Modified polynucleotides and uses thereof

• US 6,867,290 B2
• Filed: 10/26/2001
• Issued: 03/15/2005
• Est. Priority Date: 04/30/1999
• Status: Expired due to Fees

• Current Assignee: Cyclops Genome Sciences Limited
• Original Assignee: Cyclops Genome Sciences Limited
• Inventors: Goldsborough, Andrew Simon
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Kim, Young J.
• Application Number:
  US10/011,495
• Publication Number:
  US 20030039985A1
• Time in Patent Office: 1,236 Days
• Field of Search: 435/6, 435/7.72, 435/91.1, 435/91.2, 435/91.3, 435/91.32, 435/307.1, 536/23.1, 536/24.3
• US Class Current: 536/23.1
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids

Abstract

Provided is a polynucleotide comprising mRNA, rRNA or viral RNA, comprising ribose rings that are covalently modified at the 2′-OH position. Further provided are methods for producing a double-stranded oligo- or polynucleotide from a template comprising an oligo- or polyribonucleotide, a proportion of the ribose rings of which are covalently modified at the 2′-OH position to bear a substituent which enables replication of the template by the nucleic acid polymerase. Also provided is use of a poly-nucleotide comprising mRNA, rRNA or viral RNA, a proportion of the ribose rings of which are covalently modified at the 2′-OH position, in a hybridisation reaction.

Claims

1. A method for producing a composition comprising a modified poly-ribonucleotide, wherein said modified poly-ribonucleotide is an mRNA, rRNA, or viral RNA, said method comprising the steps of:

(i) contacting a poly-ribonucleotide in a reaction medium comprising at least 80% v/v organic solvent with a reactant capable of covalently modifying the 2′

-OH position of the ribose rings of said poly-ribonucleotide; and

(ii) reacting said poly-ribonucleotide to produce a modified poly-ribonucleotide comprising greater than 25% of ribose rings bearing a C1-C10 alkanoyl at their 2′

-OH position.

2. The method of claim 1, wherein said method comprises the step of separating the modified poly-ribonucleotide from said reaction medium.

3. The method of claim 1, wherein said reaction medium further comprises a catalyst.

4. The method of claim 1, wherein said covalent modification is achieved in one hour or less.

5. The method of claim 1, wherein said poly-ribonucleotide is a full length mRNA.

6. The method of claim 1, wherein said poly-ribonucleotide is a full length rRNA.

7. The method of claim 1, wherein said poly-ribonucleotide is a full length viral RNA.

8. The method of claim 1, wherein said poly-ribonucleotide is at least 1000 bases in length.

9. The method of claim 1, wherein at least 50% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

10. The method of claim 1, wherein at least 75% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

11. The method of claim 1, wherein at least 80% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

12. The method of claim 1, wherein at least 85% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

13. The method of claim 1, wherein at least 90% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

14. The method of claim 1, wherein at least 95% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

15. The method of claim 1, wherein at least 99% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

16. The method of claim 1, wherein said C1-C10 is acetyl.

17. The method of claim 1, wherein said 2′

-OH position of the ribose rings is covalently modified so that a single strand of the poly-ribonucleotide is replicable by a nucleic acid polymerase to generate a second strand of a polynucleotide complementary to the single strand.

18. The method of claim 3, wherein said catalyst is an acylation catalyst.

19. The method of claim 18, wherein said acylation catalyst is an aminopyridine.

20. The method of claim 18, wherein said acylation catalyst is 1-methylimidazole.

21. The method of claim 1, wherein said poly-ribonucleotide is reacted with an acid anhydride.

22. The method of claim 1, wherein said poly-ribonucleotide is reacted with an acid chloride.

23. The method of claim 1, wherein said poly-ribonucleotide is reacted with N-acylimidazole.

24. The method of claim 1, wherein said reaction medium comprises water.

25. The method of claim 24, wherein said water and organic solvent are in a water:

organic solvent weight ratio in a range from 1;

50 to 1;

10.

