Use of phosphorothiolate polynucleotides in ligating nucleic acids

• US 20030165841A1
• Filed: 06/15/2001
• Published: 09/04/2003
• Est. Priority Date: 05/10/2001
• Status: Active Grant

• Current Assignee: Emerald BioStructures, Inc. (Beryllium LLC), San Diego State University Foundation
• Original Assignee: Emerald BioStructures, Inc. (Beryllium LLC)
• Inventors: Burgin, Alex B. Jr., Stewart, Lance J.
• Granted Patent: US 6,951,720 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• 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 9/90 Isomerases (5.)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention provides a method of nonenzymatic ligation of a nucleic acid. The method consists of contacting a polynucleotide-3′ phosphorothiolate with an acceptor polynucleotide under conditions that allow formation of a phosphodiester bond between the polynucleotide-3′ phosphorothiolate and the acceptor polynucleotide. The invention also provides methods of molecular cloning. In one embodiment, the method consists of contacting an insert comprising a polynucleotide-3′ phosphorothiolate with an acceptor vector under conditions that allow formation of a phosphodiester bond between the insert and the acceptor vector to generate a vector comprising an insert polynucleotide. The invention further provides a compound consisting of a polynucleotide-3′ phosphorothiolate and a kit containing a polynucleotide-3′ phosphorothiolate. Also provided is a method of ligating a nucleic acid using a non-sequence specific topoisomerase.

Claims

1. A method of non-enzymatic ligation of a nucleic acid, comprising contacting a polynucleotide-3′

phosphorothiolate with an acceptor polynucleotide under conditions that allow formation of a phosphodiester bond between said polynucleotide-3′

phosphorothiolate and said acceptor polynucleotide.

2. The method of claim 1, wherein said polynucleotide-3′

phosphorothiolate further comprises a 3′

SNP moiety.

3. The method of claim 1, wherein said polynucleotide-3′

phosphorothiolate further comprises a duplex polynucleotide.

4. The method of claim 1, wherein said acceptor polynucleotide further comprises a duplex polynucleotide.

5. The method of claim 1, further comprising transducing into a host cell a polynucleotide-3′

phosphorothiolate having a phosphodiester bond with said acceptor polynucleotide.

6. The method of claim 1, further comprising the step:

contacting a polynucleotide-3′

phosphorothiolate precursor and an activator under conditions sufficient to react said polynucleotide-3′

phosphorothiolate precursor and said activator to produce said polynucleotide-3′

phosphorothiolate.

7. The method of claim 6, wherein said activator is iodonitrobenzene.

8. A method of molecular cloning comprising, contacting an insert comprising a polynucleotide-3′

phosphorothiolate with an acceptor vector under conditions that allow formation of a phosphodiester bond between said insert and said acceptor vector to generate a vector comprising an insert polynucleotide.

9. The method of claim 8, further comprising transforming said vector comprising an insert polynucleotide into a host cell.

10. The method of claim 8, wherein said polynucleotide-3′

phosphorothiolate further comprises a 3′

SNP moiety.

11. The method of claim 8, further comprising the step:

contacting a polynucleotide-3′

phosphorothiolate precursor and iodonitrobenzene under conditions sufficient to react said polynucleotide-3′

phosphorothiolate precursor and said iodonitrobenzene to produce said polynucleotide-3′

phosphorothiolate.

12. A method of molecular cloning comprising, contacting a vector comprising a polynucleotide-3′

phosphorothiolate with an acceptor polynucleotide, under conditions that allow formation of a phosphodiester bond between said vector and said acceptor polynucleotide to generate a vector comprising said acceptor polynucleotide.

13. The method of claim 12, further comprising transforming said vector comprising said acceptor polynucleotide into a host cell.

14. The method of claim 12, wherein said polynucleotide-3′

phosphorothiolate further comprises a 3′

SNP moiety.

15. The method of claim 12, wherein said vector further comprises a 3′

phosphorothiolate moiety at one or more terminal ends.

16. The method of claim 12, further comprising the step:

contacting a polynucleotide-3′

phosphorothiolate precursor and an activator under conditions sufficient to react said polynucleotide-3′

phosphorothiolate precursor and said activator to produce said polynucleotide-3′

phosphorothiolate.

17. A kit, comprising:

(a) a polynucleotide-3′

phosphorothiolate; and

(b) a buffer in an aqueous solution.

18. The kit of claim 17, wherein said polynucleotide-3′

phosphorothiolate further comprises one or more 3′

phosphorothiolate moieties.

19. The kit of claim 17, wherein said polynucleotide-3′

phosphorothiolate further comprises a single stranded polynucleotide.

20. The kit of claim 18, wherein said single stranded polynucleotide further comprises an oligonucleotide.

21. The kit of claim 17, wherein said polynucleotide-3′

phosphorothiolate further comprises a duplex polynucleotide.

22. The kit of claim 17, wherein said polynucleotide-3′

phosphorothiolate comprises a 3′

-SNP moiety.

