Rna Interference Mediated Inhibition of Severe Acute Respiratory Syndrome (Sars) Gene Expression Using Short Interfering Nucleic Acid

FIELD OF THE INVENTION

The present invention concerns compounds, compositions, and methods for the study, diagnosis, and treatment of diseases and conditions that respond to the modulation of severe acute respiratory syndrome (SARS) associated cornavirus (SARS virus) gene expression and/or activity. The present invention also concerns compounds, compositions, and methods relating to conditions and diseases that respond to the modulation of expression and/or activity of genes involved in SARS virus pathways of gene expression, including cellular genes that are involved in SARS virus infection. Specifically, the invention comprises small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against severe acute respiratory syndrome (SARS) associated cornavirus gene expression.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.

RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). The corresponding process in plants (Heifetz et al., International PCT Publication No. WO 99/61631) is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized. This mechanism appears to be different from other known mechanisms involving double stranded RNA-specific ribonucleases, such as the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094; 5,898,031; Clemens et al., 1997, J Interferon & Cytokine Res., 17, 503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101, 235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000, Nature, 404, 293). Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also been implicated in the excision of 21 and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283 and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., International PCT Publication No. WO 01/75164, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J, 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing 3′-terminal dinucleotide overhangs. Furthermore, complete substitution of one or both siRNA strands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3′-terminal siRNA overhang nucleotides with 2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end of the guide sequence (Elbashir et al., 2001, EMBO J, 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhanging segments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated, whereas complete substitution with deoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al., International PCT Publication No. WO 01/75164). In addition, Elbashir et al., supra, also report that substitution of siRNA with 2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and Beach et al., International PCT Publication-No. WO 01/68836 preliminarily suggest that siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom, however, neither application postulates to what extent such modifications would be tolerated in siRNA molecules, nor provides any further guidance or examples of such modified siRNA. Kreutzer et al., Canadian Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNA-dependent protein kinase PKR, specifically 2′-amino or 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-C methylene bridge. However, Kreutzer et al. similarly fails to provide examples or guidance as to what extent these modifications would be tolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certain chemical modifications targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and observed that RNAs with two phosphorothioate modified bases also had substantial decreases in effectiveness as RNAi. Further, Parrish et al. reported that phosphorothioate modification of more than two residues greatly destabilized the RNAs in vitro such that interference activities could not be assayed. Id. at 1081. The authors also tested certain modifications at the 2′-position of the nucleotide sugar in the long siRNA transcripts and found that substituting deoxynucleotides for ribonucleotides produced a substantial decrease in interference activity, especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting, in sense and antisense strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine for guanosine. Whereas 4-thiouracil and 5-bromouracil substitution appeared to be tolerated, Parrish reported that inosine produced a substantial decrease in interference activity when incorporated in either strand. Parrish also reported that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al., International PCT Publication No. WO 01/68836, describes specific methods for attenuating gene expression using endogenously-derived dsRNA. Tuschl et al., International PCT Publication No. WO 01/75164, describe a Drosophila in vitro RNAi system and the use of specific siRNA molecules for certain functional genomic and certain therapeutic applications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubts that RNAi can be used to cure genetic diseases or viral infection due to the danger of activating interferon response. Li et al., International PCT Publication No. WO 00/44914, describe the use of specific long (141 bp-488 bp) enzymatically synthesized or vector expressed dsRNAs for attenuating the expression of certain target genes. Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describe certain methods for inhibiting the expression of particular genes in mammalian cells using certain long (550 bp-714 bp), enzymatically synthesized or vector expressed dsRNA molecules. Fire et al., International PCT Publication No. WO 99/32619, describe particular methods for introducing certain long dsRNA molecules into cells for use in inhibiting gene expression in nematodes. Plaetinck et al., International PCT Publication No. WO 00/01846, describe certain methods for identifying specific genes responsible for conferring a particular phenotype in a cell using specific long dsRNA molecules. Mello et al., International PCT Publication No. WO 01/29058, describe the identification of specific genes involved in dsRNA-mediated RNAi. Pachuck et al., International PCT Publication No. WO 00/63364, describe certain long (at least 200 nucleotide) dsRNA constructs. Deschamps Depaillette et al., International PCT Publication No. WO 99/07409, describe specific compositions consisting of particular dsRNA molecules combined with certain anti-viral agents. Waterhouse et al., International PCT Publication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describe certain methods for decreasing the phenotypic expression of a nucleic acid in plant cells using certain dsRNAs. Driscoll et al., International PCT Publication No. WO 01/49844, describe specific DNA expression constructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. For example, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describe specific chemically-modified dsRNA constructs targeting the unc-22 gene of C. elegans. Grossniklaus, International PCT Publication No. WO 01/38551, describes certain methods for regulating polycomb gene expression in plants using certain dsRNAs. Churikov et al., International PCT Publication No. WO 01/42443, describe certain methods for modifying genetic characteristics of an organism using certain dsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475, describe certain methods for isolating a Neurospora silencing gene and uses thereof. Reed et al., International PCT Publication No. WO 01/68836, describe certain methods for gene silencing in plants. Honer et al., International PCT Publication No. WO 01/70944, describe certain methods of drug screening using transgenic nematodes as Parkinson's Disease models using certain dsRNAs. Deak et al., International PCT Publication No. WO 01/72774, describe certain Drosophila-derived gene products that may be related to RNAi in Drosophila. Arndt et al., International PCT Publication No. WO 01/92513 describe certain methods for mediating gene suppression by using factors that enhance RNAi. Tuschl et al., International PCT Publication No. WO 02/44321, describe certain synthetic siRNA constructs. Pachuk et al., International PCT Publication No. WO 00/63364, and Satishchandran et al., International PCT Publication No. WO 01/04313, describe certain methods and compositions for inhibiting the function of certain polynucleotide sequences using certain long (over 250 bp), vector expressed dsRNAs. Echeverri et al., International PCT Publication No. WO 02/38805, describe certain C. elegans genes identified via RNAi. Kreutzer et al. International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP 1144623 B1 describes certain methods for inhibiting gene expression using dsRNA. Graham et al., International PCT Publications Nos. WO 99/49029 and WO 01/70949, and AU 4037501 describe certain vector expressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559, describe certain methods for inhibiting gene expression in vitro using certain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi. Martinez et al., 2002, Cell, 110, 563-574, describe certain single stranded siRNA constructs, including certain 5′-phosphorylated single stranded siRNAs that mediate RNA interference in Hela cells. Harborth et al., 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105, describe certain chemically and structurally modified siRNA molecules. Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically and structurally modified siRNA molecules.

McCaffrey et al., 2002, Nature, 418, 38-39, describes the use of certain siRNA constructs targeting a chimeric SARS NS5B protein/luciferase transcript in mice.

Randall et al., 2003, PNAS USA, 100, 235-240, describe certain siRNA constructs targeting SARS RNA in Huh7 hepatoma cell lines.

SUMMARY OF THE INVENTION

This invention comprises compounds, compositions, and methods useful for modulating the expression of genes associated with the development or maintenance of SARS virus infection, acute respiratory failure, viral pneumonia, and/or other disease states associated with SARS virus infection, using short interfering nucleic acid (siNA) molecules. This invention also comprises compounds, compositions, and methods useful for modulating the expression and activity of severe acute respiratory syndrome (SARS) associated cornavirus or genes involved in severe acute respiratory syndrome (SARS) associated comavirus gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of severe acute respiratory syndrome (SARS) associated comavirus. For convenience, all forms of the small nucleic acid molecules of the invention (e.g., siRNA, dsRNA, micro-RNA, etc.) are referred to herein as “siNA,” unless expressly stated otherwise.

A siNA of the invention can be unmodified or chemically-modified. A siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The instant invention also features various chemically-modified synthetic short interfering nucleic acid (siNA) molecules capable of modulating repeat expansion gene expression or activity in cells by RNA interference (RNAi). The use of chemically-modified siNA improves various properties of native siNA molecules through increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Further, contrary to earlier published studies, siNA having multiple chemical modifications retains its RNAi activity. The siNA molecules of the instant invention are useful reagents and are useful in methods for a variety of therapeutic, diagnostic, target validation, genomic discovery, genetic engineering, and pharmacogenomic applications.

In one embodiment, the invention comprises one or more siNA molecules (and methods of using them) that independently or in combination modulate the expression of gene(s) encoding SARS virus. Specifically, the present invention comprises siNA molecules that modulate the expression of SARS proteins, for example, proteins encoded by SARS virus genome, such as Genbank Accession Nos. in Table I.

In one embodiment, the invention comprises one or more siNA molecules (and methods of using them) that independently or in combination modulate the expression of genes representing cellular targets for SARS virus infection, such as cellular receptors, cell surface molecules, cellular enzymes, cellular transcription factors, and/or cytokines, second messengers, and cellular accessory molecules.

Due to the high sequence variability of the SARS genome, selection of siNA molecules for broad therapeutic applications preferably involve the conserved regions of the SARS genome. In one embodiment, the present invention comprises siNA molecules that target the conserved regions of the SARS genome, such as the polymerase encoding region of the SARS virus genomic RNA. Therefore, siNA molecules of the invention are designed to target all the different isolates of SARS. siNA molecules designed to target conserved regions of various SARS isolates enable efficient inhibition of SARS replication in diverse patient populations and ensure the effectiveness of the siNA molecules against SARS quasi species that evolve due to mutations in the non-conserved regions of the SARS genome. Therefore, a single siNA molecule can be targeted against all isolates of SARS by designing the siNA molecule to interact with conserved nucleotide sequences of SARS (such conserved sequences are expected to be present in the RNA of all SARS isolates).

In one embodiment, a siNA molecule is designed to target the 3′-untranslated region and/or the shared leader sequence of genomic SARS RNA transcripts. Because SARS cornavirus mRNAs are nested with the genomic RNA and share common 3′ region and polyA region, a single siNA targeting the 3′-end can target all transcripts plus the genomic RNA.

In one embodiment, a siNA molecule of the invention targets both the plus (genomic) strand RNA and minus strand RNA of the SARS virus. Because the SARS virus generates a minus strand RNA from plus strand genomic RNA, a double stranded siNA molecule targeting the plus strand will also target the minus strand, thus allowing a single double-stranded siNA to target both the plus (genomic) and the minus strand of the SARS virus. For example, a double stranded siNA molecule targeting the 3′-end of the SARS virus genomic strand will also target the 3′-end of the minus strand, thus allowing a single double-stranded siNA to target both the plus and the minus strand of the SARS virus.

In one embodiment, the invention comprises one or more siNA molecules (and methods of using them) that independently or in combination modulate the expression of gene(s) encoding SARS virus and/or cellular proteins associated with the maintenance or development of SARS virus infection and/or acute respiratory failure, viral pneumonia, such as genes encoding sequences comprising those sequences referred to by GenBank Accession Nos. shown in Table I, referred to herein generally as SARS. The description below of the various aspects and embodiments of the invention is provided with reference to exemplary severe acute respiratory syndrome (SARS) associated cornavirus genes, generally referred to herein as SARS. However, such reference is meant to be exemplary only and the various aspects and embodiments of the invention are also directed to other genes that express alternate SARS genes, such as mutant SARS genes, splice variants of SARS genes, and genes encoding different strains of SARS, as well as as cellular targets for SARS, such as those described herein. The various aspects and embodiments are also directed to other genes involved in SARS pathways, including genes that encode cellular proteins involved in the maintenance and/or development of SARS virus infection and/or acute respiratory failure or other genes that express other proteins associated with SARS virus infection, such as cellular proteins that are utilized in the SARS life-cycle. Such additional genes can be analyzed for target sites using the methods described herein for SARS. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein. In other words, the term “SARS” as it is defined herein below and recited in the described embodiments, is meant to encompass genes associated with the development or maintenance of SARS virus infection, such as genes which encode SARS polypeptides, including polypeptides of different strains of SARS, mutant SARS genes, and splice variants of SARS genes, as well as cellular genes involved in SARS pathways of gene expression, replication, and/or SARS activity. Also, the term “SARS” as it is defined herein and recited in the described embodiments, is meant to encompass SARS viral gene products and cellular gene products involved in SARS virus infection, such as those described herein. Thus, each of the embodiments described herein with reference to the term “SARS” are applicable to all of the virus, cellular and viral protein, peptide, polypeptide, and/or polynucleotide molecules covered by the term “SARS” as that term is defined herein.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a severe acute respiratory syndrome virus (e.g., SARS) gene, wherein said siNA molecule comprises about 19 to about 23 base pairs. Preferably the number of based pairs in the siNA molecule is 18, 19, 20, 21, 22, 23, or 24.

In one embodiment, the invention features a siNA molecule that down-regulates expression of a SARS gene, for example, wherein the SARS gene comprises SARS encoding sequence. In one embodiment, the invention features a siNA molecule that down-regulates expression of a SARS gene, for example, wherein the SARS gene comprises SARS non-coding sequence or regulatory elements involved in SARS gene expression.

In one embodiment, the invention features a siNA molecule having RNAi activity against SARS RNA, wherein the siNA molecule comprises a sequence complementary to any RNA having SARS encoding sequence, such as those sequences having GenBank Accession Nos. shown in Table I. In another embodiment, the invention features a siNA molecule having RNAi activity against SARS RNA, wherein the siNA molecule comprises a sequence complementary to an RNA having other SARS encoding sequence, for example other mutant SARS genes not shown in Table I but known in the art to be associated with respiratory and/or pulmonary disease, SARS virus infection and/or acute respiratory failure, viral pneumonia, impeded respiration, respiratory distress syndrome, pulmonary hypertension, or pulmonary vasoconstriction. Chemical modifications as shown in Tables III and IV or otherwise described herein can be applied to any siNA construct of the invention. In another embodiment, a siNA molecule of the invention includes nucleotide sequence that can interact with nucleotide sequence of a SARS gene and thereby mediate silencing of SARS gene expression, for example, wherein the siNA mediates regulation of SARS gene expression by cellular processes that modulate the chromatin structure of the SARS gene and prevent transcription of the SARS gene.

In another embodiment, the invention features a siNA molecule comprising nucleotide sequence, for example, nucleotide sequence in the antisense region of the siNA molecule that is complementary to a nucleotide sequence or portion of sequence of a SARS gene. In another embodiment, the invention features a siNA molecule comprising a region, for example, the antisense region of the siNA construct, complementary to a sequence comprising a SARS gene sequence or a portion thereof.

In one embodiment, the antisense region of SARS siNA constructs can comprise a sequence complementary to sequence having any of SEQ ID NOs. 1-1651 or 3303-3318. In one embodiment, the antisense region can also comprise sequence having any of SEQ ID NOs. 1652-3302, 3319-3326, 3335-3342, 3351-3358, 3367-3374, 3376, 3378, 3380, 3383, 3385, 3387, 3389, or 3392. In another embodiment, the sense region of the SARS constructs can comprise sequence having any of SEQ ID NOs. 1-1651, 3303-3310, 3311-3318, 3327-3334, 3343-3350, 3359-3366, 3375, 3377, 3379, 3381, 3382, 3384, 3386, 3388, 3390, or 3391.

In one embodiment, a siNA molecule of the invention comprises any of SEQ ID NOs. 1-3392. The sequences shown in SEQ ID NOs: 1-3392 are not limiting. A siNA molecule of the invention can comprise any contiguous SARS sequence (e.g., about 19 to about 25, or about 19, 20, 21, 22, 23, 24 or 25 contiguous SARS nucleotides).

In yet another embodiment, the invention features a siNA molecule comprising a sequence, for example, the antisense sequence of the siNA construct, complementary to a sequence or portion of sequence comprising sequence represented by GenBank Accession Nos. shown in Table I. Chemical modifications in Tables III and IV and described herein can be applied to any siNA construct of the invention. siNA molecules of the invention are unmodified or have up to all nucleotides modified with modifications according to Tables III and IV.

In one embodiment of the invention a siNA molecule comprises an antisense strand having about 19 to about 29 (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense strand is complementary to a RNA sequence encoding a SARS protein, and wherein said siNA further comprises a sense strand having about 19 to about 29 (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and wherein said sense strand and said antisense strand are distinct nucleotide sequences with at least about 19 complementary nucleotides.

In another embodiment of the invention a siNA molecule of the invention comprises an antisense region having about 19 to about 29 (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region is complementary to a RNA sequence encoding a SARS protein, and wherein said siNA further comprises a sense region having about 19 to about 29 (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or more) nucleotides, wherein said sense region and said antisense region comprise a linear molecule with at least about 19 complementary nucleotides.

In one embodiment of the invention a siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a SARS protein. The siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a SARS gene or a portion thereof.

In another embodiment, a siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a SARS protein or a portion thereof. The siNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of a SARS gene or a portion thereof.

In one embodiment, a siNA molecule of the invention has RNAi activity that modulates expression of RNA encoded by a SARS gene. Because SARS genes can share some degree of sequence homology with each other, siNA molecules can be designed to target a class of SARS genes or alternately specific SARS genes by selecting sequences that are either shared among different SARS targets (e.g., different viral strains) or alternatively that are unique for a specific SARS target (e.g., a particular viral strain). Therefore, in one embodiment, the siNA molecule can be designed to target conserved regions of SARS RNA sequences having homology among several SARS genes so as to target several SARS genes (e.g., different SARS isoforms, splice variants, mutant genes etc.) with one siNA molecule. In another embodiment, the siNA molecule can be designed to target a sequence that is unique to a specific SARS RNA sequence due to the high degree of specificity that the siNA molecule requires to mediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act as mediators of the RNA interference gene silencing response are double-stranded nucleic acid molecules. In another embodiment, the siNA molecules of the invention consist of duplexes containing about 19 base pairs between oligonucleotides comprising about 19 to about 25 (e.g., 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides. In yet another embodiment, siNA molecules of the invention comprise duplexes with overhanging ends of about 1 to about 3 (e.g., 1, 2, 3, or 4) nucleotides, for example, about 21-nucleotide duplexes with about 19 base pairs and 3′-terminal mononucleotide, dinucleotide, or trinucleotide overhangs.

In one embodiment, the invention features one or more chemically-modified siNA constructs having specificity for SARS expressing nucleic acid molecules, such as RNA encoding a SARS protein. Non-limiting examples of such chemical modifications include without limitation phosphorothioate internucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation. These chemical modifications, when used in various siNA constructs, are shown to preserve RNAi activity in cells while at the same time, dramatically increasing the serum stability of these compounds. Furthermore, contrary to the data published by Parrish et al., supra, applicant demonstrates that multiple (greater than one) phosphorothioate substitutions are well-tolerated and confer substantial increases in serum stability for modified siNA constructs.

In one embodiment, a siNA molecule of the invention comprises modified nucleotides while maintaining the ability to mediate RNAi. The modified nucleotides can be used to improve in vitro or in vivo characteristics such as stability, activity, and/or bioavailability. For example, a siNA molecule of the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siNA molecule. As such, a siNA molecule of the invention can generally comprise about 5% to about 100% modified nucleotides (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentage of modified nucleotides present in a given siNA molecule will depend on the total number of nucleotides present in the siNA. If the siNA molecule is single stranded, the percent modification can be based upon the total number of nucleotides present in the single stranded siNA molecules. Likewise, if the siNA molecule is double stranded, the percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.

One aspect of the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a SARS gene. In one embodiment, a double stranded siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 21 nucleotides long. In one embodiment, the double-stranded siNA molecule does not contain any ribonucleotides. In another embodiment, the double-stranded siNA molecule comprises one or more ribonucleotides. In one embodiment, each strand of the double-stranded siNA molecule comprises about 19 to about 23 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, wherein each strand comprises about 19 nucleotides that are complementary to the nucleotides of the other strand. In one embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof of the SARS gene, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the SARS gene or a portion thereof.

In another embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a SARS gene comprising an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of the SARS gene or a portion thereof, and a sense region, wherein the sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of the SARS gene or a portion thereof. In one embodiment, the antisense region and the sense region each comprise about 19 to about 23 (e.g. about 19, 20, 21, 22, or 23) nucleotides, wherein the antisense region comprises about 19 nucleotides that are complementary to nucleotides of the sense region.

In another embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a SARS gene comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the SARS gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region.

In one embodiment, the SARS virus RNA comtemplated by the invention comprises SARS virus minus strand RNA. In another embodiment, the SARS virus RNA comtemplated by the invention comprises SARS virus plus strand RNA.

In one embodiment, a siNA molecule of the invention comprises blunt ends, i.e., ends that do not include any overhanging nucleotides. For example, a siNA molecule of the invention comprising modifications described herein (e.g., comprising nucleotides having Formulae I-VII or siNA constructs comprising Stab00-Stab22 or any combination thereof (see Table IV)) and/or any length described herein can comprise blunt ends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise one or more blunt ends, i.e., where a blunt end does not have any overhanging nucleotides. In a non-limiting example, a blunt ended siNA molecule has a number of base pairs equal to the number of nucleotides present in each strand of the siNA molecule. In another example, a siNA molecule comprises one blunt end, for example wherein the 5′-end of the antisense strand and the 3′-end of the sense strand do not have any overhanging nucleotides. In another example, a siNA molecule comprises one blunt end, for example wherein the 3′-end of the antisense strand and the 5′-end of the sense strand do not have any overhanging nucleotides. In another example, a siNA molecule comprises two blunt ends, for example wherein the 3′-end of the antisense strand and the 5′-end of the sense strand as well as the 5′-end of the antisense strand and 3′-end of the sense strand do not have any overhanging nucleotides. A blunt ended siNA molecule can comprise, for example, from about 18 to about 30 nucleotides (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). Other nucleotides present in a blunt ended siNA molecule can comprise mismatches, bulges, loops, or wobble base pairs, for example, to modulate the activity of the siNA molecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini or termini of a double stranded siNA molecule having no overhanging nucleotides. The two strands of a double stranded siNA molecule align with each other without over-hanging nucleotides at the termini. For example, a blunt ended siNA construct comprises terminal nucleotides that are complementary between the sense and antisense regions of the siNA molecule.

In one embodiment, the invention features double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a SARS gene, wherein the siNA molecule comprises about 19 to about 21 base pairs, and wherein each strand of the siNA molecule comprises one or more chemical modifications. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a SARS gene or a portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the SARS gene. In another embodiment, one of the strands of the double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a SARS gene or a portion thereof, and the second strand of the double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of the SARS gene. In another embodiment, each strand of the siNA molecule comprises about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand. The SARS gene can comprise, for example, sequences referred to Table I.

In one embodiment, a siNA molecule of the invention comprises no ribonucleotides. In another embodiment, a siNA molecule of the invention comprises ribonucleotides.

In one embodiment, a siNA molecule of the invention comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of a SARS gene or a portion thereof, and the siNA further comprises a sense region comprising a nucleotide sequence substantially similar to the nucleotide sequence of the SARS gene or a portion thereof. In another embodiment, the antisense region and the sense region each comprise about 19 to about 23 nucleotides and the antisense region comprises at least about 19 nucleotides that are complementary to nucleotides of the sense region. The SARS gene can comprise, for example, sequences referred to Table 1.

In one embodiment, a siNA molecule of the invention comprises a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by a SARS gene, or a portion thereof, and the sense region comprises a nucleotide sequence that is complementary to the antisense region. In another embodiment, the siNA molecule is assembled from two separate oligonucleotide fragments, wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule. In another embodiment, the sense region is connected to the antisense region via a linker molecule, such as a nucleotide or non-nucleotide linker. The SARS gene can comprise, for example, sequences referred to Table I.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a SARS gene comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the SARS gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the siNA molecule has one or more modified pyrimidine and/or purine nucleotides. In one embodiment, the pyrimidine nucleotides in the sense region are 2′-O-methylpyrimidine nucleotides or 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In another embodiment, the pyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In one embodiment, the pyrimidine nucleotides in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotides present in the antisense region are 2′-O-methyl or 2′-deoxy purine nucleotides. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a non-complementary region of the sense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a siNA molecule comprising at least one modified nucleotide, wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide. The siNA can be, for example, of length between about 12 and about 36 nucleotides. In another embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In another embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In another embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In another embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In another embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In another embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as phosphorothioate linkage. In another embodiment, the 2′-deoxy-2′-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a method of increasing the stability of a siNA molecule against cleavage by ribonucleases comprising introducing at least one modified nucleotide into the siNA molecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide. In another embodiment, all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In another embodiment, the modified nucleotides in the siNA include at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, the modified nucleotides in the siNA include at least one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In another embodiment, all uridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In another embodiment, all cytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In another embodiment, all adenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In another embodiment, all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. The siNA can further comprise at least one modified internucleotidic linkage, such as phosphorothioate linkage. In another embodiment, the 2′-deoxy-2′-fluoronucleotides are present at specifically selected locations in the siNA that are sensitive to cleavage by ribonucleases, such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a SARS gene comprising a sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by the SARS gene or a portion thereof and the sense region comprises a nucleotide sequence that is complementary to the antisense region, and wherein the purine nucleotides present in the antisense region comprise 2′-deoxy-purine nucleotides. In an alternative embodiment, the purine nucleotides present in the antisense region comprise 2′-O-methyl purine nucleotides. In either of the above embodiments, the antisense region can comprise a phosphorothioate internucleotide linkage at the 3′ end of the antisense region. Alternatively, in either of the above embodiments, the antisense region can comprise a glyceryl modification at the 3′ end of the antisense region. In another embodiment of any of the above-described siNA molecules, any nucleotides present in a non-complementary region of the antisense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the antisense region of a siNA molecule of the invention comprises sequence complementary to a portion of a SARS transcript having sequence unique to a particular SARS disease related allele, such as sequence comprising a SNP associated with the disease specific allele. As such, the antisense region of a siNA molecule of the invention can comprise sequence complementary to sequences that are unique to a particular allele to provide specificity in mediating selective RNAi against the disease related allele.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a SARS gene, wherein the siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the siNA molecule. In another embodiment about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule and wherein at least two 3′ terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule. In one embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, such as a 2′-deoxy-thymidine. In another embodiment, all 21 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule. In another embodiment, about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the SARS gene. In another embodiment, about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the SARS gene. In any of the above embodiments, the 5′-end of the fragment comprising said antisense region can optionally includes a phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of a SARS RNA sequence (e.g., wherein said target RNA sequence is encoded by a SARS gene involved in the SARS pathway), wherein the siNA molecule does not contain any ribonucleotides and wherein each strand of the double-stranded siNA molecule is about 21 nucleotides long. Examples of non-ribonucleotide containing siNA constructs are combinations of stabilization chemistries shown in Table IV in any combination of Sense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, or Stab 18/20.

In one embodiment, the invention features a chemically synthesized double stranded RNA molecule that directs cleavage of a SARS RNA via RNA interference, wherein each strand of said RNA molecule is about 21 to about 23 nucleotides in length; one strand of the RNA molecule comprises nucleotide sequence having sufficient complementarity to the SARS RNA for the RNA molecule to direct cleavage of the SARS RNA via RNA interference; and wherein at least one strand of the RNA molecule comprises one or more chemically modified nucleotides described herein, such as deoxynucleotides, 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucloetides, 2′-O-methoxyethyl nucleotides etc.

In one embodiment, the invention features a medicament comprising a siNA molecule of the invention.

In one embodiment, the invention features an active ingredient comprising a siNA molecule of the invention.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule to down-regulate expression of a SARS gene, wherein the siNA molecule comprises one or more chemical modifications and each strand of the double-stranded siNA is about 18 to about 28 or more (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more) nucleotides long.

In one embodiment, the invention features the use of a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a SARS gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of SARS RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a SARS gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of SARS RNA that encodes a protein or portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a SARS gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of SARS RNA or a portion thereof, the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification. In one embodiment, each strand of the siNA molecule comprises about 18 to about 29 or more (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or more) nucleotides, wherein each strand comprises at least about 18 nucleotides that are complementary to the nucleotides of the other strand. In another embodiment, the siNA molecule is assembled from two oligonucleotide fragments, wherein one fragment comprises the nucleotide sequence of the antisense strand of the siNA molecule and a second fragment comprises nucleotide sequence of the sense region of the siNA molecule. In yet another embodiment, the sense strand is connected to the antisense strand via a linker molecule, such as a polynucleotide linker or a non-nucleotide linker.

In a further embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-deoxy purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidine nucleotides and the purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides. In still another embodiment, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2′-deoxy purine nucleotides. In another embodiment, the antisense strand comprises one or more 2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methyl purine nucleotides. In another embodiment, the pyrimidine nucleotides present in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotides present in the antisense strand are 2′-O-methyl purine nucleotides. In a further embodiment the sense strand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety (e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotide moiety such as inverted thymidine) is present at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the sense strand. In another embodiment, the antisense strand comprises a phosphorothioate internucleotide linkage at the 3′ end of the antisense strand. In another embodiment, the antisense strand comprises a glyceryl modification at the 3′ end. In another embodiment, the 5′-end of the antisense strand optionally includes a phosphate group.

In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) molecule that inhibits expression of a SARS gene, wherein one of the strands of the double-stranded siNA molecule is an antisense strand which comprises nucleotide sequence that is complementary to nucleotide sequence of SARS RNA or a portion thereof, wherein the other strand is a sense strand which comprises nucleotide sequence that is complementary to a nucleotide sequence of the antisense strand and wherein a majority of the pyrimidine nucleotides present in the double-stranded siNA molecule comprises a sugar modification, and wherein each of the two strands of the siNA molecule comprises about 21 nucleotides. In one embodiment, about 21 nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule. In another embodiment, about 19 nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule, wherein at least two 3′ terminal nucleotides of each strand of the siNA molecule are not base-paired to the nucleotides of the other strand of the siNA molecule. In another embodiment, each of the two 3′ terminal nucleotides of each fragment of the siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine. In another embodiment, each strand of the siNA molecule is base-paired to the complementary nucleotides of the other strand of the siNA molecule. In another embodiment, about 19 nucleotides of the antisense strand are base-paired to the nucleotide sequence of the SARS RNA or a portion thereof. In another embodiment, about 21 nucleotides of the antisense strand are base-paired to the nucleotide sequence of the SARS RNA or a portion thereof.

In one embodiment, the invention features a composition comprising a siNA molecule of the invention in a pharmaceutically acceptable carrier or diluent.

In a non-limiting example, the introduction of chemically-modified nucleotides into nucleic acid molecules provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA molecules that are delivered exogenously. For example, the use of chemically-modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically-modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically-modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example, when compared to an all-RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than that of the native molecule due to improved stability and/or delivery of the molecule. Unlike native unmodified siNA, chemically-modified siNA can also minimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, the antisense region of a siNA molecule of the invention can comprise a phosphorothioate internucleotide linkage at the 3′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the antisense region can comprise about one to about five phosphorothioate internucleotide linkages at the 5′-end of said antisense region. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs of a siNA molecule of the invention can comprise ribonucleotides or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar, base, or backbone. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more universal base ribonucleotides. In any of the embodiments of siNA molecules described herein, the 3′-terminal nucleotide overhangs can comprise one or more acyclic nucleotides.

One embodiment of the invention provides an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention in a manner that allows expression of the nucleic acid molecule. Another embodiment of the invention provides a mammalian cell comprising such an expression vector. The mammalian cell can be a human cell. The siNA molecule of the expression vector can comprise a sense region and an antisense region. The antisense region can comprise sequence complementary to a RNA or DNA sequence encoding SARS and the sense region can comprise sequence complementary to the antisense region. The siNA molecule can comprise two distinct strands having complementary sense and antisense regions. The siNA molecule can comprise a single strand having complementary sense and antisense regions.

In one embodiment, the nucleotide sequence of the antisense strand or a portion thereof of a siNA molecule of the invention is complementary to the nucleotide sequence of a SARS RNA or a portion thereof that is present in the RNA of all SARS isolates.

wherein each R1 and R2 is independently any nucleotide, non-nucleotide, or polynucleotide which can be naturally-occurring or chemically-modified, each X and Y is independently O, S, N, alkyl, or substituted alkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl and wherein W, X, Y, and Z are optionally not all O. In another embodiment, a backbone modification of the invention comprises a phosphonoacetate and/or thiophosphonoacetate internucleotide linkage (see for example Sheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, for example, wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modified internucleotide linkages having Formula I at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide linkages having Formula I at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically-modified internucleotide linkages having Formula I in the sense strand, the antisense strand, or both strands. In another embodiment, a siNA molecule of the invention having internucleotide linkage(s) of Formula I also comprises a chemically-modified nucleotide or non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against SARS inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO₂, NO₂, N3, NH₂, aminoalkyl, aminoacid, aminoacyl, ONH₂, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH₂, S═O, CHF, or CF₂, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula II can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotide or non-nucleotide of Formula II at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 5′-end of the sense strand, the antisense strand, or both strands. In anther non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-nucleotides of Formula II at the 3′-end of the sense strand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against SARS inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotides having Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-naturally occurring base that can be employed to be complementary or non-complementary to target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally occurring universal base that can be complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula III can be present in one or both oligonucleotide strands of the siNA duplex, for example, in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) or non-nucleotide(s) of Formula III at the 5′-end of the sense strand, the antisense strand, or both strands. In anther non-limiting example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide of Formula III at the 3′-end of the sense strand, the antisense strand, or both strands.

In another embodiment, a siNA molecule of the invention comprises a nucleotide having Formula II or III, wherein the nucleotide having Formula II or III is in an inverted configuration. For example, the nucleotide having Formula II or III is connected to the siNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand, for example, a strand complementary to a target RNA, wherein the siNA molecule comprises an all RNA siNA molecule. In another embodiment, the invention features a siNA molecule having a 5′-terminal phosphate group having Formula IV on the target-complementary strand wherein the siNA molecule also comprises about 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminal nucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or 4) deoxyribonucleotides on the 3′-end of one or both strands. In another embodiment, a 5′-terminal phosphate group having Formula IV is present on the target-complementary strand of a siNA molecule of the invention, for example a siNA molecule having chemical modifications having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) against SARS inside a cell or reconstituted in vitro system, wherein the chemical modification comprises one or more phosphorothioate internucleotide linkages. For example, in a non-limiting example, the invention features a chemically-modified short interfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in one siNA strand. In yet another embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in both siNA strands. The phosphorothioate internucleotide linkages can be present in one or both oligonucleotide strands of the siNA duplex, for example in the sense strand, the antisense strand, or both strands. The siNA molecules of the invention can comprise one or more phosphorothioate internucleotide linkages at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand, the antisense strand, or both strands. For example, an exemplary siNA molecule of the invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate internucleotide linkages at the 5′-end of the sense strand, the antisense strand, or both strands. In another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands. In yet another non-limiting example, an exemplary siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the sense strand, the antisense strand, or both strands.

In one embodiment, the invention features a siNA molecule, wherein the sense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, wherein the sense strand comprises about 1 to about 5, specifically about 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a siNA molecule, wherein the antisense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand; and wherein the antisense strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisense strand. In another embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule having about 1 to about 5, specifically about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features a siNA molecule comprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one or both siNA sequence strands. In addition, the 2′-5′ internucleotide linkage(s) can be present at various other positions within one or both siNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a pyrimidine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine nucleotide in one or both strands of the siNA molecule can comprise a 2′-5′ internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified, wherein each strand is about 18 to about 27 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides in length, wherein the duplex has about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the chemical modification comprises a structure having any of Formulae I-VII. For example, an exemplary chemically-modified siNA molecule of the invention comprises a duplex having two strands, one or both of which can be chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein each strand consists of about 21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotide overhang, and wherein the duplex has about 19 base pairs. In another embodiment, a siNA molecule of the invention comprises a single stranded hairpin structure, wherein the siNA is about 36 to about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 19 base pairs and a 2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. For example, a linear hairpin siNA molecule of the invention is designed such that degradation of the loop portion of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises a hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about 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, or 50) nucleotides in length having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms a hairpin structure having about 3 to about 23 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) base pairs and a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV). In another embodiment, a linear hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In another embodiment, a linear hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises an asymmetric hairpin structure, wherein the siNA is about 25 to about 50 (e.g., about 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, or 50) nucleotides in length having about 3 to about 20 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) base pairs, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a linear oligonucleotide having about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-modified with one or more chemical modifications having any of Formulae I-VII or any combination thereof, wherein the linear oligonucleotide forms an asymmetric hairpin structure having about 3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18) base pairs and a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV). In another embodiment, an asymmetric hairpin siNA molecule of the invention contains a stem loop motif, wherein the loop portion of the siNA molecule is biodegradable. In another embodiment, an asymmetric hairpin siNA molecule of the invention comprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 16 to about 25 (e.g., about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length, wherein the sense region is about 3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides in length, wherein the sense region and the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises an asymmetric double stranded structure having separate polynucleotide strands comprising sense and antisense regions, wherein the antisense region is about 18 to about 22 (e.g., about 18, 19, 20, 21, or 22) nucleotides in length and wherein the sense region is about 3 to about 15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, II, 12, 13, 14, or 15) nucleotides in length, wherein the sense region the antisense region have at least 3 complementary nucleotides, and wherein the siNA can include one or more chemical modifications comprising a structure having any of Formulae I-VII or any combination thereof. In another embodiment, the asymmetic double stranded siNA molecule can also have a 5′-terminal phosphate group that can be chemically modified as described herein (for example a 5′-terminal phosphate group having Formula IV).

In another embodiment, a siNA molecule of the invention comprises a circular nucleic acid molecule, wherein the siNA is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein the siNA can include a chemical modification, which comprises a structure having any of Formulae I-VII or any combination thereof. For example, an exemplary chemically-modified siNA molecule of the invention comprises a circular oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically-modified with a chemical modification having any of Formulae I-VII or any combination thereof, wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the invention contains two loop motifs, wherein one or both loop portions of the siNA molecule is biodegradable. For example, a circular siNA molecule of the invention is designed such that degradation of the loop portions of the siNA molecule in vivo can generate a double-stranded siNA molecule with 3′-terminal overhangs, such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety, for example a compound having

Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO₂, N3, NH₂, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH₂, S═O, CHF, or CF₂.

In one embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic moiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or group having Formula I or II; R9 is O, S, CH₂, S═O, CHF, or CF₂, and either R2, R3, R8 or R13 serve as points of attachment to the siNA molecule of the invention.

In another embodiment, a siNA molecule of the invention comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituted polyalkyl moieties, for example a compound having Formula VII:

wherein each n is independently an integer from 1 to 12, each R1, R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl, or a group having Formula I, and R1, R2 or R3 serves as points of attachment to the siNA molecule of the invention.

In another embodiment, the invention features a compound having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises O and is the point of attachment to the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both strands of a double-stranded siNA molecule of the invention or to a single-stranded siNA molecule of the invention. This modification is referred to herein as “glyceryl” (for example modification 6 in FIG. 10).

In another embodiment, a moiety having any of Formula V, VI or VII of the invention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of a siNA molecule of the invention. For example, a moiety having Formula V, VI or VII can be present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense strand, the sense strand, or both antisense and sense strands of the siNA molecule. In addition, a moiety having Formula VII can be present at the 3′-end or the 5′-end of a hairpin siNA molecule as described herein.

In another embodiment, a siNA molecule of the invention comprises an abasic residue having Formula V or VI, wherein the abasic residue having Formula VI or VI is connected to the siNA construct in a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA) nucleotides, for example at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, a siNA molecule of the invention comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides, for example at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising a sense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), wherein any (e.g., one or more or all) purine nucleotides present in the sense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and wherein any nucleotides comprising a 3′-terminal nucleotide overhang that are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention comprising an antisense region, wherein any (e.g., one or more or all) pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more or all) purine nucleotides present in the antisense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified short interfering nucleic acid (siNA) molecule of the invention capable of mediating RNA interference (RNAi) against SARS inside a cell or reconstituted in vitro system comprising a sense region, wherein one or more pyrimidine nucleotides present in the sense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purine nucleotides present in the sense region are 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides), and an antisense region, wherein one or more pyrimidine nucleotides present in the antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides). The sense region and/or the antisense region can have a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/or antisense sequence. The sense and/or antisense region can optionally further comprise a 3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhang nucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages. Non-limiting examples of these chemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III and IV herein.

In any of these described embodiments, the purine nucleotides present in the sense region are alternatively 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides) and one or more purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides). Also, in any of these embodiments, one or more purine nucleotides present in the sense region are alternatively purine ribonucleotides (e.g., wherein all purine nucleotides are purine ribonucleotides or alternately a plurality of purine nucleotides are purine ribonucleotides) and any purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides). Additionally, in any of these embodiments, one or more purine nucleotides present in the sense region and/or present in the antisense region are alternatively selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g., wherein all purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides or alternately a plurality of purine nucleotides are selected from the group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the siNA molecules of the invention, preferably in the antisense strand of the siNA molecules of the invention, but also optionally in the sense and/or both antisense and sense strands, are resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi. Non-limiting examples of nucleotides having a northern configuration include locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double stranded siNA molecule of the invention comprises a terminal cap moiety, (see for example FIG. 10) such as an inverted deoxyabaisc moiety, at the 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified short interfering nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi) against SARS inside a cell or reconstituted in vitro system, wherein the chemical modification comprises a conjugate covalently attached to the chemically-modified siNA molecule. Non-limiting examples of conjugates contemplated by the invention include conjugates and ligands described in Vargeese et al, U.S. Ser. No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein in its entirety, including the drawings. In another embodiment, the conjugate is covalently attached to the chemically-modified siNA molecule via a biodegradable linker. In one embodiment, the conjugate molecule is attached at the 3′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In another embodiment, the conjugate molecule is attached at the 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule. In yet another embodiment, the conjugate molecule is attached both the 3′-end and 5′-end of either the sense strand, the antisense strand, or both strands of the chemically-modified siNA molecule, or any combination thereof. In one embodiment, a conjugate molecule of the invention comprises a molecule that facilitates delivery of a chemically-modified siNA molecule into a biological system, such as a cell. In another embodiment, the conjugate molecule attached to the chemically-modified siNA molecule is a polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that can mediate cellular uptake. Examples of specific conjugate molecules contemplated by the instant invention that can be attached to chemically-modified siNA molecules are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002, incorporated by reference herein. The type of conjugates used and the extent of conjugation of siNA molecules of the invention can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of siNA constructs while at the same time maintaining the ability of the siNA to mediate RNAi activity. As such, one skilled in the art can screen siNA constructs that are modified with various conjugates to determine whether the siNA conjugate complex possesses improved properties while maintaining the ability to mediate RNAi, for example in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule of the invention, wherein the siNA further comprises a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense region of the siNA to the antisense region of the siNA. In one embodiment, a nucleotide linker of the invention can be a linker of >2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In another embodiment, the nucleotide linker can be a nucleic acid aptamer. By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule where the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art. (See, for example, Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the invention comprises abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al., International Publication No. WO 89/02439; Usman et al, International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by reference herein. A “non-nucleotide” further means any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound can be abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine, for example at the C1 position of the sugar.

In one embodiment, the invention features a short interfering nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi) inside a cell or reconstituted in vitro system, wherein one or both strands of the siNA molecule that are assembled from two separate oligonucleotides do not comprise any ribonucleotides. For example, a siNA molecule can be assembled from a single oligonculeotide where the sense and antisense regions of the siNA comprise separate oligonucleotides not having any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotides. In another example, a siNA molecule can be assembled from a single oligonucleotide where the sense and antisense regions of the siNA are linked or circularized by a nucleotide or non-nucleotide linker as described herein, wherein the oligonucleotide does not have any ribonucleotides (e.g., nucleotides having a 2′-OH group) present in the oligonucleotide. Applicant has surprisingly found that the presence of ribonucleotides (e.g., nucleotides having a 2′-hydroxyl group) within the siNA molecule is not required or essential to support RNAi activity. As such, in one embodiment, all positions within the siNA can include chemically modified nucleotides and/or non-nucleotides such as nucleotides and or non-nucleotides having Formula I, II, III, IV, V, VI, or VII or any combination thereof to the extent that the ability of the siNA molecule to support RNAi activity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single stranded siNA molecule that mediates RNAi activity in a cell or reconstituted in vitro system comprising a single stranded polynucleotide having complementarity to a target nucleic acid sequence, wherein one or more pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purine nucleotides present in the antisense region are 2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-O-methyl purine nucleotides), and a terminal cap modification, such as any modification described herein or shown in FIG. 10, that is optionally present at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the antisense sequence. The siNA optionally further comprises about 1 to about 4 or more (e.g., about 1, 2, 3, 4 or more) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages, and wherein the siNA optionally further comprises a terminal phosphate group, such as a 5′-terminal phosphate group. In any of these embodiments, any purine nucleotides present in the antisense region are alternatively 2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides or alternately a plurality of purine nucleotides are 2′-deoxy purine nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA (i.e., purine nucleotides present in the sense and/or antisense region) can alternatively be locked nucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides or alternately a plurality of purine nucleotides are LNA nucleotides). Also, in any of these embodiments, any purine nucleotides present in the siNA are alternatively 2′-methoxyethyl purine nucleotides (e.g., wherein all purine nucleotides are 2′-methoxyethyl purine nucleotides or alternately a plurality of purine nucleotides are 2′-methoxyethyl purine nucleotides). In another embodiment, any modified nucleotides present in the single stranded siNA molecules of the invention comprise modified nucleotides having properties or characteristics similar to naturally occurring ribonucleotides. For example, the invention features siNA molecules including modified nucleotides having a Northern conformation (e.g., Northern pseudorotation cycle, see for example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemically modified nucleotides present in the single stranded siNA molecules of the invention are preferably resistant to nuclease degradation while at the same time maintaining the capacity to mediate RNAi.

In one embodiment, the invention features a method for modulating the expression of a SARS gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the SARS gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the SARS gene in the cell.

In one embodiment, the invention features a method for modulating the expression of a SARS gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the SARS gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the SARS gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one SARS gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the SARS genes; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate the expression of the SARS genes in the cell.

In another embodiment, the invention features a method for modulating the expression of two or more SARS genes within a cell comprising: (a) synthesizing one or more siNA molecules of the invention, which can be chemically-modified, wherein the siNA strands comprise sequences complementary to RNA of the SARS genes and wherein the sense strand sequences of the siNAs comprise sequences identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecules into a cell under conditions suitable to modulate the expression of the SARS genes in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one SARS gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the SARS gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequences of the target RNAs; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the SARS genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagents in ex vivo applications. For example, siNA reagents are introduced into tissue or cells that are transplanted into a subject for therapeutic effect. The cells and/or tissue can be derived from an organism or subject that later receives the explant, or can be derived from another organism or subject prior to transplantation. The siNA molecules can be used to modulate the expression of one or more genes in the cells or tissue, such that the cells or tissue obtain a desired phenotype or are able to perform a function when transplanted in vivo. In one embodiment, certain target cells from a patient are extracted. These extracted cells are contacted with siNAs targeting a specific nucleotide sequence within the cells under conditions suitable for uptake of the siNAs by these cells (e.g. using delivery reagents such as cationic lipids, liposomes and the like or using techniques such as electroporation to facilitate the delivery of siNAs into cells). The cells are then reintroduced back into the same patient or other patients. In one embodiment, the invention features a method of modulating the expression of a SARS gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the SARS gene; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the SARS gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the SARS gene in that organism.

In one embodiment, the invention features a method of modulating the expression of a SARS gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the SARS gene and wherein the sense strand sequence of the siNA comprises a sequence identical or substantially similar to the sequence of the target RNA; and (b) introducing the siNA molecule into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the SARS gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the SARS gene in that organism.

In another embodiment, the invention features a method of modulating the expression of more than one SARS gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the SARS genes; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the SARS genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the SARS genes in that organism.

In one embodiment, the invention features a method of modulating the expression of a SARS gene in an organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the SARS gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the SARS gene in the organism. The level of SARS protein or RNA can be determined as is known in the art.

In another embodiment, the invention features a method of modulating the expression of more than one SARS gene in an organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein one of the siNA strands comprises a sequence complementary to RNA of the SARS genes; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the SARS genes in the organism. The level of SARS protein or RNA can be determined as is known in the art.

In one embodiment, the invention features a method for modulating the expression of a SARS gene within a cell comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the SARS gene; and (b) introducing the siNA molecule into a cell under conditions suitable to modulate the expression of the SARS gene in the cell.

In another embodiment, the invention features a method for modulating the expression of more than one SARS gene within a cell comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the SARS gene; and (b) contacting the cell in vitro or in vivo with the siNA molecule under conditions suitable to modulate the expression of the SARS genes in the cell.

In one embodiment, the invention features a method of modulating the expression of a SARS gene in a tissue explant comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the SARS gene; and (b) contacting the cell of the tissue explant derived from a particular organism with the siNA molecule under conditions suitable to modulate the expression of the SARS gene in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the SARS gene in that organism.

In another embodiment, the invention features a method of modulating the expression of more than one SARS gene in a tissue explant comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the SARS gene; and (b) introducing the siNA molecules into a cell of the tissue explant derived from a particular organism under conditions suitable to modulate the expression of the SARS genes in the tissue explant. In another embodiment, the method further comprises introducing the tissue explant back into the organism the tissue was derived from or into another organism under conditions suitable to modulate the expression of the SARS genes in that organism.

In one embodiment, the invention features a method of modulating the expression of a SARS gene in an organism comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the SARS gene; and (b) introducing the siNA molecule into the organism under conditions suitable to modulate the expression of the SARS gene in the organism.

In another embodiment, the invention features a method of modulating the expression of more than one SARS gene in an organism comprising: (a) synthesizing siNA molecules of the invention, which can be chemically-modified, wherein the siNA comprises a single stranded sequence having complementarity to RNA of the SARS gene; and (b) introducing the siNA molecules into the organism under conditions suitable to modulate the expression of the SARS genes in the organism.

In one embodiment, the invention features a method of modulating the expression of a SARS gene in an organism comprising contacting the organism with a siNA molecule of the invention under conditions suitable to modulate the expression of the SARS gene in the organism.

In another embodiment, the invention features a method of modulating the expression of more than one SARS gene in an organism comprising contacting the organism with one or more siNA molecules of the invention under conditions suitable to modulate the expression of the SARS genes in the organism.

The siNA molecules of the invention can be designed to down regulate or inhibit target (e.g., SARS) gene expression through RNAi targeting of a variety of RNA molecules. In one embodiment, the siNA molecules of the invention are used to target various RNAs corresponding to a target gene. Non-limiting examples of such RNAs include messenger RNA (mRNA), alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates. If alternate splicing produces a family of transcripts that are distinguished by usage of appropriate exons, the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members. For example, a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms. Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein. Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siNA molecules of the invention. Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used to target conserved sequences corresponding to a gene family or gene families such as SARS family genes. As such, siNA molecules targeting multiple SARS targets can provide increased therapeutic effect. In addition, siNA can be used to characterize pathways of gene function in a variety of applications. For example, the present invention can be used to inhibit the activity of target gene(s) in a pathway to determine the function of uncharacterized gene(s) in gene function analysis, mRNA function analysis, or translational analysis. The invention can be used to determine potential target gene pathways involved in various diseases and conditions toward pharmaceutical development. The invention can be used to understand pathways of gene expression involved in, for example, the progression and/or maintenance of SARS virus infection, acute respiratory failure, viral pneumonia, and other indications that can respond to the level of SARS in a cell or tissue.

In one embodiment, siNA molecule(s) and/or methods of the invention are used to down regulate the expression of gene(s) that encode RNA referred to by Genbank Accession, for example SARS genes encoding RNA sequence(s) referred to herein by Genbank Accession number, for example, Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a) generating a library of siNA constructs having a predetermined complexity; and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target RNA sequence. In one embodiment, the siNA molecules of (a) have strands of a fixed length, for example, about 23 nucleotides in length. In another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: (a) generating a randomized library of siNA constructs having a predetermined complexity, such as of 4^N, where N represents the number of base paired nucleotides in each of the siNA construct strands (e.g. for a siNA construct having 21 nucleotide sense and antisense strands with 19 base pairs, the complexity would be 4¹⁹); and (b) assaying the siNA constructs of (a) above, under conditions suitable to determine RNAi target sites within the target SARS RNA sequence. In another embodiment, the siNA molecules of (a) have strands of a fixed length, for example about 23 nucleotides in length. In yet another embodiment, the siNA molecules of (a) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described in Example 7 herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. In another embodiment, fragments of SARS RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target SARS RNA sequence. The target SARS RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by cellular expression in in vivo systems.

In another embodiment, the invention features a method comprising: (a) analyzing the sequence of a RNA target encoded by a target gene; (b) synthesizing one or more sets of siNA molecules having sequence complementary to one or more regions of the RNA of (a); and (c) assaying the siNA molecules of (b) under conditions suitable to determine RNAi targets within the target RNA sequence. In one embodiment, the siNA molecules of (b) have strands of a fixed length, for example about 23 nucleotides in length. In another embodiment, the siNA molecules of (b) are of differing length, for example having strands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. In one embodiment, the assay can comprise a reconstituted in vitro siNA assay as described herein. In another embodiment, the assay can comprise a cell culture system in which target RNA is expressed. Fragments of target RNA are analyzed for detectable levels of cleavage, for example by gel electrophoresis, northern blot analysis, or RNAse protection assays, to determine the most suitable target site(s) within the target RNA sequence. The target RNA sequence can be obtained as is known in the art, for example, by cloning and/or transcription for in vitro systems, and by expression in in vivo systems.

By “target site” is meant a sequence within a target RNA that is “targeted” for cleavage mediated by a siNA construct which contains sequences within its antisense region that are complementary to the target sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the background for most methods of detection.

In one embodiment, the invention features a composition comprising a siNA molecule of the invention, which can be chemically-modified, in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a pharmaceutical composition comprising siNA molecules of the invention, which can be chemically-modified, targeting one or more genes in a pharmaceutically acceptable carrier or diluent. In another embodiment, the invention features a method for diagnosing a disease or condition in a subject comprising administering to the subject a composition of the invention under conditions suitable for the diagnosis of the disease or condition in the subject. In another embodiment, the invention features a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease or condition in the subject, alone or in conjunction with one or more other therapeutic compounds. In yet another embodiment, the invention features a method for reducing or preventing tissue rejection in a subject comprising administering to the subject a composition of the invention under conditions suitable for the reduction or prevention of tissue rejection in the subject.

In another embodiment, the invention features a method for validating a SARS gene target, comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a SARS target gene; (b) introducing the siNA molecule into a cell, tissue, or organism under conditions suitable for modulating expression of the SARS target gene in the cell, tissue, or organism; and (c) determining the function of the gene by assaying for any phenotypic change in the cell, tissue, or organism.

In another embodiment, the invention features a method for validating a SARS target comprising: (a) synthesizing a siNA molecule of the invention, which can be chemically-modified, wherein one of the siNA strands includes a sequence complementary to RNA of a SARS target gene; (b) introducing the siNA molecule into a biological system under conditions suitable for modulating expression of the SARS target gene in the biological system; and (c) determining the function of the gene by assaying for any phenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurified form, from biological sources, including but not limited to human or animal, wherein the system comprises the components required for RNAi activity. The term “biological system” includes, for example, a cell, tissue, or organism, or extract thereof. The term biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.

