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Accurate force calibration is essential for describing complex reactions, which can be hindered by sample heterogeneity, such as bead-to-bead differences in magnetic content. Here, we describe an in-situ force calibration methodology for high-throughput magnetic tweezers that enables the calibration for each individual bead with a precision of up to 3%, limited only by the statistical resolution. Using this approach, we investigated the mechanism of polar roadblocking by the mitochondrial transcription termination factor 1 (MTERF1) bound to the termination site. Establishing a SpyCatcher surface-attachment strategy, we performed force-jump experiments on the same tethers for up to 11 h. We showed that directional DNA unwinding is sufficient to explain the polar roadblock activity of MTERF1. Accurate force spectroscopy further reveals that the unlocking transition is rate-limited by a single kinetic barrier, with a transition-state distance consistent with structural interpretations. Together, these results provide a mechanistic and broadly applicable model for the asymmetric stability of MTERF1 and other nucleic acid polar roadblocks and establish a robust force spectroscopy framework for high-throughput magnetic tweezers experiments.<\/jats:p>","DOI":"10.1093\/nar\/gkag963","type":"journal-article","created":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T12:17:20Z","timestamp":1790425040000},"source":"Crossref","is-referenced-by-count":0,"title":["MTERF1 polar roadblock mechanism revealed using high-throughput magnetic tweezers and accurate single-bead force calibration"],"prefix":"10.1093","volume":"54","author":[{"given":"Pim P B","family":"America","sequence":"first","affiliation":[{"name":"Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam, De Boelelaan 1100, Amsterdam 1081 HZ ,","place":["The 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,","place":["The Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jamie J","family":"Arnold","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, University of North Carolina School of Medicine , Chapel Hill, NC 27599 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Craig E","family":"Cameron","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, University of North Carolina School of Medicine , Chapel Hill, NC 27599 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4209-0377","authenticated-orcid":false,"given":"David","family":"Dulin","sequence":"additional","affiliation":[{"name":"Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam, De Boelelaan 1100, Amsterdam 1081 HZ ,","place":["The Netherlands"]},{"name":"Junior Research 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