Recognition of Non-standard Base Pairs by Triplex-Forming Oligonucleotides Containing an Expanded Genetic Alphabet

Recognition of Non-standard Base Pairs by Triplex-Forming Oligonucleotides Containing an Expanded Genetic Alphabet | 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 Recognition of Non-standard Base Pairs by Triplex-Forming Oligonucleotides Containing an Expanded Genetic Alphabet David Rusling, Michael Brazzill, Ruolin Ma, Kieron Munn, Lena Prestifilippo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8652009/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The sequence-specific recognition of double-stranded DNA by biocompatible molecules is fundamental to molecular medicine and synthetic biology. Triplex-forming oligonucleotides (TFOs) enable programmable major-groove recognition via Hoogsteen base pairing; however, the limited repertoire of natural nucleobases imposes strict constraints on target sequences and requires acidic conditions for stability. Here, we have expanded the triplex recognition space using nucleobases from an artificially expanded genetic information system (AEGIS). Through a systematic evaluation of 120 base triplet combinations, we identify at least 12 new modular triplets that can be combined interchangeably to target duplex DNA containing standard, damaged, or synthetic base pairs with nanomolar affinity at neutral pH. We further demonstrate the versatility of this expanded recognition code by detecting oxidative lesions or AEGIS base pairs in enzymatically assembled duplex constructs using both chemically and enzymatically synthesized TFOs. This generalized framework provides a robust platform for precision gene-targeting, molecular sensing, and nucleic acid nanotechnology. Biological sciences/Chemical biology Biological sciences/Biotechnology Full Text Additional Declarations Yes there is potential Competing Interest. Many of the AEGIS components described in the manuscript are commercially available from Firebird Biomolecular Sciences, LLC ( www.firebirdbio.com ), which operates under intellectual property owned by S.A.B. and the Foundation for Applied Molecular Evolution. Certain authors (C.C., H.J.K., and S.H.) are employed at Firebird. The remaining authors declare no competing interests. Supplementary Files NatCommSI.docx SI Cite Share Download PDF Status: Under Review 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8652009","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":592123838,"identity":"b73c395b-927a-4f41-ac78-6681e66cbcfc","order_by":0,"name":"David Rusling","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYDCCA0DEA2KwMzYwMFQgJAwIa2EGaTkDZLARoYUBogWIGduI0MJ3/IzhgTcM9xLnNzO3Pfw6746c+fwGxg8/GA4b49IieSbH4OAchuLEDYcZ241ltz0zljnGwCzZw3DYDJcWgwNpCYd5GBISNzAztklLbjucOAPoMGkGhsM2OLWcfwbRMr8ZpGUOWAvzb7xabiQfAGtpOMzYJvmxAayFDWQLTodJ3nh84OAcgwRjoF/apBmOPTOWYEtss+wxSMfpfb7zic0f3lQkyM5vb38m+aPmjpwE8+HDN35UWBs24NIDcR6EYuYBRxMoTnFHJCpg/AHWMgpGwSgYBaMAFQAAEYRZGTp2O38AAAAASUVORK5CYII=","orcid":"","institution":"University of Portsmouth","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"","lastName":"Rusling","suffix":""},{"id":592123839,"identity":"f938a138-556f-47e2-925c-d81bdaf3f1af","order_by":1,"name":"Michael Brazzill","email":"","orcid":"","institution":"University of Portsmouth","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Brazzill","suffix":""},{"id":592123840,"identity":"3c749725-1278-4e6c-bb4b-061938c7e448","order_by":2,"name":"Ruolin Ma","email":"","orcid":"","institution":"University of Portsmouth","correspondingAuthor":false,"prefix":"","firstName":"Ruolin","middleName":"","lastName":"Ma","suffix":""},{"id":592123841,"identity":"fc02f4ee-0b34-4acf-a9cd-89ece350de86","order_by":3,"name":"Kieron Munn","email":"","orcid":"","institution":"University of Portsmouth","correspondingAuthor":false,"prefix":"","firstName":"Kieron","middleName":"","lastName":"Munn","suffix":""},{"id":592123842,"identity":"5e63151d-542e-496d-8639-2c5ce5f565da","order_by":4,"name":"Lena Prestifilippo","email":"","orcid":"","institution":"Polytech Angers","correspondingAuthor":false,"prefix":"","firstName":"Lena","middleName":"","lastName":"Prestifilippo","suffix":""},{"id":592123843,"identity":"fb28c73f-89a4-4af4-8594-1b411cb4c498","order_by":5,"name":"Andrew Pickford","email":"","orcid":"https://orcid.org/0000-0002-7237-0030","institution":"University of Portsmouth","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Pickford","suffix":""},{"id":592123844,"identity":"3411dd94-634b-4375-b664-ed482f216e07","order_by":6,"name":"Hyo-Joong Kim","email":"","orcid":"","institution":"Firebird Biomolecular Sciences