Oligoribonucleotides and ribonucleases for cleaving RNA

• US 7,695,902 B2
• Filed: 02/20/2002
• Issued: 04/13/2010
• Est. Priority Date: 06/06/1996
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Crooke, Stanley T.
• Primary Examiner(s): MCGARRY, SEAN
• Application Number:
  US10/078,949
• Publication Number:
  US 20020165189A1
• Time in Patent Office: 2,974 Days
• Field of Search: 435/6, 435/375, 435/377, 435/455, 536/24.1, 536/24.5, 514/44
• US Class Current: 435/6.12
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• 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 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/311 Phosphotriesters
• CPC Class Codes: C12N 2310/312 Phosphonates
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/316 Phosphonothioates
• CPC Class Codes: C12N 2310/318 where the PO2 is completely replaced, e.g. MMI or formacetal
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/335 Modified T or U
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/346 : having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3525 : MOE, methoxyethoxy
• CPC Class Codes: C12N 2310/3527 : Other alkyl chain
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• CPC Class Codes: Y02P 20/582 : Recycling of unreacted starting or intermediate materials

Abstract

Oligomeric compounds including oligoribonucleotides and oligoribonucleosides are provided that have subsequences of 2′-pentoribofuranosyl nucleosides that activate dsRNase. The oligoribonucleotides and oligoribonucleosides can include substituent groups for increasing binding affinity to complementary nucleic acid strand as well as substituent groups for increasing nuclease resistance. The oligomeric compounds are useful for diagnostics and other research purposes, for modulating the expression of a protein in organisms, and for the diagnosis, detection and treatment of other conditions susceptible to oligonucleotide therapeutics. Also included in the invention are mammalian ribonucleases, i.e., enzymes that degrade RNA, and substrates for such ribonucleases. Such a ribonuclease is referred to herein as a dsRNase, wherein “ds” indicates the RNase'"'"'s specificity for certain double-stranded RNA substrates. The artificial substrates for the dsRNases described herein are useful in preparing affinity matrices for purifying mammalian ribonuclease as well as non-degradative RNA-binding proteins.

Claims

1. A method of activating a double-stranded RNA nuclease, comprising:

(i) contacting the nuclease with a double-stranded oligomeric compound comprising a first oligonucleotide and a second oligonucleotide, wherein;

at least one of said first and said second oligonucleotides comprises at least four consecutive 2′

-hydroxyl ribonucleosides and at least one chemical modification;

said first and said second oligonucleotides are hybridized to each other;

said first and said second oligonucleotides are not covalently linked to each other; and

wherein

said first and said second oligonucleotides are each independently from 15 to 25 nucleoside subunits in length; and

(ii) detecting activation of said double-stranded RNA nuclease.

2. The method of claim 1, wherein the at least one chemical modification increases resistance of said oligonucleotide to single-stranded nucleases and/or increases the affinity of said oligonucleotide to the other oligonucleotide.

3. Tbe method of claim 2, wherein at least one modification is 2′

-methoxy.

4. The method of claim 2, wherein at least one modification is 2′

-fluoro.

5. The method of claim 2, wherein at least one modification is 2′

-O-methoxyethyl.

6. The method of claim 2, wherein at least one modification is a phosphorothioate internucleoside linkage.

7. The method of claim 1, wherein said first oligonucleotide and said second oligonucleotide each have at least four consecutive 2′

-hydroxyl ribonucleosides.

8. The method of claim 7, wherein the 2′

-hydroxyl residues of said first and said second oligonucleotides have phosphodiester linkages.

9. The method of claim 7, wherein the 2′

-hydroxyl residues of said first and said second oligonucleotides have phosphorothioate linkages.

10. The method of claim 7, wherein the 2′

-hydroxyl residues of said first oligonucleotide have phosphodiester linkages and the 2′

-hydroxyl residues of said second oligonucleotide have phosphorothioate linkages.

11. The method of claim 7 or claim 10, wherein said first and said second oligonucleotides further comprise flanking residues 5′

and 3′

of the 2′

-hydroxyl ribonucleosides, wherein said flanking residues have phosphorothioate linkages.

12. The method of claim 11, wherein said flanking residues of at least one of said first and said second oligonucleotides further comprises 2′

-methoxynucleosides.

13. The method of claim 11, wherein said flanking residues of each of said first and said second oligonucleotides further comprise 2′

-methoxynucleosides.

