Exploring cleavage activity of NSP15 using single molecule PIE-FRET

The NSP15 endoribonuclease is used by coronaviruses (CoVs) to avoid detection by host immune systems by cleaving viral RNA and therefore can be considered a possible therapeutic target for CoV diseases. Cryo-EM structures from prior studies have indicated NSP15 has a strong preference for cleaving uridine, but a more dynamic picture of enzyme kinetics would be useful for a complete understanding of enzyme activity. To explore real time cleavage dynamics of NSP15 in solution, we designed a single molecule assay using Pulsed Interleaved Excitation Fluorescence Resonance Energy Transfer (PIE-FRET) to monitor the cleavage of engineered fluorescently-labeled RNA substrates containing either A, U, or C at a specific site. We also used Fluorescence Correlation Spectroscopy (FCS) to determine diffusion times of RNA sub-populations during the cleavage process. To probe cleavage activity of individual complexes of NSP15 and RNA, we immobilized the RNA substrate and measured changes in the fluorescence intensity and lifetime over several minutes and performed FRET analysis. We found that PIE-FRET and FCS of the RNA substrates incubated with NSP15 suggest cleavage happens quickly for equimolar ratios of substrate and protein. Furthermore, analysis from both techniques indicates folding at the U base, which prior research has suggested may be a possible marker that host immune systems use for identifying viral RNA. Detailed analysis of the dynamics of the NSP15-RNA substrate interaction improves our understanding of NSP15 cleavage activity and may serve as a basis for exploring the kinetics of other enzymes implicated in human diseases.