Low sequence bias single-stranded DNA ligation

• US 9,816,120 B2
• Filed: 01/09/2014
• Issued: 11/14/2017
• Est. Priority Date: 01/09/2013
• Status: Active Grant

• Current Assignee: Penn State Research Foundation (Pennsylvania State University)
• Original Assignee: Penn State Research Foundation (Pennsylvania State University)
• Inventors: Bevilacqua, Philip C., Assmann, Sarah M., Ding, Yiliang, Kwok, Chun Kit
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US14/151,491
• Publication Number:
  US 20140193860A1
• Time in Patent Office: 1,405 Days
• Field of Search: None
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Y 605/01001 DNA ligase (ATP) (6.5.1.1)

Abstract

The invention provides compositions and methods for ligating single stranded nucleic acids wherein the ligation is based on fast, efficient, and low-sequence bias hybridization of an acceptor molecule with a donor molecule. In one embodiment, the structure of the donor molecule comprises a stem-loop intramolecular nucleotide base pairing (i.e., hairpin) and a 3′-overhang region such that the overhang is able to hybridize to nucleotides present in the 3′ end of the acceptor molecule.

Claims

1. A method of producing a ligated single stranded nucleic acid molecule, the method comprising:

a) contacting a single-stranded acceptor nucleic acid molecule with a population of donor nucleic acid molecules, wherein each of the donor nucleic acid molecules comprises a stem region, and a single-stranded 3′

terminal overhang region, wherein the sequence of the donor nucleic acid is designed to contain at least one mismatch internally in the stem region, and wherein at least the four 5′

terminal nucleotides of the donor molecule form Watson-Crick base pairs as part of the stem region, and wherein the donor molecule comprises at least one randomized templating nucleotide in the 3′

terminal overhang region at the position nearest the stem loop, thereby allowing unbiased hybridization with a multiplicity of different acceptor molecules;

b) hybridizing the single stranded 3′

terminal overhang region of the donor nucleic acid molecule to the acceptor molecule under conditions where the stem structure is formed in the donor molecule, wherein the acceptor molecule may comprise any nucleotide sequence at the 3′

end, thereby forming an acceptor-donor hybrid molecule comprising a nick or gap between the acceptor nucleic acid and donor nucleic acid molecule; and

c) ligating the 5′

end of the donor nucleic acid molecule to the 3′

end of the acceptor nucleic acid molecule with a ligase that is active under conditions where the stem structure is formed in the donor nucleic acid molecule, thereby generating a ligated product, whereby the yield of ligated product is at least 65%.

2. The method of claim 1, wherein the ligation in step c) is accomplished after the hybridization in step b), wherein the hybridization step positions the acceptor and donor molecule in a way that ligation occurs under conditions that allow for ligation between the 5′

end of the donor nucleic acid molecule and the 3′

end of the acceptor nucleic acid molecule.

3. The method of claim 1, wherein the ligase comprises a DNA ligase.

4. The method of claim 3, wherein the DNA ligase is T4 DNA ligase.

5. The method of claim 1, wherein the 3′

terminal overhang region of donor molecule comprises at least 6 base(s).

6. The method of claim 1, wherein the stem region of the donor molecule is double stranded and comprises at least 6 nucleotide pairs.

7. The method of claim 1, wherein the donor molecule further comprises a loop structure, wherein the loop structure comprises at least 3 bases.

8. The method of claim 7, wherein the loop structure comprises a portion of a primer binding site.

9. The method of claim 1, wherein at least one donor molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

28 and SEQ ID NO;

29, and wherein the ligation is performed using T4 DNA ligase.

10. The method of claim 1, wherein the stem of at least one donor molecule comprises a restriction enzyme cleavage site.

11. The method of claim 1, wherein at least one donor molecule comprises at least six randomized templating nucleotides in the 3′

overhang, and wherein the yield is at least 90%.

12. The method of claim 11, wherein at least one donor molecule comprises the nucleotide sequence as set forth in SEQ ID NO:

29.