Working So Hard: Double‐Stranded RNAs That Can Perform Both Gene Activation and Gene Silencing

Double‐stranded RNAs (dsRNAs) are powerful tools with therapeutic potential because they can upregulate or downregulate proteins of interest within biological systems. Small activating RNAs (saRNAs) are a type of dsRNA, identical in structure and composition to small interfering RNAs (siRNAs). We previously observed that RNA duplexes with modifications such as 2′‐ O ‐methyl (2′‐ O ‐Me), 2′‐fluoro (2′‐F), locked (LNA), and unlocked nucleic acids (UNA), and a C ₃ ‐propyl spacer linker affect gene upregulation potency, where some of these chemical modifications are commonly used in siRNA design. In this study, we tested whether some of these same chemically modified saRNAs, along with new duplex variants, could also modulate gene silencing of the same target gene, STING , using an endogenous plasmid system. We demonstrate that saRNAs, shown to induce gene expression via RNA activation (RNAa), can also perform gene silencing as siRNAs. We show that the type and number of chemical modifications on the guide strand have strong influence on siRNA and saRNA activities, but do not identify any that specifically bias one over the other. In exploring the effects of chemical modifications on short RNAs, our long‐term goal aims to reveal how specific chemical modifications influence the RNA interference (RNAi) or RNAa pathway.