Probing the hAGO2-siRNA Interactions: A Molecular Dynamics Study on the Impact of Chemical Modifications in the Seed...

The seed region (positions from g2 to g8) in the guide strand of the small interfering RNA (siRNA) plays a pivotal role in the selective recognition and binding of target mRNA during the human Argonaute 2 (hAGO2)-facilitated gene silencing. Strategic incorporation of specific chemical modifications in the seed region is crucial for enhancing specificity toward the target mRNA and minimizing off-target effects. Using microsecond-scale molecular dynamics (MD) simulations across three different states of hAGO2-siRNA complexes (guide-only, seed-paired guide-target, and fully paired guide-target), we show that 2'-OMe and 2'-F modifications stabilize the C3'-endo sugar conformation in the seed region. The conformational changes enhance Watson-Crick base pairing with the target RNA while maintaining the hAGO2 binding interactions, explaining the improved RNA interference (RNAi) activity. (S)-GNA modification at the g7 position stabilizes the intrinsic kink between the g6 and g7 nucleotides. Additionally, it reduces the base pairing occupancies at the end of the seed region, which rationalizes the mitigation of off-target effects and improved RNAi activity. At the g2 position, (S)-GNA induces large deviations in the backbone torsion angles at the g1-g2 clover-leaf geometry, which could affect the guide strand loading into the RNA-induced silencing complex (RISC). ANA modification at the g7 position maintains the A-type geometry of RNA, Watson-Crick H-bond occupancy, and binding interactions with hAGO2. However, when inserted at the g2 position, ANA induces steric clashes with MID domain residues, which likely reduce the loading of the guide strand into the RISC. Overall, the results demonstrate position-dependent structural and functional effects of seed-region modifications, including 2'-OMe, 2'-F, (S)-GNA, and ANA, providing critical insights for the design and development of next-generation siRNA-based therapeutics.