Fig. 3: Incorporation of AT-9010 and Sofosbuvir triphosphate (STP) by the SARS-CoV-2 RTC, and excision by the SARS-CoV-2 nsp14/10 exonuclease complex (ExoN).

a Timecourse of AT-9010 incorporation (red dots) as a substitute for GTP (left panel, 50 µM each of ATP, UTP, and CTP), or in competition with GTP (right panel, 50 μM each NTP) at indicated concentrations of AT-9010 (0–250 µM). Fold-preference for GTP over AT-9010 incorporation is calculated by comparing the amount of AT-9010 insertion (red dots) relative to full-length product at two concentrations and at three time-points. b Timecourse of Sofosbuvir triphosphate (STP) incorporation (green dots) as a substitute for UTP (left panel, 50 µM each of ATP, GTP and CTP), or in competition with UTP (50 μM each NTP) at indicated concentrations (0 or 250 µM). For both a and b, reactions were run at least in duplicate at multiple nucleotide/analog concentrations for two different RNA substrates, with consistent results. c Incorporation of ATP+GTP control (black, left panel), ATP + AT-9010 (red, middle panel) and ATP + GTP + STP (green, right panel) by the polymerase complex (POL), followed by excision time-course with the nsp14 ExoN (EXO). For both AT-9010 and STP experiments, the next templated nucleotide for incorporation was additionally added. d Quantitation of remaining product after ExoN excision shown in c, a representative gel of experiments done in duplicate for two separate RNAs. The RNA marked at position 0 on each gel corresponds to the size of the fluorescently labeled primer shown above each gel, prior to elongation. Source data are provided as a Source Data file.