A homogeneous, high-throughput fluorescence anisotropy-based DNA supercoiling assay

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• DNA supercoiling is crucial for cellular processes like replication and transcription.
• DNA topoisomerases regulate DNA topology and are key targets for anticancer and antibacterial drugs.
• Existing methods for assessing DNA supercoiling, such as gel electrophoresis, are low-throughput and labor-intensive.

Purpose of the Study:

• To develop a homogeneous, high-throughput screening (HTS) compatible biochemical assay for measuring DNA supercoiling.
• To facilitate the discovery of novel topoisomerase inhibitors for therapeutic applications.

Main Methods:

• Development of a homogeneous assay utilizing fluorescence anisotropy.
• The assay is based on the principle of DNA triplex formation between a fluorescently labeled oligonucleotide and plasmid DNA.
• No immobilization, filtration, or plate washing steps are required.

Main Results:

• The assay successfully measures changes in DNA supercoiling.
• Demonstrated utility with Escherichia coli topoisomerase I relaxation and E. coli DNA gyrase supercoiling.
• Successfully identified inhibition of DNA gyrase by fluoroquinolones and nalidixic acid.

Conclusions:

• The developed homogeneous fluorescence anisotropy assay is well-suited for HTS of topoisomerase inhibitors.
• This assay offers a significant improvement over traditional methods, enabling efficient drug discovery.
• The assay's simplicity and adaptability hold promise for identifying new therapeutic agents targeting DNA topoisomerases.