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

Herein, we describe the synthesis of 2′-alkoxy/fluoro-m3C phosphoramidites and their incorporation into siRNAs. These modifications, particularly at the central position of the siRNA duplex, caused significant thermal destabilization due to disrupted Watson–Crick hydrogen bonding. 2′-Alkoxy-m3C conferred improved resistance toward 5′- and 3′-exonucleases compared to their 2′-fluoro-m3C and m3C counterparts. Gene silencing studies demonstrated that incorporating 2′-alkoxy-m3U/m3C modifications as a canonical or mismatched base pair at the passenger strand cleavage site and the first base pair from the 3′-end exhibited an ∼2-fold increase in RNAi activity. Importantly, incorporating 2′-alkoxy-m3C/m3U 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.