Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

15-POS Board 7 SMFRET-BASED VISUALISATION OF BETACORONAVIRUS RNA POLYMERASE ACTIVITY AND INHIBITION Rory F Cunnison 1 ; Sonu S Philip 1 ; Danielle Groves 1,4 ; Adrian Deng 2 ; Jeremy Keown 2 ; Chris Coleman 3 ; Nicole Robb 1 ; 1 University of Warwick, Warwick Medical School, Coventry, United Kingdom 2 University of Warwick, School of Life Sciences, Coventry, United Kingdom 3 University of Nottingham, School of Life Sciences, Nottingham, United Kingdom 4 University of Chicago, Microbiology Department, Chicago, IL, USA Betacoronaviruses such as SARS-CoV-2 and MERS-CoV continue to pose significant pandemic threats due to their high rates of transmissibility and mutagenesis, as demonstrated during the COVID-19 pandemic and outbreaks of Middle East respiratory syndrome (MERS). There are few antiviral treatments available for betacoronavirus infections, with remdesivir triphosphate (Remdesivir-TP) being one of the only approved antiviral treatments for SARS-CoV-2 infection. Here, we present an established smFRET screening assay for the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). By attaching fluorophores to an RNA template–primer pair containing an overhang, we have developed a robust assay in which the addition of transcriptionally active SARS-CoV-2 RdRp changes the system's FRET efficiency from a high- to a low-FRET state as the primer is extended. This reporter assay can be used to measure the effects of both nucleoside and non-nucleoside inhibitors. Furthermore, we have successfully used it to identify the mechanisms of action of inhibitors that induce chain termination as a secondary mode of inhibition. Through the incorporation of accessory proteins into the assay, we have enhanced its inhibitor-screening capability by identifying novel exonuclease-resistant inhibitors. We have also applied a comparable approach to visualise MERS-CoV RdRp extension activity. Extending this assay to a second member of the betacoronavirus genus broadens its potential for identifying inhibitors of betacoronavirus RdRps. We anticipate that this screening platform will facilitate the development of novel antiviral treatments for betacoronavirus infections while expanding our understanding of the viral RdRps encoded by this group. In turn, this will deepen our knowledge of betacoronavirus RdRp initiation and extension dynamics through the application of smFRET based techniques.

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