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While the design and ligand incorporation into DNA origami nanostructures is well\u2010established, the study of dynamic interactions with cell surfaces is still in the explorative phase, where an in\u2010depth fundamental understanding of the molecular interaction dynamics remains underexplored. This study uniquely captures real\u2010time encounters between DNA origami and cells in situ using single particle tracking (SPT). We functionalized DNA nanorods (NRs) with antibodies or aptamers specific to the epidermal growth factor receptor (EGFR) and used them to target EGFR\u2010overexpressing cells. SPT data revealed that ligand\u2010coated NRs selectively bind to the receptors expressed in target cancer cells, while non functionalized NRs only display negligible cell interactions. Furthermore, the effect of ligand density is explored on the DNA origami, which revealed that aptamer\u2010decorated NRs exhibit nonlinear binding characteristics, whereas this effect in antibody\u2010decorated NRs is less pronounced. This study provides new mechanistic insights into the fundamental understanding of DNA origami behavior at the cell interface, with unprecedented spatiotemporal resolution, aiding the rational design of ligand\u2010targeted DNA origami for biomedical applications.<\/jats:p>","DOI":"10.1002\/smll.202502496","type":"journal-article","created":{"date-parts":[[2025,6,30]],"date-time":"2025-06-30T08:01:11Z","timestamp":1751270471000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Real\u2010Time Monitoring of DNA Origami\u2010Cell Interactions via Single Particle Tracking"],"prefix":"10.1002","volume":"21","author":[{"given":"Indra","family":"Van Zundert","sequence":"first","affiliation":[{"name":"Biomedical Engineering Department Technische Universiteit Eindhoven  Het Kranenveld 14, 5612 AZ Eindhoven The Netherlands"}]},{"given":"Elena","family":"Spezzani","sequence":"additional","affiliation":[{"name":"Biomedical Engineering Department Technische Universiteit Eindhoven  Het Kranenveld 14, 5612 AZ Eindhoven The Netherlands"}]},{"given":"Roger R.","family":"Brillas","sequence":"additional","affiliation":[{"name":"Biomedical Engineering Department Technische Universiteit Eindhoven  Het Kranenveld 14, 5612 AZ Eindhoven The Netherlands"}]},{"given":"Lars","family":"Paffen","sequence":"additional","affiliation":[{"name":"Biomedical Engineering Department Technische Universiteit Eindhoven  Het Kranenveld 14, 5612 AZ Eindhoven The Netherlands"}]},{"given":"Angelina","family":"Yurchenko","sequence":"additional","affiliation":[{"name":"Biomedical Engineering Department Technische Universiteit Eindhoven  Het Kranenveld 14, 5612 AZ Eindhoven The Netherlands"}]},{"given":"Tom F. 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