(a) Structure of 2-methylimidazole-activated guanosine-5′-monophosphate, and its schematic representation. (b) Schematic of the RNA primer extension reaction. N1 represents an individual ribonucleotide monomer in position to react with the primer, N2 represents either a monomer or an oligomer downstream, with a leaving group capable of interacting with N1. Dashed lines: Potential interactions between leaving groups or between the downstream leaving group and the upstream nucleotide N1. (c) Schematics of the RNA primers, templates, monomers and oligomers used in d–f. Templates are complementary to the displayed monomers and oligomers. Template 2 has a U following the C to which the G monomer is bound to prevent downstream binding of G. (d) Primer extension by polymerization or ligation on templates 1 or 3, respectively. Fits describe ln(fraction primer remaining) vs time, giving an apparent first-order rate constant (Figure 1—figure supplement 1). (e) Primer extension assay for the experiments described in c, showing reaction progress after 10 min. In lane 6, a primer + 4 band can be observed, representing the slow addition of the activated trimer after the monomer. (f) Pseudo-first order rates of the reactions described in c. Error bars indicate S.E.M; all experiments were performed in triplicate or greater. Reaction conditions: 10 μM primer, 11 μM template, 200 mM CHES pH 9.0, 200 mM MgCl2, 50 mM monomer, 1 mM trimer.