A combined PIE-FRET and FCS assay to monitor RNA dynamics and cleavage by SARS-CoV-2 Nsp15

Nsp15 is an endoribonuclease, highly conserved among coronaviruses, that helps the virus evade detection in host cells by cleaving uridine-rich viral RNA sequences. Those sequences would otherwise trigger immune response pathways. Its essential role in pathogenesis and highly conserved nature make Nsp15 an attractive target for therapeutic intervention. While its crystal structure and uridine specificity are well-established, the influence of RNA structural dynamics and divalent cations on Nsp15 activity remains less understood. Leveraging single-molecule Pulsed Interleaved Excitation (PIE)-FRET and Fluorescence Correlation Spectroscopy (FCS) in combination, we developed an assay to track RNA cleavage by Nsp15 variants in real time and monitor the conformational dynamics of hybrid RNA/DNA substrates. Using our methodology and analysis strategies, we obtained clear indicators RNA cleavage with both PIE-FRET and FCS data analysis. Our assay also revealed signatures of unique dynamic behavior in uridine-containing RNA substrates, indicating that divalent cations enhance substrate flexibility, which increases the reaction rate of RNA cleavage by Nsp15. We propose that divalent cations induce a more favorable conformation in single-stranded RNA substrates that mediate the efficient nuclease activity of Nsp15.