LLC","correspondingAuthor":false,"prefix":"","firstName":"Hyo-Joong","middleName":"","lastName":"Kim","suffix":""},{"id":592123845,"identity":"d9501dde-90a6-4df6-8a8a-c2358782ed39","order_by":7,"name":"Cen Chen","email":"","orcid":"https://orcid.org/0000-0002-6883-9369","institution":"Foundation for Applied Molecular Evolution","correspondingAuthor":false,"prefix":"","firstName":"Cen","middleName":"","lastName":"Chen","suffix":""},{"id":592123846,"identity":"4dafb6ef-20e6-4023-9b60-381573ba03fa","order_by":8,"name":"Shuichi Hoshika","email":"","orcid":"https://orcid.org/0000-0001-9128-1183","institution":"Foundation for Applied Molecular Evolution","correspondingAuthor":false,"prefix":"","firstName":"Shuichi","middleName":"","lastName":"Hoshika","suffix":""},{"id":592123847,"identity":"ad1e5c50-5951-4f2b-8107-4ecf4219b83e","order_by":9,"name":"Steven Benner","email":"","orcid":"","institution":"Foundation for Applied Molecular Evolution","correspondingAuthor":false,"prefix":"","firstName":"Steven","middleName":"","lastName":"Benner","suffix":""}],"badges":[],"createdAt":"2026-01-20 18:23:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8652009/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8652009/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102963158,"identity":"d71970d9-fcf2-4f5c-af78-beb19fcda043","added_by":"auto","created_at":"2026-02-19 04:14:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1173337,"visible":true,"origin":"","legend":"","description":"","filename":"NatCommMaintext.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8652009/v1_covered_223f2939-a047-4b94-a411-e98de717b9e5.pdf"},{"id":102851642,"identity":"d3f26201-106a-44f4-a2af-8a4e01bcc55b","added_by":"auto","created_at":"2026-02-17 14:28:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7070840,"visible":true,"origin":"","legend":"SI","description":"","filename":"NatCommSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-8652009/v1/cfe6f6a5e91be151671392fd.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nMany of the AEGIS components described in the manuscript are commercially available from Firebird Biomolecular Sciences, LLC (www.firebirdbio.com), which operates under intellectual property owned by S.A.B. and the Foundation for Applied Molecular Evolution. Certain authors (C.C., H.J.K., and S.H.) are employed at Firebird. The remaining authors declare no competing interests.","formattedTitle":"Recognition of Non-standard Base Pairs by Triplex-Forming Oligonucleotides Containing an Expanded Genetic Alphabet","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8652009/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8652009/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe sequence-specific recognition of double-stranded DNA by biocompatible molecules is fundamental to molecular medicine and synthetic biology. Triplex-forming oligonucleotides (TFOs) enable programmable major-groove recognition \u003cem\u003evia\u003c/em\u003e Hoogsteen base pairing; however, the limited repertoire of natural nucleobases imposes strict constraints on target sequences and requires acidic conditions for stability. Here, we have expanded the triplex recognition space using nucleobases from an artificially expanded genetic information system (AEGIS). Through a systematic evaluation of 120 base triplet combinations, we identify at least 12 new modular triplets that can be combined interchangeably to target duplex DNA containing standard, damaged, or synthetic base pairs with nanomolar affinity at neutral pH. We further demonstrate the versatility of this expanded recognition code by detecting oxidative lesions or AEGIS base pairs in enzymatically assembled duplex constructs using both chemically and enzymatically synthesized TFOs. This generalized framework provides a robust platform for precision gene-targeting, molecular sensing, and nucleic acid nanotechnology.\u003c/p\u003e","manuscriptTitle":"Recognition of Non-standard Base Pairs by Triplex-Forming Oligonucleotides Containing an Expanded Genetic Alphabet","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 14:28:35","doi":"10.21203/rs.3.rs-8652009/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"57833ee6-8972-4d7b-99fc-ff9d9f7d6c14","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63006850,"name":"Biological sciences/Chemical biology"},{"id":63006851,"name":"Biological sciences/Biotechnology"}],"tags":[],"updatedAt":"2026-04-24T13:50:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 14:28:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8652009","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8652009","identity":"rs-8652009","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}