Patent ID: 8524680

Claim:
A method for attenuating the expression of a target gene in a cell comprising: introducing siRNA into the cell in an amount sufficient to attenuate expression of the target gene wherein the siRNA is from 15 to 30 nucleotides in length and contains a duplex of from 15 to 30 contiguous nucleotides, and the siRNA is made by a method comprising: obtaining a first polynucleotide template comprising a first promoter operatively linked to a first target sequence that has 5′ and 3′ ends and that is substantially identical to at least a portion of the target gene; obtaining a second polynucleotide template comprising a second promoter operatively linked to a second target sequence that has 5′ and 3′ ends and that is substantially the reverse complement of the first target sequence of the first template; enzymatically incorporating nucleotides into RNA by contacting the first template with a reaction mixture comprising an RNA polymerase and nucleotides to transcribe the first template to form a first RNA product; enzymatically incorporating nucleotides into RNA by contacting the second template with a reaction mixture comprising an RNA polymerase and nucleotides to transcribe the second template to form a second RNA product; annealing the first and second RNA products to form a siRNA containing a duplex of from 15 to 30 contiguous nucleotides, and contacting the siRNA with a single strand specific ribonuclease, wherein the siRNA has a sequence that is substantially identical to at least a portion of the target gene, wherein the nucleotides of at least one incorporating step comprise at least one modified nucleotide analog selected from the group consisting of alpha-S ATP, alpha-S CTP, alpha-S GTP, and alpha-S UTP, wherein the at least one modified nucleotide analog is incorporated into the siRNA, and wherein fewer molecules of the siRNA are effective in achieving attenuation of gene expression when compared to the number of standard siRNA molecules required to achieve the same level of attenuation of target gene expression.