Patent Application
20190284621
The present invention relates to the detection of therapeutic modified oligonucleotides in biological samples and an adaptor oligonucleotide (capture probe) which enables a quantitative PCR based detection method and the sequencing of stereodefined or sugar-modified oligonucleotides. The invention provides novel adaptor probes for use in detecting therapeutic oligonucleotides and for in vivo discovery of preferred therapeutic oligonucleotide sequences.
Modified oligonucleotides, such as antisense oligonucleotides, siRNAs and aptamers are being developed as therapeutic agents. The qualitative and quantitative detection of these oligonucleotides in samples like cell cultures, tissue, blood, plasma or urine is a prerequisite to assess their use and to monitor their intracellular uptake, bio-distribution, metabolism and stability in vivo and/or in vitro.
Therapeutic modified oligonucleotide assert their function when delivered to target cells and tissues. However, upon delivery of such therapeutic oligonucleotide, it is often difficult to determine the level of exposure as the extent of oligonucleotide uptake varies in cells and tissues. As a result, accurately determining an effective dose of a therapeutic oligonucleotide can be challenging. Current methods to measure oligonucleotide exposure in target cells and tissues include either MS-based or ELISA-like methods, which are routinely used in the development and clinical evaluation of therapeutic oligonucleotides. The sensitivity of those methods is in the range of nM to pM concentration for detection of oligonucleotides and the assay development time can be several months. Furthermore, MS- and ELISA-like methods will only be able to measure a single antisense oligonucleotide per sample. Here, we present a PCR-based method that is 1) orders of magnitude more sensitive compared to current methods, 2) has a short development time (weeks) and 3) will allow evaluation of multiple oligonucleotides per sample. Further advantages are disclosed herein and are illustrated in the examples.
However, as is detailed herein, such traditional cloning methods do not function effectively for sugar modified oligonucleotides such as LNA or MOE, this is thought to be due to the lack of effective chain elongation from terminal modified nucleosides, as well as an inhibitory effect of modified nucleoside templates, particularly high affinity nucleoside templates, on DNA polymerases.
The present invention provides an enhanced capture probe for use in the detection, quantification, sequencing, amplification, or cloning of a sugar modified oligonucleotides and stereodefined oligonucleotide, such as an LNA modified oligonucleotides. The method also provides a method of capturing, detecting, quantifying, amplifying, and cloning nucleoside modified oligonucleotides and stereodefined oligonucelotides.
The present invention overcomes obstacles in applying qPCR to the detection of modified oligonucleotides. This has been achieved by employing a uniquely designed oligonucleotide capture probe combined with a T4DNA ligase step prior to chain elongation.
The invention provides for a capture probe oligonucleotide, comprising 5′-3′;
See
The capture probe may be for use in or detecting, quantifying, amplify, sequencing or cloning a nucleoside modified oligonucleotide. The capture probe may be for use in or detecting, quantifying, amplify, sequencing or cloning an oligonucleotide which comprises a Rp phosphorothioate internucleoside linkage between the two 3′ terminal nucleosides on the oligonucleotide.
The invention provides for the use of the capture probe oligonucleotide for use in detecting, quantifying, sequencing, amplifying or cloning a nucleoside modified oligonucleotide.
The invention provides for the use of the capture probe oligonucleotide for use in detecting, quantifying, sequencing, amplifying or cloning an oligonucleotide which comprises an Rp phosphorothioate internucleoside linkage between the two 3′ terminal nucleosides on the oligonucleotide.
The invention provides for the use of T4DNA ligase to ligate the 3′ terminus of a nucleoside modified oligonucleotide to the 5′ terminus of a DNA oligonucleotide, wherein the 3′ nucleoside of the nucleoside modified oligonucleotide is a LNA nucleoside.
The invention provides for the use of T4DNA ligase to ligate the 3′ terminus of a stereodefined oligonucleotide to the 5′ terminus of a DNA oligonucleotide, wherein the stereodefined oligonucleotide comprises a Rp phosphorothioate internucleoside linkage between the two 3′ terminal nucleosides on the oligonucleotide.
In some embodiments of the methods or uses of the invention, the ligation between the 3′ terminus of the modified oligonucleotide and the 5′ terminus of a DNA oligonucleotide, such as the capture probe, is performed in the presence of a polyethyleneglocol polymer, such as PEG 4000. The concentration of the PEG polymer, such as PEG 4000 is, in some embodiments between about 10% and about 30%, such as between about 12% and about 25%, such as between about 15% and about 20%, such as about 15% or about 20%.
The invention provides for a method for detecting, quantifying, amplifying, sequencing or cloning a nucleoside modified oligonucleotide in a sample, said method comprising the steps of;
The sample may for example be a purified nucleic acid fraction, obtained from a biological sample, such as a patient sample.
The invention provides for a method for detecting, quantifying, sequencing or cloning a nucleoside modified oligonucleotide in a sample, said method comprising the steps of;
The invention provides for a method for identifying a nucleoside modified oligonucleotide which is enriched in a target cell or tissue, e.g. in a mammal or a human, said method comprising;
A: 5′ end is phosphorylated to enable ligation.
B: 3′ end is blocked for ligation to avoid self-ligation. A 3′ amino modification is illustrated but other 3′ blocking groups may be used.
C: Stretch of nucleotides base pairing to make intracellular loop, stabilizing the positioning the target LNA towards the 5′phosphate to enhance ligation. 6 complementary base pairs are shown—other complementary regions, as described herein as regions 1A and 2A may be used.
D: Internal hexa-ethyleneglycol-spacer. Flexible spacer allowing easy self baseparing and preventing read-through of polymerase. Other linker groups may be used as described herein.
E: An overhang free for base-pairing used to capture and bind the LNA-oligonucleotide temporarily to promote the double strand dependent ligation. The overhang can be sequence specific to capture a specific sequence or as illustrated here be comprised of 6 mixed basepairs enabling the capture of the oligonucleotide sequence. The length of the overhang can be varied as described herein, but is typically at least 2 or 3 nucleotides.
The term “oligonucleotide” as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. In the context of the present invention, oligonucleotides are man-made, and are chemically synthesized, and are typically purified or isolated.
A nucleoside modified oligonucleotide is an oligonucleotide which comprises modified nucleosides, typically sugar modified nucleosides. In some embodiments the nucleoside modified oligonucleotide comprises at least one sugar modified nucleoside. In some embodiments the nucleoside modified oligonucleotide comprises at least one modified nucleoside at the 3′ end of the oligonucleotide, for example an LNA or a 2′substituted modified nucleoside, such as the at least two 3′ terminal nucleosides of the oligonucleotide are modified nucleosides, such as LNA or 2′ substituted modified nucleosides. In some embodiments the 3′ terminal nucleoside as a high affinity nucleoside analogue. In some embodiments the two 3′ terminal nucleosides are both high affinity nucleoside analogues. In some embodiments the two 3′ terminal nucleosides are both LNA nucleosides. In some embodiments the two 3′ terminal nucleosides are both 2′ substituted modified nucleosides, in particular 2′-O-MOE nucleosides. In some embodiments the oligonucleotide does not comprise a 5′ phosphate group.
A capture probe is an oligonucleotide which comprises at least one 5′ DNA nucleoside which is used to “capture” the nucleoside modified oligonucleotide. The capture may occur by the ligation of the 5′ end of the capture probe to the 3′ modified nucleotide of the modified nucleoside oligonucleotide. However, it is advantageous that the capture probe comprises a region which is complementary to a target nucleic acid sequence which is used to capture the target nucleic acid sequence via nucleic acid hybridization (Watson-Crick base pairing) prior to the ligation step. The invention provides optimized capture probes (e.g. as illustrated in
A degenerate nucleotide refers to a position on a nucleic acid sequence that can have multiple alternative bases (as used in the IUPAC notation of nucleic acids) at a defined position. It should be recognized that for an individual molecule there will be a specific nucleotide at the defined position, but within the population of molecules in the oligonucleotide sample, the nucleotide at the defined position will be degenerate. In effect, the incorporation of the degenerate sequence results in the randomization of nucleotide sequence at the defined positions between each members of a population of oligonucleotides. In some embodiments, degenerate nucleotides (a degenerate nucleotide sequence) may be used in the capture probes of the invention to form the 3′ overhang (region 2C), for example in the event that the sequence of the oligonucleotide to be captured is not known or is not defined. Alternatively or in addition, a degenerate nucleotide sequence may be used down-stream of region 1A (e.g. optional region 1B) where it can, for example, act as a molecular “bar code” allowing the identification of unique ligation products.
A predetermine nucleotide is a nucleotide that comprises a defined base (e.g. one of A, T, C or G) at a defined position within the oligonucleotide. A predetermined sequence is a sequence of predetermined nucleotides which has a known (designed) sequence. The capture probe of the invention comprises a predetermined sequence of nucleotides which form the universal primer binding site (1C) and the complementary regions 1A and 2A. Region 2B may also optionally comprise predetermined sequence, for example for use as a nested primer binding site or as a predetermined identifier sequence.
A blocked 3′ terminal group refers to a 3′ position on the 3′ terminal nucleoside of an oligonucleotide which does not comprise a —OH group. The blocked 3′ group does not therefore support enzymatic ligation (e.g. T4DNA ligase) or polymerase elongation (e.g. via Taq polymerse) from the 3′ end of the oligonucleotide. Numerous 3′ blocking groups are known in the art such as a nucleotidic modification which does not comprise a 3′-OH group, such as 3′deoxyribose, 2,3-dideoxyribose, 1,3-dideoxyribose, 1,2,3-trideoxyribose, and inverted ribose, a 3′ phosphate, 3′ amino, 3′ labels such as 3′ biotin, or a 3′fluorophore; or a non-nucleosidic modification, such as a non-ribose sugar, an abasic furan, a linker group (e.g. such as those described under region D herein), a thiol modifier (eg. C6SH, C3SH), an amino modifier, glycerol, or a conjugate group, such as fluorophores (fluorescein, AlexaFluor dyes, Atto dyes, cyanine dyes), digoxigenin, alkyne, azide, or cholesterol.
In some embodiments the 3′ blocking group is 3AmMO (3′ amino modification). In some embodiments the 3′ blocking group is a label, such as a fluorophore. In some embodiments the 3′ blocking group is not a fluorophore or is not a fluorescence quencher.
In the capture probe of the invention, region 1C comprises a universal primer binding site. This is a region of nucleotides with a predetermined nucleobase sequence which is used as a primer binding site (the Universal Primer) for first strand synthesis prior to PCR amplification: In the method of the invention, once the nucleoside modified oligonucleotide has been captured by the capture probe and the 5′ end of the capture probe has been ligated to the 3′ end of the sugar-modified oligonucleotide, a universal primer is hybridized to the universal primer binding site (region 1C) which is subsequently used for a DNA polymerase or reverse transcriptase mediated 5′-3′ chain elongation from the 3′ end of the universal primer across the length of the sugar-modified oligonucleotide, creating the first strand, or template molecule for PCR. A universal primer/universal primer binding site is typically at least 6 nucleotides in length (Ryu et al., Mol Biotechnol. 2000 January; 14(1):1-3), and may be for example 10-50 or 14-25 nucleotides in length.
