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Updated: Jun 15, 2025
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Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
Published on: May 6, 2020
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Force-enhanced sensitive and specific detection of DNA-intercalative agents directly from microorganisms at
Tianyu Liu1, Teng Cai1, Junfeng Huo1
1Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Nucleic Acids Research
|August 28, 2024
Summary
A novel single-molecule stretching assay (SMSA) directly detects DNA-intercalating agents in microbial cultures. This method enhances sensitivity for discovering potential cancer chemotherapy compounds, even weak ones, without extensive purification.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Microorganisms produce bioactive secondary metabolites with anticancer potential, such as DNA-intercalating agents.
- Discovering novel DNA intercalators is hindered by conventional assay limitations requiring large-scale purification and lacking sensitivity.
- Existing methods often overlook weak DNA intercalators, limiting the discovery of new chemotherapeutic agents.
Purpose of the Study:
- To introduce and validate a single-molecule stretching assay (SMSA) for direct discovery of DNA-intercalating agents from microbial sources.
- To enhance the sensitivity and specificity of detecting DNA intercalators, including weak binders, from complex biological samples.
- To identify novel DNA intercalator-producing microbial strains and isolate potent anticancer compounds.
Main Methods:
- Utilized single-molecule stretching assay (SMSA) applied directly to small volumes (5 μl) of microbial cultures or extracts.
- Analyzed changes in double-stranded DNA (dsDNA) contour length and overstretching transitions to detect intercalators.
- Employed force application to dsDNA to increase ligand binding affinity (Ka) and intercalation quantity, enhancing sensitivity.
Main Results:
- SMSA enabled specific detection of DNA intercalators from complex samples without extensive purification.
- Identified Streptomyces tanashiensis and Talaromyces funiculosus as producers of DNA intercalators.
- Isolated three compounds: medermycin, kalafungin, and ligustrone B, with medermycin and kalafungin showing potent in vitro anticancer activity against HCT-116 cells.
Conclusions:
- SMSA is an effective, sensitive, and specific method for discovering DNA-intercalating agents directly from microbial sources.
- The assay facilitates the identification of weak intercalators, expanding the scope for novel anticancer drug discovery.
- Medermycin and kalafungin, identified via SMSA, demonstrate significant potential as novel anticancer therapeutics.
