In some embodiments, the present disclosure demonstrated that a HF-base complex can be utilized, such as HF-NR₃, to control cleavage during removal of chiral reagent (or product formed therefrom during oligonucleotide synthesis). In some embodiments, HF-NR₃is HF-NEt₃. In some embodiments, HF-NR₃enables use of traditional linkers, e.g., succinyl linker.

In some embodiments, as described herein, G²comprises an electron-withdrawing group, e.g., at its α position. In some embodiments, G²is methyl substituted with one or more electron-withdrawing groups. In some embodiments, an electronic-withdrawing group comprises and/or is connected to the carbon atom through, e.g., —S(O)—, —S(O)₂—, —P(O)(R¹)—, —P(S)R¹—, or —C(O)—. In some embodiments, an electron-withdrawing group is —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂. In some embodiments, an electron-withdrawing group is aryl or heteroaryl, e.g., phenyl, substituted with one or more of —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂. In some embodiments, G²is —CH₂S(O)R′. In some embodiments, G²is —CH₂S(O)₂R′. In some embodiments, G²is —CHP(O)(R′)₂. Additional example embodiments are described, e.g., as for chiral reagents/auxiliaries.

Confirmation that a stereocontrolled oligonucleotide (e.g., one prepared by a method described herein or in the art) comprises the intended stereocontrolled (chirally controlled) internucleotidic linkage can be performed using a variety of suitable technologies. A stereocontrolled (chirally controlled) oligonucleotide comprises at least one stereocontrolled internucleotidic linkage, which can be, e.g., a stereocontrolled internucleotidic linkage comprising a phosphorus, a stereocontrolled phosphorothioate internucleotidic linkage (PS) in the Rp configuration, a PS in the Sp configuration, etc. Useful technologies include, as non-limiting examples: NMR (e.g., 1D (one-dimensional) and/or 2D (two-dimensional) ¹H-³¹P HETCOR (heteronuclear correlation spectroscopy)), HPLC, RP-HPLC, mass spectrometry. LC-MS, and/or stereospecific nucleases. In some embodiments, stereospecific nucleases include: benzonase, micrococcal nuclease, and svPDE (snake venom phosphodiesterase), which are specific for internucleotidic linkages in the Rp configuration (e.g., a PS in the Rp configuration); and nuclease P1, mung bean nuclease, and nuclease S1, which are specific for internucleotidic linkages in the Sp configuration (e.g., a PS in the Sp configuration).

In some embodiments, the present disclosure pertains to a method of confirming or identifying the stereochemistry pattern of the backbone of an oligonucleotide and/or stereochemistry of particular internucleotidic linkages. In some embodiments, an oligonucleotide comprises a stereocontrolled internucleotidic linkage comprising a phosphorus, a stereocontrolled phosphorothioate (PS) in the Rp configuration, or a PS in the Sp configuration. In some embodiments, an oligonucleotide comprises at least one stereocontrolled internucleotidic linkage and at least one internucleotidic linkage which is not stereocontrolled. In some embodiments, a method comprises digestion of an oligonucleotide with a stereospecific nuclease. In some embodiments, a stereospecific nuclease is selected from: benzonase, micrococcal nuclease, and svPDE (snake venom phosphodiesterase), which are specific for internucleotidic linkages in the Rp configuration (e.g., a PS in the Rp configuration); and nuclease P1, mung bean nuclease, and nuclease S1, which are specific for internucleotidic linkages in the Sp configuration (e.g., a PS in the Sp configuration). In some embodiments, an oligonucleotide or fragments thereof produced by digestion with a stereospecific nuclease are analyzed. In some embodiments, an oligonucleotide or fragments thereof (e.g., produced by digestion with a stereospecific nuclease) are analyzed by NMR, 1D (one-dimensional) and/or 2D (two-dimensional) ¹H-³¹P HETCOR (heteronuclear correlation spectroscopy), HPLC, RP-HPLC, mass spectrometry, LC-MS, UPLC, etc. In some embodiments, an oligonucleotide or fragments thereof are compared with chemically synthesized fragments of the oligonucleotide having a known pattern of stereochemistry.

Without wishing to be bound by any particular theory, the present disclosure notes that, in at least some cases, stereospecificity of a particular nuclease may be altered by a modification (e.g., 2′-modification) of a sugar, by a base sequence, or by a stereochemical context. For example, in some embodiments, benzonase and micrococcal nuclease, which are specific for Rp internucleotidic linkages, were both unable to cleave an isolated PS Rp internucleotidic linkage flanked by PS Sp internucleotidic linkages.

Various techniques and materials can be utilized. In some embodiments, the present disclosure provides useful combinations of technologies. For example, in some embodiments, stereochemistry of one or more particular internucleotidic linkages of an oligonucleotide can be confirmed by digestion of the oligonucleotide with a stereospecific nuclease and analysis of the resultant fragments (e.g., nuclease digestion products) by any of a variety of techniques (e.g., separation based on mass-to-charge ratio, NMR, HPLC, mass spectrometry, etc.). In some embodiments, stereochemistry of products of digesting an oligonucleotide with a stereospecific nuclease can be confirmed by comparison (e.g., NMR, HPLC, mass spectrometry, etc.) with chemically synthesized fragments (e.g., dimers, trimers, tetramers, etc.) produced, e.g., via technologies that control stereochemistry.

In one example, an oligonucleotide was confirmed to have the designed and intended pattern of stereochemistry in the backbone. The tested oligonucleotide comprises a core comprising 2′-deoxy nucleosides, wherein all of the internucleotidic linkages were PS in the Sp configuration except for one PS in the Rp configuration; and two wings, each of which comprising 2′-OMe nucleosides, wherein all the internucleotidic linkages in each wing were phosphodiester (PO) except for one PS in the Sp configuration in each wing. The oligonucleotide was digested with a stereospecific nuclease (e.g., nuclease P1). The various fragments were analyzed (e.g., by LC-MS and by comparison with chemically synthesized fragments of known stereochemistry). It was confirmed that the oligonucleotide had the intended pattern of stereochemistry in its backbone.

In another example, an oligonucleotide having a different sequence was confirmed to have the intended pattern of stereochemistry in its backbone, using digestion with a stereospecific nuclease and analysis of the resultant fragments. This oligonucleotide comprises a core comprising 2′-deoxy nucleotides, wherein all of the internucleotidic linkages were PS in the Sp configuration except for one PS in the Rp configuration; and two wings, each of which comprising 2′-Me nucleotides, wherein all the internucleotidic linkages in each wing were phosphodiester (PO) except for one PS in the Sp configuration in each wing.

In yet another example, a different oligonucleotide was tested to confirm that the internucleotidic linkages were in the intended configurations. The oligonucleotide is capable of skipping exon 51 of DMD; the majority of the nucleotides in the oligonucleotide were 2′-F and the remainder were 2′-OMe; the majority of the internucleotidic linkages in the oligonucleotide were PS in the Sp configuration and the remainder were PO. This oligonucleotide was tested by digestion with stereospecific nucleases, and the resultant digestion fragments were analyzed (e.g., by LC-MS and by comparison with chemically synthesized fragments of known stereochemistry). The results confirmed that the oligonucleotide had the intended pattern of stereocontrolled internucleotidic linkages.

In some embodiments, NMR is useful for characterization and/or confirming stereochemistry. In a set of example experiments, a set of oligonucleotides comprising a stereocontrolled CpG motif were tested to confirm the intended stereochemistry of the CpG motif. Oligonucleotides of the set comprise a motif having the structure of pCpGp, wherein C is Cytosine. G is Guanine, and p is a phosphorothioate which is stereorandom or stereocontrolled (e.g., in the Rp or Sp configuration). For example, one oligonucleotide comprises a pCpGp structure, wherein the pattern of stereochemistry of the phosphorothioates (e.g., the ppp) was RRR; in another oligonucleotide, the pattern of stereochemistry of the ppp was RSS; in another oligonucleotide, the pattern of stereochemistry of the ppp was RSR; etc. In the set, all possible patterns of stereochemistry of the ppp were represented. In the portion of the oligonucleotide outside the pCpGp structure, all the internucleotidic linkages were PO; all nucleosides in the oligonucleotides were 2′-deoxy. These various oligonucleotides were tested in NMR, without digestion with a stereospecific nuclease, and distinctive patterns of peaks were observed, indicating that each PS which was Rp or Sp produced a unique peak, and confirming that the oligonucleotides comprised stereocontrolled PS internucleotidic linkages of the intended stereochemistry.

Stereochemistry patterns of the internucleotidic linkages of various other stereocontrolled oligonucleotides were confirmed, wherein the oligonucleotides comprise a variety of chemical modifications and patterns of stereochemistry.

As those skilled in the art will appreciate, in some embodiments, a product oligonucleotide of a step, cycle or preparation is an oligonucleotide comprising O^5P, O^P, *^P, *^PDS, *^PDR, *^N, *^NS and/or *^NR as described herein, which oligonucleotide is optionally linked to a support (e.g., CPG) optionally via a linker (e.g., a CAN linker). For example, in some embodiments, after coupling and/or pre-modification capping and before modification, O^5Pis

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or a salt form thereof. In some embodiments, after modification O^5Pis L^PO, L^PA, L^PB, or a salt form thereof.

Metabolites

In some embodiments, a DMD oligonucleotide corresponds to a fragment of a different, longer DMD oligonucleotide. In some embodiments, a DMD oligonucleotide corresponds to a metabolite produced by cleavage (e.g., enzymatic cleavage by a nuclease) of a longer DMD oligonucleotide, which produces a fragment or portion of the longer DMD oligonucleotide. In some embodiments, the present disclosure pertains to an DMD oligonucleotide which corresponds to a metabolite produced by the cleavage of a DMD oligonucleotide described herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which corresponds to a portion, or fragment of a DMD oligonucleotide disclosed herein.

Several experiments were performed wherein a DMD oligonucleotide was incubated in vitro in the presence of any of various substances comprising nucleases. In various experiments, such substances include brain homogenatem, cerebrospinal fluid or plasma from Sprague-Dawley rat or Cynomolgus monkey. Plasma was heparinized. Oligonucleotides were incubated for various time points (e.g., 0, 1, 2, 3, 4 or 5 days for brain tissue homogenate, with a pre-incubation period of 0, 1 or 2 days; 0, 1, 2, 4, 8, 16, 24 or 48 hrs for cerebrospinal fluid; or 0, 1, 2, 4, 8, 16 or 24 hrs for plasma). Pre-incubation indicates that the homogenate is incubated at 37 degrees ° C. for 0, 24 or 48 hrs to activate the enzymes before adding the oligonucleotide. Final concentration and volume of oligonucleotides was 20 μM in 200 μl. Products produced by cleavage of the oligonucleotides were analyzed by LC/MS.

For one DMD oligonucleotide, which is 20 bases long, tested in rat brain homogenate, the major metabolites represented the 3′ end of the oligonucleotide, which were truncated by 4, 10, 11, 12, or 13 bases.

One test DMD oligonucleotide has a length of 20 bases and was tested in rat brain homogenate, yielding major metabolites which were truncated at the 5′ end by 4, 10, 11, 12, or 13 bases, leaving metabolites representing the 3′ end of the oligonucleotide and which were 16, 10, 9, 8 or 7 bases long, respectively. This oligonucleotide also produced a metabolite which was a 5′ fragment which was 12 bases long (truncated at the 3′ end by 8 bases).

A second test oligonucleotide has a length of 20 bases and was tested in rat brain homogenate, yielding major metabolites which were truncated at the 3′ end by 4, 8, 9 or 10 bases, leaving metabolites representing the 5′ end of the oligonucleotide and which were 16, 12, 11 or 10 bases long, respectively.

The two tested oligonucleotides comprise internucleotidic linkages which are phosphodiesters, phosphorothioate in the Rp configuration, and phosphorothioates in the Sp configuration. In some embodiments, phosphodiesters were more labile than the phosphorothioate in the Rp configuration or the phosphorothioate in the Sp configuration. In some cases, a metabolite of an oligonucleotide represents a product of a cleavage at a phosphodiester.

In some embodiments, the present disclosure pertains to a DMD oligonucleotide which corresponds to a metabolite of a DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or more bases shorter than a DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has a base sequence which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more bases shorter than that of a DMD oligonucleotide disclosed herein.

In some embodiments, a metabolite is designated as 3′-N-#, or 5′-N-#, wherein the # indicates the number of bases removed, and the 3′ or 5′ indicates which end of the molecule from which the bases were deleted. For example, 3′-N-1 indicates a fragment or metabolite wherein 1 base was removed from the 3′ end.

In some embodiments, the present disclosure perhaps to an oligonucleotide which corresponds to a fragment or metabolite of a DMD oligonucleotide disclosed herein, wherein the fragment or metabolite can be described as corresponding to 3′-N-1, 3′-N-2, 3′-N-3, 3′-N-4, 3′-N-5, 3′-N-6, 3′-N-7, 3′-N-8, 3′-N-9, 3′-N-10, 3′-N-11, 3′-N-12, 5′-N-1, 5′-N-2, 5′-N-3, 5′-N4, 5′-N-5, 5′-N-6, 5′-N-7, 5′-N-8, 5′-N-9, 5′-N-10, 5′-N-11, or 5′-N-12 of a DMD oligonucleotide described herein.

In some embodiments, the present disclosure pertains to a DMD oligonucleotide which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more bases shorter on the 5′ end than a DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has a base sequence which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more bases shorter on the 5′ end than that of a DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more bases shorter on the 3′ end than a DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has a base sequence which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more bases shorter on the 3′ end than that of a DMD oligonucleotide disclosed herein.

In some embodiments, the present disclosure pertains to a DMD which corresponds to a metabolite of a DMD oligonucleotide, wherein the metabolite is truncated on the 5′ and/or 3′ end relative to the DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD which corresponds to a metabolite of a DMD oligonucleotide, wherein the metabolite is truncated on both the 5′ and 3′ end relative to the DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more total bases shorter on the 5′ and/or 3′ end than a DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has a base sequence which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more bases total shorter on the 5′ and/or 3′ end than that of a DMD oligonucleotide disclosed herein.

In some embodiments, the present disclosure pertains to a DMD oligonucleotide which would be represented by a product of cleavage of a DMD oligonucleotide disclosed herein, which is cleaved at a phosphodiester linkage. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which would be represented by a product of cleavage of a DMD oligonucleotide disclosed herein, if such an oligonucleotide were cleaved at a phosphorothioate linkage in the Rp configuration. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which would be represented by a product of cleavage of a DMD oligonucleotide disclosed herein, if such an oligonucleotide were cleaved at one or more phosphodiester linkages and/or phosphorothioate linkages in the Rp configuration.

Biological Applications, Example Use, and Dosing Regimens

As described herein, provided compositions and methods are useful for various purposes, e.g., those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, and/or WO 2017/210647. Among other things, provided technologies can function and/or provide various benefits through a number of chemical and/or biological mechanisms, pathways, etc. (e.g., RNase H, RNAi, splicing modulation (exon skipping(e.g., for DMD in DMD subjects/samples), exon inclusion (e.g., for SMN2 in SMA subjects/samples)), etc.). In some embodiments, provided technologies reduce levels, activities, expressions, etc. of a nucleic acid and/or a product thereof. For example, in some embodiments, provided technologies reduce levels and/or activities of target transcripts and/or products encoded thereby (without the intention to be limited by any particular theory, in some embodiments, via RNase H pathway). In some embodiments, provided technologies increase levels and/or activities of target transcripts and/or products encoded thereby (without the intention to be limited by any particular theory, in some embodiments, via exon skipping). A number of oligonucleotides comprising various types of modified internucleotidic linkages, including many comprising non-negatively charged internucleotidic linkages (e.g., n001), which have various base sequences and/or target various nucleic acids (e.g., transcripts of various genes) were prepared, and various useful properties, activities, and/or advantages were demonstrated. Certain such oligonucleotides, including many comprising non-negatively charged internucleotidic linkages, target transcripts of PNPLA3, C9orf72, SMN2, etc. and have demonstrated various activities and/or benefits. Example oligonucleotides comprising non-negatively charged internucleotidic linkages and targeting various genes, and compositions and uses thereof, include those described in WO 2018/223056, WO 2019/032607, PCT/US18/55653, and WO 2019/032612, each of which is independently incorporated herein by reference.

In some embodiments, the present disclosure provides methods for modulating level of a transcript or a product encoded thereby in a system, comprising administering an effective amount of a provided oligonucleotide or a composition thereof. In some embodiments, the present disclosure provides methods for modulating level of a transcript or a product encoded thereby in a system, comprising contacting the transcript a provided oligonucleotide or a composition thereof. In some embodiments, a system is an in vitro system. In some embodiments, a system is a cell. In some embodiments, a system is a tissue. In some embodiments, a system is an organ. In some embodiments, a system is an organism. In some embodiments, a system is a subject. In some embodiments, a system is a human. In some embodiments, modulating level of a transcript decreases level of the transcript. In some embodiments, modulating level of a transcript increases level of the transcript.

In some embodiments, the present disclosure provides methods for preventing or treating a condition, disease, or disorder associated with a nucleic acid sequence or a product encoded thereby, comprising administering to a subject suffering therefrom or susceptible thereto an effective amount of a provided oligonucleotide or composition thereof, wherein the oligonucleotide or composition thereof modulate level of a transcript of the nucleic acid sequence. In some embodiments, a nucleic acid sequence is a gene. In some embodiments, modulating level of a transcript decreases level of the transcript. In some embodiments, modulating level of a transcript increases level of the transcript.

In some embodiments, change of the level of a modulated transcript, e.g., through knock-down, exon skipping, etc., is at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 200, 500, or 1000 fold.

In some embodiments, provided oligonucleotides and oligonucleotide compositions modulate splicing. In some embodiments, provided oligonucleotides and oligonucleotide compositions promote exon skipping, thereby produce a level of a transcript which has increased beneficial functions that the transcript prior to exon skipping. In some embodiments, a beneficial function is encoding a protein that has increased biological functions. In some embodiments, the present disclosure provides methods for modulating splicing, comprising administering to a splicing system a provided oligonucleotide or oligonucleotide composition, wherein splicing of at least one transcript is altered. In some embodiments, level of at least one splicing product is increased at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 200, 500, or 1000 fold. In some embodiments, the present disclosure provides methods for modulating DMD splicing, comprising administering to a splicing system a provided DMD oligonucleotide or composition thereof.

In some embodiments, the present disclosure provides methods for preventing or treating DMD, comprising administering to a subject susceptible thereto or suffering therefrom a pharmaceutical composition comprising an effective amount of a provided oligonucleotide or oligonucleotide composition.

In some embodiments, provided compositions and methods provide improved splicing patterns of transcripts compared to a reference pattern, which is a pattern from a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof. An improvement can be an improvement of any desired biological functions. In some embodiments, for example, in DMD, an improvement is production of an mRNA from which a dystrophin protein with improved biological activities is produced.

In some embodiments, particularly useful and effective are chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions, wherein the oligonucleotides (or oligonucleotides of a plurality in chirally controlled oligonucleotide compositions) optionally comprises one or more non-negatively charged internucleotidic linkages. Among other things, such oligonucleotides and oligonucleotide compositions can provide greatly improved effects, better delivery, lower toxicity, etc.

For Duchenne muscular dystrophy, example mutations and/or suitable DMD exons for skipping are widely known in the art, including but not limited to those described in U.S. Pat. Nos. 8,759,507, 8,486,907, and reference cited therein.

In some embodiments, one or more skipped exons are selected from exon 2, 29, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60. In some embodiments, exon 2 of DMD is skipped. In some embodiments, exon 29 of DMD is skipped. In some embodiments, exon 40 of DMD is skipped. In some embodiments, exon 41 of DMD is skipped. In some embodiments, exon 42 of DMD is skipped. In some embodiments, exon 43 of DMD is skipped. In some embodiments, exon 44 of DMD is skipped. In some embodiments, exon 45 of DMD is skipped. In some embodiments, exon 46 of DMD is skipped. In some embodiments, exon 47 of DMD is skipped. In some embodiments, exon 48 of DMD is skipped. In some embodiments, exon 49 of DMD is skipped. In some embodiments, exon 50 of DMD is skipped. In some embodiments, exon 51 of DMD is skipped. In some embodiments, exon 52 of DMD is skipped. In some embodiments, exon 53 of DMD is skipped. In some embodiments, exon 54 of DMD is skipped. In some embodiments, exon 50 of DMD is skipped. In some embodiments, exon 55 of DMD is skipped. In some embodiments, a skipped exon is any exon whose inclusion decreases a desired function of DMD. In some embodiments, a skipped exon is any exon whose skipping increased a desired function of DMD.

In some embodiments, more than one exon of DMD is skipped. In some embodiments, two or more exons of DMD are skipped. In some embodiments, two or more adjacent exons of DMD are skipped.

In some embodiments, for exon skipping of DMD transcript, or for treatment of DMD, a sequence of a provided plurality of oligonucleotides comprises a DMD sequence list herein. In some embodiments, a sequence comprises one of SEQ ID Nos 1-30 of U.S. Pat. No. 8,759,507. In some embodiments, a sequence comprises one of SEQ ID Nos 1-211 of U.S. Pat. No. 8,486,907. In some embodiments, for exon skipping of DMD transcript, or for treatment of DMD, a sequence of a provided plurality of oligonucleotides is a DMD sequence disclosed herein. In some embodiments, a sequence is one of SEQ ID Nos 1-30 of U.S. Pat. No. 8,759,507. In some embodiments, a sequence is one of SEQ ID Nos 1-211 of U.S. Pat. No. 8,486,907. In some embodiments, a sequence is, comprises or comprises at least 15 consecutive bases of the sequence of any oligonucleotide list herein, e.g., in Table A1. In some embodiments, a sequence is one described in Kemaladewi, et al., Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy, BMC Med Genomics. 2011 Apr 20:4:36. doi: 10.1186/1755-8794-4-36; or Malerba et al., Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy, Mol Ther Nucleic Acids. 2012 Dec 18; 1:e62. doi: 10.1038/mtna.2012.54.

In some embodiments, a provided oligonucleotide composition is administered at a dose and/or frequency lower than that of an otherwise comparable reference oligonucleotide composition with comparable effect in altering the splicing of a target transcript. In some embodiments, a stereocontrolled (chirally controlled) oligonucleotide composition is administered at a dose and/or frequency lower than that of an otherwise comparable stereorandom reference oligonucleotide composition with comparable effect in altering the splicing of the target transcript. If desired, a provided composition can also be administered at higher dose/frequency due to its lower toxicities.

In some embodiments, provided oligonucleotides, compositions and methods have low toxicities, e.g., when compared to a reference composition. As widely known in the art, oligonucleotides can induce toxicities when administered to, e.g., cells, tissues, organism, etc. In some embodiments, oligonucleotides can induce undesired immune response. In some embodiments, oligonucleotide can induce complement activation. In some embodiments, oligonucleotides can induce activation of the alternative pathway of complement. In some embodiments, oligonucleotides can induce inflammation. Among other things, the complement system has strong cytolytic activity that can damages cells and should therefore be modulated to reduce potential injuries. In some embodiments, oligonucleotide-induced vascular injury is a recurrent challenge in the development of oligonucleotides for e.g., pharmaceutical use. In some embodiments, a primary source of inflammation when high doses of oligonucleotides are administered involves activation of the alternative complement cascade. In some embodiments, complement activation is a common challenge associated with phosphorothioate-containing oligonucleotides, and there is also a potential of some sequences of phosphorothioates to induce innate immune cell activation. In some embodiments, cytokine release is associated with administration of oligonucleotides. For example, in some embodiments, increases in interleukin-6 (IL-6) monocyte chemoattractant protein (MCP-1) and/or interleukin-12 (IL-12) is observed. See, e.g., Frazier, Antisense Oligonucleotide Therapies: The Promise and the Challenges from a Toxicologic Pathologist's Perspective. Toxicol Pathol., 43: 78-89, 2015; and Engelhardt, et al., Scientific and Regulatory Policy Committee Points-to-consider Paper: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part 2. Antisense Oligonucleotides. Toxicol Pathol. 43: 935-944, 2015.

Oligonucleotide compositions as provided herein can be used as agents for modulating a number of cellular processes and machineries, including but not limited to, transcription, translation, immune responses, epigenetics, etc. In addition, oligonucleotide compositions as provided herein can be used as reagents for research and/or diagnostic purposes. One of ordinary skill in the art will readily recognize that the present disclosure herein is not limited to particular use but is applicable to any situations where the use of synthetic oligonucleitides is desirable. Among other things, provided compositions are useful in a variety of therapeutic, diagnostic, agricultural, and/or research applications.

Various dosing regimens can be utilized to administer provided chirally controlled oligonucleotide compositions, e.g., those described in in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, and/or WO 2017/210647, the dosing regimens of each of which is incorporated herein by reference.

In some embodiments, with their low toxicity, provided oligonucleotides and compositions can be administered in higher dosage and/or with higher frequency. In some embodiments, with their improved delivery (and other properties), provided compositions can be administered in lower dosages and/or with lower frequency to achieve biological effects, for example, clinical efficacy.

A single dose can contain various amounts of oligonucleotides. In some embodiments, a single dose can contain various amounts of a type of chirally controlled oligonucleotide, as desired suitable by the application. In some embodiments, a single dose contains about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more (e.g., about 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more) mg of a type of chirally controlled oligonucleotide. In some embodiments, a chirally controlled oligonucleotide is administered at a lower amount in a single dose, and/or in total dose, than a chirally uncontrolled oligonucleotide. In some embodiments, a chirally controlled oligonucleotide is administered at a lower amount in a single dose, and/or in total dose, than a chirally uncontrolled oligonucleotide due to improved efficacy. In some embodiments, a chirally controlled oligonucleotide is administered at a higher amount in a single dose, and/or in total dose, than a chirally uncontrolled oligonucleotide. In some embodiments, a chirally controlled oligonucleotide is administered at a higher amount in a single dose, and/or in total dose, than a chirally uncontrolled oligonucleotide due to improved safety.

Pharmaceutical Compositions

When used as therapeutics, a provided oligonucleotide or oligonucleotide composition described herein is administered as a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a provided oligonucleotides, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable inactive ingredient selected from pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and pharmaceutically acceptable carriers. In some embodiments, in provided compositions provided oligonucleotides may exist as salts, preferably pharmaceutically acceptable salts, e.g., sodium salts, ammonium salts, etc. In some embodiments, a salt of a provided oligonucleotide comprises two or more cations, for example, in some embodiments, up to the number of negatively charged acidic groups (e.g., phosphate, phosphorothioate, etc.) in an oligonucleotide. As appreciated by those skilled in the art, oligonucleotides described herein may be provided and/or utilized in a salt form, particularly a pharmaceutically acceptable salt form.

In some embodiments, the present disclosure provides salts of provided oligonucleotides, e.g., chirally controlled oligonucleotides, and pharmaceutical compositions thereof. In some embodiments, a salt is a pharmaceutically acceptable salt. In some embodiments, each hydrogen ion that may be donated to a base (e.g., under conditions of an aqueous solution, a pharmaceutical composition, etc.) is replaced by a non-H⁺ cation. For example, in some embodiments, a pharmaceutically acceptable salt of an oligonucleotide is an all-metal ion salt, wherein each hydrogen ion (for example, of —OH—SH, etc., acidic enough in water) of each internucleotidic linkage (e.g., a natural phosphate linkage, a phosphorothioate diester linkage, etc.) is replaced by a metal ion. In some embodiments, a provided salt is an all-sodium salt. In some embodiments, a provided pharmaceutically acceptable salt is an all-sodium salt. In some embodiments, a provided salt is an all-sodium salt, wherein each internucleotidic linkage which is a natural phosphate linkage (acid form —O—P(O)(OH)—O—), if any, exists as its sodium salt form (—O—P(O)(ONa)—O—), and each internucleotidic linkage which is a phosphorothioate diester linkage (phosphorothioate internucleotidic linkage; acid form —O—P(O)(SH)—O—), if any, exists as its sodium salt form (—O—P(O)(SNa)—O—).

In some embodiments, the pharmaceutical composition is formulated for intravenous injection, oral administration, buccal administration, inhalation, nasal administration, topical administration, ophthalmic administration or otic administration. In some embodiments, the pharmaceutical composition is a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spray solution, a suppository, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, an ointment, a lotion, an eye drop or an car drop.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising chirally controlled oligonucleotide, or composition thereof, in admixture with a pharmaceutically acceptable excipient. One of skill in the art will recognize that the pharmaceutical compositions include the pharmaceutically acceptable salts of the chirally controlled oligonucleotide, or composition thereof, described above.

A variety of supramolecular nanocarriers can be used to deliver nucleic acids. Example nanocarriers include, but are not limited to liposomes, cationic polymer complexes and various polymeric. Complexation of nucleic acids with various polycations is another approach for intracellular delivery; this includes use of PEGlyated polycations, polyethyleneamine (PEI) complexes, cationic block co-polymers, and dendrimers. Several cationic nanocarriers, including PEI and polyamidoamine dendrimers help to release contents from endosomes. Other approaches include use of polymeric nanoparticles, polymer micelles, quantum dots and lipoplexes. In some embodiments, an oligonucleotide is conjugated to another molecular.

Additional nucleic acid delivery strategies are known in addition to the example delivery strategies described herein.

In therapeutic and/or diagnostic applications, the compounds of the disclosure can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington. The Science and Practice of Pharmacy, (20th ed. 2000).

Provided oligonucleotides, and compositions thereof, are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from about 0.01 to about 1000 mg, from about 0.5 to about 100 mg, from about 1 to about 50 mg per day, and from about 5 to about 100 mg per day are examples of dosages that may be used. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.

Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art, and may include, by way of example but not limitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Other pharmaceutically acceptable salts may be found in, for example, Remington, The Science and Practice of Pharmacy (20th ed. 2000). Preferred pharmaceutically acceptable salts include, for example, acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide, hydrochloride, maleate, mesylate, napsylate, pamoate (embonate), phosphate, salicylate, succinate, sulfate, or tartrate.

As appreciated by a person having ordinary skill in the art, oligonucleotides may be formulated as a number of salts for, e.g., pharmaceutical uses. In some embodiments, a salt is a metal cation salt and/or ammonium salt. In some embodiments, a salt is a metal cation salt of an oligonucleotide. In some embodiments, a salt is an ammonium salt of an oligonucleotide. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, a salt is a sodium salt of an oligonucleotide. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed with oligonucleotides. As appreciated by a person having ordinary skill in the art, a salt of an oligonucleotide may contain more than one cations, e.g., sodium ions, as there may be more than one anions within an oligonucleotide.

Depending on the specific conditions being treated, such agents may be formulated into liquid or solid dosage forms and administered systemically or locally. The agents may be delivered, for example, in a timed- or sustained-low release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington, The Science and Practice of Pharmacy (20th ed. 2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articullar, intra-sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections or other modes of delivery.

For injection, the agents of the disclosure may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate the compounds herein disclosed for the practice of the disclosure into dosages suitable for systemic administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection.

Compounds, e.g., oligonucleotides, can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject (e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the disclosure may also be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances such as, saline, preservatives, such as benzyl alcohol, absorption promoters, and fluorocarbons.

In certain embodiments, oligonucleotides and compositions are delivered to the CNS. In certain embodiments, oligonucleotides and compositions are delivered to the cerebrospinal fluid. In certain embodiments, oligonucleotides and compositions are administered to the brain parenchyma. In certain embodiments, oligonucleotides and compositions are delivered to an animal/subject by intrathecal administration, or intracerebroventricular administration. Broad distribution of oligonucleotides and compositions, described herein, within the central nervous system may be achieved with intraparenchymal administration, intrathecal administration, or intracerebroventricular administration.

In certain embodiments, parenteral administration is by injection, by, e.g., a syringe, a pump, etc. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to a tissue, such as striatum, caudate, cortex, hippocampus and cerebellum.

In certain embodiments, methods of specifically localizing a pharmaceutical agent, such as by bolus injection, decreases median effective concentration (EC50) by a factor of 20, 25, 30, 35, 40, 45 or 50. In certain embodiments, the targeted tissue is brain tissue. In certain embodiments the targeted tissue is striatal tissue. In certain embodiments, decreasing EC50 is desirable because it reduces the dose required to achieve a pharmacological result in a patient in need thereof.

In certain embodiments, an oligonucleotide is delivered by injection or infusion once every month, every two months, every 90 days, every 3 months, every 6 months, twice a year or once a year.

Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of an active compound into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combining an active compound with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC), and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, an active compound may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may be added.

In some embodiments, any DMD oligonucleotide, or combination thereof, described herein, or any composition comprising a DMD oligonucleotide described herein, can be combined with any pharmaceutical preparation described herein or known in the art.

Certain Embodiments of Conjugates and Additional Chemical Moieties

In some embodiments, provided oligonucleotides comprise one or more additional chemical moieties (e.g., other than typical moieties of nucleobases, sugars and/or internucleotidic linkages, etc.), optionally through a linker. In some embodiments, a chemical moiety is a lipid moiety. In some embodiments, a chemical moiety is a carbohydrate moiety. In some embodiments, a chemical moiety is a targeting moiety. In some embodiments, a chemical moiety is a moiety of a ligand. In some embodiments, a chemical moiety can increase delivery of oligonucleotides to certain organelles, cells, tissues, organs, and/or organisms. In some embodiments, a chemical moiety enhances one or more of desired properties and/or activities. Certain example chemical moieties utilized in certain oligonucleotides are presented in the Tables (e.g., various Mod in Table A1). In some embodiments, a chemical moiety comprises one or more sugar moieties or derivatives thereof, e.g., glucose, mannose, etc. In some embodiments, a chemical moiety is or comprises a lipid moiety. In some embodiments, a chemical moiety is or comprises a vitamin E moiety. In some embodiments, a chemical moiety comprises one or more peptide moieties. In some embodiments, a peptide is a cell-penetrating peptide. In some embodiments, a peptide is a ligand of a protein, e.g., a cell surface receptor. In some embodiments, a peptide is a Tfr1 peptide. Certain example peptide moieties are utilized to prepare oligonucleotides described in the Tables, e.g., Table IA. In some embodiments, a chemical moiety comprises one or more basic moieties. In some embodiments, a basic moiety is positively charged at, e.g. about pH 7.4. In some embodiments, a basic moiety is or comprises a guanidine moiety. In some embodiments, a basic moiety is or comprises —N(R¹)₂, wherein each R¹is independently as described in the present disclosure. In some embodiments, a basic moiety is or comprises —N(R¹)₃, wherein each R¹is independently as described in the present disclosure. In some embodiments, a basic moiety is or comprises —N═C(N(R¹)₂)₂, wherein each R¹is independently as described in the present disclosure. In some embodiments, each R¹is independently R as described in the present disclosure. In some embodiments, each R¹is independently optionally substituted C_1-6alkyl. In some embodiments, R¹is methyl. In some embodiments, one or two R¹are the same. In some embodiments, each R¹is the same. In some embodiments, at least one R¹is different from another R¹. In some embodiments, a basic moiety is —N═C(N(CH₃)₂)₂. In some embodiments, a chemical moiety comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sugar, peptide, lipid, and/or basic moieties. In some embodiments, the number is 1. In some embodiments, the number is 2. In some embodiments, the number is 3. In some embodiments, the number is 4. In some embodiments, the number is 5. In some embodiments, the number is 6. In some embodiments, a chemical moiety comprises a ligand moiety of a protein, e.g., a receptor protein of a target cell. In some embodiments, a ligand is a ligand for a vitamin E receptor. In some embodiments, a ligand is for Tfr1 receptor. Chemical moieties as described and demonstrated in the present disclosure include and can be utilized as carbohydrate moieties, lipid moieties, targeting moieties, etc., and can provide a variety of functions, e.g., improving delivery, one or more properties, activities, etc.

In some embodiments, the present disclosure provides oligonucleotides comprising additional chemistry moieties, optionally connected to the oligonucleotide moiety through a linker. In some embodiments, the present disclosure provides oligonucleotides comprising (R))b-L^M1-L^M2-L^M3-, wherein:

each R^Dis independently a chemical moiety:

each of L^M1, L^M2, and L^M3is independently L; and

b is 1-1000.

In some embodiments, each of L^M1, L^M2, and L^M3is independently a covalent bond, or a bivalent or multivalent, optionally substituted, linear or branched group selected from a C_1-10aliphatic group and a C_1-10heteroaliphatic group having 1-5 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, —C(R′)₂—, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)— —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—. —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—. —OP(OR′)O—, —OP(SR′)O—. —OP(NR′)O—. —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—; and one or more CH or carbon atoms are optionally and independently replaced with Cy^L.

In some embodiments, L^M1comprises one or more —N(R′)— and one or more —C(O)—. In some embodiments, a linker (e.g., L, LM, etc.) or L^M1is or comprises

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wherein n is 1-8. In some embodiments, a linker or -L^M1-L^M2-L^M3- is

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or a salt form thereof, wherein n^Lis 1-8. In some embodiments, a linker or -L^M1-L^M2-L^M3- is

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or a salt form thereof, wherein

n^Lis 1-8.

each amino group independently connects to a moiety; and

the P atom connects to the 5′-OH of the oligonucleotide.

In some embodiments, the moiety and the linker, or (R^D)b-L^M1-L^M2-L^M3-, is or comprises

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In some embodiments, the moiety and the linker, or (R^D)b-L^M1-L^M2-L^M3-, is or comprises

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In some embodiments, the moiety and the linker or (R^D)b-L^M1-L^M2-L^M3-, is or comprises

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In some embodiments, the moiety and the linker, or (R^D)b-L^M1-L^M2-L^M3-, is or comprises

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In some embodiments, the moiety and the linker or RD)b-L^M1-L^M2-L^M3-, is or comprises

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In some embodiments, the moiety and the linker, or (R^D)b-L^M1-L^M2-L^M3-, is or comprises

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In some embodiments, the moiety and the linker, or (R^D)b-L^M1-L^M2-L^M3-, is or comprises

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In some embodiments, a linker, or L^M1, is or comprises

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In some embodiments, the moiety and linker, or (R^D)b-L^M1-L^M2-L^M3-, is or comprises:

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In some embodiments, the moiety and linker, or -L^M1-L^M2-L^M3-, is or comprises:

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In some embodiments, a linker is

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In some embodiments, the moiety and linker, or (RD)b-L^M1-L^M2-L^M3-, is or comprises:

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In some embodiments, the moiety and linker, or (D)b-L^M1-L^M2-L^M3-, is or comprises:

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In some embodiments, n^Lis 1-8. In some embodiments, n^Lis 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n^Lis 1. In some embodiments, n is 2. In some embodiments, n^Lis 3. In some embodiments, n^Lis 4. In some embodiments, n^Lis 5. In some embodiments, n^Lis 6. In some embodiments, n^Lis 7. In some embodiments, n^Lis 8.

In some embodiments, L^M2is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-10aliphatic group and a C_1-10heteroaliphatic group having 1-5 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, —C(R′)₂—, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(OR′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L. In some embodiments, L^M2is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-10aliphatic group and a C_1-10heteroaliphatic group having 1-5 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, —C(R′)₂—, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, or —P(O)(R′)—. In some embodiments, L^M2is a covalent bond, or a bivalent, optionally substituted, linear or branched C_1-10aliphatic wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, —C(R′)₂—, —O—, —S—, —N(R′)—, or —C(O)—. In some embodiments, L^M2is —NH—(CH₂)₆—, wherein —NH— is bonded to L^M1.

In some embodiments, L^M3is —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)—, —OP(O)(SR′)—, —OP(O)(R′)—, —OP(O)(NR′)—, —OP(S)(OR′)—, —OP(S)(SR′)—, —OP(S)(R′)—, —OP(S)(NR′)—, —OP(R′)—, —OP(OR′)—, —OP(SR′)—, —OP(NR′)—, or —OP(OR′)[B(R′)₃]—. In some embodiments, L^M3is —OP(O)(OR′)—, or —OP(O)(SR′)—, wherein —O— is bonded to L^M2. In some embodiments, the P atom is connected to a sugar unit, a nucleobase unit, or an internucleotidic linkage. In some embodiments, the P atom is connected to a —OH group through formation of a P-O bond. In some embodiments, the P atom is connected to the 5′-OH group through formation of a P-O bond.

In some embodiments, L^M1is a covalent bond. In some embodiments, L^M2is a covalent bond. In some embodiments, L^M3is a covalent bond. In some embodiments, L^M1is L^M2as described in the present disclosure. In some embodiments, L^M1is L^M3as described in the present disclosure. In some embodiments, L^M2is L^M1as described in the present disclosure. In some embodiments, L^M2is L^M3as described in the present disclosure. In some embodiments, L^M3is L^M1as described in the present disclosure. In some embodiments, L^M3is L^M2as described in the present disclosure. In some embodiments, L^Mis L^M1as described in the present disclosure. In some embodiments, L^Mis L^M2as described in the present disclosure. In some embodiments, L^Mis L^M3as described in the present disclosure. In some embodiments, L^Mis L^M1-L^M2, wherein each of L^M1and L^M2is independently as described in the present disclosure. In some embodiments, L^Mis L^M1-L^M3, wherein each of L^M1and L^M3is independently as described in the present disclosure. In some embodiments, L^Mis L^M2-L^M3, wherein each of L^M2and L^M3is independently as described in the present disclosure. In some embodiments, L^Mis L^M1-L^M2-L^M3, wherein each of L^M1, L^M2and L^M3is independently as described in the present disclosure.

In some embodiments, each R^Dis independently a chemical moiety as described in the present disclosure. In some embodiments, R^Dis an additional chemical moiety. In some embodiments, R^Dis targeting moiety. In some embodiments, R^Dis or comprises a carbohydrate moiety. In some embodiments, R^Dis or comprises a lipid moiety. In some embodiments, R^Dis or comprises a ligand moiety for, e.g., cell receptors such as a sigma receptor, an asialoglycoprotein receptor, etc. In some embodiments, a ligand moiety is or comprises an anisamide moiety, which may be a ligand moiety for a sigma receptor. In some embodiments, a ligand moiety is or comprises a lipid. In some embodiments, a ligand moiety is or comprises a GalNAc moiety, which may be a ligand moiety for an asialoglycoprotein receptor. In some embodiments, R^Dis selected from optionally substituted phenyl,

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wherein n′ is 0 or 1, and each other variable is independently as described in the present disclosure. In some embodiments, R^sis F. In some embodiments, R^sis OMe. In some embodiments, R^sis OH. In some embodiments, R^sis NHAc. In some embodiments, R^sis NHCOCF₃. In some embodiments, R′ is H. In some embodiments, R is H. In some embodiments, R^2sis NHAc, and R^5sis OH. In some embodiments, R^2sis p-anisoyl, and R^5sis OH. In some embodiments, R^2sis NHAc and R^5sis p-anisoyl. In some embodiments, R^2sis OH, and R^5sis p-anisoyl. In some embodiments, R^Dis selected from

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Further embodiments of R^Dincludes additional chemical moiety embodiments, e.g., those described in the examples.

In some embodiments, n′ is 1. In some embodiments, n′ is 0.

In some embodiments, n″ is 1. In some embodiments, n″ is 2.

In some embodiments, a provided oligonucleotide, e.g., DMD oligonucleotide, is conjugated to an additional component (chemical moiety). In some embodiments, a composition comprises any DMD oligonucleotide, or combination thereof, described herein, can be conjugated to any chemical moiety described herein or known in the art.

In some embodiments, a composition comprising a provided oligonucleotide, e.g., a DMD oligonucleotide, comprises an additional component which is any of: Sulfonamide (Carbonic Anhydrases IV inhibitor); Cleavable lipid; Transferrin Receptor 1 (CD71, TfR) ligand; OCTN2 transporter targeting (L-Cartinine); Glut4 and Glut1 Receptor ligand; Mannose Receptor C1 (Mrc1) and Mannose 6P Receptor (M6Pr) ligand; Cleavable Lipid; Cholesterol; or a Peptide (including, but not limited to, a short delivery peptide or cell-penetrating peptide (CPP)).

Variously oligonucleotides have been designed and/or constructed which comprise an additional component which is, comprises or is derived from: cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Gambogic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; CPP; Glucose (tri- and hex-antennary); and Mannose (tri- and hex-antennary, alpha and beta); and various synthesis schemes for these additional components and oligonucleotides comprising them or molecules derived from them have been devised.

In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from

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WV-DL-14 is also known as WV-DL-014. In some embodiments, gambogic acid or a derivative thereof binds to Transferrin receptor (CD71).

In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from L-carnitine, which binds to the OCTN2 transporter. In some embodiments, a composition comprising a DMD oligonucleotide comprises an additional component which is derived from

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In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is a sulfonamide or a derivative thereof.

In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from any of:

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In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is or comprises or comprises a derivative of:

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in some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is or comprises or comprises a derivative of:

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In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from any of: WV-DL-001, WV-DL-002, WV-DL-003, WV-DL-006, WV-DL-007, WV-DL-008, WV-DL-009, WV-DL-010, WV-DL-011, WV-DL-012, or WV-Dl-014, and other additional components, wherein the terminal —COOH is used to conjugate the additional component to a linker or to an oligonucleotide. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from any of: WV-DL-001, WV-DL-002, WV-DL-003, WV-DL-006, WV-DL-007, WV-DL-008, WV-DL-009. WV-DL-010, WV-DL-011, WV-DL-012, or WV-Dl-014, and other additional components, wherein the terminal —COOH is used to conjugate the additional component to a linker, wherein the conjugation process converts the —COOH to a —C(O)— which connects a linker. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from any of: WV-DL-001, WV-DL-002, WV-DL-003, WV-DL-006, WV-DL-007, WV-DL-008. WV-DL-009, WV-DL-010. WV-DL-011, WV-DL-012, or WV-D-014, and other additional components, wherein the terminal —COOH is used to conjugate the additional component to a linker, wherein the conjugation process replaces the —COOH with —C(O)— which connects to —NH— of a linker (e.g., L001). A non-limiting example of a product of this process for conjugation, using an additional component derived from WV-DL-006 is shown here:

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wherein WV-DL-005 indicates the additional component.

In some embodiments, a composition comprising an oligonucleotide. e.g., a DMD oligonucleotide comprises an additional component which is a lipid. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is a lipid, including but not limited to a lipid described herein.

In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide, comprises an additional component, wherein the additional component is conjugated to the oligonucleotide via a cleavable linker. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide, comprises an additional component which is a lipid, wherein the lipid is conjugated to the oligonucleotide via a cleavable linker. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide, comprises an additional component which is a lipid, including but not limited to a lipid described herein, wherein the lipid is conjugated to the oligonucleotide via a cleavable linker.

In some embodiments a cleavable linker comprises an ester. In some embodiments, a cleavable linker is cleavable within a cell, allowing the oligonucleotide to be physically separated from the additional component.

In some embodiments a cleavable linker is or comprises:

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Non-limiting examples of an oligonucleotide conjugated to a lipid(s) via a cleavable linker are shown here:

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A non-limiting example of an oligonucleotide comprising an additional component which is stearic acid, linked to the oligonucleotide via a cleavable linker is shown here:

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wherein stearic acid indicates the additional component.

A non-limiting reagent useful for conjugating stearic acid through a cleavable linker and it example preparation and use are shown below:

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A non-limiting reagent useful for conjugating a cholesterol derivative through a cleavable linker, and its example preparation, are shown here:

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In some embodiments, a composition comprising an oligonucleotide comprises an additional component derived from:

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In some embodiments, a composition comprising an oligonucleotide comprises an additional component derived from either of:

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In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises a mannose receptor ligand. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises a mannose receptor ligand which is a mannose receptor inhibitor. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from any of:

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where the arrow indicates a-COOH which can be used to conjugate the additional component to an oligonucleotide, optionally via a linker.

A non-limiting example of a procedure for preparing an additional component comprising a mannose receptor ligand is shown here:

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In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is a ligand (or derivative thereof) that binds to a glucose or Glut4 receptor. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is a ligand (or derivative thereof) that binds to a glucose receptor. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is a ligand (or derivative thereof) that binds to and inhibits a glucose receptor. In some embodiments, a ligand (or derivative thereof) that binds to a glucose or Glut4 receptor is mono-, bi-,tri, or hex-antennary. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from

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A non-limiting example of a procedure for synthesis of a tri-antennary glucose receptor inhibitor is shown here:

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A non-limiting example of a procedure for synthesis of a hex-antennary glucose receptor inhibitor is shown here:

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In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component increases internalization of the oligonucleotide via receptor-mediated endocytosis.

In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is an aptamer.

In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is an aptamer which is a peptide aptamer, a RNA apatamer, a DNA aptamer, or an aptamer which comprises a RNA nucleotide, a DNA nucleotide, a modified nucleotide, and/or an amino acid and/or peptide.

In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is an aptamer which binds to a receptor.

In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is an aptamer which binds to a receptor which is a mannose receptor, a mannose-6-phosphate receptor or transferrin receptor.

In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is an aptamer that increases internalization of the oligonucleotide.

In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is or comprises a peptide. In some embodiments, a peptide is a cell-penetrating peptide (CPP). In some embodiments, a CPP is arginine-rich. In some embodiments, a CPP has or comprises the amino acid sequence of RRQPPRSISSHPC or RRQPPRSISSHP.

A non-limiting example of a procedure for conjugating a peptide to a DMD oligonucleotide is shown here:

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In some embodiments, a peptide comprises the amino acid sequence of RC or RRC. In some embodiments, a peptide comprises a structure of either of:

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Provided oligonucleotides, e.g., DMD oligonucleotides, may be conjugated as PMOs to cell-penetrating peptides. Yokota et al. 2012 Nucl. Acid Ther. 22: 306; Wu et al. 2009 Mol. Ther. 17: 864-871; Goyenvalle et al. 2010 Mol. Ther. 18, 198-205; Jearawiriyapaisarn et al. 2010 Cardiovasc. Res. 85, 444-453; Crisp et al. 2011 Hum. Mol. Genet. 20, 413-421; Widrick et al. 2011; Wu et al. 2011 PLoS One 6, e19906.

In some embodiments, a composition comprising an oligonucleotide. e.g., a DMD oligonucleotide comprises one or more peptide and/or peptide tag. In some embodiments, a peptide is or comprises a muscle-targeting hepta peptide (MSP). In some embodiments, the sequence of a muscle-targeting helptapeptide is or comprises the sequence of ASSLNIAXB. In some embodiments, a peptide is or comprises a cell-penetrating peptide. In some embodiments, the sequence of a cell-penetrating peptide comprises multiple arginines. In some embodiments, the sequence of a cell-penetrating peptide is or comprises RXRRBRRXRRBRXB.

In some embodiments, the sequence of a peptide is or comprises a sequence of ASSLNIAXB, RXRRBRRXRRBRXB, RXRRXRRXRRXRXB, ASSLNIAXB-RXRRBRRXRRBRXB, RXRRBRRXRRBRXB-ASSLNIAXB, or any sequence comprising both ASSLNIAXB and either RXRRBRRXRRBRXB or RXRRXRRXRRXRXB, wherein R is L-arginine, X is 6-aminohexanoic acid, and B is beta-alanine.

A muscle-targeting hepta peptide (MSP) fused to an arginine-rich cell-penetrating peptide (B-peptide) may be conjugated to provided oligonucleotides in accordance with the present disclosure. Yin et al. 2009 Hum. Mol. Genet. 18: 4405-4414. Yokota et al. 2009 Arch. Neurol. 66: 32.

In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises anisamide or a derivative thereof.

In some embodiments, a composition comprising an oligonucleotide. e.g., a DMD oligonucleotide comprises one or more guanidinium group. vPMOs are reportedly morpholino oligomers conjugated with delivery moiety containing eight terminal guanidinium groups on a dendrimer scaffold that enable entry into cells. Morcos et al. 2008 Biotechniques 45: 613-618; Yokota et al. 2012 Nucl. Acid Ther. 22: 306.

In some embodiments, an oligonucleotide, e.g., DMD oligonucleotide is delivered using a leash. A non-limiting example of a leash is reported in: Gebski et al. 2003 Hum. Mol. Gen. 12: 1801-1811.

In some embodiments, an additional chemical moiety is cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; Gambogic acid; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).

Certain chemical moieties, e.g., lipid moieties, carbohydrate moieties, targeting moieties, etc. and linker moieties for connecting such moieties to oligonucleotide chains (e.g., via sugars, nucleobases, internucleotidic linkages, etc.) are described in the Tables as example: some of such chemical and linker moieties and related technologies for their preparation, conjugation with oligonucleotide chains, and uses are described in e.g., WO 2017/062862, WO 2017/192679, WO 2017/210647, etc.

Lipids

In some embodiments, an additional chemical moiety/component is a lipid moiety. In some embodiments, the present disclosure provided oligonucleotide compositions further comprise one or more lipids. In some embodiments, incorporation of lipid moieties into oligonucleotides can provide unexpected, greatly improved properties (e.g., activities, toxicities, distribution, pharmacokinetics, etc.).

A composition can be obtained by combining an active compound with a lipid. In some embodiments, the lipid is conjugated to an active compound. In some embodiments, the lipid is not conjugated to an active compound. In some embodiments, a lipid comprises a C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C_1-4aliphatic group. In some embodiments, a lipid comprises a C₁₀-C₆₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C₁₀-C₆₀linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C_1-4aliphatic group. In some embodiments, a lipid comprises a C₁₀-C₈₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C₁₀-C₈₀linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C_1-4aliphatic group. In some embodiments, a lipid comprises a C₁-C₁₀₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C₁₀-C₁₀₀linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C_1-4aliphatic group.

In some embodiments, a lipid comprises an optionally substituted. C₁₀-C₈₀saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆hetroaliphatic moiety, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)—, —S(O)₂N(R′)—, —N(R′)S(O)—, —SC(O)—, —C(O)S—, —OC(O)—, and —C(O)O—, wherein each variable is independently as defined and described herein. In some embodiments, a lipid comprises an optionally substituted C₁₀-C₈₀saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises an optionally substituted C₁₀-C₈₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C₁₀-C₈₀linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C_1-4aliphatic group. In some embodiments, a lipid comprises an optionally substituted, C₁₀-C₆₀saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)—, —S(O)₂N(R′)—, —N(R′)S(O)—, —SC(O)—, —C(O)S—, —OC(O)—, and —C(O)O—, wherein each variable is independently as defined and described herein. In some embodiments, a lipid comprises an optionally substituted C₁₀-C₆₀saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises an optionally substituted C₁₀-C₆₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C₁₀-C₆₀linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C_1-4aliphatic group. In some embodiments, a lipid comprises an optionally substituted, C₁₀-C₄₀saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆heteroaliphatic moiety, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂—, —SC(O)—, —C(O)S—, —OC(O)—, and —C(O)O—, wherein each variable is independently as defined and described herein. In some embodiments, a lipid comprises an optionally substituted C₁₀-C₄₀saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises an optionally substituted C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C_1-4aliphatic group. In some embodiments, a lipid comprises an unsubstituted C₁₀-C₈₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises no more than one optionally substituted C₁₀-C₈₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises two or more optionally substituted C₁₀-C₈₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises an unsubstituted C₁₀-C₆₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises no more than one optionally substituted C₁₀-C₆₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises two or more optionally substituted C₁₀-C₆₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises an unsubstituted C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises no more than one optionally substituted C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises two or more optionally substituted C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C₁₀-C₄₀linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid is selected from the group consisting of: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid and dilinoleyl. In some embodiments, a lipid is not conjugated to an oligonucleotide chain (whether through one or more linker moieties or not). In some embodiments, a lipid is conjugated to an oligonucleotide chain, optionally through one or more linker moieties.

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In some embodiments, an active compound is an oligonucleotide described herein. In some embodiments, an active compound is an oligonucleotide capable of mediating skipping of an exon in dystrophin. In some embodiments, an active compound is an oligonucleotide capable of mediating skipping of exon 51 in dystrophin. In some embodiments, an active compound is a nucleic acid of a sequence comprising or consisting of any sequence of any nucleic acid described herein. In some embodiments, an active compound is a nucleic acid of a sequence comprising or consisting of any sequence of any oligonucleotide listed in Table A1. In some embodiments, a composition comprises a lipid and an active compound, and further comprises another component selected from: another lipid, and a targeting compound or moiety. In some embodiments, a lipid includes, without limitation: an amino lipid; an amphipathic lipid; an anionic lipid; an apolipoprotein; a cationic lipid: a low molecular weight cationic lipid; a cationic lipid such as CLinDMA and DLinDMA; an ionizable cationic lipid; a cloaking component; a helper lipid; a lipopeptide; a neutral lipid; a neutral zwitterionic lipid: a hydrophobic small molecule; a hydrophobic vitamin; a PEG-lipid; an uncharged lipid modified with one or more hydrophilic polymers; phospholipid; a phospholipid such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; a stealth lipid; a sterol; a cholesterol; and a targeting lipid; and any other lipid described herein or reported in the art. In some embodiments, a composition comprises a lipid and a portion of another lipid capable of mediating at least one function of another lipid. In some embodiments, a targeting compound or moiety is capable of targeting a compound (e.g., a composition comprising a lipid and a active compound) to a particular cell or tissue or subset of cells or tissues. In some embodiments, a targeting moiety is designed to take advantage of cell- or tissue-specific expression of particular targets, receptors, proteins, or other subcellular components; In some embodiments, a targeting moiety is a ligand (e.g., a small molecule, antibody, peptide, protein, carbohydrate, aptamer, etc.) that targets a composition to a cell or tissue, and/or binds to a target, receptor, protein, or other subcellular component.

In some embodiments, incorporation of a lipid moiety for delivery of an active compound allow (e.g., do not prevent or interfere with) the function of an active compound. Non-limiting example lipids include: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid and dilinoleyl.

In some embodiments, lipid conjugation, such as conjugation with fatty acids, may improve one or more properties of oligonucleotides. In some embodiments, lipid conjugation improves delivery.

In some embodiments, as supported by experimental data, conjugation with lipids can increase skipping efficiency.

In some embodiments, a composition for delivery of an active compound is capable of targeting an active compound to particular cells or tissues, as desired. In some embodiments, a composition for delivery of an active compound is capable of targeting an active compound to a muscle cell or tissue. In some embodiments, the present disclosure pertains to compositions and methods related to delivery of active compounds, wherein the compositions comprise an active compound a lipid. In some embodiments to a muscle cell or tissue, the lipid is selected from: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid and dilinoleyl. Example compositions were prepared comprising an active compound (WV-942) and a lipid, and these compositions were capable of delivering an active compound to target cells and tissues, e.g., muscle cells and tissues. The example lipids used include stearic acid, oleic acid, alpha-linolenic acid, gamma-linolenic acids, cis-DHA, turbinaric acid and dilinoleyl acid.

Various compositions comprising an active compound and any of: stearic acid, oleic acid, alpha-linolenic acid, gamma-linolenic acid, cis-DHA or turbinaric acid, were able to deliver an active compound to various tissues, including gastrocnemius muscle tissue, heart muscle tissue, quadriceps muscle tissue, gastrocnemius muscle tissue, and diaphragm muscle tissue.

In some embodiments, a composition comprising a lipid, selected from: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid and dilinoleyl, and an active compound is capable of delivering an active compound to extra-hepatic cells and tissues, e.g., muscle cells and tissues.

In some embodiments, a lipid has the structure of R^LD—OH, wherein R^LDis an optionally substituted, C₁₀-C₈₀saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by C₁-C₆alkylene, C₁-C₆alkenylene, —C≡C—, a C₁-C₆heteroaliphatic moiety, —C(R′)₂-, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—. —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —N(R′)S(O)₂— —SC(O)—, —C(O)S—, —OC(O)—, and —C(O)O—. In some embodiments, a lipid has the structure of R^LD—C(O)OH. In some embodiments, R^LDis

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Example oligonucleotides comprising such R^LDgroups are described herein and in WO 2017/062862, the description of R^LDis incorporated herein by reference.

In some embodiments, a lipid is conjugated to an active compound optionally through a linker moiety. In some embodiments, a linker is L^M. In some embodiments, a linker is L. In some embodiments, -L- comprises a bivalent aliphatic chain. In some embodiments, -L- comprises a phosphate group. In some embodiments, -L- comprises a phosphorothioate group. In some embodiments, -L- has the structure of —C(O)NH—(CH₂)₆—OP(═O)(S⁻)—. In some embodiments, -L- has the structure of —C(O)NH—(CH₂)₆—OP(═O)(O⁻)—.

Lipids, optionally through linkers, can be conjugated to oligonucleotides at various suitable locations. In some embodiments, lipids are conjugated through the 5′-OH group. In some embodiments, lipids are conjugated through the 3′-OH group. In some embodiments, lipids are conjugated through one or more sugar moieties. In some embodiments, lipids are conjugated through one or more bases. In some embodiments, lipids are incorporated through one or more internucleotidic linkages. In some embodiments, an oligonucleotide may contain multiple conjugated lipids which are independently conjugated through its 5′-OH, 3′-OH, sugar moieties, base moieties and/or internucleotidic linkages.

In some embodiments, a composition comprises an oligonucleotide, e.g., DMD oligonucleotide and a lipid selected from: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid, arachidonic acid, and dilinoleyl, wherein the lipid is directly conjugated to the biologically active agent (without a linker interposed between the lipid and the biologically active agent). In some embodiments, a composition comprises an oligonucleotide and a lipid selected from: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid and dilinoleyl, wherein the lipid is directly conjugated to the biologically active agent (without a linker interposed between the lipid and the biologically active agent).

In some embodiments, a composition comprises a DMD oligonucleotide and any lipid known in the art, wherein the lipid is conjugated or not conjugated to the oligonucleotide.

Non-limiting examples of lipids, and methods of making them and conjugating them are provided in, for example, WO 2017/062862, the lipids and related methods of which are incorporated herein by reference.

Targeting Moieties

In some embodiments, an additional chemical moiety/component is a targeting moiety. In some embodiments, a provided composition further comprises a targeting moiety. In some embodiments, a targeting moiety is conjugated to an oligonucleotide chain. In some embodiments, a biologically active agent is conjugated to both a lipid and an oligonucleotide chain. Various targeting moieties can be used in accordance with the present disclosure, e.g., lipids, antibodies, peptides, carbohydrates, etc.

Targeting moieties can be incorporated into provided technologies through many types of methods in accordance with the present disclosure. In some embodiments, targeting moieties are chemically conjugated with oligonucleotides.

In some embodiments, provided compositions comprise two or more targeting moieties. In some embodiments, provided oligonucleotides comprise two or more conjugated targeting moieties. In some embodiments, the two or more conjugated targeting moieties are the same. In some embodiments, the two or more conjugated targeting moieties are different. In some embodiments, provided oligonucleotides comprise no more than one targeting moiety. In some embodiments, oligonucleotides of a provided composition comprise different types of conjugated targeting moieties. In some embodiments, oligonucleotides of a provided composition comprise the same type of targeting moieties.

Targeting moieties can be conjugated to oligonucleotides optionally through linkers. Various types of linkers in the art can be utilized in accordance of the present disclosure. In some embodiments, a linker comprises a phosphate group, which can, for example, be used for conjugating targeting moieties through chemistry similar to those employed in oligonucleotide synthesis. In some embodiments, a linker comprises an amide, ester, or ether group. In some embodiments, a linker is LM. In some embodiments, a linker has the structure of -L-. Targeting moieties can be conjugated through either the same or different linkers compared to lipids.

