FIG 7.

Measurement of the discrimination values of ATP analogs. (A) Primer and template used to assay ATP analogs, as shown in panels B and D. (B) A representative image of the results of the analysis of D_{remdesivir-TP} and D_{6-chloropurine-TP} values. nsp12 (50 nM) and nsp8-7 (2 μM) were incubated with 5 nM P/T and 0.1 μM UTP (the first nucleotide to be incorporated) in reaction buffer for 30 min at 37°C and then rapidly mixed with different concentrations (above each lane, in micromolar units) of ATP, remdesivir-TP, or 6-chloropurine-TP. 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 is indicated at the bottom of the gel. The locations of the 30-mer primer and the 31-mer and 32-mer (first and second nucleotide extension products, respectively) are indicated on the left of the gel. (C) Quantitative analysis of ATP, remdesivir-TP, and 6-chloropurine-TP incorporation in the assay whose results are shown in panel B. The incorporation efficiency was evaluated based on the extension of 31-mer to 32-mer products. The measured K_1/2 values for ATP, remdesivir-TP, and 6-chloropurine-TP in this experiment were 0.04167 μM, 0.03305 μM, and 3.351 μM, respectively. The discrimination values were calculated as K_{1/2, ATP analog}/K_{1/2, ATP} and are shown on the right of the graph. (D) Representative image of the result of analysis of ATP analogs. Primer extension reactions were performed a described for panel B. After addition of different concentrations of ATP analog, the reactions were continued at 22°C for 15 min before the addition of stopping solution. (E) Quantitative analysis of ATP analogs incorporation in the assay whose results are shown in panel D. K_1/2 analysis was as described for panels B and C. The discrimination between ATP analog and 6-chloropurine-TP, D*_{ATP analog,} was calculated as K_{1/2, ATP analog}/K_{1/2, 6-chloropurine}, and the values are shown on the right of the graph. The discrimination between ATP analogs and natural ATP, D^cal_{ATP analog}, was calculated as D*_{ATP analog} × D_{6-chloropurine-TP}. D_{6-chloropurine-TP} is 78.0 ± 3.5 (average of 2 independent experiments; results of one are shown in panel C). For clofarabine-TP, ribavirin-TP, tenofovir-DP, favipiravir-TP, and GTP (misincorporated as ATP), calculated values of D^cal_{ATP analog} were >112,242, 24,999 ± 828, >112,242, 7,343 ± 752, and 8,683 ± 600, respectively (averages from 2 independent experiments; results of one are shown here).