In some embodiments the universal primer is a nucleotide primer, and may be a DNA primer or a modified DNA primer. In some embodiments the universal primer binding site is a region of nucleosides which are complementary to the universal primer, and may comprise DNA nucleotides and/or modified nucleotides.
First strand synthesis may be performed using a DNA polymerase or a reverse transcriptase capable of reading the modified oligo nucleotide. In some embodiments, for use in PCR amplification on the first strand template, a thermostable DNA polymerase is used. Numerous DNA polymerases (also referred to herein as polymerases) are known in the art and may be employed for first strand synthesis and/or PCR, for example, in some embodiments the DNA polymerase is a thermostable polymerase such as a DNA polymerase selected from the group consisting of Taq polymerase, Hottub polymerase, Pwo polymerase, rTth polymerase, Tfl polymerase, Ultima polymerase, Volcano2G polymerase, and Vent polymerase.
The selection of the DNA polymerase/reverse transcriptase may be performed by evaluating the relative efficiency of the polymerase to read through the modified oligonucleotide, such as sugar-modified oligonucleotides. For sugar modified oligonucleotides, this may depend on the length of contiguous sugar-modified nucleosides in the oligonucleotide, and it is recognized that for heavily modified oligonucleotides an enzyme other than Taq polymerase may be desirable. The selection of the DNA polymerase/reverse transcriptase will also depend on the purity of the sample, it is well known that some polymerase enzymes are sensitive to contaminants, such as blood (See Al-Soud et al, Appl Environ Microbiol. 1998 October; 64(10): 3748-3753 for example).
In some embodiments the DNA polymerase is a Volcano2G DNA polymerase.
In some embodiments the first strand synthesis (elongation step) is performed using a reverse transcriptase. In some embodiments, the reverse transcriptase may be selected from the group consisting of M-MuLV Reverse Transcriptase, SuperScript™ III RT, AMV Reverse Transcriptase, Maxima H Minus Reverse Transcriptase.
A modification or linker moiety which blocks DNA polymerase prevents the read through of the polymerase across the linker moiety or modification, resulting in the termination of chain elongation.
The linker moiety of the capture probe of the invention is a moiety which links region 1C and region 2A, allowing the hybridization of the complementary nucleotides of regions 1A and 1C but preventing the polymerase (or reverse transcriptase) read-through across the linking moiety. The linker moiety may, in some embodiments, consist or comprise a non-nucleotide linker such as a non-nucleotide polymer, for example a alkyl linker, a polyethylene glycol linker, a non nucleosidic carbohydrate linker, a photocleavable linker (PC spacer), or an alkyl disulfide linker; or the linker moiety may consist or comprise a (poly) ribose based meoity, such as a region of 1,2-dideoxy ribose or abasic furan, or nucleosides which comprise non-hybridising base groups. It is recognized that some polymerases, such as some reverse transcriptases have sufficient promiscuity to jump across small regions of some linkers, and as such, the linker should be one which prevents read through of the polymerase to be used. For example, HIV reverse transcriptase can read through an abasic nucleoside, all be it with low efficiency (Cancio et al., Biochemical Journal 2004, 383(3) 475-482. Non-limiting examples of linker groups (D) are provided below
An alkyl spacer, e.g of structure
wherein n is at least 2, such as between 2-26, such as 2, 3, 6, 12, 18, 24, or 36. In some embodiments n=12
(http://www.linktech.co.uk/products/modifiers/spacer_modifiers/339_spacer-ce-phosphoramidite-c12)
In some embodiments n=3 C3 spacer (n=3)
http://www.linktech.co.uk/products/modifiers/spacer_modifiers/333_spacer-ce-phosphoramidite-c3
An ethyleneglycol based spacers, e.g of structure
wherein n is at least 1, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments the linker is HEG (hexaethyleneglycol)
http://www.linktech.co.uk/products/modifiers/spacer_modifiers/337_spacer-ce-phosphoramidite-18-heg
In some embodiments the linker is a TEG (triethylene glycol) spacer.
http://www.linktech.co.uk/products/modifiers/spacer_modifiers/331_spacer-ce-phosphoramidite-9-teg
A Region of (Poly) 1,2-dideoxy ribose/Abasic Furan
Wherein the region comprises at least one of said abasic furan such as 2, 3, 4, 5, 6, 7, 8, 9, 10 such abasic furan units, such as 6-50 abasic furan units.
http://www.linktech.co.uk/products/modifiers/photocleavable_modifiers/352_pc-spacer-ce-phosphoramidite
or of formula
http://www.linktech.co.uk/products/modifiers/photocleavable_modifiers/354_pc-linker-ce-phosphoramidite
In some embodiments the linker moiety is a nucleotide based moiety but it comprises a modification which prevents polymerase read through, for example it may comprise an inverted nucleoside, or may comprise one or more modified nucleobases which do not allow (block) for hybridization.
The term “Antisense oligonucleotide” as used herein is defined as oligonucleotides capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid. The antisense oligonucleotides are not essentially double stranded and are therefore not siRNAs. Typically antisense oligonucleotides comprise modified nucleosides, and are between 7-25 nucleotides in length, such as 7-20 nucleosides in length. Numerous designs of antisense oligonucleotides are known, several of which incorporate high affinity modified nucleosides, such as LNA, for example gapmer oligonucleotides, mixmer oligonucleotides etc. In some embodiments, the nucleoside modified oligonucleotide is an antisense oligonucleotide.
Typically, oligonucleotide phosphorothioates are synthesised as a random mixture of Rp and Sp phosphorothioate linkages (also referred to as a diastereomeric mixture).
The above figure illustrates the stereochemistry of Sp and Rp phopshorothioate internucleoside linkages. R groups are nucleosides. Note the protonated form of the phosphorothaiote is shown for illustrative purposes only.
A stereodefined phosphorothioate oligonucleotide is a phosphorothioate oligonucleotide where at least one of the phosphorothioate linkages of the oligonucleotide is stereodefined, i.e. is either Rp or Sp in at least 75%, such as at least 80%, or at least 85%, or at least 90% or at least 95%, or at least 97%, such as at least 98%, such as at least 99%, or (essentially) all of the oligonucleotide molecules present in the oligonucleotide sample. Stereodefined oligonucleotides comprise at least one phosphorothioate linkage which is stereodefined. A fully stereodefined oligonucleotide is an oligonucleotide wherein all of the internucleoside linkages are stereodefined phosphorothioate internucleoside linkages. The term stereodefined, may be used to describe a defined chirality of one or more phosphorothioate internucleoside linkages as either Rp or Sp, or may be used to described a oligonucleotide which comprises such a (or more) phosphorothioate internucleoside linkage. It is recognised that a stereodefined oligonucleotide may comprise a small amount of the alternative stereoisomer at any one position, for example Wan et al reports a 98% stereoselectivity for the gapmers reported in NAR, November 2014.
The inventors have discovered that the capture probe and the methods of the invention may be used to detect, quantify, sequence, amplify, or clone a phosphorothioate oligonucleotide wherein the 3′ most internucleoside linkage of the phosphorothioate oligonucleotide is an Rp phosphorothioate internucleoside linkage.
As illustrated by the examples, the selective ability of T4DNA ligase to ligate an oligonucleotide with an Rp rather than an Sp phosphorothioate internucleoside linkage positioned between the two 3′ terminal nucleosides of a modified oligonucleotide can be used to discriminate between Rp and Sp internucleoside linkages. The method of the invention may therefore be used to identify the chirality of a phosphorothioate internucleoside linkage positioned between the two 3′ terminal nucleosides of an oligonucleotide.
It will be recognized that the method of the invention is not necessarily limited to the detection or quantification of antisense oligonucleotides, but may be employed in the detection or quantification or sequencing or cloning or other therapeutic oligonucleotides, such as siRNAs or aptamers, and may be used for the detection or quantification or sequencing or cloning of other nucleic acid sequences, e.g. microRNAs and cDNAs.
Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides comprise a deoxyribose/ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (when the phosphate group(s) is absent it is refered to as a nucleoside). Nucleosides and nucleotides may also interchangeably be referred to as “units” or “monomers”.
The term “modified nucleoside” or “nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety. In a preferred embodiment the modified nucleoside comprise a modified sugar moiety. The term modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue” or modified “units” or modified “monomers”.
In some embodiments, the nucleoside modified oligonucleotide comprises internucleoside linkages other than phosphodiester—i.e. a “modified internucleoside linkage”. In some embodiments, the modified internucleoside linkage increases the nuclease resistance of the oligonucleotide compared to a phosphodiester linkage. Modified internucleoside linkages are particularly useful in stabilizing oligonucleotides for in vivo use, and may serve to protect against nuclease cleavage at regions of DNA or RNA nucleosides in the oligonucleotide of the invention, for example within the gap region of a gapmer oligonucleotide, as well as in regions of modified nucleosides. Nuclease resistance may be determined by incubating the oligonucleotide in blood serum or by using a nuclease resistance assay (e.g. snake venom phosphodiesterase (SVPD)), both are well known in the art. Internucleoside linkages which are capable of enhancing the nuclease resistance of an oligonucleotide are referred to as nuclease resistant internucleoside linkages. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are modified. It will be recognized that, in some embodiments the nucleosides which link the oligonucleotide of the invention to a non-nucleotide functional group, such as a conjugate, may be phosphodiester. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages.
In some embodiments the modified internucleoside linkages may be phosphorothioate internucleoside linkages. In some embodiments, the modified internucleoside linkages are compatible with the RNaseH recruitment of the oligonucleotide of the invention, for example phosphorothioate.
In some embodiments the internucleoside linkage comprises sulphur (S), such as a phosphorothioate internucleoside linkage.
A phosphorothioate internucleoside linkage is particularly useful due to nuclease resistance, beneficial pharmakokinetics and ease of manufacture. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate.
The term nucleobase includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention the term nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization. In this context “nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.
In a some embodiments the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobased selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2′thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.
The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or U, wherein each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine. Optionally, for LNA gapmers, 5-methyl cytosine LNA nucleosides may be used.
