Patent ID: 8309705

Claim:
A small hairpin RNA (shRNA) comprising nucleotide sequence identical to the sense and antisense strand of an asymmetrical substituted siRNA duplex comprising one or more substituted base pairs, the duplex comprising a sense strand and an antisense strand, each strand having a 5′ end and a 3′ end, and wherein the one or more substituted base pairs are within 5 base pairs from the antisense strand 5′ (AS 5′) end and the sense strand 3′ (S 3′) end and are selected from the group consisting of a mismatched base pair, a wobble base pair, and a base pair comprising a rare nucleotide and a base pair comprising a modified amino-purine nucleotide, such that entry of the antisense strand into a RISC complex is promoted relative to the sense strand; such that the antisense strand preferentially guides cleavage of a desired target mRNA by the RISC complex, wherein the asymmetrical substituted siRNA duplex is made by: (a) selecting a first siRNA duplex comprising a sense strand and an antisense strand, each strand having a 5′ and a 3′ end, wherein the first siRNA duplex directs cleavage by a RISC complex at a phosphodiester bond within a desired target mRNA, the first siRNA duplex having a base pairing strength within 5 base pairs from the antisense strand 5′ (AS 5′) and the sense strand 3′ end (S 3′) relative to a base pairing strength within 5 base pairs from the antisense strand 3′ (AS 3′) and the sense strand 5′ end (S 5′), and (b) synthesizing the substituted siRNA duplex comprising one or more substituted base pairs with respect to the first siRNA duplex, wherein the substituted siRNA duplex comprises a sense strand and an antisense strand, each strand having a 5′ and a 3′ end, and wherein the substituted siRNA duplex directs cleavage by the RISC complex at the same phosphodiester bond within the target mRNA as the first siRNA duplex, the substituted siRNA duplex having a base pairing strength within 5 base pairs from the AS 5′ and the S 3′ end and a base pairing strength within 5 base pairs from the AS 3′ and the S 5′ end, wherein the one or more substituted base pairs are within 5 base pairs from the AS 5′ and the S 3′ end of the substituted siRNA duplex and are selected from the group consisting of a mismatched base pair, a wobble base pair, and a base pair comprising a rare nucleotide and a base pair comprising a modified amino-purine nucleotide, such that the base pairing strength within 5 base pairs from the AS 5′ and the S 3′ end of the substituted siRNA duplex relative to the base pairing strength within 5 base pairs from the AS 3′ and the S ′5 end of the substituted siRNA duplex is lessened as compared to the base pairing strength within AS 5′ and the S 3′ end of the first siRNA duplex relative to the base pairing strength between the AS 3 ′ and the S 5 ′ end of the first siRNA duplex.