Force-enhanced sensitive and specific detection of DNA-intercalative agents directly from microorganisms at single-molecule level.

Abstract

Microorganisms can produce a vast array of bioactive secondary metabolites, including DNA-intercalating agents like actinomycin D, doxorubicin, which hold great potential for cancer chemotherapy. However, discovering novel DNA-intercalating compounds remains challenging due to the limited sensitivity and specificity of conventional activity assays, which require large-scale fermentation and purification. Here, we introduced the single-molecule stretching assay (SMSA) directly to microbial cultures or extracts for discovering DNA-intercalating agents, even in trace amounts of microbial cultures (5 μl). We showed that the unique changes of dsDNA incontour length and overstretching transition enable thespecific detection of intercalators from complex samples without the need forextensive purification. Applying force to dsDNA also enhanced the sensitivity by increasing both the binding affinity Ka and the quantity of ligands intercalation, thus allowing thedetection of weak intercalators, which are often overlooked using traditional methods. We demonstrated the effectiveness of SMSA,identified two DNA intercalator-producing strains: Streptomyces tanashiensis and Talaromyces funiculosus, and isolated three DNA intercalators: medermycin, kalafungin and ligustrone B. Interestingly, both medermycin and kalafungin, classified as weak DNA intercalators (Ka ∼103 M-1), exhibited potent anti-cancer activity against HCT-116 cancer cells, with IC50 values of 52±6and 70±7 nM, respectively.