微生物可以产生大量具有生物活性的次级代谢产物，包括放线菌素 D、阿霉素等 DNA 嵌入剂，它们在癌症化疗方面具有巨大的潜力。然而，由于传统活性测定的灵敏度和特异性有限，需要大规模发酵和纯化，发现新型 DNA 嵌入化合物仍然具有挑战性。在这里，我们将单分子拉伸测定 (SMSA) 直接引入微生物培养物或提取物中，以发现 DNA 嵌入剂，甚至在微量微生物培养物 (5 μl) 中也是如此。我们证明，双链 DNA 在轮廓长度和过度拉伸转变方面的独特变化使得能够从复杂样品中特异性检测嵌入剂，而无需进行大量纯化。对 dsDNA 施加力还可以通过增加结合亲和力 Ka 和配体嵌入的数量来增强灵敏度，从而可以检测弱嵌入剂，而这在传统方法中经常被忽视。我们证明了 SMSA 的有效性，鉴定了两种 DNA 嵌入剂生产菌株：Streptomyces tanashiensis 和 Talaromyces funiculosus，并分离了三种 DNA 嵌入剂：麦德霉素、卡拉芬净和女贞酮 B。有趣的是，麦德霉素和卡拉芬净都被归类为弱 DNA 嵌入剂 (Ka ∼103 M-1)，对 HCT-116 癌细胞表现出有效的抗癌活性，IC50 值分别为 52 ± 6 和 70 ± 7 nM。© 作者 2024。由牛津大学出版社代表 Nucleic 出版酸研究。

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 in contour length and overstretching transition enable the specific detection of intercalators from complex samples without the need for extensive purification. Applying force to dsDNA also enhanced the sensitivity by increasing both the binding affinity Ka and the quantity of ligands intercalation, thus allowing the detection 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 ± 6 and 70 ± 7 nM, respectively.© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.