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A critical but often overlooked challenge is the non\u2010specific electrostatic binding of cationic peptides to the anionic DNA nanostructure, which leads to uncontrolled stoichiometry and undermines functional predictability. Here, the study systematically characterizes this issue and demonstrates a practical purification strategy to mitigate it. It is quantitatively shown that cationic peptides associate with DNA origami in vast excess of their intended binding sites, a phenomenon not observed with anionic control peptides. This non\u2010specific binding is confirmed to be electrostatic and is effectively screened by high salt. To address this, a charge\u2010dependent purification approach is evaluated using polyethylene glycol (PEG) precipitation, showing that cationic peptides require extensive purification (\u22657 cycles), whereas anionic peptides need only minimal treatment (2 cycles) to achieve precise loading. Crucially, the study provides definitive functional evidence that a therapeutic peptide (brain\u2010derived neurotrophic factor\u2010mimicking peptide) must be attached via stable, site\u2010specific hybridization to elicit a potent biological response; non\u2010specifically adsorbed peptides are largely inactive. This work provides a set of critical design guidelines and purification considerations necessary for the rational design of reliable and functionally predictable DNA nanodevices.<\/jats:p>","DOI":"10.1002\/smtd.202501936","type":"journal-article","created":{"date-parts":[[2025,12,6]],"date-time":"2025-12-06T05:47:44Z","timestamp":1765000064000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Characterization of Non\u2010Specific Electrostatic Interactions of Cationic Peptides with DNA Origami and Their Functional Consequences"],"prefix":"10.1002","volume":"10","author":[{"given":"Seung Hyun","family":"Kang","sequence":"first","affiliation":[{"name":"Medicinal Materials Research Center Biomedical Research Institute Korea Institute of Science and Technology (KIST)  Seoul 02792 Republic of Korea"},{"name":"Department of Chemical and Biological Engineering Korea University  Seoul 02481 Republic of 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