Targeting moieties, optionally through linkers, can be conjugated to oligonucleotides at various suitable locations. In some embodiments, targeting moieties are conjugated through the 5′-OH group. In some embodiments, targeting moieties are conjugated through the 3′-OH group. In some embodiments, targeting moieties are conjugated through one or more sugar moieties. In some embodiments, targeting moieties are conjugated through one or more bases. In some embodiments, targeting moieties are incorporated through one or more internucleotidic linkages. In some embodiments, an oligonucleotide may contain multiple conjugated targeting moieties which are independently conjugated through its 5′-OH, 3′-OH, sugar moieties, base moieties and/or internucleotidic linkages. Targeting moieties and lipids can be conjugated either at the same, neighboring and/or separated locations. In some embodiments, a targeting moiety is conjugated at one end of an oligonucleotide, and a lipid is conjugated at the other end.

In some embodiments, a targeting moiety interacts with a protein on the surface of targeted cells. In some embodiments, such interaction facilitates internalization into targeted cells. In some embodiments, a targeting moiety comprises a sugar moiety. In some embodiments, a targeting moiety comprises a polypeptide moiety. In some embodiments, a targeting moiety comprises an antibody. In some embodiments, a targeting moiety is an antibody. In some embodiments, a targeting moiety comprises an inhibitor. In some embodiments, a targeting moiety is a moiety from a small molecule inhibitor. In some embodiments, an inhibitor is an inhibitor of a protein on the surface of targeted cells. In some embodiments, an inhibitor is a carbonic anhydrase inhibitor. In some embodiments, an inhibitor is a carbonic anhydrase inhibitor expressed on the surface of target cells. In some embodiments, a carbonic anhydrase is I, II, III, IV, V, VI, VII, VIII, IX, X. XI, XII, XIII, XIV, XV or XVI. In some embodiments, a carbonic anhydrase is membrane bound. In some embodiments, a carbonic anhydrase is IV, IX, XII or XIV. In some embodiments, an inhibitor is for IV, IX, XI and/or XIV. In some embodiments, an inhibitor is a carbonic anhydrase III inhibitor. In some embodiments, an inhibitor is a carbonic anhydrase IV inhibitor. In some embodiments, an inhibitor is a carbonic anhydrase IX inhibitor. In some embodiments, an inhibitor is a carbonic anhydrase XII inhibitor. In some embodiments, an inhibitor is a carbonic anhydrase XIV inhibitor. In some embodiments, an inhibitor comprises or is a sulfonamide (e.g., those described in Supuran, CT. Nature Rev Drug Discover 2008, 7, 168-181, which sulfonamides are incorporated herein by reference). In some embodiments, an inhibitor is a sulfonamide. In some embodiments, targeted cells are muscle cells.

In some embodiments, a targeting moiety is R^LDor R^CDor R^TDas defined and described in the present disclosure. In some embodiments, R^CDcomprises or is

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In some embodiments, R^CDcomprises or is

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In some embodiments, R^CDcomprises or is

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In some embodiments R^TDis a sulfonamide moiety as described in the present disclosure. In some embodiments, R^TDcomprises or is

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In some embodiments, R^TDor R^CDcomprises or is

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In some embodiments, R^TDor R^CDcomprises or is

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In some embodiments, R^TDcomprises or is

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In some embodiments, R^TDor R^CDcomprises or is

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In some embodiments, R^TDor R^CDcomprises or is

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In some embodiments, R^TDcomprises or is

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In some embodiments, R^TDcomprises or is

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In some embodiments, R^TDor R^CDcomprises or is

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In some embodiments, R^TDor R^CDcomprises or is

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In some embodiments, R^TDcomprises or is

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In some embodiments, R^TDcomprises or is

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In some embodiments, R^Lis a targeting moiety that comprises or is a lipid moiety. In some embodiments, X is O. In some embodiments, X is S.

In some embodiments, the present disclosure provides technologies (e.g., reagents, methods, etc.) for conjugating various moieties to oligonucleotide chains. In some embodiments, the present disclosure provides technologies for conjugating targeting moiety to oligonucleotide chains. In some embodiments, the present disclosure provides acids comprising targeting moieties for conjugation, e.g., R^LD—COOH. In some embodiments, the present disclosure provides linkers for conjugation, e.g., L^LD. A person having ordinary skill in the art understands that many known and widely practiced technologies can be utilized for conjugation with oligonucleotide chains in accordance with the present disclosure. In some embodiments, a provided acid is

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In some embodiments, a provided acid is

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In some embodiments, a provided acid is

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In some embodiments, a provided acid is

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In some embodiments, a provided acid is a fatty acid, which can provide a lipid moiety as a targeting moiety. In some embodiments, the present disclosure provides methods and reagents for preparing such acids.

In some embodiments, an additional chemical moiety, e.g., one comprising a guanidine moiety, may be incorporated into an oligonucleotide to improve one or more properties and/or activities. In some embodiments, such an additional chemical moiety is useful for improving delivery. In some embodiments, an additional chemical moiety comprises one or more group having the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2 as described herein. In some embodiments, an additional chemical moiety comprises one or more group having the structure of formula I-n-1, I-n-2, I-n-3. I-n-4, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2 as described herein. In some embodiments, such a chemical moiety has the structure of formula R¹-[-L-L^P]n-, wherein each L^Pindependently has the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2 as described herein, and each other variable is independently as described herein. In some embodiments, R¹is —OH. In some embodiments, R¹is —H. In some embodiments, each L is independently optionally substituted bivalent C_1-10aliphatic. In some embodiments, each L is independently —(CH₂)₃— alkylene. In some embodiments, each L is independently C_1-6alkylene. In some embodiments, each L^Pis independently n00

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In some embodiments, an additional chemical moiety is

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In some embodiments, an additional chemical moiety is bonded to 5′-end carbon of an oligonucleotide chain. In some embodiments, it may be incorporated, e.g., using reagents including those illustrated below:

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In some embodiments, an additional chemical moiety may be linked to an oligonucleotide chain through a cleavable group, e.g., a phosphate group, to an oligonucleotide chain (e.g., at the 5′-end carbon):

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In some embodiments, L is a sugar moiety as described herein. For example, in some embodiments, L is

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In some embodiments, an additional chemical moiety is

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In some embodiments, it is bonded to 5′-end carbon of an oligonucleotide chain. In some embodiments, it may be incorporated, e.g., using reagents including those illustrated below:

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In some embodiments, additional chemical moieties described herein may comprise one or more alkyl chain. In some embodiments, additional chemical moieties described herein may comprise one or more lipid moieties. Those skilled in the art appreciates that many other embodiments of L^P, including neutral internucleotidic linkage moieties, may be utilized in additional chemical moieties, e.g., n009. In some embodiments, an additional chemical moiety is

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In some embodiments, an additional chemical moiety is

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As described herein, in some embodiments, an additional chemical moiety may be bonded to the 5′-end carbon of an oligonucleotide chain. In some embodiments, an additional chemical moiety may be incorporated, e.g., using reagents including those illustrated below:

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Those skilled in the art will appreciate that many other technologies, including synthetic chemical technologies, can be utilized in accordance with the present disclosure to provide compounds, e.g., oligonucleotides, reagents for incorporating additional chemical moieties, etc.

In some embodiments, provided compounds, e.g., reagents, products (e.g., oligonucleotides, amidites, etc.) etc. are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% or 99% pure. In some embodiments, the purity is at least 50%. In some embodiments, the purity is at least 75%. In some embodiments, the purity is at least 80%. In some embodiments, the purity is at least 85%. In some embodiments, the purity is at least 90%. In some embodiments, the purity is at least 95%. In some embodiments, the purity is at least 96%. In some embodiments, the purity is at least 97%. In some embodiments, the purity is at least 98%. In some embodiments, the purity is at least 99%.

Combination Therapy

In some embodiments, a subject is administered an additional treatment (including, but not limited to, a therapeutic agent or method) in additional to provided oligonucleotide or oligonucleotide composition, e.g., a composition comprising a DMD oligonucleotide. In some embodiments, a composition comprising a DMD oligonucleotide(s) (or two or more compositions, each comprising a DMD oligonucleotide) is administered to a patient along with an additional treatment.

In some embodiments, the present disclosure pertains to a method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising (a) administering to a subject susceptible thereto or suffering therefrom a composition comprising a provided oligonucleotide, and (b) administering to the subject an additional treatment which is capable of preventing, treating, ameliorating or slowing the progress of muscular dystrophy. In some embodiments, an additional treatment is a composition comprising a second oligonucleotide.

In some embodiments, an additional treatment is capable of preventing, treating, ameliorating or slowing the progress of muscular dystrophy by itself. In some embodiments, an additional treatment is capable of preventing, treating, ameliorating or slowing the progress of muscular dystrophy when administered with a provided oligonucleotide.

In some embodiments, an additional treatment is administered to the subject prior to, after or simultaneously with a composition comprising a provided oligonucleotide, e.g., a provided DMD oligonucleotide. In some embodiments, a composition comprises both a DMD oligonucleotide(s) and an additional treatment. In some embodiments, a DMD oligonucleotide(s) and an additional treatment(s) are in separate compositions. In some embodiments, the present disclosure provides technologies (e.g., compositions, methods, etc.) for combination therapy, for example, with other therapeutic agents and/or medical procedures. In some embodiments, provided oligonucleotides and/or compositions may be used together with one or more other therapeutic agents. In some embodiments, provided compositions comprise provided oligonucleotides, and one or more other therapeutic agents. In some embodiments, the one or more other therapeutic agents may have one or more different targets, and/or one or more different mechanisms toward targets, when compared to provided oligonucleotides in the composition. In some embodiments, a therapeutic agent is an oligonucleotide. In some embodiments, a therapeutic agent is a small molecule drug. In some embodiments, a therapeutic agent is a protein. In some embodiments, a therapeutic agent is an antibody. A number of therapeutic agents may be utilized in accordance with the present disclosure. For example, oligonucleotides for DMD may be used together with one or more therapeutic agents that modulate utrophin production (utrophin modulators). In some embodiments, a utrophin modulator promotes production of utrophin. In some embodiments, a utrophin modulator is ezutromid. In some embodiments, a utrophin modulator is

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or a pharmaceutically acceptable salt thereof. In some embodiments, provided oligonucleotides or compositions thereof are administered prior to, concurrently with, or subsequent to one or more other therapeutic agents and/or medical procedures. In some embodiments, provided oligonucleotides or compositions thereof are administered concurrently with one or more other therapeutic agents and/or medical procedures. In some embodiments, provided oligonucleotides or compositions thereof are administered prior to one or more other therapeutic agents and/or medical procedures. In some embodiments, provided oligonucleotides or compositions thereof are administered subsequent to one or more other therapeutic agents and/or medical procedures. In some embodiments, provide compositions comprise one or more other therapeutic agents.

In some embodiments, a composition comprising a DMD oligonucleotide is co-administered with an additional agent in order to improve skipping of a DMD exon of interest. In some embodiments, an additional agent is an antibody, oligonucleotide, protein or small molecule. In some embodiments, an additional agent interferes with a protein involved in splicing. In some embodiments, an additional agent interferes with a protein involved in splicing, wherein the protein is a SR protein.

In some embodiments, an additional agent interferes with a protein involved in splicing, wherein the protein is a SR protein, which contains a protein domain with one or more long repeats of serine (S) and arginine (R) amino acid residues. SR proteins are reportedly heavily phosphorylated in cells and are involved in constitutive and alternative splicing. Long et al. 2009 Biochem. J. 417: 15-27; Shepard et al. 2009 Genome Biol. 10: 242. In some embodiments, an additional agent is a chemical compound that inhibits or decreases a SR protein kinase. In some embodiments, a chemical compound that inhibits or decreases a SR protein kinase is SRPIN340. SRPIN340 is reported in, for example, Fukuhura et al. 2006 Proc. Natl. Acad. Sci. USA 103: 11329-11333. In some embodiments, a chemical compound is a kinase inhibitor specific for Cdc-like kinases (Clks) that are also able to phosphorylate SR proteins. In some embodiments, a kinase inhibitor specific for Cdc-like kinases (Clks) that are also able to phosphorylate SR proteins is TG003. TG003 reportedly affected splicing both in vitro and in vivo. Nowak et al. 2010 J. Biol. Chem. 285: 5532-5540; Muraki et al. 2004 J. Biol. Chem. 279: 24246-24254; Yomoda et al. 2008 Genes Cells 13: 233-244; and Nishida et al. 2011 Nat Commun. 2:308.

In some embodiments, in a patient afflicted with muscular dystrophy, muscle tissue is replaced by fat and connective tissue, and affected muscles may look larger due to increased fat content, a condition known as pseudohypertrophy. In some embodiments, a composition comprising a DMD oligonucleotide(s) is administered along with a treatment which reduces or prevents development of fat or fibrous or connective tissue, or replacement of muscle tissue by fat or fibrous or connective tissue.

In some embodiments, a composition comprising a DMD oligonucleotide(s) is administered along with a treatment which reduces or prevents development of fat or fibrous or connective tissue, or replacement of muscle tissue by fat or fibrous or connective tissue, wherein the treatment is an antibody to connective tissue growth factor (CTGF), a central mediator of fibrosis (e.g., FG-3019). In some embodiments, a composition comprising a DMD oligonucleotide(s) is administered along with an agent which reduces the fat content of the human body.

Additional treatments include: slowing the progression of the disease by immune modulators (eg, steroids and transforming growth factor-beta inhibitors), inducing or introducing proteins that may compensate for dystrophin deficiency in the myofiber (eg, utrophin, biglycan, and laminin), or bolstering the muscle's regenerative response (eg, myostatin and activin 2B).

In some embodiments, an additional treatment is a small molecule capable of restoring normal balance of calcium within muscle cells.

In some embodiments, an additional treatment is a small molecule capable of restoring normal balance of calcium within muscle cells by correcting the activity of a type of channel called the ryanodine receptor calcium channel complex (RyR). In some embodiments, such a small molecule is Ryca1 ARM210 (ARMGO Pharma, Tarry Town, N.Y.).

In some embodiments, an additional treatment is a flavonoid.

In some embodiments, an additional treatment is a flavonoid such as Epicatechin. Epicatechin is a flavonoid found in dark chocolate harvested from the cacao tree which has been reported in animals and humans to increase the production of new mitochondria in heart and muscle (e.g., mitochondrial biogenesis) while concurrently stimulating the regeneration of muscle tissue.

In some embodiments, an additional treatment is follistatin gene therapy.

In some embodiments, an additional treatment is adeno-associated virus delivery of follistatin 344 to increase muscle strength and prevent muscle wasting and fibrosis.

In some embodiments, an additional treatment is glucocorticoid.

In some embodiments, an additional treatment is prednisone.

In some embodiments, an additional treatment is deflazacort.

In some embodiments, an additional treatment is vamorolone (VBP15).

In some embodiments, an additional treatment is delivery of an exogenous Dystrophin gene or synthetic version or portion thereof, such as a microdystrophin gene.

In some embodiments, an additional treatment is delivery of an exogenous Dystrophin gene or portion thereof, such as a microdystrophin gene, such as SGT-001, an adeno-associated viral (AAV) vector-mediated gene transfer system for delivery of a synthetic dystrophin gene or microdystrophin (Solid BioSciences, Cambridge, Mass.).

In some embodiments, an additional treatment is stem cell treatment.

In some embodiments, an additional treatment is a steroid.

In some embodiments, an additional treatment is a corticosteroid.

In some embodiments, an additional treatment is prednisone.

In some embodiments, an additional treatment is a beta-2 agonist.

In some embodiments, an additional treatment is an ion channel inhibitor.

In some embodiments, an additional treatment is a calcium channel inhibitor.

In some embodiments, an additional treatment is a calcium channel inhibitor which is a xanthin. In some embodiments, an additional treatment is a calcium channel inhibitor which is methylxanthine. In some embodiments, an additional treatment is a calcium channel inhibitor which is pentoxifylline. In some embodiments, an additional treatment is a calcium channel inhibitor which is a methylxanthine derivative selected from: pentoxifylline, furafylline, lisofylline, propentofylline, pentifylline, theophylline, torbafylline, albifylline, enprofylline and derivatives thereof.

In some embodiments, an additional treatment is a treatment for heart disease or cardiovascular disease.

In some embodiments, an additional treatment is a blood pressure medicine.

In some embodiments, an additional treatment is surgery.

In some embodiments, an additional treatment is surgery to fix shortened muscles, straighten the spine, or treat a heart or lung problem.

In some embodiments, an additional treatment is a brace, walker, standing walker, or other mechanical aid for walking.

In some embodiments, an additional treatment is exercise and/or physical therapy.

In some embodiments, an additional treatment is assisted ventilation.

In some embodiments, an additional treatment is anticonvulsant, immunosuppressant or treatment for constipation.

In some embodiments, an additional treatment is an inhibitor of NF-κB.

In some embodiments, an additional treatment comprises salicylic acid and/or docosahexaenoic acid (DHA).

In some embodiments, an additional treatment is edasalonexent (CAT-1004, Catabasis), a conjugate of salicylic acid and docosahexaenoic acid (DHA).

In some embodiments, an additional treatment is a cell-based therapeutic.

In some embodiments, an additional treatment is comprises allogeneic cardiosphere-derived cells.

In some embodiments, an additional treatment is CAP-1002 (Capricor).

Certain Embodiments of Variables

Embodiments of variables are extensive described in the present disclosure. Those skilled in the art appreciate that an embodiment described for one variable may be optionally and independently combined with embodiments for other variables, and such combinations, wherever and whenever appropriate, are within the scope of the present disclosure. Embodiments of a variable (e.g. R) given when describing one variable that can be such variable (e.g., R¹, which can be R) are generally applicable to other variables that can be the same variable (e.g., R^s, which can be R). Various embodiments of many variables are also described in other sections of the present disclosure.

In some embodiments, P^Lis P(═W). In some embodiments, P^Lis P. In some embodiments, P^Lis a chiral P (P*). In some embodiments, P^Lis P→B(R′)₃.

In some embodiments, W is O. In some embodiments, W is S. In some embodiments, W is Se. In some embodiments, W is —N(-L-R⁵).

In some embodiments, X is O. In some embodiments, X is S. In some embodiments, X is —N(-L-R⁵)—. In some embodiments, -L-R⁵is —R, which is taken together with a R group of -L-R¹(e.g., a —C(R′)— in L) to form a double bond or a ring as described in the present disclosure. In some embodiments, X is L.

In some embodiments, Y is O. In some embodiments, Y is S. In some embodiments, Z is O. In some embodiments, Z is S. In some embodiments, Y is O and Z is O.

In some embodiments, W is O, Y is O and Z is O. In some embodiments, W is S, Y is O and Z is O.

In some embodiments, R¹is —H. In some embodiments, R¹is -L-R. In some embodiments, R¹is halogen. In some embodiments, R¹is —CN. In some embodiments, R¹is —NO₂. In some embodiments, R¹is -L-Si(R)₃. In some embodiments, R¹is —OR. In some embodiments, R¹is —SR. In some embodiments, R¹is —N(R)₂.

In some embodiments, R¹is R as described in the present disclosure.

In some embodiments, -X-L-R¹comprises or is an optionally substituted moiety of a chiral auxiliary (e.g., H-X-L-R¹is an optionally substituted (e.g., capped) chiral auxiliary), e.g., as used in chirally controlled oligonucleotide synthesis, such as those described in US 20150211006, US 20150211006, WO 2017015555, WO 2017015575, WO 2017062862, or WO 2017160741, chiral auxiliaries of each of which are incorporated herein by reference.

In some embodiments, -X-L-R¹is —OR. In some embodiments, -X-L-R¹is —OH. In some embodiments, -X-L-R¹is —SR. In some embodiments, -X-L-R¹is —SH.

In some embodiments, -X-L-R¹is —R. In some embodiments, R is —CH₃. In some embodiments, R is —CH₂CH₃. In some embodiments, R is —CH₂CH₂CH₃. In some embodiments, R is —CH₂OCH₃. In some embodiments, R is CH₃CH₂OCH₂—. In some embodiments, R is PhCH₂OCH₂—. In some embodiments, R is HC≡C—CH₂— In some embodiments, R is H₃C—C≡C—CH₂—. In some embodiments, R is CH₂═CHCH₂—. In some embodiments, R is CH₃SCH₂—. In some embodiments, R is —CH₂COOCH₃. In some embodiments, R is —CH₂COOCH₂CH. In some embodiments, R is —CH₂CONHCH₃.

In some embodiments, -X-L-R¹is comprises a guanidine moiety. In some embodiments, -X-L-R¹is or comprises

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In some embodiments, -X-L-R¹is -L-W^z, wherein W is selected from

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wherein R″ is R′ and n is 0-15. In some embodiments, R′ and R″ are independently

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In embodiments, L is —O—CH₂CH₂—. In some embodiments, n is 0-3. In some embodiments, each R^sis independently —H, —OCH₃, —F, —CN, —CH₃·—NO₂, —CF₃, or —OCF₃. In some embodiments, R′ and R″ are the same. In some embodiments, R′ and R″ are different

In some embodiments, In some embodiments, -X-L-R¹is

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wherein each R′ is independently as described in the present disclosure. In some embodiments, two R′ on two different nitrogen atoms are taken together to form an optionally substituted ring as described in the present disclosure. In some embodiments, a ring is saturated. In some embodiments, a ring is monocyclic. In some embodiments, a ring is 3-10 membered. In some embodiments, a ring is 3-membered. In some embodiments, a ring is 4-membered. In some embodiments, a ring is 5-membered. In some embodiments, a ring is 6-membered. In some embodiments, a ring is 7-membered. In some embodiments, a ring has no additional ring heteroatoms in addition to the two nitrogen atoms.

In some embodiments, R⁵is R′ as described in the present disclosure. In some embodiments, R⁵is —H. In some embodiments, R is R as described in the present disclosure.

In some embodiments, L is a bivalent optionally substituted methylene group. In some embodiments, L is —CH₂—. In some embodiments, each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C_1-6alkylene, C_1-6alkenylene, —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, —C(R′)₂-, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L.

In some embodiments, L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C_1-6alkylene, C_1-6alkenylene, —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—·—N(R′)—, —C(O)—, —C(S)—, —C(NR′)O—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L. In some embodiments, L is a covalent bond, or a bivalent, optionally substituted, linear or branched C_1-30aliphatic group, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C_1-6alkylene, C_1-6alkenylene, —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L. In some embodiments, L is a covalent bond, or a bivalent, optionally substituted, linear or branched C_1-30heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C_1-6alkylene, C_1-6alkenylene. —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L. In some embodiments, L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C_1-6alkylene, C_1-6alkenylene, —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, or —C(O)O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L. In some embodiments, L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-10aliphatic group and a C_1-10heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C_1-6alkylene, C_1-6alkenylene, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—. —C(O)S—, and —C(O)O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L. In some embodiments, L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a Co aliphatic group and a C_1-10heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from —C(R′)₂—, -Cy -, —O—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, and —C(O)O—.

In some embodiments, L is a covalent bond. In some embodiments, L is optionally substituted bivalent C_1-30aliphatic. In some embodiments, L is optionally substituted bivalent C_1-30heteroaliphatic having 1-10 heteroatoms independently selected from boron, oxygen, nitrogen, sulfur, phosphorus and silicon.

In some embodiments, aliphatic moieties, e.g. those of L, L^s, L^M, R, etc., either monovalent or bivalent or multivalent, and can contain any number of carbon atoms (before any optional substitution) within its range. e.g., C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, etc. In some embodiments, heteroaliphatic moieties, e.g. those of L, R, etc., either monovalent or bivalent or multivalent, and can contain any number of carbon atoms (before any optional substitution) within its range, e.g., C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, etc.

In some embodiments, a methylene unit of a linker, e.g., L, L^s, L^M, etc., is replaced with -Cy-, wherein -Cy- is as described in the present disclosure. In some embodiments, one or more methylene unit is optionally and independently substituted with —O—, —S—, —N(R′)—, —C(O)—, —S(O)—, —S(O)₂—, —P(O)(OR′)—, —P(O)(SR′)—, —P(S)(OR′)—, or —P(S)(OR′)—. In some embodiments, a methylene unit is replaced with —O—. In some embodiments, a methylene unit is replaced with —S—. In some embodiments, a methylene unit is replaced with —N(R′)—. In some embodiments, a methylene unit is replaced with —C(O)—. In some embodiments, a methylene unit is replaced with —S(O)—. In some embodiments, a methylene unit is replaced with —S(O)₂—. In some embodiments, a methylene unit is replaced with —P(O)(OR′)—. In some embodiments, a methylene unit is replaced with —P(O)(SR′)—. In some embodiments, a methylene unit is replaced with —P(O)(R′)—. In some embodiments, a methylene unit is replaced with —P(O)(NR′)—. In some embodiments, a methylene unit is replaced with —P(S)(OR′)—. In some embodiments, a methylene unit is replaced with —P(S)(SR′)—. In some embodiments, a methylene unit is replaced with —P(S)(R′)—. In some embodiments, a methylene unit is replaced with —P(S)NR′)—. In some embodiments, a methylene unit is replaced with —P(R′)—. In some embodiments, a methylene unit is replaced with —P(OR′)—. In some embodiments, a methylene unit is replaced with —P(SR′)—. In some embodiments, a methylene unit is replaced with —P(NR′)—. In some embodiments, a methylene unit is replaced with —P(OR′)[B(R′)₃]—. In some embodiments, one or more methylene unit is optionally and independently substituted with —O—, —S—, —N(R′)—, —C(O)—, —S(O)—, —S(O)₂—, —P(O)(OR′)—, —P(O)(SR′)—, —P(S)(OR′)—, or —P(S)(OR′)—. In some embodiments, a methylene unit is replaced with —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, each of which may independently be an internucleotidic linkage.

In some embodiments, L or L^s(e.g., when L^sis L), e.g., when connected to R^sor a sugar ring, is —CH₂—. In some embodiments, L is —C(R)₂—, wherein at least one R is not hydrogen. In some embodiments, L is —CHR—. In some embodiments, R is hydrogen. In some embodiments, L is —CHR—, wherein R is not hydrogen. In some embodiments, C of —CHR— is chiral. In some embodiments, L is -(R)-CHR—, wherein C of —CHR— is chiral. In some embodiments, L is -(S)-CHR—, wherein C of —CHR— is chiral. In some embodiments, R is optionally substituted C_1-6aliphatic. In some embodiments, R is optionally substituted C_1-6alkyl. In some embodiments, R is optionally substituted C_1-5aliphatic. In some embodiments, R is optionally substituted C_1-5alkyl. In some embodiments, R is optionally substituted C_1-4aliphatic. In some embodiments, R is optionally substituted C_1-4alkyl. In some embodiments, R is optionally substituted C_1-3aliphatic. In some embodiments, R is optionally substituted C_1-3alkyl. In some embodiments, R is optionally substituted C₂aliphatic. In some embodiments, R is optionally substituted methyl. In some embodiments, R is C_1-4aliphatic. In some embodiments, R is C_1-4alkyl. In some embodiments, R is C_1-5aliphatic. In some embodiments, R is C_1-5alkyl. In some embodiments, R is C_1-4aliphatic. In some embodiments, R is C_1-4alkyl. In some embodiments, R is C_1-3aliphatic. In some embodiments, R is C_1-3, alkyl. In some embodiments, R is C₂aliphatic. In some embodiments, R is methyl. In some embodiments, R is C_1-6haloaliphatic. In some embodiments, R is C_1-6haloalkyl. In some embodiments, R is C_1-5haloaliphatic. In some embodiments, R is C_1-4haloalkyl. In some embodiments, R is C_1-4haloaliphatic. In some embodiments, R is C_1-4haloalkyl. In some embodiments, R is C_1-3haloaliphatic. In some embodiments, R is C_1-3haloalkyl. In some embodiments, R is C₂haloaliphatic. In some embodiments, R is methyl substituted with one or more halogen. In some embodiments, R is —CF₃. In some embodiments, L is optionally substituted —CH═CH—. In some embodiments, L is optionally substituted (E)-CH═CH—. In some embodiments, L is optionally substituted (Z)—CH═CH—. In some embodiments, L is —C≡C—.

In some embodiments, L comprises at least one phosphorus atom. In some embodiments, at least one methylene unit of L is replaced with —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—.

In some embodiments, L is bonded to a phosphorus of an linkage (e.g., when X is a covalent bond), e.g., the phosphorus of a linkage having formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, I-b-1, I-b-2, I-c-1, I-c-2, 1-d-1, I-d-2, or a salt form thereof. In some embodiments, such an linkage is an internucleotidic linkage. In some embodiments, such an linkage is a chirally controlled internucleotidic linkage.

In some embodiments, L is -Cy-. In some embodiments, L is —C≡C—.

In some embodiments, Lis a bivalent, optionally substituted, linear or branched C_1-30aliphatic group wherein one or more methylene units are optionally and independently replaced as described in the present disclosure. In some embodiments, Lis a bivalent, optionally substituted, linear or branched C_1-30heteroaliphatic group having 1-10 heteroatoms wherein one or more methylene units are optionally and independently replaced as described in the present disclosure.

In some embodiments, a heteroaliphatic group in the present disclosure, e.g., of L, R (including any variable that can be R), etc., comprises a

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moiety. In some embodiments, ═N— is directly bonded to a phosphorus atom. In some embodiments, a heteroaliphatic group comprises a

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moiety. In some embodiments, a heteroaliphatic group comprises A

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moiety. In some embodiments, such a moiety is directly bonded to a phosphorus atom. In some embodiments, R is optionally substituted C_1-6aliphatic. In some embodiments, R is optionally substituted C_1-6alkyl. In some embodiments, R is isopropyl.

In some embodiments, -Cy- is optionally substituted bivalent monocyclic, bicyclic or polycyclic C_3-20cycloaliphatic. In some embodiments, -Cy- is optionally substituted bivalent monocyclic, bicyclic or polycyclic C_6-20aryl. In some embodiments, -Cy- is optionally substituted monocyclic, bicyclic or polycyclic 3-20 membered heterocyclyl ring having 1-5 heteroatoms. In some embodiments, -Cy- is optionally substituted monocyclic, bicyclic or polycyclic 5-20 membered heterocyclyl ring having 1-5 heteroatoms, wherein at least one heteroatom is oxygen. In some embodiments, -Cy- is 3-10 membered. In some embodiments, -Cy- is 3-membered. In some embodiments, -Cy- is 4-membered. In some embodiments, -Cy- is 5-membered. In some embodiments, -Cy- is 6-membered. In some embodiments, -Cy- is 7-membered. In some embodiments, -Cy- is 8-membered. In some embodiments, -Cy- is 9-membered. In some embodiments, -Cy- is 10-membered. In some embodiments, -Cy- is optionally substituted bivalent tetrahydrofuran ring. In some embodiments, -Cy- is an optionally substituted furanose moiety. In some embodiments, -Cy- is an optionally substituted bivalent 5-membered heteroaryl ring having 1-4 heteroatoms. In some embodiments, at least one heteroatom is nitrogen. In some embodiments, each heteroatom is nitrogen. In some embodiments, -Cy- is an optionally substituted bivalent triazole ring. In some embodiments, -Cy- is optionally substituted

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In some embodiments, -Cy- is

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In some embodiments, R is optionally substituted C_1-6aliphatic. In some embodiments, R is optionally substituted C_1-6alkyl. In some embodiments, R is isopropyl.

In some embodiments, Cy^Lis an optionally substituted trivalent or tetravalent group selected from a C_3-20cycloaliphatic ring, a C_6-20aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon. In some embodiments, Cy^Lis trivalent. In some embodiments, Cy^Lis tetravalent. In some embodiments, one or more CH in a moiety, e.g., L, L^s, L^M, etc. are independently substituted with a trivalent Cy^Lgroup. In some embodiments, one or more carbon atoms in a moiety, e.g., L, L^s, L^M, etc. are independently substituted with a tetravalent Cy^Lgroup. In some embodiments, one or more CH in a moiety, e.g., L, L^s, L^M, etc. are independently substituted with a trivalent Cy^Lgroup, and one or more carbon atoms in a moiety, e.g., L, L^s, L^M, etc. are independently substituted with a tetravalent Cy^Lgroup.

In some embodiments, Cy^Lis monocyclic. In some embodiments, Cy^Lis bicyclic. In some embodiments. Cy^Lis polycyclic.

In some embodiments, Cy^Lis saturated. In some embodiments, Cy^Lis partially unsaturated. In some embodiments, Cy^Lis aromatic. In some embodiments, Cy^Lis or comprises a saturated ring moiety. In some embodiments, Cy^Lis or comprises a partially unsaturated ring moiety. In some embodiments, Cy^Lis or comprises an aromatic ring moiety.

In some embodiments, Cy^Lis an optionally substituted C_3-20cycloaliphatic ring as described in the present disclosure (for example, those described for R but tetravalent). In some embodiments, a ring is an optionally substituted saturated C_3-20cycloaliphatic ring. In some embodiments, a ring is an optionally substituted partially unsaturated C_3-20cycloaliphatic ring. A cycloaliphatic ring can be of various sizes as described in the present disclosure. In some embodiments, a ring is 3, 4, 5, 6, 7, 8, 9, or 10-membered. In some embodiments, a ring is 3-membered. In some embodiments, a ring is 4-membered. In some embodiments, a ring is 5-membered. In some embodiments, a ring is 6-membered. In some embodiments, a ring is 7-membered. In some embodiments, a ring is 8-membered. In some embodiments, a ring is 9-membered. In some embodiments, a ring is 10-membered. In some embodiments, a ring is an optionally substituted cyclopropyl moiety. In some embodiments, a ring is an optionally substituted cyclobutyl moiety. In some embodiments, a ring is an optionally substituted cyclopentyl moiety. In some embodiments, a ring is an optionally substituted cyclohexyl moiety. In some embodiments, a ring is an optionally substituted cycloheptyl moiety. In some embodiments, a ring is an optionally substituted cyclooctanyl moiety. In some embodiments, a cycloaliphatic ring is a cycloalkyl ring. In some embodiments, a cycloaliphatic ring is monocyclic. In some embodiments, a cycloaliphatic ring is bicyclic. In some embodiments, a cycloaliphatic ring is polycyclic. In some embodiments, a ring is a cycloaliphatic moiety as described in the present disclosure for R with more valences.

In some embodiments, Cy^Lis an optionally substituted 6-20 membered aryl ring. In some embodiments, a ring is an optionally substituted trivalent or tetravalent phenyl moiety. In some embodiments, a ring is a tetravalent phenyl moiety. In some embodiments, a ring is an optionally substituted naphthalene moiety. A ring can be of different size as described in the present disclosure. In some embodiments, an aryl ring is 6-membered. In some embodiments, an aryl ring is 10-membered. In some embodiments, an aryl ring is 14-membered. In some embodiments, an aryl ring is monocyclic. In some embodiments, an aryl ring is bicyclic. In some embodiments, an aryl ring is polycyclic. In some embodiments, a ring is an aryl moiety as described in the present disclosure for R with more valences.

In some embodiments, Cy^Lis an optionally substituted 5-20 membered heteroaryl ring having 1-10 heteroatoms, e.g., independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, Cy^Lis an optionally substituted 5-20 membered heteroaryl ring having 1-10 heteroatoms, e.g., independently selected from oxygen, nitrogen, and sulfur. In some embodiments, Cy^Lis an optionally substituted 5-6 membered heteroaryl ring having 1-4 heteroatoms, e.g., independently selected from oxygen, nitrogen, and sulfur. In some embodiments, Cy^Lis an optionally substituted 5-membered heteroaryl ring having 1-4 heteroatoms, e.g., independently selected from oxygen, nitrogen, and sulfur. In some embodiments, Cy^Lis an optionally substituted 6-membered heteroaryl ring having 1-4 heteroatoms, e.g., independently selected from oxygen, nitrogen, and sulfur. In some embodiments, as described in the present disclosure, heteroaryl rings can be of various sizes and contain various numbers and/or types of heteroatoms. In some embodiments, a heteroaryl ring contains no more than one heteroatom. In some embodiments, a heteroaryl ring contains more than one heteroatom. In some embodiments, a heteroaryl ring contains no more than one type of heteroatom. In some embodiments, a heteroaryl ring contains more than one type of heteroatoms. In some embodiments, a heteroaryl ring is 5-membered. In some embodiments, a heteroaryl ring is 6-membered. In some embodiments, a heteroaryl ring is 8-membered. In some embodiments, a heteroaryl ring is 9-membered. In some embodiments, a heteroaryl ring is 10-membered. In some embodiments, a heteroaryl ring is monocyclic. In some embodiments, a heteroaryl ring is bicyclic. In some embodiments, a heteroaryl ring is polycyclic. In some embodiments, a heteroaryl ring is a nucleobase moiety, e.g., A, T, C, G, U, etc. In some embodiments, a ring is a heteroaryl moiety as described in the present disclosure for R with more valences. In some embodiments, as in linkers described in the present disclosure, Cy^Lis

In some embodiments, Cy^Lis a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, Cy^Lis a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, a heterocyclyl ring is saturated. In some embodiments, a heterocyclyl ring is partially unsaturated. A heterocyclyl ring can be of various sizes as described in the present disclosure. In some embodiments, a ring is 3, 4, 5, 6, 7, 8, 9, or 10-membered. In some embodiments, a ring is 3-membered. In some embodiments, a ring is 4-membered. In some embodiments, a ring is 5-membered. In some embodiments, a ring is 6-membered. In some embodiments, a ring is 7-membered. In some embodiments, a ring is 8-membered. In some embodiments, a ring is 9-membered. In some embodiments, a ring is 10-membered. Heterocyclyl rings can contain various numbers and/or types of heteroatoms. In some embodiments, a heterocyclyl ring contains no more than one heteroatom. In some embodiments, a heterocyclyl ring contains more than one heteroatom. In some embodiments, a heterocyclyl ring contains no more than one type of heteroatom. In some embodiments, a heterocyclyl ring contains more than one type of heteroatoms. In some embodiments, a heterocyclyl ring is monocyclic. In some embodiments, a heterocyclyl ring is bicyclic. In some embodiments, a heterocyclyl ring is polycyclic. In some embodiments, a ring is a heterocyclyl moiety as described in the present disclosure for R with more valences.

As readily appreciated by a person having ordinary skill in the art, many suitable ring moieties are extensively described in and can be used in accordance with the present disclosure, for example, those described for R (which may have more valences for Cy^L).

In some embodiments, Cy^Lis a sugar moiety in a nucleic acid. In some embodiments, Cy^Lis an optionally substituted furanose moiety. In some embodiments, Cy^Lis a pyranose moiety. In some embodiments, Cy^Lis an optionally substituted furanose moiety found in DNA. In some embodiments, Cy^Lis an optionally substituted furanose moiety found in RNA. In some embodiments, Cy^Lis an optionally substituted 2′-deoxyribofuranose moiety. In some embodiments, Cy^Lis an optionally substituted ribofuranose moiety. In some embodiments, substitutions provide sugar modifications as described in the present disclosure. In some embodiments, an optionally substituted 2′-deoxyribofuranose moiety and/or an optionally substituted ribofuranose moiety comprise substitution at a 2′-position. In some embodiments, a 2′-position is a 2′-modification as described in the present disclosure. In some embodiments, a 2′-modification is —F. In some embodiments, a 2′-modification is —OR, wherein R is as described in the present disclosure. In some embodiments, R is not hydrogen. In some embodiments, Cy^Lis a modified sugar moiety, such as a sugar moiety in LNA, alpha-L-LNA or GNA. In some embodiments, Cy is a modified sugar moiety, such as a sugar moiety in ENA. In some embodiments, Cy^Lis a terminal sugar moiety of an oligonucleotide, connecting an internucleotidic linkage and a nucleobase. In some embodiments, Cy^Lis a terminal sugar moiety of an oligonucleotide, for example, when that terminus is connected to a solid support optionally through a linker. In some embodiments, Cy^Lis a sugar moiety connecting two internucleotidic linkages and a nucleobase. Example sugars and sugar moieties are extensively described in the present disclosure.

In some embodiments, Cy^Lis a nucleobase moiety. In some embodiments, a nucleobase is a natural nucleobase, such as A, T, C, G, U etc. In some embodiments, a nucleobase is a modified nucleobase. In some embodiments, Cy^Lis optionally substituted nucleobase moiety selected from A, T, C, G, U. and 5mC. Example nucleobases and nucleobase moieties are extensively described in the present disclosure.

In some embodiments, two Cy^Lmoieties are bonded to each other, wherein one Cy^Lis a sugar moiety and the other is a nucleobase moiety. In some embodiments, such a sugar moiety and nucleobase moiety forms a nucleoside moiety. In some embodiments, a nucleoside moiety is natural. In some embodiments, a nucleoside moiety is modified. In some embodiments, Cy^Lis an optionally substituted natural nucleoside moiety selected from adenosine, 5-methyluridine, cytidine, guanosine, uridine, 5-methylcytidine, 2′-deoxyadenosine, thymidine, 2′-deoxycytidine, 2′-deoxyguanosine, 2′-deoxyuridine, and 5-methy-2′-deoxycytidine. Example nucleosides and nucleosides moieties are extensive described in the present disclosure.

Ring A^Lcan be either be monovalent, bivalent or polyvalent. In some embodiments, Ring A^Lis monovalent (e.g., when g is 0 and no substitution). In some embodiments, Ring A^Lis bivalent. In some embodiments, Ring A^Lis polyvalent. In some embodiments, Ring A is bivalent and is -Cy-. In some embodiments, Ring A^Lis an optionally substituted bivalent triazole ring. In some embodiments, Ring A^Lis trivalent and is Cy^L. In some embodiments, Ring A^Lis tetravalent and is Cy^L. In some embodiments, Ring A^Lis optionally substitute

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In some embodiments, -X-L-R¹is optionally substituted alkynyl. In some embodiments, -X-L-R¹is —C≡CH. In some embodiments, an alkynyl group, e.g., —C≡CH, can react with a number of reagents through various reactions to provide further modifications. For example, in some embodiments, an alkynyl group can react with azides through click chemistry. In some embodiments, an azide has the structure of R¹—N₃.

In some embodiments, each R is independently —H, halogen, —CN, —N₃, —NO, —NO₂, -L-R′, -L-Si(R)₃, -L-OR′, -L-SR′, -L-N(R′)₂, —O-L-R′, —O-L-Si(R)₃, —O-L-OR′, —O-L^sSR′, or —O-L^sN(R′)₂as described in the present disclosure.

In some embodiments, R^sis R′, wherein R′ is as described in the present disclosure. In some embodiments, R^sis R, wherein R is as described in the present disclosure. In some embodiments, R^sis optionally substituted C_1-6aliphatic. In some embodiments, R^sis methyl. In some embodiments, R^sis optionally substituted C_1-30heteroaliphatic. In some embodiments, R^scomprises one or more silicon atoms. In some embodiments, R is —CH₂Si(Ph)₂CH₃.

In some embodiments, R^sis -L-R′. In some embodiments, R^sis -L-R′ wherein -L- is a bivalent, optionally substituted C_1-3heteroaliphatic group. In some embodiments, R^sis —CH₂Si(Ph)₂CH₃.

In some embodiments, R^sis —F. In some embodiments, R^sis —Cl. In some embodiments, R^sis —Br. In some embodiments, R^sis —I. In some embodiments, R^sis —CN. In some embodiments, R^sis —N. In some embodiments, R^sis —NO. In some embodiments, R^sis —NO₂. In some embodiments, R^sis -L-Si(R)₃. In some embodiments, R^sis —Si(R)₃. In some embodiments, R^sis -L-R′. In some embodiments, R^sis —R′. In some embodiments, R^sis -L-OR′. In some embodiments. R^sis —OR′. In some embodiments, R^sis -L-SR′. In some embodiments, R^sis —SR′. In some embodiments, R^sis -L-N(R′)₂. In some embodiments, R^sis —N(R′)₂. In some embodiments, R^sis —O-L-R′. In some embodiments, R^sis —O-L-Si(R)₃. In some embodiments, R^sis —O-L-OR′. In some embodiments, R^sis —O-L-SR′. In some embodiments, R^sis —O-L-N(R′)₂. In some embodiments, R^sis a 2′-modification as described in the present disclosure. In some embodiments, R^sis —OR, wherein R is as described in the present disclosure. In some embodiments, R^sis —OR, wherein R is optionally substituted C_1-6aliphatic. In some embodiments, R^sis -OMe. In some embodiments, R is —OCH₂CH₂OMe. In some embodiments, R^sis R^1s, R^2s, R^3s, R^4s, or R^5sas described in the present disclosure.

In some embodiments, g is 0-20. In some embodiments, g is 1-20. In some embodiments, g is 1-5. In some embodiments, g is 1. In some embodiments, g is 2. In some embodiments, g is 3. In some embodiments, g is 4. In some embodiments, g is 5. In some embodiments, g is 6. In some embodiments, g is 7. In some embodiments, g is 8. In some embodiments, g is 9. In some embodiments, g is 10. In some embodiments, g is 11. In some embodiments, g is 12. In some embodiments, g is 13. In some embodiments, g is 14. In some embodiments, g is 15. In some embodiments, g is 16. In some embodiments, g is 17. In some embodiments, g is 18. In some embodiments, g is 19. In some embodiments, g is 20.

In some embodiments,

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In some embodiments,

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In some embodiments,

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In some embodiments, each Ring A is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms, e.g., independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, Ring A is an optionally substituted ring, which ring is as described in the present disclosure. In some embodiments, Ring A comprises an oxygen ring atom. In some embodiments, Ring A is or comprises a ring of a sugar moiety. In some embodiments, a ring is

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In some embodiments, a ring is

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In some embodiments, a ring is

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In some embodiments, a ring is a bicyclic ring, e.g., found in a sugar moiety of LNA.

In some embodiments, a sugar unit is of the structure

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wherein each variable is independently as described in the present disclosure. In some embodiments, a nucleoside unit is of the structure

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wherein each variable is independently as described in the present disclosure.

In some embodiments, L^sis —C(R^5s)₂— and

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is as described in the present disclosure. In some embodiments, L^sis —CHR^5s— and

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is as described in the present disclosure. In some embodiments, L^sis —C(R)₂— and

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is as described in the present disclosure. In some embodiments, L^sis —CHR— and

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is as described in the present disclosure.

In some embodiments,

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BA is connected at Cl, and each of R^1s, R^2s, R^3s, R^4sand R^5Sis independently as described in the present closure. In some embodiments,

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wherein R^2sis as described in the present disclosure. In some embodiments,

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wherein R^2sis not —OH. In some embodiments,

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wherein R^2sand R^4sare R, and the two R groups are taken together with their intervening atoms to form an optionally substituted ring. In some embodiments,

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or Ring A, is optionally substituted

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In some embodiments

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or Ring A, is

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In some embodiments,

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or Ring A, is

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In some embodiments each of R^1s, R^2s, R^3s, R^4s, and R^5sis independently R^s, wherein R^sis as described in the present disclosure.

In some embodiments, R^1sis R^swherein R^sis as described in the present disclosure. In some embodiments, R^1sis at 1′-position (BA is at 1′-position). In some embodiments, R^1sis —H. In some embodiments, R^1sis —F. In some embodiments, R^1sis —Cl. In some embodiments, R^1sis —Br. In some embodiments, R^1sis —I. In some embodiments, R^1sis —CN. In some embodiments, R^1sis —N₃. In some embodiments, R^1sis —NO. In some embodiments, R^1sis —NO₂. In some embodiments, R^1sis -L-R′. In some embodiments, R^1sis —R′. In some embodiments, R^1sis -L-OR′. In some embodiments, R^1sis —OR′. In some embodiments, R^1sis -L-SR′. In some embodiments, R^1sis —SR′. In some embodiments, R^1sis L-L-N(R′)₂. In some embodiments, R^1sis —N(R′)₂. In some embodiments, R^1sis —OR′, wherein R′ is optionally substituted C_1-3aliphatic. In some embodiments, R^1sis —OR′, wherein R′ is optionally substituted C_1-6alkyl. In some embodiments, R^1sis -OMe. In some embodiments, R^1sis -MOE. In some embodiments, R^1sis hydrogen. In some embodiments, R^sat one 1′-position is hydrogen, and R^sat the other 1′-position is not hydrogen as described herein. In some embodiments, R^sat both 1′-positions are hydrogen. In some embodiments, R^sat one 1′-position is hydrogen, and the other 1′-position is connected to an internucleotidic linkage. In some embodiments, R^1sis —F. In some embodiments, R^1sis —Cl. In some embodiments, R^1sis —Br. In some embodiments, R^1sis —I. In some embodiments, R^1sis —CN. In some embodiments, R^1sis —N. In some embodiments, R^1sis —NO. In some embodiments, R^1sis —NO₂. In some embodiments, R^1sis -L-R′. In some embodiments, R^1sis —R′. In some embodiments, R^1sis -L-OR′. In some embodiments, R^1sis —OR′. In some embodiments, R^1sis -L-SR′. In some embodiments, R^1sis —SR′. In some embodiments, R^1sis -L-N(R′)₂. In some embodiments, R^1sis —N(R′)₂. In some embodiments, R^1sis —OR′, wherein R′ is optionally substituted C_1-6aliphatic. In some embodiments, R^1sis —OR′, wherein R′ is optionally substituted C_1-6alkyl. In some embodiments, R^1sis —OH. In some embodiments, R^1sis -OMe. In some embodiments, R^1sis -MOE. In some embodiments, R^1sis hydrogen. In some embodiments, one R^1sat a 1′-position is hydrogen, and the other R^1sat the other 1′-position is not hydrogen as described herein. In some embodiments, R^1sat both 1′-positions are hydrogen. In some embodiments, R^1sis —O-L-OR′. In some embodiments, R^1sis —O-L-OR′, wherein L is optionally substituted C_1-6alkylene, and R′ is optionally substituted C_1-6aliphatic. In some embodiments, R^1sis —O-(optionally substituted C_1-6alkylene)-OR′. In some embodiments, R^1sis —O-(optionally substituted C_falkylene)-OR′, wherein R′ is optionally substituted C_1-6alkyl. In some embodiments, R^1sis —OCH₂CH₂OMe.

In some embodiments, R^2sis R^swherein R^sis as described in the present disclosure. In some embodiments, if there are two R^2sat the 2′-position, one R^2sis —H and the other is not. In some embodiments, R^2sis at 2′-position (BA is at 1′-position). In some embodiments, R^2sis —H. In some embodiments, R^2sis —F. In some embodiments, R^2sis —Cl. In some embodiments, R^2sis —Br. In some embodiments, R^2sis —I. In some embodiments, R^2sis —CN. In some embodiments, R^2sis —N₃. In some embodiments, R^2sis —NO. In some embodiments, R^2sis —NO₂. In some embodiments, R^2sis -L-R′. In some embodiments, R^2sis —R′. In some embodiments, R^2sis -L-OR′. In some embodiments, R^2sis —OR′. In some embodiments, R^2sis -L-SR′. In some embodiments, R^2sis —SR′. In some embodiments, R^2sis L-L-N(R′)₂. In some embodiments, R^2sis —N(R′)₂. In some embodiments, R^2sis —OR′, wherein R′ is optionally substituted C_1-6aliphatic. In some embodiments, R^2sis —OR′, wherein R′ is optionally substituted C_1-6alkyl. In some embodiments, R^2sis -OMe. In some embodiments, R^2sis -MOE. In some embodiments, R^2sis hydrogen. In some embodiments, R^sat one 2′-position is hydrogen, and R^sat the other 2′-position is not hydrogen as described herein. In some embodiments, R^sat both 2′-positions are hydrogen. In some embodiments, R^sat one 2′-position is hydrogen, and the other 2′-position is connected to an internucleotidic linkage. In some embodiments, R^2sis —F. In some embodiments, R^2sis —Cl. In some embodiments, R^2sis —Br. In some embodiments, R^2sis —I. In some embodiments, R^2sis —CN. In some embodiments, R^2sis —N₃. In some embodiments, R^2sis —NO. In some embodiments, R^2sis —NO₂. In some embodiments, R^2sis -L-R′. In some embodiments, R^2sis —R′. In some embodiments, R^2sis -L-OR′. In some embodiments, R^2sis —OR′. In some embodiments, R^2sis -L-SR′. In some embodiments, R^2sis —SR′. In some embodiments, R^2sis -L-N(R′)₂. In some embodiments, R^2sis —N(R′)₂. In some embodiments, R^2sis —OR′, wherein R′ is optionally substituted C_1-6aliphatic. In some embodiments, R^2sis —OR′, wherein R′ is optionally substituted C_1-6alkyl. In some embodiments, R^2sis —OH. In some embodiments, R^2sis -OMe. In some embodiments, R^2sis -MOE. In some embodiments, R^2sis hydrogen. In some embodiments, one R^2sat a 2′-position is hydrogen, and the other R^2sat the other 2′-position is not hydrogen as described herein. In some embodiments, R^2sat both 2′-positions are hydrogen. In some embodiments, R^2sis —O-L-OR′. In some embodiments, R^2sis —O-L-OR′, wherein L is optionally substituted C_1-6alkylene, and R′ is optionally substituted C_1-6aliphatic. In some embodiments, R^2sis —O-(optionally substituted C_1-6alkylene)-OR′. In some embodiments, R^2sis —O-(optionally substituted C_1-6alkylene)-OR′, wherein R′ is optionally substituted C_1-6alkyl. In some embodiments, R^2sis —OCH₂CH₂OMe.

In some embodiments, R^2scomprises a guanidine moiety. In some embodiments, R^2scomprises

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In some embodiments, R^2sis -L-W^z, wherein W^zis selected from

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wherein R″ is R′ and n is 0-15. In some embodiments, R′ and R″ are

independently

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In some embodiments, L is —O—CH₂CH₂—. In some embodiments, n is 0-3. In some embodiments, each R^sis independently —H, —OCH₃, —F, —CN, —CH₃, —NO₂, —CF₃, or —OCF₃. In some embodiments, R′ and R″ are the same. In some embodiments, R′ and R″ are different.

In some embodiments, R^3sis R^swherein R^sis as described in the present disclosure. In some embodiments, R^3sis at 3′-position (BA is at 1′-position). In some embodiments, R^3sis —H. In some embodiments, R^3sis —F. In some embodiments, R^3sis —Cl. In some embodiments, R^3sis —Br. In some embodiments, R^3sis —I. In some embodiments, R^3sis —CN. In some embodiments, R^3sis —N₃. In some embodiments, R^3sis —NO. In some embodiments, R^3sis —NO₂. In some embodiments, R^3sis -L-R′. In some embodiments, R^3sis —R′. In some embodiments, R^3sis -L-OR′. In some embodiments, R^3sis —OR′. In some embodiments, R^3sis -L-SR′. In some embodiments, R^3sis —SR′. In some embodiments. R^3sis -L-N(R′)₂. In some embodiments, R^3sis —N(R′)₂. In some embodiments, R^3sis —OR′, wherein R′ is optionally substituted C_1-6aliphatic. In some embodiments, R^3sis —OR′, wherein R′ is optionally substituted C_1-6alkyl. In some embodiments, R^3sis -OMe. In some embodiments, R^3sis -MOE. In some embodiments, R^3sis hydrogen. In some embodiments, R^sat one 3′-position is hydrogen, and R^sat the other 3′-position is not hydrogen as described herein. In some embodiments, R³at both 3′-positions are hydrogen. In some embodiments, R^sat one 3′-position is hydrogen, and the other 3′-position is connected to an internucleotidic linkage. In some embodiments, R^3sis —F. In some embodiments, R^3sis —Cl. In some embodiments, R^3sis —Br. In some embodiments, R^3sis —I. In some embodiments, R^3sis —CN. In some embodiments, R^3sis —N₃. In some embodiments, R^3sis —NO. In some embodiments, R^3sis —NO₂. In some embodiments, R^3sis -L-R′. In some embodiments, R^3sis —R′. In some embodiments, R^3sis -L-OR′. In some embodiments, R^3sis —OR′. In some embodiments, R^3sis -L-SR′. In some embodiments, R^3sis —SR′. In some embodiments, R^3sis L-L-N(R′)₂. In some embodiments, R^3sis —N(R′)₂. In some embodiments, R^3sis —OR′, wherein R′ is optionally substituted C_1-6aliphatic. In some embodiments, R^3sis —OR′, wherein R′ is optionally substituted C_1-6alkyl. In some embodiments, R^3sis —OH. In some embodiments, R^3sis -OMe. In some embodiments, R^3sis -MOE. In some embodiments, R^3sis hydrogen.

In some embodiments, R^4sis R^swherein R^sis as described in the present disclosure. In some embodiments, R^4sis at 4′-position (BA is at 1′-position). In some embodiments, R^4sis —H. In some embodiments, R^4sis —F. In some embodiments, R^4sis —Cl. In some embodiments, R^4sis —Br. In some embodiments, R^4sis —I. In some embodiments, R^4sis —CN. In some embodiments, R^4sis —N₃. In some embodiments, R^4sis —NO. In some embodiments, R^4sis —NO₂. In some embodiments, R^4sis -L-R′. In some embodiments, R^4sis —R′. In some embodiments, R^4sis -L-OR′. In some embodiments, R^4sis —OR′. In some embodiments, R^4sis -L-SR′. In some embodiments, R^4sis —SR′. In some embodiments, R^4sis -L-N(R′)₂. In some embodiments, R^4sis —N(R′)₂. In some embodiments, R^4sis —OR′, wherein R′ is optionally substituted C_1-6aliphatic. In some embodiments, R^4Sis —OR′, wherein R′ is optionally substituted C_1-6alkyl. In some embodiments, R^4sis -OMe. In some embodiments, R^4Sis -MOE. In some embodiments, R^4sis hydrogen. In some embodiments, R^Sat one 4′-position is hydrogen, and R^Sat the other 4′-position is not hydrogen as described herein. In some embodiments, R^sat both 4′-positions are hydrogen. In some embodiments, R^Sat one 4′-position is hydrogen, and the other 4′-position is connected to an internucleotidic linkage. In some embodiments, R^4Sis —F. In some embodiments, R^4Sis —Cl. In some embodiments, R^4Sis —Br. In some embodiments, R^4sis —I. In some embodiments, R^4sis —CN. In some embodiments, R^4Sis —N. In some embodiments, R^4sis —NO. In some embodiments, R^4sis —NO₂. In some embodiments, R^4sis -L-R′. In some embodiments, R^4sis —R′. In some embodiments, R^4sis -L-OR′. In some embodiments, R^4sis —OR′. In some embodiments, R^4sis -L-SR′. In some embodiments, R^4sis —SR′. In some embodiments, R^4sis L-L-N(R′)₂. In some embodiments, R^4sis —N(R′)₂. In some embodiments, R^4sis —OR′, wherein R′ is optionally substituted C_1-6aliphatic. In some embodiments, R^4sis —OR′, wherein R′ is optionally substituted C_1-6alkyl. In some embodiments, R^4Sis —OH. In some embodiments, R^4sis -OMe. In some embodiments, R^4Sis -MOE. In some embodiments, R^4Sis hydrogen.

In some embodiments, R^5sis R^swherein R^Sis as described in the present disclosure. In some embodiments, R^5sis R′ wherein R′ is as described in the present disclosure. In some embodiments, R^5sis —H. In some embodiments, two or more R^5sare connected to the same carbon atom, and at least one is not —H. In some embodiments, R^5sis not —H. In some embodiments, R^5sis —F. In some embodiments, R^5sis —Cl. In some embodiments, R^5sis —Br. In some embodiments, R^5sis —I. In some embodiments, R^5sis —CN. In some embodiments, R^5sis —N. In some embodiments, R^5sis —NO. In some embodiments, R^5sis —NO₂. In some embodiments, R^5sis -L-R′. In some embodiments, R^5sis —R′. In some embodiments, R^5sis -L-OR′. In some embodiments, R^5sis —OR′. In some embodiments, R^5sis -L-SR′. In some embodiments, R^5sis —SR′. In some embodiments, R^5sis L-L-N(R′)₂. In some embodiments, R^5sis —N(R′)₂. In some embodiments, R^5sis —OR′, wherein R′ is optionally substituted C_1-6aliphatic. In some embodiments, R^5sis —OR′, wherein R′ is optionally substituted C_1-6alkyl. In some embodiments, R^5sis —OH. In some embodiments, R^5sis -OMe. In some embodiments, R^5sis -MOE. In some embodiments, R^5sis hydrogen.

In some embodiments, R^5sis optionally substituted C_1-6aliphatic as described in the present disclosure. e.g., C_1-6aliphatic embodiments described for R or other variables. In some embodiments, R^5sis optionally substituted C_1-6alkyl. In some embodiments, R^5sis optionally substituted methyl, wherein each substituent, if any, independently comprises no more than one carbon atoms. In some embodiments, R^5sis optionally substituted methyl, wherein each substituent, if any, independently is halogen. In some embodiments, R^5sis methyl. In some embodiments, R^5sis ethyl.

In some embodiments, R^5sis a protected hydroxyl group suitable for oligonucleotide synthesis. In some embodiments, R^5sis —OR′, wherein R′ is optionally substituted C_1-6aliphatic. In some embodiments, R^5sis DMTrO-. Example protecting groups are widely known for use in accordance with the present disclosure. For additional examples, see Greene. T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991, and U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, and/or WO 2017/210647, protecting groups of each of which are hereby incorporated by reference.

In some embodiments, two or more of R^1s, R^2s, R^3s, R^4s, and R^5sare R and can be taken together with intervening atom(s) to form a ring as described in the present disclosure. In some embodiments, R^2sand R^4sare R taken together to form a ring, and a sugar moiety can be a bicyclic sugar moiety, e.g., a LNA sugar moiety.

In some embodiments, L^sis L as described in the present disclosure.

In some embodiments, L^sis —C(R^5s)₂—, wherein each R is independently as described in the present disclosure. In some embodiments, one of R^5sis H and the other is not H. In some embodiments, none of R^5sis H. In some embodiments, L^sis —CHR^5s-, wherein each R^5sis independently as described in the present disclosure. In some embodiments, the carbon atom of —C(R^5s)₂- is stereorandom. In some embodiments, it is of R configuration. In some embodiments, it is of S configuration. In some embodiments, —C(R^5s)₂- is 5′-C, optionally substituted, of a sugar moiety. In some embodiments, the C of —C(R^5s)₂- is of R configuration. In some embodiments, the C of —C(R^5s)₂-is of S configuration. As described in the present disclosure, in some embodiments, R is optionally substituted C_1-6aliphatic; in some embodiments, R^5sis methyl.

In some embodiments, provided compounds comprise one or more bivalent or multivalent optionally substituted rings, e.g., Ring A, Cy^L, those formed by two or more R groups (R and (combinations of) variables that can be R) taken together, etc. In some embodiments, a ring is a cycloaliphatic, aryl, heteroaryl, or heterocyclyl group as described for R but bivalent or multivalent. As appreciated by those skilled in the art, ring moieties described for one variable, e.g., Ring A, can also be applicable to other variables, e.g., Cy^L, if requirements of the other variables, e.g., number of heteroatoms, valence, etc., are satisfied. Example rings are extensively described in the present disclosure.

