Hierarchically Gated DNA Nanoswitches Enable Context‐Selective Activation of Antimicrobial Peptides

ABSTRACT Precise control over cytolytic peptide activity remains a central challenge in antimicrobial therapy because membrane disruption is intrinsically coupled with systemic toxicity. Here we encode a hierarchically gated molecular logic within a DNA nanoswitch to enable context‐selective activation of melittin only at infection sites. The nanoswitch integrates three cooperative control layers: bacterial tropism, microenvironmental sensing, and inflammation‐associated enzymatic activation. Maltodextrin ligands are spatially programmed to engage the bacteria‐specific maltodextrin transporter, yielding selective bacterial recognition with a 2.3‐order‐of‐magnitude enhancement in binding. Cytosine‐rich i‐motif locks convert acidic biofilm conditions into conformational opening of the scaffold, while a caspase‐1‐cleavable peptide tether provides a final enzymatic gate under inflammatory cues. This hierarchical gating confines membrane‐lytic activity with high spatiotemporal precision and suppresses off‐target hemolysis and systemic leakage. In uropathogenic Escherichia coli (UPEC)‐associated urinary tract infection models, the nanoswitch achieves effective bacterial clearance comparable to that of ciprofloxacin in vivo, demonstrating a generalizable strategy for conditional peptide activation.