14. The method of claim 1, wherein at least one of said first and said second oligonucleotides comprises at least eight consecutive 2′

-hydroxyl ribonucleosides.

15. The method of claim 14, wherein said first oligonucleotide and said second oligonucleotide each comprise at least eight consecutive 2′

-hydroxyl ribonucleotides.

16. The method of claim 1, wherein each of said first and said second oligonucleotides are about 17 to about 20 nucleoside subunits in length.

17. The method of claim 16, wherein each of said first and said second oligonucleotides are 17 subunits in length.

18. The method of claim 16, wherein each of said first and said second oligonucleotides are 20 subunits in length.

19. A method of activating a double-stranded RNA nuclease comprising contacting the double-stranded RNA nuclease with a double-stranded oligomeric compound comprising a first oligonucleotide and a second oligonucleotide, wherein:

said first and said second oligonucleotides are each independently 15 to 25 nucleoside subunits in length;

said first and said second oligonucleotides are hybridized to each other;

said first and said second oligonucleotides are not covalently linked to each other; and

at least one of said first and said second oligonucleotides comprises at least four consecutive 2′

-hydroxyl ribonucleosides and at least one chemical modification.

20. The method of claim 19 wherein at least one chemical modification increases resistance to single-stranded nucleases.

21. The method of claim 19 wherein at least one chemical modification increases affinity of said first oligonucleotide to said second oligonucleotide.

22. The method of claim 19 wherein at least one chemical modification is a modified internucleoside linkage, a modified sugar moiety or a modified nucleobase.

23. The method of claim 19 wherein at least one chemical modification is a phosphorothioate internucleoside linkage.

24. The method of claim 19 wherein at least one chemical modification is a 2′

-substituted sugar modification.

25. The method of claim 19 wherein at least one chemical modification is a 2′

-alkoxy sugar modification.

26. The method of claim 19 wherein at least one cbexnical modification is a 2′

-methoxy sugar modification.

27. The method of claim 19 wherein at least one chemical modification is a 2′

-fluoro sugar modification.

28. The method of claim 19 wherein at least one chemical modification is a 2′

-O-methoxyethyl sugar modification.

29. The method of claim 19 wherein each of said first and said second oligonucleotides comprises at least four consecutive 2′

-hydroxyl ribonucleosides.

30. The method of claim 19 wherein each of said first and said second oligonucleotides comprises at least one chemical modification.

31. The method of claim 19 wherein each of said first and said second oligonucleotides comprises at least four consecutive 2′

-hydroxyl ribonucleosides and at least one chemical modification.

32. The method of claim 19 wherein said first oligonucleotide and said second oligonucleotide comprise at least 17 contiguous nucleotides which are 100% complementary to each other.

33. The method of claim 19 wherein said first oligonucleotide is 100% complementary to said second oligonucleotide.

34. The method of claim 19 wherein said first oligonucleotide and said second oligonucleotide are independently 17 to 20 riucleoside subunits in length.

35. The method of claim 19 further comprising detecting activation of said double-stranded RNA nuclease.

36. A method of activating a double-stranded RNA nuclease comprising contacting the double-stranded RNA nuclease with a double-stranded oligomeric compound comprising a first oligonucleotide and a second oligonucleotide, wherein:

said first and said second oligonucleotides are each independently 15 to 25 nucleoside subunits in length;

said first and said second oligonucleotides are hybridized to each other;

said first and said second oligonucleotides are not covalently linked to each other; and

at least one of said first and said second oligonucleotides comprises;

a plurality of nucleoside subunits with 2′

-hydroxyl pentofuranosyl sugar moieties; and

at least one chemical modification.

37. The method of claim 36 wherein at least one chemical modification increases resistance to single-stranded nucleases.

38. The method of claim 36 wherein at least one chemical modification increases affinity of said first oligonucleotide to said second oligonucleotide.

39. The method of claim 36 wherein at least one chemical modification is a modified internucleoside linkage, a modified sugar moiety or a modified nucleobase.

40. The method of claim 36 wherein at least one chemical modification is a phosphorothioate internucleoside linkage.

41. The method of claim 36 wherein at least one chemical modification is a 2′

-substituted sugar modification.

42. The method of claim 36 wherein at least one chemical modification is a 2′

-alkoxy sugar modification.

43. The method of claim 36 wherein at least one chemical modification is a 2′

-methoxy sugar modification.

