4.16. Topoisomerase I DNA