The term modified oligonucleotide describes an oligonucleotide comprising one or more sugar-modified nucleosides and/or modified internucleoside linkages. In some embodiments the modified nucleoside comprises a stereodefined Rp phosphorothioate internucleoside linkage between the 2 3′ most (3′ terminal) nucleosides. In some embodiments, the modified oligonucleotide is a sugar-modified oligonucleotide. In some embodiments the sugar-modified oligonucleotide comprises a 3′ terminal nucleoside which is sugar-modified, such as a 2′ substituted nucleoside, such as 2′-O-MOE, or is a LNA nucleoside. In some embodiments, the sugar-modified oligonucleotide comprises a 3′ terminal LNA nucleoside, such as a beta-D-oxy LNA nucleoside or a (S) cET nucleoside.
The term “complementarity” describes the capacity for Watson-Crick base-pairing of nucleosides/nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T)/uracil (U). It will be understood that oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).
The term “hybridizing” or “hybridizes” as used herein is to be understood as two nucleic acid strands (e.g. an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex.
A high affinity modified nucleoside, also referred to as high affinity nucleoside analogues herein, is a modified nucleoside which, when incorporated into the oligonucleotide enhances the affinity of the oligonucleotide for its complementary target, for example as measured by the melting temperature (Tm). A high affinity modified nucleoside of the present invention preferably result in an increase in melting temperature between +0.5 to +12° C., more preferably between +1.5 to +10° C. and most preferably between +3 to +8° C. per modified nucleoside. Numerous high affinity modified nucleosides are known in the art and include for example, many 2′ substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).
The oligomer of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.
Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.
Such modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO2011/017521) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.
Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2′-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2′, 3′, 4′ or 5′ positions. Nucleosides with modified sugar moieties also include 2′ modified nucleosides, such as 2′ substituted nucleosides. Indeed, much focus has been spent on developing 2′ substituted nucleosides, and numerous 2′ substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity.
A 2′ sugar modified nucleoside is a nucleoside which has a substituent other than H or —OH at the 2′ position (2′ substituted nucleoside) or comprises a 2′ linked biradicle, and includes 2′ substituted nucleosides and LNA (2′-4′ biradicle bridged) nucleosides. For example, the 2′ modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide. Examples of 2′ substituted modified nucleosides are 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-RNA, and 2′-F-ANA nucleoside. For further examples, please see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrations of some 2′ substituted modified nucleosides.
LNA nucleosides are modified nucleosides which comprise a linker group (referred to as a biradicle or a bridge) between C2′ and C4′ of the ribose sugar ring of a nucleotide. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.
In some embodiments, the modified nucleoside or the LNA nucleosides of the oligomer of the invention has a general structure of the formula I or II:
wherein W is selected from —O—, —S—, —N(Ra)—, —C(RaRb)—, such as, in some embodiments —O—;
B designates a nucleobase or modified nucleobase moiety;
Z designates an internucleoside linkage to an adjacent nucleoside, or a 5′-terminal group;
Z* designates an internucleoside linkage to an adjacent nucleoside, or a 3′-terminal group;
X designates a group selected from the list consisting of —C(RaRb)—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —O—, —Si(Ra)2—, —S—, —SO2—, —N(Ra)—, and >C═Z
In some embodiments, X is selected from the group consisting of: —O—, —S—, NH—, NRaRb, —CH2−, CRaRb, —C(═CH2)—, and —C(═CRaRb)—
In some embodiments, X is —O—
Y designates a group selected from the group consisting of —C(RaRb)—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —O—, —Si(Ra)2—, —S—, —SO2—, —N(Ra)—, and >C═Z
In some embodiments, Y is selected from the group consisting of: —CH2—, —C(RaRb)—, —CH2CH2—, —C(RaRb)—C(RaRb)—, —CH2CH2CH2—, —C(RaRb)C(RaRb)C(RaRb)—, —C(Ra)═C(Rb)—, and —C(Ra)═N—
In some embodiments, Y is selected from the group consisting of: —CH2—, —CHRa—, —CHCH3—, —CRaRb—
or —X—Y— together designate a bivalent linker group (also referred to as a radicle) together designate a bivalent linker group consisting of 1, 2, 3 or 4 groups/atoms selected from the group consisting of —C(RaRb)—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —O—, —Si(Ra)2—, —S—, —SO2—, —N(Ra)—, and >C═Z,
In some embodiments, —X—Y— designates a biradicle selected from the groups consisting of: —X—CH2—, —X—CRaRb—, —X—CHRa—, —X—C(HCH3)−, —O—Y—, —O—CH2—, —S—CH2—, —O—CHCH3—, —CH2—O—CH2, —O—CH(CH3CH3)—, —O—CH2—CH2—, OCH2—CH2—CH2—, —O—CH2OCH2—, —O—NCH2—, —O(═CH2)—CH2—, —NRa—CH2—, N—O—CH2, —S—CRaRb— and —S—CHRa—.
In some embodiments —X—Y— designates —O—CH2— or —O—CH(CH3)—.
wherein Z is selected from —O—, —S—, and —N(Ra)—,
and Ra and, when present Rb, each is independently selected from hydrogen, optionally substituted C1-6-alkyl, optionally substituted C2-6-alkenyl, optionally substituted C2-6-alkynyl, hydroxy, optionally substituted C1-6-alkoxy, C2-6-alkoxyalkyl, C2-6-alkenyloxy, carboxy, C1-6-alkoxycarbonyl, C1-6-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-alkylthio, halogen, where aryl and heteroaryl may be optionally substituted and where two geminal substituents Ra and Rb together may designate optionally substituted methylene (═CH2), wherein for all chiral centers, asymmetric groups may be found in either R or S orientation.
wherein R1, R2, R3, R5 and R5* are independently selected from the group consisting of: hydrogen, optionally substituted C1-6-alkyl, optionally substituted C2-6-alkenyl, optionally substituted C2-6-alkynyl, hydroxy, C1-6-alkoxy, C2-6-alkoxyalkyl, C2-6-alkenyloxy, carboxy, C1-6-alkoxycarbonyl, C1-6-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-alkylthio, halogen, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene.
In some embodiments R1, R2, R3, R5 and R5* are independently selected from C1-6 alkyl, such as methyl, and hydrogen.
In some embodiments R1, R2, R3, R5 and R5* are all hydrogen.
In some embodiments R1, R2, R3, are all hydrogen, and either R5 and R5* is also hydrogen and the other of R5 and R5* is other than hydrogen, such as C1-6 alkyl such as methyl.
In some embodiments, Ra is either hydrogen or methyl. In some embodiments, when present, Rb is either hydrogen or methyl.
In some embodiments, one or both of Ra and Rb is hydrogen
In some embodiments, one of Ra and Rb is hydrogen and the other is other than hydrogen
In some embodiments, one of Ra and Rb is methyl and the other is hydrogen
In some embodiments, both of Ra and Rb are methyl.
In some embodiments, the biradicle —X—Y— is —O—CH2—, W is O, and all of R1, R2, R3, R5 and R5* are all hydrogen. Such LNA nucleosides are disclosed in WO99/014226, WO00/66604, WO98/039352 and WO2004/046160 which are all hereby incorporated by reference, and include what are commonly known as beta-D-oxy LNA and alpha-L-oxy LNA nucleosides.
In some embodiments, the biradicle —X—Y— is —S—CH2—, W is O, and all of R1, R2, R3, R5 and R5* are all hydrogen. Such thio LNA nucleosides are disclosed in WO99/014226 and WO2004/046160 which are hereby incorporated by reference.
In some embodiments, the biradicle —X—Y— is —NH—CH2—, W is O, and all of R1, R2, R3, R5 and R5* are all hydrogen. Such amino LNA nucleosides are disclosed in WO99/014226 and WO2004/046160 which are hereby incorporated by reference.
In some embodiments, the biradicle —X—Y— is —O—CH2—CH2— or —O—CH2—CH2—CH2—, W is O, and all of R1, R2, R3, R5 and R5* are all hydrogen. Such LNA nucleosides are disclosed in WO00/047599 and Morita et al, Bioorganic & Med. Chem. Lett. 12 73-76, which are hereby incorporated by reference, and include what are commonly known as 2′-O-4′O-ethylene bridged nucleic acids (ENA).
In some embodiments, the biradicle —X—Y— is —O—CH2—, W is O, and all of R1, R2, R3, and one of R5 and R5* are hydrogen, and the other of R5 and R5* is other than hydrogen such as C1-6 alkyl, such as methyl. Such 5′ substituted LNA nucleosides are disclosed in WO2007/134181 which is hereby incorporated by reference.
In some embodiments, the biradicle —X—Y— is —O—CRaRb—, wherein one or both of Ra and Rb are other than hydrogen, such as methyl, W is O, and all of R1, R2, R3, and one of R5 and R5* are hydrogen, and the other of R5 and R5* is other than hydrogen such as C1-6 alkyl, such as methyl. Such bis modified LNA nucleosides are disclosed in WO2010/077578 which is hereby incorporated by reference.
In some embodiments, the biradicle —X—Y— designate the bivalent linker group —O—CH(CH2OCH3)— (2′ O-methoxyethyl bicyclic nucleic acid—Seth at al., 2010, J. Org. Chem. Vol 75(5) pp. 1569-81). In some embodiments, the biradicle —X—Y— designate the bivalent linker group —O—CH(CH2CH3)— (2′O-ethyl bicyclic nucleic acid—Seth at al., 2010, J. Org. Chem. Vol 75(5) pp. 1569-81). In some embodiments, the biradicle —X—Y— is —O—CHRa—, W is O, and all of R1, R2, R3, R5 and R5* are all hydrogen. Such 6′ substituted LNA nucleosides are disclosed in WO10036698 and WO07090071 which are both hereby incorporated by reference.
In some embodiments, the biradicle —X—Y— is —O—CH(CH2OCH3)—, W is O, and all of R1, R2, R3, R5 and R5* are all hydrogen. Such LNA nucleosides are also known as cyclic MOEs in the art (cMOE) and are disclosed in WO07090071.
In some embodiments, the biradicle —X—Y— designate the bivalent linker group —O—CH(CH3)—.
In some embodiments, the biradicle —X—Y— is —O—CRaRb—, wherein in neither Ra or Rb is hydrogen, W is O, and all of R1, R2, R3, R5 and R5* are all hydrogen. In some embodiments, Ra and Rb are both methyl. Such 6′ di-substituted LNA nucleosides are disclosed in WO 2009006478 which is hereby incorporated by reference.
In some embodiments, the biradicle —X—Y— is —S—CHRa—, W is O, and all of R1, R2, R3, R5 and R5* are all hydrogen. Such 6′ substituted thio LNA nucleosides are disclosed in WO11156202 which is hereby incorporated by reference. In some 6′ substituted thio LNA embodiments Ra is methyl.
In some embodiments, the biradicle —X—Y— is —C(═CH2)—C(RaRb)—, such as —C(═CH2)—CH2—, or —C(═CH2)—CH(CH3)—W is O, and all of R1, R2, R3, R5 and R5* are all hydrogen. Such vinyl carbo LNA nucleosides are disclosed in WO08154401 and WO09067647 which are both hereby incorporated by reference.
