Search

US-20260125671-A1 - METHODS FOR GENERATING CIRCULAR NUCLEIC ACID MOLECULES

US20260125671A1US 20260125671 A1US20260125671 A1US 20260125671A1US-20260125671-A1

Abstract

Provided herein are methods for generating circular nucleic acid molecules and circular nucleic acid libraries. The methods can be used to generate clonal populations of target nucleic acid molecules for downstream applications such as sequencing.

Inventors

  • Matthew Kellinger
  • Sinan ARSLAN
  • Michael Previte
  • Junhua Zhao

Assignees

  • ELEMENT BIOSCIENCES, INC.

Dates

Publication Date
20260507
Application Date
20251107

Claims (20)

  1. 1 . A method for generating a circular nucleic acid molecule, comprising: a. providing two double-stranded enzyme recognition nucleic acid molecule, a double-stranded target nucleic acid molecule, and one or more adaptors, wherein at least one adaptor comprises a universal primer site, a surface binding site, or an index site; b. joining one of the two double-stranded enzyme recognition nucleic acid molecules to one end of the double-stranded target nucleic acid molecule, and another one of the two double-stranded enzyme recognition nucleic acid molecules to another end of the double-stranded target nucleic acid molecule, to form a joint double-stranded nucleic acid molecule, wherein the joint double-stranded nucleic acid molecule comprises the at least one adaptor between the one of the two double-stranded enzyme recognition nucleic acid molecule and the double-stranded target nucleic acid molecule; and c. contacting the joint double-stranded nucleic acid molecule to an enzyme, wherein the enzyme binds to the two double-stranded enzyme recognition nucleic acid molecules to form the circular nucleic acid molecule.
  2. 2 . The method for generating the circular nucleic acid molecule of claim 1 , wherein the enzyme cleaves the double-stranded enzyme recognition nucleic acid molecule.
  3. 3 . The method for generating the circular nucleic acid molecule of claim 2 , wherein, after the cleavage, cleavage ends of the double-stranded enzyme recognition nucleic acid molecule form hairpin structures.
  4. 4 . The method for generating the circular nucleic acid molecule of any one of claims 1-3 , wherein the enzyme is a protelomerase.
  5. 5 . The method for generating the circular nucleic acid molecule of claim 4 , wherein the protelomerase is TelN protelomerase.
  6. 6 . The method for generating the circular nucleic acid molecule of any one of claims 1-5 , wherein the joining is carried out by a nucleic acid polymerase.
  7. 7 . The method for generating the circular nucleic acid molecule of claim 5 , wherein the TelN protelomerase comprises an amino acid sequence of SEQ ID NO: 1.
  8. 8 . The method for generating the circular nucleic acid molecule of any one of claims 1-7 , wherein the surface binding site is configured to immobilize the circular nucleic acid molecule to a surface.
  9. 9 . The method for generating the circular nucleic acid molecule of claim 8 , wherein the surface is a surface of a support or a surface within a support.
  10. 10 . The method for generating the circular nucleic acid molecule of any one of claims 1-9 , wherein the at least one adaptor is inserted between the double-stranded enzyme recognition nucleic acid molecule and the double-stranded target nucleic acid molecule by a transposase.
  11. 11 . The method for generating the circular nucleic acid molecule of any one of claims 1-9 , wherein the at least one adaptor is ligated to the one double-stranded target nucleic acid molecule by a ligase before the joining.
  12. 12 . The method for generating the circular nucleic acid molecule of any one of claims 1-11 , wherein the at least one adaptor further comprises a P5 site or a P7 site.
  13. 13 . A method for generating a circular nucleic acid library, comprising: a. fragmenting a double-stranded nucleic acid sample to form a plurality of double-stranded nucleic acid fragments; b. joining a plurality of enzyme recognition nucleic acid molecules to the plurality of double-stranded nucleic acid fragments to form a plurality of joint double-stranded nucleic acid molecules, such that at least one joint double-stranded nucleic acid molecules has at least one enzyme recognition nucleic acid molecule on each end of a given double-stranded nucleic acid fragment; and c. contacting a given joint double-stranded nucleic acid molecule with at least one enzyme recognition nucleic acid molecule on each end to an enzyme, wherein the enzyme cleaves the at least one enzyme recognition nucleic acid molecule and rejoins cleavage ends of the at least one enzyme recognition nucleic acid molecule; d. repeating (c), thereby generating the circular nucleic acid library from the double stranded nucleic acid sample.
  14. 14 . The method for generating the circular nucleic acid library of claim 13 , wherein the circular nucleic acid library comprises at least 100 circular nucleic acid molecules with distinguishable sequences.
  15. 15 . The method for generating the circular nucleic acid library of claim 14 , wherein the circular nucleic acid library comprises at least 1,000 circular nucleic acid molecules with distinguishable sequences.
  16. 16 . The method for generating the circular nucleic acid library of claim 15 , wherein the circular nucleic acid library comprises at least 10,000 circular nucleic acid molecules with distinguishable sequences.
  17. 17 . The method for generating the circular nucleic acid library of claim 16 , wherein the circular nucleic acid library comprises at least 100,000 circular nucleic acid molecules with distinguishable sequences.
  18. 18 . The method for generating the circular nucleic acid library of any one of claims 13-17 , wherein the fragmenting comprises shearing, sonicating, restriction digesting, and chemical digesting.
  19. 19 . The method for generating the circular nucleic acid library of claim 18 , wherein the shearing comprises acoustic shearing, point-sink shearing, and needle shearing.
  20. 20 . The method for generating the circular nucleic acid library of any one of claims 13-19 , wherein the fragmenting further comprises end repair.

