2'-O-Methyl–Modified Anti-MDR1 Fork-siRNA Duplexes Exhibiting High Nuclease Resistance and Prolonged Silencing Activity

Abstract

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 selective modification of nuclease-sensitive sites significantly improved the stability of fork-siRNAs in the presence of serum. The selectively modified fork-siRNA duplexes provided inhibitory effect over a period of 12 days posttransfection, whereas the gene silencing activity of the nonmodified analogs expired within 6 days. Thus, selective chemical modifications and structural alteration of siRNA duplexes improve their silencing properties and significantly prolong the duration of their silencing effect.

Noncanonical Synthetic RNAi Inducers

“…Fork-siRNAs contain base substitutions in the 3′ end of the sense strand of siRNA, resulting in destabilization of the duplex [83][84][85]. The effect of fork-siRNAs is explained by the fact that thermodynamic asymmetry of the duplexes determines the orientation of siRNA in RISC.…”

“…Mismatches at the 3′ end of the sense strand, resulting in the formation of unpaired or destabilized regions, increase the silencing activity of siRNA with low or moderate concentrations [83]. The number of mismatches in fork-siRNA also plays a crucial role in its silencing activity [85]. Fork-siRNAs with one to two mismatches at the 3′ end possess silencing activity similar to that of canonical siRNAs, indicating that this number of mismatches is not enough for the efficient silencing.…”

“…Fork-siRNAs with one to two mismatches at the 3′ end possess silencing activity similar to that of canonical siRNAs, indicating that this number of mismatches is not enough for the efficient silencing. Fork-siRNA with four mismatches is the most potent, whereas fork-siRNA with six mismatches possesses reduced silencing activity [85].…”

“…On the other hand, mismatches in the 3′ part of the sense strand and long unpaired ends increase the sensitivity of fork-siRNA to nucleases. Consequently, the application of non-modified fork-siRNAs in vivo is limited by the fact that they have reduced stability in biological fluids due to the increased degradation by nucleases [83,85]. To solve this problem, the algorithm for designing nuclease-resistant fork-siRNAs that contain 2′-Omethyl modifications in nuclease-sensitive sites was developed, which allows obtaining forksiRNAs whose stability is comparable to that of canonical siRNAs [85].…”

“…BP-modified antisense siRNAs possess silencing activity comparable to unmodified double-stranded siRNAs. Partial 2′-O-methyl modification was used for the stabilization of antisense RNA, the activity resulting in singlestranded siRNA was comparable with the activity of double-stranded siRNA when used in high or intermediate concentrations, where in low concentration, canonical siRNAs were more active [85].…”