By “phenotypic change” is meant any detectable change to a cell that occurs in response to contact or treatment with a nucleic acid molecule of the invention (e.g., siNA). Such detectable changes include, but are not limited to, changes in shape, size, proliferation, motility, protein expression or RNA expression or other physical or chemical changes as can be assayed by methods known in the art. The detectable change can also include expression of reporter genes/molecules such as Green Florescent Protein (GFP) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing a siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of a SARS target gene in a biological system, including, for example, in a cell, tissue, or organism. In another embodiment, the invention features a kit containing more than one siNA molecule of the invention, which can be chemically-modified, that can be used to modulate the expression of more than one SARS target gene in a biological system, including, for example, in a cell, tissue, or organism.

In one embodiment, the invention features a cell containing one or more siNA molecules of the invention, which can be chemically-modified. In another embodiment, the cell containing a siNA molecule of the invention is a mammalian cell. In yet another embodiment, the cell containing a siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a siNA molecule of the invention, which can be chemically-modified, comprises: (a) synthesis of two complementary strands of the siNA molecule; (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siNA molecule. In another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase oligonucleotide synthesis. In yet another embodiment, synthesis of the two complementary strands of the siNA molecule is by solid phase tandem oligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence strand of the siNA molecule, wherein the first oligonucleotide sequence strand comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of the second oligonucleotide sequence strand of the siNA; (b) synthesizing the second oligonucleotide sequence strand of siNA on the scaffold of the first oligonucleotide sequence strand, wherein the second oligonucleotide sequence strand further comprises a chemical moiety than can be used to purify the siNA duplex; (c) cleaving the linker molecule of (a) under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex; and (d) purifying the siNA duplex utilizing the chemical moiety of the second oligonucleotide sequence strand. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example under hydrolysis conditions using an alkylamine base such as methylamine. In one embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place concomitantly. In another embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy as described herein. In yet another embodiment, the chemical moiety, such as a dimethoxytrityl group, is removed during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solution phase synthesis or hybrid phase synthesis wherein both strands of the siNA duplex are synthesized in tandem using a cleavable linker attached to the first sequence which acts a scaffold for synthesis of the second sequence. Cleavage of the linker under conditions suitable for hybridization of the separate siNA sequence strands results in formation of the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizing a siNA duplex molecule comprising: (a) synthesizing one oligonucleotide sequence strand of the siNA molecule, wherein the sequence comprises a cleavable linker molecule that can be used as a scaffold for the synthesis of another oligonucleotide sequence; (b) synthesizing a second oligonucleotide sequence having complementarity to the first sequence strand on the scaffold of (a), wherein the second sequence comprises the other strand of the double-stranded siNA molecule and wherein the second sequence further comprises a chemical moiety than can be used to isolate the attached oligonucleotide sequence; (c) purifying the product of (b) utilizing the chemical moiety of the second oligonucleotide sequence strand under conditions suitable for isolating the full-length sequence comprising both siNA oligonucleotide strands connected by the cleavable linker and under conditions suitable for the two siNA oligonucleotide strands to hybridize and form a stable duplex. In one embodiment, cleavage of the linker molecule in (c) above takes place during deprotection of the oligonucleotide, for example under hydrolysis conditions. In another embodiment, cleavage of the linker molecule in (c) above takes place after deprotection of the oligonucleotide. In another embodiment, the method of synthesis comprises solid phase synthesis on a solid support such as controlled pore glass (CPG) or polystyrene, wherein the first sequence of (a) is synthesized on a cleavable linker, such as a succinyl linker, using the solid support as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing the second strand can comprise similar reactivity or differing reactivity as the solid support derivatized linker, such that cleavage of the solid support derivatized linker and the cleavable linker of (a) takes place either concomitantly or sequentially. In one embodiment, the chemical moiety of (b) that can be used to isolate the attached oligonucleotide sequence comprises a trityl group, for example a dimethoxytrityl group.

In another embodiment, the invention features a method for making a double-stranded siNA molecule in a single synthetic process comprising: (a) synthesizing an oligonucleotide having a first and a second sequence, wherein the first sequence is complementary to the second sequence, and the first oligonucleotide sequence is linked to the second sequence via a cleavable linker, and wherein a terminal 5′-protecting group, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains on the oligonucleotide having the second sequence; (b) deprotecting the oligonucleotide whereby the deprotection results in the cleavage of the linker joining the two oligonucleotide sequences; and (c) purifying the product of (b) under conditions suitable for isolating the double-stranded siNA molecule, for example using a trityl-on synthesis strategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of the invention comprises the teachings of Scaringe et al., U.S. Pat. Nos. 5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein in their entirety.

In one embodiment, the invention features siNA constructs that mediate RNAi against SARS, wherein the siNA construct comprises one or more chemical modifications, for example, one or more chemical modifications having any of Formulae I-VII or any combination thereof that increases the nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generating siNA molecules with increased nuclease resistance comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased nuclease resistance.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the sense and antisense strands of the siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the sense and antisense strands of the siNA molecule.

In one embodiment, the invention features siNA constructs that mediate RNAi against SARS, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediate RNAi against SARS, wherein the siNA construct comprises one or more chemical modifications described herein that modulates the binding affinity between the antisense strand of the siNA construct and a complementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generating siNA molecules with increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having increased binding affinity between the antisense strand of the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediate RNAi against SARS, wherein the siNA construct comprises one or more chemical modifications described herein that modulate the polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA construct.

In another embodiment, the invention features a method for generating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to a chemically-modified siNA molecule comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules capable of mediating increased polymerase activity of a cellular polymerase capable of generating additional endogenous siNA molecules having sequence homology to the chemically-modified siNA molecule.

In one embodiment, the invention features chemically-modified siNA constructs that mediate RNAi against SARS in a cell, wherein the chemical modifications do not significantly effect the interaction of siNA with a target RNA molecule, DNA molecule and/or proteins or other factors that are essential for RNAi in a manner that would decrease the efficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against SARS comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against SARS target RNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method for generating siNA molecules with improved RNAi activity against SARS target DNA comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved RNAi activity against the target DNA.

In another embodiment, the invention features a method for generating siNA molecules against SARS with improved cellular uptake comprising (a) introducing nucleotides having any of Formula I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediate RNAi against SARS, wherein the siNA construct comprises one or more chemical modifications described herein that increases the bioavailability of the siNA construct, for example, by attaching polymeric conjugates such as polyethyleneglycol or equivalent conjugates that improve the pharmacokinetics of the siNA construct, or by attaching conjugates that target specific tissue types or cell types in vivo. Non-limiting examples of such conjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability, comprising (a) introducing a conjugate into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such conjugates can include ligands for cellular receptors, such as peptides derived from naturally occurring protein ligands; protein localization sequences, including cellular ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate and N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol; polyamines, such as spermine or spermidine; and others.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein the second sequence is designed or modified in a manner that prevents its entry into the RNAi pathway as a guide sequence or as a sequence that is complementary to a target nucleic acid (e.g., RNA) sequence. Such design or modifications are expected to enhance the activity of siNA and/or improve the specificity of siNA molecules of the invention. These modifications are also expected to minimize any off-target effects and/or associated toxicity.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5′-end of said second sequence. In one embodiment, the terminal cap moiety comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.

In one embodiment, the invention features a double stranded short interfering nucleic acid (siNA) molecule that comprises a first nucleotide sequence complementary to a target RNA sequence or a portion thereof, and a second sequence having complementarity to said first sequence, wherein said second sequence comprises a terminal cap moiety at the 5′-end and 3′-end of said second sequence. In one embodiment, each terminal cap moiety individually comprises an inverted abasic, inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkyl group, a heterocycle, or any other group that prevents RNAi activity in which the second sequence serves as a guide sequence or template for RNAi.

In one embodiment, the invention features a method for generating siNA molecules of the invention with improved specificity for down regulating or inhibiting the expression of a target nucleic acid (e.g., a DNA or RNA such as a gene or its corresponding RNA), comprising (a) introducing one or more chemical modifications into the structure of a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved specificity. In another embodiment, the chemical modification used to improve specificity comprises terminal cap modifications at the 5′-end, 3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal cap modifications can comprise, for example, structures shown in FIG. 10 (e.g. inverted deoxyabasic moieties) or any other chemical modification that renders a portion of the siNA molecule (e.g. the sense strand) incapable of mediating RNA interference against an off target nucleic acid sequence. In a non-limiting example, a siNA molecule is designed such that only the antisense sequence of the siNA molecule can serve as a guide sequence for RISC mediated degradation of a corresponding target RNA sequence. This can be accomplished by rendering the sense sequence of the siNA inactive by introducing chemical modifications to the sense strand that preclude recognition of the sense strand as a guide sequence by RNAi machinery. In one embodiment, such chemical modifications comprise any chemical group at the 5′-end of the sense strand of the siNA, or any other group that serves to render the sense strand inactive as a guide sequence for mediating RNA interference. These modifications, for example, can result in a molecule where the 5′-end of the sense strand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphate group (e.g., phosphate, diphosphate, triphosphate, cyclic phosphate etc.). Non-limiting examples of such siNA constructs are described herein, such as “Stab 9/10”, “Stab ⅞”, “Stab 7/19” and “Stab 17/22” chemistries and variants thereof (see Table IV) wherein the 5′-end and 3′-end of the sense strand of the siNA do not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for screening siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising (a) generating a plurality of unmodified siNA molecules, (b) screening the siNA molecules of step (a) under conditions suitable for isolating siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence, and (c) introducing chemical modifications (e.g. chemical modifications as described herein or as otherwise known in the art) into the active siNA molecules of (b). In one embodiment, the method further comprises re-screening the chemically modified siNA molecules of step (c) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.

In one embodiment, the invention features a method for screening chemically modified siNA molecules that are active in mediating RNA interference against a target nucleic acid sequence comprising (a) generating a plurality of chemically modified siNA molecules (e.g. siNA molecules as described herein or as otherwise known in the art), and (b) screening the siNA molecules of step (a) under conditions suitable for isolating chemically modified siNA molecules that are active in mediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, that is capable of interacting with another compound, such as a receptor, either directly or indirectly. The receptor that interacts with a ligand can be present on the surface of a cell or can alternately be an intercellular receptor. Interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing an excipient formulation to a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability. Such excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, nanoparticles, receptors, ligands, and others.

In another embodiment, the invention features a method for generating siNA molecules of the invention with improved bioavailability comprising (a) introducing nucleotides having any of Formulae I-VII or any combination thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions suitable for isolating siNA molecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalently attached to siNA compounds of the present invention. The attached PEG can be any molecular weight, preferably from about 2,000 to about 50,000 daltons (Da).

The present invention can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects. For example, preferred components of the kit include a siNA molecule of the invention and a vehicle that promotes introduction of the siNA into cells of interest as described herein (e.g., using lipids and other methods of transfection known in the art, see for example Beigelman et al, U.S. Pat. No. 6,395,713). The kit can be used for target validation, such as in determining gene function and/or activity, or in drug optimization, and in drug discovery (see for example Usman et al., U.S. Ser. No. 60/402,996). Such a kit can also include instructions to allow a user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interfering RNA”, “siRNA”, “short interfering nucleic acid molecule”, “short interfering oligonucleotide molecule”, or “chemically-modified short interfering nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner; see for example Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples of siNA molecules of the invention are shown in FIGS. 4-6, and Tables II and III herein. For example the siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 19 base pairs); the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s). The siNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5′-phosphate (see for example Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certain embodiments, the siNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic intercations, and/or stacking interactions. In certain embodiments, the siNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the siNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene. As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack 2′-hydroxy (2′-OH) containing nucleotides. Applicant describes in certain embodiments short interfering nucleic acids that do not require the presence of nucleotides having a 2′-hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modified short interfering nucleic acid molecules of the invention can also be referred to as short interfering modified oligonucleotides “siMON.”As used herein, the term siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, siNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et at., 2002, Science, 297, 2232-2237).

In one embodiment, a siNA molecule of the invention is a duplex forming oligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al., U.S. Ser. No. 10/727,780 filed Dec. 3, 2003).

In one embodiment, a siNA molecule of the invention is a multifunctional siNA, (see for example FIGS. 16-22 and Jadhav et at, U.S. Ser. No. 60/543,480, filed Feb. 10, 2004). The multifunctional siNA of the invention can comprise sequence targeting, for example, two regions of SARS RNA (see for example target sequences in Tables II and III) or alternately, SARS RNA and cellular RNA involved in SARS virus infection or replication. In another embodiment, a multifunctional siNA of the invention can comprise sequence targeting for example both viral genes encoding RNAi inhibitory factors and viral genes encoding viral structural proteins.

By “asymmetric hairpin” as used herein is meant a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with loop. For example, an asymmetric hairpin siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 19 to about 22 (e.g., about 19, 20, 21, or 22) nucleotides) and a loop region comprising about 4 to about 8 (e.g., about 4, 5, 6, 7, or 8) nucleotides, and a sense region having about 3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides that are complementary to the antisense region. The asymmetric hairpin siNA molecule can also comprise a 5′-terminal phosphate group that can be chemically modified. The loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric duplex siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 19 to about 22 (e.g. about 19, 20, 21, or 22) nucleotides) and a sense region having about 3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides that are complementary to the antisense region. By “modulate” is meant that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term “modulate” can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siNA) of the invention. In one embodiment, inhibition, down-regulation or reduction with an siNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with siNA molecules is below that level observed in the presence of, for example, an siNA molecule with scrambled sequence or with mismatches. In another embodiment, inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.

By “gene”, or “target gene”, is meant, a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide. A gene or target gene can also encode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-coding RNAs can serve as target nucleic acid molecules for siNA mediated RNA interference in modulating the activity of fRNA or ncRNA involved in functional or regulatory cellular processes. Abberant fRNA or ncRNA activity leading to disease can therefore be modulated by siNA molecules of the invention. siNA molecules targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of an organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, methylation etc.). The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts.

By “SARS” or “SARS virus” as used herein is meant the SARS virus or any protein, peptide, or polypeptide, having SARS virus activity or encoded by the SARS genome. The term “SARS” also includes nucleic acid molecules encoding RNA or protein(s) associated with the development and/or maintenance of SARS virus infection, such as nucleic acid molecules which encode SARS RNA or polypeptides (such as polynucleotides having Genbank Accession numbers shown in Table I), including polypeptides of different strains of SARS, mutant SARS genes, and splice variants of SARS genes, as well as genes involved in SARS pathways of gene expression and/or SARS activity. Also, the term “SARS” is meant to encompass SARS viral gene products and genes that modulate cellular targets for SARS virus infection, such as those described herein.

By “SARS protein” or “SARS virus protein” is meant, protein, peptide, or polypeptide, having SARS virus activity or encoded by the SARS genome or alternately, cellular proteins involved in SARS virus infection and/or replication.

By “homologous sequence” is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors. A homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system or organism to another biological system or organism. The polynucleotide can include both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of a siNA molecule having complementarity to an antisense region of the siNA molecule. In addition, the sense region of a siNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a siNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of a siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity.

Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

The siNA molecules of the invention represent a novel therapeutic approach to treat various diseases and conditions, including SARS virus infection, acute respiratory failure, viral pneumonia, and any other indications that can respond to the level of SARS in a cell or tissue. The reduction of SARS expression and thus reduction in the level of the respective protein relieves, to some extent, the symptoms of the disease or condition.

In one embodiment of the present invention, each sequence of a siNA molecule of the invention is independently about 18 to about 24 nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In another embodiment, the siNA duplexes of the invention independently comprise about 17 to about 23 base pairs (e.g., about 17, 18, 19, 20, 21, 22 or 23). In yet another embodiment, siNA molecules of the invention comprising hairpin or circular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to about 44 (e.g., 38, 39, 40, 41, 42, 43 or 44) nucleotides in length and comprising about 16 to about 22 (e.g., about 16, 17, 18, 19, 20, 21 or 22) base pairs. Exemplary siNA molecules of the invention are shown in Table II. Exemplary synthetic siNA molecules of the invention are shown in Table III and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human. The cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers. In particular embodiments, the nucleic acid molecules of the invention comprise sequences shown in Tables II-III and/or FIGS. 4-5. Examples of such nucleic acid molecules consist essentially of sequences defined in these tables and figures. Furthermore, the chemically modified constructs described in Table IV can be applied to any siNA sequence of the invention.

In another aspect, the invention provides mammalian cells containing one or more siNA molecules of this invention. The one or more siNA molecules can independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotide residue. By “ribonucleotide” is meant a nucleotide with a hydroxyl group at the 2′ position of a β-D-ribo-furanose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the nucleic acid molecules of the invention can be administered. A subject can be a mammal or mammalian cells, including a human or human cells.

The term “phosphorothioate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise a sulfur atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotide linkage having Formula I, wherein Z and/or W comprise an acetyl or protected acetyl group.

The term “thiophosphonoacetate” as used herein refers to an internucleotide linkage having Formula I, wherein Z comprises an acetyl or protected acetyl group and W comprises a sulfur atom or alternately W comprises an acetyl or protected acetyl group and Z comprises a sulfur atom.

The term “universal base” as used herein refers to nucleotide base analogs that form base pairs with each of the natural DNA/RNA bases with little discrimination between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbons (C1, C2, C3, C4, or C5), are independently or in combination absent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein (e.g., cancers and other proliferative conditions). For example, to treat a particular disease or condition, the siNA molecules can be administered to a subject or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combination with other known treatments to treat conditions or diseases discussed above. For example, the described molecules could be used in combination with one or more known therapeutic agents to treat a disease or condition. Non-limiting examples of other therapeutic agents that can be readily combined with a siNA molecule of the invention are enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules, and other organic and/or inorganic compounds including metals, salts and ions.

In one embodiment, the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the invention, in a manner which allows expression of the siNA molecule. For example, the vector can contain sequence(s) encoding both strands of a siNA molecule comprising a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a siNA molecule. Non-limiting examples of such expression vectors are described in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi: 10.1038/nm725.

In another embodiment, the invention features a mammalian cell, for example, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the invention comprises a sequence for a siNA molecule having complementarity to a RNA molecule referred to by a Genbank Accession numbers, for example Genbank Accession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding two or more siNA molecules, which can be the same or different.

In another aspect of the invention, siNA molecules that interact with target RNA molecules and down-regulate gene encoding target RNA molecules (for example target RNA molecules referred to by Genbank Accession numbers herein) are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecules bind and down-regulate gene function or expression via RNA interference (RNAi). Delivery of siNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis of siNA molecules. The complementary siNA sequence strands, strand 1 and strand 2, are synthesized in tandem and are connected by a cleavable linkage, such as a nucleotide succinate or abasic succinate, which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support. The synthesis can be either solid phase or solution phase, in the example shown, the synthesis is a solid phase synthesis. The synthesis is performed such that a protecting group, such as a dimethoxytrityl group, remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon cleavage and deprotection of the oligonucleotide, the two siNA strands spontaneously hybridize to form a siNA duplex, which allows the purification of the duplex by utilizing the properties of the terminal protecting group, for example by applying a trityl on purification method wherein only duplexes/oligonucleotides with the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplex synthesized by a method of the invention. The two peaks shown correspond to the predicted mass of the separate siNA sequence strands. This result demonstrates that the siNA duplex generated from tandem synthesis can be purified as a single entity using a simple trityl-on purification methodology.

FIG. 3 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double-stranded RNA (dsRNA), which is generated by RNA-dependent RNA polymerase (RdRP) from foreign single-stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme that in turn generates siNA duplexes. Alternately, synthetic or expressed siNA can be introduced directly into a cell by appropriate means. An active siNA complex forms which recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which can activate DICER and result in additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically-modified siNA constructs of the present invention. In the figure, N stands for any nucleotide (adenosine, guanosine, cytosine, uridine, or optionally thymidine, for example thymidine can be substituted in the overhanging regions designated by parenthesis (N N). Various modifications are shown for the sense and antisense strands of the siNA constructs.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all nucleotides present are ribonucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-O-methyl modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein and wherein and all purine nucleotides that may be present are 2′-deoxy nucleotides. The antisense strand comprises 21 nucleotides, optionally having a 3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotides are optionally complementary to the target RNA sequence, and having one 3′-terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides that may be present are 2′-deoxy nucleotides except for (N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical modifications described herein. A modified internucleotide linkage, such as a phosphorothioate, phosphorodithioate or other modified internucleotide linkage as described herein, shown as “s”, optionally connects the (N N) nucleotides in the antisense strand. The antisense strand of constructs A-F comprise sequence complementary to any target nucleic acid sequence of the invention. Furthermore, when a glyceryl moiety (L) is present at the 3′-end of the antisense strand for any construct shown in FIG. 4 A-F, the modified internucleotide linkage is optional.

FIG. 5A-F shows non-limiting examples of specific chemically-modified siNA sequences of the invention. A-F applies the chemical modifications described in FIG. 4A-F to a SARS virus siNA sequence. Such chemical modifications can be applied to any SARS sequence and/or SARS polymorphism sequence.

FIG. 6 shows non-limiting examples of different siNA constructs of the invention. The examples shown (constructs 1, 2, and 3) have 19 representative base pairs; however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example comprising about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siNA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or in vitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1) sequence followed by a region having sequence identical (sense region of siNA) to a predetermined SARS target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, which is followed by a loop sequence of defined sequence (X), comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence that will result in a siNA transcript having specificity for a SARS target sequence and having self-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C.) to linearize the sequence, thus allowing extension of a complementary second DNA strand using a primer to the 3′-restriction sequence of the first strand. The double-stranded DNA is then inserted into an appropriate vector for expression in cells. The construct can be designed such that a 3′-terminal nucleotide overhang results from the transcription, for example by engineering restriction sites and/or utilizing a poly-U termination region as described in Paul et al., 2002, Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double-stranded siNA constructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) site sequence followed by a region having sequence identical (sense region of siNA) to a predetermined SARS target sequence, wherein the sense region comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length, and which is followed by a 3′-restriction site (R2) which is adjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase to generate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific to R1 and R2 to generate a double-stranded DNA which is then inserted into an appropriate vector for expression in cells. The transcription cassette is designed such that a U6 promoter region flanks each side of the dsDNA which generates the separate sense and antisense strands of the siNA. Poly T termination sequences can be added to the constructs to generate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determine target sites for siNA mediated RNAi within a particular target nucleic acid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein the antisense region of the siNA constructs has complementarity to target sites across the target nucleic acid sequence, and wherein the sense region comprises sequence complementary to the antisense region of the siNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted into vectors such that (FIG. 9C) transfection of a vector into cells results in the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence.

FIG. 10 shows non-limiting examples of different stabilization chemistries (1-10) that can be used, for example, to stabilize the 3′-end of siNA sequences of the invention, including (1) [3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3) [5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5) [5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7) [3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9) [5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. In addition to modified and unmodified backbone chemistries indicated in the figure, these chemistries can be combined with different backbone modifications as described herein, for example, backbone modifications having Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to the terminal modifications shown can be another modified or unmodified nucleotide or non-nucleotide described herein, for example modifications having any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are nuclease resistance while preserving the ability to mediate RNAi activity. Chemical modifications are introduced into the siNA construct based on educated design parameters (e.g. introducing 2′-modifications, base modifications, backbone modifications, terminal cap modifications etc). The modified construct in tested in an appropriate system (e.g. human serum for nuclease resistance, shown, or an animal model for PK/delivery parameters). In parallel, the siNA construct is tested for RNAi activity, for example in a cell culture system such as a luciferase reporter assay). Lead siNA constructs are then identified which possess a particular characteristic while maintaining RNAi activity, and can be further modified and assayed once again. This same approach can be used to identify siNA-conjugate molecules with improved pharmacokinetic profiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear and duplex constructs and asymmetric derivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminal phosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design self complementary DFO constructs utilizing palidrome and/or repeat nucleic acid sequences that are identified in a target nucleic acid sequence. (i) A palindrome or repeat sequence is identified in a nucleic acid target sequence. (ii) A sequence is designed that is complementary to the target nucleic acid sequence and the palindrome sequence. (iii) An inverse repeat sequence of the non-palindrome/repeat portion of the complementary sequence is appended to the 3′-end of the complementary sequence to generate a self complementary DFO molecule comprising sequence complementary to the nucleic acid target. (iv) The DFO molecule can self-assemble to form a double stranded oligonucleotide. FIG. 14B shows a non-limiting representative example of a duplex forming oligonucleotide sequence. FIG. 14C shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence. FIG. 14D shows a non-limiting example of the self assembly schematic of a representative duplex forming oligonucleotide sequence followed by interaction with a target nucleic acid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementary DFO constructs utilizing palidrome and/or repeat nucleic acid sequences that are incorporated into the DFO constructs that have sequence complementary to any target nucleic acid sequence of interest. Incorporation of these palindrome/repeat sequences allow the design of DFO constructs that form duplexes in which each strand is capable of mediating modulation of target gene expression, for example by RNAi. First, the target sequence is identified. A complementary sequence is then generated in which nucleotide or non-nucleotide modifications (shown as X or Y) are introduced into the complementary sequence that generate an artificial palindrome (shown as XYXYXY in the Figure). An inverse repeat of the non-palindrome/repeat complementary sequence is appended to the 3′-end of the complementary sequence to generate a self complementary DFO comprising sequence complementary to the nucleic acid target. The DFO can self-assemble to form a double stranded oligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. FIG. 16A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3′-ends of each polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 16B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5′-ends of each polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences. FIG. 17A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3′-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 17B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5′-end of the polynucleotide sequence in the multifunctional siNA. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules of the invention comprising two separate polynucleotide sequences that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifunctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. FIG. 18A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 3′-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 18B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first and second complementary regions are situated at the 5′-ends of each polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules of the invention comprising a single polynucleotide sequence comprising distinct regions that are each capable of mediating RNAi directed cleavage of differing target nucleic acid sequences and wherein the multifunctional siNA construct further comprises a self complementary, palindrome, or repeat region, thus enabling shorter bifunctional siNA constructs that can mediate RNA interference against differing target nucleic acid sequences. FIG. 19A shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the second complementary region is situated at the 3′-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. FIG. 19B shows a non-limiting example of a multifunctional siNA molecule having a first region that is complementary to a first target nucleic acid sequence (complementary region 1) and a second region that is complementary to a second target nucleic acid sequence (complementary region 2), wherein the first complementary region is situated at the 5′-end of the polynucleotide sequence in the multifunctional siNA, and wherein the first and second complementary regions further comprise a self complementary, palindrome, or repeat region. The dashed portions of each polynucleotide sequence of the multifunctional siNA construct have complementarity with regard to corresponding portions of the siNA duplex, but do not have complementarity to the target nucleic acid sequences. In one embodiment, these multifunctional siNA constructs are processed in vivo or in vitro to generate multifunctional siNA constructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid molecules, such as separate RNA molecules encoding differing proteins, for example, differing viral strains, a virus and a cellular protein involved in viral infection or replication, or differing proteins involved in a common or divergent biologic pathway that is implicated in the maintenance of progression of disease. Each strand of the multifunctional siNA construct comprises a region having complementarity to separate target nucleic acid molecules. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.

FIG. 21 shows a non-limiting example of how multifunctional siNA molecules of the invention can target two separate target nucleic acid sequences within the same target nucleic acid molecule, such as alternate coding regions of a RNA, coding and non-coding regions of a RNA, or alternate splice variant regions of a RNA. Each strand of the multifunctional siNA construct comprises a region having complementarity to the separate regions of the target nucleic acid molecule. The multifunctional siNA molecule is designed such that each strand of the siNA can be utilized by the RISC complex to initiate RNA interference mediated cleavage of its corresponding target region. These design parameters can include destabilization of each end of the siNA construct (see for example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be accomplished for example by using guanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), or destabilizing chemically modified nucleotides at terminal nucleotide positions as is known in the art.

DETAILED DESCRIPTION OF THE INVENTION

Mechanism of Action of Nucleic Acid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNA interference mediated by short interfering RNA as is presently known, and is not meant to be limiting and is not an admission of prior art. Applicant demonstrates herein that chemically-modified short interfering nucleic acids possess similar or improved capacity to mediate RNAi as do siRNA molecules and are expected to possess improved stability and activity in vivo; therefore, this discussion is not meant to be limiting only to siRNA and can be applied to siNA as a whole. By “improved capacity to mediate RNAi” or “improved RNAi activity” is meant to include RNAi activity measured in vitro and/or in vivo where the RNAi activity is a reflection of both the ability of the siNA to mediate RNAi and the stability of the siNAs of the invention. In this invention, the product of these activities can be increased in vitro and/or in vivo compared to an all RNA siRNA or a siNA containing a plurality of ribonucleotides. In some cases, the activity or stability of the siNA molecule can be decreased (i.e., less than ten-fold), but the overall activity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA or viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized. This mechanism appears to be different from the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as Dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363). Short interfering RNAs derived from Dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188). In addition, RNA interference can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably though cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). As such, siNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391, 806, were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essential to mediate efficient RNAi activity. These studies have shown that 21 nucleotide siRNA duplexes are most active when containing two 2-nucleotide 3′-terminal nucleotide overhangs. Furthermore, substitution of one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of 3′-terminal siRNA nucleotides with deoxy nucleotides was shown to be tolerated. Mismatch sequences in the center of the siRNA duplex were also shown to abolish RNAi activity. In addition, these studies also indicate that the position of the cleavage site in the target RNA is defined by the 5′-end of the siRNA guide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J, 20, 6877). Other studies have indicated that a 5′-phosphate on the target-complementary strand of a siRNA duplex is required for siRNA activity and that ATP is utilized to maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNA molecules lacking a 5′-phosphate are active when introduced exogenously, suggesting that 5′-phosphorylation of siRNA constructs may occur in vivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (“small” refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure. Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) are synthesized using protocols known in the art, for example as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45 second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoro nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-fold excess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycle of deoxy residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aqueous methylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder.

The method of synthesis used for RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In a non-limiting example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5 min coupling step for alkylsilyl protected nucleotides and a 2.5 min coupling step for 2′-O-methylated nucleotides. Table V outlines the amounts and the contact times of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.) with minimal modification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycle of 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol) of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in each coupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-pot protocol.

For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant is then added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, are dried to a white powder. The base deprotected oligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300 μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mL TEA-3HF to provide a 1.4 M HF concentration) and heated to 65° C. After 1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO:1/1 (0.8 mL) at 65° C. for 15 minutes.

The vial is brought to room temperature TEA-3HF (0.1 mL) is added and the vial is heated at 65° C. for 15 minutes. The sample is cooled at −20° C. and then quenched with 1.5 M NH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1 M NaCl and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandem synthesis methodology as described in Example 1 herein, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms. The tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.

A siNA molecule can also be assembled from two distinct nucleic acid strands or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H (for a review see Usman and Cedergren, 1992, TIBS17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.

In another aspect of the invention, siNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the siNA molecules can be delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al., supra; all of which are incorporated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.

There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Eamshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of the instant invention so long as the ability of siNA to promote RNAi is cells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting in increased efficacy and higher specificity of these molecules.

Short interfering nucleic acid (siNA) molecules having chemical modifications that maintain or enhance activity are provided. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. In cases in which modulation is the goal, therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target RNA has been modulated long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19 (incorporated by reference herein)) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, see for example Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single G-clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides. The inclusion of such nucleotides in nucleic acid molecules of the invention results in both enhanced affinity and specificity to nucleic acid targets, complementary sequences, or template strands. In another embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleic acid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (see for example Wengel et al., International PCT Publication No. WO 00/66604 and WO 99/14226).

In another embodiment, the invention features conjugates and/or complexes of siNA molecules of the invention. Such conjugates and/or complexes can be used to facilitate delivery of siNA molecules into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not limited to, small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acid or non-nucleic acid linker molecule that is designed as a biodegradable linker to connect one molecule to another molecule, for example, a biologically active molecule to a siNA molecule of the invention or the sense and antisense strands of a siNA molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.

The term “biologically active molecule” as used herein, refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system. Non-limiting examples of biologically active siNA molecules either alone or in combination with other molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof. Biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example, lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic molecule comprising at least one phosphorus group. For example, a phospholipid can comprise a phosphorus-containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) delivered exogenously optimally are stable within cells until reverse transcription of the RNA has been modulated long enough to reduce the levels of the RNA transcript. The nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in RNAi are provided. Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids. Thus, in vitro and/or in vivo the activity should not be significantly lowered. Use of the nucleic acid-based molecules of the invention will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes; nucleic acid molecules coupled with known small molecule modulators; or intermittent treatment with combinations of molecules, including different motifs and/or other chemical or biological molecules). The treatment of subjects with siNA molecules can also include combinations of different types of nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate, decoys, and aptamers.

In another aspect a siNA molecule of the invention comprises one or more 5′ and/or a 3′-cap structure, for example on only the sense siNA strand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see, for example, Adamic et al., U.S. Pat. No. 5,998,203, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell. The cap may be present at the 5′-terminus (5′-cap) or at the 3′-terminal (3′-cap) or may be present on both termini. In non-limiting examples, the 5′-cap includes, but is not limited to, glyceryl, inverted deoxy abasic residue (moiety); 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety.

Non-limiting examples of the 3′-cap include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylene nucleotide; I-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).

By the term “non-nucleotide” is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino, or SH. The term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino or SH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An “aryl” group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An “amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylaryl or hydrogen.

By “nucleotide” as used herein is as recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1′ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference herein). There are several examples of modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1′ position or their equivalents.

In one embodiment, the invention features modified siNA molecules, with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithiQate, methylphosphonate, phosphotriester, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of oligonucleotide backbone modifications, see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1′ position, see for example Adamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine, guanine, thymine, or uracil joined to the 1′ carbon of β-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate. Non-limiting examples of modified nucleotides are shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the present invention, by “amino” is meant 2′—NH₂or 2′-O—NH₂, which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both incorporated by reference in their entireties.

Various modifications to nucleic acid siNA structure can be made to enhance the utility of these molecules. Such modifications will enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, e.g., to enhance penetration of cellular membranes, and confer the ability to recognize and bind to targeted cells.

Administration of Nucleic Acid Molecules

A siRNA molecule of the invention can be adapted for use to treat for example SARS virus infection, acute respiratory failure, viral pneumonia, and other indications that can respond to the level of SARS in a cell or tissue, alone or in combination with other therapies. For example, a siNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies for Antisense Oligonucleolide Therapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and US Patent Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). In another embodiment, the nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives. Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump.

In one embodiment, the nucleic acid molecules or the invention are administered via pulmonary delivery, such as by inhalation of an aerosol or spray dried formulation administered by an inhalation device or nebulizer, providing rapid local uptake of the nucleic acid molecules into relevant pulmonary tissues. Solid particulate compositions containing respirable dry particles of micronized nucleic acid compositions can be prepared by grinding dried or lyophilized nucleic acid compositions, and then passing the micronized composition through, for example, a 400 mesh screen to break up or separate out large agglomerates. A solid particulate composition comprising the nucleic acid compositions of the invention can optionally contain a dispersant which serves to facilitate the formation of an aerosol as well as other therapeutic compounds. A suitable dispersant is lactose, which can be blended with the nucleic acid compound in any suitable ratio, such as a 1 to 1 ratio by weight.

Aerosols of liquid particles comprising a nucleic acid composition of the invention can be produced by any suitable means, such as with a nebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers are commercially available devices which transform solutions or suspensions of an active ingredient into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers comprise the active ingredient in a liquid carrier in an amount of up to 40% w/w preferably less than 20% w/w of the formulation. The carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride or other suitable salts. Optional additives include preservatives if the formulation is not prepared sterile, for example, methyl hydroxybenzoate, anti-oxidants, flavorings, volatile oils, buffering agents and emulsifiers and other formulation surfactants. The aerosols of solid particles comprising the active composition and surfactant can likewise be produced with any solid particulate aerosol generator. Aerosol generators for administering solid particulate therapeutics to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a therapeutic composition at a rate suitable for human administration. One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which can be delivered by means of an insufflator. In the insufflator, the powder, e.g., a metered dose thereof effective to carry out the treatments described herein, is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 to 100 w/w of the formulation. A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquified propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation can additionally contain one or more co-solvents, for example, ethanol, emulsifiers and other formulation surfactants, such as oleic acid or sorbitan trioleate, anti-oxidants and suitable flavoring agents. Other methods for pulmonary delivery are described in, for example US Patent Application No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728; 6,565,885.

In one embodiment, a siNA molecule of the invention is complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 20010007666, incorporated by reference herein in its entirety including the drawings. In another embodiment, the membrane disruptive agent or agents and the siNA molecule are also complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Pat. No. 6,235,310, incorporated by reference herein in its entirety including the drawings.

Thus, the invention features a pharmaceutical composition comprising one or more nucleic acid(s) of the invention in an acceptable carrier, such as a stabilizer, buffer, and the like. The polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptable formulations of the compounds described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes that lead to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes exposes the siNA molecules of the invention to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cells producing excess repeat expansion genes.

By “pharmaceutically acceptable formulation” is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of polybutylcyanoacrylate. Other non-limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophtys. Acta, 1238, 86-90). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.

Pharmaceutical compositions of the invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions can also contain sweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle. Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient. It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

For administration to non-human animals, the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable for administering nucleic acid molecules of the invention to specific cell types. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). In another example, the folate receptor is overexpressed in many cancer cells. Binding of such glycoproteins, synthetic glycoconjugates, or folates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificity through the use of N-acetyl-D-galactosamine as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. This “clustering effect” has also been described for the binding and uptake of mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose, galactosamine, or folate based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to, for example, the treatment of liver disease, cancers of the liver, or other cancers. The use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment. Furthermore, therapeutic bioavailability, pharmacodynamics, and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention. Non-limiting examples of such bioconjugates are described in Vargeese et al., U.S. Ser. No. 10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser. No. 10/151,116, filed May 17, 2002. In one embodiment, nucleic acid molecules of the invention are complexed with or covalently attached to nanoparticles, such as Hepatitis B virus S, M, or IL envelope proteins (see for example Yamado et al., 2003, Nature Biotechnology, 21, 885). In one embodiment, nucleic acid molecules of the invention are delivered with specificity for human tumor cells, specifically non-apoptotic human tumor cells including for example T-cells, hepatocytes, breast carcinoma cells, ovarian carcinoma cells, melanoma cells, intestinal epithelial cells, prostate cells, testicular cells, non-small cell lung cancers, small cell lung cancers, etc.

Alternatively, certain siNA molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Nail. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; propulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45. Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser. 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the siNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siNA molecule expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510).

In one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one siNA molecule of the instant invention. The expression vector can encode one or both strands of a siNA duplex, or a single self-complementary strand that self hybridizes into a siNA duplex. The nucleic acid sequences encoding the siNA molecules of the instant invention can be operably linked in a manner that allows expression of the siNA molecule (see for example Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance online publication doi:10.1038/nm725).

In another aspect, the invention features an expression vector comprising: a) a transcription initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., eukaryotic pol I, II or III termination region); and c) a nucleic acid sequence encoding at least one of the siNA molecules of the instant invention, wherein said sequence is operably linked to said initiation region and said termination region in a manner that allows expression and/or delivery of the siNA molecule. The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the siNA of the invention; and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J, 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U. S. A, 90, 8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as siNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al. 1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al., International PCT Publication No. WO 96/18736. The above siNA transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the siNA molecules of the invention in a manner that allows expression of that siNA molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; and c) a nucleic acid sequence encoding at least one strand of the siNA molecule, wherein the sequence is operably linked to the initiation region and the termination region in a manner that allows expression and/or delivery of the siNA molecule.

In another embodiment the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an open reading frame; and d) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the open reading frame and the termination region in a manner that allows expression and/or delivery of the siNA molecule. In yet another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; and d) a nucleic acid sequence encoding at least one siNA molecule, wherein the sequence is operably linked to the initiation region, the intron and the termination region in a manner which allows expression and/or delivery of the nucleic acid molecule.

In another embodiment, the expression vector comprises: a) a transcription initiation region; b) a transcription termination region; c) an intron; d) an open reading frame; and e) a nucleic acid sequence encoding at least one strand of a siNA molecule, wherein the sequence is operably linked to the 3′-end of the open reading frame and wherein the sequence is operably linked to the initiation region, the intron, the open reading frame and the termination region in a manner which allows expression and/or delivery of the siNA molecule.

SARS Virus Biology and Biochemistry

The following discussion is adapted from the report, “Preliminary Clinical Description of Severe Acute Respiratory Syndrome”, World Health Organization, Geneva, Switzerland, available at the Centers for Disease Control and Prevention website.

Severe acute respiratory syndrome (SARS) is a viral respiratory illness caused by a coronavirus, called SARS-associated coronavirus (SARS-CoV). SARS was first reported in Asia in February 2003. Over the next few months, the illness spread to more than two dozen countries in North America, South America, Europe, and Asia before the SARS global outbreak of 2003 was contained. According to the World Health Organization (WHO), a total of 8,098 people worldwide became sick with SARS during the 2003 outbreak. Of these, 774 died.

The incubation period for SARS is typically 2-7 days; however, isolated reports have suggested an incubation period as long as 10 days. The illness begins generally with a prodrome of fever (>100.4° F. [>38.0° C.]). Fever often is high, sometimes is associated with chills and rigors, and might be accompanied by other symptoms, including headache, malaise, and myalgia. At the onset of illness, some persons have mild respiratory symptoms. Typically, rash and neurologic or gastrointestinal findings are absent; however, some patients have reported diarrhea during the febrile prodrome.

After 3-7 days, a lower respiratory phase begins with the onset of a dry, nonproductive cough or dyspnea, which might be accompanied by or progress to hypoxemia. In 10%-20% of cases, the respiratory illness is severe enough to require intubation and mechanical ventilation. Death may result from progressive respiratory failure due to alveolar damage. The case-fatality rate among persons with illness meeting the current WHO case definition of SARS is approximately 3%.

Chest radiographs might be normal during the febrile prodrome and throughout the course of illness. However, in a substantial proportion of patients, the respiratory phase is characterized by early focal interstitial infiltrates progressing to more generalized, patchy, interstitial infiltrates. Some chest radiographs from patients in the late stages of SARS also have shown areas of consolidation.

Early in the course of disease, the absolute lymphocyte count is often decreased. Overall white blood cell counts have generally been normal or decreased. At the peak of the respiratory illness, approximately 50% of patients have leukopenia and thrombocytopenia or low-normal platelet counts (50,000-150,000/mL). Early in the respiratory phase, elevated creatine phosphokinase levels (as high as 3,000 IU/L) and hepatic transaminases (two to six times the upper limits of normal) have been noted. In the majority of patients, renal function has remained normal.

The severity of illness might be highly variable, ranging from mild illness to death. Although a few close contacts of patients with SARS have developed a similar illness, the majority have remained well. Some close contacts have reported a mild, febrile illness without respiratory signs or symptoms, suggesting the illness might not always progress to the respiratory phase.

Treatment regimens have included several antibiotics to presumptively treat known bacterial agents of atypical pneumonia. In several locations, therapy also has included antiviral agents such as oseltamivir or ribavirin. Steroids have also been administered orally or intravenously to patients in combination with ribavirin and other antimicrobials. At present, the most efficacious treatment regimen, if any, is unknown.

The causative agent of SARS appears to be a novel coronavirus that was isolated from patients who met the case definition of SARS (see Ksiazek et al., 2003, New England Journal of Medicine, 10.1056/NEJMoa030781. Indirect fluorescent antibody tests and enzyme-linked immunosorbent assays made with the new coronavirus isolate have been used to demonstrate a virus-specific serologic response. Amplification of short regions of the polymerase gene, (the most strongly conserved part of the Coronavirus genome) by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing revealed that the SARS virus is a novel Coronavirus which has not previously been present in human populations. This conclusion is confirmed by serological (antigenic) investigations. The sequence of the SARS associated coronavirus was recently made available through the CDC.

Viral entry into cells occurs via endocytosis and membrane fusion. Replication occurs in the cytoplasm. Initially, the 5′ 20 kb of the (+)sense genome is translated to produce a viral polymerase, which then produces a full-length (−)sense strand. This is used as a template to produce mRNA as a nested set of transcripts, all with an identical 5′ non-translated leader sequence of 72 nt and coincident 3′ polyadenylated ends. Each mRNA is monocistronic, the genes at the 5′ end being translated from the longest mRNA. These unusual cytoplasmic structures are produced not by splicing but by the polymerase during transcription. Between each of the genes there is a repeated intergenic sequence—UCUAAAC—which interacts with the transcriptase plus cellular factors to splice the leader sequence onto the start of each ORF. Viral assembly occurs by budding into the golgi apparatus, and viral particles are transported to the surface of the cell and are subsequently released.

The SARS virus can be grown in Vero cells (a fibroblast cell line isolated in 1962 from a primate). This is a novel property for human cornaviruses which usually cannot be cultivated. In these cells, virus infection results in a cytopathic effect, and budding of Coronavirus-like particles from the endoplasmic reticulum within infected cells.

Detection of the SARS virus can be accomplished with serological testing and molecular diagnotic procedures. Serological testing for anti-Coronavirus antibodies consists of indirect fluorescent antibody testing and enzyme-linked immunosorbent assays (ELISA) which detect antibodies against the virus produced in response to infection. Molecular testing consists of reverse transcriptase-polymerase chain reaction (RT-PCR) tests specific for the RNA from the novel Coronavirus.

The use of small interfering nucleic acid molecules targeting SARS genes therefore provides a class of novel therapeutic agents that can be used in the treatment and diagnosis of SARS virus infection, acute respiratory failure, viral pneumonia, or any other disease or condition that responds to modulation of SARS genes.

EXAMPLES

The following are non-limiting examples showing the selection, isolation, synthesis and activity of nucleic acids of the instant invention.

Example 1
Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandem using a cleavable linker, for example, a succinyl-based linker. Tandem synthesis as described herein is followed by a one-step purification process that provides RNAi molecules in high yield. This approach is highly amenable to siNA synthesis in support of high throughput RNAi screening, and can be readily adapted to multi-column or multi-well synthesis platforms.

After completing a tandem synthesis of a siNA oligo and its complement in which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact (trityl on synthesis), the oligonucleotides are deprotected as described above. Following deprotection, the siNA sequence strands are allowed to spontaneously hybridize. This hybridization yields a duplex in which one strand has retained the 5′-O-DMT group while the complementary strand comprises a terminal 5′-hydroxyl. The newly formed duplex behaves as a single molecule during routine solid-phase extraction purification (Trityl-On purification) even though only one molecule has a dimethoxytrityl group. Because the strands form a stable duplex, this dimethoxytrityl group (or an equivalent group, such as other trityl groups or other hydrophobic moieties) is all that is required to purify the pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point of introducing a tandem linker, such as an inverted deoxy abasic succinate or glyceryl succinate linker (see FIG. 1) or an equivalent cleavable linker. A non-limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamine (DIPA) and/or DMAP in the presence of an activator reagent such as Bromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After the linker is coupled, standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5′-O-DMT intact. Following synthesis, the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solid phase extraction, for example using a Waters Cl₈SepPak 1 g cartridge conditioned with 1 column volume (CV) of acetonitrile, 2 CV H₂O, and 2 CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H₂O or 50 mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with 50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with 1 CV H₂O followed by on-column detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then adding a second CV of 1% aqueous TFA to the column and allowing to stand for approximately 10 minutes. The remaining TFA solution is removed and the column washed with H20 followed by 1 CV 1M NaCl and additional H2O. The siNA duplex product is then eluted, for example, using 1 CV 20% aqueous CAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of a purified siNA construct in which each peak corresponds to the calculated mass of an individual siNA strand of the siNA duplex. The same purified siNA provides three peaks when analyzed by capillary gel electrophoresis (CGE), one peak presumably corresponding to the duplex siNA, and two peaks presumably corresponding to the separate siNA sequence strands. Ion exchange HPLC analysis of the same siNA contract only shows a single peak. Testing of the purified siNA construct using a luciferase reporter assay described below demonstrated the same RNAi activity compared to siNA constructs generated from separately synthesized oligonucleotide sequence strands.

Example 2
Identification of Potential siNA Target Sites in any RNA Sequence

The sequence of an RNA target of interest, such as a viral or human mRNA transcript, is screened for target sites, for example by using a computer folding algorithm. In a non-limiting example, the sequence of a gene or RNA gene transcript derived from a database, such as Genbank, is used to generate siNA targets having complementarity to the target. Such sequences can be obtained from a database, or can be determined experimentally as known in the art. Target sites that are known, for example, those target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions, can be used to design siNA molecules targeting those sites. Various parameters can be used to determine which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. In a non-limiting example, anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the siNA construct to be used. High throughput screening assays can be developed for screening siNA molecules using methods known in the art, such as with multi-well or multi-plate assays to determine efficient reduction in target gene expression.

Example 3
Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selection of siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, contained within the target sequence. This step is typically carried out using a custom Perl script, but commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package can be employed as well.
2. In some instances the siNAs correspond to more than one target sequence; such would be the case for example in targeting different transcripts of the same gene, targeting different transcripts of more than one gene, or for targeting both the human gene and an animal homolog. In this case, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find matching sequences in each list. The subsequences are then ranked according to the number of target sequences that contain the given subsequence; the goal is to find subsequences that are present in most or all of the target sequences. Alternately, the ranking can identify subsequences that are unique to a target sequence, such as a mutant target sequence. Such an approach would enable the use of siNA to target specifically the mutant sequence and not effect the expression of the normal sequence.
3. In some instances the siNA subsequences are absent in one or more sequences while present in the desired target sequence; such would be the case if the siNA targets a gene with a paralogous family member that is to remain untargeted. As in case 2 above, a subsequence list of a particular length is generated for each of the targets, and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog.
4. The ranked siNA subsequences can be further analyzed and ranked according to GC content. A preference can be given to sites containing 30-70% GC, with a further preference to sites containing 40-60% GC.
5. The ranked siNA subsequences can be further analyzed and ranked according to self-folding and internal hairpins. Weaker internal folds are preferred; strong hairpin structures are to be avoided.
6. The ranked siNA subsequences can be further analyzed and ranked according to whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in either strand can make oligonucleotide synthesis problematic and can potentially interfere with RNAi activity, so it is avoided whenever better sequences are available. CCC is searched in the target strand because that will place GGG in the antisense strand.
7. The ranked siNA subsequences can be further analyzed and ranked according to whether they have the dinucleotide WU (uridine dinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end of the sequence (to yield 3′ UU on the antisense sequence). These sequences allow one to design siNA molecules with terminal TT thymidine dinucleotides.
8. Four or five target sites are chosen from the ranked list of subsequences as described above. For example, in subsequences having 23 nucleotides, the right 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the upper (sense) strand of the siNA duplex, while the reverse complement of the left 21 nucleotides of each chosen 23-mer subsequence are then designed and synthesized for the lower (antisense) strand of the siNA duplex (see Tables II and III). If terminal TT residues are desired for the sequence (as described in paragraph 7), then the two 3′ terminal nucleotides of both the sense and antisense strands are replaced by TT prior to synthesizing the oligos.
9. The siNA molecules are screened in an in vitro, cell culture or animal model system to identify the most active siNA molecule or the most preferred target site within the target RNA sequence.
10. Other design considerations can be used when selecting target nucleic acid sequences, see for example Reynolds et al., 2004, Nature Biotechnology Advanced Online Publication, Feb. 1, 2004, doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32, doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a SARS target sequence is used to screen for target sites in cells expressing SARS RNA, such as VERO cells and/or FRhk-4 cells. The general strategy used in this approach is shown in FIG. 9. A non-limiting example of such is a pool comprising sequences having SEQ ID NOs: 1-3392. Cells expressing SARS (e.g., VERO cells and/or FRhk-4 cells) are transfected with the pool of siNA constructs and cells that demonstrate a phenotype associated with SARS inhibition are sorted. The pool of siNA constructs can be expressed from transcription cassettes inserted into appropriate vectors (see for example FIG. 7 and FIG. 8). The siNA from cells demonstrating a positive phenotypic change (e.g., decreased proliferation, decreased SARS mRNA levels or decreased SARS protein expression), are sequenced to determine the most suitable target site(s) within the target SARS RNA sequence.

Example 4
SARS Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the SARS RNA target and optionally prioritizing the target sites on the basis of folding (structure of any given sequence analyzed to determine siNA accessibility to the target), by using a library of siNA molecules as described in Example 3, or alternately by using an in vitro siNA system as described in Example 6 herein. siNA molecules were designed that could bind each target and are optionally individually analyzed by computer folding to assess whether the siNA molecule can interact with the target sequence. Varying the length of the siNA molecules can be chosen to optimize activity. Generally, a sufficient number of complementary nucleotide bases are chosen to bind to, or otherwise interact with, the target RNA, but the degree of complementarity can be modulated to accommodate siNA duplexes or varying length or base composition. By using such methodologies, siNA molecules can be designed to target sites within any known RNA sequence, for example those RNA sequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nuclease stability for systemic administration in vivo and/or improved pharmacokinetic, localization, and delivery properties while preserving the ability to mediate RNAi activity. Chemical modifications as described herein are introduced synthetically using synthetic methods described herein and those generally known in the art. The synthetic siNA constructs are then assayed for nuclease stability in serum and/or cellular/tissue extracts (e.g. liver extracts). The synthetic siNA constructs are also tested in parallel for RNAi activity using an appropriate assay, such as a luciferase reporter assay as described herein or another suitable assay that can quantity RNAi activity. Synthetic siNA constructs that possess both nuclease stability and RNAi activity can be further modified and re-evaluated in stability and activity assays. The chemical modifications of the stabilized active siNA constructs can then be applied to any siNA sequence targeting any chosen RNA and used, for example, in target screening assays to pick lead siNA compounds for therapeutic development (see for example FIG. 11).

Example 5
Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein. The sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above. The siNA molecules can be chemically synthesized using methods described herein. Inactive siNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence. Generally, siNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al., U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein in their entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise fashion using the phosphoramidite chemistry as is known in the art. Standard phosphoramidite chemistry involves the use of nucleosides comprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl, 3′-O-2-Cyanoethyl N,N-diisopropylphosphoroamidite groups, and exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine). Alternately, 2′-O—Silyl Ethers can be used in conjunction with acid-labile 2′-O-orthoester protecting groups in the synthesis of RNA as described by Scaringe supra. Differing 2′ chemistries can require different protecting groups, for example 2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection as described by Usman et al., U.S. Pat. No. 5,631,360, incorporated by reference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′- to 5′-direction) to the solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support (e.g., controlled pore glass or polystyrene) using various linkers. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are combined resulting in the coupling of the second nucleoside phosphoramidite onto the 5′-end of the first nucleoside. The support is then washed and any unreacted 5′-hydroxyl groups are capped with a capping reagent such as acetic anhydride to yield inactive 5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized to a more stable phosphate linkage. At the end of the nucleotide addition cycle, the 5′-O-protecting group is cleaved under suitable conditions (e.g., acidic conditions for trityl-based groups and Fluoride for silyl-based groups). The cycle is repeated for each subsequent nucleotide.

