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

A combined PIE-FRET and FCS assay to monitor RNA dynamics and cleavage by SARS-CoV-2 Nsp15 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A combined PIE-FRET and FCS assay to monitor RNA dynamics and cleavage by SARS-CoV-2 Nsp15 Kenya Gordon, Zoe Wright, Meredith Frazier, Benjamin S. Clark, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7717808/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version 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 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. FRET FCS Nsp15 RNA dynamics single-molecule Full Text Additional Declarations No competing interests reported. Supplementary Files GordonetalNsp15SRPSupplementalData09252025clean.pdf 20210816cov3cov30prepgel3.png Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 05 Nov, 2025 Reviews received at journal 05 Nov, 2025 Reviews received at journal 29 Oct, 2025 Reviewers agreed at journal 26 Oct, 2025 Reviewers agreed at journal 14 Oct, 2025 Reviewers agreed at journal 14 Oct, 2025 Reviewers invited by journal 13 Oct, 2025 Editor assigned by journal 13 Oct, 2025 Editor invited by journal 09 Oct, 2025 Submission checks completed at journal 02 Oct, 2025 First submitted to journal 02 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7717808","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":526562088,"identity":"fe3af19f-16fe-41c1-9d52-615aedf0404b","order_by":0,"name":"Kenya Gordon","email":"","orcid":"","institution":"North Carolina State University","correspondingAuthor":false,"prefix":"","firstName":"Kenya","middleName":"","lastName":"Gordon","suffix":""},{"id":526562089,"identity":"17ea288b-2fd7-4c36-a436-a6eef2b18b84","order_by":1,"name":"Zoe Wright","email":"","orcid":"","institution":"National Institutes of Health","correspondingAuthor":false,"prefix":"","firstName":"Zoe","middleName":"","lastName":"Wright","suffix":""},{"id":526562090,"identity":"bb4b4c41-2503-4781-8172-890099579fe6","order_by":2,"name":"Meredith Frazier","email":"","orcid":"","institution":"National Institutes of Health","correspondingAuthor":false,"prefix":"","firstName":"Meredith","middleName":"","lastName":"Frazier","suffix":""},{"id":526562091,"identity":"26a0e1a9-c60e-44fc-a351-2b1c834a9cce","order_by":3,"name":"Benjamin S. 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Nsp15\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"FRET, FCS, Nsp15, RNA dynamics, single-molecule","lastPublishedDoi":"10.21203/rs.3.rs-7717808/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7717808/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNsp15 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.\u003c/p\u003e","manuscriptTitle":"A combined PIE-FRET and FCS assay to monitor RNA dynamics and cleavage by SARS-CoV-2 Nsp15","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-09 10:21:49","doi":"10.21203/rs.3.rs-7717808/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-05T19:59:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-05T17:45:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-29T16:33:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"239826032188307502533967996767736277697","date":"2025-10-26T08:20:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"107755415763688849700291721504268343788","date":"2025-10-14T16:45:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"19390683130034992545875473707205249602","date":"2025-10-14T06:16:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-13T18:05:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-13T17:59:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-09T18:16:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-02T21:56:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-02T21:52:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"69ed2946-9aa8-437c-9834-d73436d3f9bd","owner":[],"postedDate":"October 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T07:57:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-09 10:21:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7717808","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7717808","identity":"rs-7717808","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}