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US-12618107-B2 - Methods for forming adapter ligated nucleic acid molecules

US12618107B2US 12618107 B2US12618107 B2US 12618107B2US-12618107-B2

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 double-stranded region. Upon ligating the 3′ adapters, the single-stranded region is released by cleaving the cleavable linker.

Inventors

  • Shengxi Guan
  • Sean Maguire

Assignees

  • NEW ENGLAND BIOLABS, INC.

Dates

Publication Date
20260505
Application Date
20200424

Claims (16)

  1. 1 . A method comprising: ligating 3′ adaptor molecules to the ends of members of a population of single stranded target polynucleotides, wherein the 3′ adaptor molecules comprise: a top strand and a bottom strand, wherein: (a) the top strand comprises a nucleic acid sequence that is complementary to a portion of the nucleic acid sequence of the bottom strand, such that the top strand and bottom strand form a double-stranded region by complementary base-pairing; and (b) the bottom strand comprises: (i) a non-complementary 3′ single-stranded extension, (ii) a sequence of at least 4 degenerate nucleotides, wherein the at least 4 degenerate nucleotide sequence is a random sequence, wherein the random sequences the 3′ adaptor molecules bind to members of the population of single stranded target polynucleotides; and (iii) a site-specific cleavable sequence or nucleotide at or near the junction between the double-stranded region and the single-strand extension, suitable f removing the single-strand extension by cleavage to form a double-stranded region at t 3′ end of the target polynucleotides; cleaving the single-strand extensions of the ligated 3′ adaptors; ligating 5′ adaptors to the 5′ ends of the single stranded target polynucleotides, wherein the 5′ polynucleotide adaptors comprise a top strand and a complementary bottom strand with the bottom strand having a 5′ single-strand extension containing degenerate bases.
  2. 2 . The method according to claim 1 , wherein the single stranded target polynucleotides are RNA.
  3. 3 . The method according to claim 2 , wherein the polynucleotides are members of a library.
  4. 4 . The method according to claim 2 , wherein the polynucleotides are members of a population of RNAs that are variable in size and concentration.
  5. 5 . The method according to claim 4 , further comprising, after ligating the 3′ adaptor molecules, cleaving the single-strand extensions of the ligated 3′ adaptors, and ligating 5′ adaptors: reverse transcribing the RNA and forming a cDNA library.
  6. 6 . The method according to claim 5 , further comprising: performing the steps of ligating the 3′ adaptor molecules, ligating the 5′adaptors and reverse transcribing the RNA in a one pot workflow.
  7. 7 . The method according to claim 5 , wherein a purification step is not performed prior to reverse transcribing the RNA and forming the cDNA library.
  8. 8 . The method according to claim 2 , wherein the RNA is sRNA.
  9. 9 . The method according to claim 1 , wherein the step of cleaving the single-strand extensions of the ligated 3′ adaptors comprises cleaving a site-specific cleavable sequence or nucleotide in the 3′ adaptors with a nicking restriction endonuclease.
  10. 10 . The method according to claim 1 , wherein the step of cleaving the single-strand extensions of the ligated 3′ adaptors comprises cleaving a site-specific cleavable sequence or nucleotide in the 3′ adaptors with a glycosylase/lyase.
  11. 11 . The method according to claim 1 , wherein the single stranded target polynucleotides are RNA in a body fluid.
  12. 12 . The method according to claim 1 , wherein the single stranded target polynucleotides are RNA in a cell lysate.
  13. 13 . The method of claim 1 , wherein cleaving the single-strand extensions of the ligated 3′ adaptors is performed before ligating 5′ adaptors.
  14. 14 . The method of claim 1 , wherein cleaving the single-strand extensions of the ligated 3′ adaptors is performed after ligating 5′ adaptors.
  15. 15 . The method of claim 1 , wherein the top strand and bottom strand of the 3′ adaptor molecules are formed from two polynucleotide strands.
  16. 16 . The method of claim 1 , wherein the top strand and bottom strand of the 3′ adaptor molecules are formed from a single polynucleotide strand.

