Presence of phosphodiester backbone, but not nucleobases, in the guide’s 3′ terminal region is necessary for RISC loading and target cleavage <i>in vitro</i> and <i>in vivo</i>

Short interfering RNAs (siRNAs) represent a novel class of therapeutic modalities, where, in the context of complex chemical modification patterns, a single administration can support sustained gene silencing. Various siRNA architectures demonstrate robust activity; however, a guide strand length of 19-21 nucleotides is generally believed to be required for effective gene silencing. Here, we show that up to five terminal positions of the guide strand can be efficiently substituted with non-nucleobase-containing analogs without a measurable loss of activity in vitro or in vivo. While nucleobases are not essential at these positions, the presence of a phosphodiester backbone is critical. Both the distance between phosphate groups and the lipophilicity of the phosphodiester-linking analogs significantly influence silencing activity. Longer carbon-based chains reduce activity, whereas ethylene glycol-based chains preserve activity, highlighting the importance of backbone architecture in RISC engagement. These findings demonstrate that non-nucleobase structures can support productive RISC interactions, offering new opportunities in the chemical engineering of therapeutic siRNAs and other classes of small-RNA drugs.