Cy3 utp

Nucleotidylation reactions were performed using a 1:100 ratio of Cy3-UTP (Jena biosciences) to UTP (Merck) pH adjusted to ∼7.4. Reactions were set up for 20 min at 30 °C with final concentrations of 20 mM HEPES pH 7.4, 150 mM NaCl, 50 μM MnCl₂, 25 μM UTP mix, 1.5 μM of enterokinase-treated Nsp9_COV19, and 0.5 μM Nsp12_COV19. Following dilution of recombinant protein stocks, the final reducing agent concentration within the assay is ∼10 μM DTT and 4% v/v DMSO. Within the 1 to 12 compound series reactions, Figure 4B, 15 μM Nsp9_COV19 and 1 μM Nsp12_COV19 were instead used. The reaction was terminated through addition of 2× loading dye containing 2% w/v sodium dodecyl sulfate and immediately separated via SDS-PAGE on an 18% w/v polyacrylamide gel. After washing once in 50 mM Tris, 150 mM NaCl, the Cy3 modified bands were imaged on an Amersham Typhoon 5 using Cy3 settings. Band quantification was performed using ImageQuant TL (Cytiva) with fitting done in Prism (GraphPad). The intensity of Cy3 staining is plotted as a percentage of the signal obtained compared with in-gel DMSO controls after background subtraction. The raw data from wild-type Nsp9 and the C73S mutant were not obviously dissimilar.