Modification of synthesis conditions can be used to optimize coupling efficiency, for example by using differing coupling times, differing reagent/phosphoramidite concentrations, differing contact times, differing solid supports and solid support linker chemistries depending on the particular chemical composition of the siNA to be synthesized. Deprotection and purification of the siNA can be performed as is generally described in Deprotection and purification of the siNA can be performed as is generally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No. 6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringe supra, incorporated by reference herein in their entireties. Additionally, deprotection conditions can be modified to provide the best possible yield and purity of siNA constructs. For example, applicant has observed that oligonucleotides comprising 2′-deoxy-2′-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using aqueous methylamine at about 35° C. for 30 minutes. If the 2′-deoxy-2′-fluoro containing oligonucleotide also comprises ribonucleotides, after deprotection with aqueous methylamine at about 35° C. for 30 minutes, TEA-HF is added and the reaction maintained at about 65° C. for an additional 15 minutes.

Example 6
RNAi In Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is used to evaluate siNA constructs targeting SARS RNA targets. The assay comprises the system described by Tuschl et al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use with SARS target RNA. A Drosophila extract derived from syncytial blastoderm is used to reconstitute RNAi activity in vitro. Target RNA is generated via in vitro transcription from an appropriate SARS expressing plasmid using T7 RNA polymerase or via chemical synthesis as described herein. Sense and antisense siNA strands (for example 20 uM each) are annealed by incubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium bromide. The Drosophila lysate is prepared using zero to two-hour-old embryos from Oregon R flies collected on yeasted molasses agar that are dechorionated and lysed. The lysate is centrifuged and the supernatant isolated. The assay comprises a reaction mixture containing 50% lysate [vol/vol], RNA (10-50 μM final concentration), and 10% [vol/vol] lysis buffer containing siNA (10 nM final concentration). The reaction mixture also contains 10 mM creatine phosphate, 10 ug.ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. The final concentration of potassium acetate is adjusted to 100 mM. The reactions are pre-assembled on ice and preincubated at 25° C. for 10 minutes before adding RNA, then incubated at 25° C. for an additional 60 minutes. Reactions are quenched with 4 volumes of 1.25×Passive Lysis Buffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis or other methods known in the art and are compared to control reactions in which siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared by in vitro transcription in the presence of [alpha-³²P] CTP, passed over a G 50 Sephadex column by spin chromatography and used as target RNA without further purification. Optionally, target RNA is 5′-³²P-end labeled using T4 polynucleotide kinase enzyme. Assays are performed as described above and target RNA and the specific RNA cleavage products generated by RNAi are visualized on an autoradiograph of a gel. The percentage of cleavage is determined by PHOSPHOR IMAGER® (autoradiography) quantitation of bands representing intact control RNA or RNA from control reactions without siNA and the cleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites the SARS RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA constructs are screened for RNAi mediated cleavage of the SARS RNA target, for example, by analyzing the assay reaction by electrophoresis of labeled target RNA, or by northern blotting, as well as by other methodology well known in the art.

Example 7
Nucleic Acid Inhibition of SARS Target RNA In Vitro

siNA molecules targeted to the human SARS RNA are designed and synthesized as described above: These nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. The target sequences and the nucleotide location within the SARS RNA are given in Table II and III.

Two formats are used to test the efficacy of siNAs targeting SARS. First, the reagents are tested in cell culture using, for example, VERO cells and/or FRhk-4 cells, to determine the extent of RNA and protein inhibition. siNA reagents (e.g.; see Tables I and III) are selected against the SARS target as described herein. RNA inhibition is measured after delivery of these reagents by a suitable transfection agent to, for example, VERO cells and/or FRhk-4 cells. Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (e.g., ABI 7700 TAQMAN®). A comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized siNA control with the same overall length and chemistry, but randomly substituted at each position. Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent concentration is chosen, a RNA time-course of inhibition is performed with the lead siNA molecule. In addition, a cell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

Cells (e.g., VERO cells and/or FRhk-4 cells infected with the SARS virus) are seeded, for example, at 1×10⁵cells per well of a six-well dish in EGM-2 (BioWhittaker) the day before transfection. siNA (final concentration, for example 20 nM) and cationic lipid (e.g., final concentration 2 μg/ml) are complexed in EGM basal media (Bio Whittaker) at 37° C. for 30 minutes in polystyrene tubes. Following vortexing, the complexed siNA is added to each well and incubated for the times indicated. For initial optimization experiments, cells are seeded, for example, at 1×10³in 96 well plates and siNA complex added as described. Efficiency of delivery of siNA to cells is determined using a fluorescent siNA complexed with lipid. Cells in 6-well dishes are incubated with siNA for 24 hours, rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptake of siNA is visualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and Lightcycler Quantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example, using Qiagen RNA purification kits for 6-well or Rneasy extraction kits for 96-well assays. For TAQMAN® analysis (real-time PCR monitoring of amplification), dual-labeled probes are synthesized with the reporter dye, FAM or JOE, covalently linked at the 5′-end and the quencher dye TAMRA conjugated to the 3′-end. One-step RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence Detector using 50 μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1X TAQMAN® PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgClz, 300 μM each dATP, dCTP, dGTP, and dTTP, IOU RNase Inhibitor (Promega), 1.25 U AMPLITAQ GOLD® (DNA polymerase) (PE-Applied Biosystems) and IOU M-MLV Reverse Transcriptase (Promega). The thermal cycling conditions can consist of 30 minutes at 48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. Quantitation of mRNA levels is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 33, 11 ng/r×n) and normalizing to B-actin or GAPDH mRNA in parallel TAQMAN® reactions (real-time PCR monitoring of amplification). For each gene of interest an upper and lower primer and a fluorescently labeled probe are designed. Real time incorporation of SYBR Green I dye into a specific PCR product can be measured in glass capillary tubes using a lightcyler. A standard curve is generated for each primer pair using control cRNA. Values are represented as relative expression to GAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparation technique (see for example Andrews and Faller, 1991, Nucleic Acids Research, 19, 2499). Protein extracts from supernatants are prepared, for example using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transferred onto nitro-cellulose membranes. Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hour at 4° C. Following washes, the secondary antibody is applied, for example (1:10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).

Example 8
RNAi Mediated Inhibition of SARS RNA Expression

siNA constructs (e.g., siNA constructs shown in Table III) are tested for efficacy in reducing SARS RNA expression in, for example, VERO cells and/or FRhk-4 cells. Cells are plated approximately 24 h before transfection in 96-well plates at 5,000-7,500 cells/well, 100 μl/well, such that at the time of transfection cells are 70-90% confluent. For transfection, annealed siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50 μl/well and incubated for 20 minutes at room temperature. The siNA transfection mixtures are added to cells to give a final siNA concentration of 25 nM in a volume of 150 μl. Each siNA transfection mixture is added to 3 wells for triplicate siNA treatments. Cells are incubated at 37° for 24 h in the continued presence of the siNA transfection mixture. At 24 h, RNA is prepared from each well of treated cells. The supernatants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well. Target gene expression following treatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data is averaged and the standard deviations determined for each treatment. Normalized data are graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs is determined.

In a non-limiting example, a siNA construct comprising ribonucleotides and 3′-terminal dithymidine caps is assayed along with a chemically modified siNA construct comprising 2′-deoxy-2′-fluoro pyrimidine nucleotides and purine ribonucleotides in which the sense strand of the siNA is further modified with 5′ and 3′-terminal inverted deoxyabasic caps and the antisense strand comprises a 3′-terminal phosphorothioate internucleotide linkage. Additional stabilization chemistries as described in Table IV are similarly assayed for activity. These siNA constructs are compared to appropriate matched chemistry inverted controls. In addition, the siNA constructs are also compared to untreated cells, cells transfected with lipid and scrambled siNA constructs, and cells transfected with lipid alone (transfection control).

Example 9
Animal Models

Evaluating the efficacy of anti-SARS agents in animal models is an important prerequisite to human clinical trials. Byron et al., 2003, Nature, 425, 915, describe ferret and feline animal models of SARS virus infection. Haagmans et al., 2004, Nature Medicine, 10, 290-293, describe the use of pegylated interferon-alpha in protecting type 1 pneumocytes against SARS coronavirus infection in macaques. Gao et al., 2003, Lancet, 362, 1895-6, describe the use of a SARS virus vaccine in monkeys. All of these models can be adapted for use for pre-clinical evaluation of the efficacy of nucleic acid compositions of the invention in modulating SARS virus gene expression toward therapeutic use.

Example 10
Indications

The present body of knowledge in SARS research indicates the need for methods to assay SARS activity and for compounds that can regulate SARS expression for research, diagnostic, and therapeutic use. As described herein, the nucleic acid molecules of the present invention can be used in assays to diagnose disease state related of SARS levels. In addition, the nucleic acid molecules can be used to treat disease state related to SARS levels.

Particular degenerative and disease states that can be associated with SARS expression modulation include, but are not limited to, SARS virus infection, liver failure, hepatocellular carcinoma, cirrhosis, and/or other disease states associated with SARS virus infection.

Immunomodulators, steroids, and anti-viral compounds are non-limiting examples of pharmaceutical agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA molecules) of the instant invention. The use of ribavirin and oseltamivir are non-limiting examples of chemotherapeutic agents that can be combined with or used in conjunction with the nucleic acid molecules (e.g. siNA molecules) of the instant invention. Those skilled in the art will recognize that other anti-cancer compounds and therapies can similarly be readily combined with the nucleic acid molecules of the instant invention (e.g. siNA molecules) and are hence within the scope of the instant invention.

Example 11
Interferons

Interferons represent a non-limiting example of a class of compounds that can be used in conjunction with the siNA molecules of the invention for treating the diseases and/or conditions described herein. Type I interferons (IFN) are a class of natural cytokines that includes a family of greater than 25 IFN-α (Pesta, 1986, Methods Enzymol. 119, 3-14) as well as IFN-β, and IFN-ω. Although evolutionarily derived from the same gene (Diaz et al., 1994, Genomics 22, 540-552), there are many differences in the primary sequence of these molecules, implying an evolutionary divergence in biologic activity. All type I IFN share a common pattern of biologic effects that begin with binding of the IFN to the cell surface receptor (Pfeffer & Strulovici, 1992, Transmembrane secondary messengers for IFN-α/β. In: Interferon. Principles and Medical Applications., S. Baron, D. H. Coopenhaver, F. Dianzani, W. R. Fleischmann Jr., T. K. Hughes Jr., G. R. Kimpel, D. W. Niesel, G. J. Stanton, and S. K. Tyring, eds. 151-160). Binding is followed by activation of tyrosine kinases, including the Janus tyrosine kinases and the STAT proteins, which leads to the production of several IFN-stimulated gene products (Johnson et al., 1994, Sci. Am. 270, 68-75). The IFN-stimulated gene products are responsible for the pleotropic biologic effects of type I IFN, including antiviral, antiproliferative, and immunomodulatory effects, cytokine induction, and HLA class I and class II regulation (Pestka et al., 1987, Annu. Rev. Biochem 56, 727). Examples of IFN-stimulated gene products include 2-5-oligoadenylate synthetase (2-5 OAS), β₂-microglobulin, neopterin, p68 kinases, and the Mx protein (Chebath & Revel, 1992, The 2-5 A system: 2-5 A synthetase, isospecies and functions. In: Interferon. Principles and Medical Applications, S. Baron, D. H. Coopenhaver, F. Dianzani, W. R. Jr. Fleischmann, T. K. Jr Hughes, G. R. Kimpel, D. W. Niesel, G. J. Stanton, and S. K. Tyring, eds., pp. 225-236; Samuel, 1992, The RNA-dependent P1/eIF-2α protein kinase. In: Interferon. Principles and Medical Applications. S. Baron, D. H. Coopenhaver, F. Dianzani, W. R. Fleischmann Jr., T. K. Hughes Jr., G. R. Kimpel, D. W. Niesel, G. H. Stanton, and S. K. Tyring, eds. 237-250; Horisberger, 1992, MX protein: function and Mechanism of Action. In: Interferon. Principles and Medical Applications. S. Baron, D. H. Coopenhaver, F. Dianzani, W. R. Fleischmann Jr., T. K. Hughes Jr., G. R. Kimpel, D. W. Niesel, G. H. Stanton, and S. K. Tyring, eds. 215-224). Although all type I IFN have similar biologic effects, not all the activities are shared by each type I IFN, and in many cases, the extent of activity varies quite substantially for each IFN subtype (Fish et al, 1989, J. Interferon Res. 9, 97-114; Ozes et al., 1992, J. Interferon Res. 12, 55-59). More specifically, investigations into the properties of different subtypes of IFN-α and molecular hybrids of IFN-α have shown differences in pharmacologic properties (Rubinstein, 1987, J. Interferon Res. 7, 545-551). These pharmacologic differences can arise from as few as three amino acid residue changes (Lee et al., 1982, Cancer Res. 42, 1312-1316).

Eighty-five to 166 amino acids are conserved in the known IFN-α subtypes. Excluding the IFN-α pseudogenes, there are approximately 25 known distinct IFN-α subtypes. Pairwise comparisons of these nonallelic subtypes show primary sequence differences ranging from 2% to 23%. In addition to the naturally occurring IFNs, a non-natural recombinant type I interferon known as consensus interferon (CIFN) has been synthesized as a therapeutic compound (Tong et al., 1997, Hepatology 26, 747-754).

Interferon is currently in use for at least 12 different indications, including infectious and autoimmune diseases and cancer (Borden, 1992, N. Engl. J. Med. 326, 1491-1492). For autoimmune diseases, IFN has been utilized for treatment of rheumatoid arthritis, multiple sclerosis, and Crohn's disease. For treatment of cancer, IFN has been used alone or in combination with a number of different compounds. Specific types of cancers for which IFN has been used include squamous cell carcinomas, melanomas, hypernephromas, hemangiomas, hairy cell leukemia, and Kaposi's sarcoma. In the treatment of infectious diseases, IFNs increase the phagocytic activity of macrophages and cytotoxicity of lymphocytes and inhibits the propagation of cellular pathogens. Specific indications for which IFN has been used as treatment include hepatitis B, human papillomavirus types 6 and 11 (i.e. genital warts) (Leventhal et al., 1991, N Engl J. Med 325, 613-617), chronic granulomatous disease, and SARS virus.

Pegylated interferons, i.e., interferons conjugated with polyethylene glycol (PEG), have demonstrated improved characteristics over interferon. Advantages incurred by PEG conjugation can include an improved pharmacokinetic profile compared to interferons lacking PEG, thus imparting more convenient dosing regimes, improved tolerance, and improved antiviral efficacy. Such improvements have been demonstrated in clinical studies of both polyethylene glycol interferon alfa-2a (PEGASYS, Roche) and polyethylene glycol interferon alfa-2b (VIRAFERON PEG, PEG-INTRON, Enzon/Schering Plough).

siNA molecules in combination with interferons and polyethylene glycol interferons have the potential to improve the effectiveness of treatment of SARS or any of the other indications discussed above. siNA molecules targeting RNAs associated with SARS virus infection can be used individually or in combination with other therapies such as interferons and polyethylene glycol interferons and to achieve enhanced efficacy.

Example 12
Diagnostic Uses

The siNA molecules of the invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets (e.g., RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings. Such diagnostic use of siNA molecules involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates. siNA molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell. The close relationship between siNA activity and the structure of the target RNA allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple siNA molecules described in this invention, one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of disease or infection. In this manner, other genetic targets can be defined as important mediators of the disease. These experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple siNA molecules targeted to different genes, siNA molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations siNA molecules and/or other chemical or biological molecules). Other in vitro uses of siNA molecules of this invention are well known in the art, and include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).

In a specific example, siNA molecules that cleave only wild-type or mutant forms of the target RNA are used for the assay. The first siNA molecules (i.e., those that cleave only wild-type forms of target RNA) are used to identify wild-type RNA present in the sample and the second siNA molecules (i.e., those that cleave only mutant forms of target RNA) are used to identify mutant RNA in the sample. As reaction controls, synthetic substrates of both wild-type and mutant RNA are cleaved by both siNA molecules to demonstrate the relative siNA efficiencies in the reactions and the absence of cleavage of the “non-targeted” RNA species. The cleavage products from the synthetic substrates also serve to generate size markers for the analysis of wild-type and mutant RNAs in the sample population. Thus, each analysis requires two siNA molecules, two substrates and one unknown sample, which is combined into six reactions. The presence of cleavage products is determined using an RNase protection assay so that full-length and cleavage fragments of each RNA can be analyzed in one lane of a polyacrylamide gel. It is not absolutely required to quantify the results to gain insight into the expression of mutant RNAs and putative risk of the desired phenotypic changes in target cells. The expression of mRNA whose protein product is implicated in the development of the phenotype (i.e., disease related or infection related) is adequate to establish risk. If probes of comparable specific activity are used for both transcripts, then a qualitative comparison of RNA levels is adequate and decreases the cost of the initial diagnosis. Higher mutant form to wild-type ratios are correlated with higher risk whether RNA levels are compared qualitatively or quantitatively.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims. The present invention teaches one skilled in the art to test various combinations and/or substitutions of chemical modifications described herein toward generating nucleic acid constructs with improved activity for mediating RNAi activity. Such improved activity can comprise improved stability, improved bioavailability, and/or improved activation of cellular responses mediating RNAi. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying siNA molecules with improved RNAi activity.

The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims. In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

TABLE ISARS virus Accession NumbersLOCUS NC_004718 29736 bp ss-RNA linear VRL15-APR-2003DEFINITION SARS coronavirus, complete genome.ACCESSION NC_004718

