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. A method of preparing a double-stranded RNA molecule, comprising:
a) synthesizing two RNA strands, each RNA strand consisting of 19-25 nucleotides in length, wherein said RNA strands are capable of forming a double-stranded RNA molecule and at least one strand forms a single-stranded 3′-overhang from 1to 5 nucleotides; and
b) combining the synthesized RNA strands under conditions such that the double-stranded RNA molecule is formed,
wherein the double-stranded RNA molecule is capable of target-specific RNA interference.
2. The method of claim 1 , wherein the RNA strands are chemically synthesized.
3. The method of claim 1 , wherein the RNA strands are enzymatically synthesized.
4. The method of claim 1 , further comprising selecting a target mRNA molecule.
5. The method of claim 2 , wherein the chemical synthesis comprises solid phase synthesis.
6. The method of claim 1 , wherein a strand of the double-stranded RNA molecule is at least 70% identical to a target mRNA molecule.
7. The method of claim 6 , wherein the strand of the double-stranded RNA molecule is at least 85% identical to the target RNA molecule.
8. The method of claim 1 , wherein a strand of the RNA molecule is identical to a target mRNA molecule.
9. The method of claim 1 , wherein a strand of the RNA molecule is at least 70% identical to a target mRNA molecule in the double-stranded portion of the RNA molecule.
10. The method of claim 9 , wherein the strand of the RNA molecule is at least 85% identical to the target RNA molecule.
11. The method of claim 1 , wherein a strand of the RNA molecule is identical to a target mRNA molecule in the double-stranded portion of the RNA molecule.
12. The method of claim 1 , wherein each of the RNA strands forms a 3′-overhang from 1-5 nucleotides in length.
13. The method of claim 1 , wherein at least one of the RNA strands forms a 3′-overhang from 1-3 nucleotides in length.
14. The method of claim 1 , wherein at least one of the strands forms a 3′-overhang which is 2 nucleotides in length.
15. The method of claim 1 , wherein each strand consists of 20 to 22, or 21 to 23 nucleotides in length.
16. The method of claim 1 , wherein at least one of the strands consists of 21 nucleotides in length.
17. The method of claim 1 , wherein the double-stranded portion of the RNA molecule comprises at least one nucleotide analogue.
18. The method of claim 17 , wherein the nucleotide analogue is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.
19. The method of claim 17 , 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.
20. The method of claim 17 , wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphorothioate group.
21. The method of claim 1 , wherein the double-stranded RNA molecule comprises at least one nucleotide analogue located at the 5′-end, the 3′-end, or both, of the double stranded RNA molecule.
22. The method of claim 21 , wherein the nucleotide analogue is located in the 3′-overhang of the double-stranded RNA molecule.
23. The method of claim 22 , wherein the nucleotide analogue of the 3′-overhang is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.
24. The method of claim 22 , 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.
25. The method of claim 22 , wherein the nucleotide analogue of the 3′-overhang is a backbone-modified ribonucleotide containing a phosphorothioate group.
26. The method of claim 22 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a 2′-deoxythymidine or a nucleotide lacking a 2′-hydroxyl in a sugar.
27. The method of claim 22 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a 2′-deoxythymidine.
28. The method of claim 22 , 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.
29. The method of claim 22 , wherein the 3′-overhang of the double-stranded RNA molecule is TT.
30. The method of claim 22 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a purine nucleotide.
31. The method of claim 22 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a modified pyrimidine nucleotide.
32. The method of claim 22 , 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.
33. The method of claim 1 , wherein the double-stranded RNA molecule consists of a single double-stranded region and a single-stranded region of 1 to 3 nucleotides at the 3′ end of at least one of the strands of said double-stranded RNA molecule.
34. The method of claim 1 , wherein the only single-stranded region in said RNA molecule is the single-stranded 3′-overhang.
35. The method of claim 1 , which comprises a terminal 3′ hydroxyl group.
36. A method of preparing a double-stranded RNA molecule, comprising:
a) synthesizing two RNA strands, each RNA strand consisting of 19-23 nucleotides in length, wherein said RNA strands are capable of forming a double-stranded RNA molecule and at least one strand has a single-stranded 3′-overhang from 1 to 3 nucleotides; and
b) combining the synthesized RNA strands under conditions such that a double-stranded RNA molecule is formed,
wherein the double-stranded RNA molecule is capable of target-specific RNA interference.
37. The method of claim 36 , wherein the RNA strands are chemically synthesized.
38. The method of claim 36 , wherein the RNA strands are enzymatically synthesized.
39. The method of claim 36 , further comprising selecting a target mRNA molecule.
40. The method of claim 37 , wherein the chemical synthesis comprises solid phase synthesis.
41. The method of claim 36 , wherein a strand of the double-stranded RNA molecule is at least 70% identical to a target mRNA molecule.
