Methods and compositions for enhancing the efficacy and specificity of RNA silencing

1. A method of producing a dsRNAi agent having decreased off-target silencing activity, the method comprising:

(a) identifying an off-target silencing activity mediated by a sense strand of a first dsRNAi agent, wherein the first dsRNAi agent directs cleavage by a RISC complex at a phosphodiester bond within a desired target mRNA; and

(b) synthesizing a substituted dsRNAi agent comprising one or more substituted base pairs with respect to the first dsRNAi agent, wherein the substituted dsRNAi agent comprises a sense and an antisense strand, each strand having a 5′ end and a 3′ end, wherein the substituted dsRNAi agent directs cleavage by the RISC complex at the same phosphodiester bond within the desired target mRNA, wherein the one or more substituted base pairs are within about 5 base pairs from the 5′ end of the antisense strand (AS 5′) and the 3′ end of the sense strand (S 3′) and are selected from the group consisting of a mismatched base pair, a wobble base pair, a base pair comprising a rare nucleotide and a base pair comprising a base-modified nucleotide, such that the sense strand of the substituted dsRNAi agent is less effective than the sense strand of the first dsRNAi agent at entering the RISC complex;

such that the substituted dsRNAi agent has decreased off-target silencing activity relative to the off-target silencing activity of the first dsRNAi agent.

2. The method of claim 1 , wherein the one or more substituted base pairs comprise at least one mismatched base pair.

3. The method of claim 2 , wherein the mismatched base pair is selected from the group consisting of G:A, C:A, C:T, C:U, G:G, A:A, C:C, U:T, and U:U.

4. The method of claim 1 , wherein the one or more substituted base pairs comprise at least one wobble base pair.

5. The method of claim 4 , wherein the wobble base pair is selected from the group consisting of a G:U or G:T.

6. The method of claim 1 , wherein the one or more substituted base pairs comprise at least one base pair comprising a rare nucleotide.

7. The method of claim 6 , wherein the rare nucleotide is inosine (I).

8. The method of claim 6 , wherein the base pair is selected from the group consisting of an I:A, I:U and I:C.

9. The method of claim 1 , wherein the one or more substituted base pairs comprise at least one base pair comprising a base-modified nucleotide.

10. The method of claim 9 , wherein the base-modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

11. A method of decreasing off-target silencing of an inadvertent target mRNA by a dsRNAi agent, the method comprising:

(a) identifying off-target silencing of an inadvertent target mRNA by a sense strand of a first dsRNAi agent, wherein the first dsRNAi agent directs cleavage by a RISC complex at a phosphodiester bond within a desired target mRNA; and

(b) contacting a cell or subject having an RNAi pathway and the inadvertent target mRNA with a substituted dsRNAi agent comprising one or more substituted base pairs with respect to the first dsRNAi agent, wherein the substituted dsRNAi agent comprises a sense strand and an antisense strand, each strand having a 5′ end and a 3′ end, and wherein the substituted dsRNAi agent directs cleavage by the RISC complex at the same phosphodiester bond within the desired target mRNA, wherein the one or more substituted base pairs are within about 5 base pairs from the 5′ end of the antisense strand (AS 5′) and the 3′ end of the sense strand (S 3′) and are selected from the group consisting of a mismatched base pair, a wobble base pair, a base pair comprising a rare nucleotide and a base pair comprising a base-modified nucleotide,

such that the sense strand of the substituted dsRNAi agent is less effective than the sense strand of the first dsRNAi agent at entering the RISC complex; such that off-target silencing of the inadvertent target mRNA by the substituted dsRNAi agent is decreased relative to the off-target silencing of the inadvertent target mRNA by the first dsRNAi agent.

12. The method of claim 1 or 11 , wherein the dsRNAi agent is a siRNA duplex.

13. The method of claim 1 or 11 , wherein the dsRNAi agent is derived from an engineered precursor.

14. A method of producing an siRNA-like duplex having decreased off-target silencing activity, the method comprising:

(a) identifying an off-target silencing activity mediated by a miRNA* strand of a first siRNA-like duplex, wherein the first siRNA-like duplex directs silencing by a RISC complex of a desired target mRNA; and

(b) synthesizing a substituted siRNA-like duplex comprising one or more substituted base pairs with respect to the first siRNA-like duplex, wherein the substituted siRNA-like duplex comprises a miRNA* strand and a miRNA strand, each strand having a 5′ end and a 3′ end, wherein the substituted siRNA-like duplex directs silencing by the RISC complex of the desired target mRNA, wherein the one or more substituted base pairs are within about 5 base pairs from the 5′ end of the miRNA strand (miRNA 5′) and the 3′ end of the miRNA* strand (miRNA* 3′) and are selected from the group consisting of a mismatched base pair, a wobble base pair, a base pair comprising a rare nucleotide and a base pair comprising a base-modified nucleotide,

such that the miRNA* strand of the substituted siRNA-like duplex is less effective than the miRNA* strand of the first siRNA-duplex at entering the RISC complex;

such that the substituted siRNA-like duplex has decreased off-target silencing activity relative to the off-targeting silencing activity of the first siRNA-like duplex.

