Thermodynamic Destabilization and Structural Adaptability of 2′-Alkoxy/Fluoro-N3-Methyl Pyrimidine Nucleotides in siRNA Enhance RNA Interference Activity

U at g7 as a seed mismatch, alone or in combination with a modified passenger strand, preserved silencing efficacy. Furthermore, these modifications displayed no adverse immunostimulatory response, maintaining bioavailability and preventing cytotoxicity. Molecular modeling studies focused on the g7 position within the MID domain of hAgo2 provided insights into the structural tolerance of these modifications and their role in modulating target mRNA binding affinity, thereby influencing RNAi activity.