Search

EP-4739790-A1 - METHODS AND COMPOSITIONS FOR STABILIZING CONCATEMERS

EP4739790A1EP 4739790 A1EP4739790 A1EP 4739790A1EP-4739790-A1

Abstract

This application describes methods, compositions, and kits for forming stabilized concatemers using staple molecules. Additionally, this application provides methods of using stabilized concatemers to improve the results of downstream applications, including sequencing applications.

Inventors

  • PATTERSON, Kurt
  • SHEN, MIN-JUI RICHARD
  • ROHRMAN, Brittany Ann
  • SPARKS, ANDREW
  • HUROWITZ, EVAN

Assignees

  • Pacific Biosciences of California, Inc.

Dates

Publication Date
20260513
Application Date
20240625

Claims (20)

  1. 1. A method of forming stabilized concatemers, the method comprising: (a) providing a plurality of concatemers, wherein individual concatemers comprise multiple instances of a target sequence and multiple instances of at least one adapter sequence; (b) contacting the plurality of concatemers with staple molecules to form a plurality of stabilized concatemers; wherein individual staple molecules comprise at least a first region and a second region; and wherein the first region and the second region of staple molecules each hybridize to a different instance of the at least one adapter sequence.
  2. 2. The method of claim 1, wherein the providing a plurality of concatemers of step (a) comprises rolling circle amplification (RCA) with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template comprises the target sequence and the at least one adapter sequence.
  3. 3. The method of claim 2, further comprising circularizing a linear nucleic acid template comprising the target sequence and the at least one adapter sequence to produce the circular nucleic acid template.
  4. 4. The method of claim 3, wherein the linear nucleic acid template comprises a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence.
  5. 5. The method of claim 4, wherein the first adapter sequence and second adapter sequence of the linear nucleic acid template are ligated after hybridization to a splint oligonucleotide.
  6. 6. The method of claim 5, wherein the splint oligonucleotide is the primer hybridized to the circularized nucleic acid.
  7. 7. The method of claim 5, wherein the splint oligonucleotide is removed prior to RCA.
  8. 8. The method of claim 2, wherein the primer is immobilized on a surface during RCA.
  9. 9. The method of claim 2, wherein the primer is in solution during RCA.
  10. 10. The method of claim 9, further comprising depositing the plurality of stabilized concatemers on a surface subsequent to contacting step (b).
  11. 11. The method of claim 10, wherein the surface is a structured surface.
  12. 12. The method of claim 9, further comprising depositing the plurality of concatemers on a surface prior to contacting step (b).
  13. 13. The method of claim 12, wherein the surface is a structured surface.
  14. 14. The method of claim 2, wherein RCA produces a sense strand, and the method further comprises amplifying the sense strand to produce a plurality of antisense strands.
  15. 15. The method of claim 14, wherein at least some of the staple molecules are hybridized to adapter sequences of the plurality of antisense strands.
  16. 16. The method of claim 14, wherein step (b) further comprises hybridizing the staple molecules to the sense strand.
  17. 17. The method of claim 16, wherein at least some of the staple molecules is a staple primer used to amplify the sense strand to produce the plurality of antisense strands.
  18. 18. The method of claim 2, wherein contacting step (b) occurs during RCA step (a).
  19. 19. The method of claim 1, wherein individual instances of the at least one adapter sequence comprises a primer binding site.
  20. 20. The method of claim 19, wherein individual instances of the at least one adapter sequence further comprise a tag region, optionally wherein the tag region is a sample index.

Description

METHODS AND COMPOSITIONS FOR STABILIZING CONCATEMERS CROSS-REFERENCE TO RELATED APPLICATIONS [1] This application claims the benefit of U.S. Provisional Application No. 63/525,275 filed on July 6, 2023, the disclosure of which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION [2] The present application generally relates to molecular biology and more specifically to compositions and methods for enhancing the stability of amplifying and sequencing nucleic acids, as well as their methods of use. BRIEF SUMMARY OF THE INVENTION [3] In a general aspect Al, a method of forming stabilized concatemers, includes: (a) providing a plurality of concatemers, wherein individual concatemers include multiple instances of a target sequence and multiple instances of at least one adapter sequence; (b) contacting the plurality of concatemers with staple molecules to form a plurality of stabilized concatemers; wherein individual staple molecules include at least a first region and a second region; and wherein the first region and the second region of staple molecules each hybridize to a different instance of the at least one adapter sequence. [4] Methods of the subject application may further include any of the below aspects: [5] A2. The method of aspect Al, wherein the providing a plurality of concatemers of step (a) includes rolling circle amplification (RCA) with a strand displacing polymerase from a primer hybridized to a circular nucleic acid template, wherein the circular nucleic acid template includes the target sequence and the at least one adapter sequence. [6] A3. The method of aspect A2, further including circularizing a linear nucleic acid template including the target sequence and the at least one adapter sequence to produce the circular nucleic acid template. [7] A4. The method of aspect A3, wherein the linear nucleic acid template includes a first adapter sequence 3’ to the target sequence and a second adapter sequence 5’ to the target sequence. [8] A5. The method of aspect A4, wherein the first adapter sequence and second adapter sequence of the linear nucleic acid template are ligated after hybridization to a splint oligonucleotide. [9] A6. The method of aspect A5, wherein the splint oligonucleotide is the primer hybridized to the circularized nucleic acid. [10] A7. The method of aspect A5, wherein the splint oligonucleotide is removed prior to RCA [11] A8. The method of aspect A2, wherein the primer is immobilized on a surface during RCA. [12] A9. The method of aspect A2, wherein the primer is in solution during RCA. [13] A10. The method of aspect A9, further including depositing the plurality of stabilized concatemers on a surface subsequent to contacting step (b). [14] Al 1. The method of aspect A10, wherein the surface is a structured surface. [15] A12. The method of aspect A9, further including depositing the plurality of concatemers on a surface prior to contacting step (b). [16] A13. The method of aspect A12, wherein the surface is a structured surface. [17] A14. The method of aspect A2, wherein RCA produces a sense strand, and the method further includes amplifying the sense strand to produce a plurality of antisense strands. [18] A15. The method of aspect A14, wherein at least some of the staple molecules are hybridized to adapter sequences of the plurality of antisense strands. [19] A16. The method of aspect A14, wherein step (b) further includes hybridizing the staple molecules to the sense strand. [20] A17. The method of aspect A16, wherein at least some of the staple molecules is a staple primer used to amplify the sense strand to produce the plurality of antisense strands. [21] Al 8. The method of aspect A2, wherein contacting step (b) occurs during RCA step (a). [22] A19. The method of aspect Al, wherein individual instances of the at least one adapter sequence includes a primer binding site. [23] A20. The method of aspect A19, wherein individual instances of the at least one adapter sequence further include a tag region, optionally wherein the tag region is a sample index. [24] A21. The method of aspect A 19, wherein individual instances of the at least one adapter sequence further include a splint binding site. [25] A22. The method of aspect Al 9, wherein individual instances of the at least one adapter sequence further include a variable region, optionally wherein the variable region is a unique molecular identifier (UMI). [26] A23. The method of aspect Al, wherein the individual concatemers include a sense strand hybridized to a plurality of antisense strands. [27] A24. The method of aspect A23, wherein the first region and the second region of the staple molecule each hybridize to a different instance of the at least one adapter sequence on a different antisense strand. [28] A25. The method of aspect A23, wherein the sense strand does not include uracil and the plurality of antisense strands includes uracil. [29] A26. The method of aspect Al, wherein all in