TABLE II

SARS siNA and Target Sequences

SARS CoV NC_004718

Pos
Seq
Seq ID
UPos
Upper seq
Seq ID
LPos
Lower seq
Seq ID

3
ACCCAGGAAAAGCCAACCA
1
3
ACCCAGGAAAAGCCAACCA
1
21
UGGUUGGCUUUUCCUGGGU
1652

21
AACCUCGAUCUCUUGUAGA
2
21
AACCUCGAUCUCUUGUAGA
2
39
UCUACAAGAGAUCGAGGUU
1653

39
AUCUGUUCUCUAAACGAAC
3
39
AUCUGUUCUCUAAACGAAC
3
57
GUUCGUUUAGAGAACAGAU
1654

57
CUUUAAAAUCUGUGUAGCU
4
57
CUUUAAAAUCUGUGUAGCU
4
75
AGCUACACAGAUUUUAAAG
1655

75
UGUCGCUCGGCUGCAUGCC
5
75
UGUCGCUCGGCUGCAUGCC
5
93
GGCAUGCAGCCGAGCGACA
1656

93
CUAGUGCACCUACGCAGUA
6
93
CUAGUGCACCUACGCAGUA
6
111
UACUGCGUAGGUGCACUAG
1657

111
AUAAACAAUAAUAAAUUUU
7
111
AUAAACAAUAAUAAAUUUU
7
129
AAAAUUUAUUAUUGUUUAU
1658

129
UACUGUCGUUGACAAGAAA
8
129
UACUGUCGUUGACAAGAAA
8
147
UUUCUUGUCAACGACAGUA
1659

147
ACGAGUAACUCGUCCCUCU
9
147
ACGAGUAACUCGUCCCUCU
9
165
AGAGGGACGAGUUACUCGU
1660

165
UUCUGCAGACUGCUUACGG
10
165
UUCUGCAGACUGCUUACGG
10
183
CCGUAAGCAGUCUGCAGAA
1661

183
GUUUCGUCCGUGUUGCAGU
11
183
GUUUCGUCCGUGUUGCAGU
11
201
ACUGCAACACGGACGAAAC
1662

201
UCGAUCAUCAGCAUACCUA
12
201
UCGAUCAUCAGCAUACCUA
12
219
UAGGUAUGCUGAUGAUCGA
1663

219
AGGUUUCGUCCGGGUGUGA
13
219
AGGUUUCGUCCGGGUGUGA
13
237
UCACACCCGGACGAAACCU
1664

237
ACCGAAAGGUAAGAUGGAG
14
237
ACCGAAAGGUAAGAUGGAG
14
255
CUCCAUCUUACCUUUCGGU
1665

255
GAGCCUUGUUCUUGGUGUC
15
255
GAGCCUUGUUCUUGGUGUC
15
273
GACACCAAGAACAAGGCUC
1666

273
CAACGAGAAAACACACGUC
16
273
CAACGAGAAAACACACGUC
16
291
GACGUGUGUUUUCUCGUUG
1667

291
CCAACUCAGUUUGCCUGUC
17
291
CCAACUCAGUUUGCCUGUC
17
309
GACAGGCAAACUGAGUUGG
1668

309
CCUUCAGGUUAGAGACGUG
18
309
CCUUCAGGUUAGAGACGUG
18
327
CACGUCUCUAACCUGAAGG
1669

327
GCUAGUGCGUGGCUUCGGG
19
327
GCUAGUGCGUGGCUUCGGG
19
345
CCCGAAGCCACGCACUAGC
1670

345
GGACUCUGUGGAAGAGGCC
20
345
GGACUCUGUGGAAGAGGCC
20
363
GGCCUCUUCCACAGAGUCC
1671

363
CCUAUCGGAGGCACGUGAA
21
363
CCUAUCGGAGGCACGUGAA
21
381
UUCACGUGCCUCCGAUAGG
1672

381
ACACCUCAAAAAUGGCACU
22
381
ACACCUCAAAAAUGGCACU
22
399
AGUGCCAUUUUUGAGGUGU
1673

399
UUGUGGUCUAGUAGAGCUG
23
399
UUGUGGUCUAGUAGAGCUG
23
417
CAGCUCUACUAGACCACAA
1674

417
GGAAAAAGGCGUACUGCCC
24
417
GGAAAAAGGCGUACUGCCC
24
435
GGGCAGUACGCCUUUUUCC
1675

435
CCAGCUUGAACAGCCCUAU
25
435
CCAGCUUGAACAGCCCUAU
25
453
AUAGGGCUGUUCAAGCUGG
1676

453
UGUGUUCAUUAAACGUUCU
26
453
UGUGUUCAUUAAACGUUCU
26
471
AGAACGUUUAAUGAACACA
1677

471
UGAUGCCUUAAGCACCAAU
27
471
UGAUGCCUUAAGCACCAAU
27
489
AUUGGUGCUUAAGGCAUCA
1678

489
UCACGGCCACAAGGUCGUU
28
489
UCACGGCCACAAGGUCGUU
28
507
AACGACCUUGUGGCCGUGA
1679

507
UGAGCUGGUUGCAGAAAUG
29
507
UGAGCUGGUUGCAGAAAUG
29
525
CAUUUCUGCAACCAGCUCA
1680

525
GGACGGCAUUCAGUACGGU
30
525
GGACGGCAUUCAGUACGGU
30
543
ACCGUACUGAAUGCCGUCC
1681

543
UCGUAGCGGUAUAACACUG
31
543
UCGUAGCGGUAUAACACUG
31
561
CAGUGUUAUACCGCUACGA
1682

561
GGGAGUACUCGUGCCACAU
32
561
GGGAGUACUCGUGCCACAU
32
579
AUGUGGCACGAGUACUCCC
1683

579
UGUGGGCGAAACCCCAAUU
33
579
UGUGGGCGAAACCCCAAUU
33
597
AAUUGGGGUUUCGCCCACA
1684

597
UGCAUACCGCAAUGUUCUU
34
597
UGCAUACCGCAAUGUUCUU
34
615
AAGAACAUUGCGGUAUGCA
1685

615
UCUUCGUAAGAACGGUAAU
35
615
UCUUCGUAAGAACGGUAAU
35
633
AUUACCGUUCUUACGAAGA
1686

633
UAAGGGAGCCGGUGGUCAU
36
633
UAAGGGAGCCGGUGGUCAU
36
651
AUGACCACCGGCUCCCUUA
1687

651
UAGCUAUGGCAUCGAUCUA
37
651
UAGCUAUGGCAUCGAUCUA
37
669
UAGAUCGAUGCCAUAGCUA
1668

669
AAAGUCUUAUGACUUAGGU
38
669
AAAGUCUUAUGACUUAGGU
38
687
ACCUAAGUCAUAAGACUUU
1689

687
UGACGAGCUUGGCACUGAU
39
687
UGACGAGCUUGGCACUGAU
39
705
AUCAGUGCCAAGCUCGUCA
1690

705
UCCCAUUGAAGAUUAUGAA
40
705
UCCCAUUGAAGAUUAUGAA
40
723
UUCAUAAUCUUCAAUGGGA
1691

723
ACAAAACUGGAACACUAAG
41
723
ACAAAACUGGAACACUAAG
41
741
CUUAGUGUUCCAGUUUUGU
1692

741
GCAUGGCAGUGGUGCACUC
42
741
GCAUGGCAGUGGUGCACUC
42
759
GAGUGCACCACUGCCAUGU
1693

759
CCGUGAACUCACUCGUGAG
43
759
CCGUGAACUCACUCGUGAG
43
777
CUCACGAGUGAGUUCACGG
1694

777
GCUCAAUGGAGGUGCAGUC
44
777
GCUCAAUGGAGGUGCAGUC
44
795
GACUGCACCUCCAUUGAGC
1695

795
CACUCGCUAUGUCGACAAC
45
795
CACUCGCUAUGUCGACAAC
45
813
GUUGUCGACAUAGCGAGUG
1696

813
CAAUUUCUGUGGCCCAGAU
46
813
CAAUUUCUGUGGCCCAGAU
46
831
AUCUGGGCCACAGAAAUUG
1697

831
UGGGUACCCUCUUGAUUGC
47
831
UGGGUACCCUCUUGAUUGC
47
849
GCAAUCAAGAGGGUACCCA
1698

849
CAUCAAAGAUUUUCUCGCA
48
849
CAUCAAAGAUUUUCUCGCA
48
867
UGCGAGAAAAUCUUUGAUG
1699

867
ACGCGCGGGCAAGUCAAUG
49
867
ACGCGCGGGCAAGUCAAUG
49
885
CAUUGACUUGCCCGCGCGU
1700

885
GUGCACUCUUUCCGAACAA
50
885
GUGCACUCUUUCCGAACAA
50
903
UUGUUCGGAAAGAGUGCAC
1701

903
ACUUGAUUACAUCGAGUCG
51
903
ACUUGAUUACAUCGAGUCG
51
921
CGACUCGAUGUAAUCAAGU
1702

921
GAAGAGAGGUGUCUACUGC
52
921
GAAGAGAGGUGUCUACUGC
52
939
GCAGUAGACACCUCUCUUC
1703

939
CUGCCGUGACCAUGAGCAU
53
939
CUGCCGUGACCAUGAGCAU
53
957
AUGCUCAUGGUCACGGCAG
1704

957
UGAAAUUGCCUGGUUCACU
54
957
UGAAAUUGCCUGGUUCACU
54
975
AGUGAACCAGGCAAUUUCA
1705

975
UGAGCGCUCUGAUAAGAGC
55
975
UGAGCGCUCUGAUAAGAGC
55
993
GCUCUUAUCAGAGCGCUCA
1706

993
CUACGAGCACCAGACACCC
56
993
CUACGAGCACCAGACACCC
56
1011
GGGUGUCUGGUGCUCGUAG
1707

1011
CUUCGAAAUUAAGAGUGCC
57
1011
CUUCGAAAUUAAGAGUGCC
57
1029
GGCACUCUUAAUUUCGAAG
1708

1029
CAAGAAAUUUGACACUUUC
58
1029
CAAGAAAUUUGACACUUUC
58
1047
GAAAGUGUCAAAUUUCUUG
1709

1047
CAAAGGGGAAUGCCCAAAG
59
1047
CAAAGGGGAAUGCCCAAAG
59
1065
CUUUGGGCAUUCCCCUUUG
1710

1065
GUUUGUGUUUCCUCUUAAC
60
1065
GUUUGUGUUUCCUCUUAAC
60
1083
GUUAAGAGGAAACACAAAC
1711

1083
CUCAAAAGUCAAAGUCAUU
61
1083
CUCAAAAGUCAAAGUCAUU
61
1101
AAUGACUUUGACUUUUGAG
1712

1101
UCAACCACGUGUUGAAAAG
62
1101
UCAACCACGUGUUGAAAAG
62
1119
CUUUUCAACACGUGGUUGA
1713

1119
GAAAAAGACUGAGGGUUUC
63
1119
GAAAAAGACUGAGGGUUUC
63
1137
GAAACCCUCAGUCUUUUUC
1714

1137
CAUGGGGCGUAUACGCUCU
64
1137
CAUGGGGCGUAUACGCUCU
64
1155
AGAGCGUAUACGCCCCAUG
1715

1155
UGUGUACCCUGUUGCAUCU
65
1155
UGUGUACCCUGUUGCAUCU
65
1173
AGAUGCAACAGGGUACACA
1716

1173
UCCACAGGAGUGUAACAAU
66
1173
UCCACAGGAGUGUAACAAU
66
1191
AUUGUUACACUCCUGUGGA
1717

1191
UAUGCACUUGUCUACCUUG
67
1191
UAUGCACUUGUCUACCUUG
67
1209
CAAGGUAGACAAGUGCAUA
1718

1209
GAUGAAAUGUAAUCAUUGC
68
1209
GAUGAAAUGUAAUCAUUGC
68
1227
GCAAUGAUUACAUUUCAUC
1719

1227
CGAUGAAGUUUCAUGGCAG
69
1227
CGAUGAAGUUUCAUGGCAG
69
1245
CUGCCAUGAAACUUCAUCG
1720

1245
GACGUGCGACUUUCUGAAA
70
1245
GACGUGCGACUUUCUGAAA
70
1263
UUUCAGAAAGUCGCACGUC
1721

1263
AGCCACUUGUGAACAUUGU
71
1263
AGCCACUUGUGAACAUUGU
71
1281
ACAAUGUUCACAAGUGGCU
1722

1281
UGGCACUGAAAAUUUAGUU
72
1281
UGGCACUGAAAAUUUAGUU
72
1299
AACUAAAUUUUCAGUGCCA
1723

1299
UAUUGAAGGACCUACUACA
73
1299
UAUUGAAGGACCUACUACA
73
1317
UGUAGUAGGUCCUUCAAUA
1724

1317
AUGUGGGUACCUACCUACU
74
1317
AUGUGGGUACCUACCUACU
74
1335
AGUAGGUAGGUACCCACAU
1725

1335
UAAUGCUGUAGUGAAAAUG
75
1335
UAAUGCUGUAGUGAAAAUG
75
1353
CAUUUUCACUACAGCAUUA
1726

1353
GCCAUGUCCUGCCUGUCAA
76
1353
GCCAUGUCCUGCCUGUCAA
76
1371
UUGACAGGCAGGACAUGGC
1727

1371
AGACCCAGAGAUUGGACCU
77
1371
AGACCCAGAGAUUGGACCU
77
1389
AGGUCCAAUCUCUGGGUCU
1728

1389
UGAGCAUAGUGUUGCAGAU
78
1389
UGAGCAUAGUGUUGCAGAU
78
1407
AUCUGCAACACUAUGCUCA
1729

1407
UUAUCACAACCACUCAAAC
79
1407
UUAUCACAACCACUCAAAC
79
1425
GUUUGAGUGGUUGUGAUAA
1730

1425
CAUUGAAACUCGACUCCGC
80
1425
CAUUGAAACUCGACUCCGC
80
1443
GCGGAGUCGAGUUUCAAUG
1731

1443
CAAGGGAGGUAGGACUAGA
81
1443
CAAGGGAGGUAGGACUAGA
81
1461
UCUAGUCCUACCUCCCUUG
1732

1461
AUGUUUUGGAGGCUGUGUG
82
1461
AUGUUUUGGAGGCUGUGUG
82
1479
CACACAGCCUCCAAAACAU
1733

1479
GUUUGCCUAUGUUGGCUGC
83
1479
GUUUGCCUAUGUUGGCUGC
83
1497
GCAGCCAACAUAGGCAAAC
1734

1497
CUAUAAUAAGCGUGCCUAC
84
1497
CUAUAAUAAGCGUGCCUAC
84
1515
GUAGGCACGCUUAUUAUAG
1735

1515
CUGGGUUCCUCGUGCUAGU
85
1515
CUGGGUUCCUCGUGCUAGU
85
1533
ACUAGCACGAGGAACCCAG
1736

1533
UGCUGAUAUUGGCUCAGGC
86
1533
UGCUGAUAUUGGCUCAGGC
86
1551
GCCUGAGCCAAUAUCAGCA
1737

1551
CCAUACUGGCAUUACUGGU
87
1551
CCAUACUGGCAUUACUGGU
87
1569
ACCAGUAAUGCCAGUAUGG
1738

1569
UGACAAUGUGGAGACCUUG
88
1569
UGACAAUGUGGAGACCUUG
88
1587
CAAGGUCUCCACAUUGUCA
1739

1587
GAAUGAGGAUCUCCUUGAG
89
1587
GAAUGAGGAUCUCCUUGAG
89
1605
CUCAAGGAGAUCCUCAUUC
1740

1605
GAUACUGAGUCGUGAACGU
90
1605
GAUACUGAGUCGUGAACGU
90
1623
ACGUUCACGACUCAGUAUC
1741

1623
UGUUAACAUUAACAUUGUU
91
1623
UGUUAACAUUAACAUUGUU
91
1641
AACAAUGUUAAUGUUAACA
1742

1641
UGGCGAUUUUCAUUUGAAU
92
1641
UGGCGAUUUUCAUUUGAAU
92
1659
AUUCAAAUGAAAAUCGCCA
1743

1659
UGAAGAGGUUGCCAUCAUU
93
1659
UGAAGAGGUUGCCAUCAUU
93
1677
AAUGAUGGCAACCUCUUCA
1744

1677
UUUGGCAUCUUUCUCUGCU
94
1677
UUUGGCAUCUUUCCUGCU
94
1695
AGCAGAGAAAGAUGCCAAA
1745

1695
UUCUACAAGUGCCUUUAUU
95
1695
UUCUACAAGUGCCUUUAUU
95
1713
AAUAAAGGCACUUGUAGAA
1746

1713
UGACACUAUAAAGAGUCUU
96
1713
UGACACUAUAAAGAGUCUU
96
1731
AAGACUCUUUAUAGUGUCA
1747

1731
UGAUUACAAGUCUUUCAAA
97
1731
UGAUUACAAGUCUUUCAAA
97
1749
UUUGAAAGACUUCUAAUCA
1748

1749
AACCAUUGUUGAGUCCUGC
98
1749
AACCAUUGUUGAGUCCUGC
98
1767
GCAGGACUCAACAAUGGUU
1749

1767
CGGUAACUAUAAAGUUACC
99
1767
CGGUAACUAUAAAGUUACC
99
1785
GGUAACUUUAUAGUUACCG
1750

1785
CAAGGGAAAGCCCGUAAAA
100
1785
CAAGGGAAAGCCCGUAAAA
100
1803
UUUUACGGGCUUUCCCUUG
1751

1803
AGGUGCUUGGAACAUUGGA
101
1803
AGGUGCUUGGAACAUUGGA
101
1821
UCCAAUGUUCCAAGCACCU
1752

1821
ACAACAGAGAUCAGUUUUA
102
1821
ACAACAGAGAUCAGUUUUA
102
1839
UAAAACUGAUCUCUGUUGU
1753

1839
AACACCACUGUGUGGUUUU
103
1839
AACACCACUGUGUGGUUUU
103
1857
AAAACCACACAGUGGUGUU
1754

1857
UCCCUCACAGGCUGCUGGU
104
1857
UCCCUCACAGGCUGCUGGU
104
1875
ACCAGCAGCCUGUGAGGGA
1755

1875
UGUUAUCAGAUCAAUUUUU
105
1875
UGUUAUCAGAUCAAUUUUU
105
1893
AAAAAUUGAUCUGAUAACA
1756

1893
UGCGCGCACACUUGAUGCA
106
1893
UGCGCGCACACUUGAUGCA
106
1911
UGCAUCAAGUGUGCGCGCA
1757

1911
AGCAAACCACUCAAUUCCU
107
1911
AGCAAACCACUCAAUUCCU
107
1929
AGGAAUUGAGUGGUUUGCU
1758

1929
UGAUUUGCAAAGAGCAGCU
108
1929
UGAUUUGCAAAGAGCAGCU
108
1947
AGCUGCUCUUUGCAAAUCA
1759

1947
UGUCACCAUACUUGAUGGU
109
1947
UGUCACCAUACUUGAUGGU
109
1965
ACCAUCAAGUAUGGUGACA
1760

1965
UAUUUCUGAACAGUCAUUA
110
1965
UAUUUCUGAACAGUCAUUA
110
1983
UAAUGACUGUUCAGAAAUA
1761

1983
ACGUCUUGUCGACGCCAUG
111
1983
ACGUCUUGUCGACGCCAUG
111
2001
CAUGGCGUCGACAAGACGU
1762

2001
GGUUUAUACUUCAGACCUG
112
2001
GGUUUAUACUUCAGACCUG
112
2019
CAGGUCUGAAGUAUAAACC
1763

2019
GCUCACCAACAGUGUCAUU
113
2019
GCUCACCAACAGUGUCAUU
113
2037
AAUGACACUGUUGGUGAGC
1764

2037
UAUUAUGGCAUAUGUAACU
114
2037
UAUUAUGGCAUAUGUAACU
114
2055
AGUUACAUAUGCCAUAAUA
1765

2055
UGGUGGUCUUGUACAACAG
115
2055
UGGUGGUCUUGUACAACAG
115
2073
CUGUUGUACAAGACCACCA
1766

2073
GACUUCUCAGUGGUUGUCU
116
2073
GACUUCUCAGUGGUUGUCU
116
2091
AGACAACCACUGAGAAGUC
1767

2091
UAAUCUUUUGGGCACUACU
117
2091
UAAUCUUUUGGGCACUACU
117
2109
AGUAGUGCCCAAAAGAUUA
1768

2109
UGUUGAAAAACUCAGGCCU
118
2109
UGUUGAAAAACUCAGGCCU
118
2127
AGGCCUGAGUUUUUCAACA
1769

2127
UAUCUUUGAAUGGAUUGAG
119
2127
UAUCUUUGAAUGGAUUGAG
119
2145
CUCAAUCCAUUCAAAGAUA
1770

2145
GGCGAAACUUAGUGCAGGA
120
2145
GGCGAAACUUAGUGCAGGA
120
2163
UCCUGCACUAAGUUUCGCC
1771

2163
AGUUGAAUUUCUCAAGGAU
121
2163
AGUUGAAUUUCUCAAGGAU
121
2181
AUCCUUGAGAAAUUCAACU
1772

2181
UGCUUGGGAGAUUCUCAAA
122
2181
UGCUUGGGAGAUUCUCAAA
122
2199
UUUGAGAAUCUCCCAAGCA
1773

2199
AUUUCUCAUUACAGGUGUU
123
2199
AUUUCUCAUUACAGGUGUU
123
2217
AACACCUGUAAUGAGAAAU
1774

2217
UUUUGACAUCGUCAAGGGU
124
2217
UUUUGACAUCGUCAAGGGU
124
2235
ACCCUUGACGAUGUCAAAA
1775

2235
UCAAAUACAGGUUGCUUCA
125
2235
UCAAAUACAGGUUGCUUCA
125
2253
UGAAGCAACCUGUAUUUGA
1776

2253
AGAUAACAUCAAGGAUUGU
126
2253
AGAUAACAUCAAGGAUUGU
126
2271
ACAAUCCUUGAUGUUAUCU
1777

2271
UGUAAAAUGCUUCAUUGAU
127
2271
UGUAAAAUGCUUCAUUGAU
127
2289
AUCAAUGAAGCAUUUUACA
1778

2289
UGUUGUUAACAAGGCACUC
128
2289
UGUUGUUAACAAGGCACUC
128
2307
GAGUGCCUUGUUAACAACA
1779

2307
CGAAAUGUGCAUUGAUCAA
129
2307
CGAAAUGUGCAUUGAUCAA
129
2325
UUGAUCAAUGCACAUUUCG
1780

2325
AGUCACUAUCGCUGGCGCA
130
2325
AGUCACUAUCGCUGGCGCA
130
2343
UGCGCCAGCGAUAGUGACU
1781

2343
AAAGUUGCGAUCACUCAAC
131
2343
AAAGUUGCGAUCACUCAAC
131
2361
GUUGAGUGAUCGCAACUUU
1782

2361
CUUAGGUGAAGUCUUCAUC
132
2361
CUUAGGUGAAGUCUUCAUC
132
2379
GAUGAAGACUUCACCUAAG
1783

2379
CGCUCAAAGCAAGGGACUU
133
2379
CGCUCAAAGCAAGGGACUU
133
2397
AAGUCCCUUGCUUUGAGCG
1784

2397
UUACCGUCAGUGUAUACGU
134
2397
UUACCGUCAGUGUAUACGU
134
2415
ACGUAUACACUGACGGUAA
1785

2415
UGGCAAGGAGCAGCUGCAA
135
2415
UGGCAAGGAGCAGCUGCAA
135
2433
UUGCAGCUGCUCCUUGCCA
1786

2433
ACUACUCAUGCCUCUUAAG
136
2433
ACUACUCAUGCCUCUUAAG
136
2451
CUUAAGAGGCAUGAGUAGU
1787

2451
GGCACCAAAAGAAGUAACC
137
2451
GGCACCAAAAGAAGUAACC
137
2469
GGUUACUUCUUUUGGUGCC
1788

2469
CUUUCUUGAAGGUGAUUCA
138
2469
CUUUCUUGAAGGUGAUUCA
138
2487
UGAAUCACCUUCAAGAAAG
1789

2487
ACAUGACACAGUACUUACC
139
2487
ACAUGACACAGUACUUACC
139
2505
GGUAAGUACUGUGUCAUGU
1790

2505
CUCUGAGGAGGUUGUUCUC
140
2505
CUCUGAGGAGGUUGUUCUC
140
2523
GAGAACAACCUCCUCAGAG
1791

2523
CAAGAACGGUGAACUCGAA
141
2523
CAAGAACGGUGAACUCGAA
141
2541
UUCGAGUUCACCGUUCUUG
1792

2541
AGCACUCGAGACGCCCGUU
142
2541
AGCACUCGAGACGCCCGUU
142
2559
AACGGGCGUCUCGAGUGCU
1793

2559
UGAUAGCUUCACAAAUGGA
143
2559
UGAUAGCUUCACAAAUGGA
143
2577
UCCAUUUGUGAAGCUAUCA
1794

2577
AGCUAUCGUCGGCACACCA
144
2577
AGCUAUCGUCGGCACACCA
144
2595
UGGUGUGCCGACGAUAGCU
1795

2595
AGUCUGUGUAAAUGGCCUC
145
2595
AGUCUGUGUAAAUGGCCUC
145
2613
GAGGCCAUUUACACAGACU
1796

2613
CAUGCUCUUAGAGAUUAAG
146
2613
CAUGCUCUUAGAGAUUAAG
146
2631
CUUAAUCUCUAAGAGCAUG
1797

2631
GGACAAAGAACAAUACUGC
147
2631
GGACAAAGAACAAUACUGC
147
2649
GCAGUAUUGUUCUUUGUCC
1798

2649
CGCAUUGUCUCCUGGUUUA
148
2649
CGCAUUGUCUCCUGGUUUA
148
2667
UAAACCAGGAGACAAUGCG
1799

2667
ACUGGCUACAAACAAUGUC
149
2667
ACUGGCUACAAACAAUGUC
149
2685
GACAUUGUUUGUAGCCAGU
1800

2685
CUUUCGCUUAAAAGGGGGU
150
2685
CUUUCGCUUAAAAGGGGGU
150
2703
ACCCCCUUUUAAGCGAAAG
1801

2703
UGCACCAAUUAAAGGUGUA
151
2703
UGCACCAAUUAAAGGUGUA
151
2721
UACACCUUUAAUUGGUGCA
1802

2721
AACCUUUGGAGAAGAUACU
152
2721
AACCUUUGGAGAAGAUACU
152
2739
AGUAUCUUCUCCAAAGGUU
1803

2739
UGUUUGGGAAGUUCAAGGU
153
2739
UGUUUGGGAAGUUCAAGGU
153
2757
ACCUUGAACUUCCCAAACA
1804

2757
UUACAAGAAUGUGAGAAUC
154
2757
UUACAAGAAUGUGAGAAUC
154
2775
GAUUCUCACAUUCUUGUAA
1805

2775
CACAUUUGAGCUUGAUGAA
155
2775
CACAUUUGAGCUUGAUGAA
155
2793
UUCAUCAAGCUCAAAUGUG
1806

2793
ACGUGUUGACAAAGUGCUU
156
2793
ACGUGUUGACAAAGUGCUU
156
2811
AAGCACUUUGUCAACACGU
1807

2811
UAAUGAAAAGUGCUCUGUC
157
2811
UAAUGAAAAGUGCUCUGUC
157
2829
GACAGAGCACUUUUCAUUA
1808

2829
CUACACUGUUGAAUCCGGU
158
2829
CUACACUGUUGAAUCCGGU
158
2847
ACCGGAUUCAACAGUGUAG
1809

2847
UACCGAAGUUACUGAGUUU
159
2847
UACCGAAGUUACUGAGUUU
159
2865
AAACUCAGUAACUUCGGUA
1810

2865
UGCAUGUGUUGUAGCAGAG
160
2865
UGCAUGUGUUGUAGCAGAG
160
2883
CUCUGCUACAACACAUGCA
1811

2883
GGCUGUUGUGAAGACUUUA
161
2883
GGCUGUUGUGAAGACUUUA
161
2901
UAAAGUCUUCACAACAGCC
1812

2901
ACAACCAGUUUCUGAUCUC
162
2901
ACAACCAGUUUCUGAUCUC
162
2919
GAGAUCAGAAACUGGUUGU
1813

2919
CCUUACCAACAUGGGUAUU
163
2919
CCUUACCAACAUGGGUAUU
163
2937
AAUACCCAUGUUGGUAAGG
1814

2937
UGAUCUUGAUGAGUGGAGU
164
2937
UGAUCUUGAUGAGUGGAGU
164
2955
ACUCCACUCAUCAAGAUCA
1815

2955
UGUAGCUACAUUCUACUUA
165
2955
UGUAGCUACAUUCUACUUA
165
2973
UAAGUAGAAUGUAGCUACA
1816

2973
AUUUGAUGAUGCUGGUGAA
166
2973
AUUUGAUGAUGCUGGUGAA
166
2991
UUCACCAGCAUCAUCAAAU
1817

2991
AGAAAACUUUUCAUCACGU
167
2991
AGAAAACUUUUCAUCACGU
167
3009
ACGUGAUGAAAAGUUUUCU
1818

3009
UAUGUAUUGUUCCUUUUAC
168
3009
UAUGUAUUGUUCCUUUUAC
168
3027
GUAAAAGGAACAAUACAUA
1819

3027
CCCUCCAGAUGAGGAAGAA
169
3027
CCCUCCAGAUGAGGAAGAA
169
3045
UUCUUCCUCAUCUGGAGGG
1820

3045
AGAGGACGAUGCAGAGUGU
170
3045
AGAGGACGAUGCAGAGUGU
170
3063
ACACUCUGCAUCGUCCUCU
1821

3063
UGAGGAAGAAGAAAUUGAU
171
3063
UGAGGAAGAAGAAAUUGAU
171
3081
AUCAAUUUCUUCUUCCUCA
1822

3081
UGAAACCUGUGAACAUGAG
172
3081
UGAAACCUGUGAACAUGAG
172
3099
CUCAUGUUCACAGGUUUCA
1823

3099
GUACGGUACAGAGGAUGAU
173
3099
GUACGGUACAGAGGAUGAU
173
3117
AUCAUCCUCUGUACCGUAC
1824

3117
UUAUCAAGGUCUCCCUCUG
174
3117
UUAUCAAGGUCUCCCUCUG
174
3135
CAGAGGGAGACCUUGAUAA
1825

3135
GGAAUUUGGUGCCUCAGCU
175
3135
GGAAUUUGGUGCCUCAGCU
175
3153
AGCUGAGGCACCAAAUUCC
1826

3153
UGAAACAGUUCGAGUUGAG
176
3153
UGAAACAGUUCGAGUUGAG
176
3171
CUCAACUCGAACUGUUUCA
1827

3171
GGAAGAAGAAGAGGAAGAC
177
3171
GGAAGAAGAAGAGGAAGAC
177
3189
GUCUUCCUCUUCUUCUUCC
1828

3189
CUGGCUGGAUGAUACUACU
178
3189
CUGGCUGGAUGAUACUACU
178
3207
AGUAGUAUCAUCCAGCCAG
1829

3207
UGAGCAAUCAGAGAUUGAG
179
3207
UGAGCAAUCAGAGAUUGAG
179
3225
CUCAAUCUCUGAUUGCUCA
1830

3225
GCCAGAACCAGAACCUACA
180
3225
GCCAGAACCAGAACCUACA
180
3243
UGUAGGUUCUGGUUCUGGC
1831

3243
ACCUGAAGAACCAGUUAAU
181
3243
ACCUGAAGAACCAGUUAAU
181
3261
AUUAACUGGUUCUUCAGGU
1832

3261
UCAGUUUACUGGUUAUUUA
182
3261
UCAGUUUACUGGUUAUUUA
182
3279
UAAAUAACCAGUAAACUGA
1833

3279
AAAACUUACUGACAAUGUU
183
3279
AAAACUUACUGACAAUGUU
183
3297
AACAUUGUCAGUAAGUUUU
1834

3297
UGCCAUUAAAUGUGUUGAC
184
3297
UGCCAUUAAAUGUGUUGAC
184
3315
GUCAACACAUUUAAUGGCA
1835

3315
CAUCGUUAAGGAGGCACAA
185
3315
CAUCGUUAAGGAGGCACAA
185
3333
UUGUGCCUCCUUAACGAUG
1836

3333
AAGUGCUAAUCCUAUGGUG
186
3333
AAGUGCUAAUCCUAUGGUG
186
3351
CACCAUAGGAUUAGCACUU
1837

3351
GAUUGUAAAUGCUGCUAAC
187
3351
GAUUGUAAAUGCUGCUAAC
187
3369
GUUAGCAGCAUUUACAAUC
1838

3369
CAUACACCUGAAACAUGGU
188
3369
CAUACACCUGAAACAUGGU
188
3387
ACCAUGUUUCAGGUGUAUG
1839

3387
UGGUGGUGUAGCAGGUGCA
189
3387
UGGUGGUGUAGCAGGUGCA
189
3405
UGCACCUGCUACACCACCA
1840

3405
ACUCAACAAGGCAACCAAU
190
3405
ACUCAACAAGGCAACCAAU
190
3423
AUUGGUUGCCUUGUUGAGU
1841

3423
UGGUGCCAUGCAAAAGGAG
191
3423
UGGUGCCAUGCAAAAGGAG
191
3441
CUCCUUUUGCAUGGCACCA
1842

3441
GAGUGAUGAUUACAUUAAG
192
3441
GAGUGAUGAUUACAUUAAG
192
3459
CUUAAUGUAAUCAUCACUC
1843

3459
GCUAAAUGGCCCUCUUACA
193
3459
GCUAAAUGGCCCUCUUACA
193
3477
UGUAAGAGGGCCAUUUAGC
1844

3477
AGUAGGAGGGUCUUGUUUG
194
3477
AGUAGGAGGGUCUUGUUUG
194
3495
CAAACAAGACCCUCCUACU
1845

3495
GCUUUCUGGACAUAAUCUU
195
3495
GCUUUCUGGACAUAAUCUU
195
3513
AAGAUUAUGUCCAGAAAGC
1846

3513
UGCUAAGAAGUGUCUGCAU
196
3513
UGCUAAGAAGUGUCUGCAU
196
3531
AUGCAGACACUUCUUAGCA
1847

3531
UGUUGUUGGACCUAACCUA
197
3531
UGUUGUUGGACCUAACCUA
197
3549
UAGGUUAGGUCCAACAACA
1848

3549
AAAUGCAGGUGAGGACAUC
198
3549
AAAUGCAGGUGAGGACAUC
198
3567
GAUGUCCUCACCUGCAUUU
1849

3567
CCAGCUUCUUAAGGCAGCA
199
3567
CCAGCUUCUUAAGGCAGCA
199
3585
UGCUGCCUUAAGAAGCUGG
1850

3585
AUAUGAAAAUUUCAAUUCA
200
3585
AUAUGAAAAUUUCAAUUCA
200
3603
UGAAUUGAAAUUUUCAUAU
1851

3603
ACAGGACAUCUUACUUGCA
201
3603
ACAGGACAUCUUACUUGCA
201
3621
UGCAAGUAAGAUGUCCUGU
1852

3621
ACCAUUGUUGUCAGCAGGC
202
3621
ACCAUUGUUGUCAGCAGGC
202
3639
GCCUGCUGACAACAAUGGU
1853

3639
CAUAUUUGGUGCUAAACCA
203
3639
CAUAUUUGGUGCUAAACCA
203
3657
UGGUUUAGCACCAAAUAUG
1854

3657
ACUUCAGUCUUUACAAGUG
204
3657
ACUUCAGUCUUUACAAGUG
204
3675
CACUUGUAAAGACUGAAGU
1855

3675
GUGCGUGCAGACGGUUCGU
205
3675
GUGCGUGCAGACGGUUCGU
205
3693
ACGAACCGUCUGCACGCAC
1856

3693
UACACAGGUUUAUAUUGCA
206
3693
UACACAGGUUUAUAUUGCA
206
3711
UGCAAUAUAAACCUGUGUA
1857

3711
AGUCAAUGACAAAGCUCUU
207
3711
AGUCAAUGACAAAGCUCUU
207
3729
AAGAGCUUUGUCAUUGACU
1858

3729
UUAUGAGCAGGUUGUCAUG
208
3729
UUAUGAGCAGGUUGUCAUG
208
3747
CAUGACAACCUGCUCAUAA
1859

3747
GGAUUAUCUUGAUAACCUG
209
3747
GGAUUAUCUUGAUAACCUG
209
3765
CAGGUUAUCAAGAUAAUCC
1860

3765
GAAGCCUAGAGUGGAAGCA
210
3765
GAAGCCUAGAGUGGAAGCA
210
3783
UGCUUCCACUCUAGGCUUC
1861

3783
ACCUAAACAAGAGGAGCCA
211
3783
ACCUAAACAAGAGGAGCCA
211
3801
UGGCUCCUCUUGUUUAGGU
1862

3801
ACCAAACACAGAAGAUUCC
212
3801
ACCAAACACAGAAGAUUCC
212
3819
GGAAUCUUCUGUGUUUGGU
1863

3819
CAAAACUGAGGAGAAAUCU
213
3819
CAAAACUGAGGAGAAAUCU
213
3837
AGAUUUCUCCUCAGUUUUG
1864

3837
UGUCGUACAGAAGCCUGUC
214
3837
UGUCGUACAGAAGCCUGUC
214
3855
GACAGGCUUCUGUACGACA
1865

3855
CGAUGUGAAGCCAAAAAUU
215
3855
CGAUGUGAAGCCAAAAAUU
215
3873
AAUUUUUGGCUUCACAUCG
1866

3873
UAAGGCCUGCAUUGAUGAG
216
3873
UAAGGCCUGCAUUGAUGAG
216
3891
CUCAUCAAUGCAGGCCUUA
1867

3891
GGUUACCACAACACUGGAA
217
3891
GGUUACCACAACACUGGAA
217
3909
UUCCAGUGUUGUGGUAACC
1868

3909
AGAAACUAAGUUUCUUACC
218
3909
AGAAACUAAGUUUCUUACC
218
3927
GGUAAGAAACUUAGUUUCU
1869

3927
CAAUAAGUUACUCUUGUUU
219
3927
CAAUAAGUUACUCUUGUUU
219
3945
AAACAAGAGUAACUUAUUG
1870

3945
UGCUGAUAUCAAUGGUAAG
220
3945
UGCUGAUAUCAAUGGUAAG
220
3963
CUUACCAUUGAUAUCAGCA
1871

3963
GCUUUACCAUGAUUCUCAG
221
3963
GCUUUACCAUGAUUCUCAG
221
3981
CUGAGAAUCAUGGUAAAGC
1872

3981
GAACAUGCUUAGAGGUGAA
222
3981
GAACAUGCUUAGAGGUGAA
222
3999
UUCACCUCUAAGCAUGUUC
1873

3999
AGAUAUGUCUUUCCUUGAG
223
3999
AGAUAUGUCUUUCCUUGAG
223
4017
CUCAAGGAAAGACAUAUCU
1874

4017
GAAGGAUGCACCUUACAUG
224
4017
GAAGGAUGCACCUUACAUG
224
4035
CAUGUAAGGUGCAUCCUUC
1875

4035
GGUAGGUGAUGUUAUCACU
225
4035
GGUAGGUGAUGUUAUCACU
225
4053
AGUGAUAACAUCACCUACC
1876

4053
UAGUGGUGAUAUCACUUGU
226
4053
UAGUGGUGAUAUCACUUGU
226
4071
ACAAGUGAUAUCACCACUA
1877

4071
UGUUGUAAUACCCUCCAAA
227
4071
UGUUGUAAUACCCUCCAAA
227
4089
UUUGGAGGGUAUUACAACA
1878

4089
AAAGGCUGGUGGCACUACU
228
4089
AAAGGCUGGUGGCACUACU
228
4107
AGUAGUGCCACCAGCCUUU
1879

4107
UGAGAUGCUCUCAAGAGCU
229
4107
UGAGAUGCUCUCAAGAGCU
229
4125
AGCUCUUGAGAGCAUCUCA
1880

4125
UUUGAAGAAAGUGCCAGUU
230
4125
UUUGAAGAAAGUGCCAGUU
230
4143
AACUGGCACUUUCUUCAAA
1881

4143
UGAUGAGUAUAUAACCACG
231
4143
UGAUGAGUAUAUAACCACG
231
4161
CGUGGUUAUAUACUCAUCA
1882

4161
GUACCCUGGACAAGGAUGU
232
4161
GUACCCUGGACAAGGAUGU
232
4179
ACAUCCUUGUCCAGGGUAC
1883

4179
UGCUGGUUAUACACUUGAG
233
4179
UGCUGGUUAUACACUUGAG
233
4197
CUCAAGUGUAUAACCAGCA
1884

4197
GGAAGCUAAGACUGCUCUU
234
4197
GGAAGCUAAGACUGCUCUU
234
4215
AAGAGCAGUCUUAGCUUCC
1885

4215
UAAGAAAUGCAAAUCUGCA
235
4215
UAAGAAAUGCAAAUCUGCA
235
4233
UGCAGAUUUGCAUUUCUUA
1886

4233
AUUUUAUGUACUACCUUCA
236
4233
AUUUUAUGUACUACCUUCA
236
4251
UGAAGGUAGUACAUAAAAU
1887

4251
AGAAGCACCUAAUGCUAAG
237
4251
AGAAGCACCUAAUGCUAAG
237
4269
CUUAGCAUUAGGUGCUUCU
1888

4269
GGAAGAGAUUCUAGGAACU
238
4269
GGAAGAGAUUCUAGGAACU
238
4287
AGUUCCUAGAAUCUCUUCC
1889

4287
UGUAUCCUGGAAUUUGAGA
239
4287
UGUAUCCUGGAAUUUGAGA
239
4305
UCUCAAAUUCCAGGAUACA
1890

4305
AGAAAUGCUUGCUCAUGCU
240
4305
AGAAAUGCUUGCUCAUGCU
240
4323
AGCAUGAGCAAGCAUUUCU
1891

4323
UGAAGAGACAAGAAAAUUA
241
4323
UGAAGAGACAAGAAAAUUA
241
4341
UAAUUUUCUUGUCUCUUCA
1892

4341
AAUGCCUAUAUGCAUGGAU
242
4341
AAUGCCUAUAUGCAUGGAU
242
4359
AUCCAUGCAUAUAGGCAUU
1893

4359
UGUUAGAGCCAUAAUGGCA
243
4359
UGUUAGAGCCAUAAUGGCA
243
4377
UGCCAUUAUGGCUCUAACA
1894

4377
AACCAUCCAACGUAAGUAU
244
4377
AACCAUCCAACGUAAGUAU
244
4395
AUACUUACGUUGGAUGGUU
1895

4395
UAAAGGAAUUAAAAUUCAA
245
4395
UAAAGGAAUUAAAAUUCAA
245
4413
UUGAAUUUUAAUUCCUUUA
1896

4413
AGAGGGCAUCGUUGACUAU
246
4413
AGAGGGCAUCGUUGACUAU
246
4431
AUAGUCAACGAUGCCCUCU
1897

4431
UGGUGUCCGAUUCUUCUUU
247
4431
UGGUGUCCGAUUCUUCUUU
247
4449
AAAGAAGAAUCGGACACCA
1898

4449
UUAUACUAGUAAAGAGCCU
248
4449
UUAUACUAGUAAAGAGCCU
248
4467
AGGCUCUUUACUAGUAUAA
1899

4467
UGUAGCUUCUAUUAUUACG
249
4467
UGUAGCUUCUAUUAUUACG
249
4485
CGUAAUAAUAGAAGCUACA
1900

4485
GAAGCUGAACUCUCUAAAU
250
4485
GAAGCUGAACUCUCUAAAU
250
4503
AUUUAGAGAGUUCAGCUUC
1901

4503
UGAGCCGCUUGUCACAAUG
251
4503
UGAGCCGCUUGUCACAAUG
251
4521
CAUUGUGACAAGCGGCUCA
1902

4521
GCCAAUUGGUUAUGUGACA
252
4521
GCCAAUUGGUUAUGUGACA
252
4539
UGUCACAUAACCAAUUGGC
1903

4539
ACAUGGUUUUAAUCUUGAA
253
4539
ACAUGGUUUUAAUCUUGAA
253
4557
UUCAAGAUUAAAACCAUGU
1904

4557
AGAGGCUGCGCGCUGUAUG
254
4557
AGAGGCUGCGCGCUGUAUG
254
4575
CAUACAGCGCGCAGCCUCU
1905

4575
GCGUUCUCUUAAAGCUCCU
255
4575
GCGUUCUCUUAAAGCUCCU
255
4593
AGGAGCUUUAAGAGAACGC
1906

4593
UGCCGUAGUGUCAGUAUCA
256
4593
UGCCGUAGUGUCAGUAUCA
256
4611
UGAUACUGACACUACGGCA
1907

4611
AUCACCAGAUGCUGUUACU
257
4611
AUCACCAGAUGCUGUUACU
257
4629
AGUAACAGCAUCUGGUGAU
1908

4629
UACAUAUAAUGGAUACCUC
258
4629
UACAUAUAAUGGAUACCUC
258
4647
GAGGUAUCCAUUAUAUGUA
1909

4647
CACUUCGUCAUCAAAGACA
259
4647
CACUUCGUCAUCAAAGACA
259
4665
UGUCUUUGAUGACGAAGUG
1910

4665
AUCUGAGGAGCACUUUGUA
260
4665
AUCUGAGGAGCACUUUGUA
260
4683
UACAAAGUGCUCCUCAGAU
1911

4683
AGAAACAGUUUCUUUGGCU
261
4683
AGAAACAGUUUCUUUGGCU
261
4701
AGCCAAAGAAACUGUUUCU
1912

4701
UGGCUCUUACAGAGAUUGG
262
4701
UGGCUCUUACAGAGAUUGG
262
4719
CCAAUCUCUGUAAGAGCCA
1913

4719
GUCCUAUUCAGGACAGCGU
263
4719
GUCCUAUUCAGGACAGCGU
263
4737
ACGCUGUCCUGAAUAGGAC
1914

4737
UACAGAGUUAGGUGUUGAA
264
4737
UACAGAGUUAGGUGUUGAA
264
4755
UUCAACACCUAACUCUGUA
1915

4755
AUUUCUUAAGCGUGGUGAC
265
4755
AUUUCUUAAGCGUGGUGAC
265
4773
GUCACCACGCUUAAGAAAU
1916

4773
CAAAAUUGUGUACCACACU
266
4773
CAAAAUUGUGUACCACACU
266
4791
AGUGUGGUACACAAUUUUG
1917

4791
UCUGGAGAGCCCCGUCGAG
267
4791
UCUGGAGAGCCCCGUCGAG
267
4809
CUCGACGGGGCUCUCCAGA
1918

4809
GUUUCAUCUUGACGGUGAG
268
4809
GUUUCAUCUUGACGGUGAG
268
4827
CUCACCGUCAAGAUGAAAC
1919

4827
GGUUCUUUCACUUGACAAA
269
4827
GGUUCUUUCACUUGACAAA
269
4845
UUUGUCAAGUGAAAGAACC
1920

4845
ACUAAAGAGUCUCUUAUCC
270
4845
ACUAAAGAGUCUCUUAUCC
270
4863
GGAUAAGAGACUCUUUAGU
1921

4863
CCUGCGGGAGGUUAAGACU
271
4863
CCUGCGGGAGGUUAAGACU
271
4881
AGUCUUAACCUCCCGCAGG
1922

4881
UAUAAAAGUGUUCACAACU
272
4881
UAUAAAAGUGUUCACAACU
272
4899
AGUUGUGAACACUUUUAUA
1923

4899
UGUGGACAACACUAAUCUC
273
4899
UGUGGACAACACUAAUCUC
273
4917
GAGAUUAGUGUUGUCCACA
1924

4917
CCACACACAGCUUGUGGAU
274
4917
CCACACACAGCUUGUGGAU
274
4935
AUCCACAAGCUGUGUGUGG
1925

4935
UAUGUCUAUGACAUAUGGA
275
4935
UAUGUCUAUGACAUAUGGA
275
4953
UCCAUAUGUCAUAGACAUA
1926

4953
ACAGCAGUUUGGUCCAACA
276
4953
ACAGCAGUUUGGUCCAACA
276
4971
UGUUGGACCAAACUGCUGU
1927

4971
AUACUUGGAUGGUGCUGAU
277
4971
AUACUUGGAUGGUGCUGAU
277
4989
AUCAGCACCAUCCAAGUAU
1928

4989
UGUUACAAAAAUUAAACCU
278
4989
UGUUACAAAAAUUAAACCU
278
5007
AGGUUUAAUUUUUGUAACA
1929

5007
UCAUGUAAAUCAUGAGGGU
279
5007
UCAUGUAAAUCAUGAGGGU
279
5025
ACCCUCAUGAUUUACAUGA
1930

5025
UAAGACUUUCUUUGUACUA
280
5025
UAAGACUUUCUUUGUACUA
280
5043
UAGUACAAAGAAAGUCUUA
1931

5043
ACCUAGUGAUGACACACUA
281
5043
ACCUAGUGAUGACACACUA
281
5061
UAGUGUGUCAUCACUAGGU
1932

5061
ACGUAGUGAAGCUUUCGAG
282
5061
ACGUAGUGAAGCUUUCGAG
282
5079
CUCGAAAGCUUCACUACGU
1933

5079
GUACUACCAUACUCUUGAU
283
5079
GUACUACCAUACUCUUGAU
283
5097
AUCAAGAGUAUGGUAGUAC
1934

5097
UGAGAGUUUUCUUGGUAGG
284
5097
UGAGAGUUUUCUUGGUAGG
284
5115
CCUACCAAGAAAACUCUCA
1935

5115
GUACAUGUCUGCUUUAAAC
285
5115
GUACAUGUCUGCUUUAAAC
285
5133
GUUUAAAGCAGACAUGUAC
1936

5133
CCACACAAAGAAAUGGAAA
286
5133
CCACACAAAGAAAUGGAAA
286
5151
UUUCCAUUUCUUUGUGUGG
1937

5151
AUUUCCUCAAGUUGGUGGU
287
5151
AUUUCCUCAAGUUGGUGGU
287
5169
ACCACCAACUUGAGGAAAU
1938

5169
UUUAACUUCAAUUAAAUGG
288
5169
UUUAACUUCAAUUAAAUGG
288
5187
CCAUUUAAUUGAAGUUAAA
1939

5187
GGCUGAUAACAAUUGUUAU
289
5187
GGCUGAUAACAAUUGUUAU
289
5205
AUAACAAUUGUUAUCAGCC
1940

5205
UUUGUCUAGUGUUUUAUUA
290
5205
UUUGUCUAGUGUUUUAUUA
290
5223
UAAUAAAACACUAGACAAA
1941

5223
AGCACUUCAACAGCUUGAA
291
5223
AGCACUUCAACAGCUUGAA
291
5241
UUCAAGCUGUUGAAGUGCU
1942

5241
AGUCAAAUUCAAUGCACCA
292
5241
AGUCAAAUUCAAUGCACCA
292
5259
UGGUGCAUUGAAUUUGACU
1943

5259
AGCACUUCAAGAGGCUUAU
293
5259
AGCACUUCAAGAGGCUUAU
293
5277
AUAAGCCUCUUGAAGUGCU
1944

5277
UUAUAGAGCCCGUGCUGGU
294
5277
UUAUAGAGCCCGUGCUGGU
294
5295
ACCAGCACGGGCUCUAUAA
1945

5295
UGAUGCUGCUAACUUUUGU
295
5295
UGAUGCUGCUAACUUUUGU
295
5313
ACAAAAGUUAGCAGCAUCA
1946

5313
UGCACUCAUACUCGCUUAC
296
5313
UGCACUCAUACUCGCUUAC
296
5331
GUAAGCGAGUAUGAGUGCA
1947

5331
CAGUAAUAAAACUGUUGGC
297
5331
CAGUAAUAAAACUGUUGGC
297
5349
GCCAACAGUUUUAUUACUG
1948

5349
CGAGCUUGGUGAUGUCAGA
298
5349
CGAGCUUGGUGAUGUCAGA
298
5367
UCUGACAUCACCAAGCUCG
1949

5367
AGAAACUAUGACCCAUCUU
299
5367
AGAAACUAUGACCCAUCUU
299
5385
AAGAUGGGUCAUAGUUUCU
1950

5385
UCUACAGCAUGCUAAUUUG
300
5385
UCUACAGCAUGCUAAUUUG
300
5403
CAAAUUAGCAUGCUGUAGA
1951

5403
GGAAUCUGCAAAGCGAGUU
301
5403
GGAAUCUGCAAAGCGAGUU
301
5421
AACUCGCUUUGCAGAUUCC
1952

5421
UCUUAAUGUGGUGUGUAAA
302
5421
UCUUAAUGUGGUGUGUAAA
302
5439
UUUACACACCACAUUAAGA
1953

5439
ACAUUGUGGUCAGAAAACU
303
5439
ACAUUGUGGUCAGAAAACU
303
5457
AGUUUUCUGACCACAAUGU
1954

5457
UACUACCUUAACGGGUGUA
304
5457
UACUACCUUAACGGGUGUA
304
5475
UACACCCGUUAAGGUAGUA
1955

5475
AGAAGCUGUGAUGUAUAUG
305
5475
AGAAGCUGUGAUGUAUAUG
305
5493
CAUAUACAUCACAGCUUCU
1956

5493
GGGUACUCUAUCUUAUGAU
306
5493
GGGUACUCUAUCUUAUGAU
306
5511
AUCAUAAGAUAGAGUACCC
1957

5511
UAAUCUUAAGACAGGUGUU
307
5511
UAAUCUUAAGACAGGUGUU
307
5529
AACACCUGUCUUAAGAUUA
1958

5529
UUCCAUUCCAUGUGUGUGU
308
5529
UUCCAUUCCAUGUGUGUGU
308
5547
ACACACACAUGGAAUGGAA
1959

5547
UGGUCGUGAUGCUACACAA
309
5547
UGGUCGUGAUGCUACACAA
309
5565
UUGUGUAGCAUCACGACCA
1960

5565
AUAUCUAGUACAACAAGAG
310
5565
AUAUCUAGUACAACAAGAG
310
5583
CUCUUGUUGUACUAGAUAU
1961

5583
GUCUUCUUUUGUUAUGAUG
311
5583
GUCUUCUUUUGUUAUGAUG
311
5601
CAUCAUAACAAAAGAAGAC
1962

5601
GUCUGCACCACCUGCUGAG
312
5601
GUCUGCACCACCUGCUGAG
312
5619
CUCAGCAGGUGGUGCAGAC
1963

5619
GUAUAAAUUACAGCAAGGU
313
5619
GUAUAAAUUACAGCAAGGU
313
5637
ACCUUGCUGUAAUUUAUAC
1964

5637
UACAUUCUUAUGUGCGAAU
314
5637
UACAUUCUUAUGUGCGAAU
314
5655
AUUCGCACAUAAGAAUGUA
1965

5655
UGAGUACACUGGUAACUAU
315
5655
UGAGUACACUGGUAACUAU
315
5673
AUAGUUACCAGUGUACUCA
1966

5673
UCAGUGUGGUCAUUACACU
316
5673
UCAGUGUGGUCAUUACACU
316
5691
AGUGUAAUGACCACACUGA
1967

5691
UCAUAUAACUGCUAAGGAG
317
5691
UCAUAUAACUGCUAAGGAG
317
5709
CUCCUUAGCAGUUAUAUGA
1968

5709
GACCCUCUAUCGUAUUGAC
318
5709
GACCCUCUAUCGUAUUGAC
318
5727
GUCAAUACGAUAGAGGGUC
1969

5727
CGGAGCUCACCUUACAAAG
319
5727
CGGAGCUCACCUUACAAAG
319
5745
CUUUGUAAGGUGAGCUCCG
1970

5745
GAUGUCAGAGUACAAAGGA
320
5745
GAUGUCAGAGUACAAAGGA
320
5763
UCCUUUGUACUCUGACAUC
1971

5763
ACCAGUGACUGAUGUUUUC
321
5763
ACCAGUGACUGAUGUUUUC
321
5781
GAAAACAUCAGUCACUGGU
1972

5781
CUACAAGGAAACAUCUUAC
322
5781
CUACAAGGAAACAUCUUAC
322
5799
GUAAGAUGUUUCCUUGUAG
1973

5799
CACUACAACCAUCAAGCCU
323
5799
CACUACAACCAUCAAGCCU
323
5817
AGGCUUGAUGGUUGUAGUG
1974

5817
UGUGUCGUAUAAACUCGAU
324
5817
UGUGUCGUAUAAACUCGAU
324
5835
AUCGAGUUUAUACGACACA
1975

5835
UGGAGUUACUUACACAGAG
325
5835
UGGAGUUACUUACACAGAG
325
5853
CUCUGUGUAAGUAACUCCA
1976

5853
GAUUGAACCAAAAUUGGAU
326
5853
GAUUGAACCAAAAUUGGAU
326
5871
AUCCAAUUUUGGUUCAAUC
1977

5871
UGGGUAUUAUAAAAAGGAU
327
5871
UGGGUAUUAUAAAAAGGAU
327
5889
AUCCUUUUUAUAAUACCCA
1978

5889
UAAUGCUUACUAUACAGAG
328
5889
UAAUGCUUACUAUACAGAG
328
5907
CUCUGUAUAGUAAGCAUUA
1979

5907
GCAGCCUAUAGACCUUGUA
329
5907
GCAGCCUAUAGACCUUGUA
329
5925
UACAAGGUCUAUAGGCUGC
1980

5925
ACCAACUCAACCAUUACCA
330
5925
ACCAACUCAACCAUUACCA
330
5943
UGGUAAUGGUUGAGUUGGU
1981

5943
AAAUGCGAGUUUUGAUAAU
331
5943
AAAUGCGAGUUUUGAUAAU
331
5961
AUUAUCAAAACUCGCAUUU
1982

5961
UUUCAAACUCACAUGUUCU
332
5961
UUUCAAACUCACAUGUUCU
332
5979
AGAACAUGUGAGUUUGAAA
1983

5979
UAACACAAAAUUUGCUGAU
333
5979
UAACACAAAAUUUGCUGAU
333
5997
AUCAGCAAAUUUUGUGUUA
1984

5997
UGAUUUAAAUCAAAUGACA
334
5997
UGAUUUAAAUCAAAUGACA
334
6015
UGUCAUUUGAUUUAAAUCA
1985

6015
AGGCUUCACAAAGCCAGCU
335
6015
AGGCUUCACAAAGCCAGCU
335
6033
AGCUGGCUUUGUGAAGCCU
1986

6033
UUCACGAGAGCUAUCUGUC
336
6033
UUCACGAGAGCUAUCUGUC
336
6051
GACAGAUAGCUCUCGUGAA
1987

6051
CACAUUCUUCCCAGACUUG
337
6051
CACAUUCUUCCCAGACUUG
337
6069
CAAGUCUGGGAAGAAUGUG
1988

6069
GAAUGGCGAUGUAGUGGCU
338
6069
GAAUGGCGAUGUAGUGGCU
338
6087
AGCCACUACAUCGCCAUUC
1989

6087
UAUUGACUAUAGACACUAU
339
6087
UAUUGACUAUAGACACUAU
339
6105
AUAGUGUCUAUAGUCAAUA
1990

6105
UUCAGCGAGUUUCAAGAAA
340
6105
UUCAGCGAGUUUCAAGAAA
340
6123
UUUCUUGAAACUCGCUGAA
1991

6123
AGGUGCUAAAUUACUGCAU
341
6123
AGGUGCUAAAUUACUGCAU
341
6141
AUGCAGUAAUUUAGCACCU
1992

6141
UAAGCCAAUUGUUUGGCAC
342
6141
UAAGCCAAUUGUUUGGCAC
342
6159
GUGCCAAACAAUUGGCUUA
1993

6159
CAUUAACCAGGCUACAACC
343
6159
CAUUAACCAGGCUACAACC
343
6177
GGUUGUAGCCUGGUUAAUG
1994

6177
CAAGACAACGUUCAAACCA
344
6177
CAAGACAACGUUCAAACCA
344
6195
UGGUUUGAACGUUGUCUUG
1995

6195
AAACACUUGGUGUUUACGU
345
6195
AAACACUUGGUGUUUACGU
345
6213
ACGUAAACACCAAGUGUUU
1996

6213
UUGUCUUUGGAGUACAAAG
346
6213
UUGUCUUUGGAGUACAAAG
346
6231
CUUUGUACUCCAAAGACAA
1997

6231
GCCAGUAGAUACUUCAAAU
347
6231
GCCAGUAGAUACUUCAAAU
347
6249
AUUUGAAGUAUCUACUGGC
1998

6249
UUCAUUUGAAGUUCUGGCA
348
6249
UUCAUUUGAAGUUCUGGCA
348
6267
UGCCAGAACUUCAAAUGAA
1999

6267
AGUAGAAGACACACAAGGA
349
6267
AGUAGAAGACACACAAGGA
349
6285
UCCUUGUGUGUCUUCUACU
2000

6285
AAUGGACAAUCUUGCUUGU
350
6285
AAUGGACAAUCUUGCUUGU
350
6303
ACAAGCAAGAUUGUCCAUU
2001

6303
UGAAAGUCAACAACCCACC
351
6303
UGAAAGUCAACAACCCACC
351
6321
GGUGGGUUGUUGACUUUCA
2002

6321
CUCUGAAGAAGUAGUGGAA
352
6321
CUCUGAAGAAGUAGUGGAA
352
6339
UUCCACUACUUCUUCAGAG
2003

6339
AAAUCCUACCAUACAGAAG
353
6339
AAAUCCUACCAUACAGAAG
353
6357
CUUCUGUAUGGUAGGAUUU
2004

6357
GGAAGUCAUAGAGUGUGAC
354
6357
GGAAGUCAUAGAGUGUGAC
354
6375
GUCACACUCUAUGACUUCC
2005

6375
CGUGAAAACUACCGAAGUU
355
6375
CGUGAAAACUACCGAAGUU
355
6393
AACUUCGGUAGUUUUCACG
2006

6393
UGUAGGCAAUGUCAUACUU
356
6393
UGUAGGCAAUGUCAUACUU
356
6411
AAGUAUGACAUUGCCUACA
2007

6411
UAAACCAUCAGAUGAAGGU
357
6411
UAAACCAUCAGAUGAAGGU
357
6429
ACCUUCAUCUGAUGGUUUA
2008

6429
UGUUAAAGUAACACAAGAG
358
6429
UGUUAAAGUAACACAAGAG
358
6447
CUCUUGUGUUACUUUAACA
2009

6447
GUUAGGUCAUGAGGAUCUU
359
6447
GUUAGGUCAUGAGGAUCUU
359
6465
AAGAUCCUCAUGACCUAAC
2010

6465
UAUGGCUGCUUAUGUGGAA
360
6465
UAUGGCUGCUUAUGUGGAA
360
6483
UUCCACAUAAGCAGCCAUA
2011

6483
AAACACAAGCAUUACCAUU
361
6483
AAACACAAGCAUUACCAUU
361
6501
AAUGGUAAUGCUUGUGUUU
2012

6501
UAAGAAACCUAAUGAGCUU
362
6501
UAAGAAACCUAAUGAGCUU
362
6519
AAGCUCAUUAGGUUUCUUA
2013

6519
UUCACUAGCCUUAGGUUUA
363
6519
UUCACUAGCCUUAGGUUUA
363
6537
UAAACCUAAGGCUAGUGAA
2014

6537
AAAAACAAUUGCCACUCAU
364
6537
AAAAACAAUUGCCACUCAU
364
6555
AUGAGUGGCAAUUGUUUUU
2015

6555
UGGUAUUGCUGCAAUUAAU
365
6555
UGGUAUUGCUGCAAUUAAU
365
6573
AUUAAUUGCAGCAAUACCA
2016

6573
UAGUGUUCCUUGGAGUAAA
366
6573
UAGUGUUCCUUGGAGUAAA
366
6591
UUUACUCCAAGGAACACUA
2017

6591
AAUUUUGGCUUAUGUCAAA
367
6591
AAUUUUGGCUUAUGUCAAA
367
6609
UUUGACAUAAGCCAAAAUU
2018

6609
ACCAUUCUUAGGACAAGCA
368
6609
ACCAUUCUUAGGACAAGCA
368
6627
UGCUUGUCCUAAGAAUGGU
2019

6627
AGCAAUUACAACAUCAAAU
369
6627
AGCAAUUACAACAUCAAAU
369
6645
AUUUGAUGUUGUAAUUGCU
2020

6645
UUGCGCUAAGAGAUUAGCA
370
6645
UUGCGCUAAGAGAUUAGCA