In some embodiments, a ring, e.g., in Ring A, R, etc. which is optionally substituted, is a 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

In some embodiments, a ring can be of any size within its range, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-membered.

In some embodiments, a ring is monocyclic. In some embodiments, a ring is saturated and monocyclic. In some embodiments, a ring is monocyclic and partially saturated. In some embodiments, a ring is monocyclic and aromatic.

In some embodiments, a ring is bicyclic. In some embodiments, a ring is polycyclic. In some embodiments, a bicyclic or polycyclic ring comprises two or more monocyclic ring moieties, each of which can be saturated, partially saturated, or aromatic, and each which can contain no or 1-10 heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises a saturated monocyclic ring. In some embodiments, a bicyclic or polycyclic ring comprises a saturated monocyclic ring containing no heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises a saturated monocyclic ring comprising one or more heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises a partially saturated monocyclic ring. In some embodiments, a bicyclic or polycyclic ring comprises a partially saturated monocyclic ring containing no heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises a partially saturated monocyclic ring comprising one or more heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises an aromatic monocyclic ring. In some embodiments, a bicyclic or polycyclic ring comprises an aromatic monocyclic ring containing no heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises an aromatic monocyclic ring comprising one or more heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises a saturated ring and a partially saturated ring, each of which independently contains one or more heteroatoms. In some embodiments, a bicyclic ring comprises a saturated ring and a partially saturated ring, each of which independently comprises no, or one or more heteroatoms. In some embodiments, a bicyclic ring comprises an aromatic ring and a partially saturated ring, each of which independently comprises no, or one or more heteroatoms. In some embodiments, a polycyclic ring comprises a saturated ring and a partially saturated ring, each of which independently comprises no, or one or more heteroatoms. In some embodiments, a polycyclic ring comprises an aromatic ring and a partially saturated ring, each of which independently comprises no, or one or more heteroatoms. In some embodiments, a polycyclic ring comprises an aromatic ring and a saturated ring, each of which independently comprises no, or one or more heteroatoms. In some embodiments, a polycyclic ring comprises an aromatic ring, a saturated ring, and a partially saturated ring, each of which independently comprises no, or one or more heteroatoms. In some embodiments, a ring comprises at least one heteroatom. In some embodiments, a ring comprises at least one nitrogen atom. In some embodiments, a ring comprises at least one oxygen atom. In some embodiments, a ring comprises at least one sulfur atom.

As appreciated by those skilled in the art in accordance with the present disclosure, a ring is typically optionally substituted. In some embodiments, a ring is unsubstituted. In some embodiments, a ring is substituted. In some embodiments, a ring is substituted on one or more of its carbon atoms. In some embodiments, a ring is substituted on one or more of its heteroatoms. In some embodiments, a ring is substituted on one or more of its carbon atoms, and one or more of its heteroatoms. In some embodiments, two or more substituents can be located on the same ring atom. In some embodiments, all available ring atoms are substituted. In some embodiments, not all available ring atoms are substituted. In some embodiments, in provided structures where rings are indicated to be connected to other structures

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“optionally substituted” is to mean that, besides those structures already connected, remaining substitutable ring positions, if any, are optionally substituted.

In some embodiments, a ring is a bivalent or multivalent C_3-30cycloaliphatic ring. In some embodiments, a ring is a bivalent or multivalent C_3-20cycloaliphatic ring. In some embodiments, a ring is a bivalent or multivalent C_3-10cycloaliphatic ring. In some embodiments, a ring is a bivalent or multivalent 3-30 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent 3-7 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent 3-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent 4-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent 5-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent 6-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent 7-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent cyclohexyl ring. In some embodiments, a ring is a bivalent or multivalent cyclopentyl ring. In some embodiments, a ring is a bivalent or multivalent cyclobutyl ring. In some embodiments, a ring is a bivalent or multivalent cyclopropyl ring.

In some embodiments, a ring is a bivalent or multivalent C_6-30aryl ring. In some embodiments, a ring is a bivalent or multivalent phenyl ring.

In some embodiments, a ring is a bivalent or multivalent 8-10 membered bicyclic saturated, partially unsaturated or aryl ring. In some embodiments, a ring is a bivalent or multivalent 8-10 membered bicyclic saturated ring. In some embodiments, a ring is a bivalent or multivalent 8-10 membered bicyclic partially unsaturated ring. In some embodiments, a ring is a bivalent or multivalent 8-10 membered bicyclic aryl ring. In some embodiments, a ring is a bivalent or multivalent naphthyl ring.

In some embodiments, a ring is a bivalent or multivalent 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, a ring is a bivalent or multivalent 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, a ring is a bivalent or multivalent 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

In some embodiments, a ring is a bivalent or multivalent 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, and oxygen.

In some embodiments, a ring is a bivalent or multivalent 5-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, a ring is a bivalent or multivalent 6-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In certain embodiments, a ring is a bivalent or multivalent 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 5,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 6,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, a ring is a bivalent or multivalent 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, a ring is a bivalent or multivalent 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 5-7 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 5-6 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 5-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 6-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 7-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 3-membered heterocyclic ring having one heteroatom selected from nitrogen, oxygen or sulfur. In some embodiments, a ring is a bivalent or multivalent 4-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 5-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 6-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 7-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, a ring is a bivalent or multivalent 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, a ring is a bivalent or multivalent 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 6,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, a ring formed by two or more groups taken together, which is typically optionally substituted, is a monocyclic saturated 5-7 membered ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is a monocyclic saturated 5-membered ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is a monocyclic saturated 6-membered ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is a monocyclic saturated 7-membered ring having no additional heteroatoms in addition to intervening heteroatoms, if any.

In some embodiments, a ring formed by two or more groups taken together is a bicyclic, saturated, partially unsaturated, or aryl 5-30 membered ring having, in addition to the intervening heteroatoms, if any, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, a ring formed by two or more groups taken together is a bicyclic, saturated, partially unsaturated, or aryl 5-30 membered ring having, in addition to the intervening heteroatoms, if any, 0-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, a ring formed by two or more groups taken together is a bicyclic and saturated 8-10 membered bicyclic ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is a bicyclic and saturated 8-membered bicyclic ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is a bicyclic and saturated 9-membered bicyclic ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is a bicyclic and saturated 10-membered bicyclic ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is bicyclic and comprises a 5-membered ring fused to a 5-membered ring. In some embodiments, a ring formed by two or more groups taken together is bicyclic and comprises a 5-membered ring fused to a 6-membered ring. In some embodiments, the 5-membered ring comprises one or more intervening nitrogen, phosphorus and oxygen atoms as ring atoms. In some embodiments, a ring formed by two or more groups taken together comprises a ring system having the backbone structure of

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In some embodiments, a ring formed by two or more groups taken together is a polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening heteroatoms, if any, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, a ring formed by two or more groups taken together is a polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening heteroatoms, if any, 0-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-10 membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-9 membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-8 membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-7 membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-6 membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms.

In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 6-membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 7-membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 8-membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 9-membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 10-membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms.

In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-membered ring whose ring atoms consist of carbon atoms and the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 6-membered ring whose ring atoms consist of carbon atoms and the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 7-membered ring whose ring atoms consist of carbon atoms and the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 8-membered ring whose ring atoms consist of carbon atoms and the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 9-membered ring whose ring atoms consist of carbon atoms and the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 10-membered ring whose ring atoms consist of carbon atoms and the intervening nitrogen, phosphorus and oxygen atoms.

In some embodiments, rings described herein are unsubstituted. In some embodiments, rings described herein are substituted. In some embodiments, substituents are selected from those described in example compounds provided in the present disclosure.

In some embodiments, each BA is independently an optionally substituted group selected from C_5-30heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and C_3-30heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon:

each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-I, II-a-2, II-b-1, II-b-2, I-c-1, II-c-2, II-d-1, II-d-2, or a salt form there, wherein each variable is independently as described in the present disclosure.

each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, I-c-1, II-c-2, II-d-1, II-d-2, or salt form thereof, wherein each variable is independently as described in the present disclosure.

In some embodiments, each BA is independently an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U;

each Ring A is independently an optionally substituted 5-7 membered monocyclic or bicyclic saturated ring having one or more oxygen atoms; and

each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, 11, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or salt form thereof, wherein each variable is independently as described in the present disclosure.

In some embodiments, each BA is independently an optionally substituted or protected nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil;

each Ring A is independently an optionally substituted 5-7 membered monocyclic or bicyclic saturated ring having one or more oxygen atoms; and

each L^Pindependently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, I-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or salt form thereof, wherein each variable is independently as described in the present disclosure.

In some embodiments, R^5s-L^s-is —CH₂OH. In some embodiments, R^5s-L^s- is —CH(R^5s)—OH, wherein R^5sis as described in the present disclosure.

In some embodiments, BA is an optionally substituted group selected from C_3-30cycloaliphatic, C_6-30aryl, C_5-30heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C_3-30heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. In some embodiments, BA is an optionally substituted group selected from C_5-30heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C_3-30heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. In some embodiments, BA is an optionally substituted group selected from C_3-30heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. In some embodiments, BA is optionally substituted C_5-30heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, BA is optionally substituted natural nucleobases and tautomers thereof. In some embodiments, BA is protected natural nucleobases and tautomers thereof. Various nucleobase protecting groups for oligonucleotide synthesis are known and can be utilized in accordance with the present disclosure. In some embodiments, BA is an optionally substituted nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil, and tautomers thereof. In some embodiments, BA is an optionally protected nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil, and tautomers thereof.

In some embodiments, s is 0-20. In some embodiments, s is 1-20. In some embodiments, s is 1-5. In some embodiments, s is 1. In some embodiments, s is 2. In some embodiments, s is 3. In some embodiments, s is 4. In some embodiments, s is 5. In some embodiments, s is 6. In some embodiments, s is 7. In some embodiments, s is 8. In some embodiments, s is 9. In some embodiments, s is 10. In some embodiments, s is 11. In some embodiments, s is 12. In some embodiments, s is 13. In some embodiments, s is 14. In some embodiments, s is 15. In some embodiments, s is 16. In some embodiments, s is 17. In some embodiments, s is 18. In some embodiments, s is 19. In some embodiments, s is 20.

In some embodiments, L^Pis an internucleotidic linkage. In some embodiments, L^Pis an internucleotidic linkage of formula I, I-a, I-b. I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1. II-a-2. II-b-1, II-b-2, II-c-1, II-c-2,11-d-1,1-d-2, or a salt form thereof. In some embodiments, L^Pis a natural phosphate linkage. In some embodiments, L^Pis a non-negatively charged internucleotidic linkage. In some embodiments, L^Pis a neutral internucleotidic linkage. In some embodiments, L^Pis a negatively-charged internucleotidic linkage. In some embodiments, L^Pis a phosphorothioate internucleotidic linkage. In some embodiments, L^Pis a chirally controlled internucleotidic linkage.

In some embodiments, z is 1-1000. In some embodiments, z+1 is an oligonucleotide length as described in the present disclosure. In some embodiments, z is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000. In some embodiments, z is 10-100. In some embodiments, z is 10-50. In some embodiments, z is 15-100. In some embodiments, z is 20-50. In some embodiments, z is no less than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments, z is no less than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, z is no more than 50, 60, 70, 80, 90, 100, 150, or 200. In some embodiments, z is 5-50, 10-50, 14-50, 14-45, 1440, 14-35, 14-30, 14-25, 14-100, 14-150, 14-200, 14-250, 14-300, 15-50, 1545, 1540, 15-35, 15-30, 15-25, 15-100, 15-150, 15-200, 15-250, 15-300, 16-50, 1645, 1640, 16-35, 16-30, 16-25, 16-100, 16-150, 16-200, 16-250, 16-300, 17-50, 17-45, 1740, 17-35, 17-30, 17-25, 17-100, 17-150, 17-200, 17-250, 17-300, 18-50, 1845, 1840, 18-35, 18-30, 18-25, 18-100, 18-150, 18-200, 18-250, 18-300, 19-50, 1945, 1940, 19-35, 19-30, 19-25, 19-100, 19-150, 19-200, 19-250, or 19-300. In some embodiments, z is 10. In some embodiments, z is 11. In some embodiments, z is 12. In some embodiments, z is 13. In some embodiments, z is 14. In some embodiments, z is 15. In some embodiments, z is 16. In some embodiments, z is 17. In some embodiments, z is 18. In some embodiments, z is 19. In some embodiments, z is 20. In some embodiments, z is 21. In some embodiments, z is 22. In some embodiments, z is 23. In some embodiments, z is 24. In some embodiments, z is 25. In some embodiments, z is 26. In some embodiments, z is 27. In some embodiments, z is 28. In some embodiments, z is 29. In some embodiments, z is 30. In some embodiments, z is 31. In some embodiments, z is 32. In some embodiments, z is 33. In some embodiments, z is 34.

In some embodiments, L^3Eis -L- or -L-L-. In some embodiments, L^3Eis -L-. In some embodiments, L^3Eis -L-L-. In some embodiments, L^3Eis a covalent bond. In some embodiments, L^3Eis a linker used in oligonucleotide synthesis. In some embodiments, L^3Eis a linker used in solid phase oligonucleotide synthesis. Various types of linkers are known and can be utilized in accordance with the present disclosure. In some embodiments, a linker is a succinate linker (—O—C(O)—CH₂—CH₂—C(O)—). In some embodiments, a linker is an oxalyl linker (—O—C(O)—C(O)—). In some embodiments, L^3Eis a succinyl-piperidine linker (SP) linker. In some embodiments, L^3Eis a succinyl linker. In some embodiments, L^3Eis a Q-linker. In some embodiments, L^3Eis —O—.

In some embodiments, R^3Eis —R′, -L-R′, —OR′, or a solid support. In some embodiments, R^3Eis —R′ as described in the present disclosure. In some embodiments, R^3Eis —R as described in the present disclosure. In some embodiments, R^3Eis hydrogen. In some embodiments, R^3Eis -L-R′. In some embodiments, R^3Eis —OR′. In some embodiments, R^3Eis a support for oligonucleotide synthesis. In some embodiments, R^3Eis a solid support. In some embodiments, a solid support is a CPG support. In some embodiments, a solid support is a polystyrene support. In some embodiments, R^3Eis —H. In some embodiments, -L³-R^3Eis —H. In some embodiments, R^3Eis —OH. In some embodiments, -L³-R^3Eis —OH. In some embodiments, R^3Eis optionally substituted C_1-6aliphatic. In some embodiments, R^3Eis optionally substituted C_1-6alkyl. In some embodiments, R^3Eis —OR′. In some embodiments, R^3Eis —OH. In some embodiments, R^3Eis —OR′, wherein R′ is not hydrogen. In some embodiments, R^3Eis —OR′, wherein R′ is optionally substituted C_1-6alkyl. In some embodiments, R^3Eis a 3′-end cap (e.g., those used in RNAi technologies).

In some embodiments, R^3Eis a solid support. In some embodiments, R^3Eis a solid support for oligonucleotide synthesis. Various types of solid support are known and can be utilized in accordance with the present disclosure. In some embodiments, a solid support is HCP. In some embodiments, a solid support is CPG.

In some embodiments, R′ is —R, —C(O)R, —C(O)OR, or —S(O)₂R, wherein R is as described in the present disclosure. In some embodiments, R′ is R, wherein R is as described in the present disclosure. In some embodiments, R′ is —C(O)R, wherein R is as described in the present disclosure. In some embodiments, R′ is —C(O)OR, wherein R is as described in the present disclosure. In some embodiments, R′ is —S(O)₂R, wherein R is as described in the present disclosure. In some embodiments, R′ is hydrogen. In some embodiments, R′ is not hydrogen. In some embodiments, R′ is R, wherein R is optionally substituted C_1-3aliphatic as described in the present disclosure. In some embodiments, R′ is R, wherein R is optionally substituted C_1-20heteroaliphatic as described in the present disclosure. In some embodiments, R′ is R, wherein R is optionally substituted C_6-20aryl as described in the present disclosure. In some embodiments, R′ is R, wherein R is optionally substituted C_6-20arylaliphatic as described in the present disclosure. In some embodiments, R′ is R, wherein R is optionally substituted C_6-20arylheteroaliphatic as described in the present disclosure. In some embodiments, R′ is R, wherein R is optionally substituted 5-20 membered heteroaryl as described in the present disclosure. In some embodiments, R′ is R, wherein R is optionally substituted 3-20 membered heterocyclyl as described in the present disclosure. In some embodiments, two or more R′ are R, and are optionally and independently taken together to form an optionally substituted ring as described in the present disclosure.

In some embodiments, each R is independently —H, or an optionally substituted group selected from C_1-30aliphatic, C_1-30heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C_6-30aryl, C_6-30arylaliphatic, C_6-30arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or

two R groups are optionally and independently taken together to form a covalent bond, or:

In some embodiments, each R is independently —H, or an optionally substituted group selected from C_1-20aliphatic, C_1-20heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. C_6-20aryl, C_6-20arylaliphatic, C_6-20arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-20 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-20 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or

two R groups are optionally and independently taken together to form a covalent bond, or:

two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

In some embodiments, each R is independently —H, or an optionally substituted group selected from C_1-20aliphatic, C_1-20heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C_6-20aryl, C_6-20arylaliphatic, C_6-20arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-20 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-20 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

In some embodiments, R is hydrogen. In some embodiments, R is not hydrogen. In some embodiments, R is an optionally substituted group selected from C_1-30aliphatic, C_1-30heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C_6-30aryl, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

In some embodiments, R is hydrogen or an optionally substituted group selected from C_1-20aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated, partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R is optionally substituted C_1-30aliphatic. In some embodiments, R is optionally substituted C_1-20aliphatic. In some embodiments, R is optionally substituted C_1-15aliphatic. In some embodiments, R is optionally substituted C_1-10aliphatic. In some embodiments, R is optionally substituted C_1-6aliphatic. In some embodiments, R is optionally substituted C_1-6alkyl. In some embodiments, R is optionally substituted hexyl, pentyl, butyl, propyl, ethyl or methyl. In some embodiments, R is optionally substituted hexyl. In some embodiments, R is optionally substituted pentyl. In some embodiments, R is optionally substituted butyl. In some embodiments, R is optionally substituted propyl. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is optionally substituted methyl. In some embodiments, R is hexyl. In some embodiments, R is pentyl. In some embodiments, R is butyl. In some embodiments, R is propyl. In some embodiments, R is ethyl. In some embodiments, R is methyl. In some embodiments, R is isopropyl. In some embodiments, R is n-propyl. In some embodiments, R is tert-butyl. In some embodiments, R is sec-butyl. In some embodiments, R is n-butyl. In some embodiments, R is —(CH₂)₂CN.

In some embodiments, R is optionally substituted C_3-30cycloaliphatic. In some embodiments, R is optionally substituted C_3-20cycloaliphatic. In some embodiments, R is optionally substituted C_3-10cycloaliphatic. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is cyclohexyl. In some embodiments, R is optionally substituted cyclopentyl. In some embodiments, R is cyclopentyl. In some embodiments, R is optionally substituted cyclobutyl. In some embodiments, R is cyclobutyl. In some embodiments, R is optionally substituted cyclopropyl. In some embodiments, R is cyclopropyl.

In some embodiments, R is an optionally substituted 3-30 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 3-7 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 3-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 6-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 7-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is optionally substituted cycloheptyl. In some embodiments, R is cycloheptyl. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is cyclohexyl. In some embodiments, R is optionally substituted cyclopentyl. In some embodiments, R is cyclopentyl. In some embodiments, R is optionally substituted cyclobutyl. In some embodiments, R is cyclobutyl. In some embodiments, R is optionally substituted cyclopropyl. In some embodiments, R is cyclopropyl.

In some embodiments, when R is or comprises a ring structure, e.g., cycloaliphatic, cycloheteroaliphatic, aryl, heteroaryl, etc., the ring structure can be monocyclic, bicyclic or polycyclic. In some embodiments, R is or comprises a monocyclic structure. In some embodiments, R is or comprises a bicyclic structure. In some embodiments, R is or comprises a polycyclic structure.

In some embodiments, R is optionally substituted C_3-30heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted C_1-20heteroaliphatic having 1-10 heteroatoms. In some embodiments, R is optionally substituted C_1-20heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus or silicon, optionally including one or more oxidized forms of nitrogen, sulfur, phosphorus or selenium. In some embodiments, R is optionally substituted C_1-30heteroaliphatic comprising 1-10 groups independently selected from

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In some embodiments, R is optionally substituted C_6-30aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is substituted phenyl.

In some embodiments, R is an optionally substituted 8-10 membered bicyclic saturated, partially unsaturated or aryl ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic saturated ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic partially unsaturated ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic aryl ring. In some embodiments, R is optionally substituted naphthyl.

In some embodiments, R is optionally substituted 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is optionally substituted 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

In some embodiments, R is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is a substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, R is a substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, and oxygen.

In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, R is an optionally substituted 6-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having one heteroatom selected from nitrogen, oxygen, and sulfur. In some embodiments, R is selected from optionally substituted pyrrolyl, furanyl, or thienyl.

In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-membered heteroaryl ring having one nitrogen atom, and an additional heteroatom selected from sulfur or oxygen. Example R groups include but are not limited to optionally substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl.

In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. Example R groups include but are not limited to optionally substituted triazolyl, oxadiazolyl or thiadiazolyl.

In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. Example R groups include but are not limited to optionally substituted tetrazolyl, oxatriazolyl and thiatriazolyl.

In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-4 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-3 nitrogen atoms. In other embodiments. R is an optionally substituted 6-membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having four nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having three nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having two nitrogen atoms. In certain embodiments, R is an optionally substituted 6-membered heteroaryl ring having one nitrogen atom. Example R groups include but are not limited to optionally substituted pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.

In certain embodiments, R is an optionally substituted 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having b heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted indolyl. In some embodiments, R is an optionally substituted azabicyclo[3.2.1]octanyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted azaindolyl. In some embodiments, R is an optionally substituted benzimidazolyl. In some embodiments, R is an optionally substituted benzothiazolyl. In some embodiments, R is an optionally substituted benzoxazolyl. In some embodiments, R is an optionally substituted indazolyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted indolyl. In some embodiments, R is optionally substituted benzofuranyl. In some embodiments, R is optionally substituted benzo[b]thienyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted azaindolyl. In some embodiments, R is optionally substituted benzimidazolyl. In some embodiments, R is optionally substituted benzothiazolyl. In some embodiments, R is optionally substituted benzoxazolyl. In some embodiments, R is an optionally substituted indazolyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted oxazolopyridiyl, thiazolopyridinyl or imidazopyridinyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted purinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, oxazolopyrazinyl, thiazolopyrazinyl, imidazopyrazinyl, oxazolopyridazinyl, thiazolopyridazinyl or imidazopyridazinyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R is optionally substituted 1,4-dihydropyrrolo[3,2-b]pyrrolyl, 4H-furo[3,2-b]pyrrolyl, 4H-thieno[3,2-b]pyrrolyl, furo[3,2-b]furanyl, thieno[3,2-b]furanyl, thieno[3,2-b]thienyl, 1H-pyrrolo[1,2-a]imidazolyl, pyrrolo[2,1-b]oxazolyl or pyrrolo[2,1-b]thiazolyl. In some embodiments, R is optionally substituted dihydropyrroloimidazolyl, 1H-furoimidazolyl, 1H-thienoimidazolyl, furooxazolyl, furoisoxazolyl, 4H-pyrrolooxazolyl, 4H-pyrroloisoxazolyl, thienooxazolyl, thienoisoxazolyl, 4H-pyrrolothiazolyl, furothiazolyl, thienothiazolyl, 1H-imidazoimidazolyl, imidazooxazolyl or imidazo[5,1-b]thiazolyl.

In certain embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted quinolinyl. In some embodiments, R is an optionally substituted isoquinolinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted quinazoline or a quinoxaline.

In some embodiments, R is 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is 3-30 membered heterocyclic ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is 3-30 membered heterocyclic ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

In some embodiments, R is an optionally substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is a substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-7 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 6-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 7-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted 3-membered heterocyclic ring having one heteroatom selected from nitrogen, oxygen or sulfur. In some embodiments, R is optionally substituted 4-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted 5-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted 6-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted 7-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R is an optionally substituted 3-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 7-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than I heteroatom, wherein the heteroatom is nitrogen. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is oxygen. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 oxygen atoms. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 nitrogen atoms. In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is nitrogen. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is oxygen. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 oxygen atoms. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 nitrogen atoms.

In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is nitrogen. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is oxygen. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is sulfur. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having 2 oxygen atoms. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having 2 nitrogen atoms.

In some embodiments, R is an optionally substituted 6-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments. R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is nitrogen. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is oxygen. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 oxygen atoms. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 nitrogen atoms.

In certain embodiments, R is a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is optionally substituted oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl, aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl, thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl, piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl, oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl, tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl, azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl, oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl, dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl, thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl, tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl, oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl, oxathiolanedionyl, piperazinedionyl, morpholinedionyl, thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl.

In certain embodiments, R is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted tetrahydropyridinyl, dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R is an optionally substituted 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted indolinyl. In some embodiments, R is optionally substituted isoindolinyl. In some embodiments, R is optionally substituted 1, 2, 3, 4-tetrahydroquinolinyl. In some embodiments, R is optionally substituted 1, 2, 3, 4-tetrahydroisoquinolinyl. In some embodiments, R is an optionally substituted azabicyclo[3.2.1]octanyl.

In some embodiments, R is an optionally substituted 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted 1,4-dihydropyrrolo[3,2-b]pyrrolyl, 4H-furo[3,2-b]pyrrolyl, 4H-thieno[3,2-b]pyrrolyl, furo[3,2-b]furanyl, thieno[3,2-b]furanyl, thieno[3,2-b]thienyl, 1H-pyrrolo[1,2-a]imidazolyl, pyrrolo[2,1-b]oxazolyl or pyrrolo[2,1-b]thiazolyl. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted dihydropyrroloimidazolyl, 1H-furoimidazolyl. 1H-thienoimidazolyl, furooxazolyl, furoisoxazolyl, 4H-pyrrolooxazolyl, 4H-pyrroloisoxazolyl, thienooxazolyl, thienoisoxazolyl, 4H-pyrrolothiazolyl, furothiazolyl, thienothiazolyl, 1H-imidazoimidazolyl, imidazooxazolyl or imidazo[5,1-b]thiazolyl. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted indolyl. In some embodiments, R is optionally substituted benzofuranyl. In some embodiments, R is optionally substituted benzo[b]thienyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted azaindolyl. In some embodiments, R is optionally substituted benzimidazolyl. In some embodiments, R is optionally substituted benzothiazolyl. In some embodiments, R is optionally substituted benzoxazolyl. In some embodiments, R is an optionally substituted indazolyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted oxazolopyridiyl, thiazolopyridinyl or imidazopyridinyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted purinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, oxazolopyrazinyl, thiazolopyrazinyl, imidazopyrazinyl, oxazolopyridazinyl, thiazolopyridazinyl or imidazopyridazinyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In certain embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having one heteroatom selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted quinolinyl. In some embodiments, R is optionally substituted isoquinolinyl. In some embodiments. R is an optionally substituted 6,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted quinazolinyl, phthalazinyl, quinoxalinyl or naphthyridinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted pyridopyrimidinyl, pyridopyridazinyl, pyridopyrazinyl, or benzotriazinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted pyridotriazinyl, pteridinyl, pyrazinopyrazinyl, pyrazinopyridazinyl, pyridazinopyridazinyl, pyrimidopyridazinyl or pyrimidopyrimidinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R is optionally substituted C_6-30arylaliphatic. In some embodiments, R is optionally substituted C_6-20arylaliphatic. In some embodiments, R is optionally substituted C_6-10arylaliphatic. In some embodiments, an aryl moiety of the arylaliphatic has 6, 10, or 14 aryl carbon atoms. In some embodiments, an aryl moiety of the arylaliphatic has 6 aryl carbon atoms. In some embodiments, an aryl moiety of the arylaliphatic has 10 aryl carbon atoms. In some embodiments, an aryl moiety of the arylaliphatic has 14 aryl carbon atoms. In some embodiments, an aryl moiety is optionally substituted phenyl.

In some embodiments, R is optionally substituted C_6-30arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted C_6-30arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is optionally substituted C_6-20arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted C_6-20arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is optionally substituted C_6-10arylheteroaliphatic having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted C_6-10arylheteroaliphatic having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

In some embodiments, two R groups are optionally and independently taken together to form a covalent bond. In some embodiments, —C═O is formed. In some embodiments, —C═C— is formed. In some embodiments, —C≡C— is formed.

In some embodiments, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-10 membered monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-6 membered monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-3 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-5 membered monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-3 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted. 3-10 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-10 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted. 3-6 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-3 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-5 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-3 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

In some embodiments, heteroatoms in R groups, or in the structures formed by two or more R groups taken together, are selected from oxygen, nitrogen, and sulfur. In some embodiments, a formed ring is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-membered. In some embodiments, a formed ring is saturated. In some embodiments, a formed ring is partially saturated. In some embodiments, a formed ring is aromatic. In some embodiments, a formed ring comprises a saturated, partially saturated, or aromatic ring moiety. In some embodiments, a formed ring comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aromatic ring atoms. In some embodiments, a formed contains no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aromatic ring atoms. In some embodiments, aromatic ring atoms are selected from carbon, nitrogen, oxygen and sulfur.

In some embodiments, a ring formed by two or more R groups (or two or more groups selected from R and variables that can be R) taken together is a C_3-30cycloaliphatic, C₃₀aryl, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, ring as described for R, but bivalent or multivalent.

As appreciated by those skilled in the art, embodiments of R described in the present disclosure can also independently be embodiments for variables that can be R.

In some embodiments, b is 1-100. In some embodiments, b is 1-50. In some embodiments, b is 1-40. In some embodiments, b is 1-30. In some embodiments, b is 1-20. In some embodiments, b is 1-15. In some embodiments, b is 1-10. In some embodiments, b is 1-9. In some embodiments, b is 1-8. In some embodiments, b is 1-7. In some embodiments, b is 1-6. In some embodiments, b is 1-5. In some embodiments, b is 1-4. In some embodiments, b is 1-3. In some embodiments, b is 1-2. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3. In some embodiments, b is 4. In some embodiments, b is 5. In some embodiments, b is 6. In some embodiments, b is 7. In some embodiments, b is 8. In some embodiments, b is 9. In some embodiments, b is 10. In some embodiments, b is 1. In some embodiments, b is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more.

In some embodiments, L^LDis L. In some embodiments, L^LD- is bivalent L^M.

In some embodiments, L^Mis -L^M1-L^M2-L^M3- as described in the present disclosure. In some embodiments, L^Mis L^M1as described in the present disclosure. In some embodiments, L^Mis L^M2as described in the present disclosure. In some embodiments, L^Mis L^M3as described in the present disclosure. In some embodiments, L^Mis L as described in the present disclosure.

In some embodiments, L^M1is L. In some embodiments, L^M2is L. In some embodiments, L^M3is L. In some embodiments, L^M1is a covalent bond. In some embodiments, L^M2is a covalent bond. In some embodiments, L^M3is a covalent bond. In some embodiments, L^M1is L^M2as described in the present disclosure. In some embodiments, L^M1is L^M3as described in the present disclosure. In some embodiments, L^M2is L^M1as described in the present disclosure. In some embodiments, L^M2is L^M3as described in the present disclosure. In some embodiments, L^M3is L^M1as described in the present disclosure. In some embodiments, L^M3is L^M2as described in the present disclosure. In some embodiments, L^Mis L^M1as described in the present disclosure. In some embodiments, L^Mis L^M2as described in the present disclosure. In some embodiments, L^Mis L^M3as described in the present disclosure. In some embodiments, L^Mis L^M1-L^M2, wherein each of L^M1and L^M2is independently as described in the present disclosure. In some embodiments, L^Mis L^M1-L^M3, wherein each of L^M1and L^M3is independently as described in the present disclosure. In some embodiments, L^Mis L^M2-L^M3, wherein each of L^M2and L^M3is independently as described in the present disclosure. In some embodiments, L^Mis L^M1-L^M2-L^M3, wherein each of L^M1, L^M2and L^M3is independently as described in the present disclosure.

In some embodiments, L^M1comprises one or more —N(R′)— and one or more —C(O)—. In some embodiments, a linker or L^M1is or comprises

embedded image

wherein n^Lis 1-8. In some embodiments, a linker or -L^M1-L^M2-L^M3- is

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or a salt form thereof, wherein n^Lis 1-8. In some embodiments, a linker or -L^M1-L^M2-L^M3- is

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or a salt form thereof, wherein:

n^Lis 1-8.

each amino group independently connects to a moiety; and

the P atom connects to the 5′-OH of the oligonucleotide.

In some embodiments, the moiety and the linker, or (R^D)b-L^M1-L^M2-L^M3-, is or comprises

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In some embodiments, the moiety and the linker, or (R^D)b-L^M1-L^M2-L^M3-, is or comprises

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In some embodiments, the moiety and the linker, or (R)b-L^M1-L^M2-L^M3-, is or comprises

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In some embodiments, the moiety and the link R, or (R^D)b-L^M1-L^M2-L^M3- or comprises

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In some embodiments, the moiety and the link (R^D)b-L^M1-L^M2-L^M3- is or comprises

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In some embodiments the moiety and the linker, or (R^D)b-L^M1-L^M2-L^M3-, is or comprises

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In some embodiments, the moiety and the linker, or (R^D)b-L^M1-L^M2-L^M3-, is or comprises

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In some embodiments, the linker, or L^M1, is or comprise

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some embodiments, the moiety and linker, or (R^D)b-L^M1-L^M2-L^M3-, is or comprises:

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In some embodiments, the moiety and linker, or (R^D)b-L^M1-L^M2-L^M3-, is or comprises:

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In some embodiments, n^Lis 1-8. In some embodiments, n^Lis 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n^Lis 1. In some embodiments, n^Lis 2. In some embodiments, n^Lis 3. In some embodiments, n^Lis 4. In some embodiments, n^Lis 5. In some embodiments, n^Lis 6. In some embodiments, n^Lis 7. In some embodiments, n^Lis 8.

In some embodiments, at least one L^Mis directly bound to a sugar unit of a provided oligonucleotide. In some embodiments, a L^Mdirectly binds to a sugar unit incorporates a lipid moiety into an oligonucleotide. In some embodiments, a L^Mdirectly binds to a sugar unit incorporates a carbohydrate moiety into an oligonucleotide. In some embodiments, a L^Mdirectly binds to a sugar unit incorporates a R^LDgroup into an oligonucleotide. In some embodiments, a L^Mdirectly binds to a sugar unit incorporates a R^CDgroup into an oligonucleotide. In some embodiments, L^Mis directed bound through 5′-OH of an oligonucleotide chain. In some embodiments, L^Mis directed bound through 3′-OH of an oligonucleotide chain.

In some embodiments, at least one L^Mis directly bound to an internucleotidic linkage unit of a provided oligonucleotide. In some embodiments, a L^Mdirectly binds to an internucleotidic linkage unit incorporates a lipid moiety into an oligonucleotide. In some embodiments, a L^Mdirectly binds to an internucleotidic linkage unit incorporates a carbohydrate moiety into an oligonucleotide. In some embodiments, a L^Mdirectly binds to an internucleotidic linkage unit incorporates a R^LDgroup into an oligonucleotide. In some embodiments, a L^Mdirectly binds to an internucleotidic linkage unit incorporates a R^CDgroup into an oligonucleotide.

In some embodiments, at least one L^Mis directly bound to a nucleobase unit of a provided oligonucleotide. In some embodiments, a L^Mdirectly binds to a nucleobase unit incorporates a lipid moiety into an oligonucleotide. In some embodiments, a L^Mdirectly binds to a nucleobase unit incorporates a carbohydrate moiety into an oligonucleotide. In some embodiments, a L^Mdirectly binds to a nucleobase unit incorporates a R^LDgroup into an oligonucleotide. In some embodiments, a L^Mdirectly binds to a nucleobase unit incorporates a R group into an oligonucleotide.

In some embodiments, L^Mis bivalent. In some embodiments, L^Mis multivalent. In some embodiments, L^Mis

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wherein L^Mis directly bond to a nucleobase, for example, as in:

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In some embodiments, L^Mis

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In some embodiments, L^Mis

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In some embodiments, L^Mis

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In some embodiments, L^Mis

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In some embodiments, a linker moiety, e.g., L^M, L^M1, L^M2, L^M3, L, L^s, etc., is or comprise

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In some embodiments, a linker moiety, e.g., L^M, L^M1, L^M2, L^M3, L, L^s, etc., is or comprise

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In some embodiments, R^Dis a lipid moiety. In some embodiments, R^D, is targeting moiety. In some embodiments, R^Dis a carbohydrate moiety. In some embodiments, R^Dis a sulfonamide moiety. In some embodiments, R^Dis an antibody or a fragment thereof. In some embodiments, R^Dis R^LDas described in the present disclosure. In some embodiments, R^Dis R^CDas described in the present disclosure. In some embodiments, R^Dis R^TDas described in the present disclosure.

In some embodiments, a lipid moiety has the structure of R^LD. In some embodiments, R^LDis optionally substituted C₁₀, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, or C₂₅to C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, C₃₅, C₄₀, C₄₅, C₅₀, C₆₀, C₇₀, or C₈₀aliphatic. In some embodiments, R^LDis optionally substituted C_10-80aliphatic. In some embodiments, R^LDis optionally substituted C_20-80aliphatic. In some embodiments, R^LDis optionally substituted C_10-70aliphatic. In some embodiments, R^LDis optionally substituted C_20-70aliphatic. In some embodiments, R^LDis optionally substituted C_10-60aliphatic. In some embodiments, R^LDis optionally substituted C_20-60aliphatic. In some embodiments, R^LDis optionally substituted C_10-50aliphatic. In some embodiments, R^LDis optionally substituted C_20-50aliphatic. In some embodiments, R^LDis optionally substituted C_10-40aliphatic. In some embodiments, R^LDis optionally substituted C_20-40aliphatic. In some embodiments, R^LDis optionally substituted C_10-30aliphatic. In some embodiments, R^LDis optionally substituted C_20-30aliphatic. In some embodiments, R^LDis unsubstituted C₁₀, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, or C₂₅to C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, C₃₀, C₃₅, C₄₀, C₄₅, C₅₀, C₆₀, C₇₀, or C₈₀aliphatic. In some embodiments, R^LDis unsubstituted C_10-80aliphatic. In some embodiments, R^LDis unsubstituted C_20-80aliphatic. In some embodiments, R^LDis unsubstituted C_10-70aliphatic. In some embodiments, R^LDis unsubstituted C_20-70aliphatic. In some embodiments, R^LDis unsubstituted C_10-60aliphatic. In some embodiments, R^LDis unsubstituted C_20-60aliphatic. In some embodiments, R^LDis unsubstituted C_10-50aliphatic. In some embodiments, R^LDis unsubstituted C_20-50aliphatic. In some embodiments, R^LDis unsubstituted C_10-40aliphatic. In some embodiments, R^LDis unsubstituted C_20-40aliphatic. In some embodiments, R^LDis unsubstituted C_10-30aliphatic. In some embodiments, R^LDis unsubstituted C_20-30aliphatic.

In some embodiments, R^LDis not hydrogen. In some embodiments, R^LDis a lipid moiety. In some embodiments, R^LDis a targeting moiety. In some embodiments, R^LDis a targeting moiety comprising a carbohydrate moiety. In some embodiments, R^LDis a GalNAc moiety.

In some embodiments, R^TDis R^LD, wherein R^LDis independently as described in the present disclosure. In some embodiments, R^TDis R^CD, wherein R^CDis independently as described in the present disclosure. In some embodiments, R^TDcomprises a sulfonamide moiety. In some embodiments, a R^TDcomprises a carbohydrate moiety. In some embodiments, a R^TDcomprises a GalNAc moiety.

In some embodiments, R^CDis an optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, —C(R′)₂, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′),]O—; and one or more carbon atoms are optionally and independently replaced with Cy^L. In some embodiments, R^CDis an optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, —C(R′), —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—; and one or more carbon atoms are independently replaced with a monosaccharide, disaccharide or polysaccharide moiety. In some embodiments, R^CDis an optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, CC, —C(R′), —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—; and one or more carbon atoms are independently replaced with a GalNac moiety.

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Further embodiments of R^Dincludes additional chemical moiety embodiments, e.g., those described in the examples.

In some embodiments, R^D, R^LDor R^TDis or comprises

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In some embodiments, R^D, R^LDor R^TDis or comprises

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In some embodiments, R^D, R^LDor R^TDis or comprises

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In some embodiments, R^D, R^LDor R^TDis or comprises

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In some embodiments, R^D, R^LD, R^CDor R^TDis or comprises

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In some embodiments, R^D, R^LD, or R^TDis or comprise

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In some embodiments, R^D, R^LD, R^CDor R^TDis or comprises —N(R¹)₂, wherein each R¹is independently as described in the present disclosure. In some embodiments, R^D, R^LD, R^CDor R^TDis or comprises —N(R¹)₃, wherein each R¹is independently as described in the present disclosure. In some embodiments, R^D, R^LD, R^CDor R^TDis or comprises one or more guanidine moieties. In some embodiments, R^D, R^LD, R^CDor R^TDis or comprises —N═C(N(R¹)₂), wherein each R¹is independently as described in the present disclosure. In some embodiments, R^Dor R^TDis or comprises

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In some embodiments, R^D, R^LDor R^Tis or comprise

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In some embodiments, R^Dor R^TDis or comprises

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In some embodiments, R^Dor R^TDis or comprises

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In some embodiments, R^D, R^CD, or R^TDis or comprises

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In some embodiments, R^D, R^LD, or R^TDis or comprise

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In some embodiments, R^D, R^CD, or R^TDis or comprises

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In some embodiments, R^D, R^LD, or R^TDis or comprise

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In some embodiments, R^Dor R^TDis or comprises

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In some embodiments, R^Dor R^TDis or comprise

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In some embodiments, R^Dor R^TDis or comprises

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In some embodiments, R^Dor R^TDis or comprises

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In some embodiments, R^Dor R^TDis or comprises

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In some embodiments R^Dor R^TDis or comprises

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In some embodiments, R^D, R^CD, or R^TDis or comprises

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In some embodiments, R^D, R^CD, or R^TDis or comprises

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In some embodiments, R^D, R^CD, or R^TDis or comprises

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In some embodiments, R^D, R^LD, R^CDor R^TDcomprise

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In some embodiments, R^D, R^LD, R^CDor R^TDcomprise

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In some embodiments, n′ is 1. In some embodiments, n′ is 0.

In some embodiments, n″ is 1. In some embodiments, n″ is 2.

In some embodiments, a moiety of the present disclosure, e.g., a heteroaliphatic, heteroaryl, heterocyclyl, a ring, etc., may contain one or more heteroatoms. In some embodiments, a heteroatom is any atom that is not carbon and is not hydrogen. In some embodiments, each heteroatom is independently selected from boron, nitrogen, oxygen, sulfur, silicon and phosphorus. In some embodiments, each heteroatom is independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus. In some embodiments, each heteroatom is independently selected from boron, nitrogen, oxygen, sulfur and phosphorus. In some embodiments, each heteroatom is independently selected from boron, nitrogen, oxygen, sulfur and silicon. In some embodiments, each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In some embodiments, at least one heteroatom is nitrogen. In some embodiments, at least one heteroatom is oxygen. In some embodiments, at least one heteroatom is sulfur.

In some embodiments, y, t, n and m. e.g., in a stereochemistry pattern, each are independently 1-20 as described in the present disclosure. In some embodiments, y is 1. In some embodiments, y is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, y is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, y is 6. In some embodiments, y is 7. In some embodiments, y is 8. In some embodiments, y is 9. In some embodiments, y is 10.

In some embodiments, m is 0-50. In some embodiments, m is 1-50. In some embodiments, m is 1. In some embodiments, m is 2-50. In some embodiments, m is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, m is 2, 3, 4, 5, 6, 7 or 8. In some embodiments, m is 3, 4, 5, 6, 7 or 8. In some embodiments, m is 4, 5, 6, 7 or 8. In some embodiments, m is 5, 6, 7 or 8. In some embodiments, m is 6, 7 or 8. In some embodiments, m is 7 or 8. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, in is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11. In some embodiments, m is 12. In some embodiments, m is 13. In some embodiments, m is 14. In some embodiments, m is 15. In some embodiments, m is 16. In some embodiments, m is 17. In some embodiments, m is 18. In some embodiments, m is 19. In some embodiments, m is 20. In some embodiments, m is 21. In some embodiments, m is 22. In some embodiments, m is 23. In some embodiments, m is 24. In some embodiments, m is 25. In some embodiments, m is at least 2. In some embodiments, m is at least 3. In some embodiments, m is at least 4. In some embodiments, m is at least 5. In some embodiments, m is at least 6. In some embodiments, m is at least 7. In some embodiments, m is at least 8. In some embodiments, m is at least 9. In some embodiments, m is at least 10. In some embodiments, m is at least 11. In some embodiments, m is at least 12. In some embodiments, m is at least 13. In some embodiments, m is at least 14. In some embodiments, m is at least 15. In some embodiments, in is at least 16. In some embodiments, in is at least 17. In some embodiments, in is at least 18. In some embodiments, m is at least 19. In some embodiments, m is at least 20. In some embodiments, in is at least 21. In some embodiments, m is at least 22. In some embodiments, m is at least 23. In some embodiments, m is at least 24. In some embodiments, m is at least 25. In some embodiments, m is at least greater than 25.

As used in the present disclosure, in some embodiments, “one or more” is 1-200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, “one or more” is one. In some embodiments, “one or more” is two. In some embodiments, “one or more” is three. In some embodiments, “one or more” is four. In some embodiments, “one or more” is five. In some embodiments, “one or more” is six. In some embodiments, “one or more” is seven. In some embodiments, “one or more” is eight. In some embodiments, “one or more” is nine. In some embodiments, “one or more” is ten. In some embodiments, “one or more” is at least one. In some embodiments, “one or more” is at least two. In some embodiments, “one or more” is at least three. In some embodiments, “one or more” is at least four. In some embodiments, “one or more” is at least five. In some embodiments, “one or more” is at least six. In some embodiments, “one or more” is at least seven. In some embodiments, “one or more” is at least eight. In some embodiments, “one or more” is at least nine. In some embodiments, “one or more” is at least ten. As used in the present disclosure, in some embodiments, “at least one” is 1-200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, “at least one” is one. In some embodiments, “at least one” is two. In some embodiments, “at least one” is three. In some embodiments, “at least one” is four. In some embodiments, “at least one” is five. In some embodiments, “at least one” is six. In some embodiments, “at least one” is seven. In some embodiments, “at least one” is eight. In some embodiments, “at least one” is nine. In some embodiments, “at least one” is ten.

In some embodiments, the present disclosure provides the following embodiments:

1. An oligonucleotide composition, comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

wherein:

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages; and

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-negatively charged internucleotidic linkages.

2. The oligonucleotide composition of embodiment 1, wherein the oligonucleotide composition being characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

3. An oligonucleotide composition, comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

wherein:

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages; and

the oligonucleotide composition being characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

4. The composition of any one of the preceding embodiments, wherein each chiral internucleotidic linkage of the oligonucleotides of the plurality is independently a chirally controlled internucleotidic linkage.

5. The composition of any one of the preceding embodiments, wherein each chiral modified internucleotidic linkage independently has a stereopurity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% at its chiral linkage phosphorus.

6. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

wherein:

the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered in that level of inclusion of a nucleic acid sequence is increased relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

7. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least one Sp.

8. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least one Rp.

9. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages; and

3) pattern of backbone phosphorus modifications,

wherein:

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-negatively charged internucleotidic linkages;

the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered in that level of inclusion of a nucleic acid sequence is increased relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

10. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage is independently an internucleotidic linkage at least 50% of which exists in its non-negatively charged form at pH 7.4.

11. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage is independently a neutral internucleotidic linkage, wherein at least 50% of the internucleotidic linkage exists in its neutral form at pH 7.4.

12. The composition of any one of the preceding embodiments, wherein the neutral form of each non-negatively charged internucleotidic linkage independently has a pKa no less than 8, 9, 10, 11, 12, 13, or 14.

13. The composition of any one of the preceding embodiments, wherein the neutral form of each non-negatively charged internucleotidic linkage, when the units which it connects are replaced with —CH₃, independently has a pKa no less than 8, 9, 10, 11, 12, 13, or 14.

14. The composition of any one of the preceding embodiments, wherein the reference condition is absence of the composition.

15. The composition of any one of the preceding embodiments, wherein the reference condition is presence of a reference composition.

16. The composition of any one of the preceding embodiments, wherein the reference composition is an otherwise identical composition wherein the oligonucleotides of the plurality comprise no chirally controlled internucleotidic linkages.

17. The composition of any one of the preceding embodiments, wherein the reference composition is an otherwise identical composition wherein the oligonucleotides of the plurality comprise no non-negatively charged internucleotidic linkages.

18. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises one or more backbone linkages selected from phosphodiester, phosphorothioate and phosphodithioate linkages.

19. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise one or more sugar modifications.

20. The composition of any one of the preceding embodiments, wherein the sugar modifications comprise one or more modifications selected from: 2′-O-methyl, 2′-MOE, 2′-F, morpholino and bicyclic sugar moieties.

21. The composition of any one of the preceding embodiments, wherein one or more sugar modifications are 2′-F modifications.

22. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise a 5′-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety.

23. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise a 3′-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety.

24. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise a middle region between the 5′-end region and the 3′-region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotidic units comprising a phosphodiester linkage.

25. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages; and

3) pattern of backbone phosphorus modifications,

wherein:

oligonucleotides of the plurality comprise:

1) a 5′-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety;

2) a 3′-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety; and

3) a middle region between the 5′-end region and the 3′-region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotidic units comprising a phosphodiester linkage.

26. The composition of embodiment 25, wherein the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered in that level of inclusion of a nucleic acid sequence is increased relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

27. The composition of any one of the preceding embodiments, wherein the 5′-end region comprises 1 or more nucleoside units not comprising a 2′-F modified sugar moiety.

28. The composition of any one of the preceding embodiments, wherein the 3′-end region comprises 1 or more nucleoside units not comprising a 2′-F modified sugar moiety.

29. The composition of any one of the preceding embodiments, wherein the middle region comprises 1 or more nucleotidic units comprising no phosphodiester linkage.

30. The composition of any one of the preceding embodiments, wherein the first of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety and a modified internucleotidic linkage of the 5′-end is the first, second, third, fourth or fifth nucleoside unit of the oligonucleotide from the 5′-end, and the last of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety and a modified internucleotidic linkage of the 3′-end is the last, second last, third last, fourth last, or fifth last nucleoside unit of the oligonucleotide.

31. The composition of any one of the preceding embodiments, wherein the 5′-end region comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2′-F modified sugar moiety.

32. The composition of any one of the preceding embodiments, wherein the 5′-end region comprising 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2′-F modified sugar moiety.

33. The composition of any one of the preceding embodiments, wherein the 3′-end region comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2′-F modified sugar moiety.

34. The composition of any one of the preceding embodiments, wherein the 3′-end region comprising 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2′-F modified sugar moiety.

35. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage between two nucleoside units comprising a 2′-F modified sugar moiety in the 5′-end region is independently a modified internucleotidic linkage.

36. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage between two nucleoside units comprising a 2′-F modified sugar moiety in the 3′-end region is independently a modified internucleotidic linkage.

37. The composition of embodiment 35 or 36, wherein each modified internucleotidic linkage is independently a chiral internucleotidic linkage.

38. The composition of embodiment 35 or 36, wherein each modified internucleotidic linkage is independently a chirally controlled internucleotidic linkage.

39. The composition of embodiment 35 or 36, wherein each modified internucleotidic linkage is a phosphorothioate internucleotidic linkage.

40. The composition of embodiment 35 or 36, wherein each modified internucleotidic linkage is a chirally controlled phosphorothioate internucleotidic linkage.

41. The composition of embodiment 35 or 36, wherein each modified internucleotidic linkage is a Sp chirally controlled phosphorothioate internucleotidic linkage.

42. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural phosphate linkages.

43. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural phosphate linkages each independently between a nucleoside unit comprising a 2′-OR¹modified sugar moiety and a nucleoside unit comprising a 2′-F modified sugar moiety, or between two nucleoside units each independently comprising a 2′-OR¹modified sugar moiety, wherein R¹is optionally substituted C_1-6alkyl.

44. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.

45. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages each independently between a nucleoside unit comprising a 2′-OR¹modified sugar moiety and a nucleoside unit comprising a 2′-F modified sugar moiety, or between two nucleoside units each independently comprising a 2′-OR¹modified sugar moiety, wherein R¹is optionally substituted C_1-6alkyl.

46. The composition of embodiment 43 or 45, wherein 2′OR¹is 2′-OCH₃.

47. The composition of embodiment 43 or 45, wherein 2′OR¹is 2′-OCH_2CH₂OCH₃.

48. The composition of any one of the preceding embodiments, wherein the 5′-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral modified internucleotidic linkages.

49. The composition of any one of the preceding embodiments, wherein the 5′-nd region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive chiral modified internucleotidic linkages.

50. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage in the 5′-end region is a chiral modified internucleotidic linkage.

51. The composition of any one of the preceding embodiments, wherein the 3′-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral modified internucleotidic linkages.

52. The composition of any one of the preceding embodiments, wherein the 3′-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive chiral modified internucleotidic linkages.

53. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage in the 3′-end region is a chiral modified internucleotidic linkage.

54. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral modified internucleotidic linkages.

55. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive chiral modified internucleotidic linkages.

56. The composition of any one of embodiments 48-55, wherein each chiral modified internucleotidic linkage is independently a chirally controlled internucleotidic linkage.

57. The composition of any one of embodiments 48-55, wherein each chiral modified internucleotidic linkage is independently a chirally controlled internucleotidic linkage wherein its chirally controlled linkage phosphorus has a Sp configuration.

58. The composition of any one of embodiments 48-57, wherein each chiral modified internucleotidic linkage is independently a chirally controlled phosphorothioate internucleotidic linkage.

59. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-negatively charged internucleotidic linkages.

60. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 neutral internucleotidic linkages.

61. The composition of any one of the preceding embodiments, wherein a neutral internucleotidic linkage is a chiral internucleotidic linkage.

62. The composition of any one of the preceding embodiments, wherein a neutral internucleotidic linkage is a chirally controlled internucleotidic linkage independently of Rp or Sp at its linkage phosphorus.

63. The composition of any one of the preceding embodiments, wherein the base sequence comprises a sequence having no more than 5 mismatches from a 20 base long portion of the dystrophin gene or its complement.

64. The composition of any one of the preceding embodiments, wherein the length of the base sequence of the oligonucleotides of the plurality is no more than 50 bases.

65. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chirally controlled centers independently of Rp or Sp.

66. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 5 chirally controlled centers independently of Rp or Sp.

67. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 6 chirally controlled centers independently of Rp or Sp.

68. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 10 chirally controlled centers independently of Rp or Sp.

69. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the particular oligonucleotide type are capable of mediating skipping of one or more exons of the dystrophin gene.

70. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 45, 51 or 53 of the dystrophin gene.

71. The composition of embodiment 70, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 45 of the dystrophin gene.

72. The composition of embodiment 70, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 51 of the dystrophin gene.

73. The composition of embodiment 70, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 53 of the dystrophin gene.

74. The composition of any one of preceding embodiments, wherein the composition provides exon skipping of two or more exons.

75. The composition of embodiment 71, wherein the base sequence comprises a sequence having no more than 5 mismatches from a sequence of Table A1.

76. The composition of embodiment 71, wherein the base sequence comprises or is a sequence of Table A1.

77. The composition of embodiment 71, wherein the base sequence is a sequence of Table A1.

78. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality are oligonucleotides of an oligonucleotide selected from Table A1.

79. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.

80. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chirally controlled non-negatively charged internucleotidic linkages.

81. The composition of anyone of the preceding embodiments, wherein the oligonucleotides comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive non-negatively charged internucleotidic linkages.

82. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive chirally controlled non-negatively charged internucleotidic linkages.

83. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure.

84. The composition of any one of the preceding embodiments, wherein a wing comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.

85. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a wing comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chirally controlled non-negatively charged internucleotidic linkages.

86. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a wing comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive non-negatively charged internucleotidic linkages.

87. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a wing comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive chirally controlled non-negatively charged internucleotidic linkages.

88. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise or consist of a wing-core-wing structure, and wherein only one wing comprise one or more non-negatively charged internucleotidic linkages.

89. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.

90. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chirally controlled non-negatively charged internucleotidic linkages.

91. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive non-negatively charged internucleotidic linkages.

92. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive chirally controlled non-negatively charged internucleotidic linkages.

93. The composition of any one of the preceding embodiments, wherein 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of internucleotidic linkages of a wing is independently a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage or a Rp chiral internucleotidic linkage.

94. The composition of any one of the preceding embodiments, wherein 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of internucleotidic linkages of a wing is independently a non-negatively charged internucleotidic linkage or a natural phosphate internucleotidic linkage.

95. The composition of any one of the preceding embodiments, wherein 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of internucleotidic linkages of a wing is independently a non-negatively charged internucleotidic linkage.

96. The composition of any one of embodiments 93-95, wherein the percentage is 50% or more.

97. The composition of any one of embodiments 93-95, wherein the percentage is 60% or more.

98. The composition of any one of embodiments 93-95, wherein the percentage is 75% or more.

99. The composition of any one of embodiments 93-95, wherein the percentage is 80% or more.

100. The composition of any one of embodiments 93-95, wherein the percentage is 90% or more.

101. The composition of any one of the preceding embodiments, wherein the oligonucleotides each comprise a non-negatively charged internucleotidic linkage and a natural phosphate internucleotidic linkage.

102. The composition of any one of the preceding embodiments, wherein the oligonucleotides each comprise a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage and a Rp chiral internucleotidic linkage.

103. The composition of any one of the preceding embodiments, wherein a wing comprises a non-negatively charged internucleotidic linkage and a natural phosphate internucleotidic linkage.

104. The composition of any one of the preceding embodiments, wherein a wing comprises a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage and a Rp chiral internucleotidic linkage.

105. The composition of any one of the preceding embodiments, wherein a core comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.

106. The composition of any one of the preceding embodiments, wherein all non-negatively charged internucleotidic linkages of the same oligonucleotide have the same constitution.

107. The composition of any one of the preceding embodiments, wherein each of the non-negatively charged internucleotidic linkages independently has the structure of formula II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

108. The composition of any one of the preceding embodiments, wherein each of the non-negatively charged internucleotidic linkages independently has the structure of formula II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

109. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage which is a neutral internucleotidic linkage.

110. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the particular type are structurally identical.

111. The composition of any one of the preceding claims, wherein each of the oligonucleotides comprises a chemical moiety conjugated to the oligonucleotide chain of the oligonucleotide optionally through a linker moiety, wherein the chemical moiety comprises a carbohydrate moiety, a peptide moiety, a receptor ligand moiety, or a moiety having the structure of —N(R′)₂, —N(R′)₃, or —N═C(N(R)₂)₂.

112. The composition of any one of the preceding claims, wherein each of the oligonucleotides comprises a chemical moiety conjugated to the oligonucleotide chain of the oligonucleotide optionally through a linker moiety, wherein the chemical moiety comprises a guanidine moiety.

113. The composition of any one of the preceding claims, wherein each of the oligonucleotides comprises a chemical moiety conjugated to the oligonucleotide chain of the oligonucleotide optionally through a linker moiety, wherein the chemical moiety comprises —N═C(N(CH₃)₂)₂.

114. The composition of any one of the preceding embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the oligonucleotides in the composition that have the same constitution as oligonucleotides of the particular oligonucleotide type are oligonucleotides of the particular oligonucleotide type.

115. The composition of any one of the preceding embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the oligonucleotides in the composition that have the base sequence, pattern of backbone linkages, and pattern of backbone phosphorus modifications of the particular oligonucleotide type are oligonucleotides of the particular oligonucleotide type.

116. The composition of any one of the preceding embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the oligonucleotides in the composition that have the base sequence of the particular oligonucleotide type are oligonucleotides of the particular oligonucleotide type.

117. The composition of any one of embodiments 114-116, wherein the percentage is at least 10%.

118. The composition of any one of embodiments 114-116, wherein the percentage is at least 50%.

119. The composition of any one of embodiments 114-116, wherein the percentage is at least 80%.

120. The composition of any one of embodiments 114-116, wherein the percentage is at least 90%.

121. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage is a phosphoramidate linkage.

122. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage comprises a guanidine moiety.

123. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I:

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or a salt form thereof, wherein:

P^Lis P(═W), P, or P→B(R′)₃;

W is O, N(-L-R⁵), S or Se;

each of R¹and R is independently —H, -L-R′, halogen, —CN, —NO₂, -L-Si(R′)₃, —OR′, —SR′, or —N(R′)₂;

each of X, Y and Z is independently —O—, —S—, —N(-L-R⁵)—, or L;

each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L;

each R′ is independently —R, —C(O)R, —C(O)OR, or —S(O)₂R;

two R groups are optionally and independently taken together to form a covalent bond, or

124. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I or a salt form thereof.

125. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-1 or a salt form thereof:

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126. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-1 or a salt form thereof.

127. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-2 or a salt form thereof:

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128. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3 or a salt form thereof:

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129. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-3 or a salt form thereof.

130. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3 or a salt form thereof, wherein one R′ from one —N(R′)₂and one R′ from the other —N(R′)₂are taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

131. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-3 or a salt form thereof, wherein one R′ from one —N(R′)₂and one R′ from the other —N(R′)₂are taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

132. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3 or a salt form thereof, wherein one R′ from one —N(R′)₂and one R′ from the other —N(R′)₂are taken together with their intervening atoms to form an optionally substituted 5-membered monocyclic ring having no more than two nitrogen atoms.

133. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-3 or a salt form thereof, wherein one R′ from one —N(R′)₂and one R′ from the other —N(R′)₂are taken together with their intervening atoms to form an optionally substituted 5-membered monocyclic ring having no more than two nitrogen atoms.

134. The composition of any one of embodiments 128-131, wherein the ring formed is a saturated ring.

135. The composition of any one of embodiments 128-131, wherein the ring formed is a partially unsaturated ring.

136. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II:

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or a salt form thereof, wherein:

P^Lis P(═W), P, or P→B(R′)₃;

W is O, N(-L-R⁵), S or Se;

each of X, Y and Z is independently —O—, —S—, —N(-L-R⁵)—, or L;

R⁵is —H, -L-R′, halogen, —CN, —NO₂, -L-Si(R′)₃, —OR′, —SR′, or —N(R′)₂;

Ring A^Lis an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms;

each R^Ls is independently —H, halogen, —CN, —N₃, —NO, —NO₂, -L-R′, -L-Si(R)₃, -L-OR′, -L-SR′, -L-N(R′)₂, —O-L-R′, —O-L-Si(R)₃, —O-L-OR′, —O-L-SR′, or —O-L-N(R′)₂;

g is 0-20;

each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L.

each Cy^Lis independently an optionally substituted trivalent or tetravalent group selected from a C_3-20cycloaliphatic ring, a C₆-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;

each R′ is independently —R, —C(O)R, —C(O)OR, or —S(O)₂R;

two R groups are optionally and independently taken together to form a covalent bond, or,

137. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II, or a salt form thereof.

138. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-a-1:

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or a salt form thereof.

139. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-a-1, or a salt form thereof.

140. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-a-2:

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or a salt form thereof.

141. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-a-2, or a salt form thereof.

142. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-b-1:

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or a salt form thereof.

143. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-b-1, or a salt form thereof.

144. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-b-2:

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or a salt form thereof.

145. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-b-2, or a salt form thereof.

146. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-c-1:

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or a salt form thereof.

147. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-c-1, or a salt form thereof.

148. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-c-2:

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or a salt form thereof.

149. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-c-2, or a salt form thereof.

150. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-d-1:

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or a salt form thereof.

151. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-d-1, or a salt form thereof.

152. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-d-2:

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or a salt form thereof.

153. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-d-2, or a salt form thereof.

154. The composition of any one of embodiments 136-153, wherein each non-negatively charged internucleotidic linkage has the same structure.

155. The composition of any one of the preceding embodiments, wherein, if applicable, each internucleotidic linkage in the oligonucleotides of the plurality that is not a non-negatively charged internucleotidic linkage independently has the structure of formula I.

156. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage in the oligonucleotides of the plurality independently has the structure of formula I.

157. The composition of any one of the preceding embodiments, wherein one or more P^Lis P(═W).

158. The composition of any one of the preceding embodiments, wherein each P^Lis independently P(═W).

159. The composition of any one of the preceding embodiments, wherein one or more W is O.

160. The composition of any one of the preceding embodiments, wherein each W is O.

161. The composition of any one of the preceding embodiments, wherein one or more Y is O.

162. The composition of any one of the preceding embodiments, wherein each Y is O.

163. The composition of any one of the preceding embodiments, wherein one or more Z is O.

164. The composition of any one of the preceding embodiments, wherein each Z is O.

165. The composition of any one of the preceding embodiments, wherein one or more X is O.

166. The composition of any one of the preceding embodiments, wherein one or more X is S.

167. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of

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168. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of

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169. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of

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170. The composition of any one of the preceding embodiments, wherein for each internucleotidic linkage of formula I or a salt fore thereof that is not a non-negatively charged internucleotidic linkage, X is independently O or S, and -L^s-R⁵is —H (natural phosphate linkage or phosphorothioate linkage, respectively).

171. The composition of any one of the preceding embodiments, wherein each phosphorothioate linkage, if any, in the oligonucleotides of the plurality is independently a chirally controlled internucleotidic linkage.

172. The composition of any one of the preceding embodiments, wherein at least one non-negatively charged internucleotidic linkage is a chirally controlled oligonucleotide composition.

173. The composition of any one of the preceding embodiments, wherein at least one non-negatively charged internucleotidic linkage is a chirally controlled oligonucleotide composition.

174. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality comprise a targeting moiety wherein the targeting moiety is independently connected to an oligonucleotide backbone through a linker.

175. The composition of embodiment 174, wherein the targeting moiety is a carbohydrate moiety.

176. The composition of embodiment 174 or 175, wherein the targeting moiety comprises or is a GalNAc moiety.

177. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality comprise a lipid moiety wherein the lipid moiety is independently connected to an oligonucleotide backbone through a linker.

178. The composition of any one of the preceding embodiments, wherein the oligonucleotide of the plurality comprise a pattern of backbone chiral centers of (Np/Op)t[(Rp)n(Sp)m]y, (Np/Op)t[(Op)n(Sp)m]y, (Np/Op)t[(Op/Rp)n(Sp)m]y, (Sp)t[(Rp)n(Sp)m]y. (Sp)t[(Op)n(Sp)m]y. (Sp)t[(Op/Rp)n(Sp)m]y, [(Rp)n(Sp)m]y, [(Op)n(Sp)m]y, [(Op/Rp)n(Sp)m]y, (Rp)t(Np)n(Rp)m, (Rp)t(Sp)n(Rp)m, (Rp)t[(Np/Op)n]y(Rp)m, (Rp)t[(Sp/Np)n]y(Rp)m, (Rp)t[(Sp/Op)n]y(Rp)m, (Np/Op)t(Np)n(Np/Op)m, (Np/Op)t(Sp)n(Np/Op)m, (Np/Op)t[(Np/Op)n]y(Np/Op)m, (Np/Op)t[(Sp/Op)n]y(Np/Op)m, (Np/Op)t[(Sp/Op)n]y(Np/Op)m, (Rp/Op)t(Np)n(Rp/Op)m. (Rp/Op)t(Sp)n(Rp/Op)m, (Rp/Op)t[(Np/Op)n]y(Rp/Op)m, (Rp/Op)t[(Sp/Op)n]y(Rp/Op)m, or (Rp/Op)t[(Sp/Op)n]y(Rp/Op)m.

179. The composition of any one of the preceding embodiments, wherein the oligonucleotide of the plurality comprise a pattern of backbone chiral centers of (Sp)t[(Rp)n(Sp)m]y.

180. The composition of any one of the preceding embodiments, wherein y is 1.

181. The composition of any one of the preceding embodiments, wherein n is 1.

182. The composition of any one of the preceding embodiments, wherein t is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

183. The composition of any one of the preceding embodiments, wherein t is 4, 5, 6, 7, 8, 9 or 10.

184. The composition of any one of the preceding embodiments, wherein m is 2, 3, 4, 5, 6, 7, 8, 9 or 10.

185. The composition of any one of the preceding embodiments, wherein m is 4, 5, 6, 7, 8, 9 or 10.

186. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality has the structure of formula O-I or a salt thereof.

187. The composition of any one of the preceding embodiments, wherein L in formula O-I independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1. II-d-2, or a salt form thereof.

188. The composition of any one of the preceding embodiments, wherein a

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189. The composition of any one of the preceding embodiments, wherein a

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190. The composition of any one of the preceding embodiments, wherein a

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191. The composition of any one of the preceding embodiments, wherein a

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is optionally substituted.

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192. The composition of any one of the preceding embodiments, wherein L^sin formula O-I between L^Pand Ring A is —C(R^5s)₂—.

193. The composition of any one of the preceding embodiments, wherein L in formula O-I between L^Pand Ring A is —CH(R^5s)₂—.

194. The composition of any one of the preceding embodiments, wherein -L^3E-R^3Ein formula O-I IS —OH.

195. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality has the structure of A^c-[-L^LD-(R^LD)_a]_b, A^c-[-L^M-(R^D)_a]_b, [(A^c)_a-L^M]_b-R^D, (A^c)_a-L^M-(A^c)_b, or (A^c)_a-L^M-(R^D)_b, or a salt thereof.

196. The composition of embodiment 195, wherein H-A^c, [H]_a-A^cor [H]_b-A^cis an oligonucleotide of any one of embodiments 186-194.

197. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein one or more non-neutral internucleotidic linkages at the condition of the composition independently exist as a salt form.

198. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein one or more negatively-charged internucleotidic linkages at the condition of the composition independently exist as a salt form.

199. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein one or more negatively-charged internucleotidic linkages at the condition of the composition independently exist as a metal salt.

200. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein each negatively-charged internucleotidic linkage at the condition of the composition independently exists as a metal salt.

201. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein each negatively-charged internucleotidic linkage at the condition of the composition independently exists as sodium salt.

202. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein each negatively-charged internucleotidic linkage is independently a natural phosphate linkage (the neutral form of which is —O—P(O)(OH)—O) or phosphorothioate internucleotidic linkage (the neutral form of which is —O—P(O)(SH)—O).

203. The composition of any one of the preceding embodiments, wherein each heteroatom in heteroaliphatic, heteroalkyl, heterocyclyl, or heteroaryl is independently boron, nitrogen, oxygen, silicon, sulfur, or phosphorus.

204. The composition of any one of the preceding embodiments, wherein each heteroatom in heteroaliphatic, heteroalkyl, heterocyclyl, or heteroaryl is independently nitrogen, oxygen, silicon, sulfur, or phosphorus.

205. The composition of any one of the preceding embodiments, wherein each heteroatom in heteroaliphatic, heteroalkyl, heterocyclyl, or heteroaryl is independently nitrogen, oxygen, or sulfur.

206. A pharmaceutical composition comprising an oligonucleotide composition of any one of the preceding embodiments and a pharmaceutically acceptable carrier.

207. A method for altering splicing of a target transcript, comprising administering an oligonucleotide composition of any one of the preceding embodiments.

208. The method of embodiment 207, wherein the splicing of the target transcript is altered relative to absence of the composition.

209. The method of any one of the preceding embodiments, wherein the alteration is that one or more exon is skipped at an increased level relative to absence of the composition.

210. The method of any one of the preceding embodiments, wherein the target transcript is pre-mRNA of dystrophin.

211. The method of any one of the preceding embodiments, wherein exon 51 of dystrophin is skipped at an increased level relative to absence of the composition.

212. The method of any one of embodiments 207-210, wherein exon 53 of dystrophin is skipped at an increased level relative to absence of the composition.

213. The method of any one of embodiments 207-210, wherein exon 45 of dystrophin is skipped at an increased level relative to absence of the composition.

214. The method of any one of the preceding embodiments, wherein two or more exons of dystrophin is skipped at an increased level relative to absence of the composition

215. The method of any one of the preceding embodiments, wherein a protein encoded by the mRNA with the exon skipped provides one or more functions better than a protein encoded by the corresponding mRNA, without the exon skipping.

216. A method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising administering to a subject susceptible thereto or suffering therefrom a composition of any one of the preceding embodiments.

217. A method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising (a) administering to a subject susceptible thereto or suffering therefrom a composition of any one of the preceding embodiments, and (b) administering to the subject additional treatment.

218. The method of embodiment 217, wherein the additional treatment is capable of preventing, treating, ameliorating or slowing the progress of muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD).

219. The method of any one of the preceding embodiments, wherein the additional treatment comprises administering a composition of any one of the preceding embodiments, wherein oligonucleotides of the composition have a different base sequence.

220. The method of any one of the preceding embodiments, wherein the additional treatment comprises administering a composition of any one of the preceding embodiments, wherein oligonucleotides of the composition have a different base sequence and target a different exon.

221. The composition of any of the preceding embodiments, wherein the transcript splicing system comprises a myoblast or myotubule.

222. The composition of any of the preceding embodiments, wherein the transcript splicing system comprises a myoblast cell.

223. The composition of any of the preceding embodiments, wherein the transcript splicing system comprises a myoblast cell, which is contacted with the composition after 0, 4 or 7 days of pre-differentiation.

224. A composition comprising a combination comprising: (a) a first composition of any of the preceding embodiments; (b) a second composition of any of the preceding embodiments; and, optionally (c) a third composition of any of the preceding embodiments, wherein the first, second and third compositions are different.

EXEMPLIFICATION

The foregoing has been a description of certain non-limiting embodiments of the disclosure. Accordingly, it is to be understood that embodiments of the disclosure herein described are merely illustrative of applications of principles of the disclosure. Reference herein to details of illustrated embodiments is not intended to limit the scope of any claims.

Various methods for preparing, and for assessing properties and/or activities of, oligonucleotides and oligonucleotide compositions are widely known in the art and may be utilized in accordance with the present disclosure, including but not limited to those described in U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, 9,598,458, US 2015/0211006, US 2017/0037399, WO 2017/015555, WO 2017/192664, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and WO 2019/055951, the methods and reagents of each of which are incorporated herein by reference. In some embodiments, the present disclosure provides technologies for preparing oligonucleotides and compositions thereof, particularly chirally controlled oligonucleotides which comprise neutral backbones (e.g., n001, n002, n003, n004, n005, n006, n007, n008, n009, n010, etc.) and chirally controlled oligonucleotide compositions thereof, and technologies for assessing and using various oligonucleotides and compositions thereof. Among other things, Applicant describes herein example technologies for preparing, assessing and using provided oligonucleotides and oligonucleotide compositions.

Functions and advantage of certain embodiments of the present disclosure may be more fully understood from the examples described below. The following examples are intended to illustrate certain benefits of such embodiments.

Example 1. Example Synthesis of Oligonucleotide Compositions

Technologies for preparing oligonucleotide and compositions thereof are widely known in the art. In some embodiments, oligonucleotides and oligonucleotide compositions of the present disclosure were prepared using technologies, e.g., reagents (e.g., solid supports, coupling reagents, cleavage reagents, phosphoramidites, etc.), chiral auxiliaries, solvents (e.g., for reactions, washing, etc.), cycles, reaction conditions (e.g., time, temperature, etc.), etc., described in one or more of U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, 9,598,458, US 2015/0211006, US 2017/0037399, WO 2017/015555, WO 2017/192664, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and WO 2019/055951.

Example 2. Example Synthesis of Oligonucleotides Comprising an Internucleotidic Linkage Comprising a Triazole Moiety or an Alkyne Moiety

Various types of internucleotidic linkages can be prepared in accordance with the present disclosure. Described in this example is preparation of oligonucleotides comprising internucleotidic linkages comprising triazole moieties. As those skilled in the art appreciates, technology described herein can be readily utilized to conjugate various desirable moieties, e.g., those derived from GalNAc, lipids, peptides, ligands, etc. Among other things, such conjugation can be useful for delivery of oligonucleotides to various target systems (e.g., CNS, muscles, eye, etc.).

Example oligonucleotide comprising internucleotidic linkages comprising triazole moieties.

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Synthesis scheme for dimer preparation in solution phase.

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Synthesis scheme for dimer preparation on solid support.

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Triazole backbone oligonucleotides:

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Synthesis scheme for dimer preparation in solution phase:

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Synthesis scheme for dimer preparation on solid support:

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Alkyne backbone oligonucleotides:

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Synthesis scheme for dimer preparation on solid support:

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Example 3. Example Synthesis of Phosphoramidate Internucleotidic Linkages Comprising a Guanidine Moiety

As illustrated herein, phosphoramidate internucleotidic linkages can be readily prepared from phosphite internucleotidic linkages, including stereopure phosphite internucleotidic linkages, in accordance with the present disclosure.

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To a stirred solution of amidite (474 mg, 0.624 mmol, 1.5 equiv., pre-dried by co-evaporation with dry acetonitrile and under vacuum for a minimum of 12 h) and TBS protected alcohol (150 mg, 0.41 mmol, pre-dried by co-evaporation with dry acetonitrile and under vacuum for a minimum of 12 h) in dry acetonitrile (5.2 ml) was added 5-(ethylthio)-1H-tetrazole (ETT, 2.08 ml, 0.6M, 3 equiv.) under argon atmosphere at room temperature. The reaction mixture was stirred for 5 mins then monitored by LCMS and then a solution of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (356 mg, 1.24 mmol, 3 equiv.) in acetonitrile (1 ml) was added. Once the reaction was completed (after ˜5 mins, monitored by LCMS) then triethylamine (0.17 ml, 1.24 mmol, 3 equiv) was added and the reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure and then redissolved in dichloromethane (50 ml), washed with water (25 ml), saturated aq. sodium bicarbonate (25 ml), and brine (25 ml), and dried with magnesium sulfate. The solvent was removed under reduced pressure. The crude product was purified by silica gel column (80 g) using DCM (5% triethyl amine) and MeOH as eluent. Product-containing fractions were collected and the solvent was evaporated. The resulted product may contain Triethylamine trihydrochloride (TEA.HCl) salt. To remove the salt, the product was re-dissolved in DCM (50 ml) and washed with saturated aq. sodium bicarbonate (20 ml) and brine (20 ml) then dried with magnesium sulfate and the solvent was evaporated. A pale yellow solid was obtained. Yield: 440 mg (89%). ³¹P NMR (162 MHz, CDCl₃) δ −1.34, −1.98. MS calculated for C₅₁H₆₅FN₇O₁₄PSi [M]⁺ 1078.17 Observed: 1078.57 M+H⁺.

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Synthesis of Stereopure (Rp) Dimer.

To a stirred solution of L-DPSE chiral amidite (1.87 g, 2.08 mmol, 1.5 equiv., pre-dried by co-evaporation with dry acetonitrile and under vacuum for a minimum of 12 h) and TBS protected alcohol (500 mg, 1.38 mmol, pre-dried by co-evaporation with dry acetonitrile and under vacuum for a minimum of 12 h) in dry acetonitrile (18 mL) was added 2-(1H-imidazol-1-yl) acetonitrile trifluoromethanesulfonate (CMIMT, 5.54 mL, 0.5M, 2 equiv.) under argon atmosphere at room temperature. The resulting reaction mixture was stirred for 5 mins then monitored by LCMS and then a solution of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (1.18 g, 4.16 mmol, 3 equiv.) in acetonitrile (2 mL) was added. Once the reaction was completed (after ˜5 mins, monitored by LCMS), the reaction mixture was concentrated under reduced pressure and then redissolved in dichloromethane (70 mL), washed with water (40 mL), saturated aq. sodium bicarbonate (40 mL) and brine (40 mL), and dried with magnesium sulfate. The solvent was removed under reduced pressure. The crude product was purified by silica gel column (120 g) using DCM (5% triethyl amine) and MeOH as eluent. Product containing fractions were collected and the solvent was evaporated. The resulted product contained TEA.HCl salt. To remove the salt, the product was re-dissolved in DCM (50 mL) and washed with saturated aq. sodium bicarbonate (20 mL) and brine (20 mL) and then dried with magnesium sulfate and the solvent was evaporated. A pale yellow foamy solid was obtained. Yield: 710 mg (47%). ¹P NMR (162 MHz, CDCl₃) δ −1.38. MS calculated for C₅₁H₆₅FN₇O₁₄PSi [M]⁺1078.17, Observed: 1078.19.

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Synthesis of Stereopure (Sp) Dimer

The same procedure was followed as for the Rp dimer. In place of L-DPSE chiral amidite, D-DPSE chiral amidite was used. A pale yellow foamy solid was obtained. Yield: 890 mg (59%). ³¹P NMR (162 MHz, CDCl₃) δ −1.93. MS calculated for C₅₁H₆₅FN₇O₁₄PSi [M]⁺ 1078.17, Observed: 1078.00.

In an example ³¹P NMR (internal standard of phosphoric acid at δ 0.0), the stereorandom preparation showed two peaks at −1.34 and −1.98, respectively; the stereopure Rp preparation showed a peak at −1.93, and the stereopure Sp preparation showed a peak at −1.38.

Example 4A. Preparation of Oligonucleotides with Internucleotidic Linkages Comprising Neutral Guanidinium Group

In accordance with technologies described in the present disclosure, oligonucleotides with various neutral and/or cationic internucleotidic linkages (e.g., at physiological pH) can be prepared. Illustrated below are preparation of oligonucleotides comprising representative such internucleotidic linkages.

WV-1237 is an oligonucleotide comprising four internucleotidic linkages having the structure of

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(n00) to introduce a neutral nature to the backbone and reduce the overall negative charges of the backbone. Expected molecular weight: 7113.4.

As an example, one preparation of WV-11237, including certain synthetic conditions and analytical results, is described below. Briefly, stereopure internucleotidic linkages were constructed using L-DPSE amidites and typical DPSE coupling cycles comprising Detritylation->Coupling->Pre-Cap->Thiolation->Post-Cap. Cycles for the n001 internucleotidic linkages were modified and comprised Detritylation->Coupling->Dimethyl imidazolium treatment->Post-cap. Compared to certain oxidation cycles, oxidation steps of oxidizing the P(III), e.g., with I₂-Pyridine (pyr)-water, was replaced with the dimethyl imidazolium treatment.

Certain conditions and/or results of an example preparation.

Synthetic scale: 127 μmol

Synthetic conditions (stereopure internucleotidic linkages)

Synthetic Steps
Conditions

Detritylation
3% DCA in Toluene; 300 cm/hr, 436 UV watch

Coupling
2.5 eq. of 0.2M chiral amidite, 67% of 0.6M CMIMT

Recycle time: 10 min

Pre-Cap B
Reagent: 20:30:50::Acetic anhydride:Lutidine:Acetonitrile

1.5 CV, 3 min CT

Thiolation
Reagent: 0.2M Xanthane Hydride

0.6 CV, 6 mm CT

Capping (1:1 Cap A + Cap B)
0.4 CV, 0.8 min CT

Cap A=N-Methylimidazole in acetonitrile, 20/80, v/v (20%:80%=NMI:ACN (v/v))

Cap B=Acetic anhydride/2,6-Lutidine/Acetonitrile, 20/30/50, v/v/v, 20%:30%:50%=Ac₂O:2,6-Lutidine:ACN (v/v/v)

Synthetic conditions (stereorandom n001)

Synthetic Steps
Conditions

Detritylation
3% DCA in Toluene; 300 cm/hr, 436 UV watch

Coupling
2.5 eq. of 0.2M standard amidite, 67% of 0.6M ETT

Recycle time: 8 min

Dimethyl imidazolium treatment:
2.30 CV, 5 mm CT, 3.5 eq.

Capping (1:1 Cap A + Cap B)
0.4 CV, 0.8 min CT

Synthesis Process Parameters:
Synthesizer: AKTA Oligopilot 100

Solid Support: CPG 2′Fluoro-U, (85 umol/g)

Synthetic scale: 127 umol; 1.5 gm

Column diameter: 20 mm

Column volume: 6.3 mL

Stereopure Coupling Reagents:

Monomer: 0.2M in MeCN (2′Fluoro-dA-L-DPSE, 2′Fluoro-dG-L-DPSE, 2′-OMe-A-L-DPSE); 0.2M in 20% isobutyronitrle/MeCN (2′Fluoro-dC-L-DPSE, 2′Fluoro-U-L-DPSE)

Deblocking: 3% Dichloroacetic acid (DCA) in Toluene

Activator: 0.6M CMIMT in MeCN

Sulfurization: 0.2M Xanthane Hydride in pyridine

Cap A: N-Methylimidazole in acetonitrile, 20/80, v/v (20% NMI in MeCN)

Cap B: Acetic anhydride/2,6-Lutidine/Acetonitrile, 20/30/50, v/v/v, (Acetic anhydride. Lutidine, MeCN (20:30:50))

Pre-Cap: Neat Cap B
Stereorandom Coupling Reagents:
Monomer: 0.2M in MeCN (2′OMeA and 2′OMeG)
Deblocking: 3% DCA in Toluene
Activator: 0.6M ETT in MeCN

2-Azido-1,3-dimethylimidazolinium-hexafluorophosphate: 0.1M in MeCN

Cap A: 20% NMI in MeCN

Cap B: Acetic anhydride, Lutidine, MeCN

Deprotection Condition:

One pot deprotection by first treating the support with 5M Triethylamine trihydrofluoride (TEA.HF) in Dimethylsulfoxid (DMSO), H₂O, Triethylamine (pH 6.8). Incubation: 3 h, room temperature, 80 μL/μmol. Followed by addition of aqueous ammonia (200 μL/μmol). Incubation: 24 h, 35° C. The deprotected material was sterile filtered using 0.45 μm filters.

Yield: 72 O.D./μmol
Recipe for 5× Solution of TEA.HF in DMSO/Water, 5/1, v/v:

Solvents/
Volume
Total Volume

Reagent
Reagents
(mL)
(mL)

(5X) TEA.HF in
DMSO
55.0
100

DMSO/Water,
Water
11.0

5/1, v/v
Triethylamine (TEA)
9.0

Triethylamine
25.0

trihydrofluoride

(TEA.3HF)

In an example crude UPLC chromatogram, there were four distinct peaks all having same desired molecular weight of 7113.2:

RT
Area
% Area
Height

9
7.843
402732
16.75
212901

10
7.884
941388
39.14
327190

11
7.968
595232
24.75
275741

12
8.025
353090
14.68
150141

The example final QC UPLC chromatogram showed four distinct peaks all having the desired molecular weight of 7113.2 (% Purity 95.32). Crude LC-MS showed a single peak of desired molecular weight of 113.2 (data not shown). The example final QC LC-MS showed a major peak with the desired molecular weight of 7113.1.

Other oligonucleotides may be prepared using similar cycle conditions or variants thereof depending on specific chemistries of each oligonucleotides. MS data of certain oligonucleotides are listed below:

ID
Average
Observed

WV-11237
7113.40288
7113.1

WV-11340
6967.19736
6967.4

WV-11341
6876.08178
6875.6

WV-11342
6888.1173
6887.7

WV-11343
7072.39402
7072.4

WV-11344
6981.27844
6981.6

WV-11345
6981.27844
6981.6

WV-11346
6981.27844
6981.6

WV-11347
6981.27844
6981.6

WV-11532
6905.78632
6905

WV-11533
7098.86298
7099

WV-12116
7909.88196
7909.4

WV-12117
7909.88196
7909.8

WV-12118
7909.88196
7910.2

WV-12119
7909.88196
7909.4

WV-12120
7909.88196
7909.8

WV-12121
7909.88196
7909.8

WV-12123
7125.35748
7125

WV-12124
6967.19736
6967

WV-12125
6967.19736
6967

WV-12126
6967.19736
6967

WV-12127
7046.27742
7046

WV-12128
7046.27742
7046

WV-12129
7046.27742
7046

WV-12504
8887.86402
8887.5

WV-12505
7278.017
7278.2

WV-12506
8944.9584
8945.2

WV-12507
7335.11138
7334.4

WV-12508
7155.95736
7156.3

WV-12539
7171.78104
7171

WV-12540
7171.78104
7171

WV-12541
7457.21802
7457

WV-12542
7219.97784
7219

WV-12543
7235.97724
7236

WV-12544
7112.86454
7113

WV-12553
6872.0517
6872

WV-12555
6876.08178
6875.8

WV-12556
6888.1173
6887.8

WV-12558
6876.08178
6875.6

WV-12559
6888.1173
6887.7

WV-12876
7204.43754
7204.4

WV-12877
7113.32196
7113.5

WV-12878
7125.35748
7125.4

WV-12879
6919.00056
6919.1

WV-12880
6923.03064
6923.2

WV-12881
6935.06616
6935.3

WV-12882
7094.4195
7094.1

WV-12883
7410.73974
7411.1

Example 4B. Chirally Controlled Non-Negatively Charged Internucleotidic Linkages

Dimer Synthesis.

This procedure is to make stereopure dimer phosphate backbone followed by incorporating it to the selective sites of oligonucleotides (e.g., antisense oligonucleotide or ASO, single-stranded RNAi agent or ssRNA, etc.). A second approach is to synthesize molecules using an automated oligonucleotide synthesizer to introduce anon-negatively charged internucleotidic linkage. e.g., a neutral internucleotidic linkage, at a specific site or full oligonucleotide.

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General experimental procedure (A): To a stirred solution of stereorandom amidite (474 mg, 0.624 mmol, 1.5 equiv., pre-dried by co-evaporation with dry acetonitrile and kept it under vacuum for minimum 12 h) and TBS protected alcohol (150 mg, 0.41 mmol, pre-dried by co-evaporation with dry acetonitrile and kept it under vacuum for minimum 12 h) in dry acetonitrile (5.2 mL) was added 5-(Ethylthio)-1H-tetrazole (ETT, 2.08 ml, 0.6M, 3 equiv.) under argon atmosphere at room temperature. Resulting reaction mixture was stirred for 5 mins then monitored by LCMS and then a solution of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (356 mg, 1.24 mmol, 3 equiv.) in acetonitrile (1 mL) was added. Once the reaction was completed (after ˜5 mins, monitored by LCMS) then triethylamine (0.17 mL, 1.24 mmol, 3 equiv.) was added and monitored LCMS. Reaction mixture was concentrated under reduced pressure and then re-dissolved in dichloromethane (50 mL) washed with water (25 mL), saturated aq. Sodium bicarbonate (25 mL) and brine (25 mL) dried with magnesium sulfate. Solvent was removed under reduced pressure. The crude product was purified by silica gel column (80 g) using DCM (2% triethylamine) and MeOH as eluent. Product containing fractions collected and evaporated. Pale yellow solid 1001 obtained. Yield: 440 mg (89%). ³¹P NMR (162 MHz, CDCl₃) δ −1.34, −1.98. MS (ES) m/z calculated for C₅₁H₆₅FN₇O₁₄PSi [M]⁺ 1077.40. Observed: 1078.57 [M+H]⁺.

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General experimental procedure (B) for stereopure (Rp) dimer: To a stirred solution of L (or) D-DPSE chiral amidite (1.87 g, 2.08 mmol, 1.5 equiv., pre-dried by co-evaporation with dry acetonitrile and kept it under vacuum for minimum 12 h) and TBS protected alcohol (500 mg, 1.38 mmol, pre-dried by co-evaporation with dry acetonitrile and kept it under vacuum for minimum 12 h) in dry acetonitrile (18 mL) was added 2-(H-imidazol-1-yl) acetonitrile trifluoromethanesulfonate (CMIMT, 5.54 mL, 0.5M, 2 equiv.) under argon atmosphere at room temperature. Resulting reaction mixture was stirred for 5 mins then monitored by LCMS and then a solution of 2-azido-, 3-dimethylimidazolinium hexafluorophosphate (1.18 g, 4.16 mmol, 3 equiv.) in acetonitrile (2 mL) was added. Once the reaction was completed (after ˜5 mins, monitored by LCMS) then the reaction mixture was concentrated under reduced pressure and then redissolved in dichloromethane (70 mL) washed with water (40 mL), saturated aq. sodium bicarbonate (40 mL) and brine (40 mL) dried with magnesium sulfate. Solvent was removed under reduced pressure. The crude product was purified by silica gel column (120 g) using DCM (2% triethyl amine) and MeOH as eluent. Product containing fractions are evaporated. Pale yellow foamy solid 1002 was obtained. Yield: 710 mg (47%). ³¹P NMR (162 MHz, CDCl₃) δ −1.38. MS (ES) m/z calculated for C₅₁H₆₅FN₇O₁₄PSi[M]⁺ 1077.40, Observed: 1078.19 [M+H]⁺.

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Stereopure (Sp) dimer 1003: The procedure B was followed as shown above. D-DPSE chiral amidite was used. Pale yellow foamy solid was obtained. Yield: 890 mg (59%). ³¹P NMR (162 MHz, CDCl₃) δ −1.93. MS (ES) m/z calculated for C₅₁H₆₅FN₇O₁₄PSi [M]⁺ 1077.40. Observed: 1078.00 [M+H]⁺.

General experimental procedure (C) for deprotection of TBS group: To a stirred solution of TBS protected compound (9.04 mmol) in trihydrofluoride (THF) (70 mL), was added TBAF (1.0 M, 13.6 mmol) at rt. The reaction mixture was stirred at room temperature for 2-4 h. LCMS showed there was no starting material left, then concentrated followed by purification using ISCO-combiflash system (330 g gold rediSep high performance silica column pre-equilibrated 3 CV with 2% TEA in DCM) and DCM/Methanol/2% TEA as a gradient eluent. Product containing column fractions were pooled together and evaporated followed by drying under high vacuum afforded the pure product.

General experimental procedure (D) for chiral amidites: The TBS deprotected compound (2.5 mmol) was dried by co-evaporation with 80 mL of anhydrous toluene (30 mL×2) at 35° C. and dried under at high vacuum for overnight. Then dried it was dissolved in dry THF (30 mL), followed by the addition of triethylamine (17.3 mmol) then the reaction mixture was cooled to −65° C. [for Guanine flavors: TMS-Cl, 2.5 mmol was added at −65° C., for non-Guanine flavors no TMS-Cl was added]. The THF solution of [(1R,3S,3aS)-1-chloro-3-((methyldiphenylsilyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole (or) (1S,3R,3aR)-1-chloro-3-((methyldiphenylsilyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole (1.8 equiv.) was added through syringe to the above reaction mixture over 2 min then gradually warmed to room temperature. After 20-30 min, at rt, TLC as well as LCMS indicated starting material was converted to product (reaction time: 1 h). Then the reaction mixture was filtered under argon using air free filter tube, washed with THF and dried under rotary evaporation at 26° C. afforded crude solid material, which was purified by ISCO-combiflash system (40 g gold rediSep high performance silica column (pre-equilibrated 3 CV with CH₃CN/5% TEA then 3 CV with DCM/5% TEA) using DCM/CH₃CN/5% TEA as a solvent (compound eluted at 10-40 DCM/CH₃CN/5% TEA). After evaporation of column fractions pooled together was dried under high vacuum afforded white solid to give isolated yield.

³¹P NMR (internal standard of Phosphoric acid at δ 0.0): 1001: −1.34 and −1.98. 1002: −1.93. 1003: −1.38. ¹H NMR of 1001, 1002, and 1003 demonstrated different chemical shifts for multiple hydrogens of the diastereomers. LCMS showed different retention times for the two diastereomers as well. Under one condition, the following retention times were observed: 1.90 and 2.15 for 1001, 1.92 for one diastereomer, and 2.17 for the other.

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Compound 1004: Procedures B and C followed, Off-white foamy solid, Yield: (36%). ³¹P NMR (162 MHz, CDCl₃) δ −1.23. MS (ES) m/z calculated for C₄₇H₅₄FN₈O₁₄P [M]⁺ 1004.34. Observed: 1043.21 [M+K]⁺.

Compound 1005: Procedure D used, Off-white foamy solid, Yield: (81%). ³¹P NMR (162 MHz, CDCl₃) δ154.43, −2.52. MS (ES) m/z calculated for C₆₆H₇₆FN₉O₁₅P₂Si [M]⁺ 1343.46, Observed: 1344.85 [M+H]⁺.

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Compound 1006: Procedures B and C followed, Off-white foamy solid, Yield: (47%). ³¹P NMR (162 MHz, CDCl₃) δ−2.54. MS (ES) m/z calculated for C₄₇H₅₄FN₈O₁₄P [M]⁺ 1004.34, Observed: 1043.12 [M+K]⁺.

Compound 1007: Procedures D used, Off-white foamy solid, yield (81%). ³¹P NMR (162 MHz, CDCl₃)_δ153.55, −2.20. MS(ES) m/z calculated for C₆₆H₇₆FN₉O₁₅P₂Si [m]⁺ 1343.46, Observed: 1344.75 [M+H]⁺.

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Compound 1008: Procedures B and C followed, Off-white foamy solid, Yield: (36%). ³¹NMR (162 MHz, CDCl₃) δ−1.38. MS (ES) m/z calculated for C₅₈H₆₃FN₁₃O₁₃P [M]⁺ 1199.43, Observed: 1200.76 [M+H]⁺.

Compound 1009: Procedure D used, Off-white foamy solid, Yield: (60%). ³¹P NMR (162 MHz, CDCl₃) δ157.26, −2.86. MS (ES) m/z calculated for C₇₇H₈₅FN₁₄O₁₄P₂Si [M]⁺ 1538.55, Observed: 1539.93 [M+H]⁺.

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Compound 1010: Procedures B and C followed, Off-white foamy solid, Yield: (36%). ³¹P NMR (162 MHz, CDCl₃) δ −2.82. MS (ES) m/z calculated for C₅₈H₆₃FN₁₃O₁₃P [M]⁺ 1199.43, Observed: 1200.19 [M+H]⁺.

Compound 1011: Procedure D used, Off-white foamy solid, Yield: (63%). ³¹P NMR (162 MHz, CDCl₃) δ 159.56, −2.99. MS (ES) m/z calculated for C₇₇H₈₅FN₁₄O₁₄P₂Si [M]⁺ 1538.55. Observed: 1539.83 [M+H]⁺.

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Compound 1012: Procedures B and C followed, Off-white foamy solid, Yield: (36%). [α]_D²³=−25.74 (c 1.06, CHCl₃). ³¹P NMR (162 MHz, Chloroform-d) δ −1.83. ¹H NMR (400 MHz, Chloroform-d) δ 12.14 (s, 1H), 11.28 (s, 1H), 9.15 (s, 1H), 8.56 (s, 1H), 8.25-7.94 (m, 2H), 7.90 (s, 1H), 7.72-7.48 (m, 2H), 7.44 (dd, J=8.2, 6.7 Hz, 2H), 7.35-7.26 (m, 2H), 7.24-7.02 (m, 8H), 6.81-6.56 (m, 4H), 6.04 (d, J=5.2 Hz, 1H), 5.67 (d, J=5.5 Hz, 1H), 4.83 (dt, J=8.6, 4.4 Hz, 1H), 4.71-4.54 (m, 2H), 4.49 (dt, J=14.2, 4.8 Hz, 2H), 4.35 (ddt, J=11.0, 5.1, 3.2 Hz, 1H), 4.28-4.09 (m, 2H), 3.68 (s, 6H), 3.37 (d, J=3.3 Hz, 7H), 3.33-3.17 (m, 5H), 2.82 (s, 5H), 2.74-2.60 (m, 1H), 1.92 (s, 2H), 1.72-1.50 (m, 1H), 1.08 (d, J=6.9 Hz, 3H), 0.94 (d, J=6.9 Hz, 3H). MS (ES) m/z calculated for C₅₉H₆₆N₁₃O₁₄P 1211.45 [M]⁺, Observed: 1212.42 [M+H]⁺.

Compound 1013: Procedure D used, Off-white foamy solid, Yield: (78%). [α]_D²³=−15.48 (c 0.96, CHCl₃). ³¹P NMR (162 MHz, Chloroform-d) δ 159.42, −2.47. MS (ES) m/z calculated for C₇₈H₈₈N₁₄O₁₅P₂Si 1550.57 [M]⁺, Observed: 1551.96 [M+H]⁺.

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Compound 1014: Procedures Band C followed, Off-white foamy solid, Yield: (30%). [α]_D²³=−21.45 (c 0.55, CHCl₃). MS(ES) m/z calculated for C₅₉H₆₆N₁₃O₁₄P 1211.45 [M]⁺, Observed: 1212.80[M+H]⁺.

Compound 1015: Procedure D used, Off-white foamy solid, Yield: (68%). [α]_D²³=−15.63 (c 1.44, CHCl₃). MS (ES) m/z Calculated for C₇₈H₈₈N₁₄O₁₅P₂Si 1550.571[M]⁺,Observed: 1551.77 [M+H]⁺.

Compound 1016: Procedure D used, Off-white foamy solid, Yield: (64%). ³¹P NMR (162 MHz, CDCl₃)_δ156.64, −2.67. MS (ES)m/z Calculated for C₇₈H₈₈N₁₄O₁₅P₂Si 1550.57[M]⁺, Observed: 1551.77 [M+H]⁺.

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General experimental procedure (E) for stereopure dimer using sulfonyl amidite: To a stirred solution of steropure sulfonyl amidite 1017 (259 mg, 0.275 mmol, 1.5 equiv) and TBS protected alcohol (100 mg, 0.18 mmol) in dry acetonitrile (2 mL) was added 2-(1H-imidazol-1-yl) acetonitrile trifluoromethanesulfonate (CMIMT, 0.73 mL, 0.36 mmol, 0.5M, 2 equiv.) under argon atmosphere at room temperature. Resulting reaction mixture was stirred for 5 mins and monitored by LCMS then a mixture of acetic anhydride (2M in ACN, 0.18 ml, 0.36 mmol, 2 equ) and lutidine (2M in ACN, 0.18 ml, 0.36 mmol, 2 equ) was added then stirred for ˜5 mins then a solution of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (104.7 mg, 0.367 mmol, 2 equiv.) in acetonitrile (1 mL) was added. Once the reaction was completed (after ˜5 mins, monitored by LCMS) then triethylamine (0.13 mL, 0.91 mmol, 5 equiv.) was added and monitored by LCMS. Once the reaction was completed, it was concentrated under reduced pressure and then re-dissolved in dichloromethane (50 mL) washed with water (25 mL), saturated aq. Sodium bicarbonate (25 mL) and brine (25 mL) dried with magnesium sulfate. Solvent was removed under reduced pressure. The crude product was purified by silica gel column (80 g) using DCM (2% triethylamine) and MeOH as eluent. Product containing fractions collected and evaporated. Off white solid 1018 obtained. Yield: 204 mg (82%). ³¹P NMR (162 MHz, CDCl₃) δ −1.87. MS (ES) m/z calculated for C₇₄H₇₅FN₁₀P [M]⁺ 1359.44. Observed: 1360.39 [M+H]⁺.

Additional phosphoramidites that may be utilized for synthesis include:

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Additional useful chiral auxiliaries include:

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Other phosphoramidites and chiral auxiliaries, such as those described in U.S. Pat. Nos. 9,695,211, 9,605,019, U.S. Pat. No. 9,598,458, US 2013/0178612, US 20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/098264, WO 2018/223056, and/or WO 2018/237194, the chiral auxiliaries and phosphoramidites of each of which is incorporated by reference.

Example 4C. Synthesis of N²,N⁶-bis(4-sulfamoylbenzoyl)-L-lysine

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Step 1. To a solution of 4-sulfamoylbenzoic acid (10.00 g, 49.70 mmol) and HOSu (6.29 g, 54.67 mmol) in DMF (300 mL) was added DCC (10.25 g, 49.70 mmol) at 0° C. The mixture was stirred at 0° C. for 16 hours. LCMS showed compound was consumed. The resulting mixture was combined and workup with another batch of crude (1 g scale). The white suspension of N,N′-dicyclohexylurea (DCU) was filtered and removed white solid. The filtrate was concentrated to give an oil. This crude product was washed with hot 2-propanol (50 mL*3) to afford an off-white solid. Compound (2,5-dioxopyrrolidin-1-yl) 4-sulfamoylbenzoate (11.80 g, 38.66 mmol, 77.78% yield, 97.713% purity) (yield from conversion rate for 10 g batch) was obtained as a white solid. Compound (2,5-dioxopyrrolidin-1-yl) 4-sulfamoylbenzoate (13 g) was totally obtained as a white solid for two batches of reactions. ¹H NMR (400 MHz, CHLOROFORM-d) δ=8.30 (d, J=8.4 Hz, 2H), 8.08 (d, J=8.3 Hz, 2H), 7.70 (s, 2H), 2.96-2.87 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆) δ=170.62, 161.47, 150.32, 131.40, 127.65, 127.18, 26.04; HPLC purity: 97.71%.

Step 2. To a solution of (2,5-dioxopyrrolidin-1-yl) 4-sulfamoylbenzoate (5.00 g, 16.76 mmol) and (2S)-2,6-diaminohexanoic acid (1.23 g, 8.38 mmol) in H₂O (50 mL) and DMF (50.00 mL) was added NaHCO₃(2.11 g, 25.14 mmol). The mixture was stirred at 15° C. for 16 hours. LCMS showed MS with desired compound was detected. The mixture concentrated under reduced pressure to give a crude (6 g). The crude (3.5 g) was purified by prep-HPLC(column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water(0.1% TFA)-ACN]; B %: 1%-30%, 20 min). N²,N⁶-bis(4-sulfamoylbenzoyl)-L-lysine (1.40 g, 30.40% yield, 93.268% purity) was obtained as a white solid and 2.5 g crude as a yellow solid. ¹H NMR (400 MHz, DMSO-d) δ=12.64 (br s, 1H), 8.80 (br d, J=7.5 Hz, 1H), 8.65 (br t, J=5.3 Hz, 1H), 8.04 (d, J=8.2 Hz, 2H), 7.99-7.95 (m, 2H), 7.95-7.84 (m, 4H), 7.48 (br d, J=11.6 Hz, 4H), 4.44-4.32 (m, 1H), 3.28 (br d, J=6.1 Hz, 2H), 1.94-1.71 (m, 3H), 1.63-1.36 (m, 4H); ¹³C NMR (101 MHz, DMSO-d) δ=174.04, 166.08, 165.58, 146.89, 146.57, 138.05, 137.36, 128.60, 128.26, 126.05, 53.21, 30.77, 29.11, 23.84. LCMS (M−H⁺); 511.0 (M+H)⁺ HPLC purity: 93.268%.

Example 4D. Example Technologies for Chirally Controlled Oligonucleotide Preparation—Example Useful Chiral Auxiliaries

Among other things, the present disclosure provides technologies (e.g., chiral auxiliaries, phosphoramidites, cycles, conditions, reagents, etc.) that are useful for preparing chirally controlled internucleotidic linkages. In some embodiments, provided technologies are particularly useful for preparing certain internucleotidic linkages, e.g., non-negatively charged internucleotidic linkages, neutral internucleotidic linkages, etc., comprising P-N═ wherein P is the linkage. In some embodiments, the linkage phosphorus is trivalent. In some embodiments, the linkage phosphorus is pentavalent. In some embodiments, such internucleotidic linkages have the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, I-b-1, II-b-2, I-c-1, II-c-2, I-d-1, II-d-2, or a salt form thereof. Certain example technologies (chiral auxiliaries and their preparations, phosphoramidites and their preparations, cycles, conditions, reagents, etc.) are described in the Examples herein. Among other things, such chiral auxiliaries provide milder reaction conditions, higher functional group compatibility, alternative deprotection and/or cleavage conditions, higher crude and/or purified yields, higher crude purity, higher product purity, and/or higher (or substantially the same or comparable) stereoselectivity when compared to a reference chiral auxiliary (e.g., of formula 0, P, Q, R or DPSE).

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Two batches in parallel: To a solution of methylsulfonylbenzene (102.93 g, 658.96 mmol, 1.5 eq.) in THF (600 mL) was added KHMDS (1 M, 658.96 mL, 1.5 eq.) dropwise at −70° C., and warmed to −30° C. slowly over 30 min. The mixture was then cooled to −70° C. A solution of compound 1 (150 g, 439.31 mmol, 1 eq.) in THF (400 mL) was added dropwise at −70° C. The mixture was stirred at −70° C. for 3 hr. TLC (Petroleum ether:Ethyl acetate=3:1, Rf=0.1) indicated compound 1 was consumed completely and one major new spot with larger polarity was detected. Combined 2 batches. The reaction mixture was quenched by added to the sat. NH₄Cl (aq. 1000 mL), and then extracted with EtOAc (1000 mL×3). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give 1000 mL solution. Then added the MeOH (600 mL), concentrated under reduced pressure to give 1000 mL solution, then filtered the residue and washed with MeOH (150 mL); the residue was dissolved with THF (1000 mL) and MeOH (600 mL), then concentrated under reduced pressure to give 1000 mL solution. Then filtered to give a residue and washed with MeOH (150 mL). And repeat one more time. Compound 2 (248 g, crude) was obtained as a white solid. And the combined mother solution was concentrated under reduced pressure to give compound 3 (200 g, crude) as yellow oil.

Compound 2: ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.80 (d, J=7.5 Hz, 2H), 7.74-7.66 (m, 1H), 7.61-7.53 (m, 2H), 7.47 (d, J=7.5 Hz, 6H), 7.24-7.12 (m, 9H), 4.50-4.33 (m, 1H), 3.33 (s, 1H), 3.26 (ddd, J=2.9, 5.2, 8.2 Hz, 1H), 3.23-3.10 (m, 2H), 3.05-2.91 (m, 2H), 1.59-1.48 (m, 1H), 1.38-1.23 (m, 1H), 1.19-1.01 (m, 1H), 0.31-0.12 (m, 1H).

Preparation of Compound WV-CA-108

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To a solution of compound 2 (248 g, 498.35 mmol, 1 eq.) in THF (1 L) was added HCl (5M, 996.69 mL, 10 eq.). The mixture was stirred at 15° C. for 1 hr. TLC (Petroleum ether:Ethyl acetate=3:1, Rf=0.03) indicated compound 2 was consumed completely and one major new spot with larger polarity was detected. The resulting mixture was washed with MTBE (500 mL×3). The combined organic layers were back-extracted with water (100 mL). The combined aqueous layer was adjusted to pH 12 with 5M NaOH aq. and extracted with DCM (500 mL×3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated to afford a white solid. WV-CA-108 (122.6 g, crude) was obtained as a white solid.

¹H NMR (400 MHz, CHLOROFORM-d) δ=7.95 (d, J=7.5 Hz, 2H), 7.66 (t, J=7.5 Hz, 1H), 7.57 (t, J=7.7 Hz, 2H), 4.03 (ddd, J=2.6, 5.3, 8.3 Hz, 1H), 3.37-3.23 (m, 2H), 3.20-3.14 (m, 1H), 2.91-2.75 (m, 3H), 2.69 (br s, 1H), 1.79-1.54 (m, 5H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ=139.58, 133.83, 129.28, 127.98, 67.90, 61.71, 59.99, 46.88, 25.98, 25.84; LCMS [M+H]⁺: 256.1. LCMS purity: 100%. SFC 100% purity.

Among other things, the present disclosure encompasses the recognition that bases utilized in reactions (e.g., from compound 1 to compound 2)can impact stereoselectivity of such reactions. Certain example results are described below:

Chiral Auxiliary

S. No
Aldehyde
Nucleophile
Base
(Diastereoselectivity, cis/trans)

1
1

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n-BuLi
WV-CA-108 (87:13)

2
1

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LiHMDS
WV-CA-108 (1.85:1)

3
1

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LDA
WV-CA-108 (1.85:1)

4
1

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KHMDS
WV-CA-108 (10:1)

5
1

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t-BuOK
WV-CA-108 (10:1)

6
4

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n-BuLi
WV-CA-242 (2:1)

7
4

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KHMDS
WV-CA-242 (8:1)

8
4

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n-BuLi
WV-CA-243 (2:1)

9
4

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KHMDS
WV-CA-243 (8:1)

10
4

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n-BuLi
WV-CA-347 (5.5:1)

11
4

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KHMDS
WV-CA-347 (10:1)

12
4

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KHMDS
WV-CA-247 (43:57)

13
4

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n-BuLi
WV-CA-247 (~1:1)

14
4

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LiHMDS
WV-CA-247 (~39:51)

15
4

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NaHMDS
WV-CA-247 (~40:66)

Preparation of compound WV-CA-237

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To a solution of compound 3 (400.00 g, 803.78 mmol) in THF (1.5 L) was added HCl (5M, 1.61 L). The mixture was stirred at 15° C. for 2 hr. TLC indicated compound 3 was consumed completely and one major new spot with larger polarity was detected. The resulting mixture was washed with MTBE (500 mL×3). The combined aqueous layer was adjusted to pH 12 with 5M NaOH aq. and extracted with DCM (500 mL×1) and EtOAc (1000 mL×2). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated to afford as a brown solid. WV-CA-237 (100 g, crude) was obtained as a brown solid.

The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=3/1 to Ethyl acetate:Methanol=1: 2) to give 24 g crude. Then the 4 g residue was purified by prep-HPLC (column: Phenomenex luna C18 250×50 mm×10 um; mobile phase: [water (0.05% HCl)-ACN]; B %: 2%→20%, 15 min) to give desired compound (2.68 g, yield 65%) as a white solid. WV-CA-237 (2.68 g) was obtained as a white solid. WV-CA-237; ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.98-7.88 (m, 2H), 7.68-7.61 (m, 1H), 7.60-7.51 (m, 2H), 4.04 (dt, J=2.4, 5.6 Hz, 1H), 3.85 (ddd, J=3.1, 5.6, 8.4 Hz, 1H), 3.37-3.09 (m, 3H), 2.95-2.77 (m, 3H), 1.89-1.53 (m, 4H), 1.53-1.39 (m, 1H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ=139.89, 133.81, 133.70, 129.26, 129.16, 128.05, 127.96, 68.20, 61.77, 61.61, 61.01, 60.05, 46.67, 28.02, 26.24, 25.93; LCMS [M+H]⁺; 256.1. LCMS purity: 80.0%. SFC dr=77.3:22.7.

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To a solution of compound 4 (140 g, 410.02 mmol) in THF (1400 mL) was added methylsulfonylbenzene (96.07 g, 615.03 mmol), then added KHMDS (1 M, 615.03 mL) in 0.5 hr. The mixture was stirred at −70˜−40° C. for 3 hr. TLC indicated compound 4 was consumed and one new spot formed. The reaction mixture was quenched by addition sat. NH₄Cl aq. 3000 mL at 0° C., and then diluted with EtOAc (3000 mL) and extracted with EtOAc (2000 mL×3). Dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. To the crude was added THF (1000 mL) and MeOH (1500 mL), concentrated under reduced pressure at 45° C. until about 1000 mL residue remained, filtered the solid. Repeat 3 times. Compound 5 (590 g, 72.29% yield) was obtained as a yellow solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.81 (d, J=7.5 Hz, 2H), 7.75-7.65 (m, 1H), 7.62-7.53 (m, 2H), 7.48 (br d, J=7.2 Hz, 6H), 7.25-7.11 (m, 9H), 4.50-4.37 (m, 1H), 3.31-3.11 (m, 3H), 3.04-2.87 (m, 2H), 1.60-1.48 (m, 1H), 1.39-1.24 (m, 1H), 1.11 (dtd, J=4.5, 8.8, 12.8 Hz, 1H), 0.32-0.12 (m, 1H).

Preparation of compound WV-CA-236

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To a solution of compound 5 (283 g, 568.68 mmol) in THF (1100 mL) was added HCl (5M, 1.14 L). The mixture was stirred at 25° C. for 2 hr. TLC indicated compound 5 was consumed and two new spots formed. The reaction mixture was washed with MTBE (1000 mL×3), then the aqueous phase was basified by addition NaOH (5M) until pH=12 at 0° C., and then extracted with DCM (1000 mL×3) to give a residue, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. Compound WV-CA-236 (280 g, 1.10 mol, 96.42% yield) was obtained as a yellow solid.

The crude product was added HCl/EtOAc (1400 mL, 4M) at 0° C., 2 hr later, filtered the white solid and washed the solid with MeOH (1000 mL×3). LCMS showed the solid contained another peak (MS=297). Then the white solid was added H₂O (600 mL) and washed with DCM (300 mL×3). The aqueous phase was added NaOH (5 M) until pH=12. Then diluted with DCM (800 mL) and extracted with DCM (800 mL×4). The combined organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the product. Compound WV-CA-236 (280 g) was obtained as a yellow solid. ¹H NMR (400 MHz, CHLOROFORM-d) 6=8.01-7.89 (m, 2H), 7.69-7.62 (m, 1H), 7.61-7.51 (m, 2H), 4.05 (ddd, J=2.8, 5.2, 8.4 Hz, 11H), 3.38-3.22 (m, 2H), 3.21-3.08 (m, 1H), 2.95-2.72 (m, 4H), 1.85-1.51 (m, 4H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ=139.75, 133.76, 129.25, 127.94, 67.57, 61.90, 60.16, 46.86, 25.86. LCMS [M+H]⁺: 256. LCMS purity: 95.94. SFC purity:

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To a solution of -methoxy-4-methylsulfonyl-benzene (36.8 g, 197.69 mmol) in THF (500 mL) was added KHMDS (1 M, 197.69 mL) at −70° C., 0.5 hr later added compound 4 (45 g, 131.79 mmol) in THF (400 mL) at −70° C. The mixture was stirred at −70→−30° C. for 4 hr, and then the mixture was added with KHMDS (1M, 131.79 mL) at −70° C. The mixture was stirred at −70° C. for 1 hr. TLC indicated compound 4 was remained, and two new spots were detected. The reaction mixture was quenched by sat. NH₄Cl (aq. 300 mL), and then extracted with EtOAc (500 mL×3). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was dissolved in THF (800 mL) and MeOH (500 mL), and then concentrated under reduced pressure until 200 mL solvent left. The mixture was added with MeOH (500 mL) and concentrated under reduced pressure to 200 mL solvent left and solid appeared. The solid was filtered to give product. Repeated the trituration 2 times. Compound 6 (49.8 g, 71.61% yield) was obtained as a brown solid. ¹H NMR (400 MHz, CHLOROFORM-d)=7.73-7.66 (m, 2H), 7.46 (d, J=7.5 Hz, 6H), 7.24-7.11 (m, 9H), 7.04-6.96 (m, 2H), 4.37 (td, J=3.1, 8.3 Hz, 1H), 3.94-3.88 (m, 3H), 3.36 (s, 1H), 3.26-3.10 (m, 3H), 3.00-2.89 (m, 2H), 1.58-1.45 (m, 1H), 1.37-1.23 (m, 1H), 1.15-1.00 (m, 1H), 0.26-0.10 (m, 1H).

Preparation of compound WV-CA-241

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To a solution of compound 6 (50 g, 94.76 mmol) in THF (250 mL) was added HCl (5 M, 189.51 mL). The mixture was stirred at 20° C. for 3 hr. TLC indicated compound 6 was consumed and two new spots formed. The reaction mixture was extracted with MTBE (200 mL×3) and the MTBE phases were discarded. And then the water phase was added with 5 M NaOH (aq.) to pH=9 and extracted with DCM (200 mL×5). The combined organic layers were washed with brine (100 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the product. WV-CA-241 (27 g, 98.10% yield, LCMS purity: 98.24% purity) was obtained as a colorless oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.83-7.76 (m, 2H), 6.98-6.91 (m, 2H), 4.00 (ddd, J=2.9, 5.0, 8.4 Hz, 1H), 3.81 (s, 3H), 3.33-3.07 (m, 5H), 2.87-2.75 (m, 2H), 1.74-1.49 (m, 4H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ=163.79, 131.10, 130.21, 114.44, 67.66, 61.88, 60.25, 55.69, 46.85, 25.84, 25.81. LCMS [M+H]⁺; 286.1. LCMS purity: 98.24%. SFC:dr=0.18:99.82. LCMS purity: 99.9%; SFC purity: 99.82%.

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To a solution of 2-methylsufonylpropane (32.21 g, 263.59 mmol) in THF (1200 mL) was added KHMDS (1 M, 263.59 mL) dropwise at −60° C., and warm to −30° C., slowly over 30 min. The mixture was then cooled to −70° C. A solution of compound 4 (60 g, 175.72 mmol) in THF (300 mL) was added dropwise at −70° C.→60° C., over 30 min. The mixture was stirred at −70° C.→60° C. for 2 hr. TLC showed compound 4 was consumed and new spot was detected. The reaction mixture was quenched with sat. aq. NH₄Cl (800 mL), and then extracted with EtOAc (1 L×3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated. Compound 7 (95 g, crude) was obtained as a yellow oil.

Preparation of Compound WV-CA-242

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To a solution of compound 7 (95 g, 204.90 mmol) in THF (400 mL) was added HCl (5M, 409.81 mL). The mixture was stirred at 0→+25° C. for 2 hr. TLC indicated compound 7 was consumed and one new spot formed. The reaction mixture was washed with MTBE (300 mL×3), then the aqueous phase was basified by addition NaOH (5 M) until pH=12 at 0° C., and then extracted with DCM (300 mL×3) to give a residue dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. Compound WV-CA-242 (45 g, 99.23% yield) was obtained as a yellow oil. LCMS [M+H]⁺: 222.0.

Purification of Compound WV-CA-242

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A solution of WV-CA-242 (45 g, 203.33 mmol), (E)-3-phenylprop-2-enoic acid (30.12 g, 203.33 mmol) in EtOH (450 mL) was stirred at 80° C. for 1 hr. The reaction was concentrated in vacuo. The residue was dissolved in TBME (400 mL), and then stirred at 80° C. for 15 min, and then to the mixture was added EtOH (20 mL) and MeCN (30 mL), and then the mixture was filtered, and the filtered cake was washed with TBME (30 mL×2) and then did this for 8 times. The salt (35 g, crude) was obtained as a red solid.

To a solution of salt (34 g, 92.02 mmol) in H₂O (20 mL) was added aq. 5N NaOH (5 M, 36.81 mL). The mixture was stirred at 25° C. for 10 min. The reaction was extracted with DCM (100 mL×8), and then the organic phase was concentrated in vacuo. Compound WV-CA-242 (18.9 g, 91.09% yield. LCMS purity: 98.16%) was obtained as an off-white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=4.13 (ddd, J=2.1, 4.6, 9.5 Hz, 1H), 3.38 (spt, J=6.9 Hz, 1H), 3.23-3.14 (m, 2H), 3.01 (dd, J=2.1, 14.4 Hz, 1H), 2.95-2.91 (m, 2H), 1.83-1.60 (m, 4H), 1.40 (dd, J=4.0, 6.8 Hz, 6H); ¹³C NMR (101 MHz, CHLOROFORM-d) 6=67.45, 61.71, 53.93, 53.42, 46.80, 25.86, 5.43, 16.03, 14.17. LCMS [M+H]⁺; 222.1. LCMS purity: 98.17%.

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To a solution 2-methyl-2-(methylsulfonyl)propane (14.96 g, 109.83 mmol) in THF (150 mL) was added KHMDS (1 M, 109.83 mL) dropwise at −70° C., and warm to −30° C. slowly over 30 min. The mixture was then cooled to −70° C. A solution of compound 4 (25.00 g, 73.22 mmol) in THF (100 mL) was added dropwise at −70° C. The mixture was stirred at −70° C. for 4 hr. TLC (Petroleum ether:Ethyl acetate=3:1 Rf=0.3) showed compound 4 was remained a little, and one major new spot with larger polarity was detected. The reaction mixture was quenched by added to the sat. NH₄Cl (aq. 100 mL), and then extracted with EtOAc (100 mL×3). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give 30 mL solution. Then added MeOH (30 mL), concentrated under reduced pressure to give 30 mL solution, then filtered the residue and washed with MeOH (10 mL); the residue was dissolved with THF (30 mL) and MeOH (30 mL), and then concentrated under reduced pressure to give 30 mL solution. Then filtered to give a residue and washed with MeOH (10 mL). And repeat one more time to give 21 g white solid and 20 g brown oil. Compound 8 (21 g, crude) was obtained as a white solid, and Compound 8A (20 g, crude) as a brown oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.56 (d, J=7.5 Hz, 6H), 7.32-7.23 (m, 6H), 7.21-7.14 (m, 3H), 4.85-4.68 (m, 1H), 3.52-3.43 (m, 4H), 3.41 (td, J=3.8, 8.1 Hz, 1H), 3.28 (td, J=8.5, 11.9 Hz, 1H), 3.09-2.91 (m, 2H), 2.78 (dd, J=2.6, 13.6 Hz, 1H), 1.65-1.50 (m, 1H), 1.37 (s, 10H), 1.16-0.98 (m, 2H), 0.39-0.21 (m, 1H). LCMS [M+H]⁺: 235.9.

Preparation of Compound WV-CA-243

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To a solution of compound 8 (20 g, 41.87 mmol) in THF (200 mL) was added HCl (5 M, 83.74 mL). The mixture was stirred at 15° C. for 3 hr. TLC indicated compound 8 was consumed completely and one major new spot with larger polarity was detected. The resulting mixture was washed with MTBE (100 mL×3). The combined aqueous layer was adjusted to pH 12 with 5M NaOH aq. and extracted with DCM (50 mL×3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated to afford a white solid. WV-CA-243 (9 g, 90.42% yield, 99% purity) was obtained as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ 4.18 (ddd, J=2.8, 5.8, 8.2 Hz, 1H), 3.29-3.21 (m, 1H), 3.19 (d, J=2.6 Hz, 1H), 3.16-3.08 (m, 1H), 2.92 (t, J=6.6 Hz, 2H), 2.74 (br s, 1H), 1.92-1.81 (m, 1H), 1.81-1.61 (m, 3H), 1.42 (s, 10H); ¹³CNMR (101 MHz, CHLOROFORM-d) δ=68.01, 62.00, 59.73, 49.79, 46.96, 26.77, 25.80, 23.22. LCMS [M+H]⁺: 236.1. LCMS purity: 99.46%.

