Preparation of RNA and DNA Hybrid Substrates

In brief, of the two strands, one was labeled at the 5′ end with hexachloro-fluorescein (HEX), and the other strand was unlabeled. HEX-labeled oligonucleotide strands were purchased from TaKaRa (Dalian, China). Unlabeled DNA strands were synthesized by Invitrogen, and unlabeled RNA strands were in vitro transcribed using T7 RNA polymerase (Promega, Madison, WI). The in vitro transcribed RNA strands were purified by Poly-Gel RNA Extraction Kit (Omega bio-tek, Guangzhou, China) according to the manufacturer's instruction. The two strands were mixed in a proper ratio, and annealed through heating and gradually cooling as previously described [30 (link),40 (link)]. The standard RNA helix substrate with both 5′ and 3′ protrusions was annealed with RNA1 and RNA2, the R*/D substrate was annealed with RNA1 and DNA1, the D*/D substrate was annealed with DNA2 and DNA3, the D*/R substrate was annealed with DNA2 and RNA3, the 3′-protruded RNA helix was annealed with RNA1 and RNA4, the 5′-protruded RNA helix was annealed with RNA1 and RNA5, the blunt-ended substrate was annealed with RNA1 and RNA6, and the 49 matched bps substrate was annealed with RNA7 and RNA8. All oligonucleotides used in this study are listed in S2 Table.