FIG 9.

Influence of single remdesivir-TP incorporation on incorporation of the next nucleotide in the presence of different templates. (A) P/T complex used in the experiment whose results are shown in panel B. (B) Influence of single remdesivir-TP incorporation on incorporation of the next nucleotide. nsp12 (50 nM) and nsp8-7 (2 μM) were incubated with 5 nM P/T complex (A), 0.1 μM GTP (the first nucleotide to be incorporated), and 0.1 μM ATP or remdesivir-TP (the second nucleotide to be incorporated) in reaction buffer for 30 min at 37°C and then rapidly mixed with different concentrations (indicated above the gel, in micromolar units) of both UTP and CTP. The reactions were continued at 22°C for 20 s before the addition of stopping solution, and the products were resolved by denaturing PAGE. The identity of the tested nucleotide (ATP or remdesivir-TP) is indicated at the bottom of the gel. The locations of the 30-mer primer and the 31-mer (GTP incorporation), 32-mer (single ATP or remdesivir-TP incorporation), and 40-mer (full-length extension products) are indicated on the left of the gel. (C and D) Influence of single remdesivir-TP incorporation on incorporation of the next nucleotide on a different template was analyzed using the method described for Fig. 8.