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

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.