The extension of 1a using 1b as the template is used for the description. The solution of 1a (20 µM), 1b (20 µM), Tris-HCl (10 mM) and KCl (100 mM) with indicated final concentration was prepared. The solution was heated at 94 o C for 5 min, and then cooled to rt slowly to give the duplex solution of 1a-b. The RNA extension reaction solution (35 µL) containing SC2RC (1.2 µM), Tris-HCl (20 mM, pH 8), KCl (50 mM), MgCl 2 (6 mM), DTT (1 mM), RNase inhibitor (1.12 U/µL), ATP (0.5 mM ), CTP (0.5 mM ), GTP (0.5 mM ), UTP (0.5 mM) and the RNA duplex of 1a-b (1.5 µM) with indicated final concentration was prepared. The RNA duplex solution was added last, and upon its addition, the mixture was immediately agitated by a brief vortex and spin. The solution was equally aliquoted into seven PCR tubes. The tubes were immediately placed into a PCR instrument, and heated at 37 o C for 0 min (not placed in PCR instrument), 5 min, 10 min, 20 min, 40 min, 2 h and 6 h, respectively. The reactions were quenched with an EDTA solution (final concentration 50 µM) followed by RNA loading dye (final concentration 1 ×). The samples were immediately stored at -80 o C until analysis by gel electrophoresis. A portion of the samples (equivalent to 0.0331 µg 1b) was analyzed with electrophoresis using 10% Mini-PROTEAN ® TBE-Urea Gel in a Mini-PROTEAN Tetra Cell at 200 V for 32 min. The gel was first imaged without staining using UVP GelDoc-IT Imaging System 2UV Transilluminator at 302 nm. The same gel was then stained with GelRed (final concentration 300 ×) for 32 min and imaged again at 302 nm. The extension reactions involving templates 1cg and their analyses were performed under the same conditions except that in the case of 1f more samples (equivalent to 0.0946 µg 1f ) were used for gel electrophoresis analysis.