METHODS AND COMPOSITIONS FOR ENHANCING THE EFFICACY AND SPECIFICITY OF SINGLE AND DOUBLE BLUNT-ENDED siRNA

Abstract

The present invention provides methods of enhancing the efficacy and specificity of RNAi using single or double blunt-ended siRNA. The invention also provides single and double-blunt ended siRNA compositions, vectors, and transgenes containing the same for mediating silencing of a target gene. Therapeutic methods are also featured.

1 - 51 . (canceled) 
     
     
         52 . A method of enhancing silencing of a target mRNA comprising providing a double stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the base pair strength between the antisense strand 5′ end (AS 5′) and the sense strand 3′ end (S 3′) is less than the base pair strength between the antisense strand 3′ end (AS 3′) and the sense strand 5′ end (S 5′), wherein said dsRNA comprises a frayed AS 5′, such that the antisense strand preferentially guides cleavage of a target mRNA, and wherein said dsRNA is double-blunt ended. 
     
     
         53 . The method of  claim 52 , wherein the base-pair strength is less due to fewer G:C base pairs between the AS 5′ and the S 3′ than between the AS 3′ and the S 5′. 
     
     
         54 . The method of  claim 52 , wherein the base pair strength is less due to at least one mismatched base pair between the AS 5′ and the S 3′ than between the AS 3′ and the S 5′. 
     
     
         55 . The method of  claim 54 , wherein the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C, U:U, I:A, I:U, and I:C. 
     
     
         56 . The method of  claim 52 , wherein the base pair strength is less due to at least one wobble base pair between the AS 5′ and the S 3′ than between the AS 3′ and the S 5′. 
     
     
         57 . The method of  claim 56 , wherein the wobble base pair is G:U. 
     
     
         57 . The method of  claim 56 , wherein the wobble base pair is G:T. 
     
     
         58 . The method of  claim 52 , wherein the base pair strength is less due to at least one base pair comprising a modified nucleotide. 
     
     
         59 . The method of  claim 58 , wherein the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A. 
     
     
         60 . The method of  claim 52 , wherein each strand of the dsRNA has a length of between about 19 nucleotides and about 22 nucleotides. 
     
     
         61 . The method of  claim 52 , wherein said dsRNA is provided as an shRNA which is processed by a cell to yield the dsRNA. 
     
     
         62 . The method of  claim 61 , wherein a viral construct encodes the shRNA.