370
6663
UGCUAAUCUCUUAGCGCAA
2021

6663
ACAACGUGUGUUUAACAAU
371
6663
ACAACGUGUGUUUAACAAU
371
6681
AUUGUUAAACACACGUUGU
2022

6681
UUAUAUGCCUUAUGUGUUU
372
6681
UUAUAUGCCUUAUGUGUUU
372
6699
AAACACAUAAGGCAUAUAA
2023

6699
UACAUUAUUGUUCCAAUUG
373
6699
UACAUUAUUGUUCCAAUUG
373
6717
CAAUUGGAACAAUAAUGUA
2024

6717
GUGUACUUUUACUAAAAGU
374
6717
GUGUACUUUUACUAAAAGU
374
6735
ACUUUUAGUAAAAGUACAC
2025

6735
UACCAAUUCUAGAAUUAGA
375
6735
UACCAAUUCUAGAAUUAGA
375
6753
UCUAAUUCUAGAAUUGGUA
2026

6753
AGCUUCACUACCUACAACU
376
6753
AGCUUCACUACCUACAACU
376
6771
AGUUGUAGGUAGUGAAGCU
2027

6771
UAUUGCUAAAAAUAGUGUU
377
6771
UAUUGCUAAAAAUAGUGUU
377
6789
AACACUAUUUUUAGCAAUA
2028

6789
UAAGAGUGUUGCUAAAUUA
378
6789
UAAGAGUGUUGCUAAAUUA
378
6807
UAAUUUAGCAACACUCUUA
2029

6807
AUGUUUGGAUGCCGGCAUU
379
6807
AUGUUUGGAUGCCGGCAUU
379
6825
AAUGCCGGCAUCCAAACAU
2030

6825
UAAUUAUGUGAAGUCACCC
380
6825
UAAUUAUGUGAAGUCACCC
380
6843
GGGUGACUUCACAUAAUUA
2031

6843
CAAAUUUUCUAAAUUGUUC
381
6843
CAAAUUUUCUAAAUUGUUC
381
6861
GAACAAUUUAGAAAAUUUG
2032

6861
CACAAUCGCUAUGUGGCUA
382
6861
CACAAUCGCUAUGUGGCUA
382
6879
UAGCCACAUAGCGAUUGUG
2033

6879
AUUGUUGUUAAGUAUUUGC
383
6879
AUUGUUGUUAAGUAUUUGC
383
6897
GCAAAUACUUAACAACAAU
2034

6897
CUUAGGUUCUCUAAUCUGU
384
6897
CUUAGGUUCUCUAAUCUGU
384
6915
ACAGAUUAGAGAACCUAAG
2035

6915
UGUAACUGCUGCUUUUGGU
385
6915
UGUAACUGCUGCUUUUGGU
385
6933
ACCAAAAGCAGCAGUUACA
2036

6933
UGUACUCUUAUCUAAUUUU
386
6933
UGUACUCUUAUCUAAUUUU
386
6951
AAAAUUAGAUAAGAGUACA
2037

6951
UGGUGCUCCUUCUUAUUGU
387
6951
UGGUGCUCCUUCUUAUUGU
387
6969
ACAAUAAGAAGGAGCACCA
2038

6969
UAAUGGCGUUAGAGAAUUG
388
6969
UAAUGGCGUUAGAGAAUUG
388
6987
CAAUUCUCUAACGCCAUUA
2039

6987
GUAUCUUAAUUCGUCUAAC
389
6987
GUAUCUUAAUUCGUCUAAC
389
7005
GUUAGACGAAUUAAGAUAC
2040

7005
CGUUACUACUAUGGAUUUC
390
7005
CGUUACUACUAUGGAUUUC
390
7023
GAAAUCCAUAGUAGUAACG
2041

7023
CUGUGAAGGUUCUUUUCCU
391
7023
CUGUGAAGGUUCUUUUCCU
391
7041
AGGAAAAGAACCUUCACAG
2042

7041
UUGCAGCAUUUGUUUAAGU
392
7041
UUGCAGCAUUUGUUUAAGU
392
7059
ACUUAAACAAAUGCUGCAA
2043

7059
UGGAUUAGACUCCCUUGAU
393
7059
UGGAUUAGACUCCCUUGAU
393
7077
AUCAAGGGAGUCUAAUCCA
2044

7077
UUCUUAUCCAGCUCUUGAA
394
7077
UUCUUAUCCAGCUCUUGAA
394
7095
UUCAAGAGCUGGAUAAGAA
2045

7095
AACCAUUCAGGUGACGAUU
395
7095
AACCAUUCAGGUGACGAUU
395
7113
AAUCGUCACCUGAAUGGUU
2046

7113
UUCAUCGUACAAGCUAGAC
396
7113
UUCAUCGUACAAGCUAGAC
396
7131
GUCUAGCUUGUACGAUGAA
2047

7131
CUUGACAAUUUUAGGUCUG
397
7131
CUUGACAAUUUUAGGUCUG
397
7149
CAGACCUAAAAUUGUCAAG
2048

7149
GGCCGCUGAGUGGGUUUUG
398
7149
GGCCGCUGAGUGGGUUUUG
398
7167
CAAAACCCACUCAGCGGCC
2049

7167
GGCAUAUAUGUUGUUCACA
399
7167
GGCAUAUAUGUUGUUCACA
399
7185
UGUGAACAACAUAUAUGCC
2050

7185
AAAAUUCUUUUAUUUAUUA
400
7185
AAAAUUCUUUUAUUUAUUA
400
7203
UAAUAAAUAAAAGAAUUUU
2051

7203
AGGUCUUUCAGCUAUAAUG
401
7203
AGGUCUUUCAGCUAUAAUG
401
7221
CAUUAUAGCUGAAAGACCU
2052

7221
GCAGGUGUUCUUUGGCUAU
402
7221
GCAGGUGUUCUUUGGCUAU
402
7239
AUAGCCAAAGAACACCUGC
2053

7239
UUUUGCUAGUCAUUUCAUC
403
7239
UUUUGCUAGUCAUUUCAUC
403
7257
GAUGAAAUGACUAGCAAAA
2054

7257
CAGCAAUUCUUGGCUCAUG
404
7257
CAGCAAUUCUUGGCUCAUG
404
7275
CAUGAGCCAAGAAUUGCUG
2055

7275
GUGGUUUAUCAUUAGUAUU
405
7275
GUGGUUUAUCAUUAGUAUU
405
7293
AAUACUAAUGAUAAACCAC
2056

7293
UGUACAAAUGGCACCCGUU
406
7293
UGUACAAAUGGCACCCGUU
406
7311
AACGGGUGCCAUUUGUACA
2057

7311
UUCUGCAAUGGUUAGGAUG
407
7311
UUCUGCAAUGGUUAGGAUG
407
7329
CAUCCUAACCAUUGCAGAA
2058

7329
GUACAUCUUCUUUGCUUCU
408
7329
GUACAUCUUCUUUGCUUCU
408
7347
AGAAGCAAAGAAGAUGUAC
2059

7347
UUUCUACUACAUAUGGAAG
409
7347
UUUCUACUACAUAUGGAAG
409
7365
CUUCCAUAUGUAGUAGAAA
2060

7365
GAGCUAUGUUCAUAUCAUG
410
7365
GAGCUAUGUUCAUAUCAUG
410
7383
CAUGAUAUGAACAUAGCUC
2061

7383
GGAUGGUUGCACCUCUUCG
411
7383
GGAUGGUUGCACCUCUUCG
411
7401
CGAAGAGGUGCAACCAUCC
2062

7401
GACUUGCAUGAUGUGCUAU
412
7401
GACUUGCAUGAUGUGCUAU
412
7419
AUAGCACAUCAUGCAAGUC
2063

7419
UAAGCGCAAUCGUGCCACA
413
7419
UAAGCGCAAUCGUGCCACA
413
7437
UGUGGCACGAUUGCGCUUA
2064

7437
ACGCGUUGAGUGUACAACU
414
7437
ACGCGUUGAGUGUACAACU
414
7455
AGUUGUACACUCAACGCGU
2065

7455
UAUUGUUAAUGGCAUGAAG
415
7455
UAUUGUUAAUGGCAUGAAG
415
7473
CUUCAUGCCAUUAACAAUA
2066

7473
GAGAUCUUUCUAUGUCUAU
416
7473
GAGAUCUUUCUAUGUCUAU
416
7491
AUAGACAUAGAAAGAUCUC
2067

7491
UGCAAAUGGAGGCCGUGGC
417
7491
UGCAAAUGGAGGCCGUGGC
417
7509
GCCACGGCCUCCAUUUGCA
2068

7509
CUUCUGCAAGACUCACAAU
418
7509
CUUCUGCAAGACUCACAAU
418
7527
AUUGUGAGUCUUGCAGAAG
2069

7527
UUGGAAUUGUCUCAAUUGU
419
7527
UUGGAAUUGUCUCAAUUGU
419
7545
ACAAUUGAGACAAUUCCAA
2070

7545
UGACACAUUUUGCACUGGU
420
7545
UGACACAUUUUGCACUGGU
420
7563
ACCAGUGCAAAAUGUGUCA
2071

7563
UAGUACAUUCAUUAGUGAU
421
7563
UAGUACAUUCAUUAGUGAU
421
7581
AUCACUAAUGAAUGUACUA
2072

7581
UGAAGUUGCUCGUGAUUUG
422
7581
UGAAGUUGCUCGUGAUUUG
422
7599
CAAAUCACGAGCAACUUCA
2073

7599
GUCACUCCAGUUUAAAAGA
423
7599
GUCACUCCAGUUUAAAAGA
423
7617
UCUUUUAAACUGGAGUGAC
2074

7617
ACCAAUCAACCCUACUGAC
424
7617
ACCAAUCAACCCUACUGAC
424
7635
GUCAGUAGGGUUGAUUGGU
2075

7635
CCAGUCAUCGUAUAUUGUU
425
7635
CCAGUCAUCGUAUAUUGUU
425
7653
AACAAUAUACGAUGACUGG
2076

7653
UGAUAGUGUUGCUGUGAAA
426
7653
UGAUAGUGUUGCUGUGAAA
426
7671
UUUCACAGCAACACUAUCA
2077

7671
AAAUGGCGCGCUUCACCUC
427
7671
AAAUGGCGCGCUUCACCUC
427
7689
GAGGUGAAGCGCGCCAUUU
2078

7689
CUACUUUGACAAGGCUGGU
428
7689
CUACUUUGACAAGGCUGGU
428
7707
ACCAGCCUUGUCAAAGUAG
2079

7707
UCAAAAGACCUAUGAGAGA
429
7707
UCAAAAGACCUAUGAGAGA
429
7725
UCUCUCAUAGGUCUUUUGA
2080

7725
ACAUCCGCUCUCCCAUUUU
430
7725
ACAUCCGCUCUCCCAUUUU
430
7743
AAAAUGGGAGAGCGGAUGU
2081

7743
UGUCAAUUUAGACAAUUUG
431
7743
UGUCAAUUUAGACAAUUUG
431
7761
CAAAUUGUCUAAAUUGACA
2082

7761
GAGAGCUAACAACACUAAA
432
7761
GAGAGCUAACAACACUAAA
432
7779
UUUAGUGUUGUUAGCUCUC
2083

7779
AGGUUCACUGCCUAUUAAU
433
7779
AGGUUCACUGCCUAUUAAU
433
7797
AUUAAUAGGCAGUGAACCU
2084

7797
UGUCAUAGUUUUUGAUGGC
434
7797
UGUCAUAGUUUUUGAUGGC
434
7815
GCCAUCAAAAACUAUGACA
2085

7815
CAAGUCCAAAUGCGACGAG
435
7815
CAAGUCCAAAUGCGACGAG
435
7833
CUCGUCGCAUUUGGACUUG
2086

7833
GUCUGCUUCUAAGUCUGCU
436
7833
GUCUGCUUCUAAGUCUGCU
436
7851
AGCAGACUUAGAAGCAGAC
2087

7851
UUCUGUGUACUACAGUCAG
437
7851
UUCUGUGUACUACAGUCAG
437
7869
CUGACUGUAGUACACAGAA
2088

7869
GCUGAUGUGCCAACCUAUU
438
7869
GCUGAUGUGCCAACCUAUU
438
7887
AAUAGGUUGGCACAUCAGC
2089

7887
UCUGUUGCUUGACCAAGCU
439
7887
UCUGUUGCUUGACCAAGCU
439
7905
AGCUUGGUCAAGCAACAGA
2090

7905
UCUUGUAUCAGACGUUGGA
440
7905
UCUUGUAUCAGACGUUGGA
440
7923
UCCAACGUCUGAUACAAGA
2091

7923
AGAUAGUACUGAAGUUUCC
441
7923
AGAUAGUACUGAAGUUUCC
441
7941
GGAAACUUCAGUACUAUCU
2092

7941
CGUUAAGAUGUUUGAUGCU
442
7941
CGUUAAGAUGUUUGAUGCU
442
7959
AGCAUCAAACAUCUUAACG
2093

7959
UUAUGUCGACACCUUUUCA
443
7959
UUAUGUCGACACCUUUUCA
443
7977
UGAAAAGGUGUCGACAUAA
2094

7977
AGCAACUUUUAGUGUUCCU
444
7977
AGCAACUUUUAGUGUUCCU
444
7995
AGGAACACUAAAAGUUGCU
2095

7995
UAUGGAAAAACUUAAGGCA
445
7995
UAUGGAAAAACUUAAGGCA
445
8013
UGCCUUAAGUUUUUCCAUA
2096

8013
ACUUGUUGCUACAGCUCAC
446
8013
ACUUGUUGCUACAGCUCAC
446
8031
GUGAGCUGUAGCAACAAGU
2097

8031
CAGCGAGUUAGCAAAGGGU
447
8031
CAGCGAGUUAGCAAAGGGU
447
8049
ACCCUUUGCUAACUCGCUG
2098

8049
UGUAGCUUUAGAUGGUGUC
448
8049
UGUAGCUUUAGAUGGUGUC
448
8067
GACACCAUCUAAAGCUACA
2099

8067
CCUUUCUACAUUCGUGUCA
449
8067
CCUUUCUACAUUCGUGUCA
449
8085
UGACACGAAUGUAGAAAGG
2100

8085
AGCUGCCCGACAAGGUGUU
450
8085
AGCUGCCCGACAAGGUGUU
450
8103
AACACCUUGUCGGGCAGCU
2101

8103
UGUUGAUACCGAUGUUGAC
451
8103
UGUUGAUACCGAUGUUGAC
451
8121
GUCAACAUCGGUAUCAACA
2102

8121
CACAAAGGAUGUUAUUGAA
452
8121
CACAAAGGAUGUUAUUGAA
452
8139
UUCAAUAACAUCCUUUGUG
2103

8139
AUGUCUCAAACUUUCACAU
453
8139
AUGUCUCAAACUUUCACAU
453
8157
AUGUGAAAGUUUGAGACAU
2104

8157
UCACUCUGACUUAGAAGUG
454
8157
UCACUCUGACUUAGAAGUG
454
8175
CACUUCUAAGUCAGAGUGA
2105

8175
GACAGGUGACAGUUGUAAC
455
8175
GACAGGUGACAGUUGUAAC
455
8193
GUUACAACUGUCACCUGUC
2106

8193
CAAUUUCAUGCUCACCUAU
456
8193
CAAUUUCAUGCUCACCUAU
456
8211
AUAGGUGAGCAUGAAAUUG
2107

8211
UAAUAAGGUUGAAAACAUG
457
8211
UAAUAAGGUUGAAAACAUG
457
8229
CAUGUUUUCAACCUUAUUA
2108

8229
GACGCCCAGAGAUCUUGGC
458
8229
GACGCCCAGAGAUCUUGGC
458
8247
GCCAAGAUCUCUGGGCGUC
2109

8247
CGCAUGUAUUGACUGUAAU
459
8247
CGCAUGUAUUGACUGUAAU
459
8265
AUUACAGUCAAUACAUGCG
2110

8265
UGCAAGGCAUAUCAAUGCC
460
8265
UGCAAGGCAUAUCAAUGCC
460
8283
GGCAUUGAUAUGCCUUGCA
2111

8283
CCAAGUAGCAAAAAGUCAC
461
8283
CCAAGUAGCAAAAAGUCAC
461
8301
GUGACUUUUUGCUACUUGG
2112

8301
CAAUGUUUCACUCAUCUGG
462
8301
CAAUGUUUCACUCAUCUGG
462
8319
CCAGAUGAGUGAAACAUUG
2113

8319
GAAUGUAAAAGACUACAUG
463
8319
GAAUGUAAAAGACUACAUG
463
8337
CAUGUAGUCUUUUACAUUC
2114

8337
GUCUUUAUCUGAACAGCUG
464
8337
GUCUUUAUCUGAACAGCUG
464
8355
CAGCUGUUCAGAUAAAGAC
2115

8355
GCGUAAACAAAUUCGUAGU
465
8355
GCGUAAACAAAUUCGUAGU
465
8373
ACUACGAAUUUGUUUACGC
2116

8373
UGCUGCCAAGAAGAACAAC
466
8373
UGCUGCCAAGAAGAACAAC
466
8391
GUUGUUCUUCUUGGCAGCA
2117

8391
CAUACCUUUUAGACUAACU
467
8391
CAUACCUUUUAGACUAACU
467
8409
AGUUAGUCUAAAAGGUAUG
2118

8409
UUGUGCUACAACUAGACAG
468
8409
UUGUGCUACAACUAGACAG
468
8427
CUGUCUAGUUGUAGCACAA
2119

8427
GGUUGUCAAUGUCAUAACU
469
8427
GGUUGUCAAUGUCAUAACU
469
8445
AGUUAUGACAUUGACAACC
2120

8445
UACUAAAAUCUCACUCAAG
470
8445
UACUAAAAUCUCACUCAAG
470
8463
CUUGAGUGAGAUUUUAGUA
2121

8463
GGGUGGUAAGAUUGUUAGU
471
8463
GGGUGGUAAGAUUGUUAGU
471
8481
ACUAACAAUCUUACCACCC
2122

8481
UACUUGUUUUAAACUUAUG
472
8481
UACUUGUUUUAAACUUAUG
472
8499
CAUAAGUUUAAAACAAGUA
2123

8499
GCUUAAGGCCACAUUAUUG
473
8499
GCUUAAGGCCACAUUAUUG
473
8517
CAAUAAUGUGGCCUUAAGC
2124

8517
GUGCGUUCUUGCUGCAUUG
474
8517
GUGCGUUCUUGCUGCAUUG
474
8535
CAAUGCAGCAAGAACGCAC
2125

8535
GGUUUGUUAUAUCGUUAUG
475
8535
GGUUUGUUAUAUCGUUAUG
475
8553
CAUAACGAUAUAACAAACC
2126

8553
GCCAGUACAUACAUUGUCA
476
8553
GCCAGUACAUACAUUGUCA
476
8571
UGACAAUGUAUGUACUGGC
2127

8571
AAUCCAUGAUGGUUACACA
477
8571
AAUCCAUGAUGGUUACACA
477
8589
UGUGUAACCAUCAUGGAUU
2128

8589
AAAUGAAAUCAUUGGUUAC
478
8589
AAAUGAAAUCAUUGGUUAC
478
8607
GUAACCAAUGAUUUCAUUU
2129

8607
CAAAGCCAUUCAGGAUGGU
479
8607
CAAAGCCAUUCAGGAUGGU
479
8625
ACCAUCCUGAAUGGCUUUG
2130

8625
UGUCACUCGUGACAUCAUU
480
8625
UGUCACUCGUGACAUCAUU
480
8643
AAUGAUGUCACGAGUGACA
2131

8643
UUCUACUGAUGAUUGUUUU
481
8643
UUCUACUGAUGAUUGUUUU
481
8661
AAAACAAUCAUCAGUAGAA
2132

8661
UGCAAAUAAACAUGCUGGU
482
8661
UGCAAAUAAACAUGCUGGU
482
8679
ACCAGCAUGUUUAUUUGCA
2133

8679
UUUUGACGCAUGGUUUAGC
483
8679
UUUUGACGCAUGGUUUAGC
483
8697
GCUAAACCAUGCGUCAAAA
2134

8697
CCAGCGUGGUGGUUCAUAC
484
8697
CCAGCGUGGUGGUUCAUAC
484
8715
GUAUGAACCACCACGCUGG
2135

8715
CAAAAAUGACAAAAGCUGC
485
8715
CAAAAAUGACAAAAGCUGC
485
8733
GCAGCUUUUGUCAUUUUUG
2136

8733
CCCUGUAGUAGCUGCUAUC
486
8733
CCCUGUAGUAGCUGCUAUC
486
8751
GAUAGCAGCUACUACAGGG
2137

8751
CAUUACAAGAGAGAUUGGU
487
8751
CAUUACAAGAGAGAUUGGU
487
8769
ACCAAUCUCUCUUGUAAUG
2138

8769
UUUCAUAGUGCCUGGCUUA
488
8769
UUUCAUAGUGCCUGGCUUA
488
8787
UAAGCCAGGCACUAUGAAA
2139

8787
ACCGGGUACUGUGCUGAGA
489
8787
ACCGGGUACUGUGCUGAGA
489
8805
UCUCAGCACAGUACCCGGU
2140

8805
AGCAAUCAAUGGUGACUUC
490
8805
AGCAAUCAAUGGUGACUUC
490
8823
GAAGUCACCAUUGAUUGCU
2141

8823
CUUGCAUUUUCUACCUCGU
491
8823
CUUGCAUUUUCUACCUCGU
491
8841
ACGAGGUAGAAAAUGCAAG
2142

8841
UGUUUUUAGUGCUGUUGGC
492
8841
UGUUUUUAGUGCUGUUGGC
492
8859
GCCAACAGCACUAAAAACA
2143

8859
CAACAUUUGCUACACACCU
493
8859
CAACAUUUGCUACACACCU
493
8877
AGGUGUGUAGCAAAUGUUG
2144

8877
UUCCAAACUCAUUGAGUAU
494
8877
UUCCAAACUCAUUGAGUAU
494
8895
AUACUCAAUGAGUUUGGAA
2145

8895
UAGUGAUUUUGCUACCUCU
495
8895
UAGUGAUUUUGCUACCUCU
495
8913
AGAGGUAGCAAAAUCACUA
2146

8913
UGCUUGCGUUCUUGCUGCU
496
8913
UGCUUGCGUUCUUGCUGCU
496
8931
AGCAGCAAGAACGCAAGCA
2147

8931
UGAGUGUACAAUUUUUAAG
497
8931
UGAGUGUACAAUUUUUAAG
497
8949
CUUAAAAAUUGUACACUCA
2148

8949
GGAUGCUAUGGGCAAACCU
498
8949
GGAUGCUAUGGGCAAACCU
498
8967
AGGUUUGCCCAUAGCAUCC
2149

8967
UGUGCCAUAUUGUUAUGAC
499
8967
UGUGCCAUAUUGUUAUGAC
499
8985
GUCAUAACAAUAUGGCACA
2150

8985
CACUAAUUUGCUAGAGGGU
500
8985
CACUAAUUUGCUAGAGGGU
500
9003
ACCCUCUAGCAAAUUAGUG
2151

9003
UUCUAUUUCUUAUAGUGAG
501
9003
UUCUAUUUCUUAUAGUGAG
501
9021
CUCACUAUAAGAAAUAGAA
2152

9021
GCUUCGUCCAGACACUCGU
502
9021
GCUUCGUCCAGACACUCGU
502
9039
ACGAGUGUCUGGACGAAGC
2153

9039
UUAUGUGCUUAUGGAUGGU
503
9039
UUAUGUGCUUAUGGAUGGU
503
9057
ACCAUCCAUAAGCACAUAA
2154

9057
UUCCAUCAUACAGUUUCCU
504
9057
UUCCAUCAUACAGUUUCCU
504
9075
AGGAAACUGUAUGAUGGAA
2155

9075
UAACACUUACCUGGAGGGU
505
9075
UAACACUUACCUGGAGGGU
505
9093
ACCCUCCAGGUAAGUGUUA
2156

9093
UUCUGUUAGAGUAGUAACA
506
9093
UUCUGUUAGAGUAGUAACA
506
9111
UGUUACUACUCUAACAGAA
2157

9111
AACUUUUGAUGCUGAGUAC
507
9111
AACUUUUGAUGCUGAGUAC
507
9129
GUACUCAGCAUCAAAAGUU
2158

9129
CUGUAGACAUGGUACAUGC
508
9129
CUGUAGACAUGGUACAUGC
508
9147
GCAUGUACCAUGUCUACAG
2159

9147
CGAAAGGUCAGAAGUAGGU
509
9147
CGAAAGGUCAGAAGUAGGU
509
9165
ACCUACUUCUGACCUUUCG
2160

9165
UAUUUGCCUAUCUACCAGU
510
9165
UAUUUGCCUAUCUACCAGU
510
9183
ACUGGUAGAUAGGCAAAUA
2161

9183
UGGUAGAUGGGUUCUUAAU
511
9183
UGGUAGAUGGGUUCUUAAU
511
9201
AUUAAGAACCCAUCUACCA
2162

9201
UAAUGAGCAUUACAGAGCU
512
9201
UAAUGAGCAUUACAGAGCU
512
9219
AGCUCUGUAAUGCUCAUUA
2163

9219
UCUAUCAGGAGUUUUCUGU
513
9219
UCUAUCAGGAGUUUUCUGU
513
9237
ACAGAAAACUCCUGAUAGA
2164

9237
UGGUGUUGAUGCGAUGAAU
514
9237
UGGUGUUGAUGCGAUGAAU
514
9255
AUUCAUCGCAUCAACACCA
2165

9255
UCUCAUAGCUAACAUCUUU
515
9255
UCUCAUAGCUAACAUCUUU
515
9273
AAAGAUGUUAGCUAUGAGA
2166

9273
UACUCCUCUUGUGCAACCU
516
9273
UACUCCUCUUGUGCAACCU
516
9291
AGGUUGCACAAGAGGAGUA
2167

9291
UGUGGGUGCUUUAGAUGUG
517
9291
UGUGGGUGCUUUAGAUGUG
517
9309
CACAUCUAAAGCACCCACA
2168

9309
GUCUGCUUCAGUAGUGGCU
518
9309
GUCUGCUUCAGUAGUGGCU
518
9327
AGCCACUACUGAAGCAGAC
2169

9327
UGGUGGUAUUAUUGCCAUA
519
9327
UGGUGGUAUUAUUGCCAUA
519
9345
UAUGGCAAUAAUACCACCA
2170

9345
AUUGGUGACUUGUGCUGCC
520
9345
AUUGGUGACUUGUGCUGCC
520
9363
GGCAGCACAAGUCACCAAU
2171

9363
CUACUACUUUAUGAAAUUC
521
9363
CUACUACUUUAUGAAAUUC
521
9381
GAAUUUCAUAAAGUAGUAG
2172

9381
CAGACGUGUUUUUGGUGAG
522
9381
CAGACGUGUUUUUGGUGAG
522
9399
CUCACCAAAAACACGUCUG
2173

9399
GUACAACCAUGUUGUUGCU
523
9399
GUACAACCAUGUUGUUGCU
523
9417
AGCAACAACAUGGUUGUAC
2174

9417
UGCUAAUGCACUUUUGUUU
524
9417
UGCUAAUGCACUUUUGUUU
524
9435
AAACAAAAGUGCAUUAGCA
2175

9435
UUUGAUGUCUUUCACUAUA
525
9435
UUUGAUGUCUUUCACUAUA
525
9453
UAUAGUGAAAGACAUCAAA
2176

9453
ACUCUGUCUGGUACCAGCU
526
9453
ACUCUGUCUGGUACCAGCU
526
9471
AGCUGGUACCAGACAGAGU
2177

9471
UUACAGCUUUCUGCCGGGA
527
9471
UUACAGCUUUCUGCCGGGA
527
9489
UCCCGGCAGAAAGCUGUAA
2178

9489
AGUCUACUCAGUCUUUUAC
528
9489
AGUCUACUCAGUCUUUUAC
528
9507
GUAAAAGACUGAGUAGACU
2179

9507
CUUGUACUUGACAUUCUAU
529
9507
CUUGUACUUGACAUUCUAU
529
9525
AUAGAAUGUCAAGUACAAG
2180

9525
UUUCACCAAUGAUGUUUCA
530
9525
UUUCACCAAUGAUGUUUCA
530
9543
UGAAACAUCAUUGGUGAAA
2181

9543
AUUCUUGGCUCACCUUCAA
531
9543
AUUCUUGGCUCACCUUCAA
531
9561
UUGAAGGUGAGCCAAGAAU
2182

9561
AUGGUUUGCCAUGUUUUCU
532
9561
AUGGUUUGCCAUGUUUUCU
532
9579
AGAAAACAUGGCAAACCAU
2183

9579
UCCUAUUGUGCCUUUUUGG
533
9579
UCCUAUUGUGCCUUUUUGG
533
9597
CCAAAAAGGCACAAUAGGA
2184

9597
GAUAACAGCAAUCUAUGUA
534
9597
GAUAACAGCAAUCUAUGUA
534
9615
UACAUAGAUUGCUGUUAUC
2185

9615
AUUCUGUAUUUCUCUGAAG
535
9615
AUUCUGUAUUUCUCUGAAG
535
9633
CUUCAGAGAAAUACAGAAU
2186

9633
GCACUGCCAUUGGUUCUUU
536
9633
GCACUGCCAUUGGUUCUUU
536
9651
AAAGAACCAAUGGCAGUGC
2187

9651
UAACAACUAUCUUAGGAAA
537
9651
UAACAACUAUCUUAGGAAA
537
9669
UUUCCUAAGAUAGUUGUUA
2188

9669
AAGAGUCAUGUUUAAUGGA
538
9669
AAGAGUCAUGUUUAAUGGA
538
9687
UCCAUUAAACAUGACUCUU
2189

9687
AGUUACAUUUAGUACCUUC
539
9687
AGUUACAUUUAGUACCUUC
539
9705
GAAGGUACUAAAUGUAACU
2190

9705
CGAGGAGGCUGCUUUGUGU
540
9705
CGAGGAGGCUGCUUUGUGU
540
9723
ACACAAAGCAGCCUCCUCG
2191

9723
UACCUUUUUGCUCAACAAG
541
9723
UACCUUUUUGCUCAACAAG
541
9741
CUUGUUGAGCAAAAAGGUA
2192

9741
GGAAAUGUACCUAAAAUUG
542
9741
GGAAAUGUACCUAAAAUUG
542
9759
CAAUUUUAGGUACAUUUCC
2193

9759
GCGUAGCGAGACACUGUUG
543
9759
GCGUAGCGAGACACUGUUG
543
9777
CAACAGUGUCUCGCUACGC
2194

9777
GCCACUUACACAGUAUAAC
544
9777
GCCACUUACACAGUAUAAC
544
9795
GUUAUACUGUGUAAGUGGC
2195

9795
CAGGUAUCUUGCUCUAUAU
545
9795
CAGGUAUCUUGCUCUAUAU
545
9813
AUAUAGAGCAAGAUACCUG
2196

9813
UAACAAGUACAAGUAUUUC
546
9813
UAACAAGUACAAGUAUUUC
546
9831
GAAAUACUUGUACUUGUUA
2197

9831
CAGUGGAGCCUUAGAUACU
547
9831
CAGUGGAGCCUUAGAUACU
547
9849
AGUAUCUAAGGCUCCACUG
2198

9849
UACCAGCUAUCGUGAAGCA
548
9849
UACCAGCUAUCGUGAAGCA
548
9867
UGCUUCACGAUAGCUGGUA
2199

9867
AGCUUGCUGCCACUUAGCA
549
9867
AGCUUGCUGCCACUUAGCA
549
9885
UGCUAAGUGGCAGCAAGCU
2200

9885
AAAGGCUCUAAAUGACUUU
550
9885
AAAGGCUCUAAAUGACUUU
550
9903
AAAGUCAUUUAGAGCCUUU
2201

9903
UAGCAACUCAGGUGCUGAU
551
9903
UAGCAACUCAGGUGCUGAU
551
9921
AUCAGCACCUGAGUUGCUA
2202

9921
UGUUCUCUACCAACCACCA
552
9921
UGUUCUCUACCAACCACCA
552
9939
UGGUGGUUGGUAGAGAACA
2203

9939
ACAGACAUCAAUCACUUCU
553
9939
ACAGACAUCAAUCACUUCU
553
9957
AGAAGUGAUUGAUGUCUGU
2204

9957
UGCUGUUCUGCAGAGUGGU
554
9957
UGCUGUUCUGCAGAGUGGU
554
9975
ACCACUCUGCAGAACAGCA
2205

9975
UUUUAGGAAAAUGGCAUUC
555
9975
UUUUAGGAAAAUGGCAUUC
555
9993
GAAUGCCAUUUUCCUAAAA
2206

9993
CCCGUCAGGCAAAGUUGAA
556
9993
CCCGUCAGGCAAAGUUGAA
556
10011
UUCAACUUUGCCUGACGGG
2207

10011
AGGGUGCAUGGUACAAGUA
557
10011
AGGGUGCAUGGUACAAGUA
557
10029
UACUUGUACCAUGCACCCU
2208

10029
AACCUGUGGAACUACAACU
558
10029
AACCUGUGGAACUACAACU
558
10047
AGUUGUAGUUCCACAGGUU
2209

10047
UCUUAAUGGAUUGUGGUUG
559
10047
UCUUAAUGGAUUGUGGUUG
559
10065
CAACCACAAUCCAUUAAGA
2210

10065
GGAUGACACAGUAUACUGU
560
10065
GGAUGACACAGUAUACUGU
560
10083
ACAGUAUACUGUGUCAUCC
2211

10083
UCCAAGACAUGUCAUUUGC
561
10083
UCCAAGACAUGUCAUUUGC
561
10101
GCAAAUGACAUGUCUUGGA
2212

10101
CACAGCAGAAGACAUGCUU
562
10101
CACAGCAGAAGACAUGCUU
562
10119
AAGCAUGUCUUCUGCUGUG
2213

10119
UAAUCCUAACUAUGAAGAU
563
10119
UAAUCCUAACUAUGAAGAU
563
10137
AUCUUCAUAGUUAGGAUUA
2214

10137
UCUGCUCAUUCGCAAAUCC
564
10137
UCUGCUCAUUCGCAAAUCC
564
10155
GGAUUUGCGAAUGAGCAGA
2215

10155
CAACCAUAGCUUUCUUGUU
565
10155
CAACCAUAGCUUUCUUGUU
565
10173
AACAAGAAAGCUAUGGUUG
2216

10173
UCAGGCUGGCAAUGUUCAA
566
10173
UCAGGCUGGCAAUGUUCAA
566
10191
UUGAACAUUGCCAGCCUGA
2217

10191
ACUUCGUGUUAUUGGCCAU
567
10191
ACUUCGUGUUAUUGGCCAU
567
10209
AUGGCCAAUAACACGAAGU
2218

10209
UUCUAUGCAAAAUUGUCUG
568
10209
UUCUAUGCAAAAUUGUCUG
568
10227
CAGACAAUUUUGCAUAGAA
2219

10227
GCUUAGGCUUAAAGUUGAU
569
10227
GCUUAGGCUUAAAGUUGAU
569
10245
AUCAACUUUAAGCCUAAGC
2220

10245
UACUUCUAACCCUAAGACA
570
10245
UACUUCUAACCCUAAGACA
570
10263
UGUCUUAGGGUUAGAAGUA
2221

10263
ACCCAAGUAUAAAUUUGUC
571
10263
ACCCAAGUAUAAAUUUGUC
571
10281
GACAAAUUUAUACUUGGGU
2222

10281
CCGUAUCCAACCUGGUCAA
572
10281
CCGUAUCCAACCUGGUCAA
572
10299
UUGACCAGGUUGGAUACGG
2223

10299
AACAUUUUCAGUUCUAGCA
573
10299
AACAUUUUCAGUUCUAGCA
573
10317
UGCUAGAACUGAAAAUGUU
2224

10317
AUGCUACAAUGGUUCACCA
574
10317
AUGCUACAAUGGUUCACCA
574
10335
UGGUGAACCAUUGUAGCAU
2225

10335
AUCUGGUGUUUAUCAGUGU
575
10335
AUCUGGUGUUUAUCAGUGU
575
10353
ACACUGAUAAACACCAGAU
2226

10353
UGCCAUGAGACCUAAUCAU
576
10353
UGCCAUGAGACCUAAUCAU
576
10371
AUGAUUAGGUCUCAUGGCA
2227

10371
UACCAUUAAAGGUUCUUUC
577
10371
UACCAUUAAAGGUUCUUUC
577
10389
GAAAGAACCUUUAAUGGUA
2228

10389
CCUUAAUGGAUCAUGUGGU
578
10389
CCUUAAUGGAUCAUGUGGU
578
10407
ACCACAUGAUCCAUUAAGG
2229

10407
UAGUGUUGGUUUUAACAUU
579
10407
UAGUGUUGGUUUUAACAUU
579
10425
AAUGUUAAAACCAACACUA
2230

10425
UGAUUAUGAUUGCGUGUCU
580
10425
UGAUUAUGAUUGCGUGUCU
580
10443
AGACACGCAAUCAUAAUCA
2231

10443
UUUCUGCUAUAUGCAUCAU
581
10443
UUUCUGCUAUAUGCAUCAU
581
10461
AUGAUGCAUAUAGCAGAAA
2232

10461
UAUGGAGCUUCCAACAGGA
582
10461
UAUGGAGCUUCCAACAGGA
582
10479
UCCUGUUGGAAGCUCCAUA
2233

10479
AGUACACGCUGGUACUGAC
583
10479
AGUACACGCUGGUACUGAC
583
10497
GUCAGUACCAGCGUGUACU
2234

10497
CUUAGAAGGUAAAUUCUAU
584
10497
CUUAGAAGGUAAAUUCUAU
584
10515
AUAGAAUUUACCUUCUAAG
2235

10515
UGGUCCAUUUGUUGACAGA
585
10515
UGGUCCAUUUGUUGACAGA
585
10533
UCUGUCAACAAAUGGACCA
2236

10533
ACAAACUGCACAGGCUGCA
586
10533
ACAAACUGCACAGGCUGCA
586
10551
UGCAGCCUGUGCAGUUUGU
2237

10551
AGGUACAGACACAACCAUA
587
10551
AGGUACAGACACAACCAUA
587
10569
UAUGGUUGUGUCUGUACCU
2238

10569
AACAUUAAAUGUUUUGGCA
588
10569
AACAUUAAAUGUUUUGGCA
588
10587
UGCCAAAACAUUUAAUGUU
2239

10587
AUGGCUGUAUGCUGCUGUU
589
10587
AUGGCUGUAUGCUGCUGUU
589
10605
AACAGCAGCAUACAGCCAU
2240

10605
UAUCAAUGGUGAUAGGUGG
590
10605
UAUCAAUGGUGAUAGGUGG
590
10623
CCACCUAUCACCAUUGAUA
2241

10623
GUUUCUUAAUAGAUUCACC
591
10623
GUUUCUUAAUAGAUUCACC
591
10641
GGUGAAUCUAUUAAGAAAC
2242

10641
CACUACUUUGAAUGACUUU
592
10641
CACUACUUUGAAUGACUUU
592
10659
AAAGUCAUUCAAAGUAGUG
2243

10659
UAACCUUGUGGCAAUGAAG
593
10659
UAACCUUGUGGCAAUGAAG
593
10677
CUUCAUUGCCACAAGGUUA
2244

10677
GUACAACUAUGAACCUUUG
594
10677
GUACAACUAUGAACCUUUG
594
10695
CAAAGGUUCAUAGUUGUAC
2245

10695
GACACAAGAUCAUGUUGAC
595
10695
GACACAAGAUCAUGUUGAC
595
10713
GUCAACAUGAUCUUGUGUC
2246

10713
CAUAUUGGGACCUCUUUCU
596
10713
CAUAUUGGGACCUCUUUCU
596
10731
AGAAAGAGGUCCCAAUAUG
2247

10731
UGCUCAAACAGGAAUUGCC
597
10731
UGCUCAAACAGGAAUUGCC
597
10749
GGCAAUUCCUGUUUGAGCA
2248

10749
CGUCUUAGAUAUGUGUGCU
598
10749
CGUCUUAGAUAUGUGUGCU
598
10767
AGCACACAUAUCUAAGACG
2249

10767
UGCUUUGAAAGAGCUGCUG
599
10767
UGCUUUGAAAGAGCUGCUG
599
10785
CAGCAGCUCUUUCAAAGCA
2250

10785
GCAGAAUGGUAUGAAUGGU
600
10785
GCAGAAUGGUAUGAAUGGU
600
10803
ACCAUUCAUACCAUUCUGC
2251

10803
UCGUACUAUCCUUGGUAGC
601
10803
UCGUACUAUCCUUGGUAGC
601
10821
GCUACCAAGGAUAGUACGA
2252

10821
CACUAUUUUAGAAGAUGAG
602
10821
CACUAUUUUAGAAGAUGAG
602
10839
CUCAUCUUCUAAAAUAGUG
2253

10839
GUUUACACCAUUUGAUGUU
603
10839
GUUUACACCAUUUGAUGUU
603
10857
AACAUCAAAUGGUGUAAAC
2254

10857
UGUUAGACAAUGCUCUGGU
604
10857
UGUUAGACAAUGCUCUGGU
604
10875
ACCAGAGCAUUGUCUAACA
2255

10875
UGUUACCUUCCAAGGUAAG
605
10875
UGUUACCUUCCAAGGUAAG
605
10893
CUUACCUUGGAAGGUAACA
2256

10893
GUUCAAGAAAAUUGUUAAG
606
10893
GUUCAAGAAAAUUGUUAAG
606
10911
CUUAACAAUUUUCUUGAAC
2257

10911
GGGCACUCAUCAUUGGAUG
607
10911
GGGCACUCAUCAUUGGAUG
607
10929
CAUCCAAUGAUGAGUGCCC
2258

10929
GCUUUUAACUUUCUUGACA
608
10929
GCUUUUAACUUUCUUGACA
608
10947
UGUCAAGAAAGUUAAAAGC
2259

10947
AUCACUAUUGAUUCUUGUU
609
10947
AUCACUAUUGAUUCUUGUU
609
10965
AACAAGAAUCAAUAGUGAU
2260

10965
UCAAAGUACACAGUGGUCA
610
10965
UCAAAGUACACAGUGGUCA
610
10983
UGACCACUGUGUACUUUGA
2261

10983
ACUGUUUUUCUUUGUUUAC
611
10983
ACUGUUUUUCUUUGUUUAC
611
11001
GUAAACAAAGAAAAACAGU
2262

11001
CGAGAAUGCUUUCUUGCCA
612
11001
CGAGAAUGCUUUCUUGCCA
612
11019
UGGCAAGAAAGCAUUCUCG
2263

11019
AUUUACUCUUGGUAUUAUG
613
11019
AUUUACUCUUGGUAUUAUG
613
11037
CAUAAUACCAAGAGUAAAU
2264

11037
GGCAAUUGCUGCAUGUGCU
614
11037
GGCAAUUGCUGCAUGUGCU
614
11055
AGCACAUGCAGCAAUUGCC
2265

11055
UAUGCUGCUUGUUAAGCAU
615
11055
UAUGCUGCUUGUUAAGCAU
615
11073
AUGCUUAACAAGCAGCAUA
2266

11073
UAAGCACGCAUUCUUGUGC
616
11073
UAAGCACGCAUUCUUGUGC
616
11091
GCACAAGAAUGCGUGCUUA
2267

11091
CUUGUUUCUGUUACCUUCU
617
11091
CUUGUUUCUGUUACCUUCU
617
11109
AGAAGGUAACAGAAACAAG
2268

11109
UCUUGCAACAGUUGCUUAC
618
11109
UCUUGCAACAGUUGCUUAC
618
11127
GUAAGCAACUGUUGCAAGA
2269

11127
CUUUAAUAUGGUCUACAUG
619
11127
CUUUAAUAUGGUCUACAUG
619
11145
CAUGUAGACCAUAUUAAAG
2270

11145
GCCUGCUAGCUGGGUGAUG
620
11145
GCCUGCUAGCUGGGUGAUG
620
11163
CAUCACCCAGCUAGCAGGC
2271

11163
GCGUAUCAUGACAUGGCUU
621
11163
GCGUAUCAUGACAUGGCUU
621
11181
AAGCCAUGUCAUGAUACGC
2272

11181
UGAAUUGGCUGACACUAGC
622
11181
UGAAUUGGCUGACACUAGC
622
11199
GCUAGUGUCAGCCAAUUCA
2273

11199
CUUGUCUGGUUAUAGGCUU
623
11199
CUUGUCUGGUUAUAGGCUU
623
11217
AAGCCUAUAACCAGACAAG
2274

11217
UAAGGAUUGUGUUAUGUAU
624
11217
UAAGGAUUGUGUUAUGUAU
624
11235
AUACAUAACACAAUCCUUA
2275

11235
UGCUUCAGCUUUAGUUUUG
625
11235
UGCUUCAGCUUUAGUUUUG
625
11253
CAAAACUAAAGCUGAAGCA
2276

11253
GCUUAUUCUCAUGACAGCU
626
11253
GCUUAUUCUCAUGACAGCU
626
11271
AGCUGUCAUGAGAAUAAGC
2277

11271
UCGCACUGUUUAUGAUGAU
627
11271
UCGCACUGUUUAUGAUGAU
627
11289
AUCAUCAUAAACAGUGCGA
2278

11289
UGCUGCUAGACGUGUUUGG
628
11289
UGCUGCUAGACGUGUUUGG
628
11307
CCAAACACGUCUAGCAGCA
2279

11307
GACACUGAUGAAUGUCAUU
629
11307
GACACUGAUGAAUGUCAUU
629
11325
AAUGACAUUCAUCAGUGUC
2280

11325
UACACUUGUUUACAAAGUC
630
11325
UACACUUGUUUACAAAGUC
630
11343
GACUUUGUAAACAAGUGUA
2281

11343
CUACUAUGGUAAUGCUUUA
631
11343
CUACUAUGGUAAUGCUUUA
631
11361
UAAAGCAUUACCAUAGUAG
2282

11361
AGAUCAAGCUAUUUCCAUG
632
11361
AGAUCAAGCUAUUUCCAUG
632
11379
CAUGGAAAUAGCUUGAUCU
2283

11379
GUGGGCCUUAGUUAUUUCU
633
11379
GUGGGCCUUAGUUAUUUCU
633
11397
AGAAAUAACUAAGGCCCAC
2284

11397
UGUAACCUCUAACUAUUCU
634
11397
UGUAACCUCUAACUAUUCU
634
11415
AGAAUAGUUAGAGGUUACA
2285

11415
UGGUGUCGUUACGACUAUC
635
11415
UGGUGUCGUUACGACUAUC
635
11433
GAUAGUCGUAACGACACCA
2286

11433
CAUGUUUUUAGCUAGAGCU
636
11433
CAUGUUUUUAGCUAGAGCU
636
11451
AGCUCUAGCUAAAAACAUG
2287

11451
UAUAGUGUUUGUGUGUGUU
637
11451
UAUAGUGUUUGUGUGUGUU
637
11469
AACACACACAAACACUAUA
2288

11469
UGAGUAUUACCCAUUGUUA
638
11469
UGAGUAUUACCCAUUGUUA
638
11487
UAACAAUGGGUAAUACUCA
2289

11487
AUUUAUUACUGGCAACACC
639
11487
AUUUAUUACUGGCAACACC
639
11505
GGUGUUGCCAGUAAUAAAU
2290

11505
CUUACAGUGUAUCAUGCUU
640
11505
CUUACAGUGUAUCAUGCUU
640
11523
AAGCAUGAUACACUGUAAG
2291

11523
UGUUUAUUGUUUCUUAGGC
641
11523
UGUUUAUUGUUUCUUAGGC
641
11541
GCCUAAGAAACAAUAAACA
2292

11541
CUAUUGUUGCUGCUGCUAC
642
11541
CUAUUGUUGCUGCUGCUAC
642
11559
GUAGCAGCAGCAACAAUAG
2293

11559
CUUUGGCCUUUUCUGUUUA
643
11559
CUUUGGCCUUUUCUGUUUA
643
11577
UAAACAGAAAAGGCCAAAG
2294

11577
ACUCAACCGUUACUUCAGG
644
11577
ACUCAACCGUUACUUCAGG
644
11595
CCUGAAGUAACGGUUGAGU
2295

11595
GCUUACUCUUGGUGUUUAU
645
11595
GCUUACUCUUGGUGUUUAU
645
11613
AUAAACACCAAGAGUAAGC
2296

11613
UGACUACUUGGUCUCUACA
646
11613
UGACUACUUGGUCUCUACA
646
11631
UGUAGAGACCAAGUAGUCA
2297

11631
ACAAGAAUUUAGGUAUAUG
647
11631
ACAAGAAUUUAGGUAUAUG
647
11649
CAUAUACCUAAAUUCUUGU
2298

11649
GAACUCCCAGGGGCUUUUG
648
11649
GAACUCCCAGGGGCUUUUG
648
11667
CAAAAGCCCCUGGGAGUUC
2299

11667
GCCUCCUAAGAGUAGUAUU
649
11667
GCCUCCUAAGAGUAGUAUU
649
11685
AAUACUACUCUUAGGAGGC
2300

11685
UGAUGCUUUCAAGCUUAAC
650
11685
UGAUGCUUUCAAGCUUAAC
650
11703
GUUAAGCUUGAAAGCAUCA
2301

11703
CAUUAAGUUGUUGGGUAUU
651
11703
CAUUAAGUUGUUGGGUAUU
651
11721
AAUACCCAACAACUUAAUG
2302

11721
UGGAGGUAAACCAUGUAUC
652
11721
UGGAGGUAAACCAUGUAUC
652
11739
GAUACAUGGUUUACCUCCA
2303

11739
CAAGGUUGCUACUGUACAG
653
11739
CAAGGUUGCUACUGUACAG
653
11757
CUGUACAGUAGCAACCUUG
2304

11757
GUCUAAAAUGUCUGACGUA
654
11757
GUCUAAAAUGUCUGACGUA
654
11775
UACGUCAGACAUUUUAGAC
2305

11775
AAAGUGCACAUCUGUGGUA
655
11775
AAAGUGCACAUCUGUGGUA
655
11793
UACCACAGAUGUGCACUUU
2306

11793
ACUGCUCUCGGUUCUUCAA
656
11793
ACUGCUCUCGGUUCUUCAA
656
11811
UUGAAGAACCGAGAGCAGU
2307

11811
ACAACUUAGAGUAGAGUCA
657
11811
ACAACUUAGAGUAGAGUCA
657
11829
UGACUCUACUCUAAGUUGU
2308

11829
AUCUUCUAAAUUGUGGGCA
658
11829
AUCUUCUAAAUUGUGGGCA
658
11847
UGCCCACAAUUUAGAAGAU
2309

11847
ACAAUGUGUACAACUCCAC
659
11847
ACAAUGUGUACAACUCCAC
659
11865
GUGGAGUUGUACACAUUGU
2310

11865
CAAUGAUAUUCUUCUUGCA
660
11865
CAAUGAUAUUCUUCUUGCA
660
11883
UGCAAGAAGAAUAUCAUUG
2311

11883
AAAAGACACAACUGAAGCU
661
11883
AAAAGACACAACUGAAGCU
661
11901
AGCUUCAGUUGUGUCUUUU
2312

11901
UUUCGAGAAGAUGGUUUCU
662
11901
UUUCGAGAAGAUGGUUUCU
662
11919
AGAAACCAUCUUCUCGAAA
2313

11919
UCUUUUGUCUGUUUUGCUA
663
11919
UCUUUUGUCUGUUUUGCUA
663
11937
UAGCAAAACAGACAAAAGA
2314

11937
AUCCAUGCAGGGUGCUGUA
664
11937
AUCCAUGCAGGGUGCUGUA
664
11955
UACAGCACCCUGCAUGGAU
2315

11955
AGACAUUAAUAGGUUGUGC
665
11955
AGACAUUAAUAGGUUGUGC
665
11973
GCACAACCUAUUAAUGUCU
2316

11973
CGAGGAAAUGCUCGAUAAC
666
11973
CGAGGAAAUGCUCGAUAAC
666
11991
GUUAUCGAGCAUUUCCUCG
2317

11991
CCGUGCUACUCUUCAGGCU
667
11991
CCGUGCUACUCUUCAGGCU
667
12009
AGCCUGAAGAGUAGCACGG
2318

12009
UAUUGCUUCAGAAUUUAGU
668
12009
UAUUGCUUCAGAAUUUAGU
668
12027
ACUAAAUUCUGAAGCAAUA
2319

12027
UUCUUUACCAUCAUAUGCC
669
12027
UUCUUUACCAUCAUAUGCC
669
12045
GGCAUAUGAUGGUAAAGAA
2320

12045
CGCUUAUGCCACUGCCCAG
670
12045
CGCUUAUGCCACUGCCCAG
670
12063
CUGGGCAGUGGCAUAAGCG
2321

12063
GGAGGCCUAUGAGCAGGCU
671
12063
GGAGGCCUAUGAGCAGGCU
671
12081
AGCCUGCUCAUAGGCCUCC
2322

12081
UGUAGCUAAUGGUGAUUCU
672
12081
UGUAGCUAAUGGUGAUUCU
672
12099
AGAAUCACCAUUAGCUACA
2323

12099
UGAAGUCGUUCUCAAAAAG
673
12099
UGAAGUCGUUCUCAAAAAG
673
12117
CUUUUUGAGAACGACUUCA
2324

12117
GUUAAAGAAAUCUUUGAAU
674
12117
GUUAAAGAAAUCUUUGAAU
674
12135
AUUCAAAGAUUUCUUUAAC
2325

12135
UGUGGCUAAAUCUGAGUUU
675
12135
UGUGGCUAAAUCUGAGUUU
675
12153
AAACUCAGAUUUAGCCACA
2326

12153
UGACCGUGAUGCUGCCAUG
676
12153
UGACCGUGAUGCUGCCAUG
676
12171
CAUGGCAGCAUCACGGUCA
2327

12171
GCAACGCAAGUUGGAAAAG
677
12171
GCAACGCAAGUUGGAAAAG
677
12189
CUUUUCCAACUUGCGUUGC
2328

12189
GAUGGCAGAUCAGGCUAUG
678
12189
GAUGGCAGAUCAGGCUAUG
678
12207
CAUAGCCUGAUCUGCCAUC
2329

12207
GACCCAAAUGUACAAACAG
679
12207
GACCCAAAUGUACAAACAG
679
12225
CUGUUUGUACAUUUGGGUC
2330

12225
GGCAAGAUCUGAGGACAAG
680
12225
GGCAAGAUCUGAGGACAAG
680
12243
CUUGUCCUCAGAUCUUGCC
2331

12243
GAGGGCAAAAGUAACUAGU
681
12243
GAGGGCAAAAGUAACUAGU
681
12261
ACUAGUUACUUUUGCCCUC
2332

12261
UGCUAUGCAAACAAUGCUC
682
12261
UGCUAUGCAAACAAUGCUC
682
12279
GAGCAUUGUUUGCAUAGCA
2333

12279
CUUCACUAUGCUUAGGAAG
683
12279
CUUCACUAUGCUUAGGAAG
683
12297
CUUCCUAAGCAUAGUGAAG
2334

12297
GCUUGAUAAUGAUGCACUU
684
12297
GCUUGAUAAUGAUGCACUU
684
12315
AAGUGCAUCAUUAUCAAGC
2335

12315
UAACAACAUUAUCAACAAU
685
12315
UAACAACAUUAUCAACAAU
685
12333
AUUGUUGAUAAUGUUGUUA
2336

12333
UGCGCGUGAUGGUUGUGUU
686
12333
UGCGCGUGAUGGUUGUGUU
686
12351
AACACAACCAUCACGCGCA
2337

12351
UCCACUCAACAUCAUACCA
687
12351
UCCACUCAACAUCAUACCA
687
12369
UGGUAUGAUGUUGAGUGGA
2338

12369
AUUGACUACAGCAGCCAAA
688
12369
AUUGACUACAGCAGCCAAA
688
12387
UUUGGCUGCUGUAGUCAAU
2339

12387
ACUCAUGGUUGUUGUCCCU
689
12387
ACUCAUGGUUGUUGUCCCU
689
12405
AGGGACAACAACCAUGAGU
2340

12405
UGAUUAUGGUACCUACAAG
690
12405
UGAUUAUGGUACCUACAAG
690
12423
CUUGUAGGUACCAUAAUCA
2341

12423
GAACACUUGUGAUGGUAAC
691
12423
GAACACUUGUGAUGGUAAC
691
12441
GUUACCAUCACAAGUGUUC
2342

12441
CACCUUUACAUAUGCAUCU
692
12441
CACCUUUACAUAUGCAUCU
692
12459
AGAUGCAUAUGUAAAGGUG
2343

12459
UGCACUCUGGGAAAUCCAG
693
12459
UGCACUCUGGGAAAUCCAG
693
12477
CUGGAUUUCCCAGAGUGCA
2344

12477
GCAAGUUGUUGAUGCGGAU
694
12477
GCAAGUUGUUGAUGCGGAU
694
12495
AUCCGCAUCAACAACUUGC
2345

12495
UAGCAAGAUUGUUCAACUU
695
12495
UAGCAAGAUUGUUCAACUU
695
12513
AAGUUGAACAAUCUUGCUA
2346

12513
UAGUGAAAUUAACAUGGAC
696
12513
UAGUGAAAUUAACAUGGAC
696
12531
GUCCAUGUUAAUUUCACUA
2347

12531
CAAUUCACCAAAUUUGGCU
697
12531
CAAUUCACCAAAUUUGGCU
697
12549
AGCCAAAUUUGGUGAAUUG
2348

12549
UUGGCCUCUUAUUGUUACA
698
12549
UUGGCCUCUUAUUGUUACA
698
12567
UGUAACAAUAAGAGGCCAA
2349

12567
AGCUCUAAGAGCCAACUCA
699
12567
AGCUCUAAGAGCCAACUCA
699
12585
UGAGUUGGCUCUUAGAGCU
2350

12585
AGCUGUUAAACUACAGAAU
700
12585
AGCUGUUAAACUACAGAAU
700
12603
AUUCUGUAGUUUAACAGCU
2351

12603
UAAUGAACUGAGUCCAGUA
701
12603
UAAUGAACUGAGUCCAGUA
701
12621
UACUGGACUCAGUUCAUUA
2352

12621
AGCACUACGACAGAUGUCC
702
12621
AGCACUACGACAGAUGUCC
702
12639
GGACAUCUGUCGUAGUGCU
2353

12639
CUGUGCGGCUGGUACCACA
703
12639
CUGUGCGGCUGGUACCACA
703
12657
UGUGGUACCAGCCGCACAG
2354

12657
ACAAACAGCUUGUACUGAU
704
12657
ACAAACAGCUUGUACUGAU
704
12675
AUCAGUACAAGCUGUUUGU
2355

12675
UGACAAUGCACUUGCCUAC
705
12675
UGACAAUGCACUUGCCUAC
705
12693
GUAGGCAAGUGCAUUGUCA
2356

12693
CUAUAACAAUUCGAAGGGA
706
12693
CUAUAACAAUUCGAAGGGA
706
12711
UCCCUUCGAAUUGUUAUAG
2357

12711
AGGUAGGUUUGUGCUGGCA
707
12711
AGGUAGGUUUGUGCUGGCA
707
12729
UGCCAGCACAAACCUACCU
2358

12729
AUUACUAUCAGACCACCAA
708
12729
AUUACUAUCAGACCACCAA
708
12747
UUGGUGGUCUGAUAGUAAU
2359

12747
AGAUCUCAAAUGGGCUAGA
709
12747
AGAUCUCAAAUGGGCUAGA
709
12765
UCUAGCCCAUUUGAGAUCU
2360

12765
AUUCCCUAAGAGUGAUGGU
710
12765
AUUCCCUAAGAGUGAUGGU
710
12783
ACCAUCACUCUUAGGGAAU
2361

12783
UACAGGUACAAUUUACACA
711
12783
UACAGGUACAAUUUACACA
711
12801
UGUGUAAAUUGUACCUGUA
2362

12801
AGAACUGGAACCACCUUGU
712
12801
AGAACUGGAACCACCUUGU
712
12819
ACAAGGUGGUUCCAGUUCU
2363

12819
UAGGUUUGUUACAGACACA
713
12819
UAGGUUUGUUACAGACACA
713
12837
UGUGUCUGUAACAAACCUA
2364

12837
ACCAAAAGGGCCUAAAGUG
714
12837
ACCAAAAGGGCCUAAAGUG
714
12855
CACUUUAGGCCCUUUUGGU
2365

12855
GAAAUACUUGUACUUCAUC
715
12855
GAAAUACUUGUACUUCAUC
715
12873
GAUGAAGUACAAGUAUUUC
2366

12873
CAAAGGCUUAAACAACCUA
716
12873
CAAAGGCUUAAACAACCUA
716
12891
UAGGUUGUUUAAGCCUUUG
2367

12891
AAAUAGAGGUAUGGUGCUG
717
12891
AAAUAGAGGUAUGGUGCUG
717
12909
CAGCACCAUACCUCUAUUU
2368

12909
GGGCAGUUUAGCUGCUACA
718
12909
GGGCAGUUUAGCUGCUACA
718
12927
UGUAGCAGCUAAACUGCCC
2369

12927
AGUACGUCUUCAGGCUGGA
719
12927
AGUACGUCUUCAGGCUGGA
719
12945
UCCAGCCUGAAGACGUACU
2370

12945
AAAUGCUACAGAAGUACCU
720
12945
AAAUGCUACAGAAGUACCU
720
12963
AGGUACUUCUGUAGCAUUU
2371

12963
UGCCAAUUCAACUGUGCUU
721
12963
UGCCAAUUCAACUGUGCUU
721
12981
AAGCACAGUUGAAUUGGCA
2372

12981
UUCCUUCUGUGCUUUUGCA
722
12981
UUCCUUCUGUGCUUUUGCA
722
12999
UGCAAAAGCACAGAAGGAA
2373

12999
AGUAGACCCUGCUAAAGCA
723
12999
AGUAGACCCUGCUAAAGCA
723
13017
UGCUUUAGCAGGGUCUACU
2374

13017
AUAUAAGGAUUACCUAGCA
724
13017
AUAUAAGGAUUACCUAGCA
724
13035
UGCUAGGUAAUCCUUAUAU
2375

13035
AAGUGGAGGACAACCAAUC
725
13035
AAGUGGAGGACAACCAAUC
725
13053
GAUUGGUUGUCCUCCACUU
2376

13053
CACCAACUGUGUGAAGAUG
726
13053
CACCAACUGUGUGAAGAUG
726
13071
CAUCUUCACACAGUUGGUG
2377

13071
GUUGUGUACACACACUGGU
727
13071
GUUGUGUACACACACUGGU
727
13089
ACCAGUGUGUGUACACAAC
2378

13089
UACAGGACAGGCAAUUACU
728
13089
UACAGGACAGGCAAUUACU
728
13107
AGUAAUUGCCUGUCCUGUA
2379

13107
UGUAACACCAGAAGCUAAC
729
13107
UGUAACACCAGAAGCUAAC
729
13125
GUUAGCUUCUGGUGUUACA
2380

13125
CAUGGACCAAGAGUCCUUU
730
13125
CAUGGACCAAGAGUCCUUU
730
13143
AAAGGACUCUUGGUCCAUG
2381