44. The method of claim 36 wherein at least one chemical modification is a 2′

-fluoro sugar modification.

45. The method of claim 36 wherein at least one chemical modification is a 2′

O-methoxyethyl sugar modification.

46. The method of claim 36 wherein each of said first and said second oligonucleotides comprises a plurality of nucleoside subunits with 2′

-hydroxyl pentofuranosyl sugar moieties.

47. The method of claim 36 wherein each of said first and said second oligonucleotides comprises at least one chemical modification.

48. The method of claim 36 wherein each of said first and said second oligonucleotides comprises a plurality of nucleoside subunits with 2′

-hydroxyl pentofuranosyl sugar moieties and at least one chemical modification.

49. The method of claim 36 wherein said first oligonucleotide and said second oligonucleotide comprise at least 17 contiguous nucleotides which are 100% complementary to each other.

50. The method of claim 36 wherein said first oligonucleotide is 100% complementary to said second oligonucleotide.

51. The method of claim 36 wherein said first oligonucleotide and said second oligonucleotide are independently 17 to 20 nucleoside subunits in length.

52. The method of claim 36 further comprising detecting activation of said double-stranded RNA nuclease.

75. The method of claim 36, wherein the double-stranded RNA nuclease is not in a cell.

53. A method of activating a double-stranded RNA nuclease comprising contacting the double-stranded RNA nuclease with a double-stranded oligomeric compound comprising a first oligonucleotide and a second oligonucleotide, wherein:

said first and said second oligonucleotides are hybridized to each other;

said first and said second oligonucleotides are not covalently linked to each other;

said first and said second oligonucleotides are each independently from 15 to 25 nucleoside subunits in length; and

each of said first and said second oligonucleotides comprises at least four consecutive 2′

-hydroxyl ribonucleosides and at least one chemical modification.

54. The method of claim 53 wherein at least one chemical modification increases resistance to single-stranded nucleases.

55. The method of claim 53 wherein at least one chemical modification increases affinity of said first oligonucteotide to said second oligonucleotide.

56. The method of claim 53 wherein at least one chemical modification is a modified internuoleoside linkage, a modified sugar moiety or a modified nucleobase.

57. The method of claim 53 wherein at least one chemical modification is a phosphorothioat internucleoside linkage.

58. The method of claim 53 wherein at least one chemical modification is a 2′

-substituted sugar modification.

59. The method of claim 53 wherein at least one chemical modification is a 2′

-alkoxy sugar modification.

60. The method of claim 53 wherein at least one chemical modification is a 2′

-methoxy sugar modification.

61. The method of claim 53 wherein at least one chemical modification is a 2′

-fiuoro sugar modification.

62. The method of claim 53 wherein at least one chemical modification is a 2′

-O-methoxyethyl sugar modification.

63. The method of claim 53 further comprising detecting activation of said double-stranded RNA nuclease.

64. A method of activating a double-stranded RNA nuclease comprising contacting the double-stranded RNA nuclease with a double-stranded oligomeric compound comprising a first oliganucleotide and a second oligonucleotide, wherein:

said first and said second oligonucleotides are hybridized to each other;

said first and said second oligonucleotides are not covalently linked to each other;

said first and said second oligonucleotides are 100% complementary to each other; and

at least one of said first and said second oligonucleotides comprises at least four consecutive 2′

-hydroxyl ribonucleosides and at least one chemical modification.

65. The method of claim 64 wherein at least one chemical modification increases resistance to single-stranded nucleases.

66. The method of claim 64 wherein at least one chemical modification increases affinity of said first oligonucleotide to said second oligonucleotide.

67. The method of claim 64 wherein at least one chemical modification is a modified internucleoside linkage, a modified sugar moiety or a modified nucleobase.

68. The method of claim 64 wherein at least one chemical modification is a phosphorothioate internucleoside linkage.

69. The method of claim 64 wherein at least one chemical modification is a 2′

-substituted sugar modification.

70. The method of claim 64 wherein at least one chemical modification is a 2′

-alkoxy sugar modification.

71. The method of claim 64 wherein at least one chemical modification is a 2′

-methoxy sugar modification.

72. The method of claim 64 wherein at least one chemical modification is a 2′

-fluoro sugar modification.

73. The method of claim 64 wherein at least one chemical modification is a 2′

-O-methoxyethyl sugar modification.

74. The method of claim 64 further comprising detecting activation of said double-stranded RNA nuclease.