In some embodiments the biradicle —X—Y— is —N(—ORa)—, W is O, and all of R1, R2, R3, R5 and R5* are all hydrogen. In some embodiments Ra is C1-6 alkyl such as methyl. Such LNA nucleosides are also known as N substituted LNAs and are disclosed in WO2008/150729 which is hereby incorporated by reference. In some embodiments, the biradicle —X—Y— together designate the bivalent linker group —O—NRa—CH3— (Seth at al., 2010, J. Org. Chem). In some embodiments the biradicle —X—Y— is —N(Ra)—, W is O, and all of R1, R2, R3, R5 and R5* are all hydrogen. In some embodiments Ra is C1-6 alkyl such as methyl.
In some embodiments, one or both of R5 and R5* is hydrogen and, when substituted the other of R5 and R5* is C1-6 alkyl such as methyl. In such an embodiment, R1, R2, R3, may all be hydrogen, and the biradicle —X—Y— may be selected from —O—CH2— or —O—C(HCRa)—, such as —O—C(HCH3)—.
In some embodiments, the biradicle is —CRaRb—O—CRaRb—, such as CH2—O—CH2—, W is O and all of R1, R2, R3, R5 and R5* are all hydrogen. In some embodiments Ra is C1-6 alkyl such as methyl. Such LNA nucleosides are also known as conformationally restricted nucleotides (CRNs) and are disclosed in WO2013036868 which is hereby incorporated by reference.
In some embodiments, the biradicle is —O—CRaRb—O—CRaRb—, such as O—CH2—O—CH2—, W is O and all of R1, R2, R3, R5 and R5* are all hydrogen. In some embodiments Ra is C1-6 alkyl such as methyl. Such LNA nucleosides are also known as COC nucleotides and are disclosed in Mitsuoka et al., Nucleic Acids Research 2009 37(4), 1225-1238, which is hereby incorporated by reference.
It will be recognized than, unless specified, the LNA nucleosides may be in the beta-D or alpha-L stereoisoform.
Certain examples of LNA nucleosides are presented in Scheme 1.
As illustrated in the examples, in some embodiments of the invention the LNA nucleosides in the oligonucleotides are beta-D-oxy-LNA nucleosides.
In some embodiments the nucleoside modified oligonucleotide comprises at least one 2′ substituted nucleoside, such as at least one 3′ terminal 2′ substituted nucleoside. In some embodiments the 2′ substituted oligonucleotide is a gapmer oligonucleotide, a mixmer oligonucleotide or a totalmer oligonucleotide. In some embodiments the 2′ substitution is selected from the group consisting of 2′methoxyethyl (2′-O-MOE) or 2′O-methyl. In some embodiments, the 3′ nucleotide of the nucleoside modified oligonucleotide is a 2′ substituted nucleoside such as 2′-O-MOE or 2′-O-methyl. In some embodiments the oligonucleotide does not comprise more than four consecutive nucleoside modified nucleosides. In some embodiments the oligonucleotide does not comprise more than three consecutive nucleoside modified nucleosides nucleosides. In some embodiments the oligonucleotide comprises 2 2′-O-MOE modified nucleotides at the 3′ terminal. In some embodiments the nucleoside modified oligonucleotide comprises phosphorothioate internucleoside linkages, and in some embodiments at least 75% of the internucleoside linkages present in the oligonucleotide are phosphorothioate internucleoside linkages. In some embodiments all of the internucleoside linkages of the modified nucleoside oligonucleotide are phosphorothioate internucleoside linkages. Phosphorotioate linked oligonucleotides are widely used for in vivo application in mammals, including their use as therapeutics.
In some embodiments the sugar modified oligonucleotide has a length of 7-30 nucleotides, such as 8-25 nucleotides. In some embodiments the length of the sugar modified oligonucleotide is 10-20 nucleotides, such as 12-18 nucleotides.
Nucleoside oligonucelotides may optionally be conjugated, e.g. with a GalNaC conjugate. If they are conjugated then it is preferable that the conjugate group is positioned other than at the 3′ position of the oligonucleotide, for example the conjugation may be at the 5′ terminal.
In some embodiments the nucleoside modified oligonucleotide comprises at least one LNA nucleoside, such as at least one 3′ terminal LNA nucleoside. In some embodiments the LNA oligonucleotide is a gapmer oligonucleotide, a mixmer oligonucleotide or a totalmer oligonucleotide. In some embodiments the LNA oligonucleotide does not comprise more than four consecutive LNA nucleosides. In some embodiments the LNA oligonucleotide does not comprise more than three consecutive LNA nucleosides. In some embodiments the LNA oligonucleotide comprises 2 LNA nucleotides at the 3′ terminal.
The nucleoside modified oligonucleotide may, in some embodiments be a gapmer oligonucleotide.
The term gapmer as used herein refers to an antisense oligonucleotide which comprises a region of RNase H recruiting oligonucleotides (gap—‘G’) which is flanked 5′ and 3′ by flanking regions (‘F’) which comprise one or more nucleoside modified nucleotides, such as affinity enhancing modified nucleosides (in the flanks or wings). Gapmers are typically 12-26 nucleotides in length and may, in some embodiments comprise a central region (G) of 6-14 DNA nucleosides, flanked either side by flanking regions F which comprises at least one nucleoside modified nucleotide such as 1-6 nucleoside modified nucleosides (F1-6G6-14 F1-6). The nucleoside in each flank positioned adjacent to the gap region (e.g. DNA nucleoside region) is a nucleoside modified nucleotide, such as an LNA or 2′-O-MOE nucleoside. In some embodiments all the nucleosides in the flanking regions are nucleoside modified nucleosides, such as LNA and/or 2′-O-MOE nucleosides, however the flanks may comprise DNA nucleosides in addition to the nucleoside modified nucleosides, which, in some embodiments are not the terminal nucleosides.
The term LNA gapmer is a gapmer oligonucleotide wherein at least one of the affinity enhancing modified nucleosides in the flanks is an LNA nucleoside. In some embodiments, the nucleoside modified oligonucleotide is a LNA gapmer wherein the 3′ terminal nucleoside of the oligonucleotide is a LNA nucleoside. In some embodiments the 2 3′ most nucleosides of the oligonucleotide are LNA nucleosides. In some embodiments, both the 5′ and 3′ flanks of the LNA gapmer comprise LNA nucleosides, and in some embodiments the nucleoside modified oligonucleotide is a LNA oligonucleotide, such as a gapmer oligonucleotide, wherein all the nucleosides of the oligonucleotide are either LNA or DNA nucleosides.
The term mixed wing gapmer or mixed flank gapmer refers to a LNA gapmer wherein at least one of the flank regions comprise at least one LNA nucleoside and at least one non-LNA modified nucleoside, such as at least one 2′ substituted modified nucleoside, such as, for example, 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-RNA and 2′-F-ANA nucleoside(s). In some embodiments the mixed wing gapmer has one flank which comprises only LNA nucleosides (e.g. 5′ or 3′) and the other flank (3′ or 5′ respectfully) comprises 2′ substituted modified nucleoside(s) and optionally LNA nucleosides. In some embodiments the mixed wing gapmer comprises LNA and 2′-O-MOE nucleosides in the flanks.
A mixmer is an oligonucleotide which comprises both nucleoside modified nucleosides and DNA nucleosides, wherein the oligonucleotides does not comprise more than 4 consecutive DNA nucleosides. Mixmer oligonucleotides are often used for non RNAseH mediated modulation of a nucleic acid target, for example for inhibition of a microRNA or for splice switching modulation or pre-mRNAs.
A totalmer is a nucleoside modified oligonucleotide wherein all the nucleosides present in the oligonucleotide are nucleoside modified. The totalmer may comprise of only one type of nucleoside modification, for example may be a full 2′-O-MOE or fully 2′-O-methyl modified oligonucleotide, or a fully LNA modified oligonucleotide, or may comprise a mixture of different nucleoside modifications, for example a mixture of LNA and 2′-O-MOE nucleosides. In some embodiments the totoalmer may comprise one or two 3′ terminal LNA nucleosides.
A tiny oligonucleotide is an oligonucleotide 7-10 nucleotides in length wherein each of the nucleosides within the oligonucleotide is an LNA nucleoside. Tiny oligonucelotides are known to be particularly effective designs for targeting microRNAs.
The inventors have identified that T4DNA ligase is capable of ligating a 5′ phosphorylated DNA nucleoside to the 3′ termini of a nucleoside modified oligonucleotide. The invention provides for the use of T4DNA ligase to ligate the 3′ terminus of a nucleoside modified oligonucleotide to the 5′ terminus of a DNA oligonucleotide, wherein the 3′ nucleoside of the nucleoside modified oligonucleotide is a modified nucleoside, such as a LNA nucleoside. The DNA oligonucleotide comprises at least one terminal DNA nucleoside, and may comprise 2 or 3 contiguous 5′ DNA nucleosides. Designs of such DNA oligonucleotides are disclosed herein, for example as illustrated in
The inventors have also identified that DNA polymerases, for example thermostable DNA polymerases such as Taq polymerase, and reverse transcriptases, can effectively use a nucleoside modified template for (e.g. first) strand synthesis. The invention therefore provides for the use of DNA polymerase or reverse transcriptase, for first strand synthesis of a complementary nucleic acid from a nucleoside modified oligonucleotide. As described herein this use may be combined with the use of T4DNA ligase. The invention therefore provides for a method for producing a complementary DNA (cDNA) molecule from a nucleoside modified oligonucleotide, said method comprising the step of ligating a DNA oligonucleotide adaptor (e.g. a capture probe) to the 3′ end of the nucleoside modified oligonucleotide, followed by the step of hybridizing a primer to the DNA oligonucleotide adaptor, and then performing 5′-3′ DNA polymerase, or reverse transcriptase, mediated elongation from the primer to produce a cDNA which comprises a nucleobase sequence which is complementary to the nucleoside modified oligonucleotide. The method may further comprise the subsequent step of performing PCR amplification of the cDNA. This method may be used for detection, quantification, amplification, sequencing or cloning of the nucleoside modified oligonucleotide. In some embodiments, the nucleoside modified oligonucleotide comprises at least one (such as 1, 2, 3, 4 or 5) 3′ terminal modified nucleosides, such as at least one (such as 1, 2, 3, 4 or 5) LNA or at least one (such as 1, 2, 3, 4 or 5) 2′ substituted nucleosides, such as 2′O-MOE. In some embodiments, the nucleoside modified oligonucleotide comprises at least one non terminal modified nucleosides, such as LNA or a 2′ substituted nucleoside, such as 2′-O-MOE.