13143
UGGUGGUGCUUCAUGUUGU
731
13143
UGGUGGUGCUUCAUGUUGU
731
13161
ACAACAUGAAGCACCACCA
2382

13161
UCUGUAUUGUAGAUGCCAC
732
13161
UCUGUAUUGUAGAUGCCAC
732
13179
GUGGCAUCUACAAUACAGA
2383

13179
CAUUGACCAUCCAAAUCCU
733
13179
CAUUGACCAUCCAAAUCCU
733
13197
AGGAUUUGGAUGGUCAAUG
2384

13197
UAAAGGAUUCUGUGACUUG
734
13197
UAAAGGAUUCUGUGACUUG
734
13215
CAAGUCACAGAAUCCUUUA
2385

13215
GAAAGGUAAGUACGUCCAA
735
13215
GAAAGGUAAGUACGUCCAA
735
13233
UUGGACGUACUUACCUUUC
2386

13233
AAUACCUACCACUUGUGCU
736
13233
AAUACCUACCACUUGUGCU
736
13251
AGCACAAGUGGUAGGUAUU
2387

13251
UAAUGACCCAGUGGGUUUU
737
13251
UAAUGACCCAGUGGGUUUU
737
13269
AAAACCCACUGGGUCAUUA
2388

13269
UACACUUAGAAACACAGUC
738
13269
UACACUUAGAAACACAGUC
738
13287
GACUGUGUUUCUAAGUGUA
2389

13287
CUGUACCGUCUGCGGAAUG
739
13287
CUGUACCGUCUGCGGAAUG
739
13305
CAUUCCGCAGACGGUACAG
2390

13305
GUGGAAAGGUUAUGGCUGU
740
13305
GUGGAAAGGUUAUGGCUGU
740
13323
ACAGCCAUAACCUUUCCAC
2391

13323
UAGUUGUGACCAACUCCGC
741
13323
UAGUUGUGACCAACUCCGC
741
13341
GCGGAGUUGGUCACAACUA
2392

13341
CGAACCCUUGAUGCAGUCU
742
13341
CGAACCCUUGAUGCAGUCU
742
13359
AGACUGCAUCAAGGGUUCG
2393

13359
UGCGGAUGCAUCAACGUUU
743
13359
UGCGGAUGCAUCAACGUUU
743
13377
AAACGUUGAUGCAUCCGCA
2394

13377
UUUAAACGGGUUUGCGGUG
744
13377
UUUAAACGGGUUUGCGGUG
744
13395
CACCGCAAACCCGUUUAAA
2395

13395
GUAAGUGCAGCCCGUCUUA
745
13395
GUAAGUGCAGCCCGUCUUA
745
13413
UAAGACGGGCUGCACUUAC
2396

13413
ACACCGUGCGGCACAGGCA
746
13413
ACACCGUGCGGCACAGGCA
746
13431
UGCCUGUGCCGCACGGUGU
2397

13431
ACUAGUACUGAUGUCGUCU
747
13431
ACUAGUACUGAUGUCGUCU
747
13449
AGACGACAUCAGUACUAGU
2398

13449
UACAGGGCUUUUGAUAUUU
748
13449
UACAGGGCUUUUGAUAUUU
748
13467
AAAUAUCAAAAGCCCUGUA
2399

13467
UACAACGAAAAAGUUGCUG
749
13467
UACAACGAAAAAGUUGCUG
749
13485
CAGCAACUUUUUCGUUGUA
2400

13485
GGUUUUGCAAAGUUCCUAA
750
13485
GGUUUUGCAAAGUUCCUAA
750
13503
UUAGGAACUUUGCAAAACC
2401

13503
AAAACUAAUUGCUGUCGCU
751
13503
AAAACUAAUUGCUGUCGCU
751
13521
AGCGACAGCAAUUAGUUUU
2402

13521
UUCCAGGAGAAGGAUGAGG
752
13521
UUCCAGGAGAAGGAUGAGG
752
13539
CCUCAUCCUUCUCCUGGAA
2403

13539
GAAGGCAAUUUAUUAGACU
753
13539
GAAGGCAAUUUAUUAGACU
753
13557
AGUCUAAUAAAUUGCCUUC
2404

13557
UCUUACUUUGUAGUUAAGA
754
13557
UCUUACUUUGUAGUUAAGA
754
13575
UCUUAACUACAAAGUAAGA
2405

13575
AGGCAUACUAUGUCUAACU
755
13575
AGGCAUACUAUGUCUAACU
755
13593
AGUUAGACAUAGUAUGCCU
2406

13593
UACCAACAUGAAGAGACUA
756
13593
UACCAACAUGAAGAGACUA
756
13611
UAGUCUCUUCAUGUUGGUA
2407

13611
AUUUAUAACUUGGUUAAAG
757
13611
AUUUAUAACUUGGUUAAAG
757
13629
CUUUAACCAAGUUAUAAAU
2408

13629
GAUUGUCCAGCGGUUGCUG
758
13629
GAUUGUCCAGCGGUUGCUG
758
13647
CAGCAACCGCUGGACAAUC
2409

13647
GUCCAUGACUUUUUCAAGU
759
13647
GUCCAUGACUUUUUCAAGU
759
13665
ACUUGAAAAAGUCAUGGAC
2410

13665
UUUAGAGUAGAUGGUGACA
760
13665
UUUAGAGUAGAUGGUGACA
760
13683
UGUCACCAUCUACUCUAAA
2411

13683
AUGGUACCACAUAUAUCAC
761
13683
AUGGUACCACAUAUAUCAC
761
13701
GUGAUAUAUGUGGUACCAU
2412

13701
CGUCAGCGUCUAACUAAAU
762
13701
CGUCAGCGUCUAACUAAAU
762
13719
AUUUAGUUAGACGCUGACG
2413

13719
UACACAAUGGCUGAUUUAG
763
13719
UACACAAUGGCUGAUUUAG
763
13737
CUAAAUCAGCCAUUGUGUA
2414

13737
GUCUAUGCUCUACGUCAUU
764
13737
GUCUAUGCUCUACGUCAUU
764
13755
AAUGACGUAGAGCAUAGAC
2415

13755
UUUGAUGAGGGUAAUUGUG
765
13755
UUUGAUGAGGGUAAUUGUG
765
13773
CACAAUUACCCUCAUCAAA
2416

13773
GAUACAUUAAAAGAAAUAC
766
13773
GAUACAUUAAAAGAAAUAC
766
13791
GUAUUUCUUUUAAUGUAUC
2417

13791
CUCGUCACAUACAAUUGCU
767
13791
CUCGUCACAUACAAUUGCU
767
13809
AGCAAUUGUAUGUGACGAG
2418

13809
UGUGAUGAUGAUUAUUUCA
768
13809
UGUGAUGAUGAUUAUUUCA
768
13827
UGAAAUAAUCAUCAUCACA
2419

13827
AAUAAGAAGGAUUGGUAUG
769
13827
AAUAAGAAGGAUUGGUAUG
769
13845
CAUACCAAUCCUUCUUAUU
2420

13845
GACUUCGUAGAGAAUCCUG
770
13845
GACUUCGUAGAGAAUCCUG
770
13863
CAGGAUUCUCUACGAAGUC
2421

13863
GACAUCUUACGCGUAUAUG
771
13863
GACAUCUUACGCGUAUAUG
771
13881
CAUAUACGCGUAAGAUGUC
2422

13881
GCUAACUUAGGUGAGCGUG
772
13881
GCUAACUUAGGUGAGCGUG
772
13899
CACGCUCACCUAAGUUAGC
2423

13899
GUACGCCAAUCAUUAUUAA
773
13899
GUACGCCAAUCAUUAUUAA
773
13917
UUAAUAAUGAUUGGCGUAC
2424

13917
AAGACUGUACAAUUCUGCG
774
13917
AAGACUGUACAAUUCUGCG
774
13935
CGCAGAAUUGUACAGUCUU
2425

13935
GAUGCUAUGCGUGAUGCAG
775
13935
GAUGCUAUGCGUGAUGCAG
775
13953
CUGCAUCACGCAUAGCAUC
2426

13953
GGCAUUGUAGGCGUACUGA
776
13953
GGCAUUGUAGGCGUACUGA
776
13971
UCAGUACGCCUACAAUGCC
2427

13971
ACAUUAGAUAAUCAGGAUC
777
13971
ACAUUAGAUAAUCAGGAUC
777
13989
GAUCCUGAUUAUCUAAUGU
2428

13989
CUUAAUGGGAACUGGUACG
778
13989
CUUAAUGGGAACUGGUACG
778
14007
CGUACCAGUUCCCAUUAAG
2429

14007
GAUUUCGGUGAUUUCGUAC
779
14007
GAUUUCGGUGAUUUCGUAC
779
14025
GUACGAAAUCACCGAAAUC
2430

14025
CAAGUAGCACCAGGCUGCG
780
14025
CAAGUAGCACCAGGCUGCG
780
14043
CGCAGCCUGGUGCUACUUG
2431

14043
GGAGUUCCUAUUGUGGAUU
781
14043
GGAGUUCCUAUUGUGGAUU
781
14061
AAUCCACAAUAGGAACUCC
2432

14061
UCAUAUUACUCAUUGCUGA
782
14061
UCAUAUUACUCAUUGCUGA
782
14079
UCAGCAAUGAGUAAUAUGA
2433

14079
AUGCCCAUCCUCACUUUGA
783
14079
AUGCCCAUCCUCACUUUGA
783
14097
UCAAAGUGAGGAUGGGCAU
2434

14097
ACUAGGGCAUUGGCUGCUG
784
14097
ACUAGGGCAUUGGCUGCUG
784
14115
CAGCAGCCAAUGCCCUAGU
2435

14115
GAGUCCCAUAUGGAUGCUG
785
14115
GAGUCCCAUAUGGAUGCUG
785
14133
CAGCAUCCAUAUGGGACUC
2436

14133
GAUCUCGCAAAACCACUUA
786
14133
GAUCUCGCAAAACCACUUA
786
14151
UAAGUGGUUUUGCGAGAUC
2437

14151
AUUAAGUGGGAUUUGCUGA
787
14151
AUUAAGUGGGAUUUGCUGA
787
14169
UCAGCAAAUCCCACUUAAU
2438

14169
AAAUAUGAUUUUACGGAAG
788
14169
AAAUAUGAUUUUACGGAAG
788
14187
CUUCCGUAAAAUCAUAUUU
2439

14187
GAGAGACUUUGUCUCUUCG
789
14187
GAGAGACUUUGUCUCUUCG
789
14205
CGAAGAGACAAAGUCUCUC
2440

14205
GACCGUUAUUUUAAAUAUU
790
14205
GACCGUUAUUUUAAAUAUU
790
14223
AAUAUUUAAAAUAACGGUC
2441

14223
UGGGACCAGACAUACCAUC
791
14223
UGGGACCAGACAUACCAUC
791
14241
GAUGGUAUGUCUGGUCCCA
2442

14241
CCCAAUUGUAUUAACUGUU
792
14241
CCCAAUUGUAUUAACUGUU
792
14259
AACAGUUAAUACAAUUGGG
2443

14259
UUGGAUGAUAGGUGUAUCC
793
14259
UUGGAUGAUAGGUGUAUCC
793
14277
GGAUACACCUAUCAUCCAA
2444

14277
CUUCAUUGUGCAAACUUUA
794
14277
CUUCAUUGUGCAAACUUUA
794
14295
UAAAGUUUGCACAAUGAAG
2445

14295
AAUGUGUUAUUUUCUACUG
795
14295
AAUGUGUUAUUUUCUACUG
795
14313
CAGUAGAAAAUAACACAUU
2446

14313
GUGUUUCCACCUACAAGUU
796
14313
GUGUUUCCACCUACAAGUU
796
14331
AACUUGUAGGUGGAAACAC
2447

14331
UUUGGACCACUAGUAAGAA
797
14331
UUUGGACCACUAGUAAGAA
797
14349
UUCUUACUAGUGGUCCAAA
2448

14349
AAAAUAUUUGUAGAUGGUG
798
14349
AAAAUAUUUGUAGAUGGUG
798
14367
CACCAUCUACAAAUAUUUU
2449

14367
GUUCCUUUUGUUGUUUCAA
799
14367
GUUCCUUUUGUUGUUUCAA
799
14385
UUGAAACAACAAAAGGAAC
2450

14385
ACUGGAUACCAUUUUCGUG
800
14385
ACUGGAUACCAUUUUCGUG
800
14403
CACGAAAAUGGUAUCCAGU
2451

14403
GAGUUAGGAGUCGUACAUA
801
14403
GAGUUAGGAGUCGUACAUA
801
14421
UAUGUACGACUCCUAACUC
2452

14421
AAUCAGGAUGUAAACUUAC
802
14421
AAUCAGGAUGUAAACUUAC
802
14439
GUAAGUUUACAUCCUGAUU
2453

14439
CAUAGCUCGCGUCUCAGUU
803
14439
CAUAGCUCGCGUCUCAGUU
803
14457
AACUGAGACGCGAGCUAUG
2454

14457
UUCAAGGAACUUUUAGUGU
804
14457
UUCAAGGAACUUUUAGUGU
804
14475
ACACUAAAAGUUCCUUGAA
2455

14475
UAUGCUGCUGAUCCAGCUA
805
14475
UAUGCUGCUGAUCCAGCUA
805
14493
UAGCUGGAUCAGCAGCAUA
2456

14493
AUGCAUGCAGCUUCUGGCA
806
14493
AUGCAUGCAGCUUCUGGCA
806
14511
UGCCAGAAGCUGCAUGCAU
2457

14511
AAUUUAUUGCUAGAUAAAC
807
14511
AAUUUAUUGCUAGAUAAAC
807
14529
GUUUAUCUAGCAAUAAAUU
2458

14529
CGCACUACAUGCUUUUCAG
808
14529
CGCACUACAUGCUUUUCAG
808
14547
CUGAAAAGCAUGUAGUGCG
2459

14547
GUAGCUGCACUAACAAACA
809
14547
GUAGCUGCACUAACAAACA
809
14565
UGUUUGUUAGUGCAGCUAC
2460

14565
AAUGUUGCUUUUCAAACUG
810
14565
AAUGUUGCUUUUCAAACUG
810
14583
CAGUUUGAAAAGCAACAUU
2461

14583
GUCAAACCCGGUAAUUUUA
811
14583
GUCAAACCCGGUAAUUUUA
811
14601
UAAAAUUACCGGGUUUGAC
2462

14601
AAUAAAGACUUUUAUGACU
812
14601
AAUAAAGACUUUUAUGACU
812
14619
AGUCAUAAAAGUCUUUAUU
2463

14619
UUUGCUGUGUCUAAAGGUU
813
14619
UUUGCUGUGUCUAAAGGUU
813
14637
AACCUUUAGACACAGCAAA
2464

14637
UUCUUUAAGGAAGGAAGUU
814
14637
UUCUUUAAGGAAGGAAGUU
814
14655
AACUUCCUUCCUUAAAGAA
2465

14655
UCUGUUGAACUAAAACACU
815
14655
UCUGUUGAACUAAAACACU
815
14673
AGUGUUUUAGUUCAACAGA
2466

14673
UUCUUCUUUGCUCAGGAUG
816
14673
UUCUUCUUUGCUCAGGAUG
816
14691
CAUCCUGAGCAAAGAAGAA
2467

14691
GGCAACGCUGCUAUCAGUG
817
14691
GGCAACGCUGCUAUCAGUG
817
14709
CACUGAUAGCAGCGUUGCC
2468

14709
GAUUAUGACUAUUAUCGUU
818
14709
GAUUAUGACUAUUAUCGUU
818
14727
AACGAUAAUAGUCAUAAUC
2469

14727
UAUAAUCUGCCAACAAUGU
819
14727
UAUAAUCUGCCAACAAUGU
819
14745
ACAUUGUUGGCAGAUUAUA
2470

14745
UGUGAUAUCAGACAACUCC
820
14745
UGUGAUAUCAGACAACUCC
820
14763
GGAGUUGUCUGAUAUCACA
2471

14763
CUAUUCGUAGUUGAAGUUG
821
14763
CUAUUCGUAGUUGAAGUUG
821
14781
CAACUUCAACUACGAAUAG
2472

14781
GUUGAUAAAUACUUUGAUU
822
14781
GUUGAUAAAUACUUUGAUU
822
14799
AAUCAAAGUAUUUAUCAAC
2473

14799
UGUUACGAUGGUGGCUGUA
823
14799
UGUUACGAUGGUGGCUGUA
823
14817
UACAGCCACCAUCGUAACA
2474

14817
AUUAAUGCCAACCAAGUAA
824
14817
AUUAAUGCCAACCAAGUAA
824
14835
UUACUUGGUUGGCAUUAAU
2475

14835
AUCGUUAACAAUCUGGAUA
825
14835
AUCGUUAACAAUCUGGAUA
825
14853
UAUCCAGAUUGUUAACGAU
2476

14853
AAAUCAGCUGGUUUCCCAU
826
14853
AAAUCAGCUGGUUUCCCAU
826
14871
AUGGGAAACCAGCUGAUUU
2477

14871
UUUAAUAAAUGGGGUAAGG
827
14871
UUUAAUAAAUGGGGUAAGG
827
14889
CCUUACCCCAUUUAUUAAA
2478

14889
GCUAGACUUUAUUAUGACU
828
14889
GCUAGACUUUAUUAUGACU
828
14907
AGUCAUAAUAAAGUCUAGC
2479

14907
UCAAUGAGUUAUGAGGAUC
829
14907
UCAAUGAGUUAUGAGGAUC
829
14925
GAUCCUCAUAACUCAUUGA
2480

14925
CAAGAUGCACUUUUCGCGU
830
14925
CAAGAUGCACUUUUCGCGU
830
14943
ACGCGAAAAGUGCAUCUUG
2481

14943
UAUACUAAGCGUAAUGUCA
831
14943
UAUACUAAGCGUAAUGUCA
831
14961
UGACAUUACGCUUAGUAUA
2482

14961
AUCCCUACUAUAACUCAAA
832
14961
AUCCCUACUAUAACUCAAA
832
14979
UUUGAGUUAUAGUAGGGAU
2483

14979
AUGAAUCUUAAGUAUGCCA
833
14979
AUGAAUCUUAAGUAUGCCA
833
14997
UGGCAUACUUAAGAUUCAU
2484

14997
AUUAGUGCAAAGAAUAGAG
834
14997
AUUAGUGCAAAGAAUAGAG
834
15015
CUCUAUUCUUUGCACUAAU
2485

15015
GCUCGCACCGUAGCUGGUG
835
15015
GCUCGCACCGUAGCUGGUG
835
15033
CACCAGCUACGGUGCGAGC
2486

15033
GUCUCUAUCUGUAGUACUA
836
15033
GUCUCUAUCUGUAGUACUA
836
15051
UAGUACUACAGAUAGAGAC
2487

15051
AUGACAAAUAGACAGUUUC
837
15051
AUGACAAAUAGACAGUUUC
837
15069
GAAACUGUCUAUUUGUCAU
2488

15069
CAUCAGAAAUUAUUGAAGU
838
15069
CAUCAGAAAUUAUUGAAGU
838
15087
ACUUCAAUAAUUUCUGAUG
2489

15087
UCAAUAGCCGCCACUAGAG
839
15087
UCAAUAGCCGCCACUAGAG
839
15105
CUCUAGUGGCGGCUAUUGA
2490

15105
GGAGCUACUGUGGUAAUUG
840
15105
GGAGCUACUGUGGUAAUUG
840
15123
CAAUUACCACAGUAGCUCC
2491

15123
GGAACAAGCAAGUUUUACG
841
15123
GGAACAAGCAAGUUUUACG
841
15141
CGUAAAACUUGCUUGUUCC
2492

15141
GGUGGCUGGCAUAAUAUGU
842
15141
GGUGGCUGGCAUAAUAUGU
842
15159
ACAUAUUAUGCCAGCCACC
2493

15159
UUAAAAACUGUUUACAGUG
843
15159
UUAAAAACUGUUUACAGUG
843
15177
CACUGUAAACAGUUUUUAA
2494

15177
GAUGUAGAAACUCCACACC
844
15177
GAUGUAGAAACUCCACACC
844
15195
GGUGUGGAGUUUCUACAUC
2495

15195
CUUAUGGGUUGGGAUUAUC
845
15195
CUUAUGGGUUGGGAUUAUC
845
15213
GAUAAUCCCAACCCAUAAG
2496

15213
CCAAAAUGUGACAGAGCCA
846
15213
CCAAAAUGUGACAGAGCCA
846
15231
UGGCUCUGUCACAUUUUGG
2497

15231
AUGCCUAACAUGCUUAGGA
847
15231
AUGCCUAACAUGCUUAGGA
847
15249
UCCUAAGCAUGUUAGGCAU
2498

15249
AUAAUGGCCUCUCUUGUUC
848
15249
AUAAUGGCCUCUCUUGUUC
848
15267
GAACAAGAGAGGCCAUUAU
2499

15267
CUUGCUCGCAAACAUAACA
849
15267
CUUGCUCGCAAACAUAACA
849
15285
UGUUAUGUUUGCGAGCAAG
2500

15285
ACUUGCUGUAACUUAUCAC
850
15285
ACUUGCUGUAACUUAUCAC
850
15303
GUGAUAAGUUACAGCAAGU
2501

15303
CACCGUUUCUACAGGUUAG
851
15303
CACCGUUUCUACAGGUUAG
851
15321
CUAACCUGUAGAAACGGUG
2502

15321
GCUAACGAGUGUGCGCAAG
852
15321
GCUAACGAGUGUGCGCAAG
852
15339
CUUGCGCACACUCGUUAGC
2503

15339
GUAUUAAGUGAGAUGGUCA
853
15339
GUAUUAAGUGAGAUGGUCA
853
15357
UGACCAUCUCACUUAAUAC
2504

15357
AUGUGUGGCGGCUCACUAU
854
15357
AUGUGUGGCGGCUCACUAU
854
15375
AUAGUGAGCCGCCACACAU
2505

15375
UAUGUUAAACCAGGUGGAA
855
15375
UAUGUUAAACCAGGUGGAA
855
15393
UUCCACCUGGUUUAACAUA
2506

15393
ACAUCAUCCGGUGAUGCUA
856
15393
ACAUCAUCCGGUGAUGCUA
856
15411
UAGCAUCACCGGAUGAUGU
2507

15411
ACAACUGCUUAUGCUAAUA
857
15411
ACAACUGCUUAUGCUAAUA
857
15429
UAUUAGCAUAAGCAGUUGU
2508

15429
AGUGUCUUUAACAUUUGUC
858
15429
AGUGUCUUUAACAUUUGUC
858
15447
GACAAAUGUUAAAGACACU
2509

15447
CAAGCUGUUACAGCCAAUG
859
15447
CAAGCUGUUACAGCCAAUG
859
15465
CAUUGGCUGUAACAGCUUG
2510

15465
GUAAAUGCACUUCUUUCAA
860
15465
GUAAAUGCACUUCUUUCAA
860
15483
UUGAAAGAAGUGCAUUUAC
2511

15483
ACUGAUGGUAAUAAGAUAG
861
15483
ACUGAUGGUAAUAAGAUAG
861
15501
CUAUCUUAUUACCAUCAGU
2512

15501
GCUGACAAGUAUGUCCGCA
862
15501
GCUGACAAGUAUGUCCGCA
862
15519
UGCGGACAUACUUGUCAGC
2513

15519
AAUCUACAACACAGGCUCU
863
15519
AAUCUACAACACAGGCUCU
863
15537
AGAGCCUGUGUUGUAGAUU
2514

15537
UAUGAGUGUCUCUAUAGAA
864
15537
UAUGAGUGUCUCUAUAGAA
864
15555
UUCUAUAGAGACACUCAUA
2515

15555
AAUAGGGAUGUUGAUCAUG
865
15555
AAUAGGGAUGUUGAUCAUG
865
15573
CAUGAUCAACAUCCCUAUU
2516

15573
GAAUUCGUGGAUGAGUUUU
866
15573
GAAUUCGUGGAUGAGUUUU
866
15591
AAAACUCAUCCACGAAUUC
2517

15591
UACGCUUACCUGCGUAAAC
867
15591
UACGCUUACCUGCGUAAAC
867
15609
GUUUACGCAGGUAAGCGUA
2518

15609
CAUUUCUCCAUGAUGAUUC
868
15609
CAUUUCUCCAUGAUGAUUC
868
15627
GAAUCAUCAUGGAGAAAUG
2519

15627
CUUUCUGAUGAUGCCGUUG
869
15627
CUUUCUGAUGAUGCCGUUG
869
15645
CAACGGCAUCAUCAGAAAG
2520

15645
GUGUGCUAUAACAGUAACU
870
15645
GUGUGCUAUAACAGUAACU
870
15663
AGUUACUGUUAUAGCACAC
2521

15663
UAUGCGGCUCAAGGUUUAG
871
15663
UAUGCGGCUCAAGGUUUAG
871
15681
CUAAACCUUGAGCCGCAUA
2522

15681
GUAGCUAGCAUUAAGAACU
872
15681
GUAGCUAGCAUUAAGAACU
872
15699
AGUUCUUAAUGCUAGCUAC
2523

15699
UUUAAGGCAGUUCUUUAUU
873
15699
UUUAAGGCAGUUCUUUAUU
873
15717
AAUAAAGAACUGCCUUAAA
2524

15717
UAUCAAAAUAAUGUGUUCA
874
15717
UAUCAAAAUAAUGUGUUCA
874
15735
UGAACACAUUAUUUUGAUA
2525

15735
AUGUCUGAGGCAAAAUGUU
875
15735
AUGUCUGAGGCAAAAUGUU
875
15753
AACAUUUUGCCUCAGACAU
2526

15753
UGGACUGAGACUGACCUUA
876
15753
UGGACUGAGACUGACCUUA
876
15771
UAAGGUCAGUCUCAGUCCA
2527

15771
ACUAAAGGACCUCACGAAU
877
15771
ACUAAAGGACCUCACGAAU
877
15789
AUUCGUGAGGUCCUUUAGU
2528

15789
UUUUGCUCACAGCAUACAA
878
15789
UUUUGCUCACAGCAUACAA
878
15807
UUGUAUGCUGUGAGCAAAA
2529

15807
AUGCUAGUUAAACAAGGAG
879
15807
AUGCUAGUUAAACAAGGAG
879
15825
CUCCUUGUUUAACUAGCAU
2530

15825
GAUGAUUACGUGUACCUGC
880
15825
GAUGAUUACGUGUACCUGC
880
15843
GCAGGUACACGUAAUCAUC
2531

15843
CCUUACCCAGAUCCAUCAA
881
15843
CCUUACCCAGAUCCAUCAA
881
15861
UUGAUGGAUCUGGGUAAGG
2532

15861
AGAAUAUUAGGCGCAGGCU
882
15861
AGAAUAUUAGGCGCAGGCU
882
15879
AGCCUGCGCCUAAUAUUCU
2533

15879
UGUUUUGUCGAUGAUAUUG
883
15879
UGUUUUGUCGAUGAUAUUG
883
15897
CAAUAUCAUCGACAAAACA
2534

15897
GUCAAAACAGAUGGUACAC
884
15897
GUCAAAACAGAUGGUACAC
884
15915
GUGUACCAUCUGUUUUGAC
2535

15915
CUUAUGAUUGAAAGGUUCG
885
15915
CUUAUGAUUGAAAGGUUCG
885
15933
CGAACCUUUCAAUCAUAAG
2536

15933
GUGUCACUGGCUAUUGAUG
886
15933
GUGUCACUGGCUAUUGAUG
886
15951
CAUCAAUAGCCAGUGACAC
2537

15951
GCUUACCCACUUACAAAAC
887
15951
GCUUACCCACUUACAAAAC
887
15969
GUUUUGUAAGUGGGUAAGC
2538

15969
CAUCCUAAUCAGGAGUAUG
888
15969
CAUCCUAAUCAGGAGUAUG
888
15987
CAUACUCCUGAUUAGGAUG
2539

15987
GCUGAUGUCUUUCACUUGU
889
15987
GCUGAUGUCUUUCACUUGU
889
16005
ACAAGUGAAAGACAUCAGC
2540

16005
UAUUUACAAUACAUUAGAA
890
16005
UAUUUACAAUACAUUAGAA
890
16023
UUCUAAUGUAUUGUAAAUA
2541

16023
AAGUUACAUGAUGAGCUUA
891
16023
AAGUUACAUGAUGAGCUUA
891
16041
UAAGCUCAUCAUGUAACUU
2542

16041
ACUGGCCACAUGUUGGACA
892
16041
ACUGGCCACAUGUUGGACA
892
16059
UGUCCAACAUGUGGCCAGU
2543

16059
AUGUAUUCCGUAAUGCUAA
893
16059
AUGUAUUCCGUAAUGCUAA
893
16077
UUAGCAUUACGGAAUACAU
2544

16077
ACUAAUGAUAACACCUCAC
894
16077
ACUAAUGAUAACACCUCAC
894
16095
GUGAGGUGUUAUCAUUAGU
2545

16095
CGGUACUGGGAACCUGAGU
895
16095
CGGUACUGGGAACCUGAGU
895
16113
ACUCAGGUUCCCAGUACCG
2546

16113
UUUUAUGAGGCUAUGUACA
896
16113
UUUUAUGAGGCUAUGUACA
896
16131
UGUACAUAGCCUCAUAAAA
2547

16131
ACACCACAUACAGUCUUGC
897
16131
ACACCACAUACAGUCUUGC
897
16149
GCAAGACUGUAUGUGGUGU
2548

16149
CAGGCUGUAGGUGCUUGUG
898
16149
CAGGCUGUAGGUGCUUGUG
898
16167
CACAAGCACCUACAGCCUG
2549

16167
GUAUUGUGCAAUUCACAGA
899
16167
GUAUUGUGCAAUUCACAGA
899
16185
UCUGUGAAUUGCACAAUAC
2550

16185
ACUUCACUUCGUUGCGGUG
900
16185
ACUUCACUUCGUUGCGGUG
900
16203
CACCGCAACGAAGUGAAGU
2551

16203
GCCUGUAUUAGGAGACCAU
901
16203
GCCUGUAUUAGGAGACCAU
901
16221
AUGGUCUCCUAAUACAGGC
2552

16221
UUCCUAUGUUGCAAGUGCU
902
16221
UUCCUAUGUUGCAAGUGCU
902
16239
AGCACUUGCAACAUAGGAA
2553

16239
UGCUAUGACCAUGUCAUUU
903
16239
UGCUAUGACCAUGUCAUUU
903
16257
AAAUGACAUGGUCAUAGCA
2554

16257
UCAACAUCACACAAAUUAG
904
16257
UCAACAUCACACAAAUUAG
904
16275
CUAAUUUGUGUGAUGUUGA
2555

16275
GUGUUGUCUGUUAAUCCCU
905
16275
GUGUUGUCUGUUAAUCCCU
905
16293
AGGGAUUAACAGACAACAC
2556

16293
UAUGUUUGCAAUGCCCCAG
906
16293
UAUGUUUGCAAUGCCCCAG
906
16311
CUGGGGCAUUGCAAACAUA
2557

16311
GGUUGUGAUGUCACUGAUG
907
16311
GGUUGUGAUGUCACUGAUG
907
16329
CAUCAGUGACAUCACAACC
2558

16329
GUGACACAACUGUAUCUAG
908
16329
GUGACACAACUGUAUCUAG
908
16347
CUAGAUACAGUUGUGUCAC
2559

16347
GGAGGUAUGAGCUAUUAUU
909
16347
GGAGGUAUGAGCUAUUAUU
909
16365
AAUAAUAGCUCAUACCUCC
2560

16365
UGCAAGUCACAUAAGCCUC
910
16365
UGCAAGUCACAUAAGCCUC
910
16383
GAGGCUUAUGUGACUUGCA
2561

16383
CCCAUUAGUUUUCCAUUAU
911
16383
CCCAUUAGUUUUCCAUUAU
911
16401
AUAAUGGAAAACUAAUGGG
2562

16401
UGUGCUAAUGGUCAGGUUU
912
16401
UGUGCUAAUGGUCAGGUUU
912
16419
AAACCUGACCAUUAGCACA
2563

16419
UUUGGUUUAUACAAAAACA
913
16419
UUUGGUUUAUACAAAAACA
913
16437
UGUUUUUGUAUAAACCAAA
2564

16437
ACAUGUGUAGGCAGUGACA
914
16437
ACAUGUGUAGGCAGUGACA
914
16455
UGUCACUGCCUACACAUGU
2565

16455
AAUGUCACUGACUUCAAUG
915
16455
AAUGUCACUGACUUCAAUG
915
16473
CAUUGAAGUCAGUGACAUU
2566

16473
GCGAUAGCAACAUGUGAUU
916
16473
GCGAUAGCAACAUGUGAUU
916
16491
AAUCACAUGUUGCUAUCGC
2567

16491
UGGACUAAUGCUGGCGAUU
917
16491
UGGACUAAUGCUGGCGAUU
917
16509
AAUCGCCAGCAUUAGUCCA
2568

16509
UACAUACUUGCCAACACUU
918
16509
UACAUACUUGCCAACACUU
918
16527
AAGUGUUGGCAAGUAUGUA
2569

16527
UGUACUGAGAGACUCAAGC
919
16527
UGUACUGAGAGACUCAAGC
919
16545
GCUUGAGUCUCUCAGUACA
2570

16545
CUUUUCGCAGCAGAAACGC
920
16545
CUUUUCGCAGCAGAAACGC
920
16563
GCGUUUCUGCUGCGAAAAG
2571

16563
CUCAAAGCCACUGAGGAAA
921
16563
CUCAAAGCCACUGAGGAAA
921
16581
UUUCCUCAGUGGCUUUGAG
2572

16581
ACAUUUAAGCUGUCAUAUG
922
16581
ACAUUUAAGCUGUCAUAUG
922
16599
CAUAUGACAGCUUAAAUGU
2573

16599
GGUAUUGCCACUGUACGCG
923
16599
GGUAUUGCCACUGUACGCG
923
16617
CGCGUACAGUGGCAAUACC
2574

16617
GAAGUACUCUCUGACAGAG
924
16617
GAAGUACUCUCUGACAGAG
924
16635
CUCUGUCAGAGAGUACUUC
2575

16635
GAAUUGCAUCUUUCAUGGG
925
16635
GAAUUGCAUCUUUCAUGGG
925
16653
CCCAUGAAAGAUGCAAUUC
2576

16653
GAGGUUGGAAAACCUAGAC
926
16653
GAGGUUGGAAAACCUAGAC
926
16671
GUCUAGGUUUUCCAACCUC
2577

16671
CCACCAUUGAACAGAAACU
927
16671
CCACCAUUGAACAGAAACU
927
16689
AGUUUCUGUUCAAUGGUGG
2578

16689
UAUGUCUUUACUGGUUACC
928
16689
UAUGUCUUUACUGGUUACC
928
16707
GGUAACCAGUAAAGACAUA
2579

16707
CGUGUAACUAAAAAUAGUA
929
16707
CGUGUAACUAAAAAUAGUA
929
16725
UACUAUUUUUAGUUACACG
2580

16725
AAAGUACAGAUUGGAGAGU
930
16725
AAAGUACAGAUUGGAGAGU
930
16743
ACUCUCCAAUCUGUACUUU
2581

16743
UACACCUUUGAAAAAGGUG
931
16743
UACACCUUUGAAAAAGGUG
931
16761
CACCUUUUUCAAAGGUGUA
2582

16761
GACUAUGGUGAUGCUGUUG
932
16761
GACUAUGGUGAUGCUGUUG
932
16779
CAACAGCAUCACCAUAGUC
2583

16779
GUGUACAGAGGUACUACGA
933
16779
GUGUACAGAGGUACUACGA
933
16797
UCGUAGUACCUCUGUACAC
2584

16797
ACAUACAAGUUGAAUGUUG
934
16797
ACAUACAAGUUGAAUGUUG
934
16815
CAACAUUCAACUUGUAUGU
2585

16815
GGUGAUUACUUUGUGUUGA
935
16815
GGUGAUUACUUUGUGUUGA
935
16833
UCAACACAAAGUAAUCACC
2586

16833
ACAUCUCACACUGUAAUGC
936
16833
ACAUCUCACACUGUAAUGC
936
16851
GCAUUACAGUGUGAGAUGU
2587

16851
CCACUUAGUGCACCUACUC
937
16851
CCACUUAGUGCACCUACUC
937
16869
GAGUAGGUGCACUAAGUGG
2588

16869
CUAGUGCCACAAGAGCACU
938
16869
CUAGUGCCACAAGAGCACU
938
16887
AGUGCUCUUGUGGCACUAG
2589

16887
UAUGUGAGAAUUACUGGCU
939
16887
UAUGUGAGAAUUACUGGCU
939
16905
AGCCAGUAAUUCUCACAUA
2590

16905
UUGUACCCAACACUCAACA
940
16905
UUGUACCCAACACUCAACA
940
16923
UGUUGAGUGUUGGGUACAA
2591

16923
AUCUCAGAUGAGUUUUCUA
941
16923
AUCUCAGAUGAGUUUUCUA
941
16941
UAGAAAACUCAUCUGAGAU
2592

16941
AGCAAUGUUGCAAAUUAUC
942
16941
AGCAAUGUUGCAAAUUAUC
942
16959
GAUAAUUUGCAACAUUGCU
2593

16959
CAAAAGGUCGGCAUGCAAA
943
16959
CAAAAGGUCGGCAUGCAAA
943
16977
UUUGCAUGCCGACCUUUUG
2594

16977
AAGUACUCUACACUCCAAG
944
16977
AAGUACUCUACACUCCAAG
944
16995
CUUGGAGUGUAGAGUACUU
2595

16995
GGACCACCUGGUACUGGUA
945
16995
GGACCACCUGGUACUGGUA
945
17013
UACCAGUACCAGGUGGUCC
2596

17013
AAGAGUCAUUUUGCCAUCG
946
17013
AAGAGUCAUUUUGCCAUCG
946
17031
CGAUGGCAAAAUGACUCUU
2597

17031
GGACUUGCUCUCUAUUACC
947
17031
GGACUUGCUCUCUAUUACC
947
17049
GGUAAUAGAGAGCAAGUCC
2598

17049
CCAUCUGCUCGCAUAGUGU
948
17049
CCAUCUGCUCGCAUAGUGU
948
17067
ACACUAUGCGAGCAGAUGG
2599

17067
UAUACGGCAUGCUCUCAUG
949
17067
UAUACGGCAUGCUCUCAUG
949
17085
CAUGAGAGCAUGCCGUAUA
2600

17085
GCAGCUGUUGAUGCCCUAU
950
17085
GCAGCUGUUGAUGCCCUAU
950
17103
AUAGGGCAUCAACAGCUGC
2601

17103
UGUGAAAAGGCAUUAAAAU
951
17103
UGUGAAAAGGCAUUAAAAU
951
17121
AUUUUAAUGCCUUUUCACA
2602

17121
UAUUUGCCCAUAGAUAAAU
952
17121
UAUUUGCCCAUAGAUAAAU
952
17139
AUUUAUCUAUGGGCAAAUA
2603

17139
UGUAGUAGAAUCAUACCUG
953
17139
UGUAGUAGAAUCAUACCUG
953
17157
CAGGUAUGAUUCUACUACA
2604

17157
GCGCGUGCGCGCGUAGAGU
954
17157
GCGCGUGCGCGCGUAGAGU
954
17175
ACUCUACGCGCGCACGCGC
2605

17175
UGUUUUGAUAAAUUCAAAG
955
17175
UGUUUUGAUAAAUUCAAAG
955
17193
CUUUGAAUUUAUCAAAACA
2606

17193
GUGAAUUCAACACUAGAAC
956
17193
GUGAAUUCAACACUAGAAC
956
17211
GUUCUAGUGUUGAAUUCAC
2607

17211
CAGUAUGUUUUCUGCACUG
957
17211
CAGUAUGUUUUCUGCACUG
957
17229
CAGUGCAGAAAACAUACUG
2608

17229
GUAAAUGCAUUGCCAGAAA
958
17229
GUAAAUGCAUUGCCAGAAA
958
17247
UUUCUGGCAAUGCAUUUAC
2609

17247
ACAACUGCUGACAUUGUAG
959
17247
ACAACUGCUGACAUUGUAG
959
17265
CUACAAUGUCAGCAGUUGU
2610

17265
GUCUUUGAUGAAAUCUCUA
960
17265
GUCUUUGAUGAAAUCUCUA
960
17283
UAGAGAUUUCAUCAAAGAC
2611

17283
AUGGCUACUAAUUAUGACU
961
17283
AUGGCUACUAAUUAUGACU
961
17301
AGUCAUAAUUAGUAGCCAU
2612

17301
UUGAGUGUUGUCAAUGCUA
962
17301
UUGAGUGUUGUCAAUGCUA
962
17319
UAGCAUUGACAACACUCAA
2613

17319
AGACUUCGUGCAAAACACU
963
17319
AGACUUCGUGCAAAACACU
963
17337
AGUGUUUUGCACGAAGUCU
2614

17337
UACGUCUAUAUUGGCGAUC
964
17337
UACGUCUAUAUUGGCGAUC
964
17355
GAUCGCCAAUAUAGACGUA
2615

17355
CCUGCUCAAUUACCAGCCC
965
17355
CCUGCUCAAUUACCAGCCC
965
17373
GGGCUGGUAAUUGAGCAGG
2616

17373
CCCCGCACAUUGCUGACUA
966
17373
CCCCGCACAUUGCUGACUA
966
17391
UAGUCAGCAAUGUGCGGGG
2617

17391
AAAGGCACACUAGAACCAG
967
17391
AAAGGCACACUAGAACCAG
967
17409
CUGGUUCUAGUGUGCCUUU
2618

17409
GAAUAUUUUAAUUCAGUGU
968
17409
GAAUAUUUUAAUUCAGUGU
968
17427
ACACUGAAUUAAAAUAUUC
2619

17427
UGCAGACUUAUGAAAACAA
969
17427
UGCAGACUUAUGAAAACAA
969
17445
UUGUUUUCAUAAGUCUGCA
2620

17445
AUAGGUCCAGACAUGUUCC
970
17445
AUAGGUCCAGACAUGUUCC
970
17463
GGAACAUGUCUGGACCUAU
2621

17463
CUUGGAACUUGUCGCCGUU
971
17463
CUUGGAACUUGUCGCCGUU
971
17481
AACGGCGACAAGUUCCAAG
2622

17481
UGUCCUGCUGAAAUUGUUG
972
17481
UGUCCUGCUGAAAUUGUUG
972
17499
CAACAAUUUCAGCAGGACA
2623

17499
GACACUGUGAGUGCUUUAG
973
17499
GACACUGUGAGUGCUUUAG
973
17517
CUAAAGCACUCACAGUGUC
2624

17517
GUUUAUGACAAUAAGCUAA
974
17517
GUUUAUGACAAUAAGCUAA
974
17535
UUAGCUUAUUGUCAUAAAC
2625

17535
AAAGCACACAAGGAUAAGU
975
17535
AAAGCACACAAGGAUAAGU
975
17553
ACUUAUCCUUGUGUGCUUU
2626

17553
UCAGCUCAAUGCUUCAAAA
976
17553
UCAGCUCAAUGCUUCAAAA
976
17571
UUUUGAAGCAUUGAGCUGA
2627

17571
AUGUUCUACAAAGGUGUUA
977
17571
AUGUUCUACAAAGGUGUUA
977
17589
UAACACCUUUGUAGAACAU
2628

17589
AUUACACAUGAUGUUUCAU
978
17589
AUUACACAUGAUGUUUCAU
978
17607
AUGAAACAUCAUGUGUAAU
2629

17607
UCUGCAAUCAACAGACCUC
979
17607
UCUGCAAUCAACAGACCUC
979
17625
GAGGUCUGUUGAUUGCAGA
2630

17625
CAAAUAGGCGUUGUAAGAG
980
17625
CAAAUAGGCGUUGUAAGAG
980
17643
CUCUUACAACGCCUAUUUG
2631

17643
GAAUUUCUUACACGCAAUC
981
17643
GAAUUUCUUACACGCAAUC
981
17661
GAUUGCGUGUAAGAAAUUC
2632

17661
CCUGCUUGGAGAAAAGCUG
982
17661
CCUGCUUGGAGAAAAGCUG
982
17679
CAGCUUUUCUCCAAGCAGG
2633

17679
GUUUUUAUCUCACCUUAUA
983
17679
GUUUUUAUCUCACCUUAUA
983
17697
UAUAAGGUGAGAUAAAAAC
2634

17697
AAUUCACAGAACGCUGUAG
984
17697
AAUUCACAGAACGCUGUAG
984
17715
CUACAGCGUUCUGUGAAUU
2635

17715
GCUUCAAAAAUCUUAGGAU
985
17715
GCUUCAAAAAUCUUAGGAU
985
17733
AUCCUAAGAUUUUUGAAGC
2636

17733
UUGCCUACGCAGACUGUUG
986
17733
UUGCCUACGCAGACUGUUG
986
17751
CAACAGUCUGCGUAGGCAA
2637

17751
GAUUCAUCACAGGGUUCUG
987
17751
GAUUCAUCACAGGGUUCUG
987
17769
CAGAACCCUGUGAUGAAUC
2638

17769
GAAUAUGACUAUGUCAUAU
988
17769
GAAUAUGACUAUGUCAUAU
988
17787
AUAUGACAUAGUCAUAUUC
2639

17787
UUCACACAAACUACUGAAA
989
17787
UUCACACAAACUACUGAAA
989
17805
UUUCAGUAGUUUGUGUGAA
2640

17805
ACAGCACACUCUUGUAAUG
990
17805
ACAGCACACUCUUGUAAUG
990
17823
CAUUACAAGAGUGUGCUGU
2641

17823
GUCAACCGCUUCAAUGUGG
991
17823
GUCAACCGCUUCAAUGUGG
991
17841
CCACAUUGAAGCGGUUGAC
2642

17841
GCUAUCACAAGGGCAAAAA
992
17841
GCUAUCACAAGGGCAAAAA
992
17859
UUUUUGCCCUUGUGAUAGC
2643

17859
AUUGGCAUUUUGUGCAUAA
993
17859
AUUGGCAUUUUGUGCAUAA
993
17877
UUAUGCACAAAAUGCCAAU
2644

17877
AUGUCUGAUAGAGAUCUUU
994
17877
AUGUCUGAUAGAGAUCUUU
994
17895
AAAGAUCUCUAUCAGACAU
2645

17895
UAUGACAAACUGCAAUUUA
995
17895
UAUGACAAACUGCAAUUUA
995
17913
UAAAUUGCAGUUUGUCAUA
2646

17913
ACAAGUCUAGAAAUACCAC
996
17913
ACAAGUCUAGAAAUACCAC
996
17931
GUGGUAUUUCUAGACUUGU
2647

17931
CGUCGCAAUGUGGCUACAU
997
17931
CGUCGCAAUGUGGCUACAU
997
17949
AUGUAGCCACAUUGCGACG
2648

17949
UUACAAGCAGAAAAUGUAA
998
17949
UUACAAGCAGAAAAUGUAA
998
17967
UUACAUUUUCUGCUUGUAA
2649

17967
ACUGGACUUUUUAAGGACU
999
17967
ACUGGACUUUUUAAGGACU
999
17985
AGUCCUUAAAAAGUCCAGU
2650

17985
UGUAGUAAGAUCAUUACUG
1000
17985
UGUAGUAAGAUCAUUACUG
1000
18003
CAGUAAUGAUCUUACUACA
2651

18003
GGUCUUCAUCCUACACAGG
1001
18003
GGUCUUCAUCCUACACAGG
1001
18021
CCUGUGUAGGAUGAAGACC
2652

18021
GCACCUACACACCUCAGCG
1002
18021
GCACCUACACACCUCAGCG
1002
18039
CGCUGAGGUGUGUAGGUGC
2653

18039
GUUGAUAUAAAGUUCAAGA
1003
18039
GUUGAUAUAAAGUUCAAGA
1003
18057
UCUUGAACUUUAUAUCAAC
2654

18057
ACUGAAGGAUUAUGUGUUG
1004
18057
ACUGAAGGAUUAUGUGUUG
1004
18075
CAACACAUAAUCCUUCAGU
2655

18075
GACAUACCAGGCAUACCAA
1005
18075
GACAUACCAGGCAUACCAA
1005
18093
UUGGUAUGCCUGGUAUGUC
2656

18093
AAGGACAUGACCUACCGUA
1006
18093
AAGGACAUGACCUACCGUA
1006
18111
UACGGUAGGUCAUGUCCUU
2657

18111
AGACUCAUCUCUAUGAUGG
1007
18111
AGACUCAUCUCUAUGAUGG
1007
18129
CCAUCAUAGAGAUGAGUCU
2658

18129
GGUUUCAAAAUGAAUUACC
1008
18129
GGUUUCAAAAUGAAUUACC
1008
18147
GGUAAUUCAUUUUGAAACC
2659

18147
CAAGUCAAUGGUUACCCUA
1009
18147
CAAGUCAAUGGUUACCCUA
1009
18165
UAGGGUAACCAUUGACUUG
2660

18165
AAUAUGUUUAUCACCCGCG
1010
18165
AAUAUGUUUAUCACCCGCG
1010
18183
CGCGGGUGAUAAACAUAUU
2661

18183
GAAGAAGCUAUUCGUCACG
1011
18183
GAAGAAGCUAUUCGUCACG
1011
18201
CGUGACGAAUAGCUUCUUC
2662

18201
GUUCGUGCGUGGAUUGGCU
1012
18201
GUUCGUGCGUGGAUUGGCU
1012
18219
AGCCAAUCCACGCACGAAC
2663

18219
UUUGAUGUAGAGGGCUGUC
1013
18219
UUUGAUGUAGAGGGCUGUC
1013
18237
GACAGCCCUCUACAUCAAA
2664

18237
CAUGCAACUAGAGAUGCUG
1014
18237
CAUGCAACUAGAGAUGCUG
1014
18255
CAGCAUCUCUAGUUGCAUG
2665

18255
GUGGGUACUAACCUACCUC
1015
18255
GUGGGUACUAACCUACCUC
1015
18273
GAGGUAGGUUAGUACCCAC
2666

18273
CUCCAGCUAGGAUUUUCUA
1016
18273
CUCCAGCUAGGAUUUUCUA
1016
18291
UAGAAAAUCCUAGCUGGAG
2667

18291
ACAGGUGUUAACUUAGUAG
1017
18291
ACAGGUGUUAACUUAGUAG
1017
18309
CUACUAAGUUAACACCUGU
2668

18309
GCUGUACCGACUGGUUAUG
1018
18309
GCUGUACCGACUGGUUAUG
1018
18327
CAUAACCAGUCGGUACAGC
2669

18327
GUUGACACUGAAAAUAACA
1019
18327
GUUGACACUGAAAAUAACA
1019
18345
UGUUAUUUUCAGUGUCAAC
2670

18345
ACAGAAUUCACCAGAGUUA
1020
18345
ACAGAAUUCACCAGAGUUA
1020
18363
UAACUCUGGUGAAUUCUGU
2671

18363
AAUGCAAAACCUCCACCAG
1021
18363
AAUGCAAAACCUCCACCAG
1021
18381
CUGGUGGAGGUUUUGCAUU
2672

18381
GGUGACCAGUUUAAACAUC
1022
18381
GGUGACCAGUUUAAACAUC
1022
18399
GAUGUUUAAACUGGUCACC
2673

18399
CUUAUACCACUCAUGUAUA
1023
18399
CUUAUACCACUCAUGUAUA
1023
18417
UAUACAUGAGUGGUAUAAG
2674

18417
AAAGGCUUGCCCUGGAAUG
1024
18417
AAAGGCUUGCCCUGGAAUG
1024
18435
CAUUCCAGGGCAAGCCUUU
2675

18435
GUAGUGCGUAUUAAGAUAG
1025
18435
GUAGUGCGUAUUAAGAUAG
1025
18453
CUAUCUUAAUACGCACUAC
2676

18453
GUACAAAUGCUCAGUGAUA
1026
18453
GUACAAAUGCUCAGUGAUA
1026
18471
UAUCACUGAGCAUUUGUAC
2677

18471
ACACUGAAAGGAUUGUCAG
1027
18471
ACACUGAAAGGAUUGUCAG
1027
18489
CUGACAAUCCUUUCAGUGU
2678

18489
GACAGAGUCGUGUUCGUCC
1028
18489
GACAGAGUCGUGUUCGUCC
1028
18507
GGACGAACACGACUCUGUC
2679

18507
CUUUGGGCGCAUGGCUUUG
1029
18507
CUUUGGGCGCAUGGCUUUG
1029
18525
CAAAGCCAUGCGCCCAAAG
2680

18525
GAGCUUACAUCAAUGAAGU
1030
18525
GAGCUUACAUCAAUGAAGU
1030
18543
ACUUCAUUGAUGUAAGCUC
2681

18543
UACUUUGUCAAGAUUGGAC
1031
18543
UACUUUGUCAAGAUUGGAC
1031
18561
GUCCAAUCUUGACAAAGUA
2682

18561
CCUGAAAGAACGUGUUGUC
1032
18561
CCUGAAAGAACGUGUUGUC
1032
18579
GACAACACGUUCUUUCAGG
2683

18579
CUGUGUGACAAACGUGCAA
1033
18579
CUGUGUGACAAACGUGCAA
1033
18597
UUGCACGUUUGUCACACAG
2684

18597
ACUUGCUUUUCUACUUCAU
1034
18597
ACUUGCUUUUCUACUUCAU
1034
18615
AUGAAGUAGAAAAGCAAGU
2685

18615
UCAGAUACUUAUGCCUGCU
1035
18615
UCAGAUACUUAUGCCUGCU
1035
18633
AGCAGGCAUAAGUAUCUGA
2686

18633
UGGAAUCAUUCUGUGGGUU
1036
18633
UGGAAUCAUUCUGUGGGUU
1036
18651
AACCCACAGAAUGAUUCCA
2687

18651
UUUGACUAUGUCUAUAACC
1037
18651
UUUGACUAUGUCUAUAACC
1037
18669
GGUUAUAGACAUAGUCAAA
2688

18669
CCAUUUAUGAUUGAUGUUC
1038
18669
CCAUUUAUGAUUGAUGUUC
1038
18687
GAACAUCAAUCAUAAAUGG
2689

18687
CAGCAGUGGGGCUUUACGG
1039
18687
CAGCAGUGGGGCUUUACGG
1039
18705
CCGUAAAGCCCCACUGCUG
2690

18705
GGUAACCUUCAGAGUAACC
1040
18705
GGUAACCUUCAGAGUAACC
1040
18723
GGUUACUCUGAAGGUUACC
2691

18723
CAUGACCAACAUUGCCAGG
1041
18723
CAUGACCAACAUUGCCAGG
1041
18741
CCUGGCAAUGUUGGUCAUG
2692

18741
GUACAUGGAAAUGCACAUG
1042
18741
GUACAUGGAAAUGCACAUG
1042
18759
CAUGUGCAUUUCCAUGUAC
2693

18759
GUGGCUAGUUGUGAUGCUA
1043
18759
GUGGCUAGUUGUGAUGCUA
1043
18777
UAGCAUCACAACUAGCCAC
2694

18777
AUCAUGACUAGAUGUUUAG
1044
18777
AUCAUGACUAGAUGUUUAG
1044
18795
CUAAACAUCUAGUCAUGAU
2695

18795
GCAGUCCAUGAGUGCUUUG
1045
18795
GCAGUCCAUGAGUGCUUUG
1045
18813
CAAAGCACUCAUGGACUGC
2696

18813
GUUAAGCGCGUUGAUUGGU
1046
18813
GUUAAGCGCGUUGAUUGGU
1046
18831
ACCAAUCAACGCGCUUAAC
2697

18831
UCUGUUGAAUACCCUAUUA
1047
18831
UCUGUUGAAUACCCUAUUA
1047
18849
UAAUAGGGUAUUCAACAGA
2698

18849
AUAGGAGAUGAACUGAGGG
1048
18849
AUAGGAGAUGAACUGAGGG
1048
18867
CCCUCAGUUCAUCUCCUAU
2699

18867
GUUAAUUCUGCUUGCAGAA
1049
18867
GUUAAUUCUGCUUGCAGAA
1049
18885
UUCUGCAAGCAGAAUUAAC
2700

18885
AAAGUACAACACAUGGUUG
1050
18885
AAAGUACAACACAUGGUUG
1050
18903
CAACCAUGUGUUGUACUUU
2701

18903
GUGAAGUCUGCAUUGCUUG
1051
18903
GUGAAGUCUGCAUUGCUUG
1051
18921
CAAGCAAUGCAGACUUCAC
2702

18921
GCUGAUAAGUUUCCAGUUC
1052
18921
GCUGAUAAGUUUCCAGUUC
1052
18939
GAACUGGAAACUUAUCAGC
2703

18939
CUUCAUGACAUUGGAAAUC
1053
18939
CUUCAUGACAUUGGAAAUC
1053
18957
GAUUUCCAAUGUCAUGAAG
2704

18957
CCAAAGGCUAUCAAGUGUG
1054
18957
CCAAAGGCUAUCAAGUGUG
1054
18975
CACACUUGAUAGCCUUUGG
2705

18975
GUGCCUCAGGCUGAAGUAG
1055
18975
GUGCCUCAGGCUGAAGUAG
1055
18993
CUACUUCAGCCUGAGGCAC
2706

18993
GAAUGGAAGUUCUACGAUG
1056
18993
GAAUGGAAGUUCUACGAUG
1056
19011
CAUCGUAGAACUUCCAUUC
2707

19011
GCUCAGCCAUGUAGUGACA
1057
19011
GCUCAGCCAUGUAGUGACA
1057
19029
UGUCACUACAUGGCUGAGC
2708

19029
AAAGCUUACAAAAUAGAGG
1058
19029
AAAGCUUACAAAAUAGAGG
1058
19047
CCUCUAUUUUGUAAGCUUU
2709

19047
GAACUCUUCUAUUCUUAUG
1059
19047
GAACUCUUCUAUUCUUAUG
1059
19065
CAUAAGAAUAGAAGAGUUC
2710

19065
GCUACACAUCACGAUAAAU
1060
19065
GCUACACAUCACGAUAAAU
1060
19083
AUUUAUCGUGAUGUGUAGC
2711

19083
UUCACUGAUGGUGUUUGUU
1061
19083
UUCACUGAUGGUGUUUGUU
1061
19101
AACAAACACCAUCAGUGAA
2712

19101
UUGUUUUGGAAUUGUAACG
1062
19101
UUGUUUUGGAAUUGUAACG
1062
19119
CGUUACAAUUCCAAAACAA
2713

19119
GUUGAUCGUUACCCAGCCA
1063
19119
GUUGAUCGUUACCCAGCCA
1063
19137
UGGCUGGGUAACGAUCAAC
2714

19137
AAUGCAAUUGUGUGUAGGU
1064
19137
AAUGCAAUUGUGUGUAGGU
1064
19155
ACCUACACACAAUUGCAUU
2715

19155
UUUGACACAAGAGUCUUGU
1065
19155
UUUGACACAAGAGUCUUGU
1065
19173
ACAAGACUCUUGUGUCAAA
2716

19173
UCAAACUUGAACUUACCAG
1066
19173
UCAAACUUGAACUUACCAG
1066
19191
CUGGUAAGUUCAAGUUUGA
2717

19191
GGCUGUGAUGGUGGUAGUU
1067
19191
GGCUGUGAUGGUGGUAGUU
1067
19209
AACUACCACCAUCACAGCC
2718

19209
UUGUAUGUGAAUAAGCAUG
1068
19209
UUGUAUGUGAAUAAGCAUG
1068
19227
CAUGCUUAUUCACAUACAA
2719

19227
GCAUUCCACACUCCAGCUU
1069
19227
GCAUUCCACACUCCAGCUU
1069
19245
AAGCUGGAGUGUGGAAUGC
2720

19245
UUCGAUAAAAGUGCAUUUA
1070
19245
UUCGAUAAAAGUGCAUUUA
1070
19263
UAAAUGCACUUUUAUCGAA
2721

19263
ACUAAUUUAAAGCAAUUGC
1071
19263
ACUAAUUUAAAGCAAUUGC
1071
19281
GCAAUUGCUUUAAAUUAGU
2722

19281
CCUUUCUUUUACUAUUCUG
1072
19281
CCUUUCUUUUACUAUUCUG
1072
19299
CAGAAUAGUAAAAGAAAGG
2723

19299
GAUAGUCCUUGUGAGUCUC
1073
19299
GAUAGUCCUUGUGAGUCUC
1073
19317
GAGACUCACAAGGACUAUC
2724

19317
CAUGGCAAACAAGUAGUGU
1074
19317
CAUGGCAAACAAGUAGUGU
1074
19335
ACACUACUUGUUUGCCAUG
2725

19335
UCGGAUAUUGAUUAUGUUC
1075
19335
UCGGAUAUUGAUUAUGUUC
1075
19353
GAACAUAAUCAAUAUCCGA
2726

19353
CCACUCAAAUCUGCUACGU
1076
19353
CCACUCAAAUCUGCUACGU
1076
19371
ACGUAGCAGAUUUGAGUGG
2727

19371
UGUAUUACACGAUGCAAUU
1077
19371
UGUAUUACACGAUGCAAUU
1077
19389
AAUUGCAUCGUGUAAUACA
2728

19389
UUAGGUGGUGCUGUUUGCA
1078
19389
UUAGGUGGUGCUGUUUGCA
1078
19407
UGCAAACAGCACCACCUAA
2729

19407
AGACACCAUGCAAAUGAGU
1079
19407
AGACACCAUGCAAAUGAGU
1079
19425
ACUCAUUUGCAUGGUGUCU
2730

19425
UACCGACAGUACUUGGAUG
1080
19425
UACCGACAGUACUUGGAUG
1080
19443
CAUCCAAGUACUGUCGGUA
2731

19443
GCAUAUAAUAUGAUGAUUU
1081
19443
GCAUAUAAUAUGAUGAUUU
1081
19461
AAAUCAUCAUAUUAUAUGC
2732

19461
UCUGCUGGAUUUAGCCUAU
1082
19461
UCUGCUGGAUUUAGCCUAU
1082
19479
AUAGGCUAAAUCCAGCAGA
2733

19479
UGGAUUUACAAACAAUUUG
1083
19479
UGGAUUUACAAACAAUUUG
