A unifying model for microRNA-guided silencing of messenger RNAs

A unifying model for microRNA-guided silencing of messenger RNAs | 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 unifying model for microRNA-guided silencing of messenger RNAs Nils Walter, Shankar Mandal, Tanmay Chatterjee, Sujay Ray, Alexander Johnson-Buck This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6422368/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Silencing by the miRNA-guided RNA induced silencing complex (miRISC) is dependent on Ago2-chaperoned base pairing between the miRNA 5′ seed (5′S) and a complementary sequence in the 3′ untranslated region of an mRNA. Prevailing mechanistic understanding posits that initial 5′S pairing can further allow functional base pair expansion into the 3′ non-seed (3′NS), while functionally distinct non-canonical pairing was reported between only the 3′NS and the mRNA coding sequence. We developed single-molecule kinetics through equilibrium Poisson sampling (SiMKEPS) to measure highly precise binding and dissociation rate constants of varying-length target sequences to 5′S and 3′NS in a paradigmatic miRISC isolated from human cells, revealing distinct stable states of miRISC with mutually exclusive 5′S and 3′NS pairing. Our data suggest conformational rearrangements of the Ago2-bound miRNA that regulate alternative 5′S- and 3′NS-driven target recognition. The resulting model reconciles previously disparate observations and deepens our acumen for successfully marshaling RNA silencing therapies. Biological sciences/Biophysics/Single-molecule biophysics Biological sciences/Chemical biology/RNA Full Text Additional Declarations Yes there is potential Competing Interest. The authors declare the following competing financial interests: A.J.-B. and N.G.W. are inventors on multiple patent applications related to SiMKEPS, and equity holders of aLight Sciences Inc., a startup company aiming to commercialize related technology. Supplementary Files SiMKEPSManuscriptSI2025FinalFinal.pdf Supplemntary Data Cite Share Download PDF Status: Published Journal Publication published 08 Dec, 2025 Read the published version in Nature Communications → Version 1 posted 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-6422368","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":445623335,"identity":"c4704297-817d-454a-91f8-33065df2a320","order_by":0,"name":"Nils 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