REUSABLE INITIATORS FOR SYNTHESIZING NUCLEIC ACIDS

• US 20160046974A1
• Filed: 08/18/2015
• Published: 02/18/2016
• Est. Priority Date: 04/02/2013
• Status: Active Grant

• Current Assignee: Molecular Assemblies, Inc.
• Original Assignee: Molecular Assemblies, Inc.
• Inventors: Efcavitch, J. William, Siddiqi, Suhaib
• Granted Patent: US 10,683,536 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template.

Claims

1. A reusable substrate for nucleic acid synthesis, comprising:

a solid support; and

an polynucleotide initiator coupled to the solid support at its 5′

end and comprising a renewable 3′

moiety,

wherein the initiator is a substrate for a terminal transferase.

2. The reusable substrate of claim 1, wherein the initiator comprises a deoxyoligonucleotide.

3. The reusable substrate of claim 1, wherein the initiator comprises three or more bases.

4. The reusable substrate of claim 1, wherein the initiator comprises eight or fewer bases.

5. The reusable substrate of claim 1, wherein the 3′

moiety comprises a ribonucleotide.

6. The reusable substrate of claim 1, wherein the 3′

moiety comprises an abasic deoxyribose.

7. The reusable substrate of claim 1, wherein the 3′

moiety comprises an abasic ribose.

8. The reusable substrate of claim 1, wherein the 3′

moiety comprises a non-nucleoside 5′

-monophosphate.

9. A method for synthesizing a nucleic acid, the method comprising the steps of:

exposing a solid support coupled to an initiator comprising an oligonucleotide and a renewable 3′

moiety to a terminal transferase and at least one nucleotide or nucleotide analog, thereby allowing the nucleotide or nucleotide analog to be coupled to the 3′

moiety.

10. The method of claim 9, further comprising exposing the nucleotide or nucleotide analog coupled to the 3′

moiety to a releasing agent, thereby allowing the nucleotide or nucleotide analog to be released from the 3′

moiety.

11. The method of claim 10, wherein the nucleotide analog comprises a blocking group that prevents an additional nucleotide or an additional nucleotide analog from being coupled to the nucleotide analog by a terminal transferase.

12. The method of claim 11, further comprising removing the blocking group.

13. The method of claim 12, further comprising allowing the terminal transferase to couple the additional nucleotide or nucleotide analog to the nucleotide analog with the blocking group removed.

14. The method of claim 11, wherein the nucleotide analog has an unmodified 3′

-OH.

15. The method of claim 11, wherein the nucleotide analog has an unmodified 3′

-OH and a cleavable terminating group, and wherein the cleavable terminating group blocks subsequent nucleotidyl transferase activity but results in a nucleotide substrate for nucleotidyl transferase upon cleavage of the terminating group.

16. The method of claim 11, wherein the nucleotide analog comprises a removable blocking group protecting the 3′

oxygen.

17. The method of claim 9, wherein exposing is done in the absence of a nucleic acid template.

18. The method of claim 9, wherein the 3′

moiety comprises a ribonucleotide.

19. The method of claim 9, wherein the 3′

moiety comprises an abasic deoxyribose.

20. The method of claim 9, wherein the 3′

moiety comprises an abasic ribose.

21. The method of claim 9, wherein the 3′

moiety comprises a non-nucleoside 5′

-monophosphate.

22. A method for reversibly coupling a nucleotide analog to a nucleic acid comprising:

exposing a nucleic acid attached to a solid support to a nucleotide analog in the presence of a polymerase, and in the absence of a template,

wherein said nucleic acid attached to a solid support comprises a 3′

moiety that is a substrate for said polymerase, and said polymerase couples said nucleotide analog to said nucleic acid attached to a solid support comprising a 3′

moiety, and

wherein said 3′

moiety is adapted to release said nucleotide analog when contacted with a releasing agent.

23. The method of claim 22, further comprising contacting said nucleotide analog coupled to said nucleic acid attached to a solid support comprising a 3′

moiety with a releasing agent, thereby causing the nucleotide analog to release from said nucleic acid attached to a solid support comprising a 3′

moiety.

24. The method of claim 22, further comprising exposing said nucleotide analog coupled to said nucleic acid attached to a solid support comprising a 3′

moiety to an additional nucleotide analog in the presence of the polymerase, and in the absence of a template, thereby creating an oligonucleotide analog coupled to said nucleic acid attached to a solid support comprising a 3′

moiety.

25. The method of claim 24, further comprising contacting said oligonucleotide analog coupled to said nucleic acid attached to a solid support comprising a 3′

moiety with a releasing agent, thereby causing the oligonucleotide analog to release from said nucleic acid attached to a solid support comprising a 3′

moiety.

26. The method of claim 22, wherein said solid support, nucleic acid, and 3′

moiety are reusable.

27. The method of claim 22, wherein the polymerase is a nucleotidyl transferase or a modified nucleotidyl transferase.

28. The method of claim 22, wherein the polymerase is terminal deoxynucleotidyl transferase (TdT) or a modified TdT.

29. The method of claim 22, wherein the polymerase is poly-A polymerase or a modified poly-A polymerase.

30. The method of claim 22, wherein the polymerase is poly-U polymerase or a modified poly-U polymerase.

31. The method of claim 22, wherein the nucleotide analog has an unmodified 3′

-OH.

32. The method of claim 22, wherein the nucleotide analog has an unmodified 3′

-OH and a cleavable terminating group, and wherein the cleavable terminating group blocks subsequent nucleotidyl transferase activity but results in a nucleotide substrate for nucleotidyl transferase upon cleavage of the terminating group.

33. The method of claim 22, wherein the nucleotide analog comprises a removable blocking moiety protecting the 3′

oxygen.

34. The method of claim 22, wherein said 3′

moiety is a ribonucleotide.

35. The method of claim 22, wherein said 3′

moiety is an abasic deoxyribose.

36. The method of claim 22, wherein said 3′

moiety is an abasic ribose.

37. The method of claim 22, wherein said 3′

moiety is a non-nucleoside 5′

-monophosphate.

38. The method of claim 22, wherein said releasing agent is basic.

39. The method of claim 22, wherein said releasing agent comprises a metal ion.

40. The method of claim 22, wherein said releasing agent comprises an enzyme.

41. The method of claim 22, wherein said releasing agent is a type II restriction nuclease.

42. A composition comprising:

a solid support;

an initiator comprising an oligonucleotide and a renewable 3′

moiety; and

a terminal transferase,

wherein the 5′

end of the oligonucleotide is coupled to the solid support, and

wherein the initiator is a substrate for a terminal transferase.

43. The composition of claim 42, wherein the terminal transferase is a terminal deoxynucleotidyl transferase (TdT).

44. The composition of claim 42, further comprising a nucleotide analog.

45. The composition of claim 44, wherein the nucleotide analog comprises an unmodified 3′

-OH.

46. The composition of claim 44, wherein the nucleotide analog has an unmodified 3′

-OH and a cleavable terminating group, and wherein the cleavable terminating group blocks subsequent nucleotidyl transferase activity but results in a nucleotide substrate for nucleotidyl transferase upon cleavage of the terminating group.

47. The composition of claim 44, wherein the nucleotide analog comprises a removable blocking group protecting the 3′

oxygen.

48. A composition comprising a solid support and an oligonucleotide,

wherein the 5′

end of the oligonucleotide is coupled to the solid support, and

wherein the oligonucleotide is synthesized directly on the solid support.