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Updated: May 14, 2026

Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples
Published on: December 2, 2022
A high-throughput-compatible, fluorescence anisotropy-based assay for ATP-dependent supercoiled DNA relaxation by
1Bioscience Department, Infection Innovative Medicines, AstraZeneca R&D Boston, Waltham, MA 02451 USA. adam.shapiro@astrazeneca.com
Abstract:
A novel, high-throughput-compatible assay for the ATP-dependent supercoiled DNA relaxing activity of human topoisomerase IIα (hTopoIIα) is described. The principle of detection is the preferential binding of the oligodeoxyribonucleotide BODIPY-TMR-5'-TTCTTCTTCT-3' to relaxed double-stranded plasmid containing the triplex forming sequence (TTC)9 versus the supercoiled plasmid. Binding of the oligonucleotide to the plasmid increases the fluorescence anisotropy of the BODIPY-TMR label. Optimization of the assay conditions was conducted to maximize the signal and the activity of the topoisomerase. The multiwell assay plate-based fluorescence anisotropy assay gave the same values for the potencies of several previously reported inhibitors of hTopoIIα as a gel electrophoresis-based assay of DNA relaxation.
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