RNA interference mediating small RNA molecules
Double-stranded RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 19-23 nt short RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the produced siRNP complex.
1. An isolated double-stranded RNA molecule, which is a non-enzymatically processed RNA molecule, wherein:
(i) each RNA strand independently consists of 19-25 nucleotides in length, and
(ii) at least one RNA strand forms a single-stranded 3′-overhang from 1 to 5 nucleotides,
wherein said RNA molecule is capable of target-specific RNA interference.
2. The double-stranded RNA molecule of claim 1 , wherein the RNA molecule is chemically synthesized.
3. An isolated double-stranded RNA molecule, which is a non-enzymatically cleaved RNA molecule, wherein:
(i) each RNA strand independently consists of 19-25 nucleotides in length, and
(ii) at least one strand has a single-stranded 3′-overhang from 1 to 5 nucleotides,
wherein said double-stranded RNA molecule is capable of target-specific RNA interference.
4. The RNA molecule of claim 3 , wherein the RNA molecule is chemically synthesized.
5. An isolated double-stranded RNA molecule, which is a non-enzymatically processed RNA molecule, wherein:
(i) each RNA strand independently consists of 19-23 nucleotides in length, and
(ii) at least one strand has a single-stranded 3′-overhang from 1 to 3 nucleotides,
wherein said double-stranded RNA molecule is capable of target-specific RNA interference.
6. An isolated double-stranded RNA molecule, which is a non-enzymatically cleaved RNA molecule, wherein:
(i) each RNA strand independently consists of 19-23 nucleotides in length, and
(ii) at least one strand has a single-stranded 3′-overhang from 1 to 3 nucleotides,
wherein said double-stranded RNA molecule is capable of target-specific RNA interference.
7. The double-stranded RNA molecule of claim 5 , wherein the RNA molecule is chemically synthesized.
8. The double-stranded RNA molecule of claim 6 , wherein the RNA molecule is chemically synthesized.
9. The double-stranded RNA molecule of claim 1 , which has a sequence having an identity of at least 70 percent to a target mRNA molecule.
10. The double-stranded RNA molecule of claim 9 , wherein the identity is at least 85% to the target RNA molecule.
11. The double-stranded RNA molecule of claim 1 , which has a sequence identity of at least 70% in the double-stranded portion of the RNA molecule to a target mRNA molecule.
12. The double-stranded RNA molecule of claim 11 , wherein the identity is at least 85% to the target RNA molecule.
13. The double-stranded RNA molecule of claim 1 , wherein the double-stranded portion of the RNA molecule is identical to a target mRNA molecule.
14. The double-stranded RNA molecule of claim 1 , wherein one RNA strand forms the 3′-overhang from 1 to 5 nucleotides in length.
15. The double-stranded RNA molecule of claim 1 , wherein each of the RNA strands forms a 3′-overhang from 1-5 nucleotides in length.
16. The double-stranded RNA molecule of claim 1 , wherein each of the RNA strands forms a 3′-overhang from 1-3 nucleotides in length.
17. The double-stranded RNA molecule of claim 1 , wherein the 3′-overhang is 2 nucleotides in length.
18. The double-stranded RNA molecule of claim 16 , wherein each strand consists of 20 to 22, or 21-23 nucleotide in length.
19. The double-stranded RNA molecule of claim 16 , wherein each strand consists of 21 to nucleotide in length.
20. The double-stranded RNA molecule of claim 1 , wherein the double-stranded portion of the RNA molecule comprises at least one nucleotide analogue.
21. The double-stranded RNA molecule of claim 20 , wherein the nucleotide analogue is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.
22. The double-stranded RNA molecule of claim 20 , wherein the nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2′-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or CN, wherein R is C 1 -C 6 alkyl, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl and halo is F, Cl, Br or I.
23. The double-stranded RNA molecule of claim 20 , wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphorothioate group.
24. The double-stranded RNA molecule of claim 1 , which comprises at least one nucleotide analogue located at the 5′-end, the 3′-end, or both, of the double stranded RNA molecule.
25. The double-stranded RNA molecule of claim 24 , wherein the nucleotide analogue is located at the 3′-end of the RNA molecule.
26. The double-stranded RNA molecule of claim 25 , wherein the nucleotide analogue of the 3′-overhang is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.
