Presence of phosphodiester backbone, but not nucleobases, in the guide's 3' terminal region is necessary for RISC loading and target cleavage in vitro and in vivo.

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.