1083
19497
CAAAUUGUUUGUAAAUCCA
2734

19497
GAUACUUAUAACCUGUGGA
1084
19497
GAUACUUAUAACCUGUGGA
1084
19515
UCCACAGGUUAUAAGUAUC
2735

19515
AAUACAUUUACCAGGUUAC
1085
19515
AAUACAUUUACCAGGUUAC
1085
19533
GUAACCUGGUAAAUGUAUU
2736

19533
CAGAGUUUAGAAAAUGUGG
1086
19533
CAGAGUUUAGAAAAUGUGG
1086
19551
CCACAUUUUCUAAACUCUG
2737

19551
GCUUAUAAUGUUGUUAAUA
1087
19551
GCUUAUAAUGUUGUUAAUA
1087
19569
UAUUAACAACAUUAUAAGC
2738

19569
AAAGGACACUUUGAUGGAC
1088
19569
AAAGGACACUUUGAUGGAC
1088
19587
GUCCAUCAAAGUGUCCUUU
2739

19587
CACGCCGGCGAAGCACCUG
1089
19587
CACGCCGGCGAAGCACCUG
1089
19605
CAGGUGCUUCGCCGGCGUG
2740

19605
GUUUCCAUCAUUAAUAAUG
1090
19605
GUUUCCAUCAUUAAUAAUG
1090
19623
CAUUAUUAAUGAUGGAAAC
2741

19623
GCUGUUUACACAAAGGUAG
1091
19623
GCUGUUUACACAAAGGUAG
1091
19641
CUACCUUUGUGUAAACAGC
2742

19641
GAUGGUAUUGAUGUGGAGA
1092
19641
GAUGGUAUUGAUGUGGAGA
1092
19659
UCUCCACAUCAAUACCAUC
2743

19659
AUCUUUGAAAAUAAGACAA
1093
19659
AUCUUUGAAAAUAAGACAA
1093
19677
UUGUCUUAUUUUCAAAGAU
2744

19677
ACACUUCCUGUUAAUGUUG
1094
19677
ACACUUCCUGUUAAUGUUG
1094
19695
CAACAUUAACAGGAAGUGU
2745

19695
GCAUUUGAGCUUUGGGCUA
1095
19695
GCAUUUGAGCUUUGGGCUA
1095
19713
UAGCCCAAAGCUCAAAUGC
2746

19713
AAGCGUAACAUUAAACCAG
1096
19713
AAGCGUAACAUUAAACCAG
1096
19731
CUGGUUUAAUGUUACGCUU
2747

19731
GUGCCAGAGAUUAAGAUAC
1097
19731
GUGCCAGAGAUUAAGAUAC
1097
19749
GUAUCUUAAUCUCUGGCAC
2748

19749
CUCAAUAAUUUGGGUGUUG
1098
19749
CUCAAUAAUUUGGGUGUUG
1098
19767
CAACACCCAAAUUAUUGAG
2749

19767
GAUAUCGCUGCUAAUACUG
1099
19767
GAUAUCGCUGCUAAUACUG
1099
19785
CAGUAUUAGCAGCGAUAUC
2750

19785
GUAAUCUGGGACUACAAAA
1100
19785
GUAAUCUGGGACUACAAAA
1100
19803
UUUUGUAGUCCCAGAUUAC
2751

19803
AGAGAAGCCCCAGCACAUG
1101
19803
AGAGAAGCCCCAGCACAUG
1101
19821
CAUGUGCUGGGGCUUCUCU
2752

19821
GUAUCUACAAUAGGUGUCU
1102
19821
GUAUCUACAAUAGGUGUCU
1102
19839
AGACACCUAUUGUAGAUAC
2753

19839
UGCACAAUGACUGACAUUG
1103
19839
UGCACAAUGACUGACAUUG
1103
19857
CAAUGUCAGUCAUUGUGCA
2754

19857
GCCAAGAAACCUACUGAGA
1104
19857
GCCAAGAAACCUACUGAGA
1104
19875
UCUCAGUAGGUUUCUUGGC
2755

19875
AGUGCUUGUUCUUCACUUA
1105
19875
AGUGCUUGUUCUUCACUUA
1105
19893
UAAGUGAAGAACAAGCACU
2756

19893
ACUGUCUUGUUUGAUGGUA
1106
19893
ACUGUCUUGUUUGAUGGUA
1106
19911
UACCAUCAAACAAGACAGU
2757

19911
AGAGUGGAAGGACAGGUAG
1107
19911
AGAGUGGAAGGACAGGUAG
1107
19929
CUACCUGUCCUUCCACUCU
2758

19929
GACCUUUUUAGAAACGCCC
1108
19929
GACCUUUUUAGAAACGCCC
1108
19947
GGGCGUUUCUAAAAAGGUC
2759

19947
CGUAAUGGUGUUUUAAUAA
1109
19947
CGUAAUGGUGUUUUAAUAA
1109
19965
UUAUUAAAACACCAUUACG
2760

19965
ACAGAAGGUUCAGUCAAAG
1110
19965
ACAGAAGGUUCAGUCAAAG
1110
19983
CUUUGACUGAACCUUCUGU
2761

19983
GGUCUAACACCUUCAAAGG
1111
19983
GGUCUAACACCUUCAAAGG
1111
20001
CCUUUGAAGGUGUUAGACC
2762

20001
GGACCAGCACAAGCUAGCG
1112
20001
GGACCAGCACAAGCUAGCG
1112
20019
CGCUAGCUUGUGCUGGUCC
2763

20019
GUCAAUGGAGUCACAUUAA
1113
20019
GUCAAUGGAGUCACAUUAA
1113
20037
UUAAUGUGACUCCAUUGAC
2764

20037
AUUGGAGAAUCAGUAAAAA
1114
20037
AUUGGAGAAUCAGUAAAAA
1114
20055
UUUUUACUGAUUCUCCAAU
2765

20055
ACACAGUUUAACUACUUUA
1115
20055
ACACAGUUUAACUACUUUA
1115
20073
UAAAGUAGUUAAACUGUGU
2766

20073
AAGAAAGUAGACGGCAUUA
1116
20073
AAGAAAGUAGACGGCAUUA
1116
20091
UAAUGCCGUCUACUUUCUU
2767

20091
AUUCAACAGUUGCCUGAAA
1117
20091
AUUCAACAGUUGCCUGAAA
1117
20109
UUUCAGGCAACUGUUGAAU
2768

20109
ACCUACUUUACUCAGAGCA
1118
20109
ACCUACUUUACUCAGAGCA
1118
20127
UGCUCUGAGUAAAGUAGGU
2769

20127
AGAGACUUAGAGGAUUUUA
1119
20127
AGAGACUUAGAGGAUUUUA
1119
20145
UAAAAUCCUCUAAGUCUCU
2770

20145
AAGCCCAGAUCACAAAUGG
1120
20145
AAGCCCAGAUCACAAAUGG
1120
20163
CCAUUUGUGAUCUGGGCUU
2771

20163
GAAACUGACUUUCUCGAGC
1121
20163
GAAACUGACUUUCUCGAGC
1121
20181
GCUCGAGAAAGUCAGUUUC
2772

20181
CUCGCUAUGGAUGAAUUCA
1122
20181
CUCGCUAUGGAUGAAUUCA
1122
20199
UGAAUUCAUCCAUAGCGAG
2773

20199
AUACAGCGAUAUAAGCUCG
1123
20199
AUACAGCGAUAUAAGCUCG
1123
20217
CGAGCUUAUAUCGCUGUAU
2774

20217
GAGGGCUAUGCCUUCGAAC
1124
20217
GAGGGCUAUGCCUUCGAAC
1124
20235
GUUCGAAGGCAUAGCCCUC
2775

20235
CACAUCGUUUAUGGAGAUU
1125
20235
CACAUCGUUUAUGGAGAUU
1125
20253
AAUCUCCAUAAACGAUGUG
2776

20253
UUCAGUCAUGGACAACUUG
1126
20253
UUCAGUCAUGGACAACUUG
1126
20271
CAAGUUGUCCAUGACUGAA
2777

20271
GGCGGUCUUCAUUUAAUGA
1127
20271
GGCGGUCUUCAUUUAAUGA
1127
20289
UCAUUAAAUGAAGACCGCC
2778

20289
AUAGGCUUAGCCAAGCGCU
1128
20289
AUAGGCUUAGCCAAGCGCU
1128
20307
AGCGCUUGGCUAAGCCUAU
2779

20307
UCACAAGAUUCACCACUUA
1129
20307
UCACAAGAUUCACCACUUA
1129
20325
UAAGUGGUGAAUCUUGUGA
2780

20325
AAAUUAGAGGAUUUUAUCC
1130
20325
AAAUUAGAGGAUUUUAUCC
1130
20343
GGAUAAAAUCCUCUAAUUU
2781

20343
CCUAUGGACAGCACAGUGA
1131
20343
CCUAUGGACAGCACAGUGA
1131
20361
UCACUGUGCUGUCCAUAGG
2782

20361
AAAAAUUACUUCAUAACAG
1132
20361
AAAAAUUACUUCAUAACAG
1132
20379
CUGUUAUGAAGUAAUUUUU
2783

20379
GAUGCGCAAACAGGUUCAU
1133
20379
GAUGCGCAAACAGGUUCAU
1133
20397
AUGAACCUGUUUGCGCAUC
2784

20397
UCAAAAUGUGUGUGUUCUG
1134
20397
UCAAAAUGUGUGUGUUCUG
1134
20415
CAGAACACACACAUUUUGA
2785

20415
GUGAUUGAUCUUUUACUUG
1135
20415
GUGAUUGAUCUUUUACUUG
1135
20433
CAAGUAAAAGAUCAAUCAC
2786

20433
GAUGACUUUGUCGAGAUAA
1136
20433
GAUGACUUUGUCGAGAUAA
1136
20451
UUAUCUCGACAAAGUCAUC
2787

20451
AUAAAGUCACAAGAUUUGU
1137
20451
AUAAAGUCACAAGAUUUGU
1137
20469
ACAAAUCUUGUGACUUUAU
2788

20469
UCAGUGAUUUCAAAAGUGG
1138
20469
UCAGUGAUUUCAAAAGUGG
1138
20487
CCACUUUUGAAAUCACUGA
2789

20487
GUCAAGGUUACAAUUGACU
1139
20487
GUCAAGGUUACAAUUGACU
1139
20505
AGUCAAUUGUAACCUUGAC
2790

20505
UAUGCUGAAAUUUCAUUCA
1140
20505
UAUGCUGAAAUUUCAUUCA
1140
20523
UGAAUGAAAUUUCAGCAUA
2791

20523
AUGCUUUGGUGUAAGGAUG
1141
20523
AUGCUUUGGUGUAAGGAUG
1141
20541
CAUCCUUACACCAAAGCAU
2792

20541
GGACAUGUUGAAACCUUCU
1142
20541
GGACAUGUUGAAACCUUCU
1142
20559
AGAAGGUUUCAACAUGUCC
2793

20559
UACCCAAAACUACAAGCAA
1143
20559
UACCCAAAACUACAAGCAA
1143
20577
UUGCUUGUAGUUUUGGGUA
2794

20577
AGUCGAGCGUGGCAACCAG
1144
20577
AGUCGAGCGUGGCAACCAG
1144
20595
CUGGUUGCCACGCUCGACU
2795

20595
GGUGUUGCGAUGCCUAACU
1145
20595
GGUGUUGCGAUGCCUAACU
1145
20613
AGUUAGGCAUCGCAACACC
2796

20613
UUGUACAAGAUGCAAAGAA
1146
20613
UUGUACAAGAUGCAAAGAA
1146
20631
UUCUUUGCAUCUUGUACAA
2797

20631
AUGCUUCUUGAAAAGUGUG
1147
20631
AUGCUUCUUGAAAAGUGUG
1147
20649
CACACUUUUCAAGAAGCAU
2798

20649
GACCUUCAGAAUUAUGGUG
1148
20649
GACCUUCAGAAUUAUGGUG
1148
20667
CACCAUAAUUCUGAAGGUC
2799

20667
GAAAAUGCUGUUAUACCAA
1149
20667
GAAAAUGCUGUUAUACCAA
1149
20685
UUGGUAUAACAGCAUUUUC
2800

20685
AAAGGAAUAAUGAUGAAUG
1150
20685
AAAGGAAUAAUGAUGAAUG
1150
20703
CAUUCAUCAUUAUUCCUUU
2801

20703
GUCGCAAAGUAUACUCAAC
1151
20703
GUCGCAAAGUAUACUCAAC
1151
20721
GUUGAGUAUACUUUGCGAC
2802

20721
CUGUGUCAAUACUUAAAUA
1152
20721
CUGUGUCAAUACUUAAAUA
1152
20739
UAUUUAAGUAUUGACACAG
2803

20739
ACACUUACUUUAGCUGUAC
1153
20739
ACACUUACUUUAGCUGUAC
1153
20757
GUACAGCUAAAGUAAGUGU
2804

20757
CCCUACAACAUGAGAGUUA
1154
20757
CCCUACAACAUGAGAGUUA
1154
20775
UAACUCUCAUGUUGUAGGG
2805

20775
AUUCACUUUGGUGCUGGCU
1155
20775
AUUCACUUUGGUGCUGGCU
1155
20793
AGCCAGCACCAAAGUGAAU
2806

20793
UCUGAUAAAGGAGUUGCAC
1156
20793
UCUGAUAAAGGAGUUGCAC
1156
20811
GUGCAACUCCUUUAUCAGA
2807

20811
CCAGGUACAGCUGUGCUCA
1157
20811
CCAGGUACAGCUGUGCUCA
1157
20829
UGAGCACAGCUGUACCUGG
2808

20829
AGACAAUGGUUGCCAACUG
1158
20829
AGACAAUGGUUGCCAACUG
1158
20847
CAGUUGGCAACCAUUGUCU
2809

20847
GGCACACUACUUGUCGAUU
1159
20847
GGCACACUACUUGUCGAUU
1159
20865
AAUCGACAAGUAGUGUGCC
2810

20865
UCAGAUCUUAAUGACUUCG
1160
20865
UCAGAUCUUAAUGACUUCG
1160
20883
CGAAGUCAUUAAGAUCUGA
2811

20883
GUCUCCGACGCAUAUUCUA
1161
20883
GUCUCCGACGCAUAUUCUA
1161
20901
UAGAAUAUGCGUCGGAGAC
2812

20901
ACUUUAAUUGGAGACUGUG
1162
20901
ACUUUAAUUGGAGACUGUG
1162
20919
CACAGUCUCCAAUUAAAGU
2813

20919
GCAACAGUACAUACGGCUA
1163
20919
GCAACAGUACAUACGGCUA
1163
20937
UAGCCGUAUGUACUGUUGC
2814

20937
AAUAAAUGGGACCUUAUUA
1164
20937
AAUAAAUGGGACCUUAUUA
1164
20955
UAAUAAGGUCCCAUUUAUU
2815

20955
AUUAGCGAUAUGUAUGACC
1165
20955
AUUAGCGAUAUGUAUGACC
1165
20973
GGUCAUACAUAUCGCUAAU
2816

20973
CCUAGGACCAAACAUGUGA
1166
20973
CCUAGGACCAAACAUGUGA
1166
20991
UCACAUGUUUGGUCCUAGG
2817

20991
ACAAAAGAGAAUGACUCUA
1167
20991
ACAAAAGAGAAUGACUCUA
1167
21009
UAGAGUCAUUCUCUUUUGU
2818

21009
AAAGAAGGGUUUUUCACUU
1168
21009
AAAGAAGGGUUUUUCACUU
1168
21027
AAGUGAAAAACCCUUCUUU
2819

21027
UAUCUGUGUGGAUUUAUAA
1169
21027
UAUCUGUGUGGAUUUAUAA
1169
21045
UUAUAAAUCCACACAGAUA
2820

21045
AAGCAAAAACUAGCCCUGG
1170
21045
AAGCAAAAACUAGCCCUGG
1170
21063
CCAGGGCUAGUUUUUGCUU
2821

21063
GGUGGUUCUAUAGCUGUAA
1171
21063
GGUGGUUCUAUAGCUGUAA
1171
21081
UUACAGCUAUAGAACCACC
2822

21081
AAGAUAACAGAGCAUUCUU
1172
21081
AAGAUAACAGAGCAUUCUU
1172
21099
AAGAAUGCUCUGUUAUCUU
2823

21099
UGGAAUGCUGACCUUUACA
1173
21099
UGGAAUGCUGACCUUUACA
1173
21117
UGUAAAGGUCAGCAUUCCA
2824

21117
AAGCUUAUGGGCCAUUUCU
1174
21117
AAGCUUAUGGGCCAUUUCU
1174
21135
AGAAAUGGCCCAUAAGCUU
2825

21135
UCAUGGUGGACAGCUUUUG
1175
21135
UCAUGGUGGACAGCUUUUG
1175
21153
CAAAAGCUGUCCACCAUGA
2826

21153
GUUACAAAUGUAAAUGCAU
1176
21153
GUUACAAAUGUAAAUGCAU
1176
21171
AUGCAUUUACAUUUGUAAC
2827

21171
UCAUCAUCGGAAGCAUUUU
1177
21171
UCAUCAUCGGAAGCAUUUU
1177
21189
AAAAUGCUUCCGAUGAUGA
2828

21189
UUAAUUGGGGCUAACUAUC
1178
21189
UUAAUUGGGGCUAACUAUC
1178
21207
GAUAGUUAGCCCCAAUUAA
2829

21207
CUUGGCAAGCCGAAGGAAC
1179
21207
CUUGGCAAGCCGAAGGAAC
1179
21225
GUUCCUUCGGCUUGCCAAG
2830

21225
CAAAUUGAUGGCUAUACCA
1180
21225
CAAAUUGAUGGCUAUACCA
1180
21243
UGGUAUAGCCAUCAAUUUG
2831

21243
AUGCAUGCUAACUACAUUU
1181
21243
AUGCAUGCUAACUACAUUU
1181
21261
AAAUGUAGUUAGCAUGCAU
2832

21261
UUCUGGAGGAACACAAAUC
1182
21261
UUCUGGAGGAACACAAAUC
1182
21279
GAUUUGUGUUCCUCCAGAA
2833

21279
CCUAUCCAGUUGUCUUCCU
1183
21279
CCUAUCCAGUUGUCUUCCU
1183
21297
AGGAAGACAACUGGAUAGG
2834

21297
UAUUCACUCUUUGACAUGA
1184
21297
UAUUCACUCUUUGACAUGA
1184
21315
UCAUGUCAAAGAGUGAAUA
2835

21315
AGCAAAUUUCCUCUUAAAU
1185
21315
AGCAAAUUUCCUCUUAAAU
1185
21333
AUUUAAGAGGAAAUUUGCU
2836

21333
UUAAGAGGAACUGCUGUAA
1186
21333
UUAAGAGGAACUGCUGUAA
1186
21351
UUACAGCAGUUCCUCUUAA
2837

21351
AUGUCUCUUAAGGAGAAUC
1187
21351
AUGUCUCUUAAGGAGAAUC
1187
21369
GAUUCUCCUUAAGAGACAU
2838

21369
CAAAUCAAUGAUAUGAUUU
1188
21369
CAAAUCAAUGAUAUGAUUU
1188
21387
AAAUCAUAUCAUUGAUUUG
2839

21387
UAUUCUCUUCUGGAAAAAG
1189
21387
UAUUCUCUUCUGGAAAAAG
1189
21405
CUUUUUCCAGAAGAGAAUA
2840

21405
GGUAGGCUUAUCAUUAGAG
1190
21405
GGUAGGCUUAUCAUUAGAG
1190
21423
CUCUAAUGAUAAGCCUACC
2841

21423
GAAAACAACAGAGUUGUGG
1191
21423
GAAAACAACAGAGUUGUGG
1191
21441
CCACAACUCUGUUGUUUUC
2842

21441
GUUUCAAGUGAUAUUCUUG
1192
21441
GUUUCAAGUGAUAUUCUUG
1192
21459
CAAGAAUAUCACUUGAAAC
2843

21459
GUUAACAACUAAACGAACA
1193
21459
GUUAACAACUAAACGAACA
1193
21477
UGUUCGUUUAGUUGUUAAC
2844

21477
AUGUUUAUUUUCUUAUUAU
1194
21477
AUGUUUAUUUUCUUAUUAU
1194
21495
AUAAUAAGAAAAUAAACAU
2845

21495
UUUCUUACUCUCACUAGUG
1195
21495
UUUCUUACUCUCACUAGUG
1195
21513
CACUAGUGAGAGUAAGAAA
2846

21513
GGUAGUGACCUUGACCGGU
1196
21513
GGUAGUGACCUUGACCGGU
1196
21531
ACCGGUCAAGGUCACUACC
2847

21531
UGCACCACUUUUGAUGAUG
1197
21531
UGCACCACUUUUGAUGAUG
1197
21549
CAUCAUCAAAAGUGGUGCA
2848

21549
GUUCAAGCUCCUAAUUACA
1198
21549
GUUCAAGCUCCUAAUUACA
1198
21567
UGUAAUUAGGAGCUUGAAC
2849

21567
ACUCAACAUACUUCAUCUA
1199
21567
ACUCAACAUACUUCAUCUA
1199
21585
UAGAUGAAGUAUGUUGAGU
2850

21585
AUGAGGGGGGUUUACUAUC
1200
21585
AUGAGGGGGGUUUACUAUC
1200
21603
GAUAGUAAACCCCCCUCAU
2851

21603
CCUGAUGAAAUUUUUAGAU
1201
21603
CCUGAUGAAAUUUUUAGAU
1201
21621
AUCUAAAAAUUUCAUCAGG
2852

21621
UCAGACACUCUUUAUUUAA
1202
21621
UCAGACACUCUUUAUUUAA
1202
21639
UUAAAUAAAGAGUGUCUGA
2853

21639
ACUCAGGAUUUAUUUCUUC
1203
21639
ACUCAGGAUUUAUUUCUUC
1203
21657
GAAGAAAUAAAUCCUGAGU
2854

21657
CCAUUUUAUUCUAAUGUUA
1204
21657
CCAUUUUAUUCUAAUGUUA
1204
21675
UAACAUUAGAAUAAAAUGG
2855

21675
ACAGGGUUUCAUACUAUUA
1205
21675
ACAGGGUUUCAUACUAUUA
1205
21693
UAAUAGUAUGAAACCCUGU
2856

21693
AAUCAUACGUUUGGCAACC
1206
21693
AAUCAUACGUUUGGCAACC
1206
21711
GGUUGCCAAACGUAUGAUU
2857

21711
CCUGUCAUACCUUUUAAGG
1207
21711
CCUGUCAUACCUUUUAAGG
1207
21729
CCUUAAAAGGUAUGACAGG
2858

21729
GAUGGUAUUUAUUUUGCUG
1208
21729
GAUGGUAUUUAUUUUGCUG
1208
21747
CAGCAAAAUAAAUACCAUC
2859

21747
GCCACAGAGAAAUCAAAUG
1209
21747
GCCACAGAGAAAUCAAAUG
1209
21765
CAUUUGAUUUCUCUGUGGC
2860

21765
GUUGUCCGUGGUUGGGUUU
1210
21765
GUUGUCCGUGGUUGGGUUU
1210
21783
AAACCCAACCACGGACAAC
2861

21783
UUUGGUUCUACCAUGAACA
1211
21783
UUUGGUUCUACCAUGAACA
1211
21801
UGUUCAUGGUAGAACCAAA
2862

21801
AACAAGUCACAGUCGGUGA
1212
21801
AACAAGUCACAGUCGGUGA
1212
21819
UCACCGACUGUGACUUGUU
2863

21819
AUUAUUAUUAACAAUUCUA
1213
21819
AUUAUUAUUAACAAUUCUA
1213
21837
UAGAAUUGUUAAUAAUAAU
2864

21837
ACUAAUGUUGUUAUACGAG
1214
21837
ACUAAUGUUGUUAUACGAG
1214
21855
CUCGUAUAACAACAUUAGU
2865

21855
GCAUGUAACUUUGAAUUGU
1215
21855
GCAUGUAACUUUGAAUUGU
1215
21873
ACAAUUCAAAGUUACAUGC
2866

21873
UGUGACAACCCUUUCUUUG
1216
21873
UGUGACAACCCUUUCUUUG
1216
21891
CAAAGAAAGGGUUGUCACA
2867

21891
GCUGUUUCUAAACCCAUGG
1217
21891
GCUGUUUCUAAACCCAUGG
1217
21909
CCAUGGGUUUAGAAACAGC
2868

21909
GGUACACAGACACAUACUA
1218
21909
GGUACACAGACACAUACUA
1218
21927
UAGUAUGUGUCUGUGUACC
2869

21927
AUGAUAUUCGAUAAUGCAU
1219
21927
AUGAUAUUCGAUAAUGCAU
1219
21945
AUGCAUUAUCGAAUAUCAU
2870

21945
UUUAAUUGCACUUUCGAGU
1220
21945
UUUAAUUGCACUUUCGAGU
1220
21963
ACUCGAAAGUGCAAUUAAA
2871

21963
UACAUAUCUGAUGCCUUUU
1221
21963
UACAUAUCUGAUGCCUUUU
1221
21981
AAAAGGCAUCAGAUAUGUA
2872

21981
UCGCUUGAUGUUUCAGAAA
1222
21981
UCGCUUGAUGUUUCAGAAA
1222
21999
UUUCUGAAACAUCAAGCGA
2873

21999
AAGUCAGGUAAUUUUAAAC
1223
21999
AAGUCAGGUAAUUUUAAAC
1223
22017
GUUUAAAAUUACCUGACUU
2874

22017
CACUUACGAGAGUUUGUGU
1224
22017
CACUUACGAGAGUUUGUGU
1224
22035
ACACAAACUCUCGUAAGUG
2875

22035
UUUAAAAAUAAAGAUGGGU
1225
22035
UUUAAAAAUAAAGAUGGGU
1225
22053
ACCCAUCUUUAUUUUUAAA
2876

22053
UUUCUCUAUGUUUAUAAGG
1226
22053
UUUCUCUAUGUUUAUAAGG
1226
22071
CCUUAUAAACAUAGAGAAA
2877

22071
GGCUAUCAACCUAUAGAUG
1227
22071
GGCUAUCAACCUAUAGAUG
1227
22089
CAUCUAUAGGUUGAUAGCC
2878

22089
GUAGUUCGUGAUCUACCUU
1228
22089
GUAGUUCGUGAUCUACCUU
1228
22107
AAGGUAGAUCACGAACUAC
2879

22107
UCUGGUUUUAACACUUUGA
1229
22107
UCUGGUUUUAACACUUUGA
1229
22125
UCAAAGUGUUAAAACCAGA
2880

22125
AAACCUAUUUUUAAGUUGC
1230
22125
AAACCUAUUUUUAAGUUGC
1230
22143
GCAACUUAAAAAUAGGUUU
2881

22143
CCUCUUGGUAUUAACAUUA
1231
22143
CCUCUUGGUAUUAACAUUA
1231
22161
UAAUGUUAAUACCAAGAGG
2882

22161
ACAAAUUUUAGAGCCAUUC
1232
22161
ACAAAUUUUAGAGCCAUUC
1232
22179
GAAUGGCUCUAAAAUUUGU
2883

22179
CUUACAGCCUUUUCACCUG
1233
22179
CUUACAGCCUUUUCACCUG
1233
22197
CAGGUGAAAAGGCUGUAAG
2884

22197
GCUCAAGACAUUUGGGGCA
1234
22197
GCUCAAGACAUUUGGGGCA
1234
22215
UGCCCCAAAUGUCUUGAGC
2885

22215
ACGUCAGCUGCAGCCUAUU
1235
22215
ACGUCAGCUGCAGCCUAUU
1235
22233
AAUAGGCUGCAGCUGACGU
2886

22233
UUUGUUGGCUAUUUAAAGC
1236
22233
UUUGUUGGCUAUUUAAAGC
1236
22251
GCUUUAAAUAGCCAACAAA
2887

22251
CCAACUACAUUUAUGCUCA
1237
22251
CCAACUACAUUUAUGCUCA
1237
22269
UGAGCAUAAAUGUAGUUGG
2888

22269
AAGUAUGAUGAAAAUGGUA
1238
22269
AAGUAUGAUGAAAAUGGUA
1238
22287
UACCAUUUUCAUCAUACUU
2889

22287
ACAAUCACAGAUGCUGUUG
1239
22287
ACAAUCACAGAUGCUGUUG
1239
22305
CAACAGCAUCUGUGAUUGU
2890

22305
GAUUGUUCUCAAAAUCCAC
1240
22305
GAUUGUUCUCAAAAUCCAC
1240
22323
GUGGAUUUUGAGAACAAUC
2891

22323
CUUGCUGAACUCAAAUGCU
1241
22323
CUUGCUGAACUCAAAUGCU
1241
22341
AGCAUUUGAGUUCAGCAAG
2892

22341
UCUGUUAAGAGCUUUGAGA
1242
22341
UCUGUUAAGAGCUUUGAGA
1242
22359
UCUCAAAGCUCUUAACAGA
2893

22359
AUUGACAAAGGAAUUUACC
1243
22359
AUUGACAAAGGAAUUUACC
1243
22377
GGUAAAUUCCUUUGUCAAU
2894

22377
CAGACCUCUAAUUUCAGGG
1244
22377
CAGACCUCUAAUUUCAGGG
1244
22395
CCCUGAAAUUAGAGGUCUG
2895

22395
GUUGUUCCCUCAGGAGAUG
1245
22395
GUUGUUCCCUCAGGAGAUG
1245
22413
CAUCUCCUGAGGGAACAAC
2896

22413
GUUGUGAGAUUCCCUAAUA
1246
22413
GUUGUGAGAUUCCCUAAUA
1246
22431
UAUUAGGGAAUCUCACAAC
2897

22431
AUUACAAACUUGUGUCCUU
1247
22431
AUUACAAACUUGUGUCCUU
1247
22449
AAGGACACAAGUUUGUAAU
2898

22449
UUUGGAGAGGUUUUUAAUG
1248
22449
UUUGGAGAGGUUUUUAAUG
1248
22467
CAUUAAAAACCUCUCCAAA
2899

22467
GCUACUAAAUUCCCUUCUG
1249
22467
GCUACUAAAUUCCCUUCUG
1249
22485
CAGAAGGGAAUUUAGUAGC
2900

22485
GUCUAUGCAUGGGAGAGAA
1250
22485
GUCUAUGCAUGGGAGAGAA
1250
22503
UUCUCUCCCAUGCAUAGAC
2901

22503
AAAAAAAUUUCUAAUUGUG
1251
22503
AAAAAAAUUUCUAAUUGUG
1251
22521
CACAAUUAGAAAUUUUUUU
2902

22521
GUUGCUGAUUACUCUGUGC
1252
22521
GUUGCUGAUUACUCUGUGC
1252
22539
GCACAGAGUAAUCAGCAAC
2903

22539
CUCUACAACUCAACAUUUU
1253
22539
CUCUACAACUCAACAUUUU
1253
22557
AAAAUGUUGAGUUGUAGAG
2904

22557
UUUUCAACCUUUAAGUGCU
1254
22557
UUUUCAACCUUUAAGUGCU
1254
22575
AGCACUUAAAGGUUGAAAA
2905

22575
UAUGGCGUUUCUGCCACUA
1255
22575
UAUGGCGUUUCUGCCACUA
1255
22593
UAGUGGCAGAAACGCCAUA
2906

22593
AAGUUGAAUGAUCUUUGCU
1256
22593
AAGUUGAAUGAUCUUUGCU
1256
22611
AGCAAAGAUCAUUCAACUU
2907

22611
UUCUCCAAUGUCUAUGCAG
1257
22611
UUCUCCAAUGUCUAUGCAG
1257
22629
CUGCAUAGACAUUGGAGAA
2908

22629
GAUUCUUUUGUAGUCAAGG
1258
22629
GAUUCUUUUGUAGUCAAGG
1258
22647
CCUUGACUACAAAAGAAUC
2909

22647
GGAGAUGAUGUAAGACAAA
1259
22647
GGAGAUGAUGUAAGACAAA
1259
22665
UUUGUCUUACAUCAUCUCC
2910

22665
AUAGCGCCAGGACAAACUG
1260
22665
AUAGCGCCAGGACAAACUG
1260
22683
CAGUUUGUCCUGGCGCUAU
2911

22683
GGUGUUAUUGCUGAUUAUA
1261
22683
GGUGUUAUUGCUGAUUAUA
1261
22701
UAUAAUCAGCAAUAACACC
2912

22701
AAUUAUAAAUUGCCAGAUG
1262
22701
AAUUAUAAAUUGCCAGAUG
1262
22719
CAUCUGGCAAUUUAUAAUU
2913

22719
GAUUUCAUGGGUUGUGUCC
1263
22719
GAUUUCAUGGGUUGUGUCC
1263
22737
GGACACAACCCAUGAAAUC
2914

22737
CUUGCUUGGAAUACUAGGA
1264
22737
CUUGCUUGGAAUACUAGGA
1264
22755
UCCUAGUAUUCCAAGCAAG
2915

22755
AACAUUGAUGCUACUUCAA
1265
22755
AACAUUGAUGCUACUUCAA
1265
22773
UUGAAGUAGCAUCAAUGUU
2916

22773
ACUGGUAAUUAUAAUUAUA
1266
22773
ACUGGUAAUUAUAAUUAUA
1266
22791
UAUAAUUAUAAUUACCAGU
2917

22791
AAAUAUAGGUAUCUUAGAC
1267
22791
AAAUAUAGGUAUCUUAGAC
1267
22809
GUCUAAGAUACCUAUAUUU
2918

22809
CAUGGCAAGCUUAGGCCCU
1268
22809
CAUGGCAAGCUUAGGCCCU
1268
22827
AGGGCCUAAGCUUGCCAUG
2919

22827
UUUGAGAGAGACAUAUCUA
1269
22827
UUUGAGAGAGACAUAUCUA
1269
22845
UAGAUAUGUCUCUCUCAAA
2920

22845
AAUGUGCCUUUCUCCCCUG
1270
22845
AAUGUGCCUUUCUCCCCUG
1270
22863
CAGGGGAGAAAGGCACAUU
2921

22863
GAUGGCAAACCUUGCACCC
1271
22863
GAUGGCAAACCUUGCACCC
1271
22881
GGGUGCAAGGUUUGCCAUC
2922

22881
CCACCUGCUCUUAAUUGUU
1272
22881
CCACCUGCUCUUAAUUGUU
1272
22899
AACAAUUAAGAGCAGGUGG
2923

22899
UAUUGGCCAUUAAAUGAUU
1273
22899
UAUUGGCCAUUAAAUGAUU
1273
22917
AAUCAUUUAAUGGCCAAUA
2924

22917
UAUGGUUUUUACACCACUA
1274
22917
UAUGGUUUUUACACCACUA
1274
22935
UAGUGGUGUAAAAACCAUA
2925

22935
ACUGGCAUUGGCUACCAAC
1275
22935
ACUGGCAUUGGCUACCAAC
1275
22953
GUUGGUAGCCAAUGCCAGU
2926

22953
CCUUACAGAGUUGUAGUAC
1276
22953
CCUUACAGAGUUGUAGUAC
1276
22971
GUACUACAACUCUGUAAGG
2927

22971
CUUUCUUUUGAACUUUUAA
1277
22971
CUUUCUUUUGAACUUUUAA
1277
22989
UUAAAAGUUCAAAAGAAAG
2928

22989
AAUGCACCGGCCACGGUUU
1278
22989
AAUGCACCGGCCACGGUUU
1278
23007
AAACCGUGGCCGGUGCAUU
2929

23007
UGUGGACCAAAAUUAUCCA
1279
23007
UGUGGACCAAAAUUAUCCA
1279
23025
UGGAUAAUUUUGGUCCACA
2930

23025
ACUGACCUUAUUAAGAACC
1280
23025
ACUGACCUUAUUAAGAACC
1280
23043
GGUUCUUAAUAAGGUCAGU
2931

23043
CAGUGUGUCAAUUUUAAUU
1281
23043
CAGUGUGUCAAUUUUAAUU
1281
23061
AAUUAAAAUUGACACACUG
2932

23061
UUUAAUGGACUCACUGGUA
1282
23061
UUUAAUGGACUCACUGGUA
1282
23079
UACCAGUGAGUCCAUUAAA
2933

23079
ACUGGUGUGUUAACUCCUU
1283
23079
ACUGGUGUGUUAACUCCUU
1283
23097
AAGGAGUUAACACACCAGU
2934

23097
UCUUCAAAGAGAUUUCAAC
1284
23097
UCUUCAAAGAGAUUUCAAC
1284
23115
GUUGAAAUCUCUUUGAAGA
2935

23115
CCAUUUCAACAAUUUGGCC
1285
23115
CCAUUUCAACAAUUUGGCC
1285
23133
GGCCAAAUUGUUGAAAUGG
2936

23133
CGUGAUGUUUCUGAUUUCA
1286
23133
CGUGAUGUUUCUGAUUUCA
1286
23151
UGAAAUCAGAAACAUCACG
2937

23151
ACUGAUUCCGUUCGAGAUC
1287
23151
ACUGAUUCCGUUCGAGAUC
1287
23169
GAUCUCGAACGGAAUCAGU
2938

23169
CCUAAAACAUCUGAAAUAU
1288
23169
CCUAAAACAUCUGAAAUAU
1288
23187
AUAUUUCAGAUGUUUUAGG
2939

23187
UUAGACAUUUCACCUUGCG
1289
23187
UUAGACAUUUCACCUUGCG
1289
23205
CGCAAGGUGAAAUGUCUAA
2940

23205
GCUUUUGGGGGUGUAAGUG
1290
23205
GCUUUUGGGGGUGUAAGUG
1290
23223
CACUUACACCCCCAAAAGC
2941

23223
GUAAUUACACCUGGAACAA
1291
23223
GUAAUUACACCUGGAACAA
1291
23241
UUGUUCCAGGUGUAAUUAC
2942

23241
AAUGCUUCAUCUGAAGUUG
1292
23241
AAUGCUUCAUCUGAAGUUG
1292
23259
CAACUUCAGAUGAAGCAUU
2943

23259
GCUGUUCUAUAUCAAGAUG
1293
23259
GCUGUUCUAUAUCAAGAUG
1293
23277
CAUCUUGAUAUAGAACAGC
2944

23277
GUUAACUGCACUGAUGUUU
1294
23277
GUUAACUGCACUGAUGUUU
1294
23295
AAACAUCAGUGCAGUUAAC
2945

23295
UCUACAGCAAUUCAUGCAG
1295
23295
UCUACAGCAAUUCAUGCAG
1295
23313
CUGCAUGAAUUGCUGUAGA
2946

23313
GAUCAACUCACACCAGCUU
1296
23313
GAUCAACUCACACCAGCUU
1296
23331
AAGCUGGUGUGAGUUGAUC
2947

23331
UGGCGCAUAUAUUCUACUG
1297
23331
UGGCGCAUAUAUUCUACUG
1297
23349
CAGUAGAAUAUAUGCGCCA
2948

23349
GGAAACAAUGUAUUCCAGA
1298
23349
GGAAACAAUGUAUUCCAGA
1298
23367
UCUGGAAUACAUUGUUUCC
2949

23367
ACUCAAGCAGGCUGUCUUA
1299
23367
ACUCAAGCAGGCUGUCUUA
1299
23385
UAAGACAGCCUGCUUGAGU
2950

23385
AUAGGAGCUGAGCAUGUCG
1300
23385
AUAGGAGCUGAGCAUGUCG
1300
23403
CGACAUGCUCAGCUCCUAU
2951

23403
GACACUUCUUAUGAGUGCG
1301
23403
GACACUUCUUAUGAGUGCG
1301
23421
CGCACUCAUAAGAAGUGUC
2952

23421
GACAUUCCUAUUGGAGCUG
1302
23421
GACAUUCCUAUUGGAGCUG
1302
23439
CAGCUCCAAUAGGAAUGUC
2953

23439
GGCAUUUGUGCUAGUUACC
1303
23439
GGCAUUUGUGCUAGUUACC
1303
23457
GGUAACUAGCACAAAUGCC
2954

23457
CAUACAGUUUCUUUAUUAC
1304
23457
CAUACAGUUUCUUUAUUAC
1304
23475
GUAAUAAAGAAACUGUAUG
2955

23475
CGUAGUACUAGCCAAAAAU
1305
23475
CGUAGUACUAGCCAAAAAU
1305
23493
AUUUUUGGCUAGUACUACG
2956

23493
UCUAUUGUGGCUUAUACUA
1306
23493
UCUAUUGUGGCUUAUACUA
1306
23511
UAGUAUAAGCCACAAUAGA
2957

23511
AUGUCUUUAGGUGCUGAUA
1307
23511
AUGUCUUUAGGUGCUGAUA
1307
23529
UAUCAGCACCUAAAGACAU
2958

23529
AGUUCAAUUGCUUACUCUA
1308
23529
AGUUCAAUUGCUUACUCUA
1308
23547
UAGAGUAAGCAAUUGAACU
2959

23547
AAUAACACCAUUGCUAUAC
1309
23547
AAUAACACCAUUGCUAUAC
1309
23565
GUAUAGCAAUGGUGUUAUU
2960

23565
CCUACUAACUUUUCAAUUA
1310
23565
CCUACUAACUUUUCAAUUA
1310
23583
UAAUUGAAAAGUUAGUAGG
2961

23583
AGCAUUACUACAGAAGUAA
1311
23583
AGCAUUACUACAGAAGUAA
1311
23601
UUACUUCUGUAGUAAUGCU
2962

23601
AUGCCUGUUUCUAUGGCUA
1312
23601
AUGCCUGUUUCUAUGGCUA
1312
23619
UAGCCAUAGAAACAGGCAU
2963

23619
AAAACCUCCGUAGAUUGUA
1313
23619
AAAACCUCCGUAGAUUGUA
1313
23637
UACAAUCUACGGAGGUUUU
2964

23637
AAUAUGUACAUCUGCGGAG
1314
23637
AAUAUGUACAUCUGCGGAG
1314
23655
CUCCGCAGAUGUACAUAUU
2965

23655
GAUUCUACUGAAUGUGCUA
1315
23655
GAUUCUACUGAAUGUGCUA
1315
23673
UAGCACAUUCAGUAGAAUC
2966

23673
AAUUUGCUUCUCCAAUAUG
1316
23673
AAUUUGCUUCUCCAAUAUG
1316
23691
CAUAUUGGAGAAGCAAAUU
2967

23691
GGUAGCUUUUGCACACAAC
1317
23691
GGUAGCUUUUGCACACAAC
1317
23709
GUUGUGUGCAAAAGCUACC
2968

23709
CUAAAUCGUGCACUCUCAG
1318
23709
CUAAAUCGUGCACUCUCAG
1318
23727
CUGAGAGUGCACGAUUUAG
2969

23727
GGUAUUGCUGCUGAACAGG
1319
23727
GGUAUUGCUGCUGAACAGG
1319
23745
CCUGUUCAGCAGCAAUACC
2970

23745
GAUCGCAACACACGUGAAG
1320
23745
GAUCGCAACACACGUGAAG
1320
23763
CUUCACGUGUGUUGCGAUC
2971

23763
GUGUUCGCUCAAGUCAAAC
1321
23763
GUGUUCGCUCAAGUCAAAC
1321
23781
GUUUGACUUGAGCGAACAC
2972

23781
CAAAUGUACAAAACCCCAA
1322
23781
CAAAUGUACAAAACCCCAA
1322
23799
UUGGGGUUUUGUACAUUUG
2973

23799
ACUUUGAAAUAUUUUGGUG
1323
23799
ACUUUGAAAUAUUUUGGUG
1323
23817
CACCAAAAUAUUUCAAAGU
2974

23817
GGUUUUAAUUUUUCACAAA
1324
23817
GGUUUUAAUUUUUCACAAA
1324
23835
UUUGUGAAAAAUUAAAACC
2975

23835
AUAUUACCUGACCCUCUAA
1325
23835
AUAUUACCUGACCCUCUAA
1325
23853
UUAGAGGGUCAGGUAAUAU
2976

23853
AAGCCAACUAAGAGGUCUU
1326
23853
AAGCCAACUAAGAGGUCUU
1326
23871
AAGACCUCUUAGUUGGCUU
2977

23871
UUUAUUGAGGACUUGCUCU
1327
23871
UUUAUUGAGGACUUGCUCU
1327
23889
AGAGCAAGUCCUCAAUAAA
2978

23889
UUUAAUAAGGUGACACUCG
1328
23889
UUUAAUAAGGUGACACUCG
1328
23907
CGAGUGUCACCUUAUUAAA
2979

23907
GCUGAUGCUGGCUUCAUGA
1329
23907
GCUGAUGCUGGCUUCAUGA
1329
23925
UCAUGAAGCCAGCAUCAGC
2980

23925
AAGCAAUAUGGCGAAUGCC
1330
23925
AAGCAAUAUGGCGAAUGCC
1330
23943
GGCAUUCGCCAUAUUGCUU
2981

23943
CUAGGUGAUAUUAAUGCUA
1331
23943
CUAGGUGAUAUUAAUGCUA
1331
23961
UAGCAUUAAUAUCACCUAG
2982

23961
AGAGAUCUCAUUUGUGCGC
1332
23961
AGAGAUCUCAUUUGUGCGC
1332
23979
GCGCACAAAUGAGAUCUCU
2983

23979
CAGAAGUUCAAUGGACUUA
1333
23979
CAGAAGUUCAAUGGACUUA
1333
23997
UAAGUCCAUUGAACUUCUG
2984

23997
ACAGUGUUGCCACCUCUGC
1334
23997
ACAGUGUUGCCACCUCUGC
1334
24015
GCAGAGGUGGCAACACUGU
2985

24015
CUCACUGAUGAUAUGAUUG
1335
24015
CUCACUGAUGAUAUGAUUG
1335
24033
CAAUCAUAUCAUCAGUGAG
2986

24033
GCUGCCUACACUGCUGCUC
1336
24033
GCUGCCUACACUGCUGCUC
1336
24051
GAGCAGCAGUGUAGGCAGC
2987

24051
CUAGUUAGUGGUACUGCCA
1337
24051
CUAGUUAGUGGUACUGCCA
1337
24069
UGGCAGUACCACUAACUAG
2988

24069
ACUGCUGGAUGGACAUUUG
1338
24069
ACUGCUGGAUGGACAUUUG
1338
24087
CAAAUGUCCAUCCAGCAGU
2989

24087
GGUGCUGGCGCUGCUCUUC
1339
24087
GGUGCUGGCGCUGCUCUUC
1339
24105
GAAGAGCAGCGCCAGCACC
2990

24105
CAAAUACCUUUUGCUAUGC
1340
24105
CAAAUACCUUUUGCUAUGC
1340
24123
GCAUAGCAAAAGGUAUUUG
2991

24123
CAAAUGGCAUAUAGGUUCA
1341
24123
CAAAUGGCAUAUAGGUUCA
1341
24141
UGAACCUAUAUGCCAUUUG
2992

24141
AAUGGCAUUGGAGUUACCC
1342
24141
AAUGGCAUUGGAGUUACCC
1342
24159
GGGUAACUCCAAUGCCAUU
2993

24159
CAAAAUGUUCUCUAUGAGA
1343
24159
CAAAAUGUUCUCUAUGAGA
1343
24177
UCUCAUAGAGAACAUUUUG
2994

24177
AACCAAAAACAAAUCGCCA
1344
24177
AACCAAAAACAAAUCGCCA
1344
24195
UGGCGAUUUGUUUUUGGUU
2995

24195
AACCAAUUUAACAAGGCGA
1345
24195
AACCAAUUUAACAAGGCGA
1345
24213
UCGCCUUGUUAAAUUGGUU
2996

24213
AUUAGUCAAAUUCAAGAAU
1346
24213
AUUAGUCAAAUUCAAGAAU
1346
24231
AUUCUUGAAUUUGACUAAU
2997

24231
UCACUUACAACAACAUCAA
1347
24231
UCACUUACAACAACAUCAA
1347
24249
UUGAUGUUGUUGUAAGUGA
2998

24249
ACUGCAUUGGGCAAGCUGC
1348
24249
ACUGCAUUGGGCAAGCUGC
1348
24267
GCAGCUUGCCCAAUGCAGU
2999

24267
CAAGACGUUGUUAACCAGA
1349
24267
CAAGACGUUGUUAACCAGA
1349
24285
UCUGGUUAACAACGUCUUG
3000

24285
AAUGCUCAAGCAUUAAACA
1350
24285
AAUGCUCAAGCAUUAAACA
1350
24303
UGUUUAAUGCUUGAGCAUU
3001

24303
ACACUUGUUAAACAACUUA
1351
24303
ACACUUGUUAAACAACUUA
1351
24321
UAAGUUGUUUAACAAGUGU
3002

24321
AGCUCUAAUUUUGGUGCAA
1352
24321
AGCUCUAAUUUUGGUGCAA
1352
24339
UUGCACCAAAAUUAGAGCU
3003

24339
AUUUCAAGUGUGCUAAAUG
1353
24339
AUUUCAAGUGUGCUAAAUG
1353
24357
CAUUUAGCACACUUGAAAU
3004

24357
GAUAUCCUUUCGCGACUUG
1354
24357
GAUAUCCUUUCGCGACUUG
1354
24375
CAAGUCGCGAAAGGAUAUC
3005

24375
GAUAAAGUCGAGGCGGAGG
1355
24375
GAUAAAGUCGAGGCGGAGG
1355
24393
CCUCCGCCUCGACUUUAUC
3006

24393
GUACAAAUUGACAGGUUAA
1356
24393
GUACAAAUUGACAGGUUAA
1356
24411
UUAACCUGUCAAUUUGUAC
3007

24411
AUUACAGGCAGACUUCAAA
1357
24411
AUUACAGGCAGACUUCAAA
1357
24429
UUUGAAGUCUGCCUGUAAU
3008

24429
AGCCUUCAAACCUAUGUAA
1358
24429
AGCCUUCAAACCUAUGUAA
1358
24447
UUACAUAGGUUUGAAGGCU
3009

24447
ACACAACAACUAAUCAGGG
1359
24447
ACACAACAACUAAUCAGGG
1359
24465
CCCUGAUUAGUUGUUGUGU
3010

24465
GCUGCUGAAAUCAGGGCUU
1360
24465
GCUGCUGAAAUCAGGGCUU
1360
24483
AAGCCCUGAUUUCAGCAGC
3011

24483
UCUGCUAAUCUUGCUGCUA
1361
24483
UCUGCUAAUCUUGCUGCUA
1361
24501
UAGCAGCAAGAUUAGCAGA
3012

24501
ACUAAAAUGUCUGAGUGUG
1362
24501
ACUAAAAUGUCUGAGUGUG
1362
24519
CACACUCAGACAUUUUAGU
3013

24519
GUUCUUGGACAAUCAAAAA
1363
24519
GUUCUUGGACAAUCAAAAA
1363
24537
UUUUUGAUUGUCCAAGAAC
3014

24537
AGAGUUGACUUUUGUGGAA
1364
24537
AGAGUUGACUUUUGUGGAA
1364
24555
UUCCACAAAAGUCAACUCU
3015

24555
AAGGGCUACCACCUUAUGU
1365
24555
AAGGGCUACCACCUUAUGU
1365
24573
ACAUAAGGUGGUAGCCCUU
3016

24573
UCCUUCCCACAAGCAGCCC
1366
24573
UCCUUCCCACAAGCAGCCC
1366
24591
GGGCUGCUUGUGGGAAGGA
3017

24591
CCGCAUGGUGUUGUCUUCC
1367
24591
CCGCAUGGUGUUGUCUUCC
1367
24609
GGAAGACAACACCAUGCGG
3018

24609
CUACAUGUCACGUAUGUGC
1368
24609
CUACAUGUCACGUAUGUGC
1368
24627
GCACAUACGUGACAUGUAG
3019

24627
CCAUCCCAGGAGAGGAACU
1369
24627
CCAUCCCAGGAGAGGAACU
1369
24645
AGUUCCUCUCCUGGGAUGG
3020

24645
UUCACCACAGCGCCAGCAA
1370
24645
UUCACCACAGCGCCAGCAA
1370
24663
UUGCUGGCGCUGUGGUGAA
3021

24663
AUUUGUCAUGAAGGCAAAG
1371
24663
AUUUGUCAUGAAGGCAAAG
1371
24681
CUUUGCCUUCAUGACAAAU
3022

24681
GCAUACUUCCCUCGUGAAG
1372
24681
GCAUACUUCCCUCGUGAAG
1372
24699
CUUCACGAGGGAAGUAUGC
3023

24699
GGUGUUUUUGUGUUUAAUG
1373
24699
GGUGUUUUUGUGUUUAAUG
1373
24717
CAUUAAACACAAAAACACC
3024

24717
GGCACUUCUUGGUUUAUUA
1374
24717
GGCACUUCUUGGUUUAUUA
1374
24735
UAAUAAACCAAGAAGUGCC
3025

24735
ACACAGAGGAACUUCUUUU
1375
24735
ACACAGAGGAACUUCUUUU
1375
24753
AAAAGAAGUUCCUCUGUGU
3026

24753
UCUCCACAAAUAAUUACUA
1376
24753
UCUCCACAAAUAAUUACUA
1376
24771
UAGUAAUUAUUUGUGGAGA
3027

24771
ACAGACAAUACAUUUGUCU
1377
24771
ACAGACAAUACAUUUGUCU
1377
24789
AGACAAAUGUAUUGUCUGU
3028

24789
UCAGGAAAUUGUGAUGUCG
1378
24789
UCAGGAAAUUGUGAUGUCG
1378
24807
CGACAUCACAAUUUCCUGA
3029

24807
GUUAUUGGCAUCAUUAACA
1379
24807
GUUAUUGGCAUCAUUAACA
1379
24825
UGUUAAUGAUGCCAAUAAC
3030

24825
AACACAGUUUAUGAUCCUC
1380
24825
AACACAGUUUAUGAUCCUC
1380
24843
GAGGAUCAUAAACUGUGUU
3031

24843
CUGCAACCUGAGCUUGACU
1381
24843
CUGCAACCUGAGCUUGACU
1381
24861
AGUCAAGCUCAGGUUGCAG
3032

24861
UCAUUCAAAGAAGAGCUGG
1382
24861
UCAUUCAAAGAAGAGCUGG
1382
24879
CCAGCUCUUCUUUGAAUGA
3033

24879
GACAAGUACUUCAAAAAUC
1383
24879
GACAAGUACUUCAAAAAUC
1383
24897
GAUUUUUGAAGUACUUGUC
3034

24897
CAUACAUCACCAGAUGUUG
1384
24897
CAUACAUCACCAGAUGUUG
1384
24915
CAACAUCUGGUGAUGUAUG
3035

24915
GAUCUUGGCGACAUUUCAG
1385
24915
GAUCUUGGCGACAUUUCAG
1385
24933
CUGAAAUGUCGCCAAGAUC
3036

24933
GGCAUUAACGCUUCUGUCG
1386
24933
GGCAUUAACGCUUCUGUCG
1386
24951
CGACAGAAGCGUUAAUGCC
3037

24951
GUCAACAUUCAAAAAGAAA
1387
24951
GUCAACAUUCAAAAAGAAA
1387
24969
UUUCUUUUUGAAUGUUGAC
3038

24969
AUUGACCGCCUCAAUGAGG
1388
24969
AUUGACCGCCUCAAUGAGG
1388
24987
CCUCAUUGAGGCGGUCAAU
3039

24987
GUCGCUAAAAAUUUAAAUG
1389
24987
GUCGCUAAAAAUUUAAAUG
1389
25005
CAUUUAAAUUUUUAGCGAC
3040

25005
GAAUCACUCAUUGACCUUC
1390
25005
GAAUCACUCAUUGACCUUC
1390
25023
GAAGGUCAAUGAGUGAUUC
3041

25023
CAAGAAUUGGGAAAAUAUG
1391
25023
CAAGAAUUGGGAAAAUAUG
1391
25041
CAUAUUUUCCCAAUUCUUG
3042

25041
GAGCAAUAUAUUAAAUGGC
1392
25041
GAGCAAUAUAUUAAAUGGC
1392
25059
GCCAUUUAAUAUAUUGCUC
3043

25059
CCUUGGUAUGUUUGGCUCG
1393
25059
CCUUGGUAUGUUUGGCUCG
1393
25077
CGAGCCAAACAUACCAAGG
3044

25077
GGCUUCAUUGCUGGACUAA
1394
25077
GGCUUCAUUGCUGGACUAA
1394
25095
UUAGUCCAGCAAUGAAGCC
3045

25095
AUUGCCAUCGUCAUGGUUA
1395
25095
AUUGCCAUCGUCAUGGUUA
1395
25113
UAACCAUGACGAUGGCAAU
3046

25113
ACAAUCUUGCUUUGUUGCA
1396
25113
ACAAUCUUGCUUUGUUGCA
1396
25131
UGCAACAAAGCAAGAUUGU
3047

25131
AUGACUAGUUGUUGCAGUU
1397
25131
AUGACUAGUUGUUGCAGUU
1397
25149
AACUGCAACAACUAGUCAU
3048

25149
UGCCUCAAGGGUGCAUGCU
1398
25149
UGCCUCAAGGGUGCAUGCU
1398
25167
AGCAUGCACCCUUGAGGCA
3049

25167
UCUUGUGGUUCUUGCUGCA
1399
25167
UCUUGUGGUUCUUGCUGCA
1399
25185
UGCAGCAAGAACCACAAGA
3050

25185
AAGUUUGAUGAGGAUGACU
1400
25185
AAGUUUGAUGAGGAUGACU
1400
25203
AGUCAUCCUCAUCAAACUU
3051

25203
UCUGAGCCAGUUCUCAAGG
1401
25203
UCUGAGCCAGUUCUCAAGG
1401
25221
CCUUGAGAACUGGCUCAGA
3052

25221
GGUGUCAAAUUACAUUACA
1402
25221
GGUGUCAAAUUACAUUACA
1402
25239
UGUAAUGUAAUUUGACACC
3053

25239
ACAUAAACGAACUUAUGGA
1403
25239
ACAUAAACGAACUUAUGGA
1403
25257
UCCAUAAGUUCGUUUAUGU
3054

25257
AUUUGUUUAUGAGAUUUUU
1404
25257
AUUUGUUUAUGAGAUUUUU
1404
25275
AAAAAUCUCAUAAACAAAU
3055

25275
UUACUCUUGGAUCAAUUAC
1405
25275
UUACUCUUGGAUCAAUUAC
1405
25293
GUAAUUGAUCCAAGAGUAA
3056

25293
CUGCACAGCCAGUAAAAAU
1406
25293
CUGCACAGCCAGUAAAAAU
1406
25311
AUUUUUACUGGCUGUGCAG
3057

25311
UUGACAAUGCUUCUCCUGC
1407
25311
UUGACAAUGCUUCUCCUGC
1407
25329
GCAGGAGAAGCAUUGUCAA
3058

25329
CAAGUACUGUUCAUGCUAC
1408
25329
CAAGUACUGUUCAUGCUAC
1408
25347
GUAGCAUGAACAGUACUUG
3059

25347
CAGCAACGAUACCGCUACA
1409
25347
CAGCAACGAUACCGCUACA
1409
25365
UGUAGCGGUAUCGUUGCUG
3060

25365
AAGCCUCACUCCCUUUCGG
1410
25365
AAGCCUCACUCCCUUUCGG
1410
25383
CCGAAAGGGAGUGAGGCUU
3061

25383
GAUGGCUUGUUAUUGGCGU
1411
25383
GAUGGCUUGUUAUUGGCGU
1411
25401
ACGCCAAUAACAAGCCAUC
3062

25401
UUGCAUUUCUUGCUGUUUU
1412
25401
UUGCAUUUCUUGCUGUUUU
1412
25419
AAAACAGCAAGAAAUGCAA
3063

25419
UUCAGAGCGCUACCAAAAU
1413
25419
UUCAGAGCGCUACCAAAAU
1413
25437
AUUUUGGUAGCGCUCUGAA
3064

25437
UAAUUGCGCUCAAUAAAAG
1414
25437
UAAUUGCGCUCAAUAAAAG
1414
25455
CUUUUAUUGAGCGCAAUUA
3065

25455
GAUGGCAGCUAGCCCUUUA
1415
25455
GAUGGCAGCUAGCCCUUUA
1415
25473
UAAAGGGCUAGCUGCCAUC
3066

25473
AUAAGGGCUUCCAGUUCAU
1416
25473
AUAAGGGCUUCCAGUUCAU
1416
25491
AUGAACUGGAAGCCCUUAU
3067

25491
UUUGCAAUUUACUGCUGCU
1417
25491
UUUGCAAUUUACUGCUGCU
1417
25509
AGCAGCAGUAAAUUGCAAA
3068

25509
UAUUUGUUACCAUCUAUUC
1418
25509
UAUUUGUUACCAUCUAUUC
1418
25527
GAAUAGAUGGUAACAAAUA
3069

25527
CACAUCUUUUGCUUGUCGC
1419
25527
CACAUCUUUUGCUUGUCGC
1419
25545
GCGACAAGCAAAAGAUGUG
3070

25545
CUGCAGGUAUGGAGGCGCA
1420
25545
CUGCAGGUAUGGAGGCGCA
1420
25563
UGCGCCUCCAUACCUGCAG
3071

25563
AAUUUUUGUACCUCUAUGC
1421
25563
AAUUUUUGUACCUCUAUGC
1421
25581
GCAUAGAGGUACAAAAAUU
3072

25581
CCUUGAUAUAUUUUCUACA
1422
25581
CCUUGAUAUAUUUUCUACA
1422
25599
UGUAGAAAAUAUAUCAAGG
3073

25599
AAUGCAUCAACGCAUGUAG
1423
25599
AAUGCAUCAACGCAUGUAG
1423
25617
CUACAUGCGUUGAUGCAUU
3074

25617
GAAUUAUUAUGAGAUGUUG
1424
25617
GAAUUAUUAUGAGAUGUUG
1424
25635
CAACAUCUCAUAAUAAUUC
3075

25635
GGCUUUGUUGGAAGUGCAA
1425
25635
GGCUUUGUUGGAAGUGCAA
1425
25653
UUGCACUUCCAACAAAGCC
3076

25653
AAUCCAAGAACCCAUUACU
1426
25653
AAUCCAAGAACCCAUUACU
1426
25671
AGUAAUGGGUUCUUGGAUU
3077

25671
UUUAUGAUGCCAACUACUU
1427
25671
UUUAUGAUGCCAACUACUU
1427
25689
AAGUAGUUGGCAUCAUAAA
3078

25689
UUGUUUGCUGGCACACACA
1428
25689
UUGUUUGCUGGCACACACA
1428
25707
UGUGUGUGCCAGCAAACAA
3079

25707
AUAACUAUGACUACUGUAU
1429
25707
AUAACUAUGACUACUGUAU
1429
25725
AUACAGUAGUCAUAGUUAU
3080

25725
UACCAUAUAACAGUGUCAC
1430
25725
