Assay for inhibition of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp)

The pre-assembled SARS-CoV-2 RdRp complex (nsp12/nsp7/nsp8)24^,25 was incubated with appropriate concentrations of aqueous daclatasvir dihydrochloride for 15 min at room temperature in reaction buffer. Then the annealed RNA template-loop-primer in reaction buffer was added to the RdRp-daclatasvir mixture and incubated for an additional 10 min at room temperature. Finally, UTP or sofosbuvir-TP was added and incubation was carried out for 1 h at 30°C. The final concentrations of the reagents in the 20 μL extension reactions were 1 μM RdRp complex (nsp12/nsp7/nsp8), 500 nM RNA template-loop-primer, 0, 1, 4, 16 or 64 μM daclatasvir and 3 μM UTP or 15 μM sofosbuvir-TP. Following desalting, the samples were subjected to MALDI-TOF MS analysis.