Abstract

Small interfering RNA (siRNA) holds considerable potential in the fields of biomedical research and therapy, primarily due to its high degree of specificity and its ability to elicit potent silencing effects. However, the application of this technology is faced with critical challenges, including low delivery efficiency, endosomal escape barriers, and toxicity/immunogenicity. Chemical modification is a core strategy for the optimization of siRNA performance. Among these chemical modifications, 2′-O-methyl (2′-OMe) was selected for its well-documented ability to enhance nuclease resistance and mitigate immunostimulatory effects. From a panel of prescreened MMP7-targeting siRNAs, a lead candidate was identified, and subsequent derivatives were generated by introducing distinct 2′-OMe modification patterns at both termini of the siRNA duplex. To evaluate silencing efficiency, subsequent assays were performed by quantifying MMP7 mRNA and protein expression levels in A549 cells. Notably, 2′-O-methyl (2′-OMe) modifications at both termini of the siRNA duplex significantly enhanced silencing activity, with the specific number and precise positions of modifications also contributing to this effect. This study provides experimental validation for a strategy involving limited dual-terminal 2′-OMe modifications in the development of RNAi-based therapeutics. Moreover, the demonstrated efficacy of this modification pattern offers valuable insights for the chemical optimization of other siRNA candidates.