Spatially Organized DNA-Templated Silver Nanoclusters as Potent Antimicrobial Agents for ESKAPE Infections

Antibiotic-resistant bacteria cause more than one million deaths annually worldwide. The rapid evolution and horizontal gene transfer among pathogens frequently render newly developed antibiotics ineffective shortly after their introduction, underscoring the urgent need for alternative therapeutic strategies. Nanoscale silver is well known for its innate antimicrobial activity but typically requires high concentrations for efficacy that causes toxicities and limits broader clinical applications. To overcome these limitations, we introduce programmable, self-assembling DNA scaffolds that template, stabilize, and spatially organize multiple copies of monodisperse silver nanoclusters (DNA-AgNCs). These nanoscale assemblies enhance the antimicrobial potency of formulations while exhibiting intrinsic fluorescence, providing a dual functionality for therapeutic and fluorescence probing applications. Comprehensive characterization revealed DNA-AgNCs with superior stability and potent activity against clinically relevant antibiotic-resistant ESKAPE pathogens. Also, DNA-AgNCs significantly reduced the intracellular bacterial burden in primary murine bone cells infected with <i>Staphylococcus aureus</i>. Mechanistic studies indicate that bacterial killing by DNA-AgNCs is mediated by reactive oxygen species, particularly singlet oxygen, in conjunction with the disruption of the bacterial membrane. Furthermore, DNA-AgNCs retained strong antibacterial activity after 4 weeks of storage at ambient temperatures, with minimal loss of efficacy. Collectively, these findings establish spatially organized DNA-AgNCs as a promising, modular platform for next-generation antibacterials with integrated real-time fluorescence probing capabilities.