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While over time, various materials and protocols have been developed, the characterization and quality assessment of the coating is either time consuming, highly invasive, or lacks detailed insights on single nanostructures. Here, a cyanine dye\u2010based molecular sensor is introduced to noninvasively probe the coating of DNA origami by either a cationic polymer or by silica, in real\u2010time and on a single nanostructure level. The cyanine dye reports changes in its local environment upon coating via increased fluorescence lifetime induced by steric restriction and water exclusion. Exploiting the addressability of DNA origami and the reversibility of the molecular sensor, the coating layer is probed at selected positions and in degrading conditions. Finally, the molecular sensor is combined with DNA PAINT super\u2010resolution imaging to investigate coating and structural integrity as well as preserved addressability of DNA nanostructures. The reported sensor presents a valuable tool to probe the coating of DNA nanodevices in complex biochemical environments in real\u2010time and at the single nanostructure level and aids the development of novel stabilization strategies.<\/jats:p>","DOI":"10.1002\/smll.202501044","type":"journal-article","created":{"date-parts":[[2025,6,29]],"date-time":"2025-06-29T10:24:34Z","timestamp":1751192674000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Monitoring the Coating of Single DNA Origami Nanostructures with a Molecular Fluorescence Lifetime Sensor"],"prefix":"10.1002","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7912-6292","authenticated-orcid":false,"given":"Michael","family":"Scheckenbach","sequence":"first","affiliation":[{"name":"Department of Chemistry and Center for NanoScience Ludwig\u2010Maximilians\u2010Universit\u00e4t  M\u00fcnchen Butenandtstr. 5\u201013 81377 M\u00fcnchen Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-5470-4394","authenticated-orcid":false,"given":"Gereon Andreas","family":"Br\u00fcggenthies","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Center for NanoScience Ludwig\u2010Maximilians\u2010Universit\u00e4t  M\u00fcnchen Butenandtstr. 5\u201013 81377 M\u00fcnchen Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2474-5395","authenticated-orcid":false,"given":"Tim","family":"Schr\u00f6der","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Center for NanoScience Ludwig\u2010Maximilians\u2010Universit\u00e4t  M\u00fcnchen Butenandtstr. 5\u201013 81377 M\u00fcnchen Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karina","family":"Betuker","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Center for NanoScience 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