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Adv."],"published-print":{"date-parts":[[2026,6,5]]},"abstract":"<jats:p>Single-molecule force spectroscopy (SMFS) has transformed our understanding of biomolecular mechanics. However, current high-throughput implementations rely on beads to apply force, introducing size and surface chemistry variability, requiring per-bead calibration, and are prone to multitether artifacts. Long handles further complicate measurements by convolving target conformational changes with handle stretching. We introduce tether force spectroscopy (TFS), a bead-free SMFS platform in which a single DNA tether serves as both the force applicator and an internal calibrator. In TFS, shear flow acting on identical DNA tethers applies piconewton-scale forces directly to surface-anchored molecules whose conformational dynamics are simultaneously monitored by single-molecule fluorescence. This guarantees single-tether results with uniform, internally calibrated forces and is inherently compatible with single-molecule fluorescence. We achieved high-resolution, high-throughput measurements across hundreds of molecules, enabling both force-extension and rupture experiments without specialized instrumentation. The combination of simplicity and simultaneous force-fluorescence capability makes TFS broadly accessible for correlating structure and function in diverse biomolecular systems.<\/jats:p>","DOI":"10.1126\/sciadv.aed1901","type":"journal-article","created":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T17:59:36Z","timestamp":1780682376000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":1,"title":["Massively parallel bead-free force spectroscopy with fluorescence"],"prefix":"10.1126","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1984-0443","authenticated-orcid":true,"given":"Adam B.","family":"Yasunaga","sequence":"first","affiliation":[{"name":"Department of Chemistry, The University of British Columbia, Kelowna, BC V1V1V7, Canada."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-2215-595X","authenticated-orcid":true,"given":"Ryan","family":"Riopel","sequence":"additional","affiliation":[{"name":"Department of Chemistry, The University of British Columbia, Kelowna, BC V1V1V7, Canada."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-5284-6205","authenticated-orcid":true,"given":"David t. 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