4′‐ThioRNA Substitution and Sense‐Strand Segmentation for the Design of Functional Fully Modified siRNAs Targeting SARS‐CoV‐2

Chemically modified small interfering RNAs (siRNAs) are proving to be highly effective as therapeutic agents, but the extent and positioning of chemical modifications must be carefully tuned because the RNA interference (RNAi) machinery imposes intrinsic constraints on siRNA architecture. Here, using a SARS-CoV-2-targeting siRNA as a model, we have systematically investigated the positional and extent-dependent tolerance of 4'-thioRNA substitution within functional siRNAs. Single 4'-thioRNA substitution was broadly tolerated across multiple positions, and complete substitution of either the sense or antisense strand preserved robust RNAi activity. In contrast, full substitution of both strands resulted in a pronounced loss of activity. Physicochemical analysis suggested that this loss arises from interference with cleavage and removal of the sense strand during loading of the RNA-induced silencing complex (RISC). Sense-strand segmentation was therefore applied to fully 4'-thio-modified siRNA to mimic the post-cleavage state during RISC loading. This design partially restored RNAi activity, facilitating the development of fully 4'-thio-modified siRNAs with antiviral activity against SARS-CoV-2. Collectively, these findings expand the chemical space available for functional siRNA design.