Extended Data Fig. 10. The NiRAN domain is specific for GDP in the capping reaction and does not transfer GMP from GTP to 5’-ppRNA.

a. Thin-layer chromatogram depicting the reaction products following incubation of nsp12 and nsp9–pRNA^LS10 with [α-³²P]GTP or [α-³²P]GDP for the indicated timepoints. Location of the cold standards (left) was visualized by UV fluorescence and the ³²P by autoradiography. b. Thin-layer chromatogram depicting [α-³²P]GTP or [α-³²P]GDP used in the assays in a. Note the presence of GDP in the GTP sample. c. Thin-layer chromatograms depicting the reaction products resulting from the incubation of [α-³²P]GTP with nsp12 or the inactive NiRAN mutant (D218A). Reactions were performed as described in²⁶ with (lower) or without (upper) 5’-pppRNA^A19C and included nsp13 or the inactive mutant (K288A) as indicated. Vaccinia capping enzyme (VCE) was used as a positive control. Reaction products were digested with nuclease P1, then treated with or without calf intestinal alkaline phosphatase (CIP) and analysed by PEI-cellulose thin-layer chromatography followed by autoradiography. The positions of the origin and standard marker compounds are indicated. d. RNA products from c were analysed by TBE urea–PAGE and visualized by toluidine blue O staining (upper) and autoradiography (lower). Markers indicate RNA size by base length.