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To a solution (chloromethyl)(phenyl)sulfane of Mg (17.08 g, 702.90 mmol, 4 eq.) and I₂(0.50 g, 1.97 mmol, 396.83 uL, 1.12-2 eq.) in THF (100 mL) was added with 1,2-dibromoethane (1.25 g, 6.63 mmol, 0.5 mL, 3.77-2 eq.). Once the mixture turned to be colorless, chloromethylsulfanylbenzene (111.51 g, 702.90 mmol, 4 eq.) in THF (100 mL) was dropwise added at 10-20° C. for 1 hr. After addition, the mixture was stirred at 10-20° C. for 1 hr, most of Mg was consumed. And then the mixture was added in the mixture of compound 1 (60 g, 175.72 mmol, 1 eq.) in THF (600 mL) at −78° C., the mixture was stirred at −78° C.-20° C. for 4 hr. TLC (Petroleum ether:Ethyl acetate=9:1, R_f=0.26) indicated compound 1 was remained and two new spots formed. The reaction mixture was quenched by addition water (100 mL) at 0° C., and then extracted with EtOAc (100 mL×3). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=200/1 to 10:1) 2 times. Compound 9 (80 g, 171.80 mmol, 97.77% yield) was obtained as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.52 (d, J=7.5 Hz, 6H), 7.31-7.09 (m, 14H), 4.24-4.14 (m, 1H), 3.54-3.44 (m, 1H), 3.30-3.18 (m, 1H), 3.08-2.96 (m, 1H), 2.91 (s, 1H), 2.80 (d, J=7.0 Hz, 2H), 1.69-1.53 (m, 1H), 1.39-1.30 (m, 1H), 1.15-1.01 (m, 1H), 0.30-0.12 (m, 1H).

Preparation of Compound WV-CA-244

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To a solution of compound 9 (80 g, 171.80 mmol, 1 eq.) in EtOAc (350 mL) was added HCl (5 M, 266.30 mL, 7.75 eq.). The mixture was stirred at 15° C. for 18 hr. TLC (Petroleum ether:Ethyl acetate=9:1, R_f=0.01) indicated compound 9 was consumed and new spots formed. The reaction mixture was extracted with MTBE (200 mL×3) and the MTBE phases were discarded. And then the water phase was added with 2 M NaOH (aq.) to pH=9 and extracted with EtOAc (200 mL×5). The combined organic layers were washed with brine (200 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the crude product. To the crude product was added EtOAc (100 mL) at 70° C. The mixture was stirred at 70° C.→20° C. for 1 hr. The reaction mixture was filtered, and the filter cake was dried to give the product. WV-CA-244 (31.9 g, 142.84 mmol, 94.66% yield) was obtained as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.37 (d, J=7.5 Hz, 2H), 7.26 (t. J=7.7 Hz, 2H), 7.20-7.12 (m, 1H), 3.74-3.65 (m, 1H), 3.24-3.15 (m, 1H), 3.13-3.00 (m, 2H), 3.00-2.21 (m, 4H), 1.77-1.59 (m, 4H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ=136.04, 129.35, 128.95, 126.15, 70.75, 61.64, 46.86, 38.54, 25.86, 25.17. LCMS [M+H]⁺: 224.1. LCMS purity: 99.57%.

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To a solution of 4-methylsulfonylbenzonitrile (47.76 g, 263.59 mmol, 1.5 eq.) in THF (800 mL) was added KHMDS (1 M, 263.59 mL, 1.5 eq.) at −70° C.→−40° C., 0.5 hr later, added compound 4 (60.00 g, 175.72 mmol, 1 eq.) in THF (400 mL) at −70° C. The mixture was stirred at −70° C. for 2.5 hr. TLC (Petroleum ether:Ethyl acetate=1:1, R_f=0.4) indicated compound 4 was consumed and one new spot formed. The reaction mixture was quenched by addition sat. NH₄Cl (20 mL) at 0° C. and extracted with DCM (600 mL×3). Dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. The residue was washed with MeOH (500 mL×5) to get compound 10 (28 g, 53.57 mmol, 30.49% yield) as a yellow solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.84-7.74 (m, 2H), 7.73-7.65 (m, 2H), 7.32 (d, J=7.2 Hz, 6H), 7.15-6.99 (m, 9H), 4.20 (td, J=2.9, 5.6 Hz, 1H), 3.22 (ddd, J=3.1, 5.7, 8.3 Hz, 1H), 3.12-3.03 (m, 2H), 3.02-2.92 (m, 1H), 2.90-2.77 (m, 2H), 1.39-1.26 (m, 1H), 1.20-0.93 (m, 2H), 0.13-0.11 (m, 1H).

Preparation of Compound WV-CA-23&

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To a solution of compound 10 (28 g, 53.57 mmol, 1 eq.) in DCM (196 mL) was added TFA (12.22 g, 107.15 mmol, 7.93 mL, 2 eq.). The mixture was stirred at 0° C. for 3 hr. TLC and LCMS indicated compound 10 was consumed and two new spots formed, the reaction mixture was washed with MTBE (100 mL×3), then the aqueous phase was basified by addition NaOH (5 M) until pH=12 at 0° C., and then extracted with DCM (50 mL×3) to give a residue dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a residue. Compound WV-CA-238 (9.5 g, 33.42 mmol, 62.38% yield, 98.62% purity) was obtained as a yellow solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=8.09 (d, J=8.4 Hz, 2H), 7.87 (d, J=8.4 Hz, 2H), 4.06 (ddd, J=2.9, 4.9, 8.3 Hz, 1H), 3.38-3.16 (m, 3H), 2.96-2.79 (m, 2H), 1.81-1.64 (m, 3H), 1.61-1.45 (m, 1H). ¹³C NMR (101 MHz, CHLOROFORM-d) δ=144.05, 132.88, 128.93, 117.48, 117.15, 67.63, 61.50, 60.09, 46.83, 25.88, 25.55. LCMS [M+H]⁺; 281.1. LCMS purity: 98.62%. SFC:dr=99.75:0.25.

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To a solution of methylsulfinylbenzene (25 g, 178.31 mmol, 1.5 eq.) in THF (400 mL) was added KHMDS (1 M, 178.31 mL, 1.5 eq.) dropwise at −60° C., and warm to −30° C. slowly over 30 min. The mixture was then cooled to −70° C. A solution of compound 4 (40.59 g, 118.88 mmol, 1 eq.) in THF (100 mL) was added dropwise at −70° C. The mixture was stirred at −70° C.→−50° C. for 2 hr. TLC (Petroleum ether:Ethyl acetate=3:1) showed compound 4 was remained. The reaction mixture was cooled to −70° C., additionally added KHMDS (M, 40 mL), and stirred at −70° C.→˜−40° C. for 2 hr. TLC (Petroleum ether:Ethyl acetate=3:1) showed compound 4 was little remained. The reaction mixture was quenched with sat. NH₄Cl (aq. 300 mL), and the separated aqueous layer was extracted with EtOAc (200 mL×3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated to afford a residue as a yellow gum, which was crystallized in MeOH (100 mL), filtered and rinsed with MeOH (50 mL) to give an off-white solid (17 g), and the filtrate was concentrated to afford a yellow gum (50 g). The white solid product (17 g) was re-dissolved in THF (150 mL), and added MeOH (80 mL), and the mixture was concentrated to remove THF, filtered and dried to give an off-white solid, which was re-dissolved in THF (150 mL), and added MeOH (80 mL), and the mixture was concentrated to remove THF filtered and dried to give the product as an off-white solid (13 g). The filtrate was concentrated to give 4 g crude. No further purification. The product compound 11 (13 g, 26.99 mmol, 22.70% yield) was obtained as an off-white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.62-7.56 (m, 2H), 7.55-7.52 (m, 3H), 7.51-7.45 (m, 6H), 7.25-7.12 (m, 9H), 4.60 (td, J=2.4, 10.1 Hz, 1H), 3.72 (s, 1H), 3.27-3.13 (m, 2H), 3.04-2.84 (m, 2H), 2.46 (dd, J=2.2, 13.5 Hz, 1H), 1.71-1.53 (m, 1H), 1.42-1.28 (m, 1H), 1.07-0.90 (m, 1H), 0.37-0.21 (m, 1H).

Preparation of Compound WV-CA-247

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To a solution of compound 11 (13 g, 26.99 mmol, 1 eq.) in THF (45 mL) was added HCl (5 M, 52.00 mL, 9.63 eq.) aqueous. The mixture was stirred at 20° C. for 2 hr. TLC (Petroleum ether:Ethyl acetate=3:1) showed the reaction was completed. The resulting mixture was washed with MTBE (60 mL×3), the combined aqueous layer was adjusted to pH 12 with 5 M NaOH aq. and extracted with DCM (80 mL×3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated to afford a white solid (5.8 g). Without further purification. The compound WV-CA-247 (5.8 g, 24.17 mmol, 89.55% yield, 99.74% purity) was obtained as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.67-7.60 (m, 2H), 7.55-7.42 (m, 3H), 4.17 (ddd, J=2.6, 4.2, 9.9 Hz, 1H), 3.74-3.23 (brs, 2H), 3.13 (dt, J=4.3, 7.3 Hz, 1H), 2.96-2.74 (m, 4H), 1.81-1.52 (m, 4H). ¹³C NMR (101 MHz, CHLOROFORM-d) δ=143.99, 130.93, 129.32, 123.92, 66.97, 62.23, 61.58, 46.86, 25.88, 25.3. LCMS [M+H]⁺: 240 LCMS purity: 99.74% SFC:dr=99.48:0.52.

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To a solution of 1,3-dithiane (13.21 g, 109.83 mmol) in THF (250 mL) was added n-BuLi (2.5 M, 29.29 mL) at −20° C., 0.5 hr later added compound 1 (25 g, 73.22 mmol) in THF (250 mL) at -70° C. The mixture was stirred at −70→20° C. for 16 hr. TLC indicated compound 4 was remained, and one new spot was detected. The reaction mixture was quenched by sat. NH₄Cl (200 mL), and then extracted with EtOAc (200 mL×5). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by MPLC (SiO₂, Petroleum ether/Ethyl acetate=50/1 to 10/1, 5% TEA) 2 times. Compound 12 (16 g, 47.33% yield) was obtained as a yellow oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.59 (d, J=7.0 Hz, 5H), 7.29-7.25 (m, 6H), 7.20-7.14 (m, 3H), 4.39 (dd, J=2.4, 10.3 Hz, 1H), 4.03 (ddd, J=2.4, 5.6, 8.2 Hz, 1H), 3.38 (d, J=10.1 Hz, 1H), 3.28 (ddd, J=7.0, 10.1, 12.3 Hz, 1H), 3.07-2.99 (m, 1H), 2.93-2.85 (m, 1H), 2.63-2.54 (m, 1H), 2.34-2.18 (m, 2H), 1.97-1.82 (m, 2H), 1.59-1.45 (m, 1H), 1.22-1.11 (m, 1H), 0.22-0.06 (m, 1H).

Preparation of Compound WV-CA-246

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To a solution of compound 12 (16 g, 34.66 mmol) in EtOAc (80 mL) was added HCl (5M, 69.31 mL). The mixture was stirred at 15° C. for 16 hr. TLC indicated compound 12 was consumed completely and new spots formed. The reaction mixture was extracted with TBME (100 mL×3) and the TBME phases were discarded. And then the water phase was added with 5 M NaOH (aq.) to pH=9 and extracted with DCM (100 mL×5). The combined organic layers were washed with brine (100 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure to give the crude product. The residue was purified by prep-HPLC (column: Phenomenex luna C18 250×50 mm×10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 0%-15%, 20 min and column: Phenomenex luna (2) C18 250×50×10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 0%-12%, 20 min). WV-CA-246 (4.2 g, 55.25% yield) was obtained as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=4.13 (d, J=7.2 Hz, 11H), 3.83 (dd, J=5.1, 7.2 Hz, 1H), 3.49 (dt, J=5.1, 7.3 Hz, 1H), 3.13-2.76 (m, 6H), 2.60 (br s, 2H), 2.20-2.05 (m, 1H), 2.04-1.90 (m, 1H), 1.89-1.62 (m, 4H). ¹³C NMR (101 MHz, CHLOROFORM-d) δ=73.76, 59.94, 50.42, 46.83, 28.95, 28.45, 25.87, 25.32. HPLC purity: 97.75%. LCMS [M+H]⁺: 220.1. SFC:dr=0.22:99.78.

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To a solution of N-methyl-N-phenyl-acetamide (18.5 g, 124.00 mmol) in THF (250 mL) was added KHMDS (1 M, 124.00 mL) dropwise at −70° C., and to warm to −30° C. slowly over 30 min. The mixture was then cooled to −70° C. A solution of compound 4 (28.23 g, 82.67 mmol) in THF (150 mL) was added dropwise at −70° C. The mixture was stirred at −70° C.˜−50° C. for 3 hr. TLC showed the reaction was almost completed. The reaction mixture was quenched with sat. NH₄Cl (aq. 30 mL), and extracted with EtOAc (25 mL×3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated to afford a residue as yellow gum. The crude was purified by column chromatography on silica gel (Petroleum ether:Ethyl acetate=10:1, 3:1, 1:1, 1:2, 5% TEA). Compound 13 (38 g, 93.7% yield) was obtained as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.53 (br d, J=7.5 Hz, 6H), 7.44-7.31 (m, 4H), 7.26-7.09 (m, 12H), 4.46-4.40 (m, 1H), 3.90 (br s, 1H), 3.31-3.19 (m, 4H), 3.15-3.07 (m, 1H), 3.00-2.91 (m, 1H), 1.48-1.26 (m, 2H), 0.86-0.74 (m, 1H), 0.33-0.19 (m, 1H).

Preparation of Compound WV-CA-24&

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To a solution of compound 13 (38 g, 77.45 mmol) in THF (125 mL) was added HCl (5M, 152.00 mL) aqueous. The mixture was stirred at 20° C. for 2 hr. TLC showed the reaction was completed. The resulting mixture was washed with MTBE (80 mL×3), EtOAc (100 mL×3), and DCM (100 mL×2) in turn. The combined aqueous layer was adjusted to pH=12 with 5M NaOH aq. and extracted with DCM (120 mL×3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated to afford a yellow gum. The crude of WV-CA-248 (15.2 g, 73.26% yield, 92.7% purity) appears a yellow gum. To a solution of WV-CA-248 (14.5 g, 58.39 mmol) in EtOH (150 mL) was added (E)-3-phenylprop-2-enoic acid (8.65 g, 58.39 mmol). The mixture was stirred at 80° C. for 1 hr. The mixture was concentrated in vacuo. The residue was dissolved in TBME (50 mL), and then the mixture was added MeCN (3 mL), the mixture was turned clear, then the solution was standed, and then solid was appeared, and the mixture was filtered, and the filtered cake was washed with TMBE (10 mL×2), and the filtered cake was desired compound. The residue (6.5 g, crude) was obtained as a yellow solid. The residue was dissolved in H₂O (10 mL) was added aq. NaOH (5 M, 6.56 mL, 2 eq.). The mixture was stirred at 25° C. for 10 min. The pH of the mixture was 13. The solution was extracted with DCM (40 mL×6), and the organic phase was concentrated in vacuo. Compound WV-CA-248 (4 g, 91.74% yield, 93.4% purity) was obtained as a brown oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.49-7.31 (m, 3H), 7.21 (br d, J=7.3 Hz, 2H), 4.00 (td, J=4.3, 8.6 Hz, 1H), 3.48 (br s, 2H), 3.28 (s, 3H), 3.10-2.98 (m, 1H), 2.97-2.80 (m, 2H), 2.36-2.17 (m, 2H), 1.79-1.47 (m, 3H), 1.79-1.47 (m, 1H). ¹³C NMR (101 MHz, CHLOROFORM-d) δ=172.38, 143.42, 129.89, 128.04, 127.27, 69.90, 62.29, 46.77, 37.98, 37.23, 25.99, 25.65. LCMS [M+H]⁺: 249.1. LCMS purity: 93.35%. SFC:SFC purity de=94.26%.

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To a solution of methylsulfonylmethane (8.27 g, 87.86 mmol) in THF (150 mL) was added KHMDS (1 M, 87.86 mL) at −70° C.˜−40° C. 0.5 hr later added compound 1 (20 g, 58.57 mmol) in THF (100 mL). The mixture was stirred at −70° C. for 1.5 hr. TLC indicated compound 4 was remained a little and one new spot formed. The reaction mixture was quenched by addition sat. NH₄Cl(aq. 200 mL) at 0° C. and then diluted with EtOAc (200 mL) and extracted with EtOAc (200 mL×3). Dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0→0:1). Compound 14 (12 g, crude, HNMR showed cis/trans isomer ratio 10:1) was obtained as a yellow oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.58-7.47 (m, 7H), 7.26-7.22 (m, 51), 7.20-7.13 (m, 3H), 4.51-4.46 (m, 1H), 3.99-3.88 (m, 1H), 3.48-3.39 (m, 1H), 3.21-2.97 (m, 4H), 2.96-2.91 (m, 3H), 2.68 (br d, J=14.6 Hz, 1H), 1.57-1.43 (m, 1H), 1.36-1.26 (m, 1H), 1.20-1.10 (m, 1H), 0.57-0.44 (m, 1H), 0.25-0.04 (m, 1H).

Preparation of WV-CA-252

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To a solution of compound 14 (18 g, 41.32 mmol) in THF (82 mL) was added HCl (5 M, 82.65 mL). The mixture was stirred at 25° C. for 3 hr. TLC indicated compound 14 was consumed and two new spots formed. The reaction mixture was washed with MTBE (50 mL×3), then the aqueous phase was basified by addition NaOH (5M) until pH=12 at 0° C. and then extracted with DCM (50 mL×6) to give a residue dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude compound WV-CA-252 (6.5 g, 81.4% yield) was obtained as a yellow solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=4.13 (ddd, J=1.8, 4.0, 9.7 Hz, 1H), 3.23 (dt, J=4.2, 7.4 Hz, 1H), 3.18-3.09 (m, 1H), 3.05 (s, 4H), 3.00-2.90 (m, 3H), 1.95-1.68 (m, 4H), 1.67-1.48 (m, 1H). LCMS [M+H]⁺: 194.0.

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A mixture of compound 1A (52.24 g, 241.62 mmol) in THF (500 mL) was degassed and purged with N₂for 3 times, and then the mixture was cooled to −70° C., and then to the mixture was added LDA (2 M, 112.76 mL). The mixture was stirred at −40° C. for 30 min, and then to the mixture was added compound 1 (55 g, 161.08 mmol) in THF (250 mL) at −70° C. The mixture was stirred at −70° C. for 2 hr under N₂atmosphere. TLC indicated compound 1 was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction was quenched by sat. aq. NH₄Cl (300 mL) and then extracted with EtOAc (100 mL×3). The combined organic phase was washed with brine (100 mL), dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The residue was dissolved in MeOH (300 mL) and filtered; the filtered cake was the desired product. Compound 2 (53 g, crude) was obtained as a white solid.

Preparation of Compound WV-CA-245

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To a solution of compound 15 (72 g, 129.11 mmol) in THF (400 mL) was added HCl (5M, 258.22 mL). The mixture was stirred at 25° C. for 1 hr. LC-MS showed compound 15 was consumed completely and one main peak with desired mass was detected. The reaction was extracted with TBME (100 mL×3), added aq. 5 N NaOH to pH=13, and then extracted with DCM (50 mL×3), and the combined organic phase was concentrated in vacuo. WV-CA-245 (38 g, 92.82% yield, 99.5% purity) was obtained as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.81-7.71 (m, 4H), 7.58-7.44 (m, 6H), 4.01-3.92 (m, 1H), 3.16-3.09 (m, 1H), 2.92-2.79 (m, 2H), 2.63-2.44 (m, 2H), 1.82-1.60 (m, 4H). ¹³C NMR (101 MHz, CHLOROFORM-d) δ=133.88, 132.89, 132.86, 131.95, 131.88, 130.73, 128.74, 68.98, 68.94, 63.79, 63.67, 47.03, 34.21, 33.49, 26.37, 25.88. LCMS [M+H]⁺: 316.1. LCMS purity: 99.45%. SFC:SFC purity de=99.5%.

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To a solution of compound 1B (13.32 g, 87.86 mmol) in THF (200 mL) was added KHMDS (1 M, 82.00 mL) at −70° C. under N₂, and then the mixture was stirred at −70° C. for 10 min, and then to the mixture was added compound 1 (20 g, 58.57 mmol) in THF (100 mL), the reaction was stirred at −70° C. for 30 min. TLC indicated compound 1 was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction mixture was quenched with sat. aq. NH₄Cl (100 mL), and then extracted with EtOAc (50 mL×3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=50:1, 20:1, 10:1, 1:1, 0:1). Compound 16 (12 g, crude) was obtained as a yellow solid.

Preparation of Compound WV-CA-249

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To a solution of compound 16 (12 g, 24.34 mmol) in THF (50 mL) was added aq. HCl (5M, 48.68 mL). The mixture was stirred at 25° C. for 30 min. TLC indicated compound 16 was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction was extracted with TBME (100 mL×3), and then to the mixture was added 5N aq. NaOH to pH=13, extracted with DCM (100 mL×3), and then the organic phase was concentrated in vacuo. WV-CA-249 (5.36 g, 87.84% yield, 100.00% purity) was obtained as a yellow solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.64 (s, 1H), 7.49 (d, J=0.9 Hz, 2H), 3.88 (td, J=3.6, 9.4 Hz, 1H), 3.24-3.16 (m, 1H), 3.02-2.89 (m, 3H), 2.78 (dd. J=9.4, 14.0 Hz, 1H), 1.84-1.70 (m, 4H). ¹³C NMR (101 MHz, CHLOROFORM-d) δ=143.11, 134.94, 132.60, 132.33, 130.12, 117.63, 111.52, 70.86, 62.02, 46.76, 37.90, 25.88, 24.21. LCMS [M+H]⁺: 251.0. LCMS purity: 100.000%. SFC:SFC purity de=98.28%.

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To a solution of nitromethane (30.59 g, 501.15 mmol) in THF (300 mL) was added KHMDS (1 M, 263.59 mL) at 20-25° C. and stirred for 1 hr. Compound 1 (30 g, 87.86 mmol) in THF (90 mL) was added to the mixture at 20-25° C. and stirred for 0.5 hr. TLC showed that the starting material was consumed mostly, and desired product was formed. The mixture was quenched by saturated aq. NH₄Cl (300 mL) and extracted with ethyl acetate (100 mL×3). The organic phase was washed by saturated aq. NaCl (100 mL×3) and dried with anhydrous Na₂SO₄, then concentrated under reduced pressure to remove the solvent. The crude product was purified by MPLC (SiO₂, Ethyl acetate/Petroleum ether=0%→20%) to obtain compound 17 (26.55 g, 75.08% yield) as yellow solid. The product was detected by ¹H NMR. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.54-7.44 (m, 6H), 7.28-7.21 (m, 6H), 7.20-7.14 (m, 3H), 4.64 (td, J=3.0, 9.4 Hz, 1H), 4.53-4.06 (m, 3H), 3.60-3.40 (m, 1H), 3.24-2.96 (m, 3H), 1.52-1.41 (m, 1H), 1.40-1.28 (m, 1H), 1.17-0.94 (m, 1H), 0.67-0.50 (m, 1H), 0.23 (quin d, J=8.8, 11.6 Hz, 1H).

Preparation of Compound WV-CA-250

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To a solution of compound 17 (7.5 g, 18.63 mmol) in EtOAc (35 mL) was added HC/EtOAc (4 M, 50 mL) at 20-25° C. and stirred for 1 hr. TLC showed that the starting material was consumed completely. Poured the supernatant liquid of the mixture, the yellow gum on the bottle wall was concentrated under reduced pressure to remove the solvent. WV-CA-250 (2.10 g, 56.70% yield, 98.927% purity, HCl salt) was obtained as yellow gum. The product was detected by ¹H NMR, ¹³C NMR and LCMS. ¹H NMR (400 MHz, DMSO-d) δ=9.89-9.54 (m, 1H), 9.03-8.75 (m, 1H), 8.94 (br s, 1H), 4.97-4.78 (m, 1H), 4.65-4.35 (m, 2H), 3.70-3.41 (m, 4H), 3.22-3.03 (m, 2H), 2.06-1.65 (m, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ=79.42, 79.00, 67.89, 66.82, 61.53, 60.77, 45.44, 45.25, 26.93, 24.57, 23.95, 23.81. LCMS [M+H]⁺: 161.1, purity: 98.92%.

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To a solution of compound benzylamine (30 g, 279.97 mmol) an TEA (56.66 g, 559.95 mmol) in DCM (60 mL) was added MsCl (38.49 g, 335.97 mmol) in DCM (30 mL) at 0° C. The mixture was stirred at 0° C. for 2 hr. LC-MS showed compound 18A was consumed and many new peaks were detected. The reaction mixture was washed with HCl (1 M, 50 mL×3) and sat. NaHCO₃(aq. 50 mL x 3). The organic layer was washed with brine (50 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. TLC showed one main spot. The residue was purified by MPLC (SiO₂, Petroleum ether/Ethyl acetate=5/1 to 1:1). Compound 18A (35 g, 67.49% yield) was obtained as a light-yellow solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.44-7.24 (m, 5H), 4.82 (br s, 1H), 4.31 (d, J=6.2 Hz, 2H), 2.85 (s, 3H).

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To a solution of compound 18A (16.28 g, 87.86 mmol) in THF (60 mL) was added with LDA (2 M, 87.86 mL) at 0° C. The mixture was stirred at 0-25° C. for 0.5 hr. And then compound 1 (15 g, 43.93 mmol) in THF (60 mL) was added to above solution at −70° C. The mixture was stirred at −70-25° C. for 4 hr. TLC indicated compound 1 was consumed completely and many new spots formed. The reaction mixture was added with sat. NH₄Cl (aq. 50 mL) and extracted with EtOAc (100 mL×3). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, Petroleum ether/Ethyl acetate=5/1, 2% TEA). Compound 18 (22 g, 95.08% yield) was obtained as a yellow oil.

Preparation of Compound WV-CA-255

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To a solution of compound 18 (22 g, 41.77 mmol) in EtOAc (15 mL) was added HCl (4M in ethyl acetate, 31.33 mL) at 0° C. The mixture was stirred at 0-25° C. for 2 hr. And solid appeared in the reaction mixture. TLC indicated compound 18 was consumed completely and many new spots formed. The reaction mixture was filtered. The filter cake was dissolved in water (10 mL), washed with MTBE (40 mL×3). The water phase was added with Na₂CO₃(powder) to pH=8-9 and extracted with DCM (50 mL×5). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. WV-CA-255 (11 g, 92.60% yield) was obtained as a brown solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.46-7.25 (m, 5H), 4.65-3.72 (m, 5H), 3.14-3.01 (m, 3H), 2.95-2.77 (m, 2H), 1.89-1.34 (m, 4H). ¹³C NMR (101 MHz, CHLOROFORM-d) δ=136.99, 128.71, 128.62, 128.19, 128.09, 127.85, 69.12, 67.58, 61.98, 61.70, 55.55, 55.36, 47.36, 47.30, 46.60, 46.28, 28.05, 26.16, 25.71, 24.92. LCMS [M+H]⁺: 285.0, LCMS purity: 99.8%. SFC:dr (trans/cis)=32.36:67.64.

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To a solution of compound dibenzylamine (30 g, 152.07 mmol) in DCM (250 mL) was added TEA (15.39 g, 152.07 mmol). The mixture was cooled to 0° C., and to the mixture was added MsCl (17.42 g, 152.07 mmol) in DCM (50 mL), and then the mixture was stirred at 25° C. for 12 hours. LC-MS showed desired mass was detected. The reaction was quenched by H₂O (100 mL) and the organic phase was extracted with H₂O (100 mL×3), the organic phase was dried by Na₂SO₄, and then concentrated in vacuum. No need further purification. Compound 19A (39 g, crude) was obtained as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.41-7.29 (m, 9H), 4.36 (s, 4H), 2.82-2.75 (m, 3H). LCMS [M+H]⁺: 298.0, purity: 86.6%.

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To a solution of compound 19A (19.36 g, 70.29 mmol) in THF (200 mL) was added KHMDS (1 M, 76.15 mL) dropwise at −78° C. to −70° C. under N₂. The mixture was warmed to −40° C. and stirred for 0.5 hr, then cooled to −78° C. To the mixture was added compound 1 (20 g, 58.57 mmol) in THF (100 mL) at −78° C. to −70° C. and stirred for 1 hr under N₂. TLC showed that the starting material was consumed completely. The mixture was quenched by saturated aq. NH₄Cl (200 mL) and extracted with ethyl acetate (70 mL×3). The organic phase was washed by saturated aq. NaCl (70 mL×3) and dried with anhydrous Na₂SO₄, then concentrated under reduced pressure to remove the solvent to obtain the crude product as yellow gum. The crude product was re-dissolved with methanol (200 mL) and standing at 20-25° C. for 12 hours. Compound 19 (20.4 g, 99.99% yield) was crystallized from the solvent as white solid, then filtered and dried in vacuum. The filtrate was concentrated under reduced pressure to remove the solvent to give compound 20 (28.4 g, crude) as brown gum. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.47-7.42 (m, 6H), 7.23-7.05 (m, 19H), 4.36 (td, J=3.0, 8.6 Hz, 1H), 4.23-4.12 (m, 4H), 3.29-3.19 (m, 1H), 3.29-3.19 (m, 1H), 3.11 (ddd, J=7.1, 9.5, 12.1 Hz, 1H), 2.97-2.82 (m, 2H), 2.59 (dd, J=3.1, 14.2 Hz, 1H), 1.37-1.27 (m, 1H), 1.24-1.14 (m, 1H), 1.00-0.92 (m, 1H), 0.16-0.02 (m, 1H).

Preparation of Compound WV-CA-263

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To a solution of compound 19 (20 g, 32.42 mmol) in THF (100 mL) was added HCl (5M, 64.85 mL) at 20-25° C. and stirred for 0.5 hr. TLC showed that the starting material was consumed completely. The mixture was extracted with TBME (80 mL×3), then adjusted the pH of the mixture with aq. NaOH (65 mL, 5M) to 11-13 and extracted with DCM (100 mL×3). The organic phase was dried with anhydrous Na₂SO₄and concentrated under reduced pressure to remove the solvent. The crude product was used for the next step without any purification. WV-CA-263 (10.04 g, 82.68% yield, 100% purity) was obtained as white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.38-7.28 (m, 10H), 4.38 (s, 4H), 4.01 (ddd, J=2.6, 5.6, 8.5 Hz, 1H), 3.20-3.13 (m, 2H), 3.10-3.02 (m, 1H), 2.91 (t, J=6.5 Hz, 2H), 1.89 (br d, J=8.6 Hz, 1H), 1.82-1.66 (m, 4H), 1.62-1.52 (m, 1H). ¹³C NMR (101 MHz, CHLOROFORM-d) δ=135.62, 128.77, 128.70, 127.98, 77.35, 76.87 (d, J=31.5 Hz, 1C), 68.84, 61.51, 57.03, 50.35, 46.96, 26.27, 25.88. LCMS [M+H]⁺: 375.1, purity: 100.00%. SFC:dr=99.55:0.45.

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To a solution of 3,3-dimethylbutan-2-one (11.00 g, 109.83 mmol) in THF (125 mL) was added LDA (2 M, 54.91 mL) dropwise at −70° C., and it was stirred at −70° C.˜−60° C. for 1 hr. A solution of compound 1 (25 g, 73.22 mmol) in THF (125 mL) was added dropwise at −70° C.˜−60° C. The mixture was stirred at −70° C. for 1.5 hr. TLC showed compound 1 was almost consumed. The reaction mixture was quenched with sat. NH₄Cl (aq., 200 mL), and the separated aqueous layer was extracted with EtOAc (150 mL×3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated to afford a residue as a light-yellow solid. The crude was purified by column chromatography on silica gel (Petroleum ether+5% TEA: Petroleum ether:Ethyl acetate (20:1)+5% TEA). Compound 21 (17 g, 52.6% yield) was obtained as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.37-7.25 (m, 6H), 7.03-6.95 (m, 6H), 6.94-6.84 (m, 3H), 4.22 (td, J=2.7, 9.2 Hz, 1H), 3.09 (td, J=4.1, 7.6 Hz, 1H), 3.04-2.92 (m, 2H), 2.75 (ddd, J=2.9, 8.5, 12.0 Hz, 1H), 2.26 (dd, J=9.3, 17.0 Hz, 1H), 2.04 (dd, J=3.4, 16.9 Hz, 1H), 1.43-1.24 (m, 2H), 1.14-1.01 (m, 1H), 0.84 (s, 9H), 0.81-0.71 (m, 1H), 0.09-−0.07 (m, 1H).

Preparation of Compound WV-CA-289

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To a solution of compound 21 (16 g, 36.23 mmol) in EtOAc (25 mL) was added 4 M HCl/EtOAc (100 mL). The mixture was stirred at 25° C. for 0.5 hr. TLC showed the reaction was completed. The resulting mixture was filtered, and the solid was stirred in EtOAc (150 mL), filtered and re-triturated with EtOAc/MeOH (150 mL/5 mL), filtered and dried to afford compound WV-CA-289 (7.5 g, 87.8% yield, HCl salt) as a white solid. ¹H NMR (400 MHz, METHANOL-d₄) δ=4.43 (ddd, J=3.5, 4.6, 7.8 Hz, 1H), 3.71 (dt, J=3.5, 8.0 Hz, 1H), 3.42-3.22 (m, 2H), 2.92 (dd, J=7.6, 17.7 Hz, 1H), 2.73 (dd, J=4.9, 17.7 Hz, 1H), 2.23-1.90 (m, 4H), 1.28-1.05 (m, 9H). [M+H]⁺: 200.1, purity: 100.00%.

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To a solution of methylsulfonylbenzene (13.72 g, 87.86 mmol) in THF (100 mL) was added LiHMDS (1 M, 87.86 mL) in 0.5 hr at −70° C.-0° C., then added compound 4 in THF (100 mL). The mixture was stirred at −70° C. in 2.5 hr. TLC indicated compound 4 was remained a little and two new spots formed. The reaction mixture was quenched by addition sat. NH₄Cl aq. (300 mL) at 0° C., extracted with DCM (200 mL×3). Dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude was added THF (100 mL) and MeOH (150 mL), concentrated under reduced pressure at 45° C. until about 100 mL residue remained, filtered the solid. Repeated 3 times. Got solid 20 g, the mother liquid was concentrated under reduced pressure to get compound 22 (20 g, crude) was obtained as a yellow oil. Compound (1R)-2-(benzenesulfonyl)-1-[(2R)-1-tritylpyrrolidin-2-yl]ethanol (20 g, 68.61% yield) was obtained as a white solid.

Preparation of Compound WV-CA-290

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To a solution of compound 22 (20 g, 40.19 mmol) in THF (80 mL) was added HCl (5 M, 80.38 mL) at 0° C. The mixture was stirred at 25° C. for 2 hr. TLC showed the compound 22 was consumed and two new spots formed. The reaction mixture was washed with MTBE (50 mL×3), then the aqueous phase was basified by addition NaOH (5M) until pH=12 at 0° C. and then extracted with DCM (50 mL×3) to give a residue dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 250×50 mm×10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 0%-15%, 20 min). Compound WV-CA-290 (0.7 g, 6.78% yield, 99.39% purity) was obtained as a yellow solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.95-7.85 (m, 2H), 7.64-7.56 (m, 1H), 7.55-7.46 (m, 2H), 3.79 (ddd, J=3.2, 5.4, 8.4 Hz, 1H), 3.28-3.05 (m, 3H), 2.92-2.72 (m, 2H), 1.84-1.54 (m, 3H), 1.51-1.37 (m, 1H). ¹³C NMR (101 MHz, CHLOROFORM-d) δ=139.81, 133.74, 129.19, 128.07, 68.15, 61.55, 60.97, 46.67, 28.03, 26.27. SFC: (AD_MeOH_IPAm_10_40_25_35_6 min), 100% purity. LCMS [M+H]⁺: 256.1. LCMS purity: 99.39%.

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Two batches in parallel: To a solution of compound tert-butyl(methyl)sulfane (25 g, 239.89 mmol) in MeOH (625 mL) was added Oxone (457.18 g, 743.67 mmol) in H₂O (625 mL) at 0° C. The mixture was stirred at 15° C. for 12 hr. HNMR showed compound tert-butyl(methyl)sulfane was consumed completely and desired compound was detected. Combined two batches of the reaction mixture, filtered and concentrated under reduced pressure to evaporate the MeOH, and then extracted with EtOAc (400 mL×4). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 23A (55 g, crude) was obtained as a colorless oil, confirmed by HNMR. ¹HNMR (400 MHz, CHLOROFORM-d) δ=7.26 (s, 1H), 5.30 (s, 8H), 2.81 (s, 3H), 1.43 (s, 9H).

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To a solution of compound 23A (50 g, 367.07 mmol) in THF (510 mL) was added KHMDS (1 M, 367.07 mL) dropwise at −70° C. and warm to −30° C. slowly over 30 min. The mixture was then cooled to −70° C. A solution of compound 1 (83.56 g, 244.72 mmol) in THF (340 mL) was added dropwise at -70° C. The mixture was stirred at −70° C. for 4 hr. TLC showed compound 1 was remained a little, and one major new spot with larger polarity was detected. The reaction mixture was quenched by added to the sat. NH₄Cl (aq. 800 mL), and then extracted with EtOAc (500 mL×3). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give brown oil. The crude was dissolved with THF (300 mL) then concentrated under reduced pressure (40° C.) to give 150 mL clarified solution. Then added to 300 mL MeOH and concentrated under reduced pressure to give 200 mL solution, then filtered to give a residue and washed with MeOH (10 mL). The mother solution was concentrated under reduced pressure to give 100 mL solution then filtered to give a residue and washed with MeOH (10 mL). Combined all the residue, repeated two times to give 60 g residue. Compound 23 (60 g, crude) was obtained as a white solid. ¹HNMR (400 MHz, CHLOROFORM-d) δ=7.56 (d, J=7.5 Hz, 6H), 7.32-7.23 (m, 6H), 7.21-7.14 (m, 3H), 4.85-4.68 (m, 1H), 3.41 (td, J=3.8, 8.1 Hz, 1H), 3.28 (td, J=8.5, 11.9 Hz, 1H), 3.09-2.91 (m, 2H), 2.78 (dd, J=2.6, 13.6 Hz, 1H), 1.65-1.50 (m, 1H), 1.37 (s, 9H), 1.16-0.98 (m, 2H), 0.39-0.21 (m, 1H).

Preparation of Compound WV-CA-240

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To a solution of compound 23 (59 g, 123.52 mmol) in THF (500 mL) was added HCl (5M, 247.04 mL). The mixture was stirred at 20° C. for 3 hr. TLC indicated compound 23 was consumed completely and one major new spot with larger polarity was detected. The resulting mixture was washed with MTBE (500 mL×3). The combined aqueous layer was adjusted to pH 12 with 5 M NaOH aq. and extracted with DCM (200 mL×3). The combined organic layers were dried over anhydrous Na₂SO₄, filtered and concentrated to afford a white solid. WV-CA-240 (23.6 g, 81.14% yield, 99.95% purity) was obtained as a white solid. ¹HNMR (400 MHz, CHLOROFORM-d) δ=4.18 (ddd, J=2.8, 5.8, 8.2 Hz, 1H), 3.29-3.21 (m, 1H), 3.19 (d, J=2.6 Hz, 1H), 3.16-3.08 (m, 1H), 2.92 (t, J=6.6 Hz, 2H), 2.74 (br s, 2H), 1.92-1.81 (m, 1H), 1.81-1.61 (m, 3H), 1.42 (s, 9H). ¹³CNMR (101 MHz, CHLOROFORM-d) δ=68.01, 62.00, 59.73, 49.79, 46.96, 26.77, 25.80, 23.22. LCMS [M+H]⁺: 236.1. LCMS purity 99.95%.

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To a solution of WV-CA-108 (37 g, 144.91 mmol, 1 eq.) in MeOH (370 mL) was added prop-2-enenitrile (7.69 g, 144.91 mmol, 9.61 mL, 1 eq.). The mixture was stirred at 20° C. for 3 hr., (TLC, Petroleum ether:Ethyl acetate=1:3, Rf=0.31) showed WV-CA-108 was consumed completely and in LCMS one main peak with desired MS was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Compound 24 (44 g, crude) was obtained as a white solid. LCMS [M+H]⁺: 308.9.

Preparation of Compound WV-CA-291

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A solution of compound 24 (44 g, 142.67 mmol, 1 eq.) in DCM (220 mL) and MeOH (220 mL) was cooled to −78° C. Then mCPBA (36.93 g, 214.01 mmol, 1.5 eq.) and K₂CO₃(29.58 g, 214.01 mmol, 1.5 eq.) was added. After addition, the mixture was stirred at −78° C. for 3 hr. And the resulting mixture was stirred at 20° C. for 12 hr. LC-MS showed compound 24 was consumed completely and one main peak with desired MS was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography. The residue was purified by flash silica gel chromatography (ISCO®; 220 g SepaFlash® Silica Flash Column. Eluent of 0-30% Ethyl acetate/Petroleum ether gradient at 100 mL/min). WV-CA-291 (12 g, 42.05 mmol, 29.47% yield, 95.08% purity) was obtained as a yellow solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=7.98-7.92 (m, 2H), 7.65 (d, J=7.5 Hz, 1H), 7.61-7.53 (m, 2H), 4.50-4.39 (m, 1H), 3.33-3.15 (m, 3H), 2.97-2.78 (m, 2H), 1.89-1.64 (m, 4H). ¹³CNMR (101 MHz, CHLOROFORM-d) δ=139.61, 133.90, 129.31, 128.02, 71.21, 64.96, 60.05, 58.12, 21.23, 20.29. LCMS [M+H]⁺: 272.0. LCMS purity 95.08%.

Example 4E. Example Technologies for Chirally Controlled Oligonucleotide Preparation—Example Useful Phosphoramidites

Among other things, the present disclosure provides phosphoramidites useful for oligonucleotide synthesis. In some embodiments, provided phosphoramidites are particularly useful for preparation of chirally controlled internucleotidic linkages. In some embodiments, provided phosphoramidites are particularly useful for preparing chirally controlled internucleotidic linkages, e.g., non-negatively charged internucleotidic linkages or neutral internucleotidic linkages, etc., that comprise P-N═. In some embodiments, the linkage phosphorus is trivalent. In some embodiments, the linkage phosphorus is pentavalent. In some embodiments, such internucleotidic linkages have the structure of formula I-n-1, I-n-2, I-n-3, I-n-4. II, II-a-1, II-a-2, I-b-1. II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

General Procedure I for Chloroderivative: In some embodiments, in an example procedure, a chiral auxiliary (174.54 mmol) was dried by azeotropic evaporation with anhydrous toluene (80 mL×3) at 35° C. in a rota-evaporator and dried under high vacuum for overnight. A solution of this dried chiral auxiliary (174.54 mmol) and 4-methylmorpholine (366.54 mmol) dissolved in anhydrous THF (200 mL) was added to an ice-cooled (isopropyl alcohol-dry ice bath) solution of trichlorophosphine (37.07 g, 16.0 mL, 183.27 mmol) in anhydrous THF (150 mL) placed in three neck round bottomed flask through cannula under Argon (start Temp: −10.0° C., Max: temp 0° C. 28 min addition) and the reaction mixture was warmed at 15° C. for 1 hr. After that the precipitated white solid was filtered by vacuum under argon using airfree filter tube (Chemglass: Filter Tube, 24/40 Inner Joints, 80 mm OD Medium Frit, Airfree, Schlenk). The solvent was removed with rota-evaporator under argon at low temperature (25° C.) and the crude semi-solid obtained was dried under vacuum overnight (-15 h) and was used for the next step directly.

General Procedure I for Chloroderivative: In some embodiments, in an example procedure, a chiral auxiliary (174.54 mmol) was dried by azeotropic evaporation with anhydrous toluene (80 mL×3) at 35° C. in a rota-evaporator and dried under high vacuum for overnight. A solution of this dried chiral auxiliary (174.54 mmol) and 4-methylmorpholine (366.54 mmol) dissolved in anhydrous THF (200 mL) was added to an ice-cooled (isopropyl alcohol-dry ice bath) solution of trichlorophosphine (37.07 g, 16.0 mL, 183.27 mmol) in anhydrous THF (150 mL) placed in three neck round bottomed flask through cannula under Argon (start Temp: −10.0° C., Max: temp 0° C., 28 min addition) and the reaction mixture was warmed at 15° C. for 1 hr. After that the precipitated white solid was filtered by vacuum under argon using airfree filter tube (Chemglass: Filter Tube, 24/40 Inner Joints, 80 mm OD Medium Frit, Airfree, Schlenk). The solvent was removed with rota-evaporator under argon at low temperature (25° C.) and the crude semi-solid obtained was dried under vacuum overnight (-15 h) and was used for the next step directly.

General Procedure III for Coupling: In some embodiments, in an example procedure, a nucleoside (9.11 mmol) was dried by co-evaporation with 60 mL of anhydrous toluene (60 mL×2) at 35° C. and dried under high vacuum for overnight. The dried nucleoside was dissolved in dry THF (78 mL), followed by the addition of triethylamine (63.80 mmol) and then cooled to −5° C. under Argon (for 2′F-dG/2′OMe-dG case 0.95 eq of TMS-Cl used). The THF solution of the crude (made from general procedure I (or) H, 14.57 mmol), was added through cannula over 3 min then gradually warmed to room temperature. After 1 hr at room temperature, TLC indicated conversion of SM to product (total reaction time 1 h), the reaction mixture was then quenched with H₂O (4.55 mmol) at 0° C., and anhydrous MgSO₄(9.11 mmol) was added and stirred for 10 min. Then the reaction mixture was filtered under argon using airfree filter tube, washed with THF, and dried under rotary evaporation at 26° C. to afford white crude solid product, which was dried under high vacuum overnight. The crude product was purified by ISCO-Combiflash system (rediSep high performance silica column pre-equilibrated with Acetonitrile) using Ethyl acetate/Hexane with 1% TEA as a solvent (compound eluted at 100% EtOAc/Hexanes/1% Et₃N) (for 2′F-dG case Acetonitrile/Ethyl acetate with 1% TEA used). After evaporation of column fractions pooled together, the residue was dried under high vacuum to afford the product as a white solid.

Preparation of Amidites (1030-1039)

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Preparation of 1030: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (73%). ³¹P NMR (162 MHz, CDCl₃) δ 153.32. (ES) m/z Calculated for C₄₇H₅₀FN₆O₁₀PS: 940.98 [M]⁺, Observed: 941.78 [M+H]⁺.

Preparation of 1031: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (78%). ³¹P NMR (162 MHz, CDCl₃) δ 153.62. (ES) m/z Calculated for C₄₂H₄₃FN₃O₁₀PS: 831.85 [M]⁺, Observed: 870.58 [M+K]⁺.

Preparation of 1032: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (68%). ³¹P NMR (162 MHz, CDCl₃) δ 153.95. (ES) m/z Calculated for C₄₁H₄₆FN₄O₁₀PS: 872.26 [M]⁺, Observed: 873.62 [M+H]⁺.

Preparation of 1033: General Procedure I followed by General Procedure III used. white foamy solid. Yield: (87%). ³¹P NMR (162 MHz, CDCl₃) δ 151.70. (ES) m/z Calculated for C₅₀H₄₈FN₆O₉PS: 958.29 [M]⁺, Observed: 959.79, 960.83 [M+H]⁺.

Preparation of 1034: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (65%). ³¹P NMR (162 MHz, CDCl₃) δ 154.80. (ES) m/z Calculated for C₅₁H₅₁N₆O₁₀PS: 971.31 [M]⁺, Observed: 971.81 [M+H]⁺.

Preparation of 1035: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (76%). ³¹P NMR (162 MHz, CDCl₃) S 156.50. (ES) m/z Calculated for C₅₃H₅₅N₆O₁₁PS: 1014.33 [M]⁺, Observed: 1015.81 [M+H]⁺.

Preparation of 1036: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (78%). ³¹P NMR (162 MHz, CDCl₃) δ 156.40. (ES) m/z Calculated for C₅₀H₅₇,N₆O₁₂PS: 996.34 [M]⁺, Observed: 997.90 [M+H]⁺.

Preparation of 1037: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (73%). ³¹P NMR (162 MHz, CDCl₃) δ 154.87. (ES) m/z Calculated for C₄₆H₅₂N₃O₁₂PS: 901.30 [M]⁺, Observed: 940.83 [M+K]⁺.

Preparation of 1038: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (75%). ³¹P NMR (162 MHz, CDCl₃) δ 154.94. (ES) m/z Calculated for C₅₃H₅₇N₄O₁₂PS: 1004.34 [M]⁺, Observed: 1005.86 [M+H]⁺.

Preparation of 1039: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (80%). ³¹P NMR (162 MHz, CDCl₃) δ 153.52. (ES) m/z Calculated for C₄₄H₄₇N₄O₁₀PS: 854.28 [M]+, Observed: 855.41 [M+H]+.

Preparation of Amidites (1040-1049)

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Preparation of 1040: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (78%). ³¹P NMR (162 MHz, CDCl₃) δ 157.80. (ES) m/z Calculated for C₄₇H₅₀FN₆O₁₀PS: 940.98 [M]⁺, Observed: 941.68 [M+H]⁺.

Preparation of 1041: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (78%). ³¹P NMR (162 MHz, CDCl₃) δ 157.79. (ES) m/z Calculated for C₄₂H₄₃FN₃OPS: 831.85 [M]⁺, Observed: 870.68 [M+K]⁺.

Preparation of 1042: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (78%). ³¹P NMR (162 MHz, CDCl₃) δ 158.07. (ES) m/z Calculated for C₄₁H₁₆FN₄O₁₀PS: 872.26 [M]⁺, Observed: 873.62 [M+H]⁺.

Preparation of 1043: General Procedure 1 followed by General Procedure III used. white foamy solid. Yield: (86%). ³¹P NMR (162 MHz, CDCl₃) δ 156.48. (ES) m/z Calculated for C₅₀H₄₈FN₆O₉PS: 958.29 [M]⁺, Observed: 959.79, 960.83 [M+H]⁺.

Preparation of 1044: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (65%). ³¹P NMR (162 MHz, CDCl₃) δ 154.80. (ES) m/z Calculated for C₅₁H₅₁N₆O₁₀PS: 971.31 [M]⁺, Observed: 971.81 [M+H]⁺.

Preparation of 1045: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (77%). ³¹P NMR (162 MHz, CDCl₃) δ 154.74. (ES) m-z Calculated for C₅₃H₅₅N₆O₁₁PS: 1014.33 [M]⁺ Observed: 1015.81 [M+H]⁺.

Preparation of 1046: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (76%). ³¹P NMR (162 MHz, CDCl₃) δ 155.05. (ES) m/z Calculated for C₅₀H₅₇N₆O₁₂PS: 996.34 [M]⁺, Observed: 997.90 [M+H]⁺.

Preparation of 1047: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (75%). ³¹P NMR (162 MHz, CDCl₃) δ 155.44. (ES) m/z Calculated for C₄₆H₅₂N₃O₁₂PS: 901.30 [M]⁺, Observed: 940.83 [M+K]⁺.

Preparation of 1048: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (73%). ¹P NMR (162 MHz, CDCl₃) δ 155.96. (ES) m/z Calculated for C₅₃H₅₇N₄O₁₂PS: 1004.34 [M]⁺, Observed: 1005.86 [M+H]⁺.

Preparation of 1049: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (80%). ³¹P NMR (162 MHz, CDCl₃) δ 156.37. (ES) m/z Calculated for C₄₄H₄₇N₄O₁₀PS: 854.28 [M]⁺, Observed: 855.31 [M+H]⁺.

Preparation of Amidites (1051)

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Preparation of 1051: General Procedure II followed by General Procedure III used. Off-white foamy solid. Yield: (72%). ³¹P NMR (162 MHz, CDCl₃) δ 154.26. (ES) m/z Calculated for C₄₂H₅₀FN₄O₁₀PS: 852.29 [M]⁺, Observed: 853.52 [M+H]⁺.

Preparation of Amidites (1052)

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Preparation of 1052: General Procedure II followed by General Procedure III used. Off-white foamy solid. Yield: (76%). ³¹P NMR (162 MHz, CDCl₃) δ 156.37. (ES) m/z Calculated for C₄₂H₅₀FN₄O₁₀PS: 852.29 [M]⁺, Observed: 853.52 [M+H]⁺.

Preparation of Amidites (1053, 1054)

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Preparation of 1053: General Procedure II followed by General Procedure III used. Off-white foamy solid. Yield: (80%). ³¹P NMR (162 MHz, CDCl₃) δ 156.62. (ES) m/z Calculated for C₄₇H₅₀FN₆O₈PS: 908.98 [M]⁺. Observed: 909.36 [M+H]⁺.

Preparation of 1054: General Procedure 11 followed by General Procedure III used. Off-white foamy solid. Yield: (79%). ³¹P NMR (162 MHz, CDCl₃) δ 157.62. (ES) m/z Calculated for C₄₄H₄₆FN₄O₈PS: 840.90 [M]⁺, Observed: 841.67 [M+H]⁺.

Preparation of Amidites (1055)

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Preparation of 1055: General Procedure 11 followed by General Procedure III used. White foamy solid. Yield: (77%). ³¹P NMR (162 MHz, CDCl₃) δ 160.00. (ES) m/z Calculated for C₄₅H₄₅FN₅O₁₀PS: 897.26 [M]⁺, Observed: 898.74 [M+H]⁺.

Preparation of Amidites (1056)

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Preparation of 1056: General Procedure II followed by General Procedure III used. Off-white foamy solid. Yield: (84%). ³¹P NMR (162 MHz, CDCl₃) δ 154.80. (ES) m/z Calculated for C₄₅H₄₄ClFN₅O₈P: 867.26 [M]⁺, Observed: 868.69 [M+H]⁺.

Preparation of Amidites (1057)

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Preparation of 1057: General Procedure II followed by General Procedure III used. white foamy solid. Yield: (91%). ³¹P NMR (162 MHz, CDCl₃) δ 154.48. (ES) m-z Calculated for C₅₂H₅₅FN₅O₁₀PS: 991.34 [M]⁺, Observed: 992.87 [M+H]⁺.

Example 4F. Example Technologies for Chirally Controlled Oligonucleotide Preparation—Example Cycles, Conditions and Reagents for Oligonucleotide Synthesis

In some embodiments, the present disclosure provides technologies (e.g., reagents, solvents, conditions, cycle parameters, cleavage methods, deprotection methods, purification methods, etc.) that are particularly useful for preparing chirally controlled internucleotidic linkages. In some embodiments, such internucleotidic linkages, e.g., non-negatively charged internucleotidic linkages or neutral internucleotidic linkages, etc., comprise P-N═, wherein P is the linkage phosphorus. In some embodiments, the linkage phosphorus is trivalent. In some embodiments, the linkage phosphorus is pentavalent. In some embodiments, such internucleotidic linkages have the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. As demonstrated herein, technologies of the present disclosure can provide mild reaction conditions, high functional group compatibility, alternative deprotection and/or cleavage conditions, high crude and/or purified yields, high crude purity, high product purity, and/or high stereoselectivity.

In some embodiments, a cycle for preparing natural phosphate linkages comprises or consists of deprotection (e.g., detritylation), coupling, oxidation (e.g., using I₂/Pyr/Water or other suitable methods available in the art) and capping (e.g., cap 2 described herein or other suitable methods available in the art). An example cycle is depicted below, wherein B1 and B2 are independently nucleobases. As appreciated by those skilled in the art, various modifications, e.g., sugar modifications, base modifications, etc. are compatible and may be included.

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In some embodiments, a cycle for preparing non-natural phosphate linkages (e.g., phosphorothioate internucleotidic linkages) comprises or consists of deprotection (e.g., detritylation), coupling, a first capping (e.g., capping-1 as described herein), modification (e.g., thiolation using XH or other suitable methods available in the art), and a second capping (e.g., capping-2 as described herein or other suitable methods available in the art). An example cycle is depicted below, wherein B1 and B2 are independently nucleobases. As appreciated by those skilled in the art, various modifications, e.g., sugar modifications, base modifications, etc. are compatible and may be included. In some embodiments, a cycle using a DPSE chiral auxiliary is referred to as a DPSE cycle or DPSE amidite cycle.

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In some embodiments, a cycle for preparing non-natural phosphate linkages (e.g., certain non-negatively charged internucleotidic linkages, neutral internucleotidic linkages, etc.), particularly those comprising P-N═, wherein P is the linkage phosphorus and/or those have the structure of formula I-n-1, I-n-2, I-n-3. I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1. II-c-2, II-d-1, II-d-2, III, or a salt form thereof, comprises or consists of deprotection (e.g., detritylation), coupling, a first capping (e.g., capping-1 as described herein), modification (e.g., using ADIH

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2-azido-1,3-dimethyl-4,5-dihydro-1H-imidazol-3-ium hexafluorophosphate(V)) or other suitable methods available in the art), and a second capping (e.g., capping-2 as described herein or other suitable methods available in the art). An example cycle is depicted below, wherein B1 and B2 are independently nucleobases. In some embodiments, a chiral auxiliary utilized in such a cycle for preparing a chirally controlled internucleotidic linkage comprises an electron-withdrawing group as described herein, e.g., various chiral auxiliaries having a G²comprising an electron-withdrawing group. In some embodiments, G²comprises a —SO₂R group as described herein (e.g., in some embodiments, R is optionally substituted phenyl; in some embodiments, R is optionally substituted alkyl (e.g., t-butyl); in some embodiments, it was observed that R being alkyl (e.g., R being t-butyl (e.g., WV-CA-240)) can provide comparable results to R being optionally substituted phenyl (e.g., R being phenyl (PSM))). As appreciated by those skilled in the art, various modifications. e.g., sugar modifications, base modifications, etc. are compatible and may be included. In some embodiments, a cycle using a PSM chiral auxiliary is referred to as a PSM cycle or PSM amidite cycle.

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Various cleavage and deprotection methods may be utilized in accordance with the present disclosure. In some embodiments, as appreciated by those skilled in the art, parameters of cleavage and deprotection (e.g., bases, solvents, temperatures, equivalents, time, etc.) can be adjusted in view of, e.g., structures of oligonucleotides to be prepared (e.g., nucleobases, sugars, internucleotidic linkages, and modifications/protections thereof), solid supports, reaction scales, etc. In some embodiments, cleavage and deprotection comprise one, or two or more, individual steps. For example, in some embodiments, a two-step cleavage and deprotection is utilized. In some embodiments, a cleavage and deprotection step comprises a fluoride-containing reagent (e.g., TEA-HF, optionally buffered with additional bases such as TEA) in a suitable solvent (e.g., DMSO/H₂O) at a suitable amount (e.g., about 100 or more (e.g., 100±5)mL/mmol) and is performed at a suitable temperature (e.g., about 0-100, 0-80, 0-50, 0-40, 0-30, 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100° C. (e.g., in one example, 27±2° C.)) for a suitable period of time (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more hours (e.g., in one example, 6±0.5 h)). In some embodiments, a cleavage and deprotection step comprises a suitable base (e.g., NR₃) in a suitable solvent (e.g., water) (e.g., conc. NH₄OH) at a suitable amount (e.g., about 200 or more (e.g., 200±5) mL/mmol) and is performed at a suitable temperature (e.g., about 0-100, 0-80, 0-50, 0-40, 0-30, 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100° C. (e.g., in one example, 37±2° C.)) for a suitable period of time (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more hours (e.g., in one example, 24±1 h)). In some embodiments, cleavage and deprotection comprises or consists of two steps, wherein one step (e.g., step 1) is 1×TEA-HF in DMSO/H₂O, 100±5 mL/mmol, 27±2° C. and 6±0.5 h, and the other step (e.g., step 2) is conc. NH₄OH, 200±5 mL/mmol, 37±2° C. and 24±1 h. Certain examples of cleavage and deprotection processes are described here.

As appreciated by those skilled in the art, oligonucleotide synthesis is often performed on solid support. Many types of solid support are commercially available and/or can be otherwise prepared/obtained and can be utilized in accordance with the present disclosure. In some embodiments, a solid support is CPG. In some embodiments, a solid support is NittoPhase HL. Types and sizes of solid support can be selected based on desired applications, and in some cases, for a specific use one type of solid support may perform better than the other. In some embodiments, it was observed that for certain preparations CPG can deliver higher crude yields and/or purities compared to certain polymer solid supports such as NittoPhase HL.

Amidites are typically dissolved in solvents at suitable concentrations. In some embodiments, amidites are dissolved in ACN. In some embodiments, amidites are dissolved in a mixture of two or more solvents. In some embodiments, amidites are dissolved in a mixture of ACN and IBN (e.g., 20% ACN/80% IBN). Various concentrations of amidites may be utilized, and may be adjusted in view of specific conditions (e.g., solid support, oligonucleotides to be prepared, reaction times, scales, etc.). In some embodiments, a concentration of about 0.01-0.5, 0.05-0.5, 0.1-0.5, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5 M is utilized. In some embodiments, a concentration of about 0.2 M is utilized. In many embodiments, amidite solutions are dried. In some embodiments, 3 Å molecular sieves are utilized to dry amidite solutions (or keep amidite solutions dry). In some embodiments, molecular sieves are utilized at about 15-20% v/v.

Various equivalents of amidites may be useful for oligonucleotide synthesis. As those skilled in the art will appreciate, equivalents of amidites can be adjusted in view of specific conditions (e.g., solid support, oligonucleotides to be prepared, reaction times, scales, etc.), and the same or different equivalents may be utilized during synthesis. In some embodiments, equivalents of amidites are about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 or more. In some embodiments, a suitable equivalent is about 2. In some embodiments, a suitable equivalent is about 2.5. In some embodiments, a suitable equivalent is about 3. In some embodiments, a suitable equivalent is about 3.5. In some embodiments, a suitable equivalent is about 4.

A number of activators are available in the art and may be utilized in accordance with the present disclosure. In some embodiments, an activator is ETT. In some embodiments, an activator is CMIMT. In some embodiments, CMIMT is utilized for chirally controlled synthesis. As appreciated by those skilled in the art, the same or different activators may be utilized for different amidites, and may be utilized at different amounts. In some embodiments, activators are utilized at about 40-100%. e.g., 40%, 50%, 60%, 70%, 80% or 90% delivery. In some embodiments, a delivery is about 60% (e.g., for ETT). In some embodiments, a delivery is about 70% (e.g., for CMIMT). In some embodiments, molar ratio of activator/amidite is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some embodiments, a molar ratio is about 3-6. In some embodiments, a molar ratio is about 1. In some embodiments, a molar ratio is about 2. In some embodiments, a molar ratio is about 3. In some embodiments, a molar ratio is about 4. In some embodiments, a molar ratio is about 5. In some embodiments, a molar ratio is about 6. In some embodiments, a molar ratio is about 7. In some embodiments, a molar ratio is about 8. In some embodiments, a molar ratio is about 9. In some embodiments, a molar ratio is about 10. In some embodiments, a molar ratio is about 2-5, 2-4 or 3-4 (e.g., for ET). In some embodiments, a molar ratio is about 3.7 (e.g., for ETT). In some embodiments, a molar ratio is about 3-8, 4-8, 4-7, 4-6, 5-7, 5-8 or 5-6 (e.g., for CMIMT). In some embodiments, a molar ratio is about 5.8 (e.g., for CMIMT).

