FIG 6.

Analysis of incorporation and chain termination effects of nucleotide analogs in primer extension assay catalyzed by SARS-CoV-2 RdRp. (A) P/T complex used in the assay whose results are shown in panel B. The first, second, third, and fourth nucleotides to be incorporated are UTP, ATP, GTP, and CTP, respectively. (B) Analysis of the incorporation and chain termination abilities of the ATP analogs. Incorporation and chain termination of tested nucleotides were measured in two separate assays. For incorporation (lanes 5, 7, 9, 11, 13, and 15), primer extension reactions were initiated by the addition of 0.1 μM UTP and 100 μM ATP analogs, as indicated under each lane. The reactions were performed at 37°C for 30 min and stopped by the addition of stopping solution. For chain termination ability (lanes 6, 8, 10, 12, 14, and 16), primer extension reactions were initiated by the addition of 0.1 μM UTP and 100 μM ATP analogs, as indicated under each lane. The reactions were performed at 37°C for 30 min, 0.1 μM GTP and 0.1 μM CTP were added to the reaction mixtures, and the reactions were continued at 37°C for another 30 min before the addition of stopping solution. The reaction products were resolved by denaturing PAGE. Analysis of incorporation and termination of GTP analogs (C and D), UTP analogs (E and F), and CTP analogs (G and H) were done by using a similar method.