27. The method of claim 1, wherein said reaction conditions are such that the covalent modification of the 2′

-OH positions of the ribose rings is substantially regiospeifiec.

28. The method of claim 1, wherein said poly-ribonucleotide is attached to a solid phase.

29. The method of claim 1, wherein said poly-ribonucleotide is from a cell or blood extract.

30. The method of claim 1, further comprising the step of reacting said modified poly-ribonucleotide with areactant capable of removing said C1-C10 alkanoyl under conditions to reinstate an —

OH group at said 2′

-OH position.

31. The method of claim 16, further comprising the step of reacting said modified polyribonucleotide with a reactant capable of removing said acetyl under conditions to reinstate an —

OH group at said 2′

-OH position.

26. The method of clain 1, wherein said organic solvent comprises an organic base.

32. A method for producing a composition comprising a modified poly-ribonucleotide at least 1000 bases in length, said method comprising the steps of:

(i) contacting a poly-ribonucleotide in a reaction medium comprising at least 80% v/v organic solvent with a reactant capable of covalently modifying the 2′

-OH position of the ribose rings of said poly-ribonucleotide; and

(ii) reacting said poly-ribonucleotide to produce a modified poly-ribonucleotide comprising greater than 25% of ribose rings bearing a C1-C10 alkanoyl at their 2′

-OH position.

33. The method of claim 32, wherein said method comprises the step of separating the modified poly-ribonucleotide from said reaction medium.

34. The method of claim 32, wherein said reaction medium further comprises a catalyst.

35. The method of claim 32, wherein said covalent modification is achieved in one hour or less.

36. The method of claim 32, wherein at least 50% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

37. The method of claim 32, wherein at least 75% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

38. The method of claim 32, wherein at least 80% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

39. The method of claim 32, wherein at least 85% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

40. The method of claim 32, wherein at least 90% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

41. The method of claim 32, wherein at least 95% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

42. The method of claim 32, wherein at least 99% of said ribose rings are covalently modified at said 2′

-OH position with a C1-C10 alkanoyl.

43. The method of claim 32, wherein said C1-C10 is acetyl.

44. The method of claim 32, wherein said 2′

-OH position of the ribose rings is covalently modified so that a single strand of the poly-ribonucleotide is replicable by a nucleic acid polymerase to generate a second strand of a polynucleotide complementary to the single strand.

45. The method of claim 34, wherein said catalyst is an acylation catalyst.

46. The method of claim 45, wherein said acylation catalyst is an aminopyridine.

47. The method of claim 45, wherein said acylation catalyst is 1-methylimidazole.

48. The method of claim 32, wherein said poly-ribonucleotide is reacted with an acid anhydride.

49. The method of claim 32, wherein said poly-ribonucleotide is reacted with an acid chloride.

50. The method of claim 32, wherein said poly-ribonucleotide is reacted with N-acylimidazole.

51. The method of claim 32, wherein said reaction medium comprises water.

52. The method of claim 51, wherein said water and organic solvent are in a water:

organic solvent weight ratio in a range from 1;

50 to 1;

10.

53. The method of claim 32, wherein said organic solvent comprises an organic base.

54. The method of claim 32, wherein said reaction conditions are such that the covalent modification of the 2′

-OH positions of the ribose rings is substantially regiospecific.

55. The method of claim 32, wherein said poly-ribonucleotide is attached to a solid phase.

56. The method of claim 32, wherein said poly-ribonucleotide is from a cell or blood extract.

57. The method of claim 32, further comprising the step of reacting said modified poly-ribonucleotide with a reactant capable of removing said C1-C10 alkanoyl under conditions to reinstate an —

OH group at said 2′

-OH position.

58. The method of claim 43, further comprising the step of reacting said modified polyribonucleotide with a reactant capable of removing said acetyl under conditions to reinstate an —

OH group at said 2′

-OH position.