To investigate the effects of epitranscriptomic modifications on the catalytic activity of SC2RC, the extension of the 20-mer RNA 1a using the 30-mer RNAs 1b-g as templates was planned. All RNAs except for 1a, which was purchased from a commercial source, were synthesized using the phosphoramidite chemistry, purified with RP HPLC and characterized with MALDI MS following reported procedures. The phosphoramidite monomers for the synthesis were from commercial sources except for the m 3 C phosphoramidite monomer, which was synthesized in consultation of reported procedures with modifications (supporting information). RNA 1a had the fluorophore FAM at its 5'-end, which was intended to provide an additional means for the analysis of the results of the extension reactions. RNAs 1b-g are complementary to 1a (Figure ). All the nucleotides in RNA 1b are canonical, while those in 1c-g include the modified nucleotides Ψ, m 5 C, m A, m 1 A and m 3 C, respectively. There are many noncanonical nucleotides; Ψ, m 5 C, m A, m 1 A and m 3 C were chosen for the study because their phosphoramidite monomers for RNA synthesis are commercially available or the procedure for the synthesis of their monomers is available. Between the RNA extension starting site (nucleotide 21) and the modified nucleotides, four or more canonical nucleotides were placed. This was intended to determine whether a failed extension, should it occurred, was due to a modified nucleotide or errors in experimental setup because if a failed extension were due to a modified nucleotide and the experimental setup were free of error, a partial extension should be observed.