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Mutations affecting MDA5 ATPase activity lead to severe pathological conditions. MDA5 nucleoprotein filament assembly\u2013disassembly dynamics are proposed to regulate dsRNA recognition, though the exact mechanism remains unclear. Here, we employed magnetic tweezers to monitor the assembly and manipulate MDA5 filaments at the single dsRNA level. Following a slow nucleation event, MDA5 assembles cooperatively and directionally into (partial) filaments and utilizes ATP hydrolysis to compact dsRNA through unwinding into single-stranded RNA (ssRNA), even against a significant opposing force. This compacted state is further stabilized by oligomerization of the caspase recruitment domain of MDA5 and requires high force to be disrupted. ssRNA gaps impaired compaction, suggesting a new mechanism for dsRNA recognition. We propose that MDA5-mediated dsRNA compaction captures viral dsRNA, preventing further usage for viral replication.<\/jats:p>","DOI":"10.1093\/nar\/gkag274","type":"journal-article","created":{"date-parts":[[2026,3,31]],"date-time":"2026-03-31T10:07:40Z","timestamp":1774951660000},"source":"Crossref","is-referenced-by-count":5,"title":["MDA5 generates compact ribonucleoprotein complexes via ATP-dependent double-stranded RNA unwinding"],"prefix":"10.1093","volume":"54","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9631-2265","authenticated-orcid":false,"given":"Salina","family":"Quack","sequence":"first","affiliation":[{"name":"Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam , De Boelelaan 1100, 1081 HZ Amsterdam ,","place":["The Netherlands"]},{"name":"Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-N\u00fcrnberg , Cauerstr. 3, 91058 Erlangen ,","place":["Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sourav","family":"Maity","sequence":"additional","affiliation":[{"name":"Moleculaire Biofysica, Zernike Instituut, Rijksuniversiteit Groningen , Nijenborgh 3, 9747 AG Groningen ,","place":["The Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pim P B","family":"America","sequence":"additional","affiliation":[{"name":"Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam , De Boelelaan 1100, 1081 HZ Amsterdam ,","place":["The Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8677-9710","authenticated-orcid":false,"given":"Misha","family":"Klein","sequence":"additional","affiliation":[{"name":"Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam , De Boelelaan 1100, 1081 HZ Amsterdam ,","place":["The 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Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam , De Boelelaan 1100, 1081 HZ Amsterdam ,","place":["The Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Quinte","family":"Smitskamp","sequence":"additional","affiliation":[{"name":"Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam , De Boelelaan 1100, 1081 HZ Amsterdam ,","place":["The Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paula","family":"Rivas","sequence":"additional","affiliation":[{"name":"Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam , De Boelelaan 1100, 1081 HZ Amsterdam ,","place":["The Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-8199-9439","authenticated-orcid":false,"given":"Flavia\u00a0S","family":"Papini","sequence":"additional","affiliation":[{"name":"Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-N\u00fcrnberg , Cauerstr. 3, 91058 Erlangen ,","place":["Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chase P","family":"Broedersz","sequence":"additional","affiliation":[{"name":"Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam , De Boelelaan 1100, 1081 HZ Amsterdam ,","place":["The Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wouter H","family":"Roos","sequence":"additional","affiliation":[{"name":"Moleculaire Biofysica, Zernike Instituut, Rijksuniversiteit Groningen , Nijenborgh 3, 9747 AG Groningen ,","place":["The Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yorgo","family":"Modis","sequence":"additional","affiliation":[{"name":"Molecular Immunity Unit, Department of Medicine, University of Cambridge, MRC Laboratory of Molecular Biology , Francis Crick Avenue, 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