The RNA extension reactions were carried out under similar conditions described in the literature. Briefly, the solution containing SC2RC, which included RdRp, NSP7 and NSP8, NTPs, KCl, MgCl 2 , DTT and an RNase inhibitor in a Tris-HCl buffer at pH 8 was prepared. The RNA extension reaction was then initiated by addition of the solution of the RNA duplex preformed from the primer 20-mer RNA 1a and a template 30-mer RNA 1b-f or 1g. The materials were mixed well and then quickly distributed equally into seven PCR tubes. The reaction in one of the tubes was quenched immediately by adding EDTA followed by RNA loading dye. The other tubes were heated at 37 o C in a PCR instrument for 5, 10, 20, 40, 120 and 360 minutes, respectively, and then quenched with EDTA and RNA loading dye. The results of the RNA extension reactions were analyzed with denatured polyacrylamide gel electrophoresis. The RNA duplex was also loaded onto the gel for comparison (Figure ). With the template being 1b, which contained no modified nucleotides, the RNA extension reaction went smoothly, and within 20 minutes, almost all primer 1a were converted to the 30-mer 1h (lane 5, Figure ). With the template being 1c, 1d and 1e, which contained the modified nucleotides Ψ, m 5 C, m 6 A, respectively, the extension reactions also went smoothly but with slower rates. At 2 hours, a small portion of primer 1a could still be observed (lane 7, Figures ). With the template being 1f, which contained the modified nucleotide m 1 A, the results were dramatically different. Even after six hours, there were still significant amounts of primer 1a left (lane 8, Figure ). Some fully extended 30-mer 1h were formed (lanes 2-8, Figure ), however, the majority of the primer were only extended to the 24-mer 1i, and the extension reaction stopped at m 1 A. RNA 1i, which had a FAM group at its 5'-end, migrated at the same rate in the electrophoresis as the 30-mer template 1f, which did not have a FAM group, and the two overlapped on the gel (Figure ). However, the existence of 1i can be unambiguously determined by visualizing the unstained gel. In this instance, only RNAs with the FAM tag could be visible (Figure ). The template 1f did not have FAM, and thus could not be observed, while 1i had FAM, and could be observed. By comparing Figures and, it is easy to find that the majority of primer 1a was converted to 1i, not to 1h. This conclusion is confirmed by the fact that similar shorter RNAs were not observed in the non-stained gels with 1c, 1d and 1e, which contained Ψ, m 5 C, m 6 A, respectively, being the template for the extension reaction (see supporting information). With the template being 1g, which contained the modified nucleotide m 3 C, the extension reaction was able to read through the modified nucleotide, and the fully extended RNA 1h could be formed (Figure ). However, the reaction was slower than the case using unmodified template, and had a similar rate as the cases using templates containing Ψ, m 5 C and m 6 A. At 2 hours, a small portion of the primer 1a could still be observable (lane 7, Figure ). Unlike the case of m 1 A, no truncated RNA, which would be a 27-mer if it were formed, was formed. In the image of the unstained gel (Figure ), between the bands of 1a and 1h, no other band was visible in all the lanes. It is noted that the relative intensity of bands of 1a and 1h in Figure does not reflect the relative quantities of RNAs in the bands due to the higher degree of fluorescent quenching by 1h than by 1a. The relative intensity of bands in the stained gel image (Figure ) is more close to the RNA quantities.