Single-turnover kinetic analysis of non-LTR retrotransposition defines the mechanism and rate constants governing each step

Single-turnover kinetic analysis of non-LTR retrotransposition defines the mechanism and rate constants governing each step | 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 Single-turnover kinetic analysis of non-LTR retrotransposition defines the mechanism and rate constants governing each step Kenneth Johnson, Tyler Dangerfield, Jun Zhou, Thomas Eickbush This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4992860/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Site specific retrotransposon-mediated gene therapy has the potential to revolutionize medicine by allowing insertion of large gene cargos. Despite decades of effort, the reaction sequence remains to be fully elucidated limiting the ability to engineer improved activity for gene insertion. Here we provide a kinetic/mechanistic framework for R2 non-LTR retrotransposition. Single turnover measurements and global data fitting defined the rate constants for each step in the pathway involving 1st -strand DNA cleavage to provide a DNA primer, reverse transcription to copy the RNA, 2nd -strand DNA cleavage to provide the second primer, and 2nd -strand synthesis to make duplex cDNA. Sequence analysis of the cDNA confirms accurate replication of the 1400 nt RNA used in this study. This represents the only complete analysis of the reaction sequence and first observation of 2nd -strand synthesis in vitro . We provide a kinetic framework to understand non-LTR retrotransposition, which provide a basis to engineer improved activity. Biological sciences/Biochemistry/Enzyme mechanisms Biological sciences/Biotechnology/Gene delivery Biological sciences/Biochemistry/DNA Full Text Additional Declarations Yes there is potential Competing Interest. K.A.J. is the President of KinTek, Corp., which provided the KinTek Explorer software used in this study. J.Z. is an employee and both J.Z. and T.H.E. are shareholders of Typewriter Therapeutics. The remaining authors declare no competing interests. Supplementary Files R2BmPaperSupplementonly8.28.24v1.pdf Cite Share Download PDF Status: Posted 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-4992860","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":348890932,"identity":"636af32f-d389-4d28-8e95-d53c533b73de","order_by":0,"name":"Kenneth Johnson","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYBACCQaGBGYwi70ByCxAyDA2ENTCcwDINADSRGhhgGiRSAASxGiRbD/w8HHhjjo5c8nXiR8eGDAk7mfvMfz4g8FGdsMB7FqkeRKSjWeeOWxsOTt3swTQYYk9PMeSpXkY0oxxaZFjSEiT5m07kLjhdu4GiBaJ5APSDAyHE3Fq4X8A0lKXuOHm2c0/wFrkHzb//MHwH6cWaQmwLcyJG27wboPawnxMgofhAE4tkjMeJBvzth02NjiTu80iwUDCuOdMWpo1jwHQhzi0SJzPSXwMdJicwfGzm2/+qLCRbW8/Ywxk2Mn24dACjIcEFCOgtAEu5SDAjtOwUTAKRsEoGAUQAABbNV1rxw8WdgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6575-2823","institution":"University of Texas at Austin","correspondingAuthor":true,"prefix":"","firstName":"Kenneth","middleName":"","lastName":"Johnson","suffix":""},{"id":348890933,"identity":"b6ad5bc1-95c5-4f77-ba69-eef8c128282c","order_by":1,"name":"Tyler Dangerfield","email":"","orcid":"https://orcid.org/0000-0003-1413-2082","institution":"University of Texas at Austin","correspondingAuthor":false,"prefix":"","firstName":"Tyler","middleName":"","lastName":"Dangerfield","suffix":""},{"id":348890934,"identity":"8346cf50-8df1-4c42-8c1f-167fd176822e","order_by":2,"name":"Jun Zhou","email":"","orcid":"","institution":"Typewriter Therapeutics","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Zhou","suffix":""},{"id":348890935,"identity":"20b3b44a-bf7d-4e21-b47a-f753c8c0cd62","order_by":3,"name":"Thomas Eickbush","email":"","orcid":"","institution":"University of Rochester","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Eickbush","suffix":""}],"badges":[],"createdAt":"2024-08-28 17:40:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4992860/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4992860/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84002722,"identity":"d267b598-dd50-4454-a15d-54b7bcde0b40","added_by":"auto","created_at":"2025-06-05 15:03:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1341315,"visible":true,"origin":"","legend":"","description":"","filename":"R2BmPaperFinal8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4992860/v1_covered_adb69fbf-83b4-47a7-ac63-7d1732643657.pdf"},{"id":63993150,"identity":"5e739391-206d-47ba-9a2b-b3767a23bd27","added_by":"auto","created_at":"2024-09-04 15:54:48","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":954162,"visible":true,"origin":"","legend":"","description":"","filename":"R2BmPaperSupplementonly8.28.24v1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4992860/v1/79c42b4cf47ccc60cbb7b615.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nK.A.J. is the President of KinTek, Corp., which provided the KinTek Explorer software used in this study. 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