15. The method of claim 14 , wherein the one or more substituted base pairs comprise at least one mismatched base pair.

16. The method of claim 14 , wherein the mismatched base pair is selected from the group consisting of G:A, C:A, C:T, C:U, G:G, A:A, C:C, U:T, and U:U.

17. The method of claim 14 , wherein the one or more substituted base pairs comprise at least one wobble base pair.

18. The method of claim 17 , wherein the wobble base pair is selected from the group consisting of a G:U or G:T.

19. The method of claim 14 , wherein the one or more substituted base pairs comprise at least one base pair comprising a rare nucleotide.

20. The method of claim 19 , wherein the rare nucleotide is inosine (I).

21. The method of claim 19 , wherein the base pair is selected from the group consisting of an I:A, I:U and I:C.

22. The method of claim 14 , wherein the one or more substituted base pairs comprise at least one base pair comprising a base-modified nucleotide.

23. The method of claim 22 , wherein the base-modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

24. A method of decreasing off-target silencing of inadvertent target mRNA by a siRNA-like duplex, the method comprising:

(a) identifying an off-target silencing activity mediated by a miRNA* strand of a first siRNA-like duplex, wherein the first siRNA-like duplex directs silencing by a RISC complex of a desired target mRNA; and

(b) contacting a cell or subject having an RNAi pathway, with a substituted siRNA-like duplex comprising one or more substituted base pairs with respect to the first siRNA-like duplex, wherein the substituted siRNA-like duplex comprises a miRNA strand and a miRNA* strand, each strand having a 5′ end and a 3′ end, and wherein the substituted siRNA-like duplex directs silencing by the RISC complex of the desired target mRNA, wherein the one or more substituted base pairs are within about 5 base pairs from the 5′ end of the miRNA strand (miRNA 5′) and the 3′ end of the miRNA* strand (miRNA* 3′) and are selected from the group consisting of a wobble base pair, a base pair comprising a rare nucleotide, and a base pair comprising a base-modified nucleotide,

such that the miRNA* strand of the substituted siRNA-like duplex is less effective than the miRNA* strand of the first siRNA-like duplex at entering the RISC complex;

such that off-target silencing of the inadvertent target mRNA by the substituted siRNA-like duplex is decreased relative to the off-target silencing of the inadvertent target mRNA by the first siRNA-like duplex.

25. The method of claim 14 or 24 , wherein the siRNA like duplex silencing agent is derived from an engineered precursor.

26. The method of claim 1 , further comprising contacting a cell or subject having an RNAi pathway and an inadvertent target mRNA with the substituted dsRNAi agent produced by step (b) such that silencing of the inadvertent target mRNA in the cell or subject by the substituted dsRNAi agent is decreased relative to silencing of the inadvertent target mRNA by the first dsRNAi agent.

27. The method of claim 14 , further comprising

contacting a cell or subject having an RNAi pathway and an inadvertent target mRNA with the substituted siRNA-like duplex of step (b) such that silencing of the inadvertent target mRNA in the cell or subject by the substituted siRNA-like duplex is decreased relative to silencing of the inadvertent target mRNA by the first dsRNAi agent.

28. The method of claim 11 or 14 , wherein the one or more substituted base pairs comprise at least one mismatched base pair.

29. The method of claim 28 , wherein the mismatched base pair is selected from the group consisting of G:A, C:A, C:T, C:U, G:G, A:A, C:C, U:T, and U:U.

30. The method of claim 11 or 14 , wherein the one or more substituted base pairs comprise at least one wobble base pair.

31. The method of claim 30 , wherein the wobble base pair is selected from the group consisting of a G:U or G:T.

32. The method of claim 11 or 14 , wherein the one or more substituted base pairs comprise at least one base pair comprising a rare nucleotide.

33. The method of claim 32 , wherein the rare nucleotide is inosine (I).

34. The method of claim 32 , wherein the base pair is selected from the group consisting of an I:A, I:U and I:C.

35. The method of claim 11 or 14 , wherein the one or more substituted base pairs comprise at least one base pair comprising a base-modified nucleotide.

36. The method of claim 35 , wherein the base-modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.