Patent application EP20171272A (EP), filed 2020-04-24
Publication number: EP3730628A1
Title: POLYNUCLEOTIDE ADAPTER DESIGN FOR REDUCED BIAS
Abstract: Compositions and methods of use are provided that among other things, allow for efficient adapter ligation to small RNAs. Embodiments of the compositions include partially double stranded polynucleotides for use as 3' adapters that contain a cleavable linker positioned between a single-stranded region and a doublestranded region. Upon ligating the 3' adapters, the single-stranded region is released by cleaving the cleavable linker.
IPC codes: C12N  15/    10            A I
C12Q   1/  6855            A I
Claims: number: 1
text: A composition comprising: a partially double-stranded polynucleotide molecule having a top strand and a bottom strand, wherein:
number: 2
text: The composition according to claim 1, wherein the bottom strand further comprises a blocking moiety at the 3' terminus;
such as wherein the blocking nucleotide prevents ligation; optionally wherein the blocking moiety comprises a modification selected from the group consisting of a 3' inverted dT, a 3' C3 spacer, a 3' amino dN, a 3' phosphorylated dN, and a dideoxynucleotide.
number: 3
text: The composition according to claim 1 or 2, wherein the top strand has a phosphorylated or pre-adenylated 5' terminus.
number: 4
text: The composition according to any of claims 1-3, wherein the top and bottom strands are part of the same oligonucleotide; or wherein the top and bottom strands are in different oligonucleotides.
number: 5
text: The composition according to any of claims 1-4, wherein the polynucleotide molecule is a DNA or wherein the polynucleotide molecule is RNA.
number: 6
text: The composition according to any of claims 1-5, wherein the third sequence has a length in the range of 4-12 nucleotides.
number: 7
text: The composition according to any of claims 1-6, wherein the site-specific cleavable sequence comprises a deoxyuridine, or wherein the site-specific cleavable nucleotide is a deoxyuridine; or wherein the site-specific cleavable sequence comprises or consists of a restriction endonuclease cleavage site.
number: 8
text: The composition according to any of claims 1-7, wherein the site-specific cleavable sequence, nucleotide or bond is positioned at the junction of the second and third sequences, or wherein the site-specific cleavable sequence, nucleotide or bond is positioned in the second sequence within 4 nucleotides of the junction between the second and third sequences; and/or wherein there is more than one cleavable nucleotide or sequence in the polynucleotide molecule, wherein each of the cleavable nucleotides, sequences or bonds is positioned within the second sequence within 8 nucleotides of the junction of the junction between the second and third sequences.
number: 9
text: A composition comprising: (i) a 5' exonuclease, such as lambda 5' exonuclease; and (ii) a nicking endonuclease.
number: 10
text: The composition according to claim 9, further comprising a deadenylase; and/or further comprising: (i) a nicking endonuclease and/or (ii) a glycosylase/lyase or a glycosylase.
number: 11
text: A method comprising:
number: 12
text: A kit comprising a composition according to any of claims 1-8, and further comprising:
number: 13
text: A method of ligating a 3' polynucleotide adapter to a target polynucleotide, comprising:
number: 14
text: The method according to claim 13, wherein the 3' adapter is DNA and the population of target polynucleotide is RNA.
number: 15
text: The method of claim 13 or 14, further comprising:
(c) ligating a 5' polynucleotide adapter molecule having a 5' single-strand extension comprising degenerate nucleotides to the product of step (b); optionally wherein the 5' adapter is RNA;
optionally wherein the method further comprises incubating the product of step (c) with a reverse transcriptase, to copy the ligated RNA into cDNA.
number: 16
text: The method according to any of claims 13-15, wherein steps (a)-(c) are performed in a single reaction vessel; and/or wherein no intermediate purification or separation steps are performed between steps (a)-(c).
number: 17
text: The method according to any of claims 13-16, further comprising priming cDNA synthesis using the bottom strand of the polynucleotide molecule, after it has been cleaved.
number: 18
text: The method according to any of claims 13-17, wherein the population of polynucleotide molecules comprises RNA molecules that are variable in size and concentration.
number: 19
text: A partially double-stranded polynucleotide molecule comprising a double-stranded region formed by a first nucleic acid strand and a second nucleic acid strand that comprises a sequence that is complementary to the sequence of the first strand, wherein:
Office action: search_report, dated 2020-10-28
Rejection reasons: novelty_epc_54
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Final disposition: pending (2020-10-28)