Abstract
Large dsRNA molecules can cause potent cytotoxic and immunostimulatory effects through the activation of pattern recognition receptors; however, synthetic versions of these molecules are mostly limited to simple sequences like poly-I:C and poly-A:U. Here we show that large RNA molecules generated by rolling circle transcription fold into periodic-shRNA (p-shRNA) structures and cause potent cytotoxicity and gene silencing when delivered to cancer cells. We determined structural requirements for the dumbbell templates used to synthesize p-shRNA, and showed that these molecules likely adopt a co-transcriptionally folded structure. The cytotoxicity of p-shRNA was robustly observed across four different cancer cell lines using two different delivery systems. Despite having a considerably different folded structure than conventional dsRNA, the cytotoxicity of p-shRNA was either equal to or substantially greater than that of poly-I:C depending on the delivery vehicle. Furthermore, p-shRNA caused greater NF-κB activation in SKOV3 cells compared to poly-I:C, indicating that it is a powerful activator of innate immunity. The tuneable sequence and combined gene silencing, immunostimulatory and cytotoxic capacity of p-shRNA make it an attractive platform for cancer immunotherapy.