Description

CROSS-REFERENCE This application is a continuation of U.S. application Ser. No. 17/320,042, filed May 13, 2021, which is a continuation of International Application No. PCT/US2019/061871, filed Nov. 15, 2019, which claims the benefit of U.S. Provisional Application No. 62/767,943, filed on Nov. 15, 2018, each of which is incorporated herein by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic Sequence Listing (ELEM-046-C03US-ST26.xml; file creation date: Nov. 6, 2025; file size: 7,886 bytes) are hereby incorporated by reference in their entirety. BACKGROUND Next-generation sequencing (NGS) techniques have become a powerful tool for acquiring sequencing data used in molecular biology techniques, taxonomy, agriscience, medical diagnostics, and the development of new therapies. For example, sequencing-by-synthesis (SBS) methods are used to extend a growing polynucleotide chain, while analyzing the identity of matching complementary nucleotides that are incorporated. However, additional methods to increase the sensitivity, accuracy, scalability, and cost efficiency of these methods are needed. SUMMARY Provided herein are methods for generating circular nucleic acid molecules and circular nucleic acid libraries for next-generation sequencing. INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “figure” and “FIG.” herein), of which: FIG. 1A depicts an example of a double-stranded enzyme recognition nucleic acid molecule. FIG. 1B depicts an example of the double-stranded enzyme recognition nucleic acid molecule after enzyme treatment. FIG. 2 depicts an example of a method for generating a circular nucleic acid molecule. FIG. 3 depicts an example of a workflow of generating a circular nucleic acid library. FIG. 4 depicts an example of a method for generating circular nucleic acid molecules. FIG. 5A depicts an example of sequencing signals generated by the method disclosed herein. FIG. 5B depicts an example of sequencing signals generated by ligation based circulation. FIG. 5C depicts an example of sequencing signals generated by uncircularized library. FIG. 6 shows a computer control system that is programmed or otherwise configured to implement methods provided herein. DETAILED DESCRIPTION Provided herein are methods for generating circular nucleic acid molecules and circular nucleic acid libraries. Some of such methods create circular nucleic acid molecules (e.g., circular DNA molecules) without using a nucleic acid ligase. Rather, some methods disclosed herein use an enzyme that identifies a nucleic acid having a target enzyme recognition sequence, cleaves the enzyme recognition nucleic acid molecule at a target site so as to generate an end having a 5′ and 3′ exposed cleavage ends, rejoins 5′ and 3′ cleavage ends of a single exposed end at the target site to form a single linear molecule from the cleaved 5′ and 3′ ends. When this reaction is performed on both ends of a double-stranded nucleic acid molecule having a target molecule added at each end, the result is a circular nucleic acid molecule. A number of enzymes or enzyme combinations are compatible with this reaction. Often, the enzyme is a protelomerase. One type of protelomerase is TelN protelomerase, such as that from E. coli phage N1. One type of the enzyme recognizes one or more enzyme recognition nucleic acid molecules attached to random linear double-stranded nucleic acid molecules to create a circular nucleic acid library suitable for sequencing. Some of the libraries generated require clonal amplification of the circular nucleic acid molecules before sequencing process. The use of the enzyme has several advantages to other nucleic acid library preparation methods. One of such advantages is that the circular nucleic acid molecule contains both the forward and reverse sequences of a target nucleic acid molecule or nucleic acid region of interest. If the circular nucleic acid molecule contains both the forward and reverse sequences, it eliminates the process to synthesize a complementary strand to obtain