Enhancing RNAi activity through 4'-O-hexadecyl-2'-deoxythymidine modification in the passenger strand

Various lipid-based siRNA drug delivery strategies are being developed for efficient delivery to extrahepatic organs. In this report, we studied the synergistic effect of 4'-O-hexadecyl-2'-dT and terminal phosphorothioate modifications on siRNA duplex stability and gene silencing efficiency. Thermal melting studies revealed that the 4'-O-C16 dT at the 3'-overhang of siRNA improved the duplex stability. Nuclease resistance assays demonstrated enhanced metabolic stability, showing ∼2-fold increase in half-life (T_1/2) for 4'-O-C16 dT modified anti-Bcl-2 siRNA relative to the unmodified duplex. Dose-dependent gene silencing studies indicated that the 4'-O-C16-dT modification is well tolerated at penultimate positions, the cleavage site, and particularly at the 6th position of the passenger strand (∼4-fold increase in RNAi activity). Western blot studies demonstrated that modified anti-Bcl-2 siRNAs exhibited similar silencing activity as native siRNAs in the presence of LF-2000. Additionally, gel retardation assay confirmed an excellent binding of C16-conjugated siRNAs to the HSA protein, suggesting improved serum stability and biodistribution. Molecular modelling study revealed that the 4'-O-C16 chain showed steric interactions with the MID domain, which may hamper the loading and activity of the siRNA duplex. Overall, these findings suggest that 4'-O-C16-dT modification could enhance both stability and silencing efficacy of siRNAs, representing a promising approach for the development of carrier-free siRNA-based therapeutics.