42. The method of claim 41 , wherein the strand of the double-stranded RNA molecule is at least 85% identical to the target RNA molecule.
43. The method of claim 36 , wherein a strand of the RNA molecule is identical to a target mRNA molecule.
44. The method of claim 36 , wherein a strand of the RNA molecule is at least 70% identical to a target mRNA molecule in the double-stranded portion of the RNA molecule.
45. The method of claim 44 , wherein the strand of the RNA molecule is at least 85% identical to the target RNA molecule.
46. The method of claim 36 , wherein a strand of the RNA molecule is identical to a target mRNA molecule in the double-stranded portion of the RNA molecule.
47. The method of claim 36 , wherein each of the RNA strands forms a 3′-overhang from 1-3 nucleotides in length.
48. The method of claim 36 , wherein at least one of the strands forms a 3′-overhang which is 2 nucleotides in length.
49. The method of claim 36 , wherein each strand consists of 20 to 22, or 21 to 23 nucleotides in length.
50. The method of claim 36 , wherein at least one strand consists of 21 nucleotides in length.
51. The method of claim 36 , wherein the double-stranded portion of the RNA molecule comprises at least one nucleotide analogue.
52. The method of claim 51 , wherein the nucleotide analogue is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.
53. The method of claim 51 , 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.
54. The method of claim 51 , wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphorothioate group.
55. The method of claim 36 , wherein the double-stranded RNA molecule comprises at least one nucleotide analogue located at the 5′-end, the 3′-end, or both, of the double stranded RNA molecule.
56. The method of claim 55 , wherein the nucleotide analogue is located in the 3′-overhang of the double-stranded RNA molecule.
57. The method of claim 56 , wherein the nucleotide analogue of the 3′-overhang is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof.
58. The method of claim 56 , 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.
59. The method of claim 56 , wherein the nucleotide analogue of the 3′-overhang is a backbone-modified ribonucleotide containing a phosphorothioate group.
60. The method of claim 56 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a 2′-deoxythymidine or a nucleotide lacking a 2′-hydroxyl in a sugar.
61. The method of claim 56 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a 2′-deoxythymidine.
62. The method of claim 56 , 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.
63. The method of claim 56 , wherein the 3′-overhang of the double-stranded RNA molecule is TT.
64. The method of claim 56 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a purine nucleotide.
65. The method of claim 56 , wherein the 3′-overhang of the double-stranded RNA molecule comprises a modified pyrimidine nucleotide.
66. The method of claim 56 , 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.
67. The method of claim 36 , wherein the double-stranded RNA molecule consists of a single double-stranded region and a single-stranded region of 1 to 3 nucleotides at the 3′ end of at least one of the strands of said double-stranded RNA molecule.
68. The method of claim 36 , wherein the only single-stranded region in said RNA molecule is the single-stranded 3′-overhang.
69. A method of preparing a double-stranded RNA molecule, comprising:
a) synthesizing two RNA strands, each RNA strand consisting of 19-23 nucleotides in length, wherein said RNA strands are capable of forming a double-stranded RNA molecule and at least one strand forms a single-stranded 3′-overhang; and
b) combining the synthesized RNA strands under conditions such that a double-stranded RNA molecule is formed,
wherein the double-stranded RNA molecule is capable of target-specific RNA interference, and a strand of the double-stranded RNA molecule is at least 70% identical to a target mRNA molecule.
70. The method of claim 69 , wherein the single-stranded 3′-overhang of the double-stranded RNA molecule is 1-3 nucleotides in length.
71. The method of claim 70 , wherein the only single-stranded regions of the RNA molecule are the single-stranded 3′-overhang.
72. The method of claim 70 , wherein the single-stranded 3′-overhang of the RNA molecule has been stabilized against degradation.
73. The method of claim 70 , wherein the single-stranded 3′-overhang comprises at least one nucleotide analogue.
74. A method of preparing a double-stranded RNA molecule, comprising:
a) synthesizing two RNA strands, each RNA strand consisting of 19-25 nucleotides in length, wherein said RNA strands are capable of forming a double-stranded RNA molecule and at least one strand forms a single-stranded 3′-overhang; and
b) combining the synthesized RNA strands under conditions such that a double-stranded RNA molecule is formed,
wherein the double-stranded RNA molecule is capable of target-specific RNA interference, and a strand of the double-stranded RNA molecule is at least 70% identical to a target mRNA molecule.
75. The method of claim 74 , wherein said single-stranded 3′-overhang of the double-stranded RNA molecule is 1-5 nucleotides in length.
76. The method of claim 1 , wherein one strand of the double-stranded RNA molecule is blunt-ended.
77. The method of claim 1 , wherein the at least one single-stranded 3′-overhang has been stabilized against degradation.
