Improved protein binder design using beta-pairing targeted RFdiffusion

Improved protein binder design using beta-pairing targeted RFdiffusion | 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 Improved protein binder design using beta-pairing targeted RFdiffusion David Baker, Isaac Sappington, Martin Toul, David Lee, Stephanie Robinson, and 25 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5473963/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jan, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Despite recent advances in the computational design of protein binders, designing proteins that bind with high affinity to polar protein targets remains an outstanding problem. Here we show that RFdiffusion can be conditioned to efficiently generate protein scaffolds that form geometrically matched extended beta-sheets with target protein edge beta-strands in which polar groups on the target are nearly perfectly complemented with hydrogen bonding groups on the design. We use this approach to design binders against a set of therapeutically relevant polar targets (KIT, PDGFRɑ, ALK-2, ALK-3, FCRL5, and NRP1) and find that beta-strand-targeted design yields higher affinities and success rates than unconditioned RFdiffusion. All by all binding experiments show that the designs have affinities ranging from 137 pM to mid nM for their targets and essentially no off target binding despite the sharing of beta-strand interactions, likely reflecting the precise customization of interacting beta-strand geometry and additional designed binder-target interactions. A co-crystal structure of one such design in complex with the KIT receptor is nearly identical to the computational design model confirming the accuracy of the design approach. The ability to robustly generate binders displaying high affinity and specificity to polar interaction surfaces with exposed beta-strands considerably increases the range and capabilities of computational binder design. Biological sciences/Computational biology and bioinformatics/Protein design Biological sciences/Biochemistry/Proteins Biological sciences/Structural biology/X-ray crystallography Biological sciences/Biological techniques/Software Biological sciences/Biophysics/Molecular biophysics Full Text Additional Declarations Yes there is potential Competing Interest. Joseph Watson and Buwei Huang are now employees at Xaira Therapeutics. Supplementary Files extendeddatastrandpairingpaperSUBMISSIONNatureBiotechnology.pdf Extended Data Cite Share Download PDF Status: Published Journal Publication published 10 Jan, 2026 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. 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-5473963","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":393005162,"identity":"5edd5f1d-9a94-40d1-b6b4-b4cb504f885f","order_by":0,"name":"David Baker","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYDCCAzwMjA0MDHJgDg8pWoxJ15LYQLQWvuNnD36cUXEvfcONBMYHb9uI0CJ5Ji9ZcsOZ4lygFmbDucRoMTiQYyD5sC0hd8PtBDZpXqK0nH9j/PPhv4R0g9sJ7L+J03Ijx0xyY0NCAlALGzNRWiRvvEuznHEswXDm/YfNknPOEaGF73zu4Zs9NQnyfGcOH/zwpowILUgAFD2jYBSMglEwCqgDAOh3PecN4JD1AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-7896-6217","institution":"University of Washington","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"","lastName":"Baker","suffix":""},{"id":393005163,"identity":"89d11699-398a-4d18-8b56-710ecfc62eb4","order_by":1,"name":"Isaac Sappington","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Isaac","middleName":"","lastName":"Sappington","suffix":""},{"id":393005164,"identity":"3136f4be-5bd5-4bd8-80e1-1869932feb48","order_by":2,"name":"Martin Toul","email":"","orcid":"https://orcid.org/0000-0002-3707-6899","institution":"VIB-UGent Center for Inflammation Research (IRC)","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Toul","suffix":""},{"id":393005165,"identity":"fe50ff4e-b9e6-4f4d-b01f-2178f9bbd9eb","order_by":3,"name":"David Lee","email":"","orcid":"https://orcid.org/0000-0001-6539-4272","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Lee","suffix":""},{"id":393005166,"identity":"9a7e207d-ae73-44c9-8ed9-004a87d991c1","order_by":4,"name":"Stephanie Robinson","email":"","orcid":"","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"","lastName":"Robinson","suffix":""},{"id":393005167,"identity":"29971635-63aa-4c84-a7f7-7b36f5cb4467","order_by":5,"name":"Inna Goreshnik","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Inna","middleName":"","lastName":"Goreshnik","suffix":""},{"id":393005168,"identity":"6d3bec50-7a63-4ece-b64d-3ef6d7d55fbd","order_by":6,"name":"Clara McCurdy","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Clara","middleName":"","lastName":"McCurdy","suffix":""},{"id":393005169,"identity":"8ebed970-9e40-4a5c-a05d-49b2e3eef0e9","order_by":7,"name":"Tung Ching Chan","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Tung","middleName":"Ching","lastName":"Chan","suffix":""},{"id":393005170,"identity":"400d53d4-08ad-487e-8ec8-d28b8c042de3","order_by":8,"name":"Nic