Characterization of Non‐Specific Electrostatic Interactions of Cationic Peptides with DNA Origami and Their Functional Consequences

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.