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Updated: May 27, 2026
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Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples
Published on: December 2, 2022
High-throughput microtitre plate-based assay for DNA topoisomerases
James A Taylor1, Nicolas P Burton, Anthony Maxwell
1Department of Biological Chemistry, John Innes Centre, Colney, Norwich NR47UH, UK.
Methods in Molecular Biology (Clifton, N.J.)
|December 2, 2011
Summary
A new assay rapidly measures DNA topoisomerase activity by exploiting supercoiled DNA
Area of Science:
- Biochemistry
- Molecular Biology
- Enzyme Assays
Background:
- DNA topoisomerases are crucial enzymes that regulate DNA topology.
- Assaying their activity is essential for understanding DNA replication, repair, and for drug discovery.
- Conventional methods are often time-consuming and labor-intensive.
Purpose of the Study:
- To develop a rapid, high-throughput assay for DNA topoisomerase activity.
- To enable efficient screening of chemical libraries for topoisomerase inhibitors.
Main Methods:
- Developed a novel assay utilizing immobilized triplex-forming oligonucleotides (TFOs).
- Employed a plasmid substrate (pNO1) with a triplex-forming region.
- Leveraged the differential triplex formation between supercoiled and relaxed DNA.
Main Results:
- Supercoiled DNA preferentially forms triplexes and is retained by the TFO.
- Relaxed DNA is washed away, allowing for activity measurement.
- The assay demonstrates high speed and reduced sample handling compared to gel-based methods.
Conclusions:
- The developed assay is a fast and efficient method for measuring DNA topoisomerase catalytic activity.
- This high-throughput assay is suitable for screening chemical libraries for novel topoisomerase inhibitors.
