Microorganisms are known to produce a variety of bioactive secondary metabolites, including DNA intercalators. However, identifying new DNA intercalators has been challenging due to the limitations of traditional activity assays, which often involve large-scale fermentation and purification processes. Manipulating individual DNA molecules has helped us gain insights into the physical properties of DNA and its interactions with small molecules, making it a crucial means for discovering antitumor drugs.

Recently, a collaborative team, led by Professors CAI Xiaofeng and YOU Huijuan from the School of Pharmacy at Tongji Medical College of HUST, published a paper titled "Force-enhanced sensitive and specific detection of DNA-intercalative agents directly from microorganisms at single-molecule level" in Nucleic Acids Research. This study originally employed single-molecule manipulation techniques for screening microorganisms that produce DNA intercalators directly in microbial cultures or extracts.

Chemotherapeutic drugs that bind to nucleic acids, such as doxorubicin, daunorubicin, and camptothecin are essential in cancer management. Statistics showed that approximately 17% of approved anticancer agents, from 1949 to 2019, are nucleic acid-binding compounds, half of which were natural products derived from microorganisms or plants. Some microorganisms evolved a competitive advantage in the environment by producing DNA-binding compounds. Nonetheless, traditional detection methods, due to limited sensitivity and specificity, often entail large-scale cultivation (>10 liters of liquid or 1 kilogram of solid culture) and complicated separation and purification to obtain sufficient quantities for further analysis and identification.

Single-molecule manipulation techniques, such as optical tweezing, magnetic tweezing, and atomic force microscopy, allow for the mechanical manipulation of individual DNA and protein molecules and have been extensively used in the study of DNA and small molecule interactions. However, these single-molecule techniques have not previously been applied for direct detection in complex samples, such as microbial and plant extracts. This study demonstrated, for the first time, the possibility to identify, at nanomolar levels, the DNA intercalators directly from 5 microliters of bacterial culture or microbial crude extracts without purification. The detection was based on the principle that DNA intercalators cause an increase in the contour length of DNA when subjected to force. This increase in DNA contour length is a unique phenomenon to intercalators and is minimally affected by solution ionic conditions, primary metabolites, or protein binding. Additionally, applying external force to DNA can enhance the binding affinity of compounds and increase the number of ligand intercalations, thereby improving detection sensitivity. Therefore, the single-molecule manipulation technique provide a new alternative for uncovering DNA intercalators from microorganisms without purification, with high sensitivity and specificity.

The study analyzed microbial cultures from 17 different species, including Gram-positive bacteria, Gram-negative bacteria, and fungi, by combining single-molecule techniques and liquid handling. Two strains producing DNA intercalators were successfully screened out: Streptomyces tanashiensis and Talaromyces funiculosus. Through secondary metabolite biosynthetic gene cluster analysis, liquid chromatography-mass spectrometry (LC-MS), and chemical separation, three DNA intercalators were identified: merdemycin, kalafungin, and ligustrone B, with kalafungin and ligustrone B being confirmed, for the first time, as natural products with DNA intercalating activity. The results of this study shows a good prospect of applying single-molecule force spectroscopy in the exploration of active natural products. Notably, the integration of magnetic tweezers with a microfluidic flow chamber allowed for buffer exchange, rendering it highly suitable for measuring multiple samples in the same chamber. This demonstrated that single-molecule manipulation techniques are capable of effectively handling complex samples, including those that may contain genotoxic DNA intercalators, highlighting their applicability in environmental monitoring.

article link: https://doi.org/10.1093/nar/gkae746