As appreciated by those skilled in the art, various suitable flowrates and reaction times may be utilized for oligonucleotide synthesis, and may be adjusted according to oligonucleotides to be prepared, scales, synthetic setups, etc. In some embodiments, a recycle flow rate utilized for synthesis is about 200 cm/h. In some embodiments, a recycle time is about 1-10 minutes. In some embodiments, a recycle time is about 8 minutes. In some embodiments, a recycle time is about 10 minutes.

Many technologies are available to modify P(III) linkages, e.g., after coupling. For example, various methods are available to convert a P(III) linkage to a P(V) P(═O)-type linkage, e.g., via oxidation. In some embodiments, I₂/Pyr/H₂O is utilized. Similarly, many methods are available to convert a P(III) linkage to a P(V) P(═S)-type linkage, e.g., via sulfurization. In some embodiments, as illustrated herein, XH is utilized as a thiolation reagent. Technologies for converting P(III) linkages to P(V) P(═N—)-type linkages are also widely available and can be utilized in accordance with the present disclosure. In some embodiments, as illustrated herein ADIH is employed. Suitable reaction parameters are described herein. In some embodiments, ADIH is used at a concentration of about 0.01-0.5, 0.05-0.5, 0.1-0.5, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5 M. In some embodiments, concentration of ADIH is about 0.25 M. In some embodiments, concentration of ADIH is about 0.3 M. In some embodiments, ADIH is utilized at about 1-50, 1-40, 1-30, 1-25, 1-20, 1-10, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45 or 50 or more equivalent. In some embodiments, equivalent of ADIH is about 7.5. In some embodiments, equivalent of ADIH is about 10. In some embodiments, equivalent of ADIH is about 15. In some embodiments, equivalent of ADIH is about 20. In some embodiments, equivalent of ADIH is about 23. In some embodiments, equivalent of ADIH is about 25. In some embodiments, equivalent of ADIH is about 30. In some embodiments, equivalent of ADIH is about 35. In some embodiments, one experiment, ADIH was utilized at 15.2 equivalent, and 15 min contact time. In some embodiments, depending on amidites, concentrations, equivalents, contact times, etc. of reagents, e.g., ADIH, may be adjusted.

Technologies of the present disclosure are suitable for preparation at various scales. In some embodiments, synthesis is performed at hundreds of umol or more. In some embodiments, a scale is about 200 umol. In some embodiments, a scale is about 300 umol. In some embodiments, a scale is about 400 umol. In some embodiments, a scale is about 500 umol. In some embodiments, a scale is about 550 umol. In some embodiments, a scale is about 600 umol. In some embodiments, a scale is about 650 umol. In some embodiments, a scale is about 700 umol. In some embodiments, a scale is about 750 umol. In some embodiments, a scale is about 800 umol. In some embodiments, a scale is about 850 umol. In some embodiments, a scale is about 900 umol. In some embodiments, a scale is about 950 umol. In some embodiments, a scale is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or more mmol. In some embodiments, a scale is about 1 mmol or more. In some embodiments, a scale is about 2 mmol or more. In some embodiments, a scale is about 5 mmol or more. In some embodiments, a scale is about 10 mmol or more. In some embodiments, a scale is about 15 mmol or more. In some embodiments, a scale is about 20 mmol or more. In some embodiments, a scale is about 25 mmol or more.

In some embodiments, observed yields were 85-90 OD/umol (e.g., 85,000 OD/mmol for a 10.2 mmol synthesis, with 58.4% crude purity (% FLP)).

Technologies of the present disclosure, among other things, can provide various advantages when utilized for preparing oligonucleotides comprising chirally controlled internucleotidic linkages, e.g., those comprising P-N═ wherein P is a linkage phosphorus (e.g., internucleotidic linkages of I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1. II-d-2, or a salt form thereof, etc.). For example, as demonstrated herein, technologies of the present disclosure can provide high crude purities and yields (e.g., in many embodiments, about 55-60% full-length product for a 20-mer oligonucleotide) with minimal amount of shorter oligonucleotides (e.g., from incomplete coupling, decomposition, etc.). Such high crude yields and/or purities, among other things, can significantly reduce downstream purification and can significantly reduce production cost and cost of goods, and in some embodiments, greatly facilitate or make possible large scale commercial production, clinical trials and/or commercial sales.

Example Procedure for Preparing Chirally Controlled Oligonucleotide Compositions—WV-13864

Described below are example procedures for preparing WV-13864 using controlled pore glass (CPG) low bulk density solid support(e.g., 2′-fC (acetyl) via CNA linker CPG (600 Å LBD)). Useful phosphoramidites include 5′-ODMTr-2′-F-dA(N6-Bz)-(L)-DPSE phosphoramidite, 5′-ODMTr-2′-F-dC(N4-Ac)-(L)-DPSE phosphoramidite, 5′-ODMTr-2′-F-dG(N2-iBu)-(L)-DPSE phosphoramidite, 5′-ODMTr-2′-F-dU-(L)-DPSE phosphoramidite, 5′-ODMTr-2′-OMe-G(N²-iBu)-(L)-DPSE phosphoramidite, 5′-ODMTr-2′-F-dC(N4-Ac)-(L)-PSM phosphoramidite, 5′-ODMTr-2′-F-dG(N2-iBu)-(L)-PSM phosphoramidite, 5′-DMT-2′-OMe-A (Bz)-p-Cyanoethyl phosphoramidite, and 5′-DMT-2′-OMe-C(Ac)-β-Cyanoethyl phosphoramidite.

0.1 M Xanthane hydride solution (XH) was used for thiolation. Neutral P^Nlinkages were formed utilizing 0.3 M of 2-azido-1,3-dimethyl-imidazolinium hexafluorophosphate (ADIH) in acetonitrile. Oxidation solution was 0.04-0.06 M iodine in pyridine/water, 90/10, v/v. Cap A was N-Methylimidazole in acetonitrile, 20/80, v/v. Cap B was acetic anhydride/2,6-Lutidine/Acetonitrile, 20/30/50, v/v/v. Deblocking was performed using 3% dichloroacetic acid in toluene. NH₄OH used was 28-30% concentrated ammonium hydroxide.

Detritylation.

To initiate the synthesis, the 5′-ODMTr-2′-F-dC(N4-Ac)-CPG solid support was subjected to acid catalyzed removal of the DMTr protecting group from the 5′-hydroxyl by treatment with 3% (DCA) in toluene. The DMTr removal step was usually visualized with strong red or orange color and can be monitored by UV watch command at the wavelength of 436 nm.

DMTr removal can be repeated at the beginning of a synthesis cycle. In every case, following detritylation, the support-bound material was washed with acetonitrile in preparation for the next step of the synthesis.

Coupling.

Amidites were dissolved either in acetonitrile (ACN) or in 20% isobutyronitrile (IBN)/80% ACN at a concentration of 0.2M without density correction. The solutions were dried over molecular sieves (3 Å) not less than 4 h before use (15-20%, v/v).

Amidite
Solvent
Concentration
MS3Å

5′-ODMTr-2′-OMe-A(N6-Bz)-CE
ACN
0.2M
15-20%, v/v

5′-ODMTr-2′-OMe-C(N4-Ac)-CE
ACN
0.2M
15-20%, v/v

5′-ODMTr-2′-F-dA(N6-Bz)-(L)-DPSE
ACN
0.2M
15-20%, v/v

5′-ODMTr-2′-F-dC(N4-Ac)-(L)-DPSE
ACN
0.2M
15-20%, v/v

5′-ODMTr-2′-F-dU-(L)-DPSE
20% IBN/80% ACN
0.2M
15-20%, v/v

5′-ODMTr-2′-F-dG(N2-iBu)-(L)-DPSE
ACN
0.2M
15-20%, v/v

5′-ODMTr-2′-OMe-G(N2-iBu)-(L)-DPSE
20% IBN/80% ACN
0.2M
15-20%, v/v

5′-ODMTr-2′-F-dC(N4-Ac)-(L)-PSM
ACN
0.2M
15-20%, v/v

5′-ODMTr-2′-F-dG(N2-iBu)-(L)-PSM
ACN
0.2M
15-20%, v/v

Dual activators (CMIMT and ET) coupling approach were utilized. Both activators were dissolved in ACN at a concentration of 0.5M. CMIMT has been used for chirally controlled coupling with CMIMT to amidite molar ratio of 5.833/1. ETT was used for the coupling of standard amidites (for natural phosphate linkages) with ETT to amidite molar ratio of 3.752/1. Recycle time for all DPSE and PSM amidites was 10 min except mG-L-DPSE which was 8 min. All standard amidites were coupled for 8 min.

Cap-1 (Capping-1, First Capping).

Cap B (Ac₂O/2,6-lutidine/MeCN (2:3:5, v/v/v)) was used. In some embodiments, Cap-1 capped secondary amine groups, e.g., on the chrial auxiliaries. In some embodiments, incomplete protection of secondary amines may lead side reaction resulting in a failed coupling or formation of one or more by-products. In some embodiments, Cap-1 may not be an efficient condition for esterification (e.g., a condition less efficient than Cap-2 (the second capping) for capping unreacted 5′-OH).

Thiolation for DPSE Cycles.

Following Cap-1, phosphite intermediates, P(III), were modified with sulfurizing reagent. In an example preparation, 1.2 CV (6-7 equivalent) of sulfurizing reagent (0.1 M XH/pyridine-ACN, 1:1, v/v) was delivered through the synthetic column via flow through mode over 6 min contact time to form P(V).

Azide Reaction for PSM Cycles.

After Cap-1, a suitable reagent (e.g., comprising —N₃such as ADIH), in ACN was used to form neutral internucleotidic linkages (P^Nlinkages). In an example preparation, 10.3 eq. of 0.25 M ADIH over 10 min contact time for fG-L-PSM and 25.8 eq. of 0.3 M ADIH over 15 min contact time for fC-L-PSM were utilized in the respective cycles.

Oxidation for Standard Nucleotide Cycles.

Cap-1 step was not necessary for standard amidite cycle. After coupling of a standard amidite onto the solid support, the phosphite intermediate, P(III), was oxidized with 0.05 M of iodine/water/pyridine solution to form P(V). In an example preparation, 3.5 eq. of oxidation solution delivered to the column by a flow through mode over 2 min contact time for efficient oxidation.

Cap-2 (Capping-2, a Second Capping).

Coupling efficiency on the solid phase oligonucleotide synthesis for each cycle was approx. 97-100% and monitored by, e.g., release of DMTr cation. Residual uncoupled 5′-hydroxyl groups, typically 1-3% by detrit monitoring, on the solid support were blocked with Cap A (20% N-Methylimidazole in acetonitrile (NMI/ACN=20/80, v/v)) and Cap B (20%:30%:50%=Ac₂O:2,6 -Lutidine: ACN (v/v/v)) reagents (e.g., 1:1). Both reagents (e.g., 0.4 CV) were delivered to the column by flow through mode over 0.8 min contact time to prevent formation of failure sequences. Uncapped amine groups may also be protected in this step.

As illustrated herein, in some embodiments, a DPSE amidite or DPSE cycle is Detritylation ->Coupling ->Cap-1 (Capping-1, first capping) ->Thiolation ->Cap-2 (Capping-L Post-capping, second capping); in some embodiments, a PSM amidite or PSM cycle is Detritylation ->Coupling ->Cap-1 (Capping-, first capping) ->Azide reaction ->Cap-2 (Capping-1, Post-capping, second capping); in some embodiments, a standard amidite or standard cycle (traditional, non-chirally controlled) is Detritylation ->Coupling ->Oxidation ->Cap-2 (Capping-1, Post-capping, second capping).

Synthetic cycles were selected and repeated until the desired length was achieved.

Amine Wash.

In some embodiments, provided technologies are particularly effective for preparing oligonucleotides comprising internucleotidic linkages that comprise P-N═, wherein P is the linkage phosphorus. In some embodiments, provided technologies comprise contacting an oligonucleotide intermediate with a base. In some embodiments, a contact is performed after desired oligonucleotide lengths have been achieved. In some embodiments, such a contact provides an oligonucleotide comprising internucleotidic linkages that comprise P-N═, wherein P is the linkage phosphorus (e.g., those of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof). In some embodiments, a contact removes a chiral auxiliary (e.g., those with a G²that is connected to the rest of the molecule through a carbon atom, and the carbon atom is connected to at least one electron-withdrawing group (e.g., WV-CA-231, WV-CA-236, WV-CA-240, etc.)). In some embodiments, a contact is performed utilizing a base or a solution of a base which is substantially free of OH or water (anhydrous). In some embodiments, a base is an amine (e.g., N(R)₃). In some embodiments, an amine has the structure of NH(R)₂, wherein each R is independently optionally substituted C1-6 aliphatic; in some embodiments, each R is independently optionally substituted C1-6 alkyl. In some embodiments, a base is N, N-diethylamine (DEA). In some embodiments, a base solution is 20% DEA/ACN. In some embodiments, such a contact with a base lowers levels of by-products which, at one or more locations of internucleotidic linkages that comprise P-N═, have instead natural phosphate linkages.

In an example preparation, an on-column amine wash was performed after completion of oligonucleotide nucleotide synthesis cycles, by five column volume of 20% DEA in acetonitrile over 15 min contact time.

In some embodiments, contact with a base may also remove 2-cyanoethyl group used for construction of standard natural phosphate linkage. In some embodiments, contact with a base provide a natural phosphate linkage (e.g., in a salt form in which the cation is the corresponding ammonium salt of the amine base).

Cleavage and Deprotection.

After contact with a base, oligonucleotides are exposed to further cleavage and deprotection. In an example preparation, auxiliary removal (e.g., DPSE), cleavage & deprotection was a two steps process. In step 1, CPG solid support with oligonucleotides was treated with 1×TEA-HF solution (DMSO:Water:TEA.3HF:TEA=43:8.6:2.8:1=v/v/v/v, 100±5 uL/umol) for 6±0.5h at 27+2° C. The bulk slurry was then treated with concentrated ammonium hydroxide (28-30%, 200±10 mL/mmol) for 24±1h at 37±2° C. (step 2) to release oligonucleotide from the solid support. Crude product was collected by filtration. Filtrates were combined with washes (e.g., water) of the solid support. In some embodiments, observed yields were about 80-90 OD/umole.

Example Procedure for Preparing Chirally Controlled Oligonucleotide Compositions—WV-13835

In an example preparation, WV-13835 was prepared at a 1.2 mmol scale starting from CPG 2′-F-U. DPSE was utilized as chiral auxiliary for chirally controlled internucleotidic linkages. The preparation comprised multiple cycles comprising a de-blocking step (detritylation under an acidic condition), a coupling step (with a DPSE phosphoramidite), a pre-modification capping step (e.g., Cap B), a modification step (e.g., thiolation using 0.1M XH in Pyr/CAN), a post-modification capping step (e.g., under a cap 2 condition (1:1 Cap A+Cap B). In some embodiments, a cycle comprises a modification step which is or comprises oxidation with I₂/Pyr/H₂O. Cleavage and deprotection included two steps, wherein step one utilized TEA-HF at 100 mL/mmol and 27±2.5° C., and step 2 utilized conc. NH₄OH at 200 mL/mmol and 37±2.5° C. Total crude yield was 91800 OD (76500 OD/mmol). Neat % FLP was 53.6% and NAP (after de-salting) % FLP was 58.3%. % FLP in crude was 1.71 g.

Example Procedure for Preparing Chirally Controlled Oligonucleotide Compositions—WV-14791

In an example preparation, WV-14791 was prepared at a 402 umol scale starting from CPG 2′-F-U. DPSE was utilized as chiral auxiliary for chirally controlled phosphorothioate internucleotidic linkages, and PSM for chirally controlled n001. The preparation comprised multiple cycles comprising a de-blocking step (detritylation under an acidic condition), a coupling step (with a DPSE (for a chirally controlled phosphorothioate internucleotidic linkage) or PSM phosphoramidites (for a chirally controlled n001 internucleotidic linkage)), a pre-modification capping step (e.g., Cap B), a modification step (e.g., thiolation using 0.1 M XH in Pyr/CAN for phosphorothioate internucleotidic linkages, 2-azido-1,3-dimethyl-imidazolinium hexafluorophosphate in CAN for n001), a post-modification capping step (e.g., under a cap 2 condition (1:1 Cap A+Cap B). In some embodiments, a cycle comprises a modification step which is or comprises oxidation with I₂/Pyr/H₂O. Total crude yield was 27000 OD (67.1 OD/umol). Neat % FLP was 45.7% and NAP (after de-salting) % FLP was 51.8%. % FLP in crude was 445 mg.

Example Procedure for Preparing Chirally Controlled Oligonucleotide Compositions—WV-14344

In an example preparation, WV-14344 was prepared at a 400 umol scale starting from CPG 2′-F-C. DPSE was utilized as chiral auxiliary for chirally controlled phosphorothioate internucleotidic linkages, and PSM for chirally controlled n001. The preparation comprised multiple cycles comprising a de-blocking step (detritylation under an acidic condition), a coupling step (with a DPSE (for a chirally controlled phosphorothioate internucleotidic linkage) or PSM phosphoramidites (for a chirally controlled n001 internucleotidic linkage)), a pre-modification capping step (e.g., Cap B), a modification step (e.g., thiolation using 0.1M XH in Pyr/CAN for phosphorothioate internucleotidic linkages, 2-azido-1,3-dimethyl-imidazolinium hexafluorophosphate in CAN for n001), a post-modification capping step (e.g., under a cap 2 condition (1:1 Cap A+Cap B). In some embodiments, a cycle comprises a modification step which is or comprises oxidation with I₂/Pyr/H₂O. Total crude yield was 32000 OD (80 OD/umol). Neat % FLP was 48.8% and NAP (after de-salting) % FLP was 59.2%. % FLP in crude was 571 mg.

Example Preparation of Additional Chirally Controlled Oligonucleotide Compositions

Various oligonucleotide compositions including chirally controlled oligonucleotide composition were prepared utilizing technologies described herein. In some embodiments, oligonucleotide compositions were prepared using automated solid-phase synthesis. Certain preparations were performed at 25 umol using TWISTτM columns 10 um/15 um column (GlenResearch, catalog #20-0040) filled with 325 mg of CNA linked nucleosides-CPG. Example cycles and azide modification reagents for chirally controlled internucleotidic linkages at 25 umol were shown below.

Waiting

Step
Operation
Reagents
Volume
time

1
Deblocking (detritylation)
3% DCA/DCM
10
mL
1
min

2
Coupling
0.2M monomer/MeCN
0.5
mL
8
min

0.6M CMIMT/MeCN
1
mL

3
Pre-modification capping (cap-1)
Cap-B
2
mL
2
min

4
Modification
0.2M XH/pyridine or
2
mL
6
min

(sulfurization or azide reaction)
0.5M azide reagent/MeCN
2
mL
10
min

5
Post-modification capping (cap-2)
Cap-A + Cap-B
2
mL
45
s

Final linkage
Azide Reagent

n001

embedded image

n003

n004

n006

n008

After cycles were completed, the CPG support was treated with 20% DEA in MeCN for 12 min, washed with dry MeCN and dried under argon and vacuum. The dried CPG support was transferred into a 15 mL plastic tube, treated with 1×solution (1M HF-TEA in H₂O-DMSO (1:5, v/v), 100 uL/umol) for 6 h at 28° C., then added cone. NH₃(200 uL/umol) and reacted for 24 h at 37° C. The mixture was cooled to mom temperature and the CPG was removed by membrane filtration, and the product was analyzed by LTQ and RP-UPLC with a linear gradient of MeCN (1-15%/15 m) in (10 mM TEA, 100 mM HFIP in water) at 55° C. at a rate of 0.8 mL/min. Crude oligonucleotides were purified by AEX-HPLC eluting with 20 mM NaOH to 2.5M NaCl, and desalted to obtain the target oligonucleotide compositions.

Example preparations were listed below, with crude UPLC purity ranging from about 9% to about 58% percent. Higher crude HPLC purities were observed for preparation of the same and/or other oligonucleotides.

Oligonucleotide
Scale (umol)
Observed Mass

WV-16006
70
6912.3

WV-16007
70
7068.9

WV-24092
24
7282

WV-24098
24
7237.1

WV-24104
24
7399.1

WV-24109
24
7355.1

WV-25536
24
6729.1

WV-25537
24
6705.2

WV-25538
24
6739.1

WV-25539
24
6702

WV-25540
24
6726.9

WV-25541
25
7012.6

WV-25542
25
7014.1

WV-25543
25
6989.9

WV-25544
25
7024.2

Among other things, provided technologies provided high crude purities and/or yields. In many preparations (various scales, reagents concentrations, reaction times, etc.), about 55-60% crude purities (% FLP) were obtained, with minimal amount of shorter oligonucleotides (e.g., from incomplete coupling, decomposition, side-reactions, etc.). In many embodiments, amounts of the most significant shorter oligonucleotide are no more than about 2-10%, often no more than 2-4% (e.g., in some embodiments, as low as about 2% (the most significant shorter oligonucleotide being N-3)).

Various technologies are available for oligonucleotide purification and can be utilized in accordance with the present disclosure. In some embodiments, crude products were further purified (e.g., over 90% purity) using, e.g., AEX purification, and/or UF/DF.

Using technologies described herein, various oligonucleotides comprising diverse base sequences, modifications (e.g., nucleobase, sugar, and internucleotidic linkage modifications) and/or patterns thereof, linkage phosphorus stereochemistry and/or patterns thereof, etc. were prepared at various scales from umol to mmol. Such oligonucleotides have various targets and may function through various mechanisms. Certain such oligonucleotides were presented in the Tables of the present disclosure.

As appreciated by those skilled in the art, examples described herein are for illustration only. Those skilled in the art will appreciate that various conditions, parameters, etc. may be adjusted according to, e.g., instrumentation, scales, reagents, reactants, desired outcomes, etc. Certain results may be further improved using various technologies in accordance with the present disclosure. Among other things, provided oligonucleotides and compositions thereof can provide significantly improved properties and/or activities, e.g., in various assays and in vivo models, and may be particularly useful for preventing and/or treating various conditions, disorders or diseases. Certain data are provided in Examples herein.

Example 4G. Synthesis of Certain Reagents for Incorporation of Mod

As described in the present disclosure, oligonucleotide of the present disclosure may comprise various additional chemical moieties (e.g., various Mods) in addition to the oligonucleotide chain moiety. In some embodiments, the present disclosure provides oligonucleotide comprising a Mod described herein. In some embodiments, such additional moieties provide improved properties, activities, deliveries, etc. In some embodiments, the present disclosure provides useful additional chemical moieties, and technologies for preparing and incorporating such additional chemical moieties. Certain examples are described below. Those skilled in the art appreciates and various technologies related to additional chemical moieties (e.g., structures, preparations, incorporation, uses, etc.), e.g., those described in U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, 9,598,458, US 2015/0211006, US 2017/0037399, WO 2017/015555, WO 2017/192664, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, WO 2019/055951, etc., such technologies of each of which are independently incorporated by reference, may be utilized in accordance with the present disclosure.

Synthesis of 5-((1,19-bis((1,3-dimethylimidazolidin-2-ylidene)amino)-10-((3-((3-((1,3-dimethylimidazolidin-2-ylidene)amino)propyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-416-diazanonadecan-10-yl)amino)-5-oxopentanoic acid

embedded image

Step 1. To a solution of benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-2-oate (5 g, 4.95 mmol, 1 eq.) in DCM (50 mL) was added TFA (16.93 g, 148.48 mmol, 10.99 mL, 30 eq.) at 0° C. The mixture was stirred at 0-25° C. for 2 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. Then added ACN (5 mL), and MTBE (40 mL), filtered the viscous liquid. The crude benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (5.21 g, crude, 3TFA) was obtained as a yellowish oil. LCMS: (M+H⁺): 710.6: (M+Na⁺): 732.7.

Step 2. To a solution of benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (5.21 g, crude, 3TFA) in DCM (35 mL) was added DIEA (6.39 g, 49.45 mmol, 8.61 mL, 10 eq.) and 2-chloro-1,3-dimethyl-4,5-dihydroimidazol-1-ium; hexafluorophosphate (4.55 g, 16.32 mmol, 3.3 eq.). The mixture was stirred at 25° C. for 15 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The crude was purified by RP-MPLC (Spec: C18, 330 g, 20-35 micron, 100 Å). The product benzyl 5-((1,19-bis((1,3-dimethylimidazolidin-2-ylidene)amino)-10-((3-((3-((1,3-dimethylimidazolidin-2-ylidene)amino)propyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (4.94 g, crude) was obtained as a yellow oil. ¹H NMR (400 MHz, METHANOL-d₄) δ=7.39-7.29 (m, 5H), 3.70-3.62 (m, 28H), 3.45 (q, J=6.6 Hz, 7H), 3.30-3.26 (m, 6H), 3.08-2.99 (m, 21H), 2.47-2.39 (m, 9H), 2.23 (t, J=7.4 Hz, 2H), 1.92-1.78 (m, 10H).

Step 3. To a solution of benzyl 5-((1,19-bis((1,3-dimethylimidazolidin-2-ylidene)amino)-10-((3-((3-((1,3-dimethylimidazolidin-2-ylidene)amino)propyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (2 g, 2.00 mmol, 1 eq.) in THF (10 mL) and H₂O (2 mL) was added LiOH.H₂O (588.51 mg, 14.02 mmol, 7 eq.). The mixture was stirred at 25° C. for 3 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by prep-HPLC (column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 0%-25%, 20 min). 5-((1,19-bis((1,3-dimethylimidazolidin-2-ylidene)amino)-10-((3-((3-((1,3-dimethylimidazolidin-2-ylidene)amino)propyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoic acid (0.6 g, 651.84 umol, 32.54% yield, 98.66% purity) was obtained as a yellow gum. ¹H NMR (400 MHz, DMSO-d6) δ=8.03 (br t, J=5.6 Hz, 3H), 7.75 (br t, J=5.6 Hz, 3H), 7.08 (s, 1H), 3.62-3.54 (m, 24H), 3.34 (q, J=6.6 Hz, 7H), 3.12 (q, J=6.2 Hz, 5H), 2.96 (s, 18H), 2.30 (br t, J=6.4 Hz, 6H), 2.23-2.03 (m, 4H), 1.79-1.59 (m, 8H); LCMS: (M/2+H): 454.9; LCMS purity: 98.66%.

Synthesis of (E)-2-methyl-14,14-bis((E)-2-methyl-3-morpholino-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-3-morpholino-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oic acid

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Step 1. To a solution of benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-2-oate (5 g, 4.95 mmol, 1 eq.) in DCM (50 mL) was added TFA (16.93 g, 148.48 mmol, 10.99 mL, 30 eq.). The mixture was stirred at 0-25° C. for 2 hr. The reaction mixture was concentrated under reduced pressure to remove solvent, then added ACN (50 mL), and MTBE (500 mL), filtered the viscous liquid. The crude benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (5.21 g, crude, 3TFA) was obtained as a yellow oil. LCMS: (M+H⁺): 710.6; (M+Na⁺): 732.5.

Step 2. To a solution of benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (3.86 g, 3.67 mmol, 1 eq., 3TFA) in DCM (35.1 mL) was added DIEA (4.73 g, 36.63 mmol, 6.38 mL, 10 eq.) and [[(Z)-(1-cyano-2-ethoxy-2-oxo-ethylidene)amino]oxy-morpholino-methylene]-dimethylammonium; hexafluorophosphate (5.18 g, 12.09 mmol, 3.3 eq.). The mixture was stirred at 25° C. for 15 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The crude was dissolved by ACN (15 mL) then input it into the reversed-phase column. The crude product was purified by reversed-phase HPLC (0.75% TFA in water, and acetonitrile). The crude compound benzyl (E)-2-methyl-14,14-bis((E)-2-methyl-3-morpholino-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-3-morpholino-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oate (4.14 g, crude) was obtained as a yellow oil. ¹H NMR (400 MHz, METHANOL-d₄) δ=7.43-7.24 (m, 5H), 3.78 (br s, 13H), 3.72-3.64 (m, 12H), 3.50-3.36 (m, 13H), 3.27 (br d, J=8.6 Hz, 11H), 3.11-2.97 (m, 18H), 2.50-2.42 (m, 8H), 2.26 (t, J=7.4 Hz, 2H), 1.93-1.78 (m, 8H).

Step 3. To a solution of benzyl (E)-2-methyl-14,14-bis((E)-2-methyl-3-morpholino-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-3-morpholino-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oate (2 g, 1.77 mmol, 1 eq.) in THF (1 mL) and H₂O (0.2 mL) was added LiOH.H₂O (519.71 mg, 12.38 mmol, 7 eq.). The mixture was stirred at 25° C. for 3 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by prep-HPLC (Phenomenex luna C18 250*50 mm *10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 0%-20%, 20 min). The compound (E)-2-methyl-14,14-bis((E)-2-methyl-3-morpholino-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-3-morpholino-9,16-dioxo-2-oxa-2,4,8,15-tetraazaicos-3-en-20-oic acid (1.2 g, 1.14 mmol, 64.65% yield, 99.16% purity) was obtained as a yellow gum. ¹H NMR (400 MHz, DMSO-d6) δ=7.99 (br s, 3H), 7.84 (br s, 3H), 7.06 (s, 1H), 3.67 (br s, 12H), 3.59-3.49 (m, 12H), 3.44-3.25 (m, 12H), 3.11 (br s, 12H), 3.02-2.81 (m, 17H), 2.31 (br t, J=6.1 Hz, 6H), 2.23-2.04 (m, 4H), 1.79-1.60 (m, 8H). LCMS: (M/2+H⁺): 521.0; LCMS purity: 99.16%.

Synthesis of (S)-3-(dimethylamino)-26-(3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-amido)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16,20,27-tetraoxo-12-oxa-2,4,8,15,21,28-hexaazatetratriacont-3-en-34-oic acid

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Step 1. To a solution of 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-meth)yl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,48,15-tetraazaicos-3-en-20-oic acid (10 g, 10.94 mmol, 5 eq.) in DMF (100 mL) was added DIPEA (2.83 g, 21.88 mmol, 3.81 mL, 10 eq.) and followed by benzyl (S)-6-(2,6-diaminohexanamido)hexanoate (924.07 mg, 2.19 mmol, 1 eq., 2HC) and then to the mixture was dropwise added HATU (1.91 g, 5.03 mmol, 2.3 eq.) in DMF (10 mL) at 0° C. The reaction mixture was stirred at 25° C. for 12 hr. The mixture was concentrated in vacuo. The residue was purified by prep-HPLC (TFA condition). Column: Phenomenex luna C18 250*50 mm*10 um, mobile phase: [water (0.1% TFA)-ACN]; B1% CH₃CN: 10%-35%, 20 min. Benzyl (S)-3-(dimethylamino)-26-(3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxo-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-amido)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16,20,27-tetraoxo-12-oxa-2,4,8,15,21,28-hexaazatetratriacont-3-en-34-oate (3.7 g, crude) was obtained as a yellow oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ=8.01-7.77 (m, 10H) 7.63 (br t, J=4.9 Hz, 6H), 7.40-7.29 (m, 5H), 7.07 (br d, J=16.5 Hz, 2H), 5.08 (s, 2H), 4.18-4.07 (m, 1H), 3.63-3.46 (m, 24H), 3.10 (br dd, J=3.2, 5.1 Hz, 25H), 3.00-2.78 (m, 79H), 2.39-2.23 (m, 18H), 2.15-1.98 (m, 20H), 1.72-1.13 (m, 31H). LCMS: M/4+H⁺=536.5.

Step 2. To a solution of compound benzyl (S)-3-(dimethylamino)-26-(3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-amido)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16,20,27-tetraoxo-12-oxa-2,4,8,15,21,28-hexaazatetratriacont-3-en-34-oate (4.4 g, 2.05 mmol, 1 eq.) in THF (40 mL) and H₂O (8 mL) was added LiOH.H₂O (603.45 mg, 14.38 mmol, 7 eq.). The mixture was stirred at 25° C. for 2 hr. The mixture was concentrated in vacuo. The residue was purified by prep-HPLC (TFA condition). Column: Phenomenex luna C 18 250*50 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 2%-30%, 20 min. Compound (S)-3-(dimethylamino)-26-(3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-amido)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16,20,27-tetraoxo-12-oxa-2,4,8,15,21,28-hexaazatetratriacont-3-n-34-oic acid (1.4 g, 678.84 umol, 33.04% yield, 99.483% purity) was obtained as a yellow oil. ¹H NMR (400 MHz, DMSO-d6) δ=8.00 (br t, J=5.5 Hz, 6H), 7.91 (br t, J=5.6 Hz, 1H), 7.87-7.79 (m, 2H), 7.67 (br t, J=4.8 Hz, 5H), 7.15-7.01 (m, 2H), 4.17-4.10 (m, 1H), 3.70-3.43 (m, 24H), 3.16-3.06 (m, 24H), 3.05-2.75 (m, 76H), 2.30 (br t, J=6.4 Hz, 12H), 2.18 (t, J=7.4 Hz, 2H), 2.15-1.98 (m, 8H), 1.66 (quin, J=6.6 Hz, 17H), 1.48 (quin, J=7.4 Hz, 3H), 1.41-1.31 (m, 4H), 1.28-1.17 (m, 4H). ¹³C NMR (101 MHz, DMSO-d6) δ=174.85, 172.67, 172.61, 172.40, 172.19, 170.87, 161.50, 158.77 (q, 0.1=35.2 Hz, 1C), 118.06, 115.15, 68.72, 67.84, 60.03, 53.08, 42.36, 38.87, 38.78, 36.40, 35.95, 35.88, 35.81, 35.25, 34.91, 34.08, 29.85, 29.40, 29.19, 26.34, 24.63, 23.47, 22.14. LCMS: M/3+H⁺=684.7, purity: 99.48%.

Synthesis of (S)-6-(4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-methoxy-5-oxopentanamido)hexanoic acid

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Step 1. To a solution of (S)-4-(((benzyloxy)carbonyl)amino)-5-methoxy-5-oxopentanoic acid (14 g, 47.41 mmol, 1 eq.) in THF (150 mL) was added TEA (14.39 g, 142.23 mmol, 19.80 mL, 3 eq.), followed by tert-butyl 6-aminohexanoate 6-aminohexanoate (11.54 g, 61.63 mmol, 1.3 eq.) at 0-5° C. and stirred for 0.5 hour. T3P (60.34 g, 94.82 mmol, 56.39 mL, 50% purity, 2 eq.) was added to the mixture at 0-5° C. and stirred at 20-25° C. for 12 hours. TLC (Petroleum ether/Ethyl acetate=1:1, R_f=0.35) showed that the starting material was consumed completely. The mixture was concentrated under reduced pressure to remove the solvent, and then re-dissolved with ethyl acetate (100 mL). The organic phase was washed by saturated aq. NaHCO₃(50 mL×3) and dried over anhydrous Na₂SO₄. The crude product was purified by MPLC (SiO₂, Petroleum ether/Ethyl acetate=1:1) to obtain tert-butyl (S)-6-(4-(((benzyloxy)carbonyl)amino)-5-methoxy-5-oxopentanamido)hexanoate (19.7 g, crude) as yellow oil.

Step 2. A mixture of tert-butyl (S)-6-(4-(((benzyloxy)carbonyl)amino)-5-methoxy-5-oxopentanamido)hexanoate (15 g, 32.29 mmol, eq.) and Pd/C (10 g, 10% purity) in THF (300 mL) was evacuated in vacuo and backfilled with H₂(15 Psi) three times, then stirred at 20-25° C. for 6 hours. TLC (Petroleum ether/Ethyl acetate=1:1, R_f=0) showed that the starting material was consumed completely. The mixture was filtered and concentrated under reduced pressure to remove the most solvent. The crude product was used for the next step without any purification, tert-butyl (S)-6-(4-amino-5-methoxy-5-oxopentanamido)hexanoate (10.67 g, 31.42 mmol, 97.31% yield, 97.303% purity) was obtained as colorless liquid (in solvent). LCMS: M+H⁺=331.2, purity: 97.70%.

Step 3. To a mixture of 4-(N-((2-Amino-4-oxo-3,4-dihydropteridin-6-yl)-methyl)-2,2,2-trifluoroacetamido)benzoic acid (8.28 g, 25.06 mmol, 1.1 eq.) and DIPEA (8.83 g, 68.33 mmol, 11.90 mL, 3 eq.) in DMSO (20 mL) was added HATU (8.66 g, 22.78 mmol, 1 eq.) and tert-butyl (S)-6-(4-amino-5-methoxy-5-oxopentanamido)hexanoate at 20-25° C. and stirred for 12 hours. The mixture was diluted with H₂O (20 mL) and extracted with ethyl acetate (20 mL×3). The organic phase was concentrated under reduced pressure to remove the solvent. The crude product was purified by MPLC (SiO₂, Methanol/Ethyl acetate=2:5) to obtain tert-butyl (S)-6-(4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-methoxy-5-oxopentanamido)hexanoate (26.2 g. crude) as brown gum. LCMS: M+H⁺=721.2.

Step 4. To a solution of tert-butyl (S)-6-(4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroactamido)benzamido)-5-methoxy-5-oxopentanamido)hexanoate (13.1 g, 11.39 mmol, 1 eq.) in DCM (100 mL) was added TFA (7.79 g, 68.35 mmol, 5.06 mL, 6 eq.) at 0-5° C. and the mixture was stirred at 35-40° C. for 12 hours. The mixture was concentrated under reduced pressure to remove the solvent. The crude product was detected by HPLC and purified by prep-HPLC (column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water (0.05% HCl)-ACN]; B %: 15%-35%, 20 min) to obtain (S)-6-(4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-methoxy-5-oxopentanamido)hexanoic acid (1.51 g, 1.88 mmol, 32.96% yield, 82.627% purity). ¹H NMR (400 MHz, DMSO-d₆) δ=8.92 (br d, J=7.1 Hz, 1H), 8.74 (s, 1H), 7.93 (br d, J=8.4 Hz, 3H), 7.83 (br t, J=5.5 Hz, 1H), 7.66 (br d, J=8.3 Hz, 2H), 5.18 (s, 2H), 5.06-4.52 (m, 3H), 4.45-4.32 (m, 1H), 3.63 (s, 2H), 3.00 (q, J=6.2 Hz, 2H), 2.25-2.13 (m, 4H), 2.12-2.03 (m, 1H), 1.99-1.87 (m, 1H), 1.46 (quin, J=7.5 Hz, 2H), 1.35 (td, J=7.4, 14.9 Hz, 2H), 1.27-1.15 (m, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ=174.91, 172.83, 171.50, 166.02, 159.47, 153.27, 149.15, 142.22, 134.71, 129.15, 128.99, 128.64, 54.27, 52.97, 52.38, 38.79, 34.05, 32.16, 29.29, 26.76, 26.40, 24.66. LCMS: M+H⁺=665.2.

Example 5. Synthesis of N6-Stearoyl-N2-(4-Sulfamoylbenzoyl)-L-Lysine

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Step 1. To a solution of stearic acid (8.00 g, 28.12 mmol) in DCM (210 m was added 1-hydroxypyrrolidine-2,5-dione (3.24 g, 28.12 mmol) followed by EDCI (5.39 g, 28.12 mmol) at 15° C. The mixture was stirred at 15° C. for 21 hr. TLC showed part of stearic acid remained. Additionally added 1-hydroxypyrrolidine-2,5-dione (0.32 g) and EDCI (1.07 g). Stirring was continued at 15° C. for 8 hr. TLC showed the reaction was completed. The solvent was evaporated under reduced pressure. The residue was dissolved in DCM (300 mL) and the solution washed with water (200 mL); the aqueous phase was then back-extracted with DCM (2*100 mL). The combined organic phase was dried (MgSO₄) and the solvent evaporated under reduced pressure to yield 2,5-dioxopyrrolidin-1-yl stearate as a white solid. No further purification. The crude product 2,5-dioxopyrrolidin-1-yl stearate (10.70 g, crude) was used into the next step without further purification. TLC (Petroleum ether:Ethyl acetate=1:1) R_f=0.79.

Step 2. To a solution of (tert-butoxycarbonyl)-L-lysine (4.49 g, 18.24 mmol) and 2,5-dioxopyrrolidin-1-yl stearate (5.80 g, 15.20 mmol) in DMF (20 mL) was added DIPEA (5.89 g, 45.60 mmol, 7.96 mL). The mixture was stirred at 20° C. for 20 hour. TLC and LCMS showed the reaction was completed. The resulting mixture was concentrated to dry under reduced pressure. The residue was combined with 9 g crude compound, partitioned between water (200 mL) and EtOAc (300 mL) and DCM (80 mL). The separated aqueous layer was extracted with EtOAc (300 mL*3). The combined organic layers were washed with water (100 mL*2), dried over anhydrous MgSO₄, filtered and concentrated to afford the product as a white solid (14.5 g). The crude product compound N²-(tert-butoxycarbonyl)-N⁶-stearoyl-L-lysine (7.70 g, crude) was used into the next step without further purification. ¹H NMR (400 MHz, CHLOROFORM-d) δ=11.29 (br s, 1H), 7.97 (s, 1H), 5.88 (br s, 1H), 5.24 (br d, J=7.3 Hz, 1H), 4.21 (br d, J=5.1 Hz, 1H), 3.17 (q, J=6.5 Hz, 2H), 2.11 (t, J=7.6 Hz, 2H), 1.79 (br s, 1H), 1.64 (dt, J=7.9, 14.0 Hz, 1H), 1.58-1.42 (m, 4H), 1.41-1.28 (m, 11H), 1.18 (br s, 29H), 0.81 (t, J=6.7 Hz, 3H); LCMS: (M+Na⁺): 535.3; TLC (Petroleum ether:Ethyl acetate=1:1) R_f=0.01.

Step 3. To a solution of N²-(tert-butoxycarbonyl)-N⁶-stearoyl-L-lysine (12.50 g, 24.38 mmol) in DCM (120 mL) was added TFA (46.20 g, 405.20 mmol, 30 mL). The mixture was stirred at 15° C. for 4.5 hr. LCMS showed the reaction was almost completed. The resulting mixture was concentrated under reduced pressure on a rotary evaporator with water pump to give a gray crude solid. The crude product compound N⁶-stearoyl-L-lysine (12.80 g, crude, TFA salt) was used into the next step without further purification. ¹H NMR (400 MHz, DMSO-d) δ=8.19 (br s, 3H), 7.77-7.65 (m, 1H), 3.88 (br d, J=4.9 Hz, 1H), 3.02 (br d, J=5.5 Hz, 2H), 2.03 (br t, J=7.3 Hz, 2H), 1.75 (br s, 2H), 1.56-1.34 (m, 6H), 1.24 (s, 28H), 0.86 (br t, J=6.4 Hz, 3H); LCMS: (M+H⁺): 413.3.

Step 4. To a solution of compound N⁶-stearoyl-L-lysine (5.00 g, 9.49 mmol, TFA salt) in DMF (150 mL) was added compound 2,5-dioxopyrrolidin-1-yl 4-sulfamoylbenzoate (3.98 g, 13.34 mmol) followed by DIPEA (9.40 g, 72.73 mmol, 12.70 mL). The mixture was stirred at 80° C. for 18 hr. LCMS showed the reaction was completed. The resulting mixture was concentrated under reduced pressure until 20 mL residue mixture left. To the residue was added DCM (80 mL) and petroleum ether (50 mL). After stood for 36 hr at 15° C., the precipitated solid was filtered and dried to give the product as a light yellow solid (1.9 g). The filtrate was concentrated to dry and triturated with ACN (100 mL), filtered and the filter cake was dried to give a crude (2.4 g). The filtrate was concentrated to give an oil messy crude. No further purification. N⁶-stearoyl-N2-(4-sulfamoylbenzoyl)-L-lysine (1.90 g, 33.60% yield) was obtained as a light yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 13.19-11.82 (m, 1H), 8.74 (br d, J=5.7 Hz, 1H), 8.04 (br d, J=6.6 Hz, 2H), 7.91 (br d, J=7.1 Hz, 2H), 7.74 (br s, 1H), 7.49 (br s, 2H), 4.35 (br s, 1H), 3.02 (br s, 2H), 2.02 (br s, 2H), 1.80 (br s, 2H), 1.23 (br s, 31H), 0.86 (br s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 174.06, 172.39, 165.94, 146.85, 137.28, 128.54, 125.99, 53.24, 38.55, 35.88, 31.76, 30.69, 29.50, 29.41, 29.24, 29.18, 25.78, 23.72, 22.55, 14.39; LCMS: (M+H⁺): 596.4, purity: 89.89%.

Example 6. Synthesis of 18-Oxo-18-((4-Sulfamoylphenethyl)Amino)Octadecanoic Acid

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To a solution of octadecanedioic acid (4.90 g, 15.58 mmol) and 4-(2-aminoethyl)benzenesulfonamide (3.12 g, 15.58 mmol) in DCM (50 mL) was added HATU (7.11 g, 18.70 mmol) and DIPEA (6.04 g, 46.74 mmol, 8.16 mL). The mixture was stirred at 10° C. for 16 hours. The resulting mixture was concentrated under reduced pressure to give a residue. The residue was washed by CH₃CN (100 mL*2) to give the crude product (II g) as white solid. 1 g crude was dissolved by DMSO/DMF (V/V=3:1, 20 mL) purified by prep-HPLC (column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water(0.1% TFA)-ACN]; B %: 45%-75%, 20 min) to give 40 mg product as a white solid. 10 g crude was added CH₃CN/H₂O (V/V=4:1, 100 mL) and stayed at ultrasonic instrument for 30 min, then filtered to give filter cake, filter cake was washed by petroleum ether (20 mL) and acetone (20 mL). Filter cake was concentrated under reduced pressure to give 6 g product as a yellow solid. Compound 18-oxo-18-((4-sulfamoylphenethyl)amino)octadecanoic acid (6.00 g, 77.53% yield) was obtained as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ=7.86 (br t, J=5.3 Hz, 1H), 7.71 (d, J=8.2 Hz, 2H), 7.35 (d, J=7.9 Hz, 2H), 7.27 (s, 2H), 3.26 (q, J=6.6 Hz, 3H), 2.75 (br t, J=7.2 Hz, 2H), 2.15 (t, J=7.3 Hz, 1H), 2.00 (br t, =7.3 Hz, 2H), 1.44 (br d, J=6.6 Hz, 4H), 1.21 (s, 23H), 1.06 (d, =6.6 Hz, 3H). LCMS: (M+H⁺): 497.3, purity 67.72%.

Example 7. Synthesis of 1,7,14-trioxo-12,12-bis((3-oxo-3-((3-(4-sulfamoylbenzamido)propyl)amino)propoxy)methyl)-1-(4-sulfamoylphenyl)-10-oxa-2,6,13-triazaoctadecan-18-oic acid

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Step 1. A solution of di-tert-butyl 3,3′-((2-amino-2-((3-(tert-butoxy)-3- oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoate (4.0 g, 7.91 mmol) and dihydro-2H-pyran-2,6(3H)-dione (0.903 g, 7.91 mmol) in THF (40 mL) was stirred at 50° C. for 3 hrs and at rt for 3 hrs. LC-MS showed desired product. Solvent was evaporated to give 5-((9-((3-(tert-butoxy)-3-oxopropoxy)methyl)-2,2,16,16-tetramethyl-4,14-dioxo-3,7,11,15-tetraoxaheptadecan-9-yl)amino)-5-oxopentanoic acid, which was directly used for next step without purification.

Step 2. To a solution of 5-((9-((3-tert-butoxy)-3-oxopropoxy)methyl)-2,2,16,16-tetramethyl-4,14-dioxo-3,7,11,15-tetraoxaheptadecan-9-yl)amino)-5-oxopentanoic acid (4.90 g, 7.91 mmol) and (bromomethyl)benzene (1.623 g, 9.49 mmol) in DMF was added anhydrous K₂CO₃(3.27 g, 23.73 mmol). The mixture was stirred at 40° C. for 4 hrs and at room temperature for overnight. Solvent was evaporated under reduced pressure. The reaction mixture was diluted with EtOAc, washed with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure to give a residue, which was purified by ISCO eluting with 10% EtOAc in hexane to 50% EtOAc in hexane to give di-tert-butyl 3,3′-((2-(5-(benzyloxy)-5-oxopentanamido)-2-((3-(tert-butoxy)-3-oxopropox)methyl)propane-1,3-diyl)bis(oxy))dipropanoate (5.43 g, 7.65 mmol, 97% yield) as a colorless oil. ¹H NMR (400 MHz, Chloroform-d)δ7.41-7.28 (m, 5H), 6.10 (s, 1H), 5.12 (s, 2H), 3.72-3.60 (m, 12H), 2.50-2.38 (in, 8H), 2.22 (t, J=7.3 Hz, 2H), 1.95 (p, J=7.4 Hz, 2H), 1.45 (s, 27H); MS(ESI), 710.5 (M+H)+.

Step 3. A solution of di-tert-butyl 3,3′-((2-(5-(benzyloxy)-5-oxopentanamido)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoate (5.43 g, 7.65 mmol) in formic acid (50 mL) was stirred at room temperature for 48 hrs. LC-MS showed the reaction was not complete. Solvent was evaporated under reduced pressure. The crude product was re-dissolved in formic acid (50 mL) and was stirred at room temperature for 6 hrs. LC-MS showed the reaction was complete. Solvent was evaporated under reduced pressure, co-evaporated with toluene (3×) under reduced pressure, and dried under vacuum to give 3,3′-((2-(5-(benzyloxy)-5-oxopentanamido)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoic acid (4.22 g, 7.79 mmol, 100% yield) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 12.11 (s, 3H), 7.41-7.27 (m, 5H), 6.97 (s, 1H), 5.07 (s, 2H), 3.55 (d, J=6.4 Hz, 6H), 2.40 (t, J=6.3 Hz, 6H), 2.37-2.26 (m, 2H), 2.08 (t, J=7.3 Hz, 2H), 1.70 (p, J=7.4 Hz, 2H): MS (ESI), 542.3 (M+H)⁺.

Step 4. A solution of 3,3′-((2-(5-(benzyloxy)-5-oxopentanamido)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoic acid (4.10 g, 7.57 mmol) and HOBt (4.60 g, 34.1 mmol) in DCM (60 mL) and DMF (15 mL) at 0° C. was added tert-butyl (3-aminopropyl)carbamate (5.94 g, 34.1 mmol), EDAC HCl salt (6.53 g, 34.1 mmol) and DIPEA (10.55 ml, 60.6 mmol). The reaction mixture was stirred at 0° C. for 15 minutes and at room temperature for 20 hrs. LC-MS showed the reaction was not complete. EDAC HCl salt (2.0 g) and tert-butyl (3-aminopropyl)carbamate (1.0 g) was added into the reaction mixture. The reaction mixture was stirred at room temperature for 4 hrs. Solvent was evaporated to give a residue, which was dissolved in EtOAc (300 mL), washed with water (1×), saturated sodium bicarbonate (2×), 10% citric acid (2×) and water, dried over sodium sulfate, and concentrated to give a residue which was purified by ISCO (80 g gold catridge) eluting with DCM to 30% MeOH in DCM to give benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-21-oate 5 (6.99 g, 6.92 mmol, 91% yield) as a white solid. ¹H NMR (500 MHz, Chloroform-d) δ 7.35 (t, J=4.7 Hz, 5H), 6.89 (s, 3H), 6.44 (s, 1H), 5.22 (d, J=6.6 Hz, 3H), 5.12 (s, 2H), 3.71-3.62 (m, 12H), 3.29 (q, J=6.2 Hz, 6H), 3.14 (q, J=6.5 Hz, 6H), 2.43 (dt, J=27.0, 6.7 Hz, 8H), 2.24 (t, J=7.2 Hz, 2H), 1.96 (p, J=7.5 Hz, 2H), 1.69-1.59 (m, 6H), 1.43 (d, J=5.8 Hz, 27H); MS (ESI): 1011.5 (M+H)+.

Step 5. A solution of benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-2-oate (1.84 g, 1.821 mmol) in DCM (40 mL) was added 2,2,2-trifluoroacetic acid (7.02 ml, 91 mmol). The reaction mixture was stirred at room temperature for overnight. Solvent was evaporated to give benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate as a colorless oil. MS (ESI), 710.6 (M+H)⁺.

Step 6. To a solution of 4-sulfamoylbenzoic acid (1.466 g, 7.28 mmol) and HATU (2.77 g, 7.28 mmol) in DCM (40 mL) followed by benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (1.293 g, 1.821 mmol) in DMF (4.0 mL). The mixture was stirred at room temperature for 5 hrs. Solvent was evaporated under reduced pressure to give a residue, which was purified by ISCO (40 g gold column) eluting with DCM to 50% MeOH in DCM to give benzyl 1,7,14-trioxo-12,12-bis((3-oxo-3-((3-(4-sulfamoylbenzamido)propyl)amino)-propoxy)methyl)-1-(4-sulfamoylphenyl)-10-oxa-2,6,13-triazaoctadecan-18-oate (0.36 g, 0.286 mmol, 16% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 8.60 (t, J=5.6 Hz, 3H), 7.96-7.81 (m, 15H), 7.44 (s, 6H), 7.35-7.23 (m, 5H), 7.04 (s, 1H), 5.02 (s, 2H), 3.50 (t, J=6.9 Hz, 6H), 3.48 (s, 6H), 3.23 (q, J=6.6 Hz, 6H), 3.06 (q, J=6.6 Hz, 6H), 2.29 (t, J=7.4 Hz, 2H), 2.24 (t, J=6.5 Hz, 6H), 2.06 (t, J=7.4 Hz, 2H), 1.69-1.57 (m, 8H).

Step 7. To a round bottom flask flushed with Ar was added 10% Pd/C (80 mg, 0.286 mmol) and EtOAc (15 mL). A solution of benzyl 1,7,14-trioxo-12,12-bis((3-oxo-3-((3-(4-sulfamoylbenzamido)propyl)amino)propoxy)methyl)-1-(4-sulfamoylphenyl)-10-oxa-2,6,13-triazaoctadecan-18-oate (360 mg) in methanol (15 mL) was added followed by diethyl(methyl)silane (0.585 g, 5.72 mmol) dropwise. The mixture was stirred at room temperature for 3 hrs. LC-MS showed the reaction was complete, diluted with EtOAc, and filtered through celite, washed with 20% MeOH in EtOAc, concentrated under reduced pressure to give 1,7,14-trioxo-12,12-bis((3-oxo-3-((3-(4-sulfamoylbenzamido)propyl)-amino)propoxy)methyl)-1-(4-sulfamoylphenyl)-10-oxa-2,6,13-triazaoctadecan-18-oic acid (360 mg, 100% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.60 (t, J=5.6 Hz, 3H), 7.94-7.81 (m, 15H), 7.44 (s, 6H), 7.04 (s, 1H), 3.50 (t, J=6.9 Hz, 6H), 3.48 (s, 6H), 3.23 (q, J=6.6 Hz, 6H), 3.06 (q, J=6.6 Hz, 6H), 2.24 (t, J=6.4 Hz, 6H), 2.14 (t, J=7.5 Hz, 2H), 2.05 (t, J=7.4 Hz, 2H), 1.66-1.57 (m, 8H); MS (ESI), 1170.4 (M+H)⁺.

Example & Synthesis of 2,5-dioxopyrrolidin-1-yl 4-oxo-4-((4-sulfamoylphenethyl)aminobutanoate

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Step 1. A solution of 4-(2-aminoethyl)benzenesulfonamide (20 g, 99.87 mmol), tetrahydrofuran-2,5-dione (9.99 g, 99.87 mmol) in THF (200 mL) was stirred at 60° C. for 16 hr. The reaction mixture was diluted with HCl (aq., 1 M, 100 mL) and extracted with EtOAc (200 mL*3). The combined organic layers were washed with brine (100 mL*2), dried over Na₂SO₄, filtered and concentrated under reduced pressure to give 4-oxo-4-((4-sulfamoylphenethyl)amino)butanoic acid (17 g, 55.60 mmol, 55.67% yield, 98.228% purity) was obtained as a white solid. ¹H NMR (400 MHz, DMSO-d₆)δ=7.94 (t, J=5.7 Hz, 1H), 7.72 (d, J=7.9 Hz, 2H), 7.37 (d, J=8.3 Hz, 2H), 3.30-3.20 (m, 22H), 2.75 (t, J=7.2 Hz, 2H), 2.53-2.44 (m, 4H), 2.44-2.35 (m, 3H), 2.32-2.23 (m, 2H). LCMS: (M+H⁺): 301.1.

Step 2. To a solution of 4-oxo-4-((4-sulfamoylphenethyl)amino)butanoic acid (17 g, 56.60 mmol) and HOSu (10.42 g, 90.57 mmol) in DMF (200 mL) was added DCC (18.69 g, 90.57 mmol, 18.32 mL) at 0° C.-5° C. The mixture was stirred at 0-5° C. for 16 hr. LCMS showed the reaction was not complete. The mixture was stirred at 15° C. for 16 hr. LCMS showed the reaction was complete and one main peak with desired MS was detected. The white suspension of N,N′-dicyclohexylurea (DCU) was filtered and removed white solid. The filtrate was concentrated to an oil. This crude product was washed with hot 2-propanol (60 mL*3), affording an off-white solid. The crude product was added THF (100 mL), and Petroleum ether (50 mL) and stirred for 30 min. then filtered to give 2,5-dioxopyrrolidin-1-yl 4-oxo-4-((4-sulfamoylphenethyl)amino)butanoate (8 g, 16.58 mmol, 29.29% yield, 82.36% purity) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ=8.12-7.96 (m, 1H), 7.71 (br d, J=7.9 Hz, 2H), 7.37 (br d, J=8.2 Hz, 2H), 3.58 (br t, J=6.7 Hz, 1H), 3.30-3.21 (m, 2H), 2.89-2.70 (m, 8H), 2.58 (s, 1H), 2.42 (br t, J=6.7 Hz, 2H); LCMS: (M+H⁺)): 398.0, LCMS purity: 82.36%.

Example 9. Synthesis of 4-oxo-4-((4-sufamoylphenyl)amino)butanoic acid

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To a solid reagent of 4-aminobenezensulfonamide (2.0 g, 11.61 mmol) and tetrahydofuran-2,5-dione (1.16 g, 11.61 mmol) was added THF (30 mL). The reaction mixture was stirred at 60° C. for 4 hrs, and white solid precipitated out. The reaction mixture was cooled to room temperature, and filtered to give a white solid. The white solid was dried under vacuum to give 4-oxo-4-(4-sulfamoylanilino)butanoic acid (2.115 g, 67% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 10.31 (s, 1H), 7.74 (s, 4H), 7.23 (s, 2H), 2.65-2.51 (m, 4H).

Example 10. Synthesis of 3-((4-nitrophenoxy)carbonyl)oxy)propyl stearate

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Step 1. A mixture of propane-1,3-diol (9.80 g, 128.75 mmol, 9.33 mL), Pyridine (2.61 g, 33.01 mmol, 2.66 mL) in CHCl₃(50 mL) was degassed and purged with N₂for 3 times, and then the mixture was dropwised stearoyl chloride (10 g, 33.01 mmol) in CHCl₃(50 mL) at 0° C. and stirred at 20° C. for 20 hr under N₂atmosphere. The mixture was extracted with EtOAc (50 mL*2), and the combined organic layers were washed with 1N HCl (50 mL*2), aq. NaHCO₃(50 mL*2), H₂O (50 mL), dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Ethyl acetate/Petroleum ether=2%, 12.5%) to afford 3-hydroxypropyl stearate (9 g) as a white gum. ¹H NMR (400 MHz, DMSO-d₆) δ=4.24 (t, J=6.06 Hz, 2H), 3.69 (t, J=5.95 Hz, 2H), 2.31 (t, J=7.50 Hz, 2H), 1.87 (q, J=6.06 Hz, 2H), 1.56-1.68 (m, 2H), 1.22-1.31 (m, 24H), 0.88 (t, J=6.73 Hz, 3H); TLC (Petroleum ether:Ethyl acetate=3:1) R_f=0.54.

Step 2. A mixture of 3-hydroxypropyl stearate (9 g, 26.27 mmol), TEA (3.99 g, 39.41 mmol, 5.49 mL) in DCM (160 mL) was dropwised the solution of 4-nitrophenyl carbonochloridate (6.35 g, 31.53 mmol) in DCM (20 mL), then degassed and purged with N₂for 3 times at 0° C., and then the mixture was stirred at 20° C. for 16 hr under N₂atmosphere. TLC indicated compound was consumed completely and many new spots formed. The reaction was clean according to TLC. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiO₂, Ethyl acetate/Petroleum ether=0%, 5%) to afford 3-(((4-nitrophenoxy)carbonyl)oxy)propyl stearate (5.73 g, 11.29 mmol, 42.96% yield) as an off-white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ=8.29 (d, J=9.21 Hz, 2H), 7.39 (d, J=9.21 Hz, 2H), 4.39 (t, J=6.36 Hz, 2H), 4.24 (t, J=6.14 Hz, 2H), 2.32 (t, J=7.45 Hz, 2H), 2.11 (t, J=6.36 Hz, 2H), 1.57-1.68 (m, 2H), 1.21-1.32 (m, 28H), 0.88 (t. J=6.80 Hz, 3H); ¹³C NMR (101 MHz, CHLOROFORM-d) δ=173.73, 155.44, 152.40, 145.37, 125.30, 121.74, 66.00, 60.22, 34.21, 31.91, 29.68, 29.67, 29.64, 29.60, 29.30, 27.92, 24.91, 22.69, 14.12; TLC (Petroleum ether:Ethyl acetate=3:1) R_f=0.72.

Example 11. Synthesis of(R)-3-(((4-nitrophenoxy)carbonyl)oxy)propane-1,2-diyl didodecanoate

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To a solution of 4-nitrophenyl carbonochloridate (69.51 mg, 0.34 mmol) in THF (3.0 ml) at room temperature was added (S)-3-hydroxypropane-1,2-diyl didodecanoate (1,2-dilaurin) and DIPEA (0.11 ml, 0.66 mmol). The reaction mixture was stirred at room temperature for 3 hrs. Solvent was evaporated under reduced pressure, diluted with EtOAc, washed with water, dried over sodium sulfate, concentrated to give the desired product (R)-3-(((4-nitrophenoxy)carbonyl)oxy)propane-1,2-diyl didodecanoate (204 mg, 100% yield). ¹H NMR (400 MHz, Chloroform-d) δ 8.22 (d, J=8.9 Hz, 2H), 7.32 (d, J=8.9 Hz, 2H), 5.32-.528 (m, 1H), 4.34-4.09 (m, 4H), 2.31-2.23 (m, 4H), 1.58-0.79 (m, 42H).