23. A kit, comprising:

(a) a polynucleotide-3′

phosphorothiolate precursor; and

(b) an activator.

24. The kit of claim 23, wherein said polynucleotide-3′

phosphorothiolate further comprises one or more 3′

phosphorothiolate moieties.

25. The kit of claim 24, wherein said polynucleotide-3′

phosphorothiolate further comprises a single stranded polynucleotide.

26. The kit of claim 24, wherein said single stranded polynucleotide further comprises an oligonucleotide.

27. The kit of claim 24, wherein said polynucleotide-3′

phosphorothiolate further comprises a duplex polynucleotide.

28. The kit of claim 24, wherein said polynucleotide-3′

phosphorothiolate comprises a 3′

-SNP moiety.

29. A method of ligating a nucleic acid, comprising contacting a polynucleotide-5′

phosphorothiolate with a non-sequence specific topoisomerase, or a fragment or modification thereof, and an acceptor polynucleotide under conditions that allow formation of a phosphodiester bond between said polynucleotide-5′

phosphorothiolate and said acceptor polynucleotide, with the proviso that said polynucleotide-5′

phosphorothiolate does not contain the nucleotide sequence G(C/T)CCTT (SEQ ID NO;

5).

30. The method of claim 29, wherein said topoisomerase is human topoisomerase I, or a fragment or modification thereof.

31. The method of claim 30, wherein said human topoisomerase I is Topo65, or a fragment or modification thereof.

32. The method of claim 29, wherein said polynucleotide-5′

phosphorothiolate further comprises a duplex polynucleotide.

33. The method of claim 29, wherein said acceptor polynucleotide further comprises a vector.

34. The method of claim 29, wherein said polynucleotide-5′

phosphorothiolate further comprises a vector.

35. The method of claim 29, wherein said polynucleotide-5′

phosphorothiolate further comprises a polynucleotide having a 5′

phosphorothiolate moiety incorporated within four base pairs from a 3′

end of said polynucleotide-5′

phosphorothiolate.

36. A kit, comprising:

(a) a polynucleotide-5′

phosphorothiolate, with the proviso that said polynucleotide-5′

phosphorothiolate does not contain a nucleotide sequence selected from the group of SEQ ID NO;

5, SEQ ID NO;

6 or SEQ ID NO;

7;

(b) a non-sequence specific topoisomerase, or fragment or modification thereof having topoisomerase activity.

37. The kit of claim 36, wherein said topoisomerase is human topoisomerase I, or a fragment or modification thereof.

38. The kit of claim 37, wherein said topoisomerase is Topo65, or a fragment or modification thereof.

39. The kit of claim 36, wherein said polynucleotide-5′

phosphorothiolate is a single stranded polynucleotide-5′

phosphorothiolate.

40. The kit of claim 37, wherein said single stranded polynucleotide-5′

phosphorothiolate further comprises an oligonucleotide.

41. The kit of claim 36, wherein said polynucleotide-5′

phosphorothiolate further comprises a duplex polynucleotide-5′

phosphorothiolate.

42. The kit of claim 41, wherein said duplex polynucleotide-5′

phosphorothiolate further comprises one or more terminal end overhangs.

43. The kit of claim 42, wherein said one or more terminal end overhangs further comprise a nucleotide sequence complementary to one or more restriction endonuclease cleavage sites.

44. A composition comprising,

(a) a polynucleotide-5′

phosphorothiolate, with the proviso that said polynucleotide-5′

phosphorothiolate does not contain a nucleotide sequence selected from the group of SEQ ID NO;

5, SEQ ID NO;

6 or SEQ ID NO;

7; and

(b) a non-sequence specific topoisomerase, or fragment or modification thereof having topoisomerase activity.

45. The composition of claim 44, wherein said topoisomerase is human topoisomerase I, or a fragment or modification thereof.

46. The composition of claim 45, wherein said topoisomerase is Topo65, or a fragment or modification thereof.

47. The composition of claim 44, wherein said polynucleotide-5′

phosphorothiolate is a single stranded polynucleotide-5′

phosphorothiolate.

48. The composition of claim 47, wherein said single stranded polynucleotide-5′

phosphorothiolate further comprises an oligonucleotide.

49. The composition of claim 44, wherein said polynucleotide-5′

phosphorothiolate further comprises a duplex polynucleotide-5′

phosphorothiolate.

50. The composition of claim 49, wherein said duplex polynucleotide-5′

phosphorothiolate further comprises one or more terminal end overhangs.

51. The composition of claim 50, wherein said one or more terminal end overhangs further comprise a nucleotide sequence complementary to one or more restriction endonuclease cleavage sites.

52. A compound of the formula:

53. The compound of claim 52, wherein R2 is H.

54. The compound of claim 52, wherein R3 is nitrophenyl.

55. The compound of claim 52, further comprising a complementary polynucleotide.

56. A compound of the formula: