Abstract
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 of 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 is associated with a faster observed reaction rate of RNA cleavage by Nsp15 in the presence of Mn2+. We propose that divalent cations induce a conformation that may be more favorable for cleavage in single-stranded RNA substrates that mediate the efficient nuclease activity of Nsp15.
Similar content being viewed by others
Acknowledgements
We thank Dr. Keith Weninger (North Carolina State University) for critical reading of the manuscript. We thank Dr. Jose F. Castaneda for critical reading of the manuscript and helpful suggestions.
Funding
NC State Startup funds and Chan Zuckerberg Initiative Science Diversity Leadership Award to S.J.L. NC State Southern Regional Education Board (SREB) and Graduate Assistance in Areas of National Need (GAANN) awards to K.G. This work was supported by the US National Institutes of Health Intramural Research Program; US National Institute of Environmental Health Sciences (NIEHS) (ZIA ES103340 to R.E.S).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Gordon, K., Wright, Z.M., Stephens, C.R. et al. A combined PIE-FRET and FCS assay to monitor RNA dynamics and cleavage by SARS-CoV-2 Nsp15. Sci Rep (2026). https://doi.org/10.1038/s41598-026-52571-y
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-026-52571-y