The inventors have designed capture probe oligonucleotides which can be used to capture, detect, amplify, quantify or sequence oligonucleotides and polynucleotides, such as nucleoside modified oligonucleotides.
The invention provides for a capture probe oligonucleotide comprising 5′-3′:
i) a first nucleotide segment comprising
ii) a second nucleotide segment, comprising
See
In some embodiments, region 1A comprises or consists of at least 3 contiguous nucleotides, of predetermined sequence, wherein the 5′ terminal nucleotide is a DNA nucleotide which comprises a 5′ phosphate group (A). The at least 3 contiguous nucleotides are complementary to and can hybridize to region 2A. In some embodiments the at least 3 contiguous nucleotides of region 1A are DNA nucleotides.
In some embodiments, region 1A comprises or consists of at least 3 contiguous nucleotides, such as 3-10 contiguous nucleotides, such as 3-10 DNA nucleotides.
Region 1B is an optional sequence of nucleotides positioned 3′ of region 1A which may comprise a predetermined sequence or a degenerate sequence, or in some embodiments both a predetermined sequence part and a degenerate sequence part. The length of region B, when present may be modulated according to use. When a degenerate sequence is used it may allow the “molecular bar coding” of amplification products in subsequent sequencing steps, allowing for the determination of whether a particular amplification product is unique. This allows for comparative quantification of different oligonucleotides present in a heterogenous mixture of oligonucleotides. In some embodiments region 1B comprises 3-30 degenerate contiguous nucleotides, such as 3-30 degenerate contiguous DNA nucleotides.
It is known that some sequences may be preferentially amplified during PCR, and as such by counting the occurance of a genetic “barcode sequence”, originating from the degenerate sequence, you can determine the pre-amplification relative quantities (see e.g. Kielpinski & Vinter, NAR (2014) 42 (8): e70.
In some embodiments region 1B introduces a semi-degenerate sequence, which allows benefit of both a bar code sequence and a predetermined sequence. Additional benefit is a quality control of the barcode sequence (see e.g. Kielpinski et al., Methods in Enzymology (2015) vol. 558, pages 153-180). A semi-degenerate sequence has a selected semi-degenerate nucleobase at each position (based upon the Need a definition of semi-degenerate—add IUPAC codes, R, Y, S, W, K, M, B, D, H and V (See table 3).
In some embodiments region 1B has both degenerate sequence and predetermined sequence, or has both degenerate sequence and semi-degenerate sequence, or has both predetermined sequence and semi-degenerate sequence, or has degenerate sequence and predetermined sequence and semi-degenerate sequence.
If region B comprises a predetermined sequence it may for example provide an alternative, or nested, primer site, upstream of the universal primer site, the use of nested primer sites is a well-known tool for reducing non-specific binding during PCR amplification. In some embodiments region 1B comprises 3-30 predetermined contiguous nucleotides, such as 3-30 predetermined contiguous DNA nucleotides.
In some embodiments the capture probe does not comprise region 1B.
Region 1C is a region of nucleotides which comprises a predetermined primer binding site (also referred to as the universal primer binding site herein).
Region 2A is a region of nucleotides which are complementary to region 1A which form a duplex with region 1A (
In some embodiments, region 2A comprises or consists of 3-10 contiguous nucleotides, such as 3-10 DNA nucleotides. In some embodiments, the nucleotides of region 1A and region 2A are DNA nucleotides. The length and composition (e.g. G/C vs NT) of the complementary sequences 1A and 2A may be used to modulate the strength of hybridization, allowing for optimization of the capture probe. It is also recognized that introduction of mismatches within a complementary sequence can be used to decrease the hybridization strength (see WO2014110272 for example). In some embodiments region 1A and 2A do not form a contiguous complementary sequence, but due to partial complementarity in some embodiments regions 1A and 2A form a duplex when admixed with the sample. The 3′ most base pair of regions 1A and 2A should be a complementary base pair, and in some embodiments the two or three most base pairs of regions 1A and 2A are complementary base pairs. In some embodiments, these 3′ base pair(s) are DNA base pairs.
Region 2B serves the purpose of hybridizing the capture probe oligonucleotide to the nucleoside modified oligonucleotide that is to be detected, captured, sequenced and/quantified.
Region 2B is a region of at least two or three nucleotides which form a 3′ overhang (E), when region 1A and 2A, of the complementary sequences thereof, are hybridized. The 3′ terminal nucleoside of region 2B is blocked at the 3′ position (
In some embodiments, region 2B has a length of at least 3 nucleotides. The optimal length of region 2B may depend, at least on the length of the oligonucleotide to be captured, and the present inventors have found that region 2B can function with an overlap of 2 nucleotides, for example when using an RNase treated sample, and preferably is at least 3 nucleotides.
In some embodiments, region 2B comprises a degenerate sequence, or a semi-degenerate sequence, which allows for the capture of oligonucleotides without prior knowledge of the oligonucleotide sequence. The capture of oligonucleotides without prior knowledge of their sequence is particularly useful in identifying specific oligonucleotides from a library of different oligonucleotide sequences which have a desired biodistribution, or for the identification of partial oligonucleotide degradation products. The probes and methods of the invention may also be applied to the capture and identification of aptamers.
In some embodiments, region 2B comprises a predetermined sequence, allowing for the capture of nucleoside modified oligonucleotides with a known sequence. The use of a predetermined capture region 2B allows for capture, detection and quantification of therapeutic oligonucleotides in vivo, for example for pre-clinical or clinical development or subsequently for determining local tissue or cellular concentration or exposure in patient derived material. The determination of compound concentration in patients can be important in optimizing the dosage of therapeutic oligonucleotides in patients.
D is a linker moiety which blocks DNA polymerase, such as a linker which comprises a non-nucleotide linker. Region D allows for the capture probe regions 1A and 2A to hybridize. The advantage of preventing read-through of the DNA polymerase from region 1C to 2A is that it prevents the formation of an alternative template molecule. Such alternative template molecules result in mispriming of the primers specific to the nucleoside modified oligonucleotide on the 5′ region of the capture probe. (
In some embodiments of the invention the linker moiety D may be a region of nucleotides which allow region 1A and 2A to hybridise. Such a linker moiety would typically act as a template for DNA polymerase activity during PCR amplification, and as such the presence of the capture probe with a contiguous nucleotide sequence between region 1B and 2A may result in the co-amplification of a competing template during the PCR cycles, a particular issue when there is a low copy number of the initial template in the PCR reaction (or a low concentration of nucleoside modified oligonucleotide in the (e.g. patient) sample). It is therefore, when region D is a region of nucleotides, the region comprises a modification which prevents the read through activity of the polymerase, thereby avoiding the production of competing template molecules during PCR amplification. Such modifications may include the use of one or more non-hybridisable base moiety within the nucleotides of region D, or the use of inverted nucleotides.
In some embodiments region D comprises a polymerase blocking linker, such as a C6-32 polyethyleneglycol linker, such as a C18 polyethyleneglycol linker or an alkyl linker. Other non-limiting exemplary linker groups which may be used are disclosed herein.
In some embodiments regions 1A and 2A form a duplex of 3-20 base pairs, such as 6-15 base pairs, such as 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 base pairs. In some embodiments regions 1A and 2A form a region of DNA base pairs. In some embodiments, regions 1A and 2A form a region of 9 DNA base pairs.
In some embodiments, region C is between 10-30 nucleotides, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. In some embodiments, region C is design to avoid significant self-complementarity either within region C or within the capture probe—such significant self-complementarity may create an undesirable secondary structure of the capture probe when used in the sample. In some embodiments, region C may consist or comprise of DNA nucleosides.
In some embodiments, when present regions 1B consists or comprises at least 3 contiguous degenerate nucleosides, such as 3, 4, 5, 6, 7, 8, 9 or 10 contiguous degenerate nucleosides.
In some embodiments, region 2B consists or comprises at least 4 contiguous degenerate nucleosides, such as 4, 5, 6, 7, 8, 9, 10, 11 or 12 contiguous degenerate nucleosides.
In some embodiments, the nucleosides of regions 1A, 2A, 2B and C, and when present are DNA nucleosides.
The capture probe oligonucleotide may be used in detecting, quantifying, sequencing, amplifying or cloning an oligonucleotide in a sample, such as nucleoside modified oligonucleotides or an oligonucleotide wherein the 3′ most internucleoside linkage is a Rp phosphorothioate internucleoside linkage. The capture probe of the invention may be used for detecting, quantifying, sequencing, amplifying or cloning an oligonucleotide which comprises 3′ terminal modified nucleoside(s), such as high affinity nucleoside analogues and/or 2′ modified nucleosides, such as LNA nucleosides. The capture probe of the invention may be used for detecting, quantifying, sequencing, amplifying or cloning an oligonucleotide wherein said oligonucleotide further comprises a Rp phosphorothioate internucleoside linkage between the two 3′ most nucleosides of the oligonucleotide. The capture probe of the invention may be used for detecting, quantifying, sequencing, amplifying or cloning an oligonucleotide which comprises 3′ terminal modified nucleoside(s), such as high affinity nucleoside analogues and/or 2′ modified nucleosides, such as LNA nucleosides, wherein said oligonucleotide further comprises a Rp phosphorothioate internucleoside linkage between the two 3′ most nucleosides of the oligonucleotide. As illustrated herein the capture probes of the invention may be used to effectively capture LNA oligonucleotides, allowing for the detecting, quantifying, sequencing or cloning. 2′ modified oligonucleotides such as 2′-O-MOE or LNA gapmers, mixmers, totalmers are being developed or have already been approved as therapeutic oligonucleotides. The capture probe of the invention may be used to detecting, quantifying, sequencing or cloning phosphorothioate modified internucleoside linkages.
In some embodiments, the use of the capture probe oligonucleotide of the invention is for detecting, amplifying or quantifying a nucleoside modified oligonucleotide in a biological sample, such as in a biopsy sample, a blood sample or a fraction thereof, such as blood serum or plasma sample.
In some embodiments, the use of the capture probe oligonucleotide of the invention is for detecting, amplifying cloning, or quantifying an oligonucleotide in a biological sample, such as in a biopsy sample, a blood sample or a fraction thereof, such as blood serum or plasma sample, wherein said oligonucleotide comprises a Rp phosphorothioate internucleoside linkage between the two 3′ most nucleosides of the oligonucleotide.