78. The method of claim 77 , wherein said single-stranded 3′-overhang comprises one or more nucleotide analogues.
79. The method of claim 78 , wherein the nucleotide analogue of said single-stranded 3′-overhang is a sugar-modified ribonucleotide, wherein the 2′ —OH group of said sugar-modified ribonucleotide is replaced by a group selected from the group consisting of H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 and CN, wherein R is C 1 -C 6 alkyl, C 2 -C 6 alkenyl or alkynyl and halo is F, Cl, Br or I.
80. The method of claim 79 , wherein said single-stranded 3′-overhang further comprises a backbone-modified ribonucleotide containing a phosphorothioate group.
81. The method of claim 1 , wherein said double-stranded RNA molecule comprises a 2′-F sugar modified ribonucleotide, a 2′-OMe sugar modified ribonucleotide and a phosphorothioate backbone modified ribonucleotide.
82. The method of claim 1 , wherein said double-stranded RNA molecule is substantially free from contaminants occurring in cell extracts.
83. The method of claim 1 , wherein said double-stranded RNA molecule is not enzymatically processed.
84. The method of claim 1 , wherein said double-stranded RNA molecule is not enzymatically cleaved.
85. The method of claim 36 , wherein one strand of the double-stranded RNA molecule is blunt-ended.
86. The method of claim 36 , wherein the at least one single-stranded 3′-overhang has been stabilized against degradation.
87. The method of claim 86 , wherein said single-stranded 3′-overhang comprises one or more nucleotide analogues.
88. The method of claim 87 , wherein the nucleotide analogue of said single-stranded 3′-overhang is a sugar-modified ribonucleotide, wherein the 2′ —OH group of said sugar-modified ribonucleotide is replaced by a group selected from the group consisting of H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 and CN, wherein R is C 1 -C 6 alkyl, C 2 -C 6 alkenyl or alkynyl and halo is F, Cl, Br or I.
89. The method of claim 88 , wherein said single-stranded 3′-overhang further comprises a backbone-modified ribonucleotide containing a phosphorothioate group.
90. The method of claim 36 , wherein said double-stranded RNA molecule comprises a 2′-F sugar modified ribonucleotide, a 2′-OMe sugar modified ribonucleotide and a phosphorothioate backbone modified ribonucleotide.
91. The method according to claim 36 , wherein said double-stranded RNA molecule is substantially free from contaminants occurring in cell extracts.
92. The method of claim 36 , wherein said double-stranded RNA molecule is not enzymatically processed.
93. The method of claim 36 , wherein said double-stranded RNA molecule is not enzymatically cleaved.
94. The method of claim 75 , wherein one strand of the double-stranded RNA molecule is blunt-ended.
95. The method of claim 75 , wherein the at least one single-stranded 3′-overhang has been stabilized against degradation.
96. The method of claim 95 , wherein said single-stranded 3′-overhang comprises one or more nucleotide analogues.
97. The method of claim 96 , wherein the nucleotide analogue of said single-stranded 3′-overhang is a sugar-modified ribonucleotide, wherein the 2′ —OH group of said sugar-modified ribonucleotide is replaced by a group selected from the group consisting of H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 and CN, wherein R is C 1 -C 6 alkyl, C 2 -C 6 alkenyl or alkynyl and halo is F, Cl, Br or I.
98. The method of claim 9 , wherein said single-stranded 3′-overhang further comprises a backbone-modified ribonucleotide containing a phosphorothioate group.
99. The method of claim 75 , wherein said double-stranded RNA molecule comprises a 2′-F sugar modified ribonucleotide, a 2′-OMe sugar modified ribonucleotide and a phosphorothioate backbone modified ribonucleotide.
100. The method according to claim 75 , wherein said double-stranded RNA molecule is substantially free from contaminants occurring in cell extracts.
101. A method of preparing a double-stranded RNA molecule, comprising:
a) synthesizing two RNA strands, each RNA strand consisting of 19-23 nucleotides in length, wherein said RNA strands are capable of forming a double-stranded RNA molecule and at least one strand forms a single-stranded 3′-overhang; and
b) combining the synthesized RNA strands under conditions such that a double-stranded RNA molecule is formed,
wherein said double-stranded RNA molecule is not enzymatically processed.
102. A method of preparing a double-stranded RNA molecule, comprising:
a) synthesizing two RNA strands, each RNA strand consisting of 19-23 nucleotides in length, wherein said RNA strands are capable of forming a double-stranded RNA molecule and at least one strand forms a single-stranded 3′-overhang; and
b) combining the synthesized RNA strands under conditions such that a double-stranded RNA molecule is formed,
wherein said double-stranded RNA molecule is not enzymatically cleaved.