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The thermodynamic asymmetry of siRNA duplexes determines their silencing activity. Favorable asymmetry can be achieved by incorporation of mismatches into the 3' part of the sense strand, providing fork-siRNAs, which exhibit higher silencing activity and higher sensitivity to nucleases. Recently, we found that selective 2'-O-methyl modifications of the nuclease-sensitive sites of siRNA significantly improve its nuclease resistance without substantial loss of silencing activity. Here, we examined the impact of nucleotide mismatches and the number and location of 2'-O-methyl modifications on the silencing activity and nuclease resistance of anti-MDR1 siRNAs. We found that both nonmodified and selectively modified fork-siRNAs with 4 mismatches at the 3' end of the sense strand suppress the expression of target gene at lower effective concentrations than the parent siRNAs with classical duplex design. The …