UACCAUAUAACAGUGUCAC
1430
25743
GUGACACUGUUAUAUGGUA
3081

25743
CAGAUACAAUUGUCGUUAC
1431
25743
CAGAUACAAUUGUCGUUAC
1431
25761
GUAACGACAAUUGUAUCUG
3082

25761
CUGAAGGUGACGGCAUUUC
1432
25761
CUGAAGGUGACGGCAUUUC
1432
25779
GAAAUGCCGUCACCUUCAG
3083

25779
CAACACCAAAACUCAAAGA
1433
25779
CAACACCAAAACUCAAAGA
1433
25797
UCUUUGAGUUUUGGUGUUG
3084

25797
AAGACUACCAAAUUGGUGG
1434
25797
AAGACUACCAAAUUGGUGG
1434
25815
CCACCAAUUUGGUAGUCUU
3085

25815
GUUAUUCUGAGGAUAGGCA
1435
25815
GUUAUUCUGAGGAUAGGCA
1435
25833
UGCCUAUCCUCAGAAUAAC
3086

25833
ACUCAGGUGUUAAAGACUA
1436
25833
ACUCAGGUGUUAAAGACUA
1436
25851
UAGUCUUUAACACCUGAGU
3087

25851
AUGUCGUUGUACAUGGCUA
1437
25851
AUGUCGUUGUACAUGGCUA
1437
25869
UAGCCAUGUACAACGACAU
3088

25869
AUUUCACCGAAGUUUACUA
1438
25869
AUUUCACCGAAGUUUACUA
1438
25887
UAGUAAACUUCGGUGAAAU
3089

25887
ACCAGCUUGAGUCUACACA
1439
25887
ACCAGCUUGAGUCUACACA
1439
25905
UGUGUAGACUCAAGCUGGU
3090

25905
AAAUUACUACAGACACUGG
1440
25905
AAAUUACUACAGACACUGG
1440
25923
CCAGUGUCUGUAGUAAUUU
3091

25923
GUAUUGAAAAUGCUACAUU
1441
25923
GUAUUGAAAAUGCUACAUU
1441
25941
AAUGUAGCAUUUUCAAUAC
3092

25941
UCUUCAUCUUUAACAAGCU
1442
25941
UCUUCAUCUUUAACAAGCU
1442
25959
AGCUUGUUAAAGAUGAAGA
3093

25959
UUGUUAAAGACCCACCGAA
1443
25959
UUGUUAAAGACCCACCGAA
1443
25977
UUCGGUGGGUCUUUAACAA
3094

25977
AUGUGCAAAUACACACAAU
1444
25977
AUGUGCAAAUACACACAAU
1444
25995
AUUGUGUGUAUUUGCACAU
3095

25995
UCGACGGCUCUUCAGGAGU
1445
25995
UCGACGGCUCUUCAGGAGU
1445
26013
ACUCCUGAAGAGCCGUCGA
3096

26013
UUGCUAAUCCAGCAAUGGA
1446
26013
UUGCUAAUCCAGCAAUGGA
1446
26031
UCCAUUGCUGGAUUAGCAA
3097

26031
AUCCAAUUUAUGAUGAGCC
1447
26031
AUCCAAUUUAUGAUGAGCC
1447
26049
GGCUCAUCAUAAAUUGGAU
3098

26049
CGACGACGACUACUAGCGU
1448
26049
CGACGACGACUACUAGCGU
1448
26067
ACGCUAGUAGUCGUCGUCG
3099

26067
UGCCUUUGUAAGCACAAGA
1449
26067
UGCCUUUGUAAGCACAAGA
1449
26085
UCUUGUGCUUACAAAGGCA
3100

26085
AAAGUGAGUACGAACUUAU
1450
26085
AAAGUGAGUACGAACUUAU
1450
26103
AUAAGUUCGUACUCACUUU
3101

26103
UGUACUCAUUCGUUUCGGA
1451
26103
UGUACUCAUUCGUUUCGGA
1451
26121
UCCGAAACGAAUGAGUACA
3102

26121
AAGAAACAGGUACGUUAAU
1452
26121
AAGAAACAGGUACGUUAAU
1452
26139
AUUAACGUACCUGUUUCUU
3103

26139
UAGUUAAUAGCGUACUUCU
1453
26139
UAGUUAAUAGCGUACUUCU
1453
26157
AGAAGUACGCUAUUAACUA
3104

26157
UUUUUCUUGCUUUCGUGGU
1454
26157
UUUUUCUUGCUUUCGUGGU
1454
26175
ACCACGAAAGCAAGAAAAA
3105

26175
UAUUCUUGCUAGUCACACU
1455
26175
UAUUCUUGCUAGUCACACU
1455
26193
AGUGUGACUAGCAAGAAUA
3106

26193
UAGCCAUCCUUACUGCGCU
1456
26193
UAGCCAUCCUUACUGCGCU
1456
26211
AGCGCAGUAAGGAUGGCUA
3107

26211
UUCGAUUGUGUGCGUACUG
1457
26211
UUCGAUUGUGUGCGUACUG
1457
26229
CAGUACGCACACAAUCGAA
3108

26229
GCUGCAAUAUUGUUAACGU
1458
26229
GCUGCAAUAUUGUUAACGU
1458
26247
ACGUUAACAAUAUUGCAGC
3109

26247
UGAGUUUAGUAAAACCAAC
1459
26247
UGAGUUUAGUAAAACCAAC
1459
26265
GUUGGUUUUACUAAACUCA
3110

26265
CGGUUUACGUCUACUCGCG
1460
26265
CGGUUUACGUCUACUCGCG
1460
26283
CGCGAGUAGACGUAAACCG
3111

26283
GUGUUAAAAAUCUGAACUC
1461
26283
GUGUUAAAAAUCUGAACUC
1461
26301
GAGUUCAGAUUUUUAACAC
3112

26301
CUUCUGAAGGAGUUCCUGA
1462
26301
CUUCUGAAGGAGUUCCUGA
1462
26319
UCAGGAACUCCUUCAGAAG
3113

26319
AUCUUCUGGUCUAAACGAA
1463
26319
AUCUUCUGGUCUAAACGAA
1463
26337
UUCGUUUAGACCAGAAGAU
3114

26337
ACUAACUAUUAUUAUUAUU
1464
26337
ACUAACUAUUAUUAUUAUU
1464
26355
AAUAAUAAUAAUAGUUAGU
3115

26355
UCUGUUUGGAACUUUAACA
1465
26355
UCUGUUUGGAACUUUAACA
1465
26373
UGUUAAAGUUCCAAACAGA
3116

26373
AUUGCUUAUCAUGGCAGAC
1466
26373
AUUGCUUAUCAUGGCAGAC
1466
26391
GUCUGCCAUGAUAAGCAAU
3117

26391
CAACGGUACUAUUACCGUU
1467
26391
CAACGGUACUAUUACCGUU
1467
26409
AACGGUAAUAGUACCGUUG
3118

26409
UGAGGAGCUUAAACAACUC
1468
26409
UGAGGAGCUUAAACAACUC
1468
26427
GAGUUGUUUAAGCUCCUCA
3119

26427
CCUGGAACAAUGGAACCUA
1469
26427
CCUGGAACAAUGGAACCUA
1469
26445
UAGGUUCCAUUGUUCCAGG
3120

26445
AGUAAUAGGUUUCCUAUUC
1470
26445
AGUAAUAGGUUUCCUAUUC
1470
26463
GAAUAGGAAACCUAUUACU
3121

26463
CCUAGCCUGGAUUAUGUUA
1471
26463
CCUAGCCUGGAUUAUGUUA
1471
26481
UAACAUAAUCCAGGCUAGG
3122

26481
ACUACAAUUUGCCUAUUCU
1472
26481
ACUACAAUUUGCCUAUUCU
1472
26499
AGAAUAGGCAAAUUGUAGU
3123

26499
UAAUCGGAACAGGUUUUUG
1473
26499
UAAUCGGAACAGGUUUUUG
1473
26517
CAAAAACCUGUUCCGAUUA
3124

26517
GUACAUAAUAAAGCUUGUU
1474
26517
GUACAUAAUAAAGCUUGUU
1474
26535
AACAAGCUUUAUUAUGUAC
3125

26535
UUUCCUCUGGCUCUUGUGG
1475
26535
UUUCCUCUGGCUCUUGUGG
1475
26553
CCACAAGAGCCAGAGGAAA
3126

26553
GCCAGUAACACUUGCUUGU
1476
26553
GCCAGUAACACUUGCUUGU
1476
26571
ACAAGCAAGUGUUACUGGC
3127

26571
UUUUGUGCUUGCUGCUGUC
1477
26571
UUUUGUGCUUGCUGCUGUC
1477
26589
GACAGCAGCAAGCACAAAA
3128

26589
CUACAGAAUUAAUUGGGUG
1478
26589
CUACAGAAUUAAUUGGGUG
1478
26607
CACCCAAUUAAUUCUGUAG
3129

26607
GACUGGCGGGAUUGCGAUU
1479
26607
GACUGGCGGGAUUGCGAUU
1479
26625
AAUCGCAAUCCCGCCAGUC
3130

26625
UGCAAUGGCUUGUAUUGUA
1480
26625
UGCAAUGGCUUGUAUUGUA
1480
26643
UACAAUACAAGCCAUUGCA
3131

26643
AGGCUUGAUGUGGCUUAGC
1481
26643
AGGCUUGAUGUGGCUUAGC
1481
26661
GCUAAGCCACAUCAAGCCU
3132

26661
CUACUUCGUUGCUUCCUUC
1482
26661
CUACUUCGUUGCUUCCUUC
1482
26679
GAAGGAAGCAACGAAGUAG
3133

26679
CAGGCUGUUUGCUCGUACC
1483
26679
CAGGCUGUUUGCUCGUACC
1483
26697
GGUACGAGCAAACAGCCUG
3134

26697
CCGCUCAAUGUGGUCAUUC
1484
26697
CCGCUCAAUGUGGUCAUUC
1484
26715
GAAUGACCACAUUGAGCGG
3135

26715
CAACCCAGAAACAAACAUU
1485
26715
CAACCCAGAAACAAACAUU
1485
26733
AAUGUUUGUUUCUGGGUUG
3136

26733
UCUUCUCAAUGUGCCUCUC
1486
26733
UCUUCUCAAUGUGCCUCUC
1486
26751
GAGAGGCACAUUGAGAAGA
3137

26751
CCGGGGGACAAUUGUGACC
1487
26751
CCGGGGGACAAUUGUGACC
1487
26769
GGUCACAAUUGUCCCCCGG
3138

26769
CAGACCGCUCAUGGAAAGU
1488
26769
CAGACCGCUCAUGGAAAGU
1488
26787
ACUUUCCAUGAGCGGUCUG
3139

26787
UGAACUUGUCAUUGGUGCU
1489
26787
UGAACUUGUCAUUGGUGCU
1489
26805
AGCACCAAUGACAAGUUCA
3140

26805
UGUGAUCAUUCGUGGUCAC
1490
26805
UGUGAUCAUUCGUGGUCAC
1490
26823
GUGACCACGAAUGAUCACA
3141

26823
CUUGCGAAUGGCCGGACAC
1491
26823
CUUGCGAAUGGCCGGACAC
1491
26841
GUGUCCGGCCAUUCGCAAG
3142

26841
CUCCCUAGGGCGCUGUGAC
1492
26841
CUCCCUAGGGCGCUGUGAC
1492
26859
GUCACAGCGCCCUAGGGAG
3143

26859
CAUUAAGGACCUGCCAAAA
1493
26859
CAUUAAGGACCUGCCAAAA
1493
26877
UUUUGGCAGGUCCUUAAUG
3144

26877
AGAGAUCACUGUGGCUACA
1494
26877
AGAGAUCACUGUGGCUACA
1494
26895
UGUAGCCACAGUGAUCUCU
3145

26895
AUCACGAACGCUUUCUUAU
1495
26895
AUCACGAACGCUUUCUUAU
1495
26913
AUAAGAAAGCGUUCGUGAU
3146

26913
UUACAAAUUAGGAGCGUCG
1496
26913
UUACAAAUUAGGAGCGUCG
1496
26931
CGACGCUCCUAAUUUGUAA
3147

26931
GCAGCGUGUAGGCACUGAU
1497
26931
GCAGCGUGUAGGCACUGAU
1497
26949
AUCAGUGCCUACACGCUGC
3148

26949
UUCAGGUUUUGCUGCAUAC
1498
26949
UUCAGGUUUUGCUGCAUAC
1498
26967
GUAUGCAGCAAAACCUGAA
3149

26967
CAACCGCUACCGUAUUGGA
1499
26967
CAACCGCUACCGUAUUGGA
1499
26985
UCCAAUACGGUAGCGGUUG
3150

26985
AAACUAUAAAUUAAAUACA
1500
26985
AAACUAUAAAUUAAAUACA
1500
27003
UGUAUUUAAUUUAUAGUUU
3151

27003
AGACCACGCCGGUAGCAAC
1501
27003
AGACCACGCCGGUAGCAAC
1501
27021
GUUGCUACCGGCGUGGUCU
3152

27021
CGACAAUAUUGCUUUGCUA
1502
27021
CGACAAUAUUGCUUUGCUA
1502
27039
UAGCAAAGCAAUAUUGUCG
3153

27039
AGUACAGUAAGUGACAACA
1503
27039
AGUACAGUAAGUGACAACA
1503
27057
UGUUGUCACUUACUGUACU
3154

27057
AGAUGUUUCAUCUUGUUGA
1504
27057
AGAUGUUUCAUCUUGUUGA
1504
27075
UCAACAAGAUGAAACAUCU
3155

27075
ACUUCCAGGUUACAAUAGC
1505
27075
ACUUCCAGGUUACAAUAGC
1505
27093
GCUAUUGUAACCUGGAAGU
3156

27093
CAGAGAUAUUGAUUAUCAU
1506
27093
CAGAGAUAUUGAUUAUCAU
1506
27111
AUGAUAAUCAAUAUCUCUG
3157

27111
UUAUGAGGACUUUCAGGAU
1507
27111
UUAUGAGGACUUUCAGGAU
1507
27129
AUCCUGAAAGUCCUCAUAA
3158

27129
UUGCUAUUUGGAAUCUUGA
1508
27129
UUGCUAUUUGGAAUCUUGA
1508
27147
UCAAGAUUCCAAAUAGCAA
3159

27147
ACGUUAUAAUAAGUUCAAU
1509
27147
ACGUUAUAAUAAGUUCAAU
1509
27165
AUUGAACUUAUUAUAACGU
3160

27165
UAGUGAGACAAUUAUUUAA
1510
27165
UAGUGAGACAAUUAUUUAA
1510
27183
UUAAAUAAUUGUCUCACUA
3161

27183
AGCCUCUAACUAAGAAGAA
1511
27183
AGCCUCUAACUAAGAAGAA
1511
27201
UUCUUCUUAGUUAGAGGCU
3162

27201
AUUAUUCGGAGUUAGAUGA
1512
27201
AUUAUUCGGAGUUAGAUGA
1512
27219
UCAUCUAACUCCGAAUAAU
3163

27219
AUGAAGAACCUAUGGAGUU
1513
27219
AUGAAGAACCUAUGGAGUU
1513
27237
AACUCCAUAGGUUCUUCAU
3164

27237
UAGAUUAUCCAUAAAACGA
1514
27237
UAGAUUAUCCAUAAAACGA
1514
27255
UCGUUUUAUGGAUAAUCUA
3165

27255
AACAUGAAAAUUAUUCUCU
1515
27255
AACAUGAAAAUUAUUCUCU
1515
27273
AGAGAAUAAUUUUCAUGUU
3166

27273
UUCCUGACAUUGAUUGUAU
1516
27273
UUCCUGACAUUGAUUGUAU
1516
27291
AUACAAUCAAUGUCAGGAA
3167

27291
UUUACAUCUUGCGAGCUAU
1517
27291
UUUACAUCUUGCGAGCUAU
1517
27309
AUAGCUCGCAAGAUGUAAA
3168

27309
UAUCACUAUCAGGAGUGUG
1518
27309
UAUCACUAUCAGGAGUGUG
1518
27327
CACACUCCUGAUAGUGAUA
3169

27327
GUUAGAGGUACGACUGUAC
1519
27327
GUUAGAGGUACGACUGUAC
1519
27345
GUACAGUCGUACCUCUAAC
3170

27345
CUACUAAAAGAACCUUGCC
1520
27345
CUACUAAAAGAACCUUGCC
1520
27363
GGCAAGGUUCUUUUAGUAG
3171

27363
CCAUCAGGAACAUACGAGG
1521
27363
CCAUCAGGAACAUACGAGG
1521
27381
CCUCGUAUGUUCCUGAUGG
3172

27381
GGCAAUUCACCAUUUCACC
1522
27381
GGCAAUUCACCAUUUCACC
1522
27399
GGUGAAAUGGUGAAUUGCC
3173

27399
CCUCUUGCUGACAAUAAAU
1523
27399
CCUCUUGCUGACAAUAAAU
1523
27417
AUUUAUUGUCAGCAAGAGG
3174

27417
UUUGCACUAACUUGCACUA
1524
27417
UUUGCACUAACUUGCACUA
1524
27435
UAGUGCAAGUUAGUGCAAA
3175

27435
AGCACACACUUUGCUUUUG
1525
27435
AGCACACACUUUGCUUUUG
1525
27453
CAAAAGCAAAGUGUGUGCU
3176

27453
GCUUGUGCUGACGGUACUC
1526
27453
GCUUGUGCUGACGGUACUC
1526
27471
GAGUACCGUCAGCACAAGC
3177

27471
CGACAUACCUAUCAGCUGC
1527
27471
CGACAUACCUAUCAGCUGC
1527
27489
GCAGCUGAUAGGUAUGUCG
3178

27489
CGUGCAAGAUCAGUUUCAC
1528
27489
CGUGCAAGAUCAGUUUCAC
1528
27507
GUGAAACUGAUCUUGCACG
3179

27507
CCAAAACUUUUCAUCAGAC
1529
27507
CCAAAACUUUUCAUCAGAC
1529
27525
GUCUGAUGAAAAGUUUUGG
3180

27525
CAAGAGGAGGUUCAACAAG
1530
27525
CAAGAGGAGGUUCAACAAG
1530
27543
CUUGUUGAACCUCCUCUUG
3181

27543
GAGCUCUACUCGCCACUUU
1531
27543
GAGCUCUACUCGCCACUUU
1531
27561
AAAGUGGCGAGUAGAGCUC
3182

27561
UUUCUCAUUGUUGCUGCUC
1532
27561
UUUCUCAUUGUUGCUGCUC
1532
27579
GAGCAGCAACAAUGAGAAA
3183

27579
CUAGUAUUUUUAAUACUUU
1533
27579
CUAGUAUUUUUAAUACUUU
1533
27597
AAAGUAUUAAAAAUACUAG
3184

27597
UGCUUCACCAUUAAGAGAA
1534
27597
UGCUUCACCAUUAAGAGAA
1534
27615
UUCUCUUAAUGGUGAAGCA
3185

27615
AAGACAGAAUGAAUGAGCU
1535
27615
AAGACAGAAUGAAUGAGCU
1535
27633
AGCUCAUUCAUUCUGUCUU
3186

27633
UCACUUUAAUUGACUUCUA
1536
27633
UCACUUUAAUUGACUUCUA
1536
27651
UAGAAGUCAAUUAAAGUGA
3187

27651
AUUUGUGCUUUUUAGCCUU
1537
27651
AUUUGUGCUUUUUAGCCUU
1537
27669
AAGGCUAAAAAGCACAAAU
3188

27669
UUCUGCUAUUCCUUGUUUU
1538
27669
UUCUGCUAUUCCUUGUUUU
1538
27687
AAAACAAGGAAUAGCAGAA
3189

27687
UAAUAAUGCUUAUUAUAUU
1539
27687
UAAUAAUGCUUAUUAUAUU
1539
27705
AAUAUAAUAAGCAUUAUUA
3190

27705
UUUGGUUUUCACUCGAAAU
1540
27705
UUUGGUUUUCACUCGAAAU
1540
27723
AUUUCGAGUGAAAACCAAA
3191

27723
UCCAGGAUCUAGAAGAACC
1541
27723
UCCAGGAUCUAGAAGAACC
1541
27741
GGUUCUUCUAGAUCCUGGA
3192

27741
CUUGUACCAAAGUCUAAAC
1542
27741
CUUGUACCAAAGUCUAAAC
1542
27759
GUUUAGACUUUGGUACAAG
3193

27759
CGAACAUGAAACUUCUCAU
1543
27759
CGAACAUGAAACUUCUCAU
1543
27777
AUGAGAAGUUUCAUGUUCG
3194

27777
UUGUUUUGACUUGUAUUUC
1544
27777
UUGUUUUGACUUGUAUUUC
1544
27795
GAAAUACAAGUCAAAACAA
3195

27795
CUCUAUGCAGUUGCAUAUG
1545
27795
CUCUAUGCAGUUGCAUAUG
1545
27813
CAUAUGCAACUGCAUAGAG
3196

27813
GCACUGUAGUACAGCGCUG
1546
27813
GCACUGUAGUACAGCGCUG
1546
27831
CAGCGCUGUACUACAGUGC
3197

27831
GUGCAUCUAAUAAACCUCA
1547
27831
GUGCAUCUAAUAAACCUCA
1547
27849
UGAGGUUUAUUAGAUGCAC
3198

27849
AUGUGCUUGAAGAUCCUUG
1548
27849
AUGUGCUUGAAGAUCCUUG
1548
27867
CAAGGAUCUUCAAGCACAU
3199

27867
GUAAGGUACAACACUAGGG
1549
27867
GUAAGGUACAACACUAGGG
1549
27885
CCCUAGUGUUGUACCUUAC
3200

27885
GGUAAUACUUAUAGCACUG
1550
27885
GGUAAUACUUAUAGCACUG
1550
27903
CAGUGCUAUAAGUAUUACC
3201

27903
GCUUGGCUUUGUGCUCUAG
1551
27903
GCUUGGCUUUGUGCUCUAG
1551
27921
CUAGAGCACAAAGCCAAGC
3202

27921
GGAAAGGUUUUACCUUUUC
1552
27921
GGAAAGGUUUUACCUUUUC
1552
27939
GAAAAGGUAAAACCUUUCC
3203

27939
CAUAGAUGGCACACUAUGG
1553
27939
CAUAGAUGGCACACUAUGG
1553
27957
CCAUAGUGUGCCAUCUAUG
3204

27957
GUUCAAACAUGCACACCUA
1554
27957
GUUCAAACAUGCACACCUA
1554
27975
UAGGUGUGCAUGUUUGAAC
3205

27975
AAUGUUACUAUCAACUGUC
1555
27975
AAUGUUACUAUCAACUGUC
1555
27993
GACAGUUGAUAGUAACAUU
3206

27993
CAAGAUCCAGCUGGUGGUG
1556
27993
CAAGAUCCAGCUGGUGGUG
1556
28011
CACCACCAGCUGGAUCUUG
3207

28011
GCGCUUAUAGCUAGGUGUU
1557
28011
GCGCUUAUAGCUAGGUGUU
1557
28029
AACACCUAGCUAUAAGCGC
3208

28029
UGGUACCUUCAUGAAGGUC
1558
28029
UGGUACCUUCAUGAAGGUC
1558
28047
GACCUUCAUGAAGGUACCA
3209

28047
CACCAAACUGCUGCAUUUA
1559
28047
CACCAAACUGCUGCAUUUA
1559
28065
UAAAUGCAGCAGUUUGGUG
3210

28065
AGAGACGUACUUGUUGUUU
1560
28065
AGAGACGUACUUGUUGUUU
1560
28083
AAACAACAAGUACGUCUCU
3211

28083
UUAAAUAAACGAACAAAUU
1561
28083
UUAAAUAAACGAACAAAUU
1561
28101
AAUUUGUUCGUUUAUUUAA
3212

28101
UAAAAUGUCUGAUAAUGGA
1562
28101
UAAAAUGUCUGAUAAUGGA
1562
28119
UCCAUUAUCAGACAUUUUA
3213

28119
ACCCCAAUCAAACCAACGU
1563
28119
ACCCCAAUCAAACCAACGU
1563
28137
ACGUUGGUUUGAUUGGGGU
3214

28137
UAGUGCCCCCCGCAUUACA
1564
28137
UAGUGCCCCCCGCAUUACA
1564
28155
UGUAAUGCGGGGGGCACUA
3215

28155
AUUUGGUGGACCCACAGAU
1565
28155
AUUUGGUGGACCCACAGAU
1565
28173
AUCUGUGGGUCCACCAAAU
3216

28173
UUCAACUGACAAUAACCAG
1566
28173
UUCAACUGACAAUAACCAG
1566
28191
CUGGUUAUUGUCAGUUGAA
3217

28191
GAAUGGAGGACGCAAUGGG
1567
28191
GAAUGGAGGACGCAAUGGG
1567
28209
CCCAUUGCGUCCUCCAUUC
3218

28209
GGCAAGGCCAAAACAGCGC
1568
28209
GGCAAGGCCAAAACAGCGC
1568
28227
GCGCUGUUUUGGCCUUGCC
3219

28227
CCGACCCCAAGGUUUACCC
1569
28227
CCGACCCCAAGGUUUACCC
1569
28245
GGGUAAACCUUGGGGUCGG
3220

28245
CAAUAAUACUGCGUCUUGG
1570
28245
CAAUAAUACUGCGUCUUGG
1570
28263
CCAAGACGCAGUAUUAUUG
3221

28263
GUUCACAGCUCUCACUCAG
1571
28263
GUUCACAGCUCUCACUCAG
1571
28281
CUGAGUGAGAGCUGUGAAC
3222

28281
GCAUGGCAAGGAGGAACUU
1572
28281
GCAUGGCAAGGAGGAACUU
1572
28299
AAGUUCCUCCUUGCCAUGC
3223

28299
UAGAUUCCCUCGAGGCCAG
1573
28299
UAGAUUCCCUCGAGGCCAG
1573
28317
CUGGCCUCGAGGGAAUCUA
3224

28317
GGGCGUUCCAAUCAACACC
1574
28317
GGGCGUUCCAAUCAACACC
1574
28335
GGUGUUGAUUGGAACGCCC
3225

28335
CAAUAGUGGUCCAGAUGAC
1575
28335
CAAUAGUGGUCCAGAUGAC
1575
28353
GUCAUCUGGACCACUAUUG
3226

28353
CCAAAUUGGCUACUACCGA
1576
28353
CCAAAUUGGCUACUACCGA
1576
28371
UCGGUAGUAGCCAAUUUGG
3227

28371
AAGAGCUACCCGACGAGUU
1577
28371
AAGAGCUACCCGACGAGUU
1577
28389
AACUCGUCGGGUAGCUCUU
3228

28389
UCGUGGUGGUGACGGCAAA
1578
28389
UCGUGGUGGUGACGGCAAA
1578
28407
UUUGCCGUCACCACCACGA
3229

28407
AAUGAAAGAGCUCAGCCCC
1579
28407
AAUGAAAGAGCUCAGCCCC
1579
28425
GGGGCUGAGCUCUUUCAUU
3230

28425
CAGAUGGUACUUCUAUUAC
1580
28425
CAGAUGGUACUUCUAUUAC
1580
28443
GUAAUAGAAGUACCAUCUG
3231

28443
CCUAGGAACUGGCCCAGAA
1581
28443
CCUAGGAACUGGCCCAGAA
1581
28461
UUCUGGGCCAGUUCCUAGG
3232

28461
AGCUUCACUUCCCUACGGC
1582
28461
AGCUUCACUUCCCUACGGC
1582
28479
GCCGUAGGGAAGUGAAGCU
3233

28479
CGCUAACAAAGAAGGCAUC
1583
28479
CGCUAACAAAGAAGGCAUC
1583
28497
GAUGCCUUCUUUGUUAGCG
3234

28497
CGUAUGGGUUGCAACUGAG
1584
28497
CGUAUGGGUUGCAACUGAG
1584
28515
CUCAGUUGCAACCCAUACG
3235

28515
GGGAGCCUUGAAUACACCC
1585
28515
GGGAGCCUUGAAUACACCC
1585
28533
GGGUGUAUUCAAGGCUCCC
3236

28533
CAAAGACCACAUUGGCACC
1586
28533
CAAAGACCACAUUGGCACC
1586
28551
GGUGCCAAUGUGGUCUUUG
3237

28551
CCGCAAUCCUAAUAACAAU
1587
28551
CCGCAAUCCUAAUAACAAU
1587
28569
AUUGUUAUUAGGAUUGCGG
3238

28569
UGCUGCCACCGUGCUACAA
1588
28569
UGCUGCCACCGUGCUACAA
1588
28587
UUGUAGCACGGUGGCAGCA
3239

28587
ACUUCCUCAAGGAACAACA
1589
28587
ACUUCCUCAAGGAACAACA
1589
28605
UGUUGUUCCUUGAGGAAGU
3240

28605
AUUGCCAAAAGGCUUCUAC
1590
28605
AUUGCCAAAAGGCUUCUAC
1590
28623
GUAGAAGCCUUUUGGCAAU
3241

28623
CGCAGAGGGAAGCAGAGGC
1591
28623
CGCAGAGGGAAGCAGAGGC
1591
28641
GCCUCUGCUUCCCUCUGCG
3242

28641
CGGCAGUCAAGCCUCUUCU
1592
28641
CGGCAGUCAAGCCUCUUCU
1592
28659
AGAAGAGGCUUGACUGCCG
3243

28659
UCGCUCCUCAUCACGUAGU
1593
28659
UCGCUCCUCAUCACGUAGU
1593
28677
ACUACGUGAUGAGGAGCGA
3244

28677
UCGCGGUAAUUCAAGAAAU
1594
28677
UCGCGGUAAUUCAAGAAAU
1594
28695
AUUUCUUGAAUUACCGCGA
3245

28695
UUCAACUCCUGGCAGCAGU
1595
28695
UUCAACUCCUGGCAGCAGU
1595
28713
ACUGCUGCCAGGAGUUGAA
3246

28713
UAGGGGAAAUUCUCCUGCU
1596
28713
UAGGGGAAAUUCUCCUGCU
1596
28731
AGCAGGAGAAUUUCCCCUA
3247

28731
UCGAAUGGCUAGCGGAGGU
1597
28731
UCGAAUGGCUAGCGGAGGU
1597
28749
ACCUCCGCUAGCCAUUCGA
3248

28749
UGGUGAAACUGCCCUCGCG
1598
28749
UGGUGAAACUGCCCUCGCG
1598
28767
CGCGAGGGCAGUUUCACCA
3249

28767
GCUAUUGCUGCUAGACAGA
1599
28767
GCUAUUGCUGCUAGACAGA
1599
28785
UCUGUCUAGCAGCAAUAGC
3250

28785
AUUGAACCAGCUUGAGAGC
1600
28785
AUUGAACCAGCUUGAGAGC
1600
28803
GCUCUCAAGCUGGUUCAAU
3251

28803
CAAAGUUUCUGGUAAAGGC
1601
28803
CAAAGUUUCUGGUAAAGGC
1601
28821
GCCUUUACCAGAAACUUUG
3252

28821
CCAACAACAACAAGGCCAA
1602
28821
CCAACAACAACAAGGCCAA
1602
28839
UUGGCCUUGUUGUUGUUGG
3253

28839
AACUGUCACUAAGAAAUCU
1603
28839
AACUGUCACUAAGAAAUCU
1603
28857
AGAUUUCUUAGUGACAGUU
3254

28857
UGCUGCUGAGGCAUCUAAA
1604
28857
UGCUGCUGAGGCAUCUAAA
1604
28875
UUUAGAUGCCUCAGCAGCA
3255

28875
AAAGCCUCGCCAAAAACGU
1605
28875
AAAGCCUCGCCAAAAACGU
1605
28893
ACGUUUUUGGCGAGGCUUU
3256

28893
UACUGCCACAAAACAGUAC
1606
28893
UACUGCCACAAAACAGUAC
1606
28911
GUACUGUUUUGUGGCAGUA
3257

28911
CAACGUCACUCAAGCAUUU
1607
28911
CAACGUCACUCAAGCAUUU
1607
28929
AAAUGCUUGAGUGACGUUG
3258

28929
UGGGAGACGUGGUCCAGAA
1608
28929
UGGGAGACGUGGUCCAGAA
1608
28947
UUCUGGACCACGUCUCCCA
3259

28947
ACAAACCCAAGGAAAUUUC
1609
28947
ACAAACCCAAGGAAAUUUC
1609
28965
GAAAUUUCCUUGGGUUUGU
3260

28965
CGGGGACCAAGACCUAAUC
1610
28965
CGGGGACCAAGACCUAAUC
1610
28983
GAUUAGGUCUUGGUCCCCG
3261

28983
CAGACAAGGAACUGAUUAC
1611
28983
CAGACAAGGAACUGAUUAC
1611
29001
GUAAUCAGUUCCUUGUCUG
3262

29001
CAAACAUUGGCCGCAAAUU
1612
29001
CAAACAUUGGCCGCAAAUU
1612
29019
AAUUUGCGGCCAAUGUUUG
3263

29019
UGCACAAUUUGCUCCAAGU
1613
29019
UGCACAAUUUGCUCCAAGU
1613
29037
ACUUGGAGCAAAUUGUGCA
3264

29037
UGCCUCUGCAUUCUUUGGA
1614
29037
UGCCUCUGCAUUCUUUGGA
1614
29055
UCCAAAGAAUGCAGAGGCA
3265

29055
AAUGUCACGCAUUGGCAUG
1615
29055
AAUGUCACGCAUUGGCAUG
1615
29073
CAUGCCAAUGCGUGACAUU
3266

29073
GGAAGUCACACCUUCGGGA
1616
29073
GGAAGUCACACCUUCGGGA
1616
29091
UCCCGAAGGUGUGACUUCC
3267

29091
AACAUGGCUGACUUAUCAU
1617
29091
AACAUGGCUGACUUAUCAU
1617
29109
AUGAUAAGUCAGCCAUGUU
3268

29109
UGGAGCCAUUAAAUUGGAU
1618
29109
UGGAGCCAUUAAAUUGGAU
1618
29127
AUCCAAUUUAAUGGCUCCA
3269

29127
UGACAAAGAUCCACAAUUC
1619
29127
UGACAAAGAUCCACAAUUC
1619
29145
GAAUUGUGGAUCUUUGUCA
3270

29145
CAAAGACAACGUCAUACUG
1620
29145
CAAAGACAACGUCAUACUG
1620
29163
CAGUAUGACGUUGUCUUUG
3271

29163
GCUGAACAAGCACAUUGAC
1621
29163
GCUGAACAAGCACAUUGAC
1621
29181
GUCAAUGUGCUUGUUCAGC
3272

29181
CGCAUACAAAACAUUCCCA
1622
29181
CGCAUACAAAACAUUCCCA
1622
29199
UGGGAAUGUUUUGUAUGCG
3273

29199
ACCAACAGAGCCUAAAAAG
1623
29199
ACCAACAGAGCCUAAAAAG
1623
29217
CUUUUUAGGCUCUGUUGGU
3274

29217
GGACAAAAAGAAAAAGACU
1624
29217
GGACAAAAAGAAAAAGACU
1624
29235
AGUCUUUUUCUUUUUGUCC
3275

29235
UGAUGAAGCUCAGCCUUUG
1625
29235
UGAUGAAGCUCAGCCUUUG
1625
29253
CAAAGGCUGAGCUUCAUCA
3276

29253
GCCGCAGAGACAAAAGAAG
1626
29253
GCCGCAGAGACAAAAGAAG
1626
29271
CUUCUUUUGUCUCUGCGGC
3277

29271
GCAGCCCACUGUGACUCUU
1627
29271
GCAGCCCACUGUGACUCUU
1627
29289
AAGAGUCACAGUGGGCUGC
3278

29289
UCUUCCUGCGGCUGACAUG
1628
29289
UCUUCCUGCGGCUGACAUG
1628
29307
CAUGUCAGCCGCAGGAAGA
3279

29307
GGAUGAUUUCUCCAGACAA
1629
29307
GGAUGAUUUCUCCAGACAA
1629
29325
UUGUCUGGAGAAAUCAUCC
3280

29325
ACUUCAAAAUUCCAUGAGU
1630
29325
ACUUCAAAAUUCCAUGAGU
1630
29343
ACUCAUGGAAUUUUGAAGU
3281

29343
UGGAGCUUCUGCUGAUUCA
1631
29343
UGGAGCUUCUGCUGAUUCA
1631
29361
UGAAUCAGCAGAAGCUCCA
3282

29361
AACUCAGGCAUAAACACUC
1632
29361
AACUCAGGCAUAAACACUC
1632
29379
GAGUGUUUAUGCCUGAGUU
3283

29379
CAUGAUGACCACACAAGGC
1633
29379
CAUGAUGACCACACAAGGC
1633
29397
GCCUUGUGUGGUCAUCAUG
3284

29397
CAGAUGGGCUAUGUAAACG
1634
29397
CAGAUGGGCUAUGUAAACG
1634
29415
CGUUUACAUAGCCCAUCUG
3285

29415
GUUUUCGCAAUUCCGUUUA
1635
29415
GUUUUCGCAAUUCCGUUUA
1635
29433
UAAACGGAAUUGCGAAAAC
3286

29433
ACGAUACAUAGUCUACUCU
1636
29433
ACGAUACAUAGUCUACUCU
1636
29451
AGAGUAGACUAUGUAUCGU
3287

29451
UUGUGCAGAAUGAAUUCUC
1637
29451
UUGUGCAGAAUGAAUUCUC
1637
29469
GAGAAUUCAUUCUGCACAA
3288

29469
CGUAACUAAACAGCACAAG
1638
29469
CGUAACUAAACAGCACAAG
1638
29487
CUUGUGCUGUUUAGUUACG
3289

29487
GUAGGUUUAGUUAACUUUA
1639
29487
GUAGGUUUAGUUAACUUUA
1639
29505
UAAAGUUAACUAAACCUAC
3290

29505
AAUCUCACAUAGCAAUCUU
1640
29505
AAUCUCACAUAGCAAUCUU
1640
29523
AAGAUUGCUAUGUGAGAUU
3291

29523
UUAAUCAAUGUGUAACAUU
1641
29523
UUAAUCAAUGUGUAACAUU
1641
29541
AAUGUUACACAUUGAUUAA
3292

29541
UAGGGAGGACUUGAAAGAG
1642
29541
UAGGGAGGACUUGAAAGAG
1642
29559
CUCUUUCAAGUCCUCCCUA
3293

29559
GCCACCACAUUUUCAUCGA
1643
29559
GCCACCACAUUUUCAUCGA
1643
29577
UCGAUGAAAAUGUGGUGGC
3294

29577
AGGCCACGCGGAGUACGAU
1644
29577
AGGCCACGCGGAGUACGAU
1644
29595
AUCGUACUCCGCGUGGCCU
3295

29595
UCGAGGGUACAGUGAAUAA
1645
29595
UCGAGGGUACAGUGAAUAA
1645
29613
UUAUUCACUGUACCCUCGA
3296

29613
AUGCUAGGGAGAGCUGCCU
1646
29613
AUGCUAGGGAGAGCUGCCU
1646
29631
AGGCAGCUCUCCCUAGCAU
3297

29631
UAUAUGGAAGAGCCCUAAU
1647
29631
UAUAUGGAAGAGCCCUAAU
1647
29649
AUUAGGGCUCUUCCAUAUA
3298

29649
UGUGUAAAAUUAAUUUUAG
1648
29649
UGUGUAAAAUUAAUUUUAG
1648
29667
CUAAAAUUAAUUUUACACA
3299

29667
GUAGUGCUAUCCCCAUGUG
1649
29667
GUAGUGCUAUCCCCAUGUG
1649
29685
CACAUGGGGAUAGCACUAC
3300

29685
GAUUUUAAUAGCUUCUUAG
1650
29685
GAUUUUAAUAGCUUCUUAG
1650
29703
CUAAGAAGCUAUUAAAAUC
3301

29703
GGAGAAUGACAAAAAAAAA
1651
29703
GGAGAAUGACAAAAAAAAA
1651
29721
UUUUUUUUUGUCAUUCUCC
3302

The 3′-ends of the Upper sequence and the Lower sequence of the siNA construct can include an overhang sequence, for example about 1, 2, 3, or 4 nucleotides in length, preferably 2 nucleotides in length, wherein the overhanging sequence of the lower sequence is optionally complementary to a portion of the target sequence. The overhang can comprise the general structure B, BNN, NN, BNsN, or NsN, where B stands for any terminal cap moiety, N stands for any nucleotide (e.g., thymidine)

# and s stands for phosphorothioate or other internucleotide linkage as described herein (e.g. internucleotide linkage having Formula I). The upper sequence is also referred to as the sense strand, whereas the lower sequence is also referred to as the antisense strand. The upper and lower sequences in the Table can further comprise a chemical modification having Formulae I-VII or any combination thereof (see for example chemical modifications as shown in Table V herein).

TABLE III

SARS synthetic siNA and Target Sequences

Target

Seq
RPI

Seq

Pos
Target
ID
#
Aliases
Sequence
ID

1655
UGAAUGAAGAGGUUGCCAUCAUU
3303

SARS:1657U21 siRNA sense
AAUGAAGAGGUUGCCAUCATT
3311

1164
UGUUGCAUCUCCACAGGAGUGUA
3304

SARS:1166U21 siRNA sense
UUGCAUCUCCACAGGAGUGTT
3312

2381
CUCAAAGCAAGGGACUUUACCGU
3305

SARS:2383U21 siRNA sense
CAAAGCAAGGGACUUUACCTT
3313

2598
CUGUGUAAAUGGCCUCAUGCUCU
3306

SARS:2600U21 siRNA sense
GUGUAAAUGGCCUCAUGCUTT
3314

26572
UUUGUGCUUGCUGCUGUCUACAG
3307

SARS:26574U21 siRNA sense
UGUGCUUGCUGCUGUCUACTT
3315

26790
ACUUGUCAUUGGUGCUGUGAUCA
3308

SARS:26792U21 siRNA sense
UUGUCAUUGGUGCUGUGAUTT
3316

28786
UUGAACCAGCUUGAGAGCAAAGU
3309

SARS:28788U21 siRNA sense
GAACCAGCUUGAGAGCAAATT
3317

26529
GCUUGUUUUCCUCUGGCUCUUGU
3310

SARS:26531U21 siRNA sense
UUGUUUUCCUCUGGCUCUUTT
3318

1655
UGAAUGAAGAGGUUGCCAUCAUU
3303

SARS:1675L21 siRNA (1657C)
UGAUGGCAACCUCUUCAUUTT
3319

antisense

1164
UGUUGCAUCUCCACAGGAGUGUA
3304

SARS:1184L21 siRNA (1166C)
CACUCCUGUGGAGAUGCAATT
3320

antisense

2381
CUCAAAGCAAGGGACUUUACCGU
3305

SARS:2401L21 siRNA (2383C)
GGUAAAGUCCCUUGCUUUGTT
3321

antisense

2598
CUGUGUAAAUGGCCUCAUGCUCU
3306

SARS:2618L21 siRNA (2600C)
AGCAUGAGGCCAUUUACACTT
3322

antisense

26572
UUUGUGCUUGCUGCUGUCUACAG
3307

SARS:26592L21 siRNA (26574C)
GUAGACAGCAGCAAGCACATT
3323

antisense

26790
ACUUGUCAUUGGUGCUGUGAUCA
3308

SARS:26810L21 siRNA (26792C)
AUCACAGCACCAAUGACAATT
3324

antisense

28786
UUGAACCAGCUUGAGAGCAAAGU
3309

SARS:28806L21 siRNA (28788C)
UUUGCUCUCAAGCUGGUUCTT
3325

antisense

26529
GCUUGUUUUCCUCUGGCUCUUGU
3310

SARS:26549L21 siRNA (26531C)
AAGAGCCAGAGGAAAACAATT
3326

antisense

1655
UGAAUGAAGAGGUUGCCAUCAUU
3303

SARS:1657U21 siRNA stab04 sense
B AAuGAAGAGGuuGccAucATT B
3327

1164
UGUUGCAUCUCCACAGGAGUGUA
3304

SARS:1166U21 siRNA stab04 sense
B uuGcAucuccAcAGGAGuGTT B
3328

2381
CUCAAAGCAAGGGACUUUACCGU
3305

SARS:2383U21 siRNA stab04 sense
B cAAAGcAAGGGAcuuuAccTT B
3329

2598
CUGUGUAAAUGGCCUCAUGCUCU
3306

SARS:2600U21 siRNA stab04 sense
B GuGuAAAuGGccucAuGcuTT B
3330

26572
UUUGUGCUUGCUGCUGUCUACAG
3307

SARS:26574U21 siRNA stab04 sense
B uGuGcuuGcuGcuGucuAcTT B
3331

26790
ACUUGUCAUUGGUGCUGUGAUCA
3308

SARS:26792U21 siRNA stab04 sense
B uuGucAuuGGuGcuGuGAuTT B
3332

28786
UUGAACCAGCUUGAGAGCAAAGU
3309

SARS:28788U21 siRNA stab04 sense
B GAAccAGcuuGAGAGcAAATT B
3333

26529
GCUUGUUUUCCUCUGGCUCUUGU
3310

SARS:26531U21 siRNA stab04 sense
B uuGuuuuccucuGGcucuuTT B
3334

1655
UGAAUGAAGAGGUUGCCAUCAUU
3303

SARS:1675L21 siRNA (1657C)
uGAuGGcAAccucuucAuuTsT
3335

stab05 antisense

1164
UGUUGCAUCUCCACAGGAGUGUA
3304

SARS:1184L21 siRNA (1166C)
cAcuccuGuGGAGAuGcAATsT
3336

stab05 antisense

2381
CUCAAAGCAAGGGACUUUACCGU
3305

SARS:2401L21 siRNA (2383C)
GGuAAAGucccuuGcuuuGTsT
3337

stab05 antisense

2598
CUGUGUAAAUGGCCUCAUGCUCU
3306

SARS:2618L21 siRNA (2600C)
AGcAuGAGGccAuuuAcAcTsT
3338

stab05 antisense

26572
UUUGUGCUUGCUGCUGUCUACAG
3307

SARS:26592L21 siRNA (26574C)
GuAGAcAGcAGcAAGcAcATsT
3339

stab05 antisense

26790
ACUUGUCAUUGGUGCUGUGAUCA
3308

SARS:26810L21 siRNA (26792C)
AucAcAGcAccAAuGAcAATsT
3340

stab05 antisense

28786
UUGAACCAGCUUGAGAGCAAAGU
3309

SARS:28806L21 siRNA (28788C)
uuuGcucucAAGcuGGuucTsT
3341

stab05 antisense

26529
GCUUGUUUUCCUCUGGCUCUUGU
3310

SARS:26549L21 siRNA (26531C)
AAGAGccAGAGGAAAAcAATsT
3342

stab05 antisense

1655
UGAAUGAAGAGGUUGCCAUCAUU
3303

SARS:1657U21 siRNA stab07 sense
B AAuGAAGAGGuuGccAucATT B
3343

1164
UGUUGCAUCUCCACAGGAGUGUA
3304

SARS:1166U21 siRNA stab07 sense
B uuGcAucuccAcAGGAGuGTT B
3344

2381
CUCAAAGCAAGGGACUUUACCGU
3305

SARS:2383U21 siRNA stab07 sense
B cAAAGcAAGGGAcuuuAccTT B
3345

2598
CUGUGUAAAUGGCCUCAUGCUCU
3306

SARS:2600U21 siRNA stab07 sense
B GuGuAAAuGGccucAuGcuTT B
3346

26572
UUUGUGCUUGCUGCUGUCUACAG
3307

SARS:26574U21 siRNA stab07 sense
B uGuGcuuGcuGcuGucuAcTT B
3347

26790
ACUUGUCAUUGGUGCUGUGAUCA
3308

SARS:26792U21 siRNA stab07 sense
B uuGucAuuGGuGcuGuGAuTT B
3348

28786
UUGAACCAGCUUGAGAGCAAAGU
3309

SARS:28788U21 siRNA stab07 sense
B GAAccAGcuuGAGAGcAAATT B
3349

26529
GCUUGUUUUCCUCUGGCUCUUGU
3310

SARS:26531U21 siRNA stab07 sense
B uuGuuuuccucuGGcucuuTT B
3350

1655
UGAAUGAAGAGGUUGCCAUCAUU
3303

SARS:1675L21 siRNA (1657C)
uGAuGGcAAccucuucAuuTsT
3351

stab11 antisense

1164
UGUUGCAUCUCCACAGGAGUGUA
3304

SARS:1184L21 siRNA (1166C)
cAcuccuGuGGAGAuGcAATsT
3352

stab11 antisense

2381
CUCAAAGCAAGGGACUUUACCGU
3305

SARS:2401L21 siRNA (2383C)

GGuAAAGucccuuGcuuuGTsT
3353

stab11 antisense

2598
CUGUGUAAAUGGCCUCAUGCUCU
3306

SARS:2618L21 siRNA (2600C)

AGcAuGAGGccAuuuAcAcTsT
3354

stab11 antisense

26572
UUUGUGCUUGCUGCUGUCUACAG
3307

SARS:26592L21 siRNA (26574C)

GuAGAcAGcAGcAAGcAcATsT
3355

stab11 antisense

26790
ACUUGUCAUUGGUGCUGUGAUCA
3308

SARS:26810L21 siRNA (26792C)

AucAcAGcAccAAuGAcAATsT
3356

stab11 antisense

28786
UUGAACCAGCUUGAGAGCAAAGU
3309

SARS:28806L21 siRNA (28788C)
uuuGcucucAAGcuGGuucTsT
3357

stab11 antisense

26529
GCUUGUUUUCCUCUGGCUCUUGU
3310

SARS:26549L21 siRNA (26531C)

AAGAGccAGAGGAAAAcAATsT
3358

stab11 antisense

1655
UGAAUGAAGAGGUUGCCAUCAUU
3303

SARS:1657U21 siRNA stab08 sense

AAuGAAGAGGuuGccAucATsT
3359

1164
UGUUGCAUCUCCACAGGAGUGUA
3304

SARS:1166U21 siRNA stab08 sense
uuGcAucuccAcAGGAGuGTsT
3360

2381
CUCAAAGCAAGGGACUUUACCGU
3305

SARS:2383U21 siRNA stab08 sense
cAAAGcAAGGGAcuuuAccTsT
3361

2598
CUGUGUAAAUGGCCUCAUGCUCU
3306

SARS:2600U21 siRNA stab08 sense

GuGuAAAuGGccucAuGcuTsT
3362

26572
UUUGUGCUUGCUGCUGUCUACAG
3307

SARS:26574U21 siRNA stab08 sense
uGuGcuuGcuGcuGucuAcTsT
3363

26790
ACUUGUCAUUGGUGCUGUGAUCA
3308

SARS:26792U21 siRNA stab08 sense
uuGucAuuGGuGcuGuGAuTsT
3364

28786
UUGAACCAGCUUGAGAGCAAAGU
3309

SARS:28788U21 siRNA stab08 sense

GAAccAGcuuGAGAGcAAATsT
3365

26529
GCUUGUUUUCCUCUGGCUCUUGU
3310

SARS:26531U21 siRNA stab08 sense
uuGuuuuccucuGGcucuuTsT
3366

1655
UGAAUGAAGAGGUUGCCAUCAUU
3303

SARS:1675L21 siRNA (1657C)
uGAuGGcAAccucuucAuuTsT
3367

stab08 antisense

1164
UGUUGCAUCUCCACAGGAGUGUA
3304

SARS:1184L21 siRNA (1166C)
cAcuccuGuGGAGAuGcAATsT
3368

stab08 antisense

2381
CUCAAAGCAAGGGACUUUACCGU
3305

SARS:2401L21 siRNA (2383C)

GGuAAAGucccuuGcuuuGTsT
3369

stab08 antisense

2598
CUGUGUAAAUGGCCUCAUGCUCU
3306

SARS:2618L21 siRNA (2600C)

AGcAuGAGGccAuuuAcAcTsT
3370

stab08 antisense

26572
UUUGUGCUUGCUGCUGUCUACAG
3307

SARS:26592L21 siRNA (26574C)

GuAGAcAGcAGcAAGcAcATsT
3371

stab08 antisense

26790
ACUUGUCAUUGGUGCUGUGAUCA
3308

SARS:26810L21 siRNA (26792C)

AucAcAGcAccAAuGAcAATsT
3372

stab08 antisense

28786
UUGAACCAGCUUGAGAGCAAAGU
3309

SARS:28806L21 siRNA (28788C)
uuuGcucucAAGcuGGuucTsT
3373

stab08 antisense

26529
GCUUGUUUUCCUCUGGCUCUUGU
3310

SARS:26549L21 siRNA (26531C)

AAGAGccAGAGGAAAAcAATsT
3374

stab08 antisense

Uppercase = ribonucleotide

u,c = 2′-deoxy-2′-fluoro U, C

A = 2′-O-methyl Adenosine

G = 2′-O-methyl Guanosine

T = thymidine

B = inverted deoxy abasic

s = phosphorothioate linkage

A = deoxy Adenosine

G = deoxy Guanosine

TABLE IV

Non-limiting examples of Stabilization Chemistries for chemically

modified siNA constructs

Chemistry
pyrimidine
Purine
cap
p = S
Strand

“Stab 00”
Ribo
Ribo
TT at

S/AS

3′-ends

“Stab 1”
Ribo
Ribo
—
5 at 5′-end
S/AS

1 at 3′-end

“Stab 2”
Ribo
Ribo
—
All
Usually AS

linkages

“Stab 3”
2′-fluoro
Ribo
—
4 at 5′-end
Usually S

4 at 3′-end

“Stab 4”
2′-fluoro
Ribo
5′ and
—
Usually S

3′-ends

“Stab 5”
2′-fluoro
Ribo
—
1 at 3′-end
Usually AS

“Stab 6”
2′-O-Methyl
Ribo
5′ and
—
Usually S

3′-ends

“Stab 7”
2′-fluoro
2′-deoxy
5′ and
—
Usually S

3′-ends

“Stab 8”
2′-fluoro
2′-O-
—
1 at 3′-end
Usually AS

Methyl

“Stab 9”
Ribo
Ribo
5′ and
—
Usually S

3′-ends

“Stab 10”
Ribo
Ribo
—
1 at 3′-end
Usually AS

“Stab 11”
2′-fluoro
2′-deoxy
—
1 at 3′-end
Usually AS

“Stab 12”
2′-fluoro
LNA
5′ and

Usually S

3′-ends

“Stab 13”
2′-fluoro
LNA

1 at 3′-end
Usually AS

“Stab 14”
2′-fluoro
2′-deoxy

2 at 5′-end
Usually AS

1 at 3′-end

“Stab 15”
2′-deoxy
2′-deoxy

2 at 5′-end
Usually AS

1 at 3′-end

“Stab 16
Ribo
2′-O-
5′ and

Usually S

Methyl
3′-ends

“Stab 17”
2′-O-Methyl
2′-O-
5′ and

Usually S

Methyl
3′-ends

“Stab 18”
2′-fluoro
2′-O-
5′ and
1 at 3′-end
Usually S

Methyl
3′-ends

“Stab 19”
2′-fluoro
2′-O-
3′-end

Usually AS

Methyl

“Stab 20”
2′-fluoro
2′-deoxy
3′-end

Usually AS

“Stab 21”
2′-fluoro
Ribo
3′-end

Usually AS

“Stab 22”
Ribo
Ribo
3′-end-

Usually AS

CAP = any terminal cap, see for example FIG. 10.

All Stab 1-22 chemistries can comprise 3′-terminal thymidine (TT) residues

All Stab 1-22 chemistries typically comprise about 21 nucleotides, but can vary as described herein.

S = sense strand

AS = antisense strand

TABLE V

Reagent
Equivalents
Amount
Wait Time* DNA
Wait Time* 2′-O-methyl
Wait Time*RNA

A. 2.5 μmol Synthesis Cycle ABI 394 Instrument

Phosphoramidites
6.5
163
μL
45
sec
2.5
min
7.5
min

S-Ethyl Tetrazole
23.8
238
μL
45
sec
2.5
min
7.5
min

Acetic Anhydride
100
233
μL
5
sec
5
sec
5
sec

N-Methyl
186
233
μL
5
sec
5
sec
5
sec

Imidazole

TCA
176
2.3
mL
21
sec
21
sec
21
sec

Iodine
11.2
1.7
mL
45
sec
45
sec
45
sec

Beaucage
12.9
645
μL
100
sec
300
sec
300
sec

Acetonitrile
NA
6.67
mL
NA
NA
NA

B. 0.2 μmol Synthesis Cycle ABI 394 Instrument

Phosphoramidites
15
31
μL
45
sec
233
sec
465
sec

S-Ethyl Tetrazole
38.7
31
μL
45
sec
233
min
465
sec

Acetic Anhydride
655
124
μL
5
sec
5
sec
5
sec

N-Methyl
1245
124
μL
5
sec
5
sec
5
sec

Imidazole

TCA
700
732
μL
10
sec
10
sec
10
sec

Iodine
20.6
244
μL
15
sec
15
sec
15
sec

Beaucage
7.7
232
μL
100
sec
300
sec
300
sec

Acetonitrile
NA
2.64
mL
NA
NA
NA

C. 0.2 μmol Synthesis Cycle 96 well Instrument

Equivalents: DNA/
Amount: DNA/2′-O-

Wait Time* 2′-O-

Reagent
2′-O-methyl/Ribo
methyl/Ribo
Wait Time* DNA
methyl
Wait Time* Ribo

Phosphoramidites
22/33/66
40/60/120
μL
60
sec
180
sec
360
sec

S-Ethyl Tetrazole
70/105/210
40/60/120
μL
60
sec
180
min
360
sec

Acetic Anhydride
265/265/265
50/50/50
μL
10
sec
10
sec
10
sec

N-Methyl
502/502/502
50/50/50
μL
10
sec
10
sec
10
sec

Imidazole

TCA
238/475/475
250/500/500
μL
15
sec
15
sec
15
sec

Iodine
6.8/6.8/6.8
80/80/80
μL
30
sec
30
sec
30
sec

Beaucage
34/51/51
80/120/120

100
sec
200
sec
200
sec

Acetonitrile
NA
1150/1150/1150
μL
NA
NA
NA

*Wait time does not include contact time during delivery.

*Tandem synthesis utilizes double coupling of linker molecule

	Number	Date	Country
Parent	10757803	Jan 2004	US
Child	10553729	Mar 2007	US
Parent	10720448	Nov 2003	US
Child	10757803	Jan 2004	US
Parent	10693059	Oct 2003	US
Child	10720448	Nov 2003	US
Parent	10444853	May 2003	US
Child	10693059	Oct 2003	US
Parent	10427160	Apr 2003	US
Child	PCT/US04/11320		US

Rna Interference Mediated Inhibition of Severe Acute Respiratory Syndrome (Sars) Gene Expression Using Short Interfering Nucleic Acid

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Parent Case Info

PCT Information

Provisional Applications (1)

Continuation in Parts (5)