Outline of the pfRCT strategy for preparation of dsRNA. (A) Schematic illustration of pfRCT using the combination of RCT and RNase H cleavage. T7 RNAP initiates transcription without direction preference at the bubble. During transcription, the RNA products bind to the ssDNA parts at the bubble to form short RNA/DNA duplexes for RNase H cleavage (Drolet et al. 1995; Hraiky et al. 2000; Amon and Koshland 2016; Chedin and Benham 2020). The synthesized two complementary RNA strands hybridize to form the desired dsRNA. RNA cleavage can be accelerated by using short DNA Helpers, which hybridize with RNA products transcribed from the bubble. The obtained dsRNA can be circularized by T4 RNA ligase 1 (T4 Rnl1). (B) An example of sequence design. The 109-bp dsDNA template (T-B15) without promoter sequence contains a 15-bp bubble.