Example 12. Synthesis of 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methy)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propy)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-2-oic acid

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Step 1: To a solution of benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-21-oate (0.95 g, 0.940 mmol) in DCM (5 mL) was added TFA (5 mL). The reaction mixture was stirred at room temperature for 4 hrs. LC-MS showed the reaction was completed. Solvent was evaporated under reduced pressure to give benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate as a colorless oil. Directly use for next step without purification.

Step 2: To a solution of benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (0.46 mmol) in DCM (6 mL) was added HOBt (62.16 mg, 0.46 mmol), HBTU (558.24 mg, 1.47 mmol), DIPEA (1.2 mL, 6.9 mmol) and a solution of 4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)ox)butanoic acid (1.10 g, 1.61 mmol) in acetonitrile (5 mL). The reaction mixture was stirred at rt for 3 hrs. Solvent was evaporated under reduced pressure to give a residue, which was diluted with EtOAc, washed with water, dried over anhydrous sodium sulfate to give a residue, which was purified by ISCO (24 g gold column) eluting with DCM to 20% MeOH in DCM to give 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-2-anoic benzyl ester (1.14 g, 91.7%). MS (ESI), 1353.6 ((M/2+H)⁺.

Step 3. To a solution of 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-21-anoic benzyl ester (1.09 g, 0.400 mmol) in EtOAc (50 mL) was added 10% Pd-C (200 mg). The reaction mixture was stirred at rt for 4 hrs under hydrogen balloon. LC-MS showed the reaction was not completed. The reaction mixture was added another 10% Pd-C (300 mg) and stirred at room temperature for 24 hrs under hydrogen balloon. The reaction mixture was filtered, washed with EtOAc/MeOH, concentrated to give 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-2-oic acid (1.055 g, 100%). MS (ESI), 1308.1 ((M/2+H).

Example 13. Synthesis of 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-29-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazanonacosyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl-5-oxopentanoic acid

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Step 1 to 2. To a solid reagent 2,4,6-trichloro-1,3,5-triazine (0.500 g, 2.71 mmol) in THF (30 mL) was added tert-butyl 3-aminopropanoate HCl salt (0.985 g, 5.42 mmol) and DIPEA (2.36 ml, 13.56 mmol). The reaction mixture was stirred at room temperature for 5 hrs. LC-MS showed the desired product; MS(ESI): 402.4 (M+H)⁺. Solvent was evaporated under reduced pressure to give a residue, which was directly used for next step. To a solution of di-tert-butyl 3,3′-((6-chloro-1,3,5-triazine-2,4-diyl)bis(azanediyl))dipropionate (1.052 g, 2.71 mmol) in aceotnitrile (50 mL) was added benzyl 5-oxo-5-(piperazin-1-yl)pentanoate (1.103 g, 3.80 mmol) and K2CO3 (2.248 g, 16.27 mmol). The reaction mixture was stirred at room temperature for overnight and at 50° C. Diluted with EtOAc, filtered and concentrated under reduced pressure to give a residue, which was purified by ISCO (40 g gold) eluting with 20% EtOAc in hexane to 50° % EtOAc in hexane to give di-tert-butyl 3,3′-((6-(4-(5-(benzyloxy)-5-oxopentanoyl)piperazin-1-yl)-1,3,5-triazine-2,4-diyl)bis(azanediyl))dipropionate (1.13 g, 64%) as a white solid. ¹H NMR (400 MHz, Chloroform-d) δ 7.43-7.30 (m, 5H), 5.15 (s, 2H), 3.75 (brs, 4H), 3.63 (brs, 6H), 3.43 (brs, 2H4), 2.51 (q, J=7.0, 6.5 Hz, 6H), 2.42 (t, J=7.4 Hz, 2H), 2.09-1.96 (m, 2H), 1.48 (s, 18H); MS (ESI): 656.6 (M+H)⁺.

Step 3. A solution of di-tert-butyl 3,3′-((6-(4-(5-(benzyloxy)-5-oxopentanoyl)piperazin-1-yl)-1,3,5-triazine-2,4-diyl)bis(azanediyl))dipropionate (1.10 g, 1.68 mmol) in formic acid (20 mL) was stirred at room temperature for overnight. LC-MS showed the reaction was not completed and solvent was evaporated. Formic acid (20 mL) was added to the reaction mixture and the reaction mixture was stirred at room temperature for 5 hrs. LC-MS showed the reaction was complete. Solvent was concentrated, co-evaporated with toluene (2×) and dried under vacuum for overnight to give 3,3′-((6-(4-(5-(benzyloxy)-5-oxopentanoyl)piperazin-1-yl)-1,3,5-triazine-2,4-diyl)bis(azanediyl))dipropionic acid (0.91 g, 100% yield) as a white solid. MS (ESI), 544.2 (M+H)⁺.

Step 4. A solution of 3,3′-((6-(4-(5-(benzyloxy)-5-oxopentanoyl)piperazin-1-yl)-1,3,5-triazine-2,4-diyl)bis(azanediyl))dipropionic acid (0.91 g, 1.68 mmol) and HOBt (0.76 g, 4.36 mmol) in DCM (30 mL) and DMF (3 mL) at 0° C. was added tert-butyl (3-aminopropyl)carbamate (0.840 g, 4.36 mmol), EDC HCl salt (0.836 g, 4.36 mmol) and DIPEA (1.460 ml, 8.39 mmol). The reaction mixture was stirred at 0° C. for 15 minutes and at room temperature for 20 hrs. Solvent was evaporated to give a residue, which was dissolved in EtOAc (300 mL), washed with water (1×), saturated sodium bicarbonate (2×), 10% citric acid (2×) and water, dried over sodium sulfate, and concentrated to give a residue which was purified by ISCO (80 g gold catridge) eluting with DCM to 30% MeOH in DCM to give benzyl 5-(4-(4,6-bis((3-((3-((tert-butoxycarbonyl)amino)propyl)amino)-3-oxopropyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)-5-oxopentanoate (1.11 g, 77% yield) as a white solid. MS (ESI): 857.5 (M+H).

Step 5. A solution of benzyl 5-(4-(4,6-bis((3-((3-((tert-butoxycarbonyl)amino)propyl)amino)-3-oxopropyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)-5-oxopentanoate (75.93 mg, 0.090 mmol) in DCM (3 mL) was added TFA (0.5 mL). The reaction mixture was stirred at room temperature for 3 hrs. Solvent was evaporated under reduced pressure, use directly for next step without purification. MS (ESI): 656.3 (M+H)⁺.

Step 6. To a solution of 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-21-oic acid (580 mg, 0.222 mmol) in DCM (10 mL) was added HBTU (84.1 mg, 0.220 mmol), HOBt (11.99 mg, 0.09 mmol) and DIPEA (0.15 ml, 0.890 mmol). The reaction mixture was stirred at rt for 5 minutes and a solution of benzyl 5-(4-(4,6-bis((3-((3-aminopropyl)amino)-3-oxopropyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)-5-oxopentanoate TFA salt (0.090 mmol) in acetonitrile was added to the reaction mixture. The reaction mixture was stirred at rt for overnight. Solvent was evaporated under reduced pressure to give a residue, which was purified by ISCO (24 g gold) eluting with DCM to 40% MeOH in DCM to give 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-29-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazanonacosyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)-5-oxopentanoic benzyl ester (300 mg, 57.8%). MS (ESI), 1950.6 ((M/3+H)⁺.

Step 7. To a solution of 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-29-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazanonacosyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)-5-oxopentanoic benzyl ester (300 mg, 0.05 mmol) in EtOAc (10 ml) was added 10% Pd-C (100 mg). The reaction mixture was stirred at rt under hydrogen balloon for overnight. LC-MS showed the reaction was not complete. The reaction mixture was added MeOH (1 mL) and triethylsilane (2 mL). The reaction mixture was stirred at mom temperature for 4 hrs. LC-MS showed the desired product. The reaction mixture was filtered, washed with EtOAc/MeOH, and concentrated under reduced pressure to give a residue, which was purified by ISCO (50 g C18 catridge) eluting with 1% TFA in water to 100% acetonitrile and lyophilized to give 5(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-29-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazanonacosyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)-5-oxopentanoic acid (120 mg, 40.6% yield) as a white solid. MS (ES), 1920 ((M/3+H)⁺.

Example 14. Synthesis of 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methy-30-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazatriacontyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)-5-oxopentanoic acid

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Step 1. To a solution of 5-(2,S4,R6)3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid(2.43 g, 5.43 mmol) in DCM was added HBTU (2.06 g, 5.43 mmol), HOBt (183.36 mg, 1.36 mmol) and DIPEA (4.73 ml, 27.14 mmol). The reaction mixture was stirred at room temperature for 10 minutes, and a solution of benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate TFA salt (1.36 mmol) in acetonitrile was added. The reaction mixture was stirred at room temperature for 3 hrs. Solvent was concentrated under reduced pressure to give a residue, which was purified by ISCO (80 g gold catridge) eluting with 5% MeOH in DCM to 60% MeOH in DCM to give 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic benzyl ester (2.22 g, 81.8%). MS (ESI): 1002 (M/2+H)⁺.

Step 2. To a solution of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic benzyl ester (2.20 g, 1.1 mmol) in EtOAc (30 mL) and MeOH (3 mL) was added 10% Pd-C (300 mg) and triethylsilane (1.8 mL, 11.3 mmol) slowly. The reaction mixture was stirred at room temperature for 1 hr. The reaction mixture was filtered through celite and concentrated to give 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid. MS (ESI), 1912 (M+H)⁺.

Step 3. To a solution of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid (1911 mg, 0.580 mmol) in DCM (30 mL) was added HBTU (266 mg, 0.700 mmol), HOBt (31.56 mg, 0.23 mmol) and DIPEA (0.81 ml, 4.67 mmol). The reaction mixture was stirred at rt for 10 minutes and a solution of benzyl 5-(4-(4,6-bis((3-((3-aminopropyl)amino)-3-oxopropyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)-5-oxopentanoate TFA salt (0.23 mmol) in acetonitrile (5 mL) was added to the reaction mixture. The reaction mixture was stirred at rt for 3 hrs. Solvent was evaporated under reduced pressure to give a residue, which was purified by ISCO (24 g gold) eluting with DCM to 50% MeOH in DCM to give 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-30-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazatriacontyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)-5-oxopentanoic benzyl ester (430 mg, 41.4%). MS (ESI), 1482.1 (M/3+H)⁺.

Step 4. A solution of 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-30-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazatriacontyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)-5-oxopentanoic benzyl ester (420 mg, 0.090 mmol) in EtOAc (15 mL) and MeOH (2 mL) was added 10% Pd-C (200 mg). The reaction mixture was stirred at room temperature under hydrogen balloon for overnight. The reaction mixture was filtered through celite, washed with 50% MeOH in EtOAc, and concentrated under reduced pressure to give 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-30-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazatriacontyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)-5-oxopentanoic acid. MS (ESI), 1452.0 (M/3+H)⁺.

Example 15. Synthesis of 3-(((4-nitrophenoxy)carbonyl)oxy)propyl (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenoate

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Step 1. To the solution of turbinaric acid (200 g, 4.992 mmol) in DCM (20 mL) was added 1,3-propanediol (1.8 mL, 24.96 mmol), EDC (1.91 g, 9.984 mmol) and DMAP (30.5 mg). The reaction mixture was stirred at rt for 5 hrs. LC-MS showed the reaction was complete. The reaction mixture was concentrated, diluted with EtOAc (100 mL), washed successively with 1N HC aq solution (20 ml), saturated NaHCO₃aq solution (20 mL), water (10 mL), and brine (5 mL), dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by ISCO (40 g gold catridge) using 0-100% EtOAc in hexane as the gradient to give 3-hydroxypropyl (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenoate (1.129 g, 49% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 5.15-5.02 (m, 5H), 4.46 (t, J=5.1 Hz, 1H), 4.06 (t, J=6.6 Hz, 2H), 3.45 (td, J=6.3, 5.1 Hz, 2H), 2.40-2.31 (m, 2H), 2.20 (t, J=7.6 Hz, 2H), 2.08-1.90 (m, 16H), 1.70 (p, J=6.4 Hz, 2H), 1.64 (d, J=1.5 Hz, 3H), 1.56 (m, 15H); MS (EST), 481.3 (M+Na)⁺.

Step 2. To a solution of 3-hydroxypropyl (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenoate (1.12 g, 2.4416 mmol) in anhydrous DCM (12.5 mL) at 0° C. was added TEA (0.68 mL), and a solution of 4-nitrophenyl chloroformate (738 mg) in anhydrous DCM (5 ml) slowly. The reaction mixture was stirred at 0° C. for 40 min, and at room temperature for overnight. The reaction mixture was concentrated to give a residue, which was purified by ISCO (40 gold catridge) eluting with using 0-50% EtOAc in hexane to give 3-(((4-nitrophenoxy)carbonyl)oxy)propyl (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenoate (1.06 g, 70% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 8.34-8.29 (m, 2H), 7.58-7.51 (m, 2H), 5.13-5.01 (m, 5H), 4.32 (t, J=6.3 Hz, 2H), 4.13 (t, J=6.3 Hz, 2H), 2.44-2.34 (m, 2H), 2.21 (t, J=7.6 Hz, 2H), 2.07-1.87 (m, 18H), 1.63 (d, J=1.5 Hz, 3H), 1.55 (m, 15H).

Example 16. Preparation of Certain Chemical Moieties and Oligonucleotides Comprising Certain Chemical Moieties

In some embodiments, the present disclosure provides chemical moieties that can be incorporated into oligonucleotides. In some embodiments, a chemical moiety is a targeting moiety. In some embodiments, a chemical moiety is a carbohydrate moiety. In some embodiments, a chemical moiety is a lipid moiety. In some embodiments, chemical moieties may be incorporated into oligonucleotides to improve one or more properties, activities, and/or delivery. Certain chemical moieties, their preparation, and oligonucleotides comprising such moieties are described in the present example. Those skilled in the art appreciate that such chemical moieties may also be incorporated into oligonucleotides having other base sequences, modifications, etc.

Synthesis of 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,48,15-tetraazaicos-3-en-20-oic acid

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Step 1. To a solution of benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,1017-trioxo-3,13-dioxa-5,9,16-triazahenicosan-21-oate (9.0 g, 8.91 mmo) in DCM (100 mL) was added TFA (30.47 g, 267.27 mmol, 19.79 mL) at 0′C. The mixture was stirred at 0-15° C. for 4 hr. The mixture was formed two phase. Lower phase was separated and concentrated under reduced pressure to give a crude, benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate TFA salt (13 g) was obtained as a yellow oil. ¹H NMR (400 MHz, METHANOL-d4) Shift=7.39-7.27 (m, 5H), 5.12 (s, 2H), 3.70-3.63 (m, 13H), 3.32-3.30 (m, 2H), 3.26 (s, 2H), 2.94 (t, J=7.3 Hz, 7H), 2.49-2.38 (m, 9H), 2.23 (t, J=7.4 Hz, 2H), 1.94-1.78 (m, 9H). LCMS: M+H⁺=710.2.

Step 2. To a solution of benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate TFA salt (13 g) in DCM (200 mL) was added DIPEA (15.97 g, 123.58 mmol, 21.53 mL) and HATU (15.51 g, 40.78 mmol). The mixture was stirred at 15° C. for 15 hr. LCMS showed compound 2 was consumed and desired MS was detected. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Agela innoval ods-2 250*80 mm; mobile phase: [water (0.1% TFA)-ACN]; B %: 8%-38%, 20 min) to give compound benzyl 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oate (6.5 g, 52.37% yield) as a brown oil. LCMS: M/2+H⁺=503.1.

Step 3. To a solution of compound benzyl 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oate (5.7 g, 5.68 mmol) in MeOH (30 mL) and H₂O (6 mL) was added LiOH.H₂O (1.67 g, 39.73 mmol). The mixture was stirred at 15° C. for 2 hr. LCMS showed compound 3 was consumed and desired MS was detected. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 250*50 mm*10 um: mobile phase: [water (0.1% TFA)-ACN]; B %: 0%-25%, 20 min). 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oic acid (2.09 g, 2.25 mmol, 40% yield) was obtained as a yellow gum. ¹HNMR (400 MHz, DMSO-d6) Shift=8.07 (br t, J=5.7 Hz, 3H), 7.75 (br t, J=5.0 Hz, 3H), 7.08 (s, 1H), 3.63-3.45 (m, 12H), 3.09 (q, J=6.1 Hz, 11H), 2.88 (br d, J=15.3 Hz, 36H), 2.29 (br t, J=6.4 Hz, 6H), 2.18 (t, J=7.5 Hz, 2H), 2.12-2.06 (m, 2H), 1.65 (br t, J=6.6 Hz, 8H). ¹³CNMR (101 MHz, DMSO-d6) Shift=173.10, 170.88, 169.27, 159.88, 157.61, 157.27, 156.93, 156.58, 119.48, 116.56, 113.63, 110.70, 67.13, 66.27, 58.46, 40.77, 34.82, 34.34, 33.88, 31.87, 28.23, 19.66, 0.00. LCMS: M+H⁺=915.7, purity: 98.265%.

Synthesis of 5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid

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Step 1. A mixture of phenylmethanol (864.10 g, 7.99 mol), compound 1 (100 g, 998.85 mmol), and cation exchange resin (1.92 g, 998.85 mmol.) was stirred at 75° C. with N₂for 4 hr, and then the mixture was stirred at 20° C. for 12 hr under N₂atmosphere. TLC showed compound 1 was consumed completely and two main peaks were detected. The reaction mixture was filtered and then the residue was washed with DCM (500 mL). The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether/Ethyl acetate=10/1 to 3:1) to get compound 2 as a colorless oil (62 g, 29.81% yield). ¹HNMR (400 MHz, CHLOROFORM-d): δ=7.41-7.27 (m, 5H), 5.11 (s, 2H), 3.62 (t, J=6.4 Hz, 2H), 2.39 (t, J=7.3 Hz, 2H), 1.77-1.70 (m, 2H), 1.65-1.51 (m, 2H); TLC (Petroleum ether/Ethyl acetate=3:1) Rf=0.20.

Step 2. To a solution of compound 3 (350 g, 896.66 mmol.) in DMF (2 L) was added acetic acid hydrazine (99.10 g, 1.08 mol). The mixture was stirred at 60° C. for Shr. TLC showed the starting material was consumed. The mixture was concentrated to move the most solvent and water (500 mL) was added, and the mixture was extracted with EtOAc (500 mL*3). The combined organic was dried over Na₂SO₄, filtered and concentrated to get the compound 4 as a brown oil (310 g, crude). ¹HNMR (400 MHz, CHLOROFORM-d): δ=5.49 (t, J=9.9 Hz, 1H), 5.39 (d, J=3.5 Hz, 1H), 5.06-4.99 (m, 1H), 4.84 (dd, J=3.5, 10.1 Hz, 1H), 4.25-4.17 (m, 2H), 4.13-4.02 (m, 2H), 2.04-1.96 (m, 12H): TLC (Petroleum ether/Ethyl acetate=1:1), Rf=0.43.

Step 3. To a solution of compound 4 (310 g, 890.03 mmol.) in DCM (1.5 L) was added 2,2,2-trichloroacetonitrile (1.16 kg, 8.01 mol) at 0° C. The mixture was added drop-wise DBU (271.00 g, 1.78 mol) dissolved in DCM (1 L) at 0° C. The mixture was stirred at 20° C. for 1h. TLC showed the starting material was consumed. The mixture was concentrated to get the crude. The mixture was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=20:1, 10:1, 5:1) to get compound 5 as a yellow oil (90 g, 20.52% yield). ¹HNMR (400 MHz, CDCl₃): δ=8.70 (s, 1H), 6.56 (br d, J=3.1 Hz, 1H), 5.57 (t, J=9.8 Hz, 1H), 5.24-5.08 (m, 2H), 4.35-4.15 (m, 2H), 2.11-1.99 (m, 12H); TLC (Petroleum ether/Ethyl acetate=1:1) Rf=0.31.

Step 4. To a solution compound 5 (89.5 g, 181.66 mmol) and compound 2 (75.66 g, 363.31 mmol) in DCM (800 mL) was added 4A MS (90 g), the mixture was stirred at −30° C. for 30 min. TMSOTf (40.37 g, 181.66 mmol.) was added to the reaction and the mixture was stirred at 25° C. for 3 hr. LCMS and TLC showed the starting material was consumed and LCMS showed the de-Ac MS was found. Sat. NaHCO₃(aq., 100 mL) was added and the mixture was extracted with DCM (150 mL*3). The combined organic was dried over Na₂SO₄, filtered and concentrated to get the crude. Totally got the mixture of benzyl compound 6 and compound 6A (98 g) as a yellow oil, the mixture was used next step directly. TLC (Petroleum ether/Ethyl acetate=2:1) Rf=0.38.

Step 5. The mixture compound 6 and compound 6A (98 g crude) was dissolved in the pyridine (150 mL) and then Ac₂O (150 mL) was added. The mixture was stirred at 20° C. for 12h. TLC showed the starting material was consumed. The mixture was concentrated to get the crude. The mixture was purified by MPLC (silica, Petroleum ether/Ethyl acetate=20:1, 10:1, 05:1) to get compound 6 as a yellow oil (41 g, 41.84% yield) and 12 g crude. ¹HNMR (400 MHz, CDCl₃): δ=7.39-7.31 (m, 5H), 5.23-4.93 (m, 3H), 4.48 (d, J=7.9 Hz, 1H), 4.37-4.22 (m, 1H), 4.17-4.05 (m, 1H), 3.92-3.81 (m, 1H), 3.71-3.63 (m, 1H), 3.48 (td, J=6.3, 9.8 Hz, 1H), 2.44-2.32 (m, 2H), 2.09-1.98 (m, 12H), 1.75-1.53 (m, 4H); LCMS: (M+Na⁺): 561.0; SFC: de %: 100%: TLC (Petroleum ether/Ethyl acetate=3:1) Rf=0.14.

Step 6. To a solution of compound 7 (19.5 g, 36.21 mmol) in EtOAc (200 mL) was added Pd/C (4 g, 17.64 mmol, 10% purity) under N₂atmosphere. The suspension was degassed and purged with H₂for 3 times. The mixture was stirred under H₂(25 Psi) at 20° C. for 2 hr. LCMS and TLC showed the starting material was consumed. The mixture was filtered, the cake was washed with MeOH (50 mL*3) and the combined filter was concentrated to get the crude. The mixture was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=3:1, 1:1, 1:3) to get 5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid 7 as a white solid (23.9 g, 51.72 mmol, 71.41% yield, 97.03% LCMS purity). ¹HNMR (400 MHz, CHLOROFORM-d): δ=5.24-5.17 (m, 1H), 5.12-4.96 (m, 2H), 4.50 (d, J=7.9 Hz, 1H), 4.26 (dd, J=4.7, 12.3 Hz, 1H), 4.20-4.02 (m, 1H), 3.95-3.85 (m, 1H), 3.75-3.64 (m, 1H), 3.55-3.46 (m, 1H), 2.42-2.32 (m, 2H), 2.15-1.99 (m, 12H), 1.76-1.57 (m, 4H); ¹³CNMR (101 MHz, CHLOROFORM-d): δ=178.85, 170.71, 170.30, 169.40, 169.35, 100.71, 72.81, 71.74, 71.25, 69.37, 68.42, 61.94, 33.36, 28.59, 21.09, 20.70, 20.56; LCMS: (M−H+): 447.1. LCMS purity: 97.03%; TLC (Petroleum ether/Ethyl acetate=1:1) Rf=0.03.

Synthesis of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid

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Step 1: To a solution of benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-2-oate (2.15 g, 2.1282 mmol) in DCM (20 mL) was added TFA (5 mL). The reaction mixture was stirred at room temperature for 4 hrs. LC-MS showed the reaction was completed. Solvent was evaporated under reduced pressure to give benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate as a colorless oil. Directly use for next step without purification.

Step 2: To a solution of 5-(((2R,3R,4S,5R6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (3.817 g, 8.51 mmol) in DMF (20 mL) was added DIPEA (5.66 mL, 31.92 mmol) and HATU (2.824 g, 7.45 mmol) followed by benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (2.1282 mmol). The reaction mixture was stirred at room temperature for 3 hrs. Solvent was evaporated under reduced pressure to give a residue, which was purified by ISCO (120 g gold column) eluting with DCM to 50% MeOH in DCM to give 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic benzyl ester (5.08 g, 120%), which containing some impurities. MS (ESI), 1001.4 ((M/2+H)⁺.

Step 3. To a solution of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic benzyl ester (5.08 g) in EtOAc (100 mL) and MeOH (10 mL) was added 10% Pd-C (500 mg). The reaction mixture was stirred at rt for 4 hrs under hydrogen balloon. LC-MS showed the reaction was completed. The reaction mixture was filtered, washed with EtOAc/MeOH, concentrated to give 45,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid (4.60 g, 95%). MS (ESI), 1912 ((M+H).

Synthesis of (S)-5,11,18,22-tetraoxo-6,16-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-28-(5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanamido)-14-oxa-6,10,17,23-tetraazanonacosan-29-oic acid

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Step 1: To a solution of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid (987 mg, 0.520 mmol) in acetonitrile (3 mL) and DCM (10 ml) was added DIPEA (0.27 mL, 1.55 mmol) and HATU (150 mg, 0.400 mmol) followed by L-lysine benzyl ester di-4-toluensulfonate salt (100 mg, 0.170 mmol). The reaction mixture was stirred at room temperature for overnight. Solvent was evaporated under reduced pressure to give a residue, which was purified by ISCO (40 g gold column) eluting with DCM to 30% MeOH in DCM to give (S)-5,11,18,22-tetraoxo-16,16-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-28-(5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanamido)-14-oxa-6,10,17,23-tetraazanonacosan-29-oic benzyl ester (433 mg, 63%), which containing some impurities. MS (ESI), 1342.0 ((M/3+H)⁺.

Step 3. To a solution of (S)-5,11,18,22-tetraoxo-16,16-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-28-(5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanamido)-14-oxa-6,10,17,23-tetraazanonacosan-29-oic benzyl ester (430 mg) in EtOAc (15 mL) and MeOH (3 mL) was added 10% Pd-C (100 mg). The reaction mixture was stirred at it for 4 hrs under hydrogen balloon. LC-MS showed the reaction was completed. The reaction mixture was filtered, washed with EtOAc/MeOH, concentrated to give (S)-5,11,18,22-tetraoxo-16,16-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-28-(5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanamido)-14-oxa-6,10,17,23-tetraazanonacosan-29-oic acid (400 mg, 94%). MS (ESI), 1968 ((M/2+H)⁺.

Synthesis of WV-12567

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To a solution of WV-12566 in 0.4 ml NMP and 0.57 ml water was added DIPEA (20 μL) and a solution of 3-(((4-nitrophenoxy)carbonyl)oxy)propyl (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenoate (20 mg) in NMP (0.40 mL). The reaction mixture was shaken for 12 hours at 35° C. LC-MS showed the starting material was disappeared. The crude product was purified on RP HPLC (C8) using 50 mM TEAA in water and acetonitrile, and desalt to obtain 1.77 mg of the conjugate WV-12567. Deconvoluted mass: 7362; Calculated molecular weight: 7360.

Synthesis of WV-12570

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To a solution of (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenoic acid (turbinaric acid) (6.4 mg, 16 μmol) and HATU (5.4 mg, 14.4 μmol) was added DIPEA (17 μL). The mixture was shaken for 30 min at rt. The reaction mixture was added into a solution of WV 12569 (12.4 mg, 1.6 μmol) in water (0.20 mL) and NMP (0.20 ml) and stirred for 2 hrs at 35° C. LC-MS showed the starting material was disappeared. The crude product was purified on RP (C-8) HPLC using 50 mM TEAA in water and acetonitrile, and desalt to obtain 2.10 mg of the conjugate WV-12570. Deconvoluted mass: 8172; Calculated molecular weight: 8170.

Synthesis of WV-14333

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A solution of 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-21-oic acid (25.4 mg, 9.72 μmol) in acetonitrile (0.50 mL) was added HATU (3.32 mg, 8.75 μmol) and DIPEA (8.5 μL). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was added into a solution of WV-12566 (16.7 mg, 2.43 μmol) in 0.5 mL water. The reaction mixture was stirred at 30° C. for 2 hrs, and LC-MS showed the reaction was complete. The reaction mixture was transferred to the pressure tube, and 4 ml 28-30% ammonium hydroxide was added. The reaction mixture was stirred at 35° C. for overnight. LC-MS showed the reaction was completely de-protected. The crude product was purified by ISCO via 30 g C18 Catridge eluting with 50 mM TEAA to acetonitrile, and desalt to obtain 12.8 mg of the conjugate WV-14333. Deconvoluted mass: 8224; Calculated molecular weight: 8221.

Synthesis of WV-14332

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A solution of 4-nitrophenyl (2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)chroman-6-yl) carbonate (7.24 mg, 12.15 μmol) and DIPEA (8.50 μL) in NMP (0.20 ml) was added to a solution of WV-12566 (16.7 mg, 2.43 μmol) in 0.5 ml DMSO and 0.05 mL water. The reaction mixture was shaken for 3 hours at 40° C. LC-MS showed the reaction was very clean. The crude product was lyophilized, purified on RP (C-8) HPLC using 50 mM TEAA in water and acetonitrile, and desalt to obtain 10 mg of the conjugate WV-14332. Deconvoluted mass: 7335; Calculated molecular weight: 7334.

Synthesis of WV-14346

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A solution of 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oic acid (75.26 mg, 82.34 μmol) in DMF (1.0 mL) was added DIPEA (123 μL, 0.823 mmol) and HATU (28.1 mg, 74.12 μmol). The reaction mixture was stirred at room temperature for 15 minutes. The reaction mixture was added to a solution of WV-12566 (113.22 mg, 16.47 μmol) in 1.50 ml DMSO and 0.50 mL water. The reaction mixture was shaken for 2 hours at rt. LC-MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 84.3 mg of the conjugate WV-14346. Deconvoluted mass: 7772; Calculated molecular weight: 7771.

Synthesis of WV-14335

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Step 1. A solution of 3-(2-Pyridyldithio)-propionic acid-OSu (9.08 mg) in DMF (1.0 mL) was added into a solution of WV-12566 (100 mg, 14.54 in 1.5 ml 0.5 M sodium phosphate buffer (pH=8). The reaction mixture was stirred at room temperature for 1 hr. LC-MS showed that reaction was completed. Diluted with water, and lyophilized to give the desired product.

Synthesis of WV-14335

Step 2. A solution of H-RRQPPRSISSHPC-OH (5.47 mg, 3.6 umol) in DMF (0.85 ml) and 0.1 M sodium bicarbonate (0.15 ml) was added to the above product (step 1) (12 mg, 1.8 μmol) in 0.1M sodium bicarbonate (0.50 mL). The reaction mixture was shaken for 1.5 hours at it. LC-MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 3.0 mg of the conjugate WV-14335. Deconvoluted mass: 8485; Calculated molecular weight: 8482.

Synthesis of WV-14347

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A solution of Ac-CHAIYPRH-OH (3.74 mg, 3.6 μmol) in DMF (0.85 mL) and 0.1 M NaHCO₀(0.15 mL) was added to SPDP oligo (step 1 product of WV-14335) (12 mg, 1.8 μmol) in 0.10 M NaHCO₃(0.50 mL). The reaction mixture was shaken for 1.5 hours at room temperature. LC-MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 8.8 mg of the conjugate WV-14347. Deconvoluted mass: 8003; Calculated molecular weight: 7999.

Synthesis of WV-14348

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A solution of Ac-CTHRPPMWSPVWP-OH (5.88 mg, 3.6 μmol) in DMF (0.85 mL) and 0.1 M NaHCO₃(0.15 mL) was added to SPDP oligo (step 1 product of WV-14335) (12 mg, 1.8 μmol) in 0.10 M NaHCO₃(0.50 mL). The reaction mixture was shaken for 1.5 hours at room temperature. LC-MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 4.1 mg of the conjugate WV-14348. Deconvoluted mass: 8602; Calculated molecular weight: 8597.

Synthesis of WV-15074

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Step 1. A solution of 2,5-dioxopyrrolidin-1-vi 4-((2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylate (8.25 mg, 24.71 μmol) in DMF (0.30 mL) was added to WV-12566 (113.22 mg, 16.47 μmol) and DIPEA (31 μL, 173 μmol) in DMSO (1.50 mL) and water (0.5 mL). The reaction mixture was stirred for 30 minutes at room temperature. LC-MS showed the reaction was almost complete.

Step 2. A solution of Ac-CHAIYPRH-OH (38.47 mg, 37.1 μmol) in DMF (0.50 mL) was added to the above reaction mixture. The reaction mixture was stirred at room temperature for 2 hr. LC_MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 66.0 mg of the conjugate WV-15074. Deconvoluted mass: 8133; Calculated molecular weight: 8132.

Synthesis of WV-15075

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Step 1. A solution of 2,5-dioxopyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylate (1.3 mg, 3.99 μmol) in DMF (0.10 mL) was added to a solution of WV-12566 (16.7 mg, 2.49 μmol) and DIPEA (3.5 μL) in DMSO (0.30 mL) and water (0.10 mL). The reaction mixture was shaken for 1 hr at room temperature. LC-MS showed the reaction was almost complete.

Step 2. A solution of Ac-CTHRPPMWSPVWP-OH (9.8 mg, 6.0 μmol) in DMF (0.20 mL) was added to the above reaction mixture. The reaction mixture was stirred at room temperature for 3 hrs. LC_MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 8.9 mg of the conjugate WV-15075. Deconvoluted mass: 8735; Calculated molecular weight: 8730.

Synthesis of WV-15076

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Step 1. A solution of 2,5-dioxopyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylate (1.3 mg, 3.99 umol) in DMF (0.10 mL) was added to a solution of WV-12566 (16.7 mg, 2.49 μmol) and DIPEA (3.5 μL) in DMSO (0.30 mL) and water (0.10 mL). The reaction mixture was shaken for 1 hr at room temperature. LC-MS showed the reaction was almost complete.

Step 2. A solution of H-RRQPPRSISSHPC-OH (9.1 mg, 6.0 μmol) in DMF (0.20 mL) was added to the above reaction mixture. The reaction mixture was stirred at room temperature for 3 hrs. LC_MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 4.7 mg of the conjugate WV-15076. Deconvoluted mass: 8735; Calculated molecular weight: 8730.

Synthesis of WV-15367

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A solution of 5,1218-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2S3S,4S5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid (13.9 mg 7.29 μmol) in DMF (0.50 mL) was added DIPEA (6.3 μL, 36.4 mol) and HATU (2.3 mg, 6.0 μmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was added to a solution of WV-12566 (16.7 mg, 2.43 μmol) in 0.30 ml DMSO and 0.10 mL water. The reaction mixture was shaken for 2 hours at rt. LC_MS showed the reaction was complete. The reaction mixture was added 28-30% ammonium hydroxide, stirred at 40° C. for 3 hrs. LC_MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 9.2 mg of the conjugate WV-15367. Deconvoluted mass: 8269; Calculated molecular weight: 8263.

Synthesis of WV-15368

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A solution of 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-30-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazatriacontyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)-5-oxopentanoic acid (31.7 mg, 7.29 μmol) in DMF (0.50 mL) was added DIPEA (6.3 μL 36.4 μmol) and HATU (2.3 mg, 6.0 μmol). The reaction mixture was stirred at room temperature for 30 minutes, the reaction mixture was added to a solution of W-12566 (16.7 mg, 2.43 μmol) in 0.30 ml DMSO and 0.10 mL water. The reaction mixture was shaken for 2 hours at t. LC_MS showed the reaction was complete. The reaction mixture was added 28-30% ammonium hydroxide (1.0 mL), stirred at 40° C. for 5 hrs. LC_MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 7.5 mg of the conjugate WV-15368. Deconvoluted mass: 10206; Calculated molecular weight: 10200.

Synthesis of WV-15882

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A solution of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid (102 mg, 53.43 μmol) in DMF (1.0 mL) was added DIPEA (46.8 μL, 266.5 μmol) and HATU (13.5 mg, 35.68 μmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was added to a solution of WV-12566 (122.65 mg, 17.84 μmol) in 1.5 ml DMSO and 0.50 mL water. The reaction mixture was shaken for 1.5 hours at rt. LC_MS showed the reaction was completed. The reaction mixture was added 28-20% ammonium hydroxide (5.0 mL) and stirred at 35° C. for 1.5 hrs. LC_MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 83.8 mg of the conjugate WV-15882. Deconvoluted mass: 8263, Calculated molecular weight: 8264.

Some of the examples reference oligonucleotides which target Malat1. Some of these oligonucleotides are described elsewhere herein and/or below.

Oligo-

nucleotide
Modified Sequence
Naked Sequence
Stereo-chemistry

WV-2809
L001 * Geo * Geo * Geo * Teo * m5Ceo
GGGTCAGCTGC
XXXXXXXXXXX

* A * G * C * T * G * C * C * A * A * T
CAATGCTAG
XXXXXXXXX

* Geo * m5Ceo * Teo * Aeo * Geo

WV-3356
L001Geo * Geo * Geo * Teo * m5Ceo *
GGGTCAGCTGC
OXXXXXXXXXXX

A * G * C * T * G * C * C * A * A * T *
CAATGCTAG
XXXXXXXX

Geo * m5Ceo * Teo * Aeo * Geo

WV-7430
ModO43L001Geo * Geo * Geo * Teo *
GGGTCAGCTGC
OXXXXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A *
CAATGCTAG
XXXXXXXX

A* T* Geo * m5Ceo * Teo * Aeo * Geo

WV-7519
Mod009L001 * Geo * Geo * Geo * Teo *
GGGTCAGCTGC
XXXXXXXXXXX

m5Ceo * A * G * C * T * G * C * C * A *
CAATGCTAG
XXXXXXXXX

A * T * Geo * m5Ceo * Teo * Aeo * Geo

WV-7557
L001mU * Geo * Geo * Geo * Teo *
UGCCAGGCTG
OXXXXXXXXXXX

* C * T * G * G * T * T * A * T * mG *
GTTATGACUC
XXXXXXXX

mA * mC * mU * mC

WV-7558
Mod027L001mU * mG * mC * mC * mA
UGCCAGGCTG
OXXXXXXXXXXX

* G * G * C * T * G * G * T * T * A * T *
GTTATGACUC
XXXXXXXX

mG * mA * mC * mU * mC

WV-7559
Mod028L001mU * mG * mC * mC * mA
UGCCAGGCTG
OXXXXXXXXXXX

* G * G * C * T * G * G * T * T * A * T *
GTTATGACUC
XXXXXXXX

mG * mA * mC * mU * mC

WV-7560
Mod007L001mU * mG * mC * mC * mA
UGCCAGGCTG
OXXXXXXXXXXX

* G * G * C * T * G * G * T * T * A * T *
GTTATGACUC
XXXXXXXX

mG * mA * mC * mU * mC

WV-8448
Mod059L001mU * mG * mC * mC * mA
UGCCAGGCTG
OXXXXXXXXXXX

* G * G * C * T * G * G * T * T * A * T *
GTTATGACUC
XXXXXXXX

mG * mA * mC * mU * mC

WV-8927
Mod053L001mU * mG * mC * mC * mA
UGCCAGGCTG
OXXXXXXXXXXX

* G * G * C * T * G * G* T * T * A * T *
GTTATGACUC
XXXXXXXX

mG * mA * mC * mU * mC

WV-8929
Mod057L001mU * mG * mC * mC * mA
UGCCAGGCTG
OXXXXXXXXXXX

* G * G * C * T * G * G * T * T * A * T *
GTTATGACUC
XXXXXXXX

mG * mA * mC * mU * mC

WV-8930
Mod058L001mU * mG * mC * mC * mA
UGCCAGGCTG
OXXXXXXXXXXX

* G * G *C * T * G * G * T * T * A * T *
GTTATGACUC
XXXXXXXX

mG * mA * mC * mU * mC

WV-8931
Mod009L001mU * mG * mC * mC * mA
UGCCAGGCTG
OXXXXXXXXXXX

* G * G *C * T * G * G * T * T * A * T *
GTTATGACUC
XXXXXXXX

mG * mA * mC * mU * mC

WV-8934
Mod050L001mU * mG * mC * mC * mA
UGCCAGGCTG
OXXXXXXXXXXX

* G * G * C * T * G * G * T * T * A * T *
GTTATGACUC
XXXXXXXX

mG * mA * mC * mU * mC

WV-9385
Mod066L001mU * mG * mC * mC * mA
UGCCAGGCTG
OXXXXXXXXXXX

* G * G * C * T * G * G * T * T * A * T *
GTTATGACUC
XXXXXXXX

mG * mA * mC * mU * mC

WV-9390
Mod074L001m1U * mG * mC * mC * mA
UGCCAGGCTG
OXXXXXXXXXXX

* G * G * C * T * G * G * T * T * A * T *
GTTATGACUC
XXXXXXXX

mG * mA * mC * mU * mC

WV-13809
Mod0971001mU *
UGCCAGGCTG
OSOOOSSRS

SGeom5Ceom5CeomA * SG * SG * RC *
GTTATGACUC
SRSSRSSSSSS

ST * SG * RG * ST * ST * RA * ST *

SmG * SmA * SmC * SmU * SmC

WV-27145
mU * SGCCmA * SG * SG * RC *
UGCCAGGCTG
SOOOSSRSnXR

STn001G * RG * ST * ST * RA * ST
GTTATGACUC
SSRSSSSSSS

* SmG * SmA * SmC * SmU * SmC *
U

SfU

The Modifications (e.g., designated by Mod followed by a number, such as Mod097, Mod074, etc.) are described in the legend to Table A11 or elsewhere herein.

Synthesis of WV-13809

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A solution of 4-nitrophenyl (2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl))chroman-6-yl) carbonate (activated vitamin E) (15 mg, 25 μmol) and DIPEA (21 μL) in NMP (0.20 ml) was added to a solution of WV-9696 in 0.5 ml DMSO and 0.05 ml water. The reaction mixture was shaken for 2 hrs at 50° C. LC-MS showed the reaction was completed. The crude product was lyophilized, purified on RP (C-8) HPLC using 50 mM TEAA in water and acetonitrile, and desalt to obtain 4.90 mg of the conjugate WV-13809. Deconvoluted mass: 7451; Calculated molecular weight: 7451.

Synthesis of WV-14349

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A solution of 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-916-dioxo-12-oxa-2,48,15-tetraazaicos-3-en-20-oic acid (19.61 mg, 21.45 μmol) in DMF (0.30 mL) was added DIPEA (75 μL) and HATU (7.32 mg, 19.31 μmol). The reaction mixture was stirred at rom temperature for 20 minutes. The reaction mixture was added to a solution of WV-9696 (30 mg, 4.29 μmol) in 0.4 ml DMSO and 0.10 mL water. The reaction mixture was shaken at rt for overnight. LC_MS showed the reaction was not complete. A solution of 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,815-tetraazaicos-3-en-20-oic acid (10 mg) in DMF (0.10 mL) was added DIPEA (38 μL) and HATU (3.7 mg). The reaction mixture was stirred at room temperature for 20 minutes. The reaction mixture was added into the above the reaction mixture with WV-9696. The reaction mixture was stirred at 30° C. for 2 hrs. LCMS showed the reaction was completed. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 9.1 mg of the conjugate WV-14349. Deconvoluted mass: 7893; Calculated molecular weight: 7889.

Synthesis of WV8448

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To solution of 4, 10, 17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R, 4S, 5R, 6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)methyl)-1-(((2R,3R,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-21-oic acid (57 mg, 21.8 μmol), HATU (7.5 mg, 19.6 μmol) and DIPEA (14.6 mg, 109 μmol) in DMF (2.0 mL) was stirred at room temperature for 15 minutes. To this solution was added 75 mg (10.9 μmol) of WV7557 in 1 ml water. Reaction mixture was stirred for 60 minutes to obtain the desired product. This product was heated at 40° C. with NH₄OH for 3 hrs. LC_MS showed the reaction was completed. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 39.73 mg of the conjugate WV-8448. Deconvoluted mass: 8233; Calculated molecular weight: 8227.

Synthesis of WV8927

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To a solution of gambogic acid (21 mg, 33.6 μmol) in 2 ml dry DMF was added HATU (11.5 mg, 30.2 μmol) and DIPEA (3.6 mg, 28 μmol) and vortexed well. This solution was added WV7557 (42 mg, 5.6 μmol) in water (1 ml) and shaken for 4 hours. LC-Analysis indicated product formation, but starting material remained. Another 6 six equivalents of Gambogic acid-HATU complex (same amount used initially) was added and shaken well for 2 hours. LC analysis indicated more product formation. The reaction mixture was diluted with water (10 ml). Excess gambogic acid precipitated out. This precipitate was filtered off and the crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 19 mg of the conjugate WV-8927. Deconvoluted mass: 7496; Calculated molecular weight: 7492.

Synthesis of WV-7558

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To a solution of 4-sulfamoylbenzoic acid (7.3 mg, 36 μmol) in DMF (2.0 mL) was added HATU (12.4 mg, 32.7 μmol) and DIPEA (46 mg, 360 μmol) and vortexed. After 2 minutes WV7557 (50 mg, 7.27 μmol) in 1 ml water was added and shaken well. After 60 minutes the reaction mixture was diluted with water (5 ml) and filtered. The filtrate was purified by RP column chromatography (C-18) and desalted to obtain the product (17 mg). Mass calculated: 7064; Deconvoluted Mass: 7068.

Synthesis of WV-7559

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To a solution of 4-oxo-4-((4-sulfamoylphenethyl)amino)butanoic acid (8.7 mg, 29 μmol) in DMF (2.0 mL) was added HATU (9.9 mg, 26 μmol) and DIPEA (37 mg, 290 μmol) and vortexed. After 2 minutes WV7557 (40 mg, 5.81 μmol) in 1 ml water was added and shaken well. After 30 minutes the reaction mixture was diluted with water (5 ml) and filtered. The filtrate was purified by RP column chromatography (C-18) and desalted to obtain the product (13 mg). Mass calculated: 7163: Deconvoluted Mass: 7166.

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To a solution of WV7557 (62 mg, 9 μmol) in water (0.5 ml) and DMF (2.5 ml) was added DIPEA (11.6 mg, 90 μmol) and stirred well. To this solution was added 3-(2-Pyridyldithio)-propionic acid-OSu (4 mg, 12.6 μmol) and stirred well for 2h. The crude product was diluted with water and purified on ISCO (C18 column) using 50 mM TEAA and acetonitrile. Amount of product obtained: 46 mg.

Synthesis of WV-8929

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To a solution of the oligo (WV7557 derivative, 23.5 mg, 33 mol) in water DMF (2 ml -20+1 ml) mixture was added DIPEA (8.52 mg, 66 μmol), and vortexed for 5 minutes. To this solution was added H-RRQPPRSISSHPC-OH (10 mg 6.6 μmol) and again vortexed for 5 minutes. After 12 hours, the reaction mixture was analyzed by LC-MS. LC_MS showed the reaction was completed. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 14 mg of the conjugate WV-8929. Deconvoluted mass: 8496; Calculated molecular weight: 8490.

Synthesis of WV-8930

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To a solution of the oligo (WV7557 derivative, 23.5 mg, 3.3 μmol) in water-DMF (2 ml+1 ml) mixture was added DIPEA (8.52 mg, 66 μmol) and vortexed for 5 minutes. To this solution was added H-Arg-Arg-Cys-OH (4 mg, 10 μmol) and vortexed for 5 minutes. After 12 hours, the reaction mixture was analyzed by LC-MS. LC_MS showed the reaction was completed. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 5 mg of the conjugate WV-8930. Deconvoluted mass: 7405; Calculated molecular weight: 7401.

Synthesis of WV8931

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To a solution of WV7557 (20 mg, 2.91 μmol) in 0.47 ml water was treated with DIPEA (3.76 mg, 29.1 μmol) and vortexed well for 5 minutes. To this solution was added a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl (4-nitrophenyl) carbonate (activated cholesterol derivative) (10.50 mg, 19 μmol) in NMP (1.0 ml). The solution turned slightly yellowish. It was shaken at 40 degrees for 12 hours. A bright yellow solution was obtained. LC-MS analysis indicated product formation. This solution was diluted to 10 ml using water, filtered and purified on a RP-HPLC using a C-8 column and desalted. Amount of product obtained: 18 mg; Deconvoluted mass: 7298; Calculated molecular weight: 7293.

Synthesis of WV8934

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L-carnitine (3 mg, 17.5 μmol) and HATU (6 mg, 16 μmol) were mixed together and made in to a 1 ml solution in DMF. DIPEA (5.7 mg, 44 μmol) was added and stirred well for 3 minutes. To this solution was added a solution of WV-7557 (30 mg, 4.4 mmol) in 0.5 ml water and stirred well for 30 minutes. LC-MS analysis of the solution indicated product formation. But starting oligo was present in the reaction mixture. 4 equivalents more L-carnitine/HATU complex was added again and stirred well for 2h. The reaction mixture was diluted with water and the crude product was purified on a RP (C-18) column to obtain the product. Amount of product obtained: 12 mg, Calculated mass: 7025; De-convoluted mass: 7029.

Synthesis of WV-9390

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To solution of 5-oxo-5-(4-(4-((2,8,12,19,25-pentaoxo-14,14-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-29-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-16-oxa-3,7,13,20,24-pentaazanonacosyl)amino)-6-((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy -6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-30 (((2S,3S,4S,5R,6R)-3,4,5-triacetoxy -6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazatriacontyl)amino)-1,3,5-triazin-2-yl)piperazin-1-yl)pentanoic acid (15 mg, 3.5 μmol) and HATU (1.33 mg, 35 μmol) in DMF (1.0 ml) was added DIPEA (4.5 mg, 35 μmol) and vortexed for 2 minutes. To this solution was added WV7557 (12 mg, 1.74 μmol) in water (0.5 ml) and shaken for 60 minutes. 5 ml water was added to it and the solvent was removed under vacuum. The crude product was purified on a RP column (C-8) obtain acetylated product (Mass calculated: 10207, Deconvoluted mass: 10212). This product was dissolved in 5 ml 30% ammonium hydroxide solution and heated at 40 degrees Celsius for 6 hours. Solvent was removed under vacuum and the crude product was purified on a RP column (C-8) to obtain the product. Amount of product obtained (10 mg). Calculated Mass: 10205; Deconvoluted Mass obtained: 10205.

Synthesis of WV 9430

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To a solution of 1,7,14-trioxo-12,12-bis((3-oxo-3-((3-(4-sulfamoylbenzamido)propyl)amino)propoxy)methyl)-1-(4-sulfamoylphenyl)-10-oxa-2,6,13-triazaoctadecan-8-oic acid (5.14 mg, 1.45 μmol) in DMF was added HATU (1.5 mg, 3.96 μmol) and DIPEA (2 mg, 15 μmol). The reaction mixture was stirred at room temperature for 2 minutes. A solution of WV7557 in 0.4 ml water was added and shaken well. After 30 minutes the reaction mixture was diluted with water (5 ml) and filtered. The filtrate was purified by RP column chromatography (C-18) and desalted to obtain the product WV-9430 (6 mg). Mass calculated: 8032; Deconvoluted Mass: 8031.

Synthesis of WV-9385

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WV7557 (48 mg, 6.9 μmol) was dissolved in 1 ml NMP and 0.5 ml water. DIPEA (14 mg, 103.5 μmol) was added to this solution. Vortexed for 5 minutes. To this solution was added 3-(((4-nitrophenoxy)carbonyl)oxy)propyl stearate (14 mg, 27.6 μmol) in 1 ml NMP. The reaction mixture was filtered and the filtrate was purified by RP column chromatography (C-8) to obtain the product. The purified material was desalted and 11 mg of product was obtained. Mass calculated: 7250; Deconvoluted Mass: 7254.

Synthesis of WV-7560

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12,12-bis((3-((3-(4-methoxybenzamido)propyl)amino)-3-oxopropoxy)methyl)-1-(4-methoxyphenyl)-1,7,14-trioxo-10-oxa-2,6,13-triazapentacosan-25-oic acid (triantennary anisamide) (32.5 mg, 29 μmol), HATU (10 mg, 26.1 μmol) and DIPEA (28 mg, 58 μmol) were dissolved in 2 ml DMF. After 2 minutes WV7557 (100 mg. 15 μmol) in 1 ml water was added and shaken well. After 60 minutes the reaction mixture was diluted with water (5 ml) and filtered. The filtrate was purified by RP column chromatography (C-8) and desalted to obtain the product (55 mg). Mass calculated: 7983; Deconvoluted Mass: 7987.

Synthesis of WV-7408

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A suspension of WV 3356 (40 mg, 5.3 μmol) and DIPEA (7 mg, 53 μmol) in 2 ml DMF was vortexed for five minutes. To this suspension was added a solution of 2,5-dioxopyrrolidin-1-yl 4-sulfamoylbenzoate (8 mg, 26.5 μmol)J in 1 ml DMF. The reaction mixture was shaken for 12 hours. Afterwards, the reaction mixture was diluted with 5 ml water and filtered. The filtrate was purified by RP (C-18) column chromatography and desalted to obtain the product (20 mg). Mass calculated: 7596; Deconvoluted mass: 7594.

Synthesis of WV7409

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To a solution of 4-oxo-4-((4-sulfamoylphenethyl)amino)butanoic acid (2.16 mg, 7.2 μmol), HATU (2.32 mg, 6.1 μmol) and DIPEA (3.1 mg, 24 μmol) were dissolved in 1 ml DMF and vortexed. After 2 minutes WV3356 (18 mg, 2.4 μmol) in 0.5 ml water was added and shaken well. After 60 minutes the reaction mixture was diluted with water (5 ml) and filtered. The filtrate was purified by RP column chromatography (C-18) and desalted to obtain the product (9 mg). Mass calculated: 7694; Deconvoluted Mass: 7695.

Synthesis of WV-7430

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To a solution of WV3356 (32 mg, 4.3 μmol) in DMF (2.0 mL) was added DIPEA (5.8 mg, 43 μmol) was added a solution of (R)-3-(((4-nitrophenoxy)carbonyl)oxy)propane-1,2-diyl didodecanoate (11 mg, 17.6 μmol) in acetonitrile (1.0 mL). Reaction mixture was shaken at 40° C. for 12 hours. LC-MS analysis indicated formation of product. The reaction mixture was diluted with water and filtered. The filtrate was purified by RP column chromatography (C-8) to obtain the product. The purified material was desalted and 11 mg of product was obtained. Mass calculated: 7895, Deconvoluted Mass:7896.

Synthesis of WV-7419

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To a suspension of WV-2809 (56 mg, 7.5 μmol, 125 mg support) in DMF (2.0 mL) was added DIPEA (19.3 mg, 150 μmol) and vortexed well for 5 minutes. To this suspension was added perfluorophenyl 18-oxo-18-((4-(N-(2,2,2-trifluoroacetyl)sulfamoyl)phenethyl)amino)octadecanoate (12 mg, 15 μmol) and shaken for 12 hours at room temperature. The solid support was washed with acetonitrile (20 ml×3) and dried. This support was treated with 20% DEA in acetonitrile (1 ml) for 10 minutes, the DEA solution was removed by filtration. The solid support was washed with acetonitrile (20 ml×3) and dried. The solid support was heated with 2 ml of 30% ammonium hydroxide for 12 hours. The support was filtered off and the filtrate was lyophilized to remove the solvent. The crude product was purified by RP column chromatography (C-8) and desalted to obtain the product (7 mg). Mass calculated:7906, Deconvoluted Mass:7909.

Synthesis of WV-7519

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To a suspension of WV2809 (60 mg, 8 μmol, 150 mg support) in 2 ml NMP was added DIPEA (11 mg, 80 μmol) and vortexed well for 5 minutes. To this suspension was added (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl carbonochloridate (15 mg, 33 μmol) and shaken for 12 hours at room temperature. The solid support was washed with acetonitrile (20 ml×3) and dried. This support was treated with 20% DEA in acetonitrile (1 ml) for 10 minutes. The DEA solution was removed by filtration. The solid support was washed with acetonitrile (20 ml×3) and dried. The solid support was heated at 50° C. with 2 ml of 30% ammonium hydroxide for 12 hours. The support was filtered off and the filtrate was lyophilized to remove the solvent. The crude product was purified by RP column chromatography (C-8) and desalted to obtain the product (20 mg). Mass calculated:7840, Deconvoluted mass: 7841.

Synthesis of WV-7422

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To a suspension of WV2809 (56 mg, 7.5 μmol, 125 mg support) in 2 ml DMF was added DIPEA (19.3 mg, 150 μmol) and vortexed well for 5 minutes. To this suspension was added perfluorophenyl 3-(4-(N-(2,2,2-trifluoroacetyl)sulfamoyl)phenyl)propanoate (37 mg, 75 μmol) and shaken for 12 hours at room temperature. The solid support was washed with acetonitrile (20 ml×3) and dried. This support was treated with 20% DEA in acetonitrile (1 ml) for 10 minutes. The DEA solution was removed by filtration. The solid support was washed with acetonitrile (20 ml×3) and dried. The solid support was heated at 50° C. with 2 ml of 30% ammonium hydroxide for 12 hours. The support was filtered off and the filtrate was lyophilized to remove the solvent. The crude product was purified by RP column chromatography (C-8) and desalted to obtain the product (18 mg). Mass calculated:7638, Deconvoluted Mass:7641.

Synthesis of WV-7421

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2-(4-sulfamoylphenyl)acetic acid (17.2 mg, 80 μmol), HATU (28 mg, 76 molμ) and DIPEA (20.6 mg, 160 μmol) in 2 ml NMP was vortexed well for 2 minutes. To this suspension was added WV2809 (60 mg, 8 μmol, 150 mg support) and shaken well for 12 hours at room temperature. The solid support was washed with acetonitrile (20 ml×3) and dried. This support was treated with 20% DEA in acetonitrile (1 ml) for 10 minutes. The DEA solution was removed by filtration. The solid support was washed with acetonitrile (20 ml×3) and dried. The solid support was heated at 50° C. with 2 ml of 30% ammonium hydroxide for 12 hours. The support was filtered off and the filtrate was lyophilized to remove the solvent. The crude product was purified by RP column chromatography (C-18) and desalted to obtain the product (20 mg). Mass calculated:7624, Deconvoluted Mass:7627.

Synthesis of WV-7417

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A suspension of 1,7,14-trioxo-12,12-bis((3-oxo-3-((3-(4-sulfamoylbenzamido)propyl)amino)propoxy)methyl)-1-(4-sulfamoylphenyl)-10-oxa-2,6,13-triazaoctadecan-18-oic acid (40 mg, 34 μmol), HATU (12 mg, 76 μmol) and DIPEA (44 mg, 340 μmol) in 2 ml NMP was vortexed well for 3 minutes. To this suspension was added WV2809 (60 mg, 8 μmol, 150 mg support) and shaken well for 12 hours at 40° C. The solid support was washed with acetonitrile (20 ml×3) and dried. This support was treated with 20% DEA in acetonitrile (1 ml) for 10 minutes. The DEA solution was removed by filtration. The solid support was washed with acetonitrile (20 ml×3) and dried. The solid support was heated at 50° C. with 2 ml of 30% ammonium hydroxide for 12 hours. The support was filtered off and the filtrate was lyophilized to remove the solvent. The crude product was purified by RP column chromatography (C-18) and desalted to obtain the product (10 mg). Mass calculated:8579, Deconvoluted Mass:8577.

Example 17. General Procedure for the Deprotection of Amine

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15.2 g of NHBoc amine was dissolved in dry DCM (100 ml) then TFA (50 ml) was added dropwise at RT. Reaction mixture was stirred at RT overnight. Solvents were removed under reduced pressure then co-evaporated with toluene (2×50 mL) then used for the next step without any further purification. NMR in CD₃OD confirmed the NHBoc deprotection.

Example 18. General Procedure for the Anisamide Formation

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Procedure-A: The crude amine from the previous step was dissolved in a mixture of DCM (100 ml) and Et₃N (10 equ.) at RT. During this process, the reaction mixture was cooled with a water bath. Then 4-Methoxybenzoyl chloride (4 equ) was added dropwise to the reaction mixture under argon atmosphere at RT, stirring continued for 3 h. Reaction mixture was diluted with water and extracted with DCM. Organic layer was extracted with aq. NaHCO₃, 1N HCl, brine then dried with magnesium sulfate evaporated to dryness. The crude product was purified by silica column chromatography using DCM-MeOH as eluent.

Procedure-B: The crude amine (0.27 equ), acid and HOBt (1 equ) were dissolved in a mixture of DCM and DMF (2:1) in an appropriate sized RBF under argon. EDAC.HCl (1.25 equ) was added portion wise to the reaction mixture under constant stirring. After 15 mins, the reaction mixture was cooled to ˜10° C. then DIEA (2.7 equ) was added over a period of 5 mins. Slowly warmed the reaction mixture to ambient temperature and stirred under argon for overnight. TLC indicated completion of the reaction TLC condition, DCM:MeOH (9.5:0.5). Solvents were removed under reduced pressure, then water was added to the residue, and a gummy solid separated out. The clear solution was decanted, and the solid residue was dissolved in EtOAc and washed successively with water, 10% aqueous citric acid, aq. NaHCO₃, followed by saturated brine. The organic layer was separated and dried over magnesium sulfate. Solvent was removed under reduced pressure then the crude product was purified with silica column to get the pure product.

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Anisamide was obtained from the amine in 32% yield over 2 steps using the above procedure-B: ¹H NMR (CDCl₃): δ=7.74 (d, 6H), 7.44 (t, 2H), 7.34 (t, 1H), 7.26 (m, 5H), 7.05 (m, 3H), 6.83 (d, 6H), 6.46 (s, 1H), 5.01 (s, 2H), 3.75 (s, 9H), 3.57 (m, 12H), 3.37 (m, 6H), 3.25 (m, 6H), 2.31 (m, 8H), 2.11 (m, 2H), 1.84 (m, 2H), 1.62 (m, 6H) ppm.

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Anisamide was obtained from the amine in 57% yield over 2 steps using the above procedure-A: ¹H NMR (CDCl₃): δ=7.75 (m, 3H), 7.73 (d, 6H), 7.43 (t, 3H), 7.25 (m, 5H), 6.80 (d, 6H), 6.51 (brs, 1H), 5.01 (s, 2H), 3.72 (s, 9H), 3.58 (m, 6H), 3.21 (m, 12H), 2.33 (t, 3H), 2.25 (t, 2H), 2.02 (t, 2H), 1.64 (q, 6H), 1.52 (p, 2H), 1.41 (q, 2H), 1.12 (m, 12H) ppm.

General Procedure for Debenzylation.

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The benzyl ester (10 g) was dissolved in a mixture of ethyl acetate (100 ml) and methanol (25 ml) then Pd/C, 1 g (10% palladium content) was added under argon atmosphere then the reaction mixture was vacuumed and flushed with hydrogen and stirred at RT under H₂atmosphere for 3 h. TLC indicated completion of the reaction, filtered through pad of celite and washed with methanol, evaporated to dryness to yield a foamy white solid.

