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While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize short specific target sequences through interactions with bases in the major groove and use this method to generate binders for five distinct DNA targets with mid-nanomolar to high-nanomolar affinities. The individual binding modules have specificity closely matching the computational models at as many as six base-pair positions and higher-order specificity can be achieved by rigidly positioning the binders along the DNA double helix using RFdiffusion. The crystal structure of a designed DBP\u2013target site complex is in close agreement with the design model and the designed DBPs function in both\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    and mammalian cells to repress and activate transcription of neighboring genes. Our method provides a route to small and, hence, readily deliverable sequence-specific DBPs for gene regulation and editing.\n                  <\/jats:p>","DOI":"10.1038\/s41594-025-01669-4","type":"journal-article","created":{"date-parts":[[2025,9,12]],"date-time":"2025-09-12T09:03:08Z","timestamp":1757667788000},"page":"2252-2261","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":42,"title":["Computational design of sequence-specific DNA-binding proteins"],"prefix":"10.1038","volume":"32","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5223-6339","authenticated-orcid":false,"given":"Cameron J.","family":"Glasscock","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert J.","family":"Pecoraro","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0291-2196","authenticated-orcid":false,"given":"Ryan","family":"McHugh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0008-473X","authenticated-orcid":false,"given":"Lindsey A.","family":"Doyle","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5255-4166","authenticated-orcid":false,"given":"Wei","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Olivier","family":"Boivin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-1778-2135","authenticated-orcid":false,"given":"Beau","family":"Lonnquist","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily","family":"Na","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuliya","family":"Politanska","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hugh K.","family":"Haddox","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Cox","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christoffer","family":"Norn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6910-6255","authenticated-orcid":false,"given":"Brian","family":"Coventry","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Inna","family":"Goreshnik","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dionne","family":"Vafeados","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9119-5303","authenticated-orcid":false,"given":"Gyu Rie","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6404-6556","authenticated-orcid":false,"given":"Raluca","family":"Gord\u00e2n","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Barry L.","family":"Stoddard","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7524-8938","authenticated-orcid":false,"given":"Frank","family":"DiMaio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7896-6217","authenticated-orcid":false,"given":"David","family":"Baker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,9,12]]},"reference":[{"key":"1669_CR1","doi-asserted-by":"publisher","DOI":"10.1186\/gb-2000-1-1-reviews001","volume":"1","author":"NM Luscombe","year":"2000","unstructured":"Luscombe, N. 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