Tianyu Liu, Teng Cai, Junfeng Huo, Hongwei Liu, Aiying Li, Meng Yin, Yan Mei, Yueyue Zhou, Sijun Fan, Yao Lu, Luosheng Wan, Huijuan You, Xiaofeng Cai
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引用次数: 0
摘要
微生物可产生大量具有生物活性的次级代谢产物,包括放线菌素 D、多柔比星等 DNA 结合剂,它们在癌症化疗方面具有巨大潜力。然而,由于传统活性测定的灵敏度和特异性有限,需要进行大规模发酵和纯化,因此发现新型DNA拮抗剂仍具有挑战性。在这里,我们在微生物培养物或提取物中直接引入了单分子拉伸测定(SMSA),以发现DNA拮抗剂,即使是痕量的微生物培养物(5 μl)。我们的研究表明,dsDNA 在轮廓长度和过度伸展转变方面的独特变化使我们能够从复杂样品中特异性地检测插入因子,而无需进行大量纯化。对dsDNA施加作用力还能提高配体的结合亲和力Ka和插入量,从而提高灵敏度,这样就能检测弱插入因子,而传统方法往往会忽略这些因子。我们证明了 SMSA 的有效性,并鉴定了两种产生 DNA 插层物的菌株:有趣的是,被归类为弱DNA插入因子(Ka∼103 M-1)的medermycin和kalafungin对HCT-116癌细胞都表现出了强大的抗癌活性,IC50值分别为52 ± 6和70 ± 7 nM。
Force-enhanced sensitive and specific detection of DNA-intercalative agents directly from microorganisms at single-molecule level.
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.
期刊介绍:
Nucleic Acids Research (NAR) is a scientific journal that publishes research on various aspects of nucleic acids and proteins involved in nucleic acid metabolism and interactions. It covers areas such as chemistry and synthetic biology, computational biology, gene regulation, chromatin and epigenetics, genome integrity, repair and replication, genomics, molecular biology, nucleic acid enzymes, RNA, and structural biology. The journal also includes a Survey and Summary section for brief reviews. Additionally, each year, the first issue is dedicated to biological databases, and an issue in July focuses on web-based software resources for the biological community. Nucleic Acids Research is indexed by several services including Abstracts on Hygiene and Communicable Diseases, Animal Breeding Abstracts, Agricultural Engineering Abstracts, Agbiotech News and Information, BIOSIS Previews, CAB Abstracts, and EMBASE.