Buccholz","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Nic","middleName":"","lastName":"Buccholz","suffix":""},{"id":393005171,"identity":"7f129892-cfbb-4d03-865b-e3724ac9dc97","order_by":9,"name":"Buwei Huang","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Buwei","middleName":"","lastName":"Huang","suffix":""},{"id":393005172,"identity":"be0d1953-3a07-4aa4-845f-947e2bd01ed9","order_by":10,"name":"Dionne Vafeados","email":"","orcid":"https://orcid.org/0000-0002-2560-8907","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Dionne","middleName":"","lastName":"Vafeados","suffix":""},{"id":393005173,"identity":"ef3c24dd-610d-46be-8923-3a8294efbb23","order_by":11,"name":"Mariana Garcia-Sanchez","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Mariana","middleName":"","lastName":"Garcia-Sanchez","suffix":""},{"id":393005174,"identity":"9d6a4235-8d29-4601-b0f5-8b51d0f430a6","order_by":12,"name":"Nicole Roullier","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"","lastName":"Roullier","suffix":""},{"id":393005175,"identity":"d30f4245-038d-4f3d-8908-d985c5acd27a","order_by":13,"name":"Matthias Glögl","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Matthias","middleName":"","lastName":"Glögl","suffix":""},{"id":393005176,"identity":"8d976b64-c2f8-48e6-9d85-82a0da185d5d","order_by":14,"name":"Christopher Kim","email":"","orcid":"https://orcid.org/0000-0003-4305-0473","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Kim","suffix":""},{"id":393005177,"identity":"5176a633-5c6b-4ead-b668-a3d1a4b91baf","order_by":15,"name":"Joseph Watson","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"","lastName":"Watson","suffix":""},{"id":393005178,"identity":"960aa594-a4ff-491a-958f-165218eb74a8","order_by":16,"name":"Susana Vázquez Torres","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Susana","middleName":"Vázquez","lastName":"Torres","suffix":""},{"id":393005179,"identity":"b1e0cbb4-3105-4cd5-8dc2-bdb3559e90e2","order_by":17,"name":"Koen Verschueren","email":"","orcid":"","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Koen","middleName":"","lastName":"Verschueren","suffix":""},{"id":393005180,"identity":"b64e10dd-392f-4e39-9405-aafe57fc7162","order_by":18,"name":"Kenneth Verstraete","email":"","orcid":"","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Kenneth","middleName":"","lastName":"Verstraete","suffix":""},{"id":393005181,"identity":"515c3cd1-3168-42d2-be4d-024a09b36b7b","order_by":19,"name":"Cynthia Hinck","email":"","orcid":"","institution":"University of Pittsburgh","correspondingAuthor":false,"prefix":"","firstName":"Cynthia","middleName":"","lastName":"Hinck","suffix":""},{"id":393005182,"identity":"b2104090-ea80-4a07-afc8-1c7720c08ee6","order_by":20,"name":"Melisa Benard Valle","email":"","orcid":"https://orcid.org/0000-0002-0042-6592","institution":"Technical University of Denmark","correspondingAuthor":false,"prefix":"","firstName":"Melisa","middleName":"Benard","lastName":"Valle","suffix":""},{"id":393005183,"identity":"08d4f4b1-bcc4-43cf-8217-602f0212c4fe","order_by":21,"name":"Brian Coventry","email":"","orcid":"https://orcid.org/0000-0002-6910-6255","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Brian","middleName":"","lastName":"Coventry","suffix":""},{"id":393005184,"identity":"88c0b7fd-0bee-46e2-8f4c-489be7af35fe","order_by":22,"name":"Jeremiah Sims","email":"","orcid":"https://orcid.org/0000-0002-0479-0367","institution":"Institute for Protein Design","correspondingAuthor":false,"prefix":"","firstName":"Jeremiah","middleName":"","lastName":"Sims","suffix":""},{"id":393005185,"identity":"f6d6f05d-81d7-4c5d-8685-1c22eff6d63e","order_by":23,"name":"Green Ahn","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Green","middleName":"","lastName":"Ahn","suffix":""},{"id":393005186,"identity":"baec4cb2-1c1c-42a2-9ca2-320d3ba25bd6","order_by":24,"name":"Xinru Wang","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Xinru","middleName":"","lastName":"Wang","suffix":""},{"id":393005187,"identity":"d7a9afc2-2a66-4b3a-8afc-00f36a9c3ada","order_by":25,"name":"Andrew Hinck","email":"","orcid":"","institution":"University of Pittsburgh","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Hinck","suffix":""},{"id":393005188,"identity":"f80a6f85-a9b6-4ca4-8884-555094e8b972","order_by":26,"name":"Timothy Jenkins","email":"","orcid":"https://orcid.org/0000-0003-2979-5663","institution":"Department of Biotechnology and Biomedicine, Technical University of Denmark","correspondingAuthor":false,"prefix":"","firstName":"Timothy","middleName":"","lastName":"Jenkins","suffix":""},{"id":393005189,"identity":"7f5e94b3-963e-4686-8310-7dd8aed82a2b","order_by":27,"name":"Hannele Ruohola-Baker","email":"","orcid":"https://orcid.org/0000-0002-5588-4531","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Hannele","middleName":"","lastName":"Ruohola-Baker","suffix":""},{"id":393005190,"identity":"d869bbb1-68ca-4962-afe8-7d0f5b5a4de9","order_by":28,"name":"Steven