Description

CROSS-REFERENCE This application is a § 371 application of International Application No. PCT/US2020/29761, filed Apr. 24, 2020, which claims the benefit of US Provisional Application 62/839,191, filed on Apr. 26, 2019, and U.S. application Ser. No. 16/796,113, filed Feb. 20, 2020. These applications are incorporated herein by reference in their entireties. BACKGROUND Preferential ligation of adapters to some single-stranded RNAs and not others in an RNA library results in inaccurate profiling of a library composition. In order to reduce bias, adapters having single-strand extensions that act as splints can be utilized. However, such adapters can readily ligate with each other in part because of their excess concentration relative to the target RNA. Adapter dimer formation is particularly problematic when the target RNAs are small because the ligation artifacts such as adapter dimers may not be readily distinguished from target RNAs based on size. As a consequence, standard size separation techniques such as electrophoresis are ineffective. Current methods are thus challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Some classes of small RNAs (sRNAs) contain a 2′-O-methylation (2′OMe) modification on the ribose moiety of the 3′ terminal nucleotide. This modification stabilizes the sRNA and is present in endogenous siRNAs, miRNAs in plants and piRNAs in animals (Ghildiyal, et al. (2009) Nature Reviews Genetics, 10, 94-108). The 2′OMe modification severely impacts ligation efficiency to single-stranded DNA (ssDNA) adapters, as well as the efficiency of the 3′ polyadenylation or polyuridylation required for template-switching approaches (Munafo, et al., (2010) RNA, 16, 2537-2552). Combined with structural and sequence biases, this modification can make sequencing and discovery of 2′OMe modified RNA difficult and bias sequencing libraries against modified sRNA (Dard-Dascot, et. al., (2018) BMC Genomics, 19, 118). sRNAs are important regulators of gene expression and are involved in human development and disease. Next-generation sequencing (NGS) allows for scalable, genome-wide studies of sRNA with the proviso that library preparations derived from sRNA populations are representative of the component RNAs. The ligation efficiency and ligation bias of existing single-stranded adapters varies according to the sequence of the target and the adapter. Different adapter sequences can cause profound changes in library content (Jayaprakash, et al., (2011) Nucleic Acids Research, 39, e141-e141; Baran-Gale, et al., (2015) Frontiers in Genetics, 6, 352 and McLaughlin, et al. (1982) 125, 639-643). SUMMARY Provided herein, among other things, is a partially double-stranded polynucleotide molecule having a top strand and a bottom strand that can be used as a 3′ adapter and thereby may be referred to as a randomized Splint adapter. This polynucleotide molecule is characterized by a first sequence in the top strand. The bottom strand is characterized by a second sequence which is complementary to the first sequence, and a third sequence that is 3′ of the second sequence and includes a sequence of at least 4 degenerate nucleotides; and a site-specific cleavable linker that may be a sequence, nucleotide or bond, where the cleavable linker is at or near the junction between the second and third sequences. An embodiment of the partially double stranded polynucleotide serving as a 3′ adapter is illustrated as Adapter 2 in FIG. 1. As shown, the nucleotide at the 5′ end of the top strand is base paired with the bottom strand such that the double-stranded polynucleotide molecule has a 3′ single-stranded extension (or “overhang”) comprising the degenerate nucleotides of the bottom strand. The top strand of the 3′ adapter may be ligated to a target polynucleotide via a ligation that is splinted by the bottom strand of the 3′ adapter, as illustrated in FIG. 1. In some embodiments, the present 3′ adapter may be used in conjunction with a 5′ adapter, an example of which is also illustrated as Adapter 1 in FIG. 1. As shown, the 5′ adapter may be a partially double-stranded polynucleotide molecule having a top strand and a bottom strand, wherein the top strand comprises a first sequence and the bottom strand comprises: a second sequence which is complementary to the first sequence and a third sequence that is 5′ of the second sequence and comprises a sequence of at least 4 degenerate nucleotides. In this adapter, the nucleotide at the 3′ end of the top strand is base paired with the bottom strand such that the double-stranded polynucleotide molecule has a 5′ single-stranded extension (or “overhang”) comprising the degenerate nucleotides of the bottom strand. The top strand of the 5′ adapter is ligated to the target polynucleotide via a ligation that is splinted by the bottom strand of the 5′ adapter, as illustrated in FIG. 1. As illustrated in FIG. 1, t