Extended Data Fig. 6: Biochemical assays probing stabilization of pre-incorporation complex.

(a) SDS-PAGE of purified SARS-CoV-2 nsp7/8 & nsp12. The experiment was performed once. (b) Size exclusion chromatography for the purified RTC complex, composed of nsp7/8₂/12 bound to the reconstituted product/template-RNA scaffold (chromatogram trace using the S4 scaffold is depicted). (c) SDS-PAGE of assembled RTC complex following size exclusion. Gel (n = 1) illustrates RTC assembly on the S4 scaffold. (d) S4 RNA scaffold utilized for the S4_GTP structure and incorporation assays. (e) Gel-based primer elongation assay in presence of physiological metal, Mg²⁺, and non-physiological metal, Ca²⁺, to investigate use of Ca²⁺ for stabilization of the pre-incorporation complex (gel depicts n = 2 with n = 3 experiments performed). Products are visualized alongside Decade RNA ladder (Invitrogen) showing separation in nucleotides (nts). (f) S3 RNA scaffold used for the S3_UTP structure and incorporation assays. (g) Gel-based primer elongation assay in presence of UTP, UMPNPP and 3′deoxy UTP (gel depicts n = 2 with n = 3 experiments performed). Products are visualized alongside Decade RNA ladder (Invitrogen) showing separation in nucleotides (nts). (h) Native mass spectrometry analysis of RNA extension in presence of ADP, RDV-DP & ATP using the S1/S2 RNA scaffold. (i) Native mass spectrometry analysis of RNA extension in presence of GDP using the S4 RNA scaffold.