In some embodiments, the use is for sequencing or cloning the oligonucleotide. Whilst for many years oligonucleotide therapeutics has provided the promise of going from target sequence to drug designed by Watson-Crick base pairing rules, in practice this has been very difficult to achieve and it has recently become apparent that individual sequences of oligonucleotides may have a profound effect on the pharmacological distribution of an oligonucleotide. It is therefore difficult to presume that a compound which has been select on the basis of its outstanding effect in vitro will have the same outstanding effect in vivo—simply put, its biodistribution may result in accumulation in non-target tissues and a low pharmacological effect in the target tissue. The present invention provides for the first time a method of cloning and sequencing nucleoside modified nucleotides, allowing the identification of the cryptic sequences which result in the uptake in the desired tissues, and avoid accumulation in non-target tissues. This may be achieved by making libraries of oligonucleotides with different sequences (e.g. degenerate oligonucleotide libraries) and using them in a method for identifying a nucleoside modified oligonucleotide (sequence) which is enriched in a target tissue in a mammal, said method comprising:
Alternatively, the method may be used to identify a nucleoside modified oligonucleotide (sequence) which has low accumulation in a non-target tissue in a mammal, said method comprising:
Typically, the step of isolation of the nucleoside modified oligonucleotides from the sample of tissue or cells obtained from the mammal (or patient) is RNase treated and may be further purified (e.g. via gel or column purification) prior to use in the oligonucleotide capture method of the invention.
The invention provides a method for detecting, quantifying, amplifying, sequencing or cloning a nucleoside modified oligonucleotide in a sample, said method comprising the steps of
In the above method the optimized capture probe of the invention may be used: The invention provides a method for detecting, quantifying, amplifying, sequencing or cloning a nucleoside modified oligonucleotide in a sample, said method comprising the steps of
The invention provides a method for detecting, quantifying, amplifying, sequencing or cloning an oligonucleotide in a sample, said method comprising the steps of
The invention provides a method for detecting, quantifying, amplifying, sequencing or cloning a nucleoside modified oligonucleotide in a sample, said method comprising the steps of
The sample may be a biological sample, such as a sample from an animal which has been administered the oligonucleotide.
In the methods disclosed herein, biological samples may be RNase and/or DNase treated prior to admixing with the oligonucleotide capture probe. Suitably RNase or DNase treatment is performed with enzymes which degrade RNA or DNA (respectively), but do not degrade nucleoside modified nucleotides or nucleotide modified nucleotides (e.g. phosphorothioates).
In the methods disclosed herein, biological samples may be RNase and/or DNase treated and an oligonucleotide fraction purified, e.g. via gel or column purification, prior to admixing with the oligonucleotide capture probe. In some embodiments, an oligonucleotide containing fraction is purified from a biological sample prior to admixing with the oligonucleotide capture probe. The sample referred to in the method(s) of the invention may therefore be an oligonucleotide enriched fraction obtained from the biological sample.
In the method(s) of the invention, prior to step a) an additional step of RNase and/or DNase treatment and/or purification of the sample may be performed. Furthermore, or alternatively, the ligation product of step b. may be purified prior to step c. Gel purification or column purification may be used for example after step b.
In some embodiments, step e. of the method(s) of the invention comprises a PCR amplification of the chain elongation product. The PCR step may utilize a primer which comprises a region which is complementary to the nucleoside modified oligonucleotide or a part thereof. The PCR may be a qPCR (quantitative PCR) method, such as droplet digital PCR (ddPCR).
For the detecting, quantifying, sequencing, amplifying or cloning of oligonucleotides with an unknown sequence, it may be necessary to utilize a 3′ adaptor ligation strategy whereby a nucleotide adaptor of known sequence is ligated to the 3′ end of the first synthesized strand including the reverse complement sequence of the nucleoside modified oligonucleotide.
In some embodiments, the method comprises an additional step, performed after step d) said additional step comprises ligating a 3′ adaptor to the product obtained in step d, and performing PCR on the product obtained using a primer which is complementary to the 3′ adaptor and a primer which is complementary to the capture probe oligonucleotide, such as the universal primer [a primer complementary to region 1C].
In some embodiments, the method further comprises the step of cloning the PCR product obtained.
In some embodiments, the method further comprises the step of sequencing the PCR product obtained.
In some embodiments, the method is for detecting or quantifying a therapeutic nucleoside modified oligonucleotide in a patient sample.
Security: DNA oligonucelotides with a unique sequence, for example have been developed to mark personal property or to contaminate thieves at the site of the theft. However, DNA oligonucleotides are inherently unstable in the environment, and as such the ability to detect the unique DNA oligonucleotides will deteriorate over time, and may be further accelerated by decontamination attempts. The use of nucleoside modified oligonucleotides in security and asset marking is therefore highly desirable as the modifications greatly enhance the stability of the oligonucleotides. The capture probe oligonucleotides of the present enable the detection of nucleoside modified oligonucleotides used in security and asset marking applications, and may be combined with PCR based detection methods.
The invention provides for:
The a4 capture probe is illustrated in
Where a mix pool of LNA oligonucleotides (LNA-mix-pool1) was used this was prepared by mixing the following LNA oligonucleotides. (O5 10 μM, O6 5 μM, O7 10 μM, O8 10 μM, O9 10 μM, O10 10 μM, O11 10 μM, O12 10 μM, O13 10 μM, O14 10 μM). Oligonucleotide O6 was present in the mix in half the molar ratio of all the other LNA oligonucleotides. For ease of writing this mix pool will always be presented as an equimolar mix in the examples with the conc. being correct for all LNA oligonucleotides but O6 that always will be present in half the described concentration.
All Ligation reactions before PCR reaction (Examples 7 to 10) were performed as follows: 2 μl LNA oligonucleotide containing sample was added to 2 μl capture probe oligonucleotide and mixed and incubated at 55° C. for 5 min. A mix containing 2 μl T4 DNA ligase (Thermo Scientific), 2 μl T4-DNA-ligase buffer, 8 μl PEG 4000 and 4 μl H2O was added to each tube and mixed. The following program was run on a thermal cycler. 2 min 37° C., 3 min 30° C., 5 min 22° C., 30 min 16° C. this cycle was repeated twice, then 10 min at 70° C. and stable at 4° C.
Sybr Green qPCR:
Sybr Green qPCR was performed using the SYBR® Green SuperMix low Rox kit from Quantabio. All reactions were performed in 10 μL with the following setup: 5 μl SYBR® Green SuperMix, 100 nM forward primer, 100 nM reverse primer, 2 μl input template and H2O up to 10 μL.
SYBR Green PCR Program:
Droplet Digital PCR (ddPCR):
qLNA-PCR was performed with droplet digital PCR (emulsion PCR) using BioRad Automatic Droplet Generator (AutoDG) together with the OX200 droplet digital PCR system. The emulsion PCR was performed with QX200™ ddPCR™ EvaGreen Supermix and the Automated Droplet Generation Oil for EvaGreen. The PCR reaction that was used as input for the AutoDG was setup as follows: 11 μl ddPCR™ EvaGreen Supermix, forward primer (final conc. 100 nM), reverse primer (final conc. 100 nM), sample 2 μL and H2O up to a total of 22 μL.
Following droplet generation the plate was sealed and run on the ddPCR program on a thermal cycler:
EvaGreen ddPCR Program:
Droplets were read on a QX200 droplet reader and the threshold was set manually.
In the present example six different ligation reactions were performed in an attempt of ligating the LNA containing, phosphorothioated oligonucleotides with capture probe oligonucleotides. Two different enzymes were tested—CircLigase II and T4 RNA Ligase. These enzymes are known to allow ligation of single stranded DNA molecules or single stranded RNA molecules, respectively. Each of those enzymes was tested for three different capture probe oligonucleotide designs: (a1) DNA oligonucleotide with fixed sequence, (a2) DNA oligonucleotide with 10 nucleotides on the 5′ end partially randomized and (a3) modified a1 oligonucleotide, designed to carry 2′-O-methyl modification on three 5′ most nucleotides. Each of the capture probe oligonucleotides was modified with 5′ phosphate which was necessary for the ligase to perform the ligation and with 3′ FAM, which was necessary to block the 3′ hydroxyl group, which would otherwise act as an undesired substrate for the ligation reaction, as well as allowing for fluorescence based detection of the molecule. The ligation reactions were performed as follows:
Ligation with CircLigase 2:
A pool of nucleoside modified oligonucleotides o1, o2 and o3 (see table 2) was prepared to contain 10 μM concentration of each species. Prior to ligation, 1 μl of the pool was mixed with a selected capture probe oligonucleotide from table 1, either 1 μl of 100 μM a1, or 1 μl of 100 μM a2, or 1 μl of 100 μM a3, followed by incubation at 50° C. for 5 min and placing on ice.
In parallel, a master mix was prepared composed of 1.5 volumes of H2O, 2 volumes of 50% PEG 4000, 0.5 volume of 50 mM MnCl2, 1 volume of CircLigase II 10× Reaction Buffer (epicentre), 2 volumes of 5 M betaine and 1 volume of CircLigase II enzyme (epicentre).
Eight μl of the master mix was added to 2 μl of each of the prepared oligonucleotide-capture probe mixes followed by incubation for 3 hours at 60° C. followed by 10 min at 80° C. and held at 4° C. until analysis using gel electrophoresis as described below.
Ligation with T4 RNA Ligase:
A Pool of LNA oligonucleotides o1, o2 and o3 (see table 2) was prepared to contain 10 μM concentration of each species. Prior to ligation, 2 μl of the pool was mixed with a selected capture probe oligonucleotide from table 1, either 2 μl of 100 μM a1, or 2 μl of of 100 μM a2, or 2 μl of of 100 μM a3, followed by incubation in 50° C. for 5 min and placing on ice.
In parallel, a master mix was prepared composed of 8 volumes of 50% PEG 4000, 2 volumes of 10× T4 RNA Ligase buffer (Thermo Fisher Scientific), 2 volumes of 1 mg/ml BSA (Thermo Fisher Scientific), 2 volumes of 10 mM ATP and 2 volumes of 10 U/μl T4 RNA Ligase (Thermo Fisher Scientific, catalog number EL0021).
Sixteen μl of the master mix was added to 4 μl of each of the prepared oligonucleotide-capture probe mixes followed by incubation for 5 hours at 4° C. followed by 10 hours at 16° C. followed by 10 min at 70° C. and held at 4° C. until analysis using gel electrophoresis as described below.
Treatment without any Ligase:
Reaction was performed identically to “Ligation with T4 RNA Ligase” but with replacement of volume of 10 mM ATP and T4 RNA Ligase with H2O.
To each of the above mentioned reactions an equal volume of 2× Novex® TBE-Urea Sample Buffer (Thermo Fisher Scientific) was added and samples were heat denatured for 2 min at 95° C. and placed on ice. Ten μl of the prepared samples were loaded onto Novex® TBE-Urea Gels, 15%, 15 well (Thermo Fisher Scientific) and the electrophoresis was conducted for 75 min with constant voltage of 180 V. Gel was visualized with ChemiDoc Touch Imaging System(Bio Rad) on a Blue Tray.
