Force-Enhanced Sensitive Detection of New DNA-Interactive Agents from Microorganisms at the Single-Molecule Level

The discovery of microbial-derived DNA-interacting agents, which hold broad therapeutic potential, is inherently challenging due to the limited sensitivity and specificity of conventional methodologies. Our study introduces a pioneering application of single-molecule stretching assay (SMSA) in natural product chemistry to identify DNA-intercalating agents directly from microbial cultures or extracts. We demonstrate that mechanical force can enhance sensitivity by increasing both the binding affinity K a and the quantity of ligands bound. The changes induced by intercalators in the counter length and overstretching transition of dsDNA yield a distinctive and highly specific signature indicative of DNA intercalative binding, thereby enabling straightforward detection of DNA intercalators even in trace amounts from microbial cultures. This methodology eliminates the need for extensive large-scale fermentation and purification processes, thus offering a more streamlined approach to DNA-intercalating natural product discovery. By applying SMSA to 17 microorganisms, we identified two DNA intercalator-producing strains: Streptomyces tanashiensis and Talaromyces funiculosus . Subsequently, three DNA intercalators, namely medermycin, kalafungin, and ligustrone B, were isolated and characterized. Among them, medermycin and kalafungin showed significant inhibitory effects against HCT-116 cancer cells, with IC ₅₀ values of 52 ± 6 nM and 70 ± 7 nM, respectively.