Banik","email":"","orcid":"https://orcid.org/0000-0002-0544-6488","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Steven","middleName":"","lastName":"Banik","suffix":""},{"id":393005191,"identity":"84ce46c7-957e-4d3c-ae8f-e7046ad56f9b","order_by":29,"name":"Savvas Savvides","email":"","orcid":"","institution":"VIB UGent – Center for Inflammation Research","correspondingAuthor":false,"prefix":"","firstName":"Savvas","middleName":"","lastName":"Savvides","suffix":""}],"badges":[],"createdAt":"2024-11-18 08:10:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5473963/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5473963/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-67866-3","type":"published","date":"2026-01-10T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101391349,"identity":"8a82957c-e00b-4b43-a3f5-f705c7abd085","added_by":"auto","created_at":"2026-01-29 08:28:38","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3226166,"visible":true,"origin":"","legend":"Article File","description":"","filename":"strandpairingbindermanuscriptsubmissionNatureBiotechnology.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5473963/v1_covered_3d97dea0-228c-4751-9d0a-a21f0717ada9.pdf"},{"id":74013593,"identity":"8dd04582-ecd2-4801-9693-016e9f7a02ce","added_by":"auto","created_at":"2025-01-17 03:15:42","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3935651,"visible":true,"origin":"","legend":"Extended Data","description":"","filename":"extendeddatastrandpairingpaperSUBMISSIONNatureBiotechnology.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5473963/v1/e6ddab9573a517f8369a8986.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nJoseph Watson and Buwei Huang are now employees at Xaira Therapeutics.","formattedTitle":"Improved protein binder design using beta-pairing targeted RFdiffusion","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":"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-5473963/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5473963/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Despite recent advances in the computational design of protein binders, designing proteins that bind with high affinity to polar protein targets remains an outstanding problem. Here we show that RFdiffusion can be conditioned to efficiently generate protein scaffolds that form geometrically matched extended beta-sheets with target protein edge beta-strands in which polar groups on the target are nearly perfectly complemented with hydrogen bonding groups on the design. We use this approach to design binders against a set of therapeutically relevant polar targets (KIT, PDGFRɑ, ALK-2, ALK-3, FCRL5, and NRP1) and find that beta-strand-targeted design yields higher affinities and success rates than unconditioned RFdiffusion. All by all binding experiments show that the designs have affinities ranging from 137 pM to mid nM for their targets and essentially no off target binding despite the sharing of beta-strand interactions, likely reflecting the precise customization of interacting beta-strand geometry and additional designed binder-target interactions. A co-crystal structure of one such design in complex with the KIT receptor is nearly identical to the computational design model confirming the accuracy of the design approach. The ability to robustly generate binders displaying high affinity and specificity to polar interaction surfaces with exposed beta-strands considerably increases the range and capabilities of computational binder design.","manuscriptTitle":"Improved protein binder design using beta-pairing targeted RFdiffusion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-17 03:15:38","doi":"10.21203/rs.3.rs-5473963/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":"4a2cd273-8214-46ba-92b7-189f68185c83","owner":[],"postedDate":"January 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41901842,"name":"Biological sciences/Computational biology and bioinformatics/Protein design"},{"id":41901843,"name":"Biological sciences/Biochemistry/Proteins"},{"id":41901844,"name":"Biological sciences/Structural biology/X-ray crystallography"},{"id":41901845,"name":"Biological sciences/Biological techniques/Software"},{"id":41901846,"name":"Biological sciences/Biophysics/Molecular biophysics"}],"tags":[],"updatedAt":"2026-01-29T08:28:25+00:00","versionOfRecord":{"articleIdentity":"rs-5473963","link":"https://doi.org/10.1038/s41467-025-67866-3","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-01-10 05:00:00","publishedOnDateReadable":"January 10th, 2026"},"versionCreatedAt":"2025-01-17 03:15:38","video":"","vorDoi":"10.1038/s41467-025-67866-3","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67866-3","workflowStages":[]},"version":"v1","identity":"rs-5473963","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5473963","identity":"rs-5473963","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}