Kang, Seung Hyun Kwon, Oheun Cho, Bo Kyung Yoo, Seungmin Wang, Jin Myeong Choi, Youngjin Yoon, Hong Yeol Choi, Jungkyu Ryu, Ju Hee 2025-12-19T07:00:44Z 2025-12-19T07:00:44Z 2025-12-19 2026-01 2366-9608 https://pubs.kist.re.kr/handle/201004/153802 The functionalization of DNA origami with peptides is a powerful strategy for creating nanodevices for therapeutic and diagnostic applications. A critical but often overlooked challenge is the non-specific 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-specific binding is confirmed to be electrostatic and is effectively screened by high salt. To address this, a charge-dependent purification approach is evaluated using polyethylene glycol (PEG) precipitation, showing that cationic peptides require extensive purification (≥7 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-derived neurotrophic factor-mimicking peptide) must be attached via stable, site-specific hybridization to elicit a potent biological response; non-specifically 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. English WILEY-V C H VERLAG GMBH Characterization of Non-Specific Electrostatic Interactions of Cationic Peptides with DNA Origami and Their Functional Consequences Article 10.1002/smtd.202501936 1 Small Methods, v.10, no.1 Small Methods 10 1 Y scie 001631306200001 2-s2.0-105023970839 Chemistry, Physical Nanoscience & Nanotechnology Materials Science, Multidisciplinary Chemistry Science & Technology - Other Topics Materials Science Article STEM-CELLS BINDING RESPONSES IMMUNITY GROWTH cationic peptide DNA origami electrostatic interaction non-specific binding stoichiometric control