SARS-CoV-2 nsp14 MTase kinetic parameters and substrate specificity.

(A) SARS-CoV-2 nsp14 enzyme activity was monitored using a titration of uncapped (RNA1) short sequence and uncapped oligo 1 but methylated at the penultimate nucleotide sugar in the presence of 50μM SAM per reaction. (B) Titration of SAM substrate using saturating concentrations of RNA substrates, 25 μM of oligo 1 and 50 μM of RNA1. The data were generated using 1ng of nsp14 per reaction at 23°C for 30min. (C) Substrate selectivity of nsp14 using different RNA sequences and proteins known to be methylated. Reactions were carried out using 1ng of nsp14 per reaction with 50μM SAM and 25μM of each Oligo RNA and 20μM for both Histones and reactions were carried out at 37°C for 80min in white solid 384LV-well plate. MTase-Glo™ Assay was performed as described in Materials and Methods section. Each point represents an average of two data points; the error bars represent the standard deviation. Data analysis was performed with GraphPad Prism® software, version 9.1.0, for Windows® using a One Site Binding (hyperbola) program (panel A and B) and with Microsoft® Excel 365 program (panel C). All substrate sequences are shown in their specific panel.