In the presented assay, the fluorescent signal after electrophoresis from samples treated with CircLigase II, T4 RNA Ligase or no ligase enzyme showed similar pattern and lacked the expected shift of the bands indicating ligation of the capture probe oligonucleotide to the LNA oligonucleotides. This indicates that within the detection limit of the utilized system there was no ligation between LNA oligonucleotides and tested capture probe oligonucleotides.
In conclusion none of the capture probe oligonucleotide-enzyme combinations yielded detectable ligation product.
To overcome difficulties with ligation a capture probe oligonucleotide to an LNA-oligonucleotide, novel designs of the capture probe have been envisioned (
In the present example we have tested multiple combinations of capture probe oligonucleotides (included in table 1) composed of DNA only (a4) or having four 5′-most nucleotides composed of RNA (the rest DNA) (a5) or having overhang composed of RNA (the rest DNA) (a6) or having both four 5′-most nucleotides composed of RNA and overhang composed of RNA (the rest DNA) (a7). An attempt of ligating these four different capture probe designs with four different ligase enzymes (T4 RNA Ligase, T4 DNA Ligase, T4 RNA Ligase 2, T7 DNA Ligase) was performed.
Five and a half μl of 10 μM of LNA oligonucleotide o4 was mixed with 5.5 μl of 100 μM capture probe oligonucleotide a4, or a5 or a6 or a7, followed by incubation at 50° C. for 5 min and placing on ice.
For Treatment without any enzyme, Master mix was prepared by combining 4 volumes of 50% PEG 4000, 3 volumes of H2O and 1 volume of 10× T4 DNA Ligase Buffer (Thermo Fisher Scientific).
For treatment with T4 RNA Ligase, master mix was prepared by combining 4 volumes of 50% PEG 4000, 1 volume of 10× T4 RNA Ligase Buffer (Thermo Fisher Scientific), 1 volume of 1 mg/ml BSA (Thermo Fisher Scientific), 1 volume of 10 mM ATP and 1 volume of 10 U/μl T4 RNA Ligase (Thermo Fisher Scientific, catalog number EL0021).
For treatment with T4 DNA Ligase, master mix was prepared by combining 4 volumes of 50% PEG 4000, 1 volume of 10× T4 DNA Ligase Buffer (Thermo Fisher Scientific), 2 volumes of H2O and 1 volume of 30 U/μl T4 DNA Ligase HC (Thermo Fisher Scientific, catalog number EL0021).
For treatment with T4 RNA Ligase 2, master mix was prepared by combining 4 volumes of 50% PEG 4000, 1 volume of 10× T4 RNA Ligase 2 Buffer (New England Biolabs), 2 volumes of H2O and 1 volume of T4 RNA Ligase 2 (New England Biolabs, catalog number M0239S). For treatment with T7 DNA Ligase, master mix was prepared by combining 2 volumes of 50% PEG 4000, 5 volumes of 2× T7 DNA Ligase Buffer (New England Biolabs) and 1 volume of T7 DNA Ligase (New England Biolabs, catalog number M0318).
Each master mix was split into 4 tubes, 8 μl to each. Two μl of the prepared substrate was added to each of the master mix, yielding in total 20 different combinations of enzyme and capture probe oligonucleotide. The mixture was incubated for (2 min at 37° C., 3 min at 30° C., 5 min at 22° C., 80 min at 16° C.)×2, hold at 4° C.
To each of the above mentioned reactions an equal volume of 2× Novex® TBE-Urea Sample Buffer (Thermo Fisher Scientific) was added and samples were heat denatured for 2 min at 95° C. and placed on ice. Ten μl of thus prepared samples have been loaded onto Novex® TBE-Urea Gels, 15%, 15 well (Thermo Fisher Scientific) and the electrophoresis was conducted for 75 min with constant voltage of 180 V. Gel was visualized with ChemiDoc Touch Imaging System(Bio Rad) on a Blue Tray.
The most efficient ligation was observed for a combination of T4 DNA ligase and the capture probe oligonucleotide a4. Detectable ligation signal has also been obtained for reaction of T7 DNA Ligase with the capture probe oligonucleotide a4 and the reaction of T4 DNA Ligase with a5 capture probe oligonucleotide. No other reactions gave any detectable signal, indicating either lack of any reactivity or very low efficiency of the reaction. The results were compared with a control reaction devoid of any added ligase.
In order to confirm that the observed band is indeed a product of ligation of the FAM labeled capture probe oligonucleotide and LNA oligonucleotide we have performed a series of reactions varying different parameters.
For reaction “1”, 2 μl of 10 μM of LNA oligonucleotide o4 was mixed with 2 μl of 10 μM capture probe oligonucleotide a4; for reactions “2”, “4” and “5”, 2 μl of 10 μM of LNA oligonucleotide o4 was mixed with 2 μl of 100 μM capture probe oligonucleotide a4; for reaction “3”, 2 μl of 100 μM of LNA oligonucleotide o4 was mixed with 2 μl of 100 μM capture probe oligonucleotide a4. Mixing of LNA oligonucleotide with the capture probe oligonucleotide was followed by incubation at 50° C. for 5 min and placing on ice.
Master mix for reactions “1”, “2” and “3” was prepared by combining 4 volumes of 50% PEG 4000, 1 volume of 10× T4 DNA Ligase Buffer (Thermo Fisher Scientific), 2 volumes of H2O and 1 volume of 30 U/μl T4 DNA Ligase HC (Thermo Fisher Scientific, catalog number EL0021).
For reaction “4”, master mix was prepared in identical way as for reactions “1”, “2” and “3”, but it was heat treated by incubating at 70° C. for 10 min to inactivate the enzyme.
For reaction “5”, master mix was prepared in identical way as for reactions “1”, “2” and “3”, but T4 DNA Ligase HC has not been added, and its volume was replaced with H2O.
Ligation reactions were initiated by transferring 16 μl of the appropriate master mix to the prepared substrate and incubating for 5 h at 4° C., 10 h at 16° C., 10 min at 70° C. and kept at 4° C.
To each of the above mentioned reactions an equal volume of 2× Novex® TBE-Urea Sample Buffer (Thermo Fisher Scientific) was added and samples were heat denatured for 2 min at 95° C. and placed on ice. Seven μl of thus prepared samples have been loaded onto Novex® TBE-Urea Gels, 15%, 15 well (Thermo Fisher Scientific) and the electrophoresis was conducted for 75 min with constant voltage of 180 V. Gel was visualized with ChemiDoc Touch Imaging System(Bio Rad) on a Blue Tray.
In reaction “1”, the amount of the capture probe oligonucleotide was reduced 10 times, yielding dramatic decrease of signal for unligated capture probe (lower band) and a slight decrease of signal for the ligated product (upper band). In reaction “3”, the amount of LNA oligonucleotide was increased 10 times, yielding dramatic increase of signal for ligated product, and slight decrease for unligated capture probe oligonucleotide. Reactions “4” and “5” were designed to investigate if the appearance of the ligated product is T4 DNA Ligase dependent, which is confirmed since the ligation product does not appear in the absence of the enzyme nor in the presence of heat inactivated enzyme.
In the present example LNA containing oligonucleotide “o15” was ligated to the capture probe oligonucleotide “a4” varying both o15 and a4 concentrations.
One μl of 2 μM (“L”) or 10 μM (“H”) LNA oligonucleotide o15 was mixed with 1 μl of either 1, 5, 10, 20, 30, 60 or 100 μM capture probe oligonucleotide a4 and with 2 μl H2O, yielding 14 different combinations.
Mixing of LNA oligonucleotide with the capture probe oligonucleotide was followed by incubation at 50° C. for 5 min and placing on ice.
Master mix was prepared by combining 4 volumes of 50% PEG 4000, 1 volume of 10× T4 DNA Ligase Buffer (Thermo Fisher Scientific) and 1 volume of 30 U/μl T4 DNA Ligase HC (Thermo Fisher Scientific, catalog number EL0021).
Ligation reactions were initiated by transferring 6 μl of the appropriate master mix to the prepared substrate and incubating for (2 min at 37° C., 3 min at 30° C., 5 min at 22° C., 80 min at 16° C.)×2, kept at 4° C.
To each of the above mentioned reactions an equal volume of 2× Novex® TBE-Urea Sample Buffer (Thermo Fisher Scientific) was added and samples were heat denatured for 2 min at 95° C. and placed on ice. Seven μl of thus prepared samples have been loaded onto Novex® TBE-Urea Gels, 15%, 15 well (Thermo Fisher Scientific) and the electrophoresis was conducted for 75 min with constant voltage of 180 V. Gel was visualized with ChemiDoc Touch Imaging System(Bio Rad) on a Blue Tray.
Quantification of the band with the ligated product revealed that the most efficient ligation for two tested concentration of o15 (final concentrations of 1 μM and 0.2 μM) occurred at the final concentration of a4 equal or higher than 2 μM.
This experiment was designed to determine the minimal time needed for efficient ligation of the a4 capture probe oligonucleotide to a random pool of LNA oligonucleotides (o15).
Samples of two different concentrations of o15 were ligated for 0 up to 6 cycles, 20 minutes each.
7.7 μl of 2 μM (“L”) or 10 μM (“H”) oligonucleotide o15 was mixed with 7.7 μl of 20 μM capture probe oligonucleotide a4 and with 15.4 μl H2O.
Mixing of LNA oligonucleotide with the capture probe oligonucleotide oligonucleotide was followed by incubation at 50° C. for 5 min and placing on ice.
4 μl of the mix was removed and combined with 10 μl 2× Novex® TBE-Urea Sample Buffer (Thermo Fisher Scientific)—to act as samples L0 and H0.
Master mix was prepared by combining 4 volumes of 50% PEG 4000, 1 volume of 10× T4 DNA Ligase Buffer (Thermo Fisher Scientific) and 1 volume of 30 U/μl T4 DNA Ligase HC (Thermo Fisher Scientific, catalog number EL0021).
Ligation reactions were initiated by transferring 40.2 μl of the appropriate master mix to the prepared substrate and incubating for (2 min at 37° C., 3 min at 30° C., 5 min at 22° C., 10 min at 16° C.) for 6 cycles, removing 10 μl after cycles 1, 2, 3, 4, 5 or 6 and combining with 10 μl 2× Novex® TBE-Urea Sample Buffer (Thermo Fisher Scientific) to stop the reaction. L0 and H0 samples, which already contained 10 μl 2× Novex® TBE-Urea Sample Buffer (Thermo Fisher Scientific) were combined with 6 μl of the master mix.
To each of the above mentioned reactions an equal volume of 2× Novex® TBE-Urea Sample Buffer (Thermo Fisher Scientific) was added and samples were heat denatured for 2 min at 95° C. and placed on ice. Seven μl of thus prepared samples have been loaded onto Novex® TBE-Urea Gels, 15%, 15 well (Thermo Fisher Scientific) and the electrophoresis was conducted for 75 min with constant voltage of 180 V. Gel was visualized with ChemiDoc Touch Imaging System(Bio Rad) on a Blue Tray.