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Yield 98% ¹H NMR (CD₃OD): δ=8.35 (t, 1H), 8.01 (t, 1H), 7.82 (d, 6H), 7.27 (d, 1H), 6.99 (d, 6H), 3.85 (s, 9H), 3.68 (m, 12H), 3.41 (m, 6H), 3.29 (m, 6H), 2.42 (m, 6H), 2.31 (q, 2H), 2.21 (td, 21), 1.80 (m, 8H) ppm.

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Yield 94%, ¹H NMR (CD₃OD): δ=8.36 (t, 2H), 8.02 (t, 2H), 7.82 (d, 6H), 7.23 (d, 1H), 6.98 (d, 6H), 3.85 (s, 911), 3.70 (s, 6H), 3.67 (t, 6H), 3.41 (q, 4H), 3.28 (m, 8H), 2.42 (t, 6H), 2.27 (t, 2H), 2.13 (t, 2H), 1.79 (p, 6H), 1.54 (dp, 4H), 1.25 (m, 12H) ppm.

Example 19. Timelines for ‘Pre-Differentiation’ of Patient Myoblasts for Gymnotic Dosing

Various technologies, e.g., those described in U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, 9,598,458, US 2015/0211006, US 2017/0037399, WO 2017/015555, WO 2017/192664, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192679, and WO 2017/210647, etc., can be utilized in accordance with the present disclosure to assess properties and/or activities of technologies of the present disclosure. In some embodiments, technologies of the present disclosure, e.g., oligonucleotides and compositions and methods of use thereof, demonstrate unexpectedly superior results compared to a suitable reference technology (e.g., a technology based on a stereorandom composition of oligonucleotides having the same base sequence but no neutral and/or cationic internucleotidic linkages at physiological pH). Described below are example technologies that can be useful for assessing properties and/or activities of oligonucleotides described in the present disclosure. Those skilled in the art understand that conditions illustrated below may be varied/modified, and additionally and/or alternatively, other suitable reagents, temperatures, conditions, time periods, amounts, etc., may be utilized in accordance with the present disclosure.

Maintenance of Patient Derived Myoblast Cell Lines:

DMD Δ52 and DMD Δ45-52 myoblast cells were maintained in complete Skeletal Muscle Growth Medium (Promocell, Heidelberg, Germany) supplemented with 5% FBS, 1× Penicillin-Streptomycin and 1× L-Glutamine. Flasks or plates were coated with Matrigel:DMEM solution (1:100) for a suitable period of time, e.g., 30 mins, after which Matrigel:DMEM solution was removed via aspiration before seeding of cells in complete Skeletal Muscle Growth Medium.

Standard Dosing Procedure (0 Days Pre-Differentiation)

On Day 1: Coat suitable cell growth containers, e.g., 6-well plates or 24-well plates, with Matrigel: DMEM Solution. Incubate at a condition, e.g., 37° C., 5% CO₂for a suitable period of time, e.g., 30 mins. Aspirate, and seed a suitable number of cells to cell growth containers, e.g., 150K cells/well in a total of 1500 μl of complete growth medium in 6-well plate, and 30K cells/well in 500 ul of growth medium in a 24-well plate. Incubate at a suitable condition for a suitable period of time, e.g., 37° C., 5% CO₂overnight.

On Day 2: Prepare a suitable Differentiation medium, e.g., DMEM+5% Horse Serum+10 μg/ml Insulin. Prepare suitable oligonucleotide dilutions in Differentiation Medium, e.g., serial dilutions of 30 uM, 10 uM, 3.33 uM, 1.11 uM, 0.37 uM. Aspirate growth medium off of adherent cells, and add oligonucleotide:Differentiation Medium solution to cells. Oligonucleotides remain on cells (no media change) until cell harvesting.

On Day 6: Obtain RNA. In a typical procedure, a suitable number of cells, e.g., cells from wells of a 24-well plate, were washed. e.g., with cold PBS, followed by addition of a suitable amount of a reagent for RNA extraction and storage of sample/RNA extraction, e.g., 500 ul/well TRIZOL in 24-well plate and freezing plate at −80° C. or continuing with RNA extraction to obtain RNA.

On Day 8: Obtain protein. In a typical procedure, a suitable number of cells, e.g., cells in wells of 6-well plate, were washed, e.g., with cold PBS. A suitable amount of a suitable lysis buffer was then added—e.g., in a typical procedure, 200 ul/well of RIPA supplemented with protease inhibitors for a 6-well plate. After lysis the sample can be stored, e.g., freezing at −80° C., or continue with protein extraction.

Other suitable procedures may be employed, for example, those described below. As appreciated by those skilled in the art, many parameters, such as reagents, temperatures, conditions, time periods, amounts, etc., may be modified.

4 Days Pre-Differentiation Dosing Procedure

On Day 1: Coat 6-well plates or 24-well plates with Matrigel: DMEM Solution. Incubate at 37° C., 5% CO₂for 30 mins. Aspirate, seed 150K cells/well in a total of 1500 μl of complete growth medium in 6-well plate, and 30K cells/well in 500 ul of growth medium in a 24-well plate. Incubate at 37° C., 5% CO₂overnight.

On Day 2: Prepare Differentiation medium as follows: DMEM+5% Horse Serum+10 μg/ml Insulin. Aspirate Growth Media and replace with Differentiation Media.

On Day 6: Cells have differentiated for 4 days. Prepare oligonucleotide dilutions in Differentiation Medium, for example serial dilutions of 30 uM, 10 uM, 3.33 uM, 1.11 uM, 0.37 uM. Aspirate Differentiation medium off of adherent cells, and add oligonucleotide:Differentiation Medium solution to cells. Oligonucleotides remain on cells (no media change) until cell harvesting.

On Day 10: Wash cells in 24-well plate with cold PBS, add 500 ul/well TRIZOL in 24-well plate and freeze plate at −80° C. or continue with RNA extraction.

On Day 12: Wash cells in 6-well plate with cold PBS. Add 200 ul/well of RIPA supplemented with protease inhibitors. Freeze plate at −80° C. or continue with protein Extraction.

7 dais Pre-Differentiation Dosing Procedure

On Day 2: Prepare Differentiation medium as follows: DMEM+5% Horse Serum+10 μg/ml Insulin. Aspirate Growth Media and replace with Differentiation Media.

On Day 9: Cells have differentiated for 7 days. Prepare oligonucleotide dilutions in Differentiation Medium, for example serial dilutions of 30 uM, 10 uM, 3.33 uM, 1.11 uM, 0.37 uM. Aspirate Differentiation medium off of adherent cells, and add oligonucleotid:Differentiation Medium solution to cells. Oligonucleotides remain on cells (no media change) until cell harvesting.

On Day 13: Wash cells in 24-well plate with cold PBS, add 500 ul/well TRIZOL in 24-well plate and freeze plate at −80° C. or continue with RNA extraction.

On Day 15: Wash cells in 6-well plate with cold PBS. Add 200 ul/well of RIPA supplemented with protease inhibitors. Freeze plate at −80° C. or continue with protein extraction.

10 Days Pre-Differentiation Dosing Procedure

On Day 2: Prepare Differentiation medium as follows: DMEM+5% Horse Serum+10 μg/ml Insulin. Aspirate Growth Media and replace with Differentiation Media.

On Day 12: Cells have differentiated for 10 days. Prepare oligonucleotide dilutions in Differentiation Medium, for example serial dilutions of 30 uM, 10 uM, 3.33 uM, 1.11 uM, 0.37 uM. Aspirate Differentiation medium off of adherent cells, and add oligonucleotide:Differentiation Medium solution to cells. Oligonucleotides remain on cells (no media change) until cell harvesting.

On Day 16: Wash cells in 24-well plate with cold PBS, add 500 ul/well TRIZOL in 24-well plate and freeze plate at −80° C. or continue with RNA extraction.

On Day 18: Wash cells in 6-well plate with cold PBS. Add 200 ul/well of RIPA supplemented with protease inhibitors. Freeze plate at −80° C. or continue with protein extraction.

Example 20. Multi-Exon Skipping Assay

The assay described herein can be adapted to detect any gene's splice-variants with frequency of each variant (quantification). DMD Exon43-Exon64 is used as an example.

Among other things, a unique feature of this assay is that an unique-molecular-identifier (UMI) is introduced in the reverse transcription primers with an unique PCR handler sequence (this can be any sequence without homology to genomic or transcriptome sequences). Therefore, each cDNA has its unique UMI (bar-code) that can be used in later sequencing analysis to eliminate PCR and sequencing bias toward smaller amplicons.

In a typical procedure, the steps include: Reverse RT primer containing a PCR handle at 5′-end, then 8-16 sequences of randomly incorporated nucleotides that create UMI/bar code and reverse complement sequence in exon 64 (Reverse RT primer in table), was used to prime the reverse transcription by a RT kit (e.g., SuperScript IV, ThermoFisher, Cambridge, Mass.). Then primary and nested PCR were run to amplify gene-specific fragments used for PacBio long range sequencing or Oxford Nanopore MinION platform.

The NGS sequences (BAM files) were mapped to reference sequence (DMD for example) to identify splice variants (exon junctions). The UMI were counted in each splice variant, and frequency of variant was calculated by UMI counts in each variant divided by total UMI counts in all variants.

An illustration of this process is shown in FIG. 2.

Example Reverse RT primer:

5'-CAGTGGTATCAACGCAGAGTACG-NNNNNNNN-

ctgagaatctgacacagg-3'

5′-capital letter=N1 binding sequence (nested secondary)

N . . . N=UMI

underline=gene specific sequence in exon64

Forward primer (exon 43):

Fnest=5′-gaagctctctcccagcttgat-3′

Among other things, the present disclosure provides the following Example Embodiments:

1. An oligonucleotide composition, comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers, and

4) pattern of backbone phosphorus modifications,

wherein:

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages; and

2. The composition of any one of the preceding embodiment, wherein the transcript is a Dystrophin transcript.

3. The composition of any one of the preceding embodiments, wherein splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.

4. The composition of any one of the preceding embodiments, wherein each chiral internucleotidic linkage of the oligonucleotides of the plurality is independently a chirally controlled internucleotidic linkage.

5. The composition of any one of the preceding embodiments, wherein each chiral modified internucleotidic linkage independently has a stereopurity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% at its chiral linkage phosphorus.

6. The composition of any one of the preceding embodiments, wherein the base sequence is or comprises or comprises 15 contiguous bases of the base sequence of any oligonucleotide in Table A1.

7. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage.

8. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage which is a neutral internucleotidic linkage.

9. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one neutral internucleotidic linkage which is or comprises a triazole, neutral triazole, alkyne, or a cyclic guanidine.

10. The composition of any one of the preceding embodiments, wherein the oligonucleotide type comprises any of: cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Gambogic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).

11. The composition of any one of the preceding embodiments, wherein the oligonucleotide type is any oligonucleotide listed in Table A1.

12. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

wherein:

13. The composition of any one of the preceding embodiments, wherein the transcript is a Dystrophin transcript.

14. The composition of any one of the preceding embodiments, wherein the exon is DMD exon 45, 51 or 53 or multiple DMD exons, and wherein the splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.

15. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least one Sp.

16. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least one Rp.

17. The composition of any one of the preceding embodiments, wherein the composition is a chirally pure composition.

18. The composition of any one of the preceding embodiments, wherein each chiral modified internucleotidic linkage independently has a stereopurity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% at its chiral linkage phosphorus.

19. The composition of any one of the preceding embodiments, wherein the base sequence is or comprises or comprises 15 contiguous bases of the base sequence of any oligonucleotide in Table A1.

20. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage.

21. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage which is a neutral internucleotidic linkage.

22. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one neutral internucleotidic linkage which is or comprises a triazole, neutral triazole, alkyne, or a cyclic guanidine.

23. The composition of any one of the preceding embodiments, wherein the oligonucleotide type comprises any of: cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Gambogic acid; Cleavable lipid (1,2-dilaurin and ester bond): Insulin receptor ligand; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).

24. The composition of any one of the preceding embodiments, wherein the oligonucleotide type is any oligonucleotide listed in Table A1.

25. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages; and

3) pattern of backbone phosphorus modifications,

wherein:

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-negatively charged internucleotidic linkages;

26. The composition of any one of the preceding embodiments, wherein the transcript is a Dystrophin transcript.

27. The composition of any one of the preceding embodiments, wherein the exon is DMD exon 45, 51, or 53 or multiple DMD exons, and the splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.

28. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage is independently an internucleotidic linkage at least 50% of which exists in its non-negatively charged form at pH 7.4.

29. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage is independently a neutral internucleotidic linkage, wherein at least 50% of the internucleotidic linkage exists in its neutral form at pH 7.4.

30. The composition of any one of the preceding embodiments, wherein the neutral form of each non-negatively charged internucleotidic linkage independently has a pKa no less than 8, 9, 10, 11, 12, 13, or 14.

31. The composition of any one of the preceding embodiments, wherein the neutral form of each non-negatively charged internucleotidic linkage, when the units which it connects are replaced with —CH₃, independently has a pKa no less than 8, 9, 10, 11, 12, 13, or 14.

32. The composition of any one of the preceding embodiments, wherein the reference condition is absence of the composition.

33. The composition of any one of the preceding embodiments, wherein the reference condition is presence of a reference composition.

34. The composition of any one of the preceding embodiments, wherein the reference composition is an otherwise identical composition wherein the oligonucleotides of the plurality comprise no chirally controlled internucleotidic linkages.

35. The composition of any one of the preceding embodiments, wherein the reference composition is an otherwise identical composition wherein the oligonucleotides of the plurality comprise no non-negatively charged internucleotidic linkages.

36. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises one or more backbone linkages selected from phosphodiester, phosphorothioate and phosphodithioate linkages.

37. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise one or more sugar modifications.

38. The composition of any one of the preceding embodiments, wherein the sugar modifications comprise one or more modifications selected from: 2′-O-methyl, 2′-MOE, 2′-F, morpholino and bicyclic sugar moieties.

39. The composition of any one of the preceding embodiments, wherein one or more sugar modifications are 2′-F modifications.

40. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise a 5′-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety.

41. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise a 3′-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety.

42. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise a middle region between the 5′-end region and the 3′-region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotidic units comprising a phosphodiester linkage.

43. The composition of any one of the preceding embodiments, wherein the base sequence is or comprises or comprises 15 contiguous bases of the base sequence of any oligonucleotide in Table A1.

44. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage.

45. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage which is a neutral internucleotidic linkage.

46. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one neutral internucleotidic linkage which is or comprises a triazole, neutral triazole, alkyne, or a cyclic guanidine.

47. The composition of any one of the preceding embodiments, wherein the oligonucleotide type comprises any of: cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Gambogic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).

48. The composition of any one of the preceding embodiments, wherein the oligonucleotide type is any oligonucleotide listed in Table A1.

49. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages; and

3) pattern of backbone phosphorus modifications,

wherein:

oligonucleotides of the plurality comprise:

1) a 5′-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety;

2) a 3′-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety; and

3) a middle region between the 5′-end region and the 3′-region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotidic units comprising a phosphodiester linkage.

50. The composition of embodiment 43 or 49, wherein the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered in that level of skipping of an exon is increased relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

51. The composition of any one of the preceding embodiments, wherein the transcript is a Dystrophin transcript.

52. The composition of any one of the preceding embodiments, wherein the exon is DMD exon 45, 51, or 53 or multiple DMD exons, and the splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.

53. The composition of any one of the preceding embodiments, wherein the 5′-end region comprises 1 or more nucleoside units not comprising a 2′-F modified sugar moiety.

54. The composition of any one of the preceding embodiments, wherein the 3′-end region comprises 1 or more nucleoside units not comprising a 2′-F modified sugar moiety.

55. The composition of any one of the preceding embodiments, wherein the middle region comprises 1 or more nucleotidic units comprising no phosphodiester linkage.

56. The composition of any one of the preceding embodiments, wherein the first of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety and a modified internucleotidic linkage of the 5′-end is the first, second, third, fourth or fifth nucleoside unit of the oligonucleotide from the 5′-end, and the last of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2′-F modified sugar moiety and a modified internucleotidic linkage of the 3′-end is the last, second last, third last, fourth last, or fifth last nucleoside unit of the oligonucleotide.

57. The composition of any one of the preceding embodiments, wherein the 5′-end region comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2′-F modified sugar moiety.

58. The composition of any one of the preceding embodiments, wherein the 5′-end region comprising 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2′-F modified sugar moiety.

59. The composition of any one of the preceding embodiments, wherein the 3′-end region comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2′-F modified sugar moiety.

60. The composition of any one of the preceding embodiments, wherein the 3′-end region comprising 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2′-F modified sugar moiety.

61. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage between two nucleoside units comprising a 2′-F modified sugar moiety in the 5′-end region is independently a modified internucleotidic linkage.

62. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage between two nucleoside units comprising a 2′-F modified sugar moiety in the 3′-end region is independently a modified internucleotidic linkage.

63. The composition of embodiment 61 or 62, wherein each modified internucleotidic linkage is independently a chiral internucleotidic linkage.

64. The composition of embodiment 61 or 62, wherein each modified internucleotidic linkage is independently a chirally controlled internucleotidic linkage.

65. The composition of embodiment 61 or 62, wherein each modified internucleotidic linkage is a phosphorothioate internucleotidic linkage.

66. The composition of embodiment 61 or 62, wherein each modified internucleotidic linkage is a chirally controlled phosphorothioate internucleotidic linkage.

67. The composition of embodiment 61 or 62, wherein each modified internucleotidic linkage is a Sp chirally controlled phosphorothioate internucleotidic linkage.

68. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural phosphate linkages.

69. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural phosphate linkages each independently between a nucleoside unit comprising a 2′-OR¹modified sugar moiety and a nucleoside unit comprising a 2′-F modified sugar moiety, or between two nucleoside units each independently comprising a 2′-OR¹modified sugar moiety, wherein R¹is optionally substituted C_1-6alkyl.

70. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.

71. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages each independently between a nucleoside unit comprising a 2′-OR¹modified sugar moiety and a nucleoside unit comprising a 2′-F modified sugar moiety, or between two nucleoside units each independently comprising a 2′-OR¹modified sugar moiety, wherein R¹is optionally substituted C_1-6alkyl.

72. The composition of embodiment 69 or 71, wherein 2′-OR¹is 2′-OCH₃.

73. The composition of embodiment 69 or 71, wherein 2′-OR¹is 2′-OCH_2CH2OCH₃.

74. The composition of any one of the preceding embodiments, wherein the 5′-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral modified internucleotidic linkages.

75. The composition of any one of the preceding embodiments, wherein the 5′-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive chiral modified internucleotidic linkages.

76. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage in the 5′-end region is a chiral modified internucleotidic linkage.

77. The composition of any one of the preceding embodiments, wherein the 3′-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral modified internucleotidic linkages.

78. The composition of any one of the preceding embodiments, wherein the 3′-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive chiral modified internucleotidic linkages.

79. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage in the 3′-end region is a chiral modified internucleotidic linkage.

80. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral modified internucleotidic linkages.

81. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive chiral modified internucleotidic linkages.

82. The composition of any one of embodiments 74-81, wherein each chiral modified internucleotidic linkage is independently a chirally controlled internucleotidic linkage.

83. The composition of any one of embodiments 74-81, wherein each chiral modified internucleotidic linkage is independently a chirally controlled internucleotidic linkage wherein its chirally controlled linkage phosphorus has a Sp configuration.

84. The composition of any one of embodiments 74-83, wherein each chiral modified internucleotidic linkage is independently a chirally controlled phosphorothioate internucleotidic linkage.

85. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-negatively charged internucleotidic linkages.

86. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 neutral internucleotidic linkages.

87. The composition of any one of the preceding embodiments, wherein a neutral internucleotidic linkage is a chiral internucleotidic linkage.

88. The composition of any one of the preceding embodiments, wherein a neutral internucleotidic linkage is a chirally controlled internucleotidic linkage independently of Rp or Sp at its linkage phosphorus.

89. The composition of any one of the preceding embodiments, wherein the base sequence comprises a sequence having no more than 5 mismatches from a 20 base long portion of the dystrophin gene or its complement.

90. The composition of any one of the preceding embodiments, wherein the length of the base sequence of the oligonucleotides of the plurality is no more than 50 bases.

91. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chirally controlled centers independently of Rp or Sp.

92. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 5 chirally controlled centers independently of Rp or Sp.

93. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 6 chirally controlled centers independently of Rp or Sp.

94. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 10 chirally controlled centers independently of Rp or Sp.

95. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the particular oligonucleotide type are capable of mediating skipping of one or more exons of the dystrophin gene.

96. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 45, 51 or 53 of the dystrophin gene.

97. The composition of embodiment 96, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 45 of the dystrophin gene.

98. The composition of embodiment 96, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 51 of the dystrophin gene.

99. The composition of embodiment 96, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 53 of the dystrophin gene.

100. The composition of embodiment 97, wherein the base sequence comprises a sequence having no more than 5 mismatches from the sequence of any oligonucleotide disclosed herein.

101. The composition of embodiment 97, wherein the base sequence comprises or is the sequence of any oligonucleotide disclosed herein.

102. The composition of embodiment 97, wherein the base sequence is that of any oligonucleotide disclosed herein.

103. The composition of embodiment 97, wherein the base sequence comprises a sequence having no more than 5 mismatches from the sequence of any oligonucleotide disclosed herein.

104. The composition of embodiment 97, wherein the base sequence comprises or is any oligonucleotide disclosed herein.

105. The composition of embodiment 97, wherein the base sequence is any oligonucleotide disclosed herein.

106. The composition of any of the preceding embodiments, wherein the oligonucleotides of the plurality are any oligonucleotide disclosed herein.

107. The composition of embodiment 18, wherein oligonucleotides of the particular oligonucleotide type are any oligonucleotide disclosed herein.

108. The composition of any one of the preceding embodiments, wherein the base sequence is or comprises or comprises 15 contiguous bases of the base sequence of any oligonucleotide in Table A1.

109. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage.

110. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.

111. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chirally controlled non-negatively charged internucleotidic linkages.

112. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive non-negatively charged internucleotidic linkages.

113. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive chirally controlled non-negatively charged internucleotidic linkages.

114. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure.

115. The composition of any one of the preceding embodiments, wherein a wing comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.

116. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a wing comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chirally controlled non-negatively charged internucleotidic linkages.

117. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a wing comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive non-negatively charged internucleotidic linkages.

118. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a wing comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive chirally controlled non-negatively charged internucleotidic linkages.

119. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise or consist of a wing-core-wing structure, and wherein only one wing comprise one or more non-negatively charged internucleotidic linkages.

120. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.

121. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chirally controlled non-negatively charged internucleotidic linkages.

122. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive non-negatively charged internucleotidic linkages.

123. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive chirally controlled non-negatively charged internucleotidic linkages.

124. The composition of any one of the preceding embodiments, wherein 40%, 45%, 50%, 55%, 600%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of internucleotidic linkages of a wing is independently a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage or a Rp chiral internucleotidic linkage.

125. The composition of any one of the preceding embodiments, wherein 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90°, 95%, or 100% of internucleotidic linkages of a wing is independently a non-negatively charged internucleotidic linkage or a natural phosphate internucleotidic linkage.

126. The composition of any one of the preceding embodiments, wherein 400, 45%, 50%, 55%, 60%0, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of internucleotidic linkages of a wing is independently a non-negatively charged internucleotidic linkage.

127. The composition of any one of embodiments 124-126, wherein the percentage is 50% or more.

128. The composition of any one of embodiments 124-126, wherein the percentage is 60% or more.

129. The composition of any one of embodiments 124-126, wherein the percentage is 75% or more.

130. The composition of any one of embodiments 124-126, wherein the percentage is 80% or more.

131. The composition of any one of embodiments 124-126, wherein the percentage is 900 or more.

132. The composition of any one of the preceding embodiments, wherein the oligonucleotides each comprise a non-negatively charged internucleotidic linkage and a natural phosphate internucleotidic linkage.

133. The composition of any one of the preceding embodiments, wherein the oligonucleotides each comprise a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage and a Rp chiral internucleotidic linkage.

134. The composition of any one of the preceding embodiments, wherein a wing comprises a non-negatively charged internucleotidic linkage and a natural phosphate internucleotidic linkage.

135. The composition of any one of the preceding embodiments, wherein a wing comprises a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage and a Rp chiral internucleotidic linkage.

136. The composition of any one of the preceding embodiments, wherein a core comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.

137. The composition of any one of the preceding embodiments, wherein all non-negatively charged internucleotidic linkages of the same oligonucleotide have the same constitution.

138. The composition of any one of the preceding embodiments, wherein each of the non-negatively charged internucleotidic linkages independently has the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, I-a-1, H-a-2, I-b-1, H-b-2, I-c-1, II-c-2, H-d-1, II-d-2, or a salt form thereof.

139. The composition of any one of the preceding embodiments, wherein each of the non-negatively charged internucleotidic linkages independently has the structure of formula I-n-1, I-n-2,1-n-3, 1-n-4, II, II-a-1,11-a-2,11-b-1,11-b-2,11-c-1,11-c-2,11-d-1, II-d-2, or a salt form thereof.

140. The composition of any one of the preceding embodiments, wherein each of the non-negatively charged internucleotidic linkages independently has the structure of formula II, I-a-1, II-a-2, I-b-1, II-b-2, I-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

141. The composition of any one of the preceding embodiments, wherein each of the non-negatively charged internucleotidic linkages independently has the structure of formula II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

142. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage which is a neutral internucleotidic linkage.

143. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one neutral internucleotidic linkage which is or comprises a triazole, neutral triazole, alkyne, or a cyclic guanidine.

144. The composition of any one of the preceding embodiments, wherein the oligonucleotide type comprises any of: cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Gambogic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).

145. The composition of any one of the preceding embodiments, wherein the oligonucleotide type is any oligonucleotide listed in Table A1.

146. The composition of any one of the preceding embodiments, wherein each of the oligonucleotides comprises a chemical moiety conjugated to the oligonucleotide chain of the oligonucleotide optionally through a linker moiety, wherein the chemical moiety comprises a carbohydrate moiety, a peptide moiety, a receptor ligand moiety, or a moiety having the structure of —N(R¹)₂, —N(R¹)₃, or —N═C(N(R¹)₂)₂.

147. The composition of any one of the preceding embodiments, wherein each of the oligonucleotides comprises a chemical moiety conjugated to the oligonucleotide chain of the oligonucleotide optionally through a linker moiety, wherein the chemical moiety comprises a guanidine moiety.

148. The composition of any one of the preceding embodiments, wherein each of the oligonucleotides comprises a chemical moiety conjugated to the oligonucleotide chain of the oligonucleotide optionally through a linker moiety, wherein the chemical moiety comprises —N═C(N(CH₃)₂)₂.

149. The composition of any one of the preceding embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the oligonucleotides in the composition that have the base sequence of the particular oligonucleotide type are oligonucleotides of the particular oligonucleotide type.

150. The composition of any one of the preceding embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the oligonucleotides in the composition that have the base sequence, pattern of backbone linkages, and pattern of backbone phosphorus modifications of the particular oligonucleotide type are oligonucleotides of the particular oligonucleotide type.

151. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the particular type are structurally identical.

152. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage is a phosphoramidate linkage.

153. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage comprises a guanidine moiety.

154. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I:

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or a salt form thereof, wherein:

P^Lis P(═W), P, or P→B(R′)₃;

W is O, N(-L-R⁵), S or Se;

each of R¹and R⁵is independently —H, -L-R′, halogen, —CN, —NO₂, -L-Si(R′)₃, —OR′, —SR′, or —N(R′)₂;

each of X. Y and Z is independently —O—, —S—, —N(-L-R⁵)—, or L;

each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L;

each R′ is independently —R. —C(O)R, —C(O)OR, or—S(O)₂R;

two R groups are optionally and independently taken together to form a covalent bond, or

155. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I or a salt form thereof.

156. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-1 or a salt form thereof:

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157. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-1 or a salt form thereof.

158. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-2 or a salt form thereof:

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159. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3 or a salt form thereof:

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160. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-3 or a salt form thereof.

161. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3 or a salt form thereof, wherein one R′ from one —N(R′)₂and one R′ from the other —N(R′)₂are taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

162. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-3 or a salt form thereof, wherein one R′ from one —N(R′)₂and one R′ from the other —N(R′)₂are taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

163. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3 or a salt form thereof, wherein one R′ from one —N(R′)₂and one R′ from the other —N(R′)₂are taken together with their intervening atoms to form an optionally substituted 5-membered monocyclic ring having no more than two nitrogen atoms.

164. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-3 or a salt form thereof, wherein one R′ from one —N(R′)₂and one R′ from the other —N(R′)₂are taken together with their intervening atoms to form an optionally substituted 5-membered monocyclic ring having no more than two nitrogen atoms.

165. The composition of any one of embodiments 159-162, wherein the ring formed is a saturated ring.

166. The composition of any one of embodiments 159-162, wherein the ring formed is a partially unsaturated ring.

167. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-4 or a salt form thereof:

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168. The composition of embodiment 167, wherein L^ais a covalent bond.

169. The composition of embodiment 167, wherein L^ais —N(R′)—.

170. The composition of embodiment 167, wherein L^ais —N(R′)—.

171. The composition of embodiment 167, wherein L^ais —N(R)—.

172. The composition of embodiment 167, wherein L^ais —S(O)—.

173. The composition of embodiment 167, wherein L^ais —S(O)₂—.

174. The composition of embodiment 167, wherein L^ais —S(O)₂N(R′)—.

175. The composition of any one of embodiments 167-174, wherein L^bis a covalent bond.

176. The composition of any one of embodiments 167-174, wherein L is —N(R)—.

177. The composition of any one of embodiments 167-174, wherein L is —N(R′)—.

178. The composition of any one of embodiments 167-174, wherein L is —N(R)—.

179. The composition of any one of embodiments 167-174, wherein L is —S(O)—.

180. The composition of any one of embodiments 167-174, wherein L^bis —S(O)₂—.

181. The composition of any one of embodiments 167-174, wherein L^bis —S(O)₂N(R′)—.

182. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II:

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or a salt form thereof, wherein:

P^Lis P(═W), P, or P→B(R′)₃;

W is O, N(-L-R⁵), S or Se;

each of X, Y and Z is independently —O—, —S—, —N(-L-R⁵)—, or L;

R⁵is —H, -L-R′, halogen, —CN, —NO₂, -L-Si(R′)₃, —OR′, —SR′, or —N(R′)₂;

Ring A^Lis an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms;

each R^sis independently —H, halogen, —CN, —N₃, —NO, —NO₂, -L-R′, -L-Si(R)₃, -L-OR′, -L-SR′, -L-N(R′)₂, —O-L-R′, -θ-L-Si(R)₃, —O-L-OR′, —O-L-SR′, or —O-L-N(R′)₂:

g is 0-20:

each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C_1-30aliphatic group and a C_1-30heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C_1-6alkylene, C_1-6alkenylene, —C≡C—, a bivalent C₁-C₆heteroaliphatic group having 1-5 heteroatoms, —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)₃]—, —OP(O)(OR′)O—, —P(O)(SR′)O—, —P(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, or —OP(OR′)[B(R′)₃]O—, and one or more CH or carbon atoms are optionally and independently replaced with Cy^L;

each R′ is independently —R, —C(O)R, —C(O)OR, or —S(O)₂R;

two R groups are optionally and independently taken together to form a covalent bond, or

183. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II, or a salt form thereof.

184. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-a-1:

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or a salt form thereof.

185. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-a-2:

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or a salt form thereof.

186. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-a-1 or II-a-2, or a salt form thereof.

187. The composition of any one of embodiments 182-186, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-b-1:

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or a salt form thereof, wherein g is 0-18.

188. The composition of any one of embodiments 182-187, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-b-2:

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or a salt form thereof, wherein g is 0-18.

189. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-b-1 or II-b-2, or a salt form thereof.

190. The composition of any one of embodiments 182-188, wherein Ring A^Lis an optionally substituted 3-20 membered monocyclic ring having 0-10 heteroatoms (in addition to the two nitrogen atoms for formula II-b-1 or II-b-2).

191. The composition of any one of embodiments 182-188, wherein Ring A^Lis an optionally substituted 5-membered monocyclic saturated ring.

192. The composition of any one of embodiments 182-191, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-c-1:

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or a salt form thereof, wherein g is 0-4.

193. The composition of any one of embodiments 182-193, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-c-2:

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or a salt form thereof, wherein g is 0-4.

194. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-c-1 or II-c-2, or a salt form thereof.

195. The composition of any one of embodiments 182-193, wherein each non-negatively charged internucleotidic linkage has the same structure.

196. The composition of any one of the preceding embodiments, wherein, if applicable, each internucleotidic linkage in the oligonucleotides of the plurality that is not a non-negatively charged internucleotidic linkage independently has the structure of formula I.

197. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage in the oligonucleotides of the plurality independently has the structure of formula I.

198. The composition of any one of the preceding embodiments, wherein one or more P^Lis P(═W).

199. The composition of any one of the preceding embodiments, wherein each P^Lis independently P(═W).

200. The composition of any one of the preceding embodiments, wherein one or more W is O.

201. The composition of any one of the preceding embodiments, wherein each W is O.

202. The composition of any one of the preceding embodiments, wherein one or more W is S.

203. The composition of any one of the preceding embodiments, wherein one or more W is independently N(-L-R⁵).

204. The composition of any one of the preceding embodiments, wherein one or more internucleotidic linkage independently has the structure of formula III or salt form thereof:

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205. The composition of embodiment 204, wherein P^Nis P(═N-L-R⁵).

206. The composition of embodiment 204, wherein P^Nis

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207. The composition of embodiment 204, wherein P^Nis

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208. The composition of embodiment 207, wherein L^ais a covalent bond.

209. The composition of embodiment 207, wherein L^ais —N(R)—.

210. The composition of embodiment 207, wherein L^ais —N(R′)—.

211. The composition of embodiment 207, wherein L^ais —N(R)—.

212. The composition of embodiment 207, wherein L^ais —S(O)—.

213. The composition of embodiment 207, wherein L^ais —S(O)₂—.

214. The composition of embodiment 207, wherein L^ais —S(O)₂N(R′)—.

215. The composition of embodiment 204, wherein P^Nis

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216. The composition of embodiment 204, wherein P^Nis

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217. The composition of embodiment 204, wherein P^Nis

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218. The composition of any one of the preceding embodiments, wherein one or more Y is O.

219. The composition of any one of the preceding embodiments, wherein each Y is O.

220. The composition of any one of the preceding embodiments, wherein one or more Z is O.

221. The composition of any one of the preceding embodiments, wherein each Z is O.

222. The composition of any one of the preceding embodiments, wherein one or more X is O.

223. The composition of any one of the preceding embodiments, wherein one or more X is S.

224. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of

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225. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of

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226. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of

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227. The composition of any one of the preceding embodiments, wherein for each internucleotidic linkage of formula I or a salt fore thereof that is not a non-negatively charged internucleotidic linkage, X is independently O or S, and -L-R¹is —H (natural phosphate linkage or phosphorothioate linkage, respectively).

228. The composition of any one of the preceding embodiments, wherein each phosphorothioate linkage, if any, in the oligonucleotides of the plurality is independently a chirally controlled internucleotidic linkage.

229. The composition of any one of the preceding embodiments, wherein at least one non-negatively charged internucleotidic linkage is a chirally controlled internucleotidic linkage.

230. The composition of any one of the preceding embodiments, wherein at least one non-negatively charged internucleotidic linkage is a chirally controlled internucleotidic linkage.

231. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality comprise a targeting moiety wherein the targeting moiety is independently connected to an oligonucleotide backbone through a linker.

232. The composition of embodiment 231, wherein the targeting moiety is a carbohydrate moiety.

233. The composition of embodiment 231 or 232, wherein the targeting moiety comprises or is a GalNac moiety.

234. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality comprise a lipid moiety wherein the lipid moiety is independently connected to an oligonucleotide backbone through a linker.

235. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein one or more non-neutral internucleotidic linkages at the condition of the composition independently exist as a salt form.

236. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein one or more negatively-charged internucleotidic linkages at the condition of the composition independently exist as a salt form.

237. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein one or more negatively-charged internucleotidic linkages at the condition of the composition independently exist as a metal salt.

238. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein each negatively-charged internucleotidic linkage at the condition of the composition independently exists as a metal salt.

239. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein each negatively-charged internucleotidic linkage at the condition of the composition independently exists as sodium salt.

240. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein each negatively-charged internucleotidic linkage is independently a natural phosphate linkage (the neutral form of which is —O—P(O)(OH)—O) or phosphorothioate internucleotidic linkage (the neutral form of which is —O—P(O)(SH)—O).

241. An oligonucleotide composition, comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

wherein:

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages; and

oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-negatively charged internucleotidic linkages.

242. The composition of any one of the preceding embodiments, wherein at least one non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage.

243. The composition of any one of the preceding embodiments, wherein a neutral internucleotidic linkage is or comprises a triazole, neutral triazole, alkyne, or a cyclic guanidine.

244. The oligonucleotide composition of any one of the preceding embodiments, wherein the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

245. The oligonucleotide composition of any one of the preceding embodiments, wherein the transcript is a Dystrophin transcript.

246. The oligonucleotide composition of any one of the preceding embodiments, wherein the splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.

247. The oligonucleotide composition of any one of the preceding embodiments, wherein the oligonucleotide composition is capable of mediating knockdown of a target gene.

248. An oligonucleotide composition, comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

1) base sequence;

2) pattern of backbone linkages;

3) pattern of backbone chiral centers; and

4) pattern of backbone phosphorus modifications,

wherein:

the oligonucleotides of the plurality comprise cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; Gambogic acid; CPP: Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).

249. The composition of embodiment 248, wherein the oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages.

250. The composition of any one of the preceding embodiments, wherein the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.

251. The composition of any one of the preceding embodiments, wherein the transcript is a Dystrophin transcript.

252. The composition of any one of the preceding embodiments, wherein the splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.

253. The composition of any one of the preceding embodiments, wherein the oligonucleotide composition is capable of mediating knockdown of a target gene.

254. The composition of any one of the preceding embodiments, wherein each heteroatom is independently boron, nitrogen, oxygen, silicon, sulfur, or phosphorus.

255. A pharmaceutical composition comprising an oligonucleotide composition of any one of the preceding embodiments and a pharmaceutically acceptable carrier.

256. A method for altering splicing of a target transcript, comprising administering an oligonucleotide composition of any one of the preceding embodiments.

257. The method of embodiment 256, wherein the splicing of the target transcript is altered relative to absence of the composition.

258. The method of any one of the preceding embodiments, wherein the alteration is that one or more exon is skipped at an increased level relative to absence of the composition.

259. The method of any one of the preceding embodiments, wherein the target transcript is pre-mRNA of dystrophin.

260. The method of any one of the preceding embodiments, wherein exon 45 of dystrophin is skipped at an increased level relative to absence of the composition.

261. The method of any one of the preceding embodiments, wherein exon 51 of dystrophin is skipped at an increased level relative to absence of the composition.

262. The method of any one of embodiments 256-259, wherein exon 53 of dystrophin is skipped at an increased level relative to absence of the composition.

263. The method of any one of the preceding embodiments, wherein a protein encoded by the mRNA with the exon skipped provides one or more functions better than a protein encoded by the corresponding mRNA without the exon skipping.

264. A method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising administering to a subject susceptible thereto or suffering therefrom a composition of any one of the preceding embodiments.

265. A method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising administering to a subject susceptible thereto or suffering therefrom a composition comprising any oligonucleotide disclosed herein.

266. A method for treating muscular dystrophy. Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising (a) administering to a subject susceptible thereto or suffering therefrom a composition comprising any oligonucleotide disclosed herein, and (b) administering to the subject additional treatment which is capable of preventing, treating, ameliorating or slowing the progress of muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD).

267. The method of embodiment 266, wherein the additional treatment is a second oligonucleotide.

268. The composition of any of the preceding embodiments, wherein the transcript splicing system comprises a myoblast or myotubule.

269. The composition of any of the preceding embodiments, wherein the transcript splicing system comprises a myoblast cell.

270. The composition of any of the preceding embodiments, wherein the transcript splicing system comprises a myoblast cell, which is contacted with the composition after 0.4 or 7 days of pre-differentiation.

271. A composition comprising a combination comprising: (a) a first composition of any of the preceding embodiments; (b) a second composition of any of the preceding embodiments; and, optionally (c) a third composition of any of the preceding embodiments, wherein the first, second and third compositions are different.

272. A method for preparing an oligonucleotide or an oligonucleotide composition thereof, comprising providing a compound having the structure of:

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or a salt thereof.

273. A method for preparing an oligonucleotide or an oligonucleotide composition thereof, comprising providing a compound having the structure of:

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or a salt thereof.

274. A method for preparing an oligonucleotide or an oligonucleotide composition thereof, comprising providing a compound having the structure of

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or salt thereof.

275. The method of any one of embodiments 272-274, wherein the compound is stereochemically pure.

276. The method of any one of embodiments 272-275, wherein the compound is a compound of Tables CA-1, CA-2, CA-3, CA-4, CA-5, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, or CA-12, or a related diastereomer or enantiomer thereof.

277. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-2 or a related diastereomer or enantiomer thereof.

278. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-3 or a related diastereomer or enantiomer thereof.

279. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-4 or a related diastereomer or enantiomer thereof.

280. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-5 or a related diastereomer or enantiomer thereof.

281. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-6 or a related diastereomer or enantiomer thereof.

282. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-7 or a related diastereomer or enantiomer thereof.

283. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-8 or a related diastereomer or enantiomer thereof.

284. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-9 or a related diastereomer or enantiomer thereof.

285. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-10 or a related diastereomer or enantiomer thereof.

286. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-11 or a related diastereomer or enantiomer thereof.

287. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-12 or a related diastereomer or enantiomer thereof.

288. A method for preparing an oligonucleotide or an oligonucleotide composition thereof, comprising providing a phosphoramidite compound comprising a chiral auxiliary moiety having the structure of

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289. A method for preparing an oligonucleotide or an oligonucleotide composition thereof, comprising providing a phosphoramidite compound having the structure of:

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or salt thereof.

290. The method of any one of embodiments 272-289, wherein W¹is -NG⁵-.

291. The method of any one of embodiments 272-290, wherein G⁵and one of G³and G⁴are taken together to form an optionally substituted 3-8 membered saturated ring having 0-3 heteroatoms in addition to the nitrogen of -NG⁵-.

292. The method of any one of embodiments 272-290, wherein G⁵and one of G³and G⁴are taken together to form an optionally substituted 5-membered saturated ring having no heteroatoms in addition to the nitrogen of -NG⁵-.

293. The method of any one of embodiments 272-292, wherein W²is —O—.

294. The method of any one of embodiments 272-293, wherein G²comprises an electron-withdrawing group.

295. The method of any one of embodiments 272-293, wherein G²is methyl substituted with one or more electron-withdrawing groups.

296. The method of any one of embodiments 294-295, wherein an electron-withdrawing group is —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R′, —S(O)₂R′, —P(W)(R′)₂, —P(O)(R′)₂, —P(O)(OR′)₂, or —P(S)(R′)₂, or aryl or heteroaryl substituted with one or more of —CN, —NO₂, halogen. —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R′)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂.

297. The method of any one of embodiments 294-295, wherein an electron-withdrawing group is —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂, or phenyl substituted with one or more of —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R′)₂, —P(O)(OR¹)₂, or —P(S)(R′)₂.

298. The method of any one of embodiments 294-295, wherein an electron-withdrawing group is —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂. —P(O)(OR′)₂, or —P(S)(R¹)₂.

299. The method of any one of embodiments 272-294, wherein G²is -L′-L″-R′, wherein L′ is —C(R)₂— or optionally substituted —CH₂—, and L″ is a covalent bond, —P(O)(R′)—, —P(O)(R′)O—, —P(O)(OR′)—, —P(O)(OR′)O—, —P(O)[N(R′)]—. —P(O)[N(R′)]O—, —P(O)[N(R′)][N(R′)]—, —P(S)(R′)—, —S(O)₂—, —S(O)₂—, —S(O)₂O—, —S(O)—, —C(O)—, or —C(O)N(R′)—.

300. The method of any one of embodiments 272-294, wherein G²is -L′-L″-R′, wherein L′ is —C(R)₂— or optionally substituted —CH₂—, and L″ is —P(O)(R′)—, —P(O)(R′)O—, —P(O)(OR′)—, —P(O)(OR′)O—, —P(O)[N(R′)]—, —P(O)[N(R′)]O—, —P(O)[N(R′)][N(R′)]—, —P(S)(R′)—, —S(O)₂—, —S(O)₂—, —S(O)₂O—, —S(O)—, —C(O)—, or —C(O)N(R′)—.

301. The method of any one of embodiments 272-300, wherein G²is -L′-S(O)₂R′.

302. The method of embodiment 301, wherein R′ is optionally substituted C_1-6aliphatic.

303. The method of embodiment 301, wherein R′ is optionally substituted C_1-6alkyl.

304. The method of embodiment 301, wherein R′ is methyl, isopropyl or t-butyl.

305. The method of embodiment 301, wherein R′ is optionally substituted phenyl.

306. The method of embodiment 301, wherein R′ is phenyl.

307. The method of embodiment 301, wherein R′ is substituted phenyl.

308. The method of any one of embodiments 272-300, wherein G²is -L′-P(O)(R′)₂.

309. The method of embodiment 308, wherein one R′ is optionally substituted C_1-6aliphatic.

310. The method of embodiment 308, wherein one R′ is optionally substituted C_1-6alkyl.

311. The method of embodiment 308, wherein one R′ is optionally substituted phenyl.

312. The method of embodiment 308, wherein one R′ is phenyl.

313. The method of embodiment 308, wherein one R′ is substituted phenyl.

314. The method of any one of embodiments 309-313, wherein the other R′ is optionally substituted C_1-6aliphatic.

315. The method of any one of embodiments 309-313, wherein the other R′ is optionally substituted C_1-6alkyl.

316. The method of any one of embodiments 309-313, wherein the other R′ is optionally substituted phenyl.

317. The method of any one of embodiments 309-313, wherein the other R′ is phenyl.

318. The method of any one of embodiments 309-313, wherein the other R′ is substituted phenyl.

319. The method of any one of embodiments 299-318, wherein L′ is —C(R′)₂—.

320. The method of any one of embodiments 299-318, wherein L′ is optionally substituted —CH₂—.

321. The method of any one of embodiments 299-318, wherein L′ is —CH₂—.

322. The method of any one of embodiments 272-321, comprising providing one or more additional compounds, wherein each compound is independently a compound of any one of embodiments 272-321.

323. The method of embodiment 322, wherein an additional compound has a different structure than the compound.

324. The method of embodiment 322, wherein in an additional compound. G²is -L′-Si(R), wherein each R is independently not —H.

325. The method of embodiment 322, wherein in an additional compound, G²is —CH₂SiCH₃Ph₂.

326. The method of any one of embodiments 272-325, comprising one or more cycles, each of which independently comprises or consisting of:

1) deblocking;

2) coupling;

3) optionally a first capping;

4) modifying; and

5) optionally a second capping.

327. A method for preparing an oligonucleotide or a composition thereof, comprising one or more cycles, each of which independently comprises or consisting of:

1) deblocking;

2) coupling;

3) optionally a first capping;

4) modifying; and

5) optionally a second capping.

328. The method of any one of embodiments 326-327, wherein at least one cycle comprises or consists of 1) to 5).

329. The method of any one of embodiments 326-328, wherein the steps are performed sequentially from 1) to 5).

330. The method of any one of embodiments 326-329, wherein the cycles are performed until a desired length of an oligonucleotide is achieved.

331. The method of any one of embodiments 326-330, wherein deblocking removes a protection group on 5′-OH and provides a free 5′-OH.

332. The method of embodiment 331, wherein the protection group is R′—C(O)—.

333. The method of embodiment 331, wherein the protection group is DMTr.

334. The method of any one of embodiments 331-333, comprising contacting the oligonucleotides to be de-blocked with an acid.

335. The method of any one of embodiments 272-334, comprising a coupling that comprises: 1) providing a phosphoramidite; and 2) reacting the phosphoramidite with an oligonucleotide, wherein a P-O bond is formed between the phosphorus of the phosphoramidite and the 5′-OH of the oligonucleotide.

336. The method of any one of embodiments 272-335, comprising a coupling that comprises: 1) providing a phosphoramidite; and 2) reacting the phosphoramidite with an oligonucleotide, wherein a P-O bond is formed between the phosphorus of the phosphoramidite and the 5′-OH of the oligonucleotide, wherein the phosphoramidite is a compound of any one of embodiments 288-321.

337. The method of any one of embodiments 272-336, comprising a coupling that comprises: 1) providing a phosphoramidite; and 2) reacting the phosphoramidite with an oligonucleotide, wherein a P-O bond is formed between the phosphorus of the phosphoramidite and the 5′-OH of the oligonucleotide, wherein the phosphoramidite is a compound of any one of embodiments 288-293, wherein G²is -L′-Si(R)₃, wherein each R is independently not —H.

338. The method of embodiment 337, wherein G²is —CH₂SiCH₃Ph₂.

339. The method of any one of embodiments 336-338, wherein the coupling forms an internucleotidic linkage with a stereoselectivity of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

340. The method of embodiment 339, wherein the internucleotidic linkage formed is an internucleotidic linkage of formula I or a salt form thereof.

341. The method of embodiment 340, wherein -X-L-R¹is

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342. The method of embodiment 340 or 341, wherein P^Lis P.

343. The method of any one of embodiments 272-342, comprising a coupling that comprises: 1) providing a phosphoramidite; and 2) reacting the phosphoramidite with an oligonucleotide, wherein a P-O bond is formed between the phosphorus of the phosphoramidite and the 5′-OH of the oligonucleotide, wherein the phosphoramidite is a standard phosphoramidite for oligonucleotide synthesis wherein the phosphorus atom is bonded to a protected nucleoside, —N(i-Pr)₂, and 2-cyanoethyl.

344. The method of any one of embodiments 272-343, comprising a first capping comprises: 1) providing an acylating reagent, and 2) contacting an oligonucleotide with the acylating reagent, wherein the first capping caps an amino group of an internucleotidic linkage.

345. The method of any one of embodiments 272-344, comprising a first capping which forms an internucleotidic linkage of formula I or a salt form thereof, wherein -X-L-R¹is

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346. The method of embodiment 345, wherein P^Lis P and R¹is —C(O)R.

347. The method of any one of embodiments 272-346, wherein a first capping is performed after each coupling of embodiment 339.

348. The method of any one of embodiments 272-347, comprising a modifying step which is or comprises sulfurization.

349. The method of embodiment 348, wherein the sulfurization installs ═S on a linkage phosphorus.

350. The method of embodiment 348 or 349, wherein the sulfurization forms an internucleotidic linkage of formula I or a salt form thereof, wherein P^Lis P(═S).

351. The method of embodiment 350, wherein -X-L-R¹is

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352. The method of embodiment 351, wherein R¹is —C(O)R.

353. The method of any one of embodiments 272-352, comprising a modifying step which is or comprises oxidation.

354. The method of embodiment 348, wherein the sulfurization installs ═O on a linkage phosphorus.

355. The method of any one of embodiments 272-354, comprising a modifying step which installs ═N-L-R⁵on a linkage phosphorus.

356. The method of any one of embodiments 272-354, comprising a modifying step which converts a linkage phosphorus into

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357. The method of any one of embodiments 272-356, comprising a modifying step which comprises contact the oligonucleotide with an azido imidazolinium salt.

358. The method of any one of embodiments 272-356, comprising a modifying step which comprises contact the oligonucleotide with a compound comprising

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359. The method of any one of embodiments 272-356, comprising a modifying step which comprises contact the oligonucleotide with a compound having the structure of

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wherein Q is an anion.

360. The method of embodiment 359, wherein Q⁻ is F⁻, Cl⁻, Br⁻, BF₄⁻, PF₆⁻, TfO⁻, Tf₂N⁻, AsF₆⁻, ClO₄⁻, or SbF₆⁻.

361. The method of embodiment 360, wherein Q⁻ is PF₆⁻.

362. The method of any one of embodiments 272-362, wherein a modifying step forms an internucleotidic linkage of formula I or a salt form thereof, wherein P^Lis P(═N-L-R⁵).

363. The method of any one of embodiments 272-362, wherein a modifying step forms an internucleotidic linkage of formula III or a salt form thereof.

364. The method of embodiment 362 or 363, wherein -X-L-R¹is

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365. The method of embodiment 364, wherein R¹is —C(O)R.

366. The method of any one of embodiments 272-365, comprising a second capping which caps free 5′-OH.

367. The method of any one of embodiments 272-366, comprising a second capping which caps free 5′-OH, wherein a second capping is performed in each cycle.

368. The method of any one of embodiments 272-366, comprising a second capping which caps free 5′-OH, wherein a second capping is performed in each cycle that is followed by another cycle.

369. The method of any one of embodiments 366-368, wherein a 5′-OH is capped as -OAc.

370. The method of any one of embodiments 272-369, wherein the oligonucleotide is attached to a solid support.

371. The method of embodiment 370, wherein the solid support is CPG.

372. The method of any one of embodiments 370-371, comprising a contact in which the oligonucleotide is contacted with a base.

373. The method of embodiment 372, wherein the contact is performed substantially absent of water.

374. The method of embodiment 372 or 373, wherein the contact is after the oligonucleotide length is achieved before deprotection and cleavage of oligonucleotide.

375. The method of any one of embodiments 372-374, wherein the base is an amine base having the structure of NR₃.

376. The method of embodiment 375, wherein the base is triethylamine.

377. The method of embodiment 375, wherein the base is N, N-diethylamine.

378. The method of any one of embodiments 372-377, wherein the contact removes a chiral auxiliary.

379. The method of any one of embodiments 372-378, wherein the contact removes a -X-L-R¹group.

380. The method of embodiment 379, wherein -X-L-R¹is

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381. The method of any one of embodiments 372-380, wherein the contact forms an internucleotidic linkage of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2, wherein P^Lis P(O).

382. The method of any one of embodiments 364-381, wherein G²comprises an electron-withdrawing group.

383. The method of any one of embodiments 364-382, wherein G²is methyl substituted with one or more electron-withdrawing groups.

384. The method of any one of embodiments 382-383, wherein an electron-withdrawing group is —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂, or aryl or heteroaryl substituted with one or more of —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R, —S(O)₂R, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂.

385. The method of any one of embodiments 382-383, wherein an electron-withdrawing group is —CN, —NO₂, halogen, —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂, or phenyl substituted with one or more of —CN, —NO₂, halogen. —C(O)R¹, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂.

386. The method of any one of embodiments 382-383, wherein an electron-withdrawing group is —CN, —NO₂, halogen, —C(O)R, —C(O)OR′, —C(O)N(R′)₂, —S(O)R¹, —S(O)₂R¹, —P(W)(R¹)₂, —P(O)(R¹)₂, —P(O)(OR′)₂, or —P(S)(R¹)₂.

387. The method of any one of embodiments 364-386, wherein G²is -L′-L″-R′, wherein L′ is —C(R)₂- or optionally substituted —CH₂—, and L″ is a covalent bond, —P(O)(R′)—, —P(O)(R′)O—, —P(O)(OR′)—, —P(O)(OR′)O—, —P(O)[N(R′)]—, —P(O)[N(R′)]O—, —P(O)[N(R′)][N(R′)]—, —P(S)(R′)—, —S(O)₂—, —S(O)₂—, —S(O)₂O—, —S(O)—, —C(O)—, or —C(O)N(R′)—.

388. The method of any one of embodiments 364-386, wherein G²is -L′-L″-R′, wherein L′ is —C(R)₂— or optionally substituted —CH₂—, and L″ is —P(O)(R′)—, —P(O)(R′)O—, —P(O)(OR′)—, —P(O)(OR′)O—, —P(O)[N(R′)]—, —P(O)[N(R′)]O—, —P(O)[N(R′)N(R′)]—, —P(S)(R′)—, —S(O)₂—, —S(O)₂—, —S(O)₂O—, —S(O)—, —C(O)—, or —C(O)N(R′)—.

389. The method of any one of embodiments 364-388, wherein G²is -L′-S(O),R′.

390. The method of embodiment 389, wherein R′ is optionally substituted C_1-6aliphatic.

391. The method of embodiment 389, wherein R′ is optionally substituted C_1-6alkyl.

392. The method of embodiment 389, wherein R′ is methyl, isopropyl or t-butyl.

393. The method of embodiment 389, wherein R′ is optionally substituted phenyl.

394. The method of embodiment 389, wherein R′ is phenyl.

395. The method of embodiment 389, wherein R′ is substituted phenyl.

396. The method of any one of embodiments 364-388, wherein G²is -L′-P(O)(R′)₂.

397. The method of embodiment 396, wherein one R′ is optionally substituted C_1-6aliphatic.

398. The method of embodiment 396, wherein one R′ is optionally substituted C_1-6alkyl.

399. The method of embodiment 396, wherein one R′ is optionally substituted phenyl.

400. The method of embodiment 396, wherein one R′ is phenyl.

401. The method of embodiment 396, wherein one R′ is substituted phenyl.

402. The method of any one of embodiments 397401, wherein the other R′ is optionally substituted C_1-6aliphatic.

403. The method of any one of embodiments 397401, wherein the other R′ is optionally substituted C_1-6alkyl.

404. The method of any one of embodiments 309-313, wherein the other R′ is optionally substituted phenyl.

405. The method of any one of embodiments 309-313, wherein the other R′ is phenyl.

406. The method of any one of embodiments 309-313, wherein the other R′ is substituted phenyl.

407. The method of any one of embodiments 387-406, wherein L′ is —C(R′)₂—.

408. The method of any one of embodiments 387406, wherein L′ is optionally substituted —CH₂—.

409. The method of any one of embodiments 387406, wherein L′ is —CH₂—.

410. The method of any one of embodiments 372409, wherein the contact removes 2′-cyanoethyl.

411. The method of any one of embodiments 372-410, wherein the contact forms a natural phosphate linkage or a salt form thereof.

412. The method of any one of embodiments 272-410, comprising removing of another chiral auxiliary or group that having a different structure than that of any one of embodiments 378-410.

413. The method of any one of embodiments 272410, comprising removing of

embedded image

wherein G²is -L′-Si(R)₃, wherein each R is independently not —H.

414. The method of embodiment 413, wherein G²is —CH₂SiCH₃Ph₂.

415. The method of any one of embodiments 412-414, comprising contacting an oligonucleotide with a fluoride.

416. The method of any one of embodiments 412414, comprising contacting an oligonucleotide with a solution comprising TEA-HF and a base.

417. The method of any one of embodiments 272416, comprising cleaving oligonucleotide from a solid support.

418. The method of any one of embodiments 272417, wherein the oligonucleotide or a composition thereof is an oligonucleotide or composition of any one of embodiments 1-254.

419. The compound of any one of embodiments 272-321, or a related diastereomer or enantiomer.

420. An oligonucleotide, wherein the oligonucleotide is, WV-20104, WV-20103, WV-20102, WV-20101, WV-20100, WV-20099, WV-20098, WV-20097, WV-20096, WV-20095, WV-20094, WV-20106, WV-20119, WV-20118, WV-13739, WV-13740, WV-9079, WV-9082, WV-9100, WV-9096, WV-9097, WV-9106, WV-9133, WV-9148, WV-9154, WV-9898, WV-9899, WV-9900, WV-9906, WV-9907. WV-9908, WV-9909, WV-9756, WV-9757, WV-9517, WV-9714, WV-9715, WV-9519, WV-9521, WV-9747, WV-9748, WV-9749, WV-9897, WV-9898, WV-9900, WV-9899, WV-9906, WV-9912, WV-9524, WV-9912, WV-9906, WV-9900, WV-9899, WV-9899, WV-9898, WV-9898, WV-9898, WV-9898, WV-9898, WV-9897, WV-9897, WV-9897, WV-9897, WV-9897, WV-9747, WV-9714, WV-9699, WV-9517. WV-9517, WV-13409, WV-13408, WV-12887, WV-12882, WV-12881. WV-12880, WV-12880, WV-WV12880, WV-12878, WV-12877, WV-12877, WV-12876, WV-12873, WV-12872, WV-12559, WV-12559, WV-12558, WV-12558, WV-12557, WV-12556, WV-12556, WV-12555, WV-12555, WV-12554, WV-12553, WV-12129, WV-12127, WV-12125, WV-12123, WV-11342, WV-11342, WV-11341, WV-11341, WV-11340, WV-10672. WV-10671, WV-10670, WV-10461, WV-10455, WV-9897, WV-9898, WV-13826, WV-13827, WV-13835, WV-12880, WV-14344, WV-13864, WV-13835, WV-14791, WV-14344, WV-13754, WV-13766, WV-11086, WV-11089, WV-17859, WV-17860, WV-20070, WV-20073, WV-20076, WV-20052, WV-20099, WV-20049, WV-20085, WV-20087, WV-20034, WV-20046, WV-20052, WV-20061, WV-20064, WV-20067, WV-20092, WV-20091. WV-20093, WV-20084, WV-9738. WV-9739, WV-9740, WV-9741, WV-15860. WV-15862, WV-11084, WV-11086, WV-11088, WV-11089, WV-14522, WV-14523, WV-17861, WV-17862, WV-13815, WV-13816, WV-13817, WV-13780, WV-17862, WV-17863, WV-17864, WV-17865, WV-17866, WV-20082, WV-20081, WV-20080, WV-20079, WV-20076, WV-20075, WV-20074, WV-20073, WV-20072, WV-20071, WV-20064, WV-20059. WV-20058, WV-20057, WV-20056, WV-20053, WV-20052, WV-20051, WV-20050, WV-20049, WV-20094, WV-20095, or a salt form thereof.

EQUIVALENTS

Having described some illustrative embodiments of the disclosure, it should be apparent to those skilled in the art that the foregoing is merely illustrative and not limiting, having been presented by way of example only. Numerous modifications and other illustrative embodiments are within the scope of one of ordinary skill in the art and are contemplated as falling within the scope of the disclosure. In particular, although many of the examples presented herein involve specific combinations of method acts or system elements, it should be understood that those acts and those elements may be combined in other ways to accomplish the same objectives. Acts, elements, and features discussed only in connection with one embodiment are not intended to be excluded from a similar role in other embodiments. Further, for the one or more means-plus-function limitations, if any, recited in the following claims, the means are not intended to be limited to the means disclosed herein for performing the recited function, but are intended to cover in scope any means, known now or later developed, for performing the recited function.

Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present disclosure is not to be limited in scope by examples provided. Examples are intended as illustration of one or more aspect of an invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

Number	Date	Country
62776432	Dec 2018	US
62723375	Aug 2018	US
62715684	Aug 2018	US
62670709	May 2018	US
62656949	Apr 2018	US

OLIGONUCLEOTIDE COMPOSITIONS AND METHODS OF USE THEREOF

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