Single-Molecule Assay Reveals Binding Dynamics of SARS-CoV-2 Polymerase Components and Provides a New Tool to Distinguish Polymerase Inhibitors

The genome replication of SARS-CoV-2, the causative agent of COVID-19, involves a multisubunit replication complex consisting of nonstructural proteins (nsps) 12, 7, and 8. While the structure of this complex is known, the dynamic behavior of the subunits interacting with RNA is missing. Here we report a single-molecule protein induced fluorescence enhancement (SM-PIFE) assay to monitor binding dynamics between the reconstituted or coexpressed replication complex and RNA. Increasing binding times were observed, in this order, with nsp7 (none), nsp8, and nsp12, in nsp8 nsp12 mixtures and in reconstituted mixtures bearing all three proteins. Unstable, unstable→stable, and stable binding modes were recorded in the latter case, indicating that complexation is dynamic and the correct conformation must be achieved before stable RNA binding can occur. Notably, the coexpressed protein yields mostly stable binding even at low concentrations, while the reconstituted proteins exhibit unstable binding indicating inefficient complexation with reduced protein. The SM-PIFE assay distinguishes inhibitors that impact protein binding from those that prevent replication, as demonstrated with suramin and remdesivir, respectively. The data reveals a correlation between binding lifetime/affinity and protein activity and underscores differences between coexpressed vs reconstituted mixtures, suggesting the existence of trapped conformations that may not evolve to productive binding.