LAUSR

Significance The innate immune system is a collection of essential frontline defenses against infectious pathogens such as viruses. Currently, however, we do not fully understand how one innate immune sensor of double-stranded RNA (dsRNA), oligoadenylate synthetase 1 (OAS1), is regulated in the absence of infection, nor the specific features of dsRNA molecules that lead to potent OAS1 activation. Here, we uncover a molecular basis for how sequence-independent features of short dsRNAs influence the extent of OAS1 activation. We also find that OAS1 is able to sense inosine-containing dsRNAs, suggesting that RNA editing may not be a protective mechanism to avoid aberrant cellular activation of OAS1 as has been proposed for other dsRNA sensing innate immune proteins. The 2’-5’-oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) that play a critical role in limiting viral infection. How these proteins are able to avoid aberrant activation by cellular RNAs is not fully understood, but adenosine-to-inosine (A-to-I) editing has been proposed to limit accumulation of endogenous RNAs that might otherwise cause stimulation of the OAS/RNase L pathway. Here, we aim to uncover whether and how such sequence modifications can restrict the ability of short, defined dsRNAs to activate the single-domain form of OAS, OAS1. Unexpectedly, we find that all tested inosine-containing dsRNAs have an increased capacity to activate OAS1, whether in a destabilizing (I•U) or standard Watson–Crick-like base pairing (I–C) context. Additional variants with strongly destabilizing A•C mismatches or stabilizing G–C pairs also exhibit increased capacity to activate OAS1, eliminating helical stability as a factor in the relative ability of the dsRNAs to activate OAS1. Using thermal difference spectra and molecular dynamics simulations, we identify both increased helical dynamics and specific local changes in helical structure as important factors in the capacity of short dsRNAs to activate OAS1. These helical features may facilitate more ready adoption of the distorted OAS1-bound conformation or stabilize important structures to predispose the dsRNA for optimal binding and activation of OAS1. These studies thus reveal the molecular basis for the greater capacity of some short dsRNAs to activate OAS1 in a sequence-independent manner.