Single-molecule FRET is a powerful tool which can be used to explore the dynamics of fluorescently labelled molecules of interest, informing structure and function. We have utilised this technique to understand the mechanisms governing interactions between viral proteins and RNA in multiple systems.
We have developed an in vitro system to directly visualize replication carried out by the SARS-CoV-2 RNA polymerase. We purified a minimal replication complex, comprising nsp12, nsp7, and nsp8, and combined it with fluorescently labelled RNA substrates, enabling real-time monitoring of RNA primer elongation at the single-molecule level. This platform allowed us to investigate the mechanisms of action of key inhibitors of SARS-CoV-2 replication. In particular, our data provides evidence for remdesivir triphosphate's mechanism of action, which involves polymerase stalling and subsequent chain termination dependent on the concentration of competing nucleotide triphosphates.
We are also applying this approach to other priority viral diseases, to further understand their replication and transcription cycles and provide a new platform for RNA polymerase-specific antiviral compound screening. Our work demonstrates the power of single-molecule FRET to provide dynamic insights into viral replication, offering a valuable tool for antiviral screening and mechanistic studies of protein activity.