As the electrophoretic analysis of the ligated samples indicates, the majority of the ligation occurs within the first cycle with only modest improvements in subsequent cycles.
This experiment was designed to determine the optimal PEG 4000 concentration in the capture probe oligonucleotide ligation reaction.
5.5 μl of 2 μM (“L”) or 10 μM (“H”) LNA oligonucleotide o15 was mixed with 5.5 μl of 20 μM capture probe oligonucleotide a4 and with 11 μl H2O.
Mixing of LNA oligonucleotide with the capture probe oligonucleotide oligonucleotide was followed by incubation at 50° C. for 5 min and placing on ice. Mixture was split into 5 tubes, 4 μl to each.
Master mix was prepared by combining 4 volumes of 0% or 10% or 20% or 30% or 40% or 50% PEG 4000, 1 volume of 10× T4 DNA Ligase Buffer (Thermo Fisher Scientific) and 1 volume of 30 U/μl T4 DNA Ligase HC (Thermo Fisher Scientific, catalog number EL0021).
Ligation reactions were initiated by transferring 6 μl of each of the master mixes to the prepared substrate and incubating for (2 min at 37° C., 3 min at 30° C., 5 min at 22° C., 10 min at 16° C.) for 4 cycles, followed by adding 10 μl Novex® TBE-Urea Sample Buffer (Thermo Fisher Scientific) to stop the reaction.
L0 and H0 samples, which already contained 10 μl 2× Novex® TBE-Urea Sample Buffer (Thermo Fisher Scientific) were combined with 6 μl of the master mix.
To each of the above mentioned reactions an equal volume of 2× Novex® TBE-Urea Sample Buffer (Thermo Fisher Scientific) was added and samples were heat denatured for 2 min at 95° C. and placed on ice. Seven μl of thus prepared samples have been loaded onto Novex® TBE-Urea Gels, 15%, 15 well (Thermo Fisher Scientific) and the electrophoresis was conducted for 75 min with constant voltage of 180 V. Gel was visualized with ChemiDoc Touch Imaging System(Bio Rad) on a Blue Tray.
Quantification of the bands in the electrophoregram indicate that the most efficient ligation occurred with the final PEG 4000 concentration of 15%, closely followed by 20%.
To illustrate that a PCR reaction can be performed on the product of the ligation between a LNA oligonucleotide and a capture probe, we setup up a ligation reaction using LNA oligonucleotide 6(O6) and O6-capture probe 1 (O6-CP1). This reaction was done at high concentration in equimolar ratio. 100 μM O6 and 100 μM O6-CP1. The ligation was performed as described in the Materials and Methods section “LNA-DNA ligation for PCR” and was setup both in the absence and presence of T4-DNA Ligase. A dilution series of the ligation mix was made (250 pM, 62.5 pM, 15.6 pM, 3.9 pM, 1 pM, 244 fM, 61 fM) and used as input in a Sybr Green PCR reaction using the PerfeCTa SYBR® Green SuperMix kit from Quantabio that utilizes a modified Taq DNA polymerase (as described in the Materials and Method section “Sybr Green qPCR”). The ligated product was detected using a primer set consisting of the O6-p1 and CP1-p1 (see table 1 at an annealing temperature of 60° C.
In order to use this new technique as a quantitative LNA detection method, it has to have a linear relation between LNA oligonucleotide input in the ligation and measured output in the PCR. We showed that this reaction is linear by setting up the experiment depicted in
We conclude that this PCR based LNA oligonucleotide detection method is linear and can therefore be used as a quantitative method to measure LNA oligonucleotide concentrations. In the following the combined method of LNA-DNA ligation followed by a quantitative PCR reaction is termed quantitative LNA-PCR (qLNA-PCR).
To examine the specificity of the qLNA-PCR method we set up an experiment to illustrate that the ligation of the capture probe oligonucleotide to a LNA oligonucleotide is sequence specific. In short, we made a 5× serial dilution of the LNA-pool-1 (1 nM, 200 pM, 40 pM, 8 pM, 1.6 pM, 320 fM, 64 fM, H20) and used it as input material in a ligation reaction with primers O5-CP1, O6-CP1 or Universal1-CP1 all done with and with-out presence of T4 DNA Ligase. Following a 9× dilution of the ligation reactions, 2 μL product of all ligations was used as input for two Sybr Green PCR reactions. A PCR using the primers O5-p1 and CP1-p1 with a 53° C. annealing temperature (O5 PCR) and a PCR using the primers O6-p2 and CP1-p1 with a 50° C. annealing temperature (O6 PCR) was conducted.
Collectively we concluded that qLNA-PCR can be very specific with specificity originating from both the ligation step and from the PCR step where a LNA specific primer is used.
To illustrate that qLNA-PCR can be used in an in vivo setting to detect and quantify LNA oligonucleotides an in vivo mouse experiment was setup.
LNA-mix-pool1 was injected intravenously in two adult female C57 black with 950 nmol/kg of LNA oligonucleotide in total (100 nmol/kg of each LNA oligonucleotide, and 50 nmol/kg of oligonucleotide o6). Two control mice were injected with PBS. Seven days after injection the mice were sacrificed and brain and liver tissue was isolated and snap frozen with dry ice. The small RNA fraction <200 bp was isolated from 50 mg of brain tissue and 25 mg of liver tissue from each mouse using the miRNeasy kit from Qiagen using there suggested “Preparation of miRNA-enriched fractions separate from larger RNAs (>200)” protocol. The frozen tissue was placed in QIAzol lysis reagent and homogenized in a final volume of 18 μl. Two μl sample was used in a ligation reaction with 2 μl (1 μM) Universal1-CP1 capture probe oligonucleotide. Following ligation the ligation-mix were diluted in H2O (Brain 50×, Liver 5000×) and 2 μl of this dilution was used as input in a EvaGreen ddPCR reaction (as described in the Materials and Methods section) with the primers O13-p1 and CP1-p1 at 56.6° C.
The used LNA oligo are displayed in table 1. A mix pool of LNAs (LNA-mix-pool1) was prepared by mixing the following LNA oligos. (O5 10 μM, O6 5 μM, O7 10 μM, O8 10 μM, O9 10 μM, O10 10 μM, O11 10 μM, O12 10 μM, O13 10 μM, O14 10 μM). Oligo 6(O6) is present in the mix in half the molar ratio of all the other LNAs. For ease of writing this mix pool will always be presented as an equimolar mix in the examples with the conc. being correct for all but O6 that always will be present in half the described concentration.
All Ligation reaction before PCR reaction was performed as follows: 2 μl Sample was added to 2 μl Capture probe mixed and incubated at 55° C. for 5 min. A mix containing 2 μl T4 DNA ligase (Thermo Scientific), 2 μl T4-DNA ligase buffer, 8 μl PEG and 4 μl H2O was added to each tube and mixed. The following program was run on a thermal cycler. 2 min 37° C., 3 min 30° C., 5 min 22° C., 30 min 16° C. this cycle was repeated twice, then 10 min at 70° C. and stable at 4° C.
Sybr Green qPCR:
Sybr Green qPCR was performed using the SYBR® Green SuperMix low Rox kit from Quantabio. All reaction was performed 10 μL with the following setup: 5 μl SYBR® Green SuperMix, 100 nM forward primer, 100 nM reverse primer, 2 μl input template and H2O up to 10 μL.
SYBR Green PCR Program:
ddPCR:
qLNA-PCR was performed with droplet digital PCR (emulsion PCR) using BioRad Automatic Droplet Generator (AutoDG) together with the OX200 droplet digital PCR system. The emulsion PCR was performed with QX200™ ddPCR™ EvaGreen Supermix and the Automated Droplet Generation Oil for EvaGreen. The PCR reaction that was used as input for the AutoDG was setup as follows: 11 μl ddPCR™ EvaGreen Supermix, forward primer (final conc. 100 nM), reverse primer (final conc. 100 nM), sample 2 μL and H2O up to a total of 22 μL.
Following droplet generation the plate was sealed and run on the ddPCR program on a thermal cycler:
EvaGreen ddPCR Program:
Droplets were read on a QX200 droplet reader and the threshold was set manually.
In vivo qLNA-PCR study: LNA-mix-pool1 was injected IV in 2 adult mice with 950 nmol/kg in total (100 nmol/kg of each oligo and 50 nmol/kg of O6). 2 mice were injected with pure PBS as control. 7 days after injection the mice were sacrificed and various tissues were harvested and snap frozen with dry ice. Small RNA was purified from 50 mg tissue or 25 mg (liver tissue) using the miRNeasy kit from Qiagen using there suggested “Preparation of miRNA-enriched fractions separate from larger RNAs (>200)” protocol. The frozen tissue was placed in QIAzol lysis reagent and homogenized.
One μl of 10 μM of the oligo O16, O17, O18, O19, O20, O21, O22, O8 or H2O was mixed with 1 μl of 100 μM O8-CP1. Mixing of LNA oligonucleotide with the capture probe was followed by incubation at 50° C. for 5 min and placing on ice.
Master mix was prepared by combining 3 volumes of 50% PEG 4000, 3 volumes of H2O, 1 volume of 10× T4 DNA Ligase Buffer (Thermo Fisher Scientific) and 1 volume of 30 U/μl T4 DNA Ligase HC (Thermo Fisher Scientific, catalog number EL0021).
Ligation reactions were initiated by transferring 8 μl of the appropriate master mix to the prepared substrate and incubating for (2 min at 37° C., 3 min at 30° C., 5 min at 22° C., 30 min at 16° C.)×3, kept at 4° C.
To each of the above mentioned reactions an equal volume of 2× Novex® TBE-Urea Sample Buffer (Thermo Fisher Scientific) has been added and samples were heat denatured for 2 min at 95° C. and placed on ice. Ten μl of thus prepared samples have been loaded onto Novex® TBE-Urea Gels, 15%, 15 well (Thermo Fisher Scientific) and the electrophoresis was conducted for 75 min with constant voltage of 180 V. Gel was visualized with ChemiDoc Touch Imaging System(Bio Rad) on a Blue Tray.
This experiment is design to evaluate the importance of the chirality of the phosphotioate backbone with respect to T4 DNA Ligase ability to ligate the LNA oligo and the DNA capture probe together. We use fully stereo defined compounds of 08 as substrate. The chirallity of the last three phophotioate bindings of the 3′ end are indicated in the figure. The band of the ligated product appears just above the strong band of the capture probe (see
| Number | Date | Country | Kind |
|---|---|---|---|
| 16198340.8 | Nov 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/078695 | 11/9/2017 | WO | 00 |
