Dual effects of duplex RNA harboring 5′-terminal triphosphate on gene silencing and RIG-I mediated innate immune response

• Gene silencing efficacy of short hairpin RNA containing 5′-triphosphate (3p-shRNA).
• 3p-shRNA is an activator of the RNA sensor retinoic acid inducible gene I (RIG-I).
• Presence of 5′-triphosphate in siRNA attenuates selective gene silencing efficacy.
• Competition for duplex RNA bearing 5′-triphosphate between RIG-I and RNAi.

Duplex RNA harboring the 5′-terminal triphosphate RNA is hypothesized to not only execute selective gene silencing via RNA interference, but also induce type I interferon (IFN) through activation of the retinoic acid inducible gene I (RIG-I). We evaluated gene silencing efficacy of the shRNA containing 5′-triphosphate (3p-shRNA) targeting the hepatitis C virus (HCV) RNA genome in hepatic cells. Gene silencing efficacy of the 3p-shRNA was diminished due to the presence of the 5′-triphosphate moiety in shRNA, whereas the shRNA counterpart without 5′-triphosphate (HO-shRNA) showed a strong antiviral activity without significant induction of type I IFN in the cells. 3p-shRNA was observed to be a better activator of the RIG-I signaling than the HO-shRNA with an elevated induction of type I IFN in cells that express RIG-I. Taken together, we suggest that competition for the duplex RNA bearing 5′-triphosphate between RIG-I and RNA interference factors may compromise efficacy of selective gene silencing.