Extended Data Fig. 9: Biochemical analysis of the role of residue S759 in RDV-TP recognition.

(a) RNA template sequence used to determine the efficiency of adenosine triphosphate (ATP) or RDV-TP incorporation at position 6 (i). G indicates incorporation of [α32P] guanosine triphosphate (GTP) at position 5 (red). (b) Migration patterns of the products of ATP or RDV-TP incorporation reactions with wild-type and S759A SARS-CoV-2 RdRp complexes are shown. Main products emerge at position 6. The 5’- 32P-labeled 4-nucleotide primer (4) is used as a size marker (m), illustrating sizing in nucleotides (nts) (c) Graphical representation of ATP or RDV-TP single-nucleotide incorporation during RNA synthesis as a function of their respective concentrations shown in (b). Best-fit lines illustrate fitting of the data points to Michaelis-Menten kinetics function using GraphPad Prism 7.0. Data shows mean values with error bars illustrating the SDs of the data. All data represent at least three independent experiments (n = 4 for wild-type and n = 3 for S759A SARS-CoV-2 RdRp complexes). (d) V_max refers to the maximum velocity required to convert substrates into products in a substrate-saturated system, reported as a product fraction of incorporated nucleotide. K_m is a parameter indicating the concentration of substrate at one-half V_max, in μM. The standard error of the linear fit is denoted (±). The V_max/K_m ratio reflects catalytic efficiency and is used here to determine the selectivity of RDV-TP by taking the ratio between the catalytic efficiency of ATP and of RDV-TP. The discrimination index is defined as the ratio between the mutant RdRp’s selectivity values in relation to that of the wild-type.