High throughput screening assay to identify DNA topoisomerase inhibitors
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United States of America
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Feb 25, 2025
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Oct 1, 2024
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Oct 1, 2024
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Abstract
The subject invention provides a novel fluorescence-based, T5 exonuclease-amplified DNA cleavage assay for gyrase poisoning inhibitor discovery. According to the assay, multiple gyrase molecules can simultaneously bind to a plasmid DNA molecule to form multiple gyrase-DNA cleavage complexes on the same plasmid. These gyrase-DNA cleavage complexes, greatly stabilized by a gyrase poisoning inhibitor, and can be trapped by a detergent such as sarkosyl. Digestion of gyrase by proteinase K results in the production of small DNA fragments, which can be digested by T5 exonuclease. This fluorescence-based DNA cleavage HTS assay is also suitable for screening large compound libraries to identify inhibitors against DNA topoisomerases, including human DNA topoisomerase IIα.
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