{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T13:40:51Z","timestamp":1791898851226,"version":"4.3.4"},"posted":{"date-parts":[[2025,6,25]]},"group-title":"Bioengineering","reference-count":45,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,6,25]],"date-time":"2025-06-25T00:00:00Z","timestamp":1750809600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"award":["CHE-2108660"],"award-info":[{"award-number":["CHE-2108660"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"award":["MCB-2306190"],"award-info":[{"award-number":["MCB-2306190"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R35GM122603"],"award-info":[{"award-number":["R35GM122603"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01GM131641"],"award-info":[{"award-number":["R01GM131641"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R35GM118119"],"award-info":[{"award-number":["R35GM118119"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100020397","name":"UCSF Cardiovascular Research Institute","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100020397","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Chan Zuckerberg Biohub San Francisco"},{"id":[{"id":"https:\/\/ror.org\/006w34k90","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,6,25]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  <jats:italic>De novo<\/jats:italic>\n                  protein design has emerged as a powerful strategy with the promise to create new tools. The practical performance of designed fluorophore binders, however, has remained far from meeting fluorescence microscopy demands. Here, we design\n                  <jats:italic>de novo<\/jats:italic>\n                  Rhodamine Binder (Rhobin) tags that combine ideal properties including size, brightness, and now adding hyperstability. Rhobin allows live and fixed cell imaging of a wide range of subcellular targets in mammalian cells. Its reversible fluorophore binding further enables live super-resolution STED microscopy with low photobleaching, as well as PAINT-type single-molecule localization microscopy. We showcase Rhobin in the extremophile\n                  <jats:italic>Sulfolobus acidocaldarius<\/jats:italic>\n                  living at 75\u00b0C, an application previously inaccessible by existing tags. Rhobin will serve as the basis for a new class of live cell fluorescent tags and biosensors.\n                <\/jats:p>","DOI":"10.1101\/2025.06.24.661379","type":"posted-content","created":{"date-parts":[[2025,6,25]],"date-time":"2025-06-25T11:35:11Z","timestamp":1750851311000},"source":"Crossref","is-referenced-by-count":8,"title":["<i>De novo<\/i>\n                  designed bright, hyperstable rhodamine binders for fluorescence microscopy"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5399-1720","authenticated-orcid":false,"given":"Yuda","family":"Chen","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA"},{"name":"Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9746-6912","authenticated-orcid":false,"given":"Klaus","family":"Yserentant","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7648-3761","authenticated-orcid":false,"given":"Kibeom","family":"Hong","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiming","family":"Kuang","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3755-8572","authenticated-orcid":false,"given":"Arghya","family":"Bhowmick","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5185-4124","authenticated-orcid":false,"given":"Arthur","family":"Charles-Orszag","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA, USA"},{"name":"Department of Microbiology and Molecular Genetics, University of California Davis, Davis, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2785-989X","authenticated-orcid":false,"given":"Samuel J.","family":"Lord","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7097-1505","authenticated-orcid":false,"given":"Lei","family":"Lu","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA"},{"name":"Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4816-7067","authenticated-orcid":false,"given":"Kaipeng","family":"Hou","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA"},{"name":"Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3392-3170","authenticated-orcid":false,"given":"Samuel I.","family":"Mann","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of California Riverside, Riverside, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0331-4200","authenticated-orcid":false,"given":"Jonathan B.","family":"Grimm","sequence":"additional","affiliation":[{"name":"Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0789-6343","authenticated-orcid":false,"given":"Luke D.","family":"Lavis","sequence":"additional","affiliation":[{"name":"Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0871-5479","authenticated-orcid":false,"given":"R. Dyche","family":"Mullins","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4745-263X","authenticated-orcid":false,"given":"William F.","family":"DeGrado","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA"},{"name":"Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1704-4141","authenticated-orcid":false,"given":"Bo","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA"},{"name":"Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA"},{"name":"Chan Zuckerberg Biohub San Francisco, San Francisco, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041210092472000_2025.06.24.661379v1.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.67.1.509"},{"key":"2026041210092472000_2025.06.24.661379v1.2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1168683"},{"key":"2026041210092472000_2025.06.24.661379v1.3","doi-asserted-by":"publisher","DOI":"10.1021\/acs.accounts.2c00098"},{"key":"2026041210092472000_2025.06.24.661379v1.4","doi-asserted-by":"publisher","DOI":"10.1021\/cb800025k"},{"key":"2026041210092472000_2025.06.24.661379v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/nbt765"},{"key":"2026041210092472000_2025.06.24.661379v1.6","doi-asserted-by":"publisher","DOI":"10.1126\/science.3043666"},{"key":"2026041210092472000_2025.06.24.661379v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/nature19946"},{"key":"2026041210092472000_2025.06.24.661379v1.8","doi-asserted-by":"crossref","unstructured":"D. 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