Structure-guided design and dynamic evaluation of VP4-targeting siRNAs against rotavirus A

Rotavirus is a major cause of severe diarrheal disease in children under the age of five, particularly in low- and middle-income countries, with no currently available antiviral therapeutics and treatment being largely supportive. In this study, we developed an integrative in silico approach to design and evaluate siRNAs targeting conserved mRNA transcript of the VP4 gene of Rotavirus A across six geographically diverse countries. Candidate siRNAs were screened using established design rules, target accessibility assessment, regression-based scoring with off-target filtering and thermodynamics analysis. Three optimized siRNAs were selected and structurally evaluated through AlphaFold 3-based modeling of RISC-associated protein–siRNA complexes with independent validation using Boltz-2. The predicted interactions showed conserved binding within TRBP and AGO2 domains, corroborating efficient RISC compatibility. Molecular dynamics simulations (100 ns) showed stable conformational behavior of AGO2–siRNA–mRNA complexes, with low RMSD values (~ 0.2–0.5 nm) and limited residue fluctuations (< 0.2 nm). MM/PBSA analysis showed that siRNA02 exhibited the most favorable AGO2 binding free energy (-453.62 kJ/mol), suggesting enhanced RISC stabilization, whereas siRNA01 demonstrated the strongest target mRNA interaction (-60.63 kJ/mol), indicating superior target recognition. Comparative dynamic analyses identified two siRNAs as the most balanced candidate for AGO2 mediated VP4 silencing based on structural compatibility, reduced conformational fluctuations, and stable interactions. These findings provide a rational computational basis for VP4-targeted siRNA development, facilitating experimental validation.