27. The double-stranded RNA molecule according to claim 25 , wherein the nucleotide analogue of the 3′-overhang is a sugar-modified ribonucleotide, wherein the 2′-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, N(R) 2 or CN, wherein R is C 1 -C 6 alkyl, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl and halo is F, Cl, Br or I.
28. The double-stranded RNA molecule of claim 25 , wherein the nucleotide analogue of the 3′-overhang is a backbone-modified ribonucleotide containing a phosphothioate group.
29. The double-stranded RNA molecule of claim 25 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a 2′-deoxythymidine or a nucleotide lacking a 2′-hydroxyl in a sugar.
30. The double-stranded RNA molecule of claim 25 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a 2′-deoxythymidine.
31. The double-stranded RNA molecule of claim 25 , wherein the 3′-overhang of the double-stranded RNA molecule is chosen from NN/UG, NN/UU, NN/TdG or NN/TT, wherein N is any nucleotide.
32. The double-stranded RNA molecule of claim 25 , wherein the 3′-overhang of the double-stranded RNA molecule is TT.
33. The double-stranded RNA molecule of claim 25 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a purine nucleotide.
34. The double-stranded RNA molecule of claim 25 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a modified pyrimidine nucleotide.
35. The double-stranded RNA molecule of claim 25 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a nucleobase-modified ribonucleotide chosen from a non-naturally-occurring nucleobase modified at the 5-position; an adenosine or a guanosine modified at the 8-position; or an O- or an N-alkylated nucleotide.
36. The double-stranded RNA molecule of claim 1 , which consists of a single double-stranded and single-stranded regions of 1 to 3 nucleotides at the 3′ ends of at least one of the strands of said double-stranded RNA molecule.
37. The double-stranded RNA molecule of claim 1 , wherein the only single-stranded region in said RNA molecule is the single-stranded 3′-overhang.
38. The double-stranded RNA molecule of claim 1 , which comprises a terminal 3′ hydroxyl group.
39. The double-stranded RNA molecule of claim 1 , which cleaves a target mRNA at a site within the region spanned by the double-stranded RNA molecule.
40. The double-stranded RNA molecule of claim 39 , which cleaves a target mRNA at a single site.
41. The double-stranded RNA molecule of claim 2 , wherein the chemical synthesis comprises solid phase synthesis.
42. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 1 and a pharmaceutical carrier.
43. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 41 and a pharmaceutical carrier.
44. The double-stranded RNA molecule of claim 5 , which has a sequence having an identity of at least 70 percent to a target mRNA molecule.
45. The double-stranded RNA molecule of claim 44 , wherein the identity is at least 85% to the target RNA molecule.
46. The double-stranded RNA molecule of claim 5 , which has a sequence identity of at least 70% in the double-stranded portion of the RNA molecule to a target mRNA molecule.
47. The double-stranded RNA molecule of claim 46 , wherein the identity is at least 85% to the target RNA molecule.
48. The double-stranded RNA molecule of claim 5 , wherein the double-stranded portion of the RNA molecule is identical to a target mRNA molecule.
49. The double-stranded RNA molecule of claim 5 , wherein one RNA strand forms the 3′-overhang from 1 to 3 nucleotides in length.
50. The double-stranded RNA molecule of claim 5 , wherein each of the RNA strands forms a 3′-overhang from 1-3 nucleotides in length.
51. The double-stranded RNA molecule of claim 5 , wherein the 3′-overhang is 2 nucleotides in length.
52. The double-stranded RNA molecule of claim 5 , wherein each strand consists of 20 to 22, or 21-23, nucleotides in length.
53. The double-stranded RNA molecule of claim 5 , wherein each strand consists of 21 nucleotide in length.
54. The double-stranded RNA molecule of claim 5 , wherein the double-stranded portion of the RNA molecule comprises at least one nucleotide analogue.
55. The double-stranded RNA molecule of claim 54 , wherein the nucleotide analogue is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.
56. The double-stranded RNA molecule of claim 54 , wherein the nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2′-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or CN, wherein R is C 1 -C 6 alkyl, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl and halo is F, Cl, Br or I.
57. The double-stranded RNA molecule of claim 54 , wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphorothioate group.
58. The double-stranded RNA molecule of claim 5 , which comprises at least one nucleotide analogue located at the 5′-end, the 3′-end, or both, of the double stranded RNA molecule.
59. The double-stranded RNA molecule of claim 5 , wherein the 3′-overhang has been stabilized against degradation.
60. The double-stranded RNA molecule of claim 59 , wherein the nucleotide analogue is located at the 3′-end of the RNA molecule.
61. The double-stranded RNA molecule of claim 60 , wherein the nucleotide analogue of the 3′-overhang is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.
62. The double-stranded RNA molecule according to claim 60 , wherein the nucleotide analogue of the 3′-overhang is a sugar-modified ribonucleotide, wherein the 2′-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, N(R) 2 or CN, wherein R is C 1 -C 6 alkyl, C 2 -C 6 alkenyl or C 2 -C 6 alkynyl and halo is F, Cl, Br or I.
63. The double-stranded RNA molecule of claim 60 , wherein the nucleotide analogue of the 3′-overhang is a backbone-modified ribonucleotide containing a phosphothioate group.
64. The double-stranded RNA molecule of claim 60 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a 2′-deoxythymidine or a nucleotide lacking a 2′-hydroxyl in a sugar.
65. The double-stranded RNA molecule of claim 60 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a 2′-deoxythymidine.
66. The double-stranded RNA molecule of claim 60 , wherein the 3′-overhang of the double-stranded RNA molecule is chosen from NN/UG, NN/UU, NN/TdG or NN/TT, wherein N is any nucleotide.
67. The double-stranded RNA molecule of claim 60 , wherein the 3′-overhang of the double-stranded RNA molecule is TT.
68. The double-stranded RNA molecule of claim 60 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a purine nucleotide.
69. The double-stranded RNA molecule of claim 60 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a modified pyrimidine nucleotide.
70. The double-stranded RNA molecule of claim 60 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a nucleobase-modified ribonucleotide chosen from a non-naturally-occurring nucleobase modified at the 5-position; an adenosine or a guanosine modified at the 8-position; or an O- or an N-alkylated nucleotide.
71. The double-stranded RNA molecule of claim 5 , which consists of a single double-stranded and single-stranded regions of 1 to 3 nucleotides at the 3′ ends of at least one of the strands of said double-stranded RNA molecule.
72. The double-stranded RNA molecule of claim 5 , wherein the only single-stranded region in said RNA molecule is the single-stranded 3′-overhang.
73. The double-stranded RNA molecule of claim 5 , which comprises a terminal 3′ hydroxyl group.
74. The double-stranded RNA molecule of claim 5 , which cleaves a target mRNA at a site within the region spanned by the double-stranded RNA molecule.
75. The double-stranded RNA molecule of claim 74 , which cleaves a target mRNA at a single site.
76. The double-stranded RNA molecule of claim 8 , wherein the chemical synthesis comprises solid phase synthesis.
77. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 5 and a pharmaceutical carrier.
78. A pharmaceutical composition comprising at least one double-stranded RNA molecule of claim 6 and a pharmaceutical carrier.
79. An isolated double-stranded RNA molecule, which is non-enzymatically cleaved, wherein:
(i) each RNA strand consists of 19-23 nucleotides in length, and
(ii) at least one strand has a single-stranded 3′-overhang,
wherein said double-stranded RNA molecule is capable of target-specific RNA interference, and has a sequence having an identity of at least 70 percent to a target mRNA molecule.
80. The double-stranded RNA molecule of claim 79 , wherein said single-stranded 3′-overhang is of 1-3 nucleotides in length.
81. The double-stranded RNA molecule of claim 80 , wherein the only single-stranded regions in said RNA molecule is the single-stranded 3′-overhang.
82. The double-stranded RNA molecule of claim 80 , wherein said single-stranded 3′-overhang has been stabilized against degradation.
83. The double-stranded RNA molecule of claim 80 , wherein said single-stranded 3′-overhang comprises at least one nucleotide analogue.
84. An isolated double-stranded RNA molecule, which is non-enzymatically cleaved, wherein:
(i) each RNA strand consists of 19-25 nucleotides in length, and
(ii) at least one strand has a single stranded 3′-overhang,
wherein said double-stranded RNA molecule is capable of target-specific RNA interference, and has a sequence having an identity of at least 70 percent to a target mRNA molecule.
85. The double-stranded RNA molecule of claim 84 , wherein said single-stranded 3′-overhang is of 1-5 nucleotides in length.