Direct dsRNA preparation by promoter-free RCT and RNase H cleavage using one circular dsDNA template with a mismatched bubble

Abstract

Double-stranded RNA (dsRNA) has aroused widespread interest due to its effects on immunity and applications based on RNAi. However, in vitro preparation of dsRNA is costly and laborious. In this study, we developed a novel and interesting method designated as pfRCT (promoter free Rolling-Circle Transcription) for direct, facile, and efficient dsRNA preparation. This method generates equal amounts of sense strands and antisense strands simultaneously from a single circular dsDNA template. To initiate transcription by T7 RNA polymerase without direction preference, a 9-15-bp bubble (mismatched duplex with strong sequence symmetry) is introduced to the template. During RCT, all the necessary reagents, including the template, NTPs, RNA polymerase, RNase H, and Helpers, are present in one pot; and the just transcribed RNA is immediately truncated by RNase H to monomers with the desired size. The ends of dsRNA product can also be simply sealed by T4 RNA ligase 1 after pfRCT. This new approach is expected to promote the applications of dsRNA.