Taming the Achilles’ Heel: A Chemical and Structural Design to Address Off-Target Effects in siRNA Therapeutics

Off-target effects represent one of the major bottlenecks for RNA interference (RNAi) technology. To address this issue, we present a novel strategy by combining seed-region chemical modification with an extended 3'-overhang on the sense strand (SS) to mitigate SS-mediated and miRNA-like nontargeted interactions. To modify the seed-region, we developed a novel 2'-diol modification that was selectively installed at different positions within the seed-region of siRNA. For this purpose, we synthesized universal 2'-diacetate phosphoramidites that yielded a free 2'-diol functionality after standard deprotection of oligonucleotides. The 2'-diol moieties with single (positions 3-7) and dual (6 + 7) insertions in the seed-region decreased the melting temperature (<i>T</i> _m) by ca. -1 to -4.0 °C, imposing thermodynamic asymmetry. To improve the end-asymmetry of siRNA, we developed a structurally unsymmetrical siRNA (US-siRNA) design (five-nucleotides at the 3'-overhang region of SS), which together with seed-region modifications significantly increased the relative RISC loading of antisense strand (AS) with respect to their canonical sense variants. Overall, our rational design of chemical modifications of the seed-region with a 2'-diol moiety, in concert with the US-siRNA design, furnishes a simple, modular strategy to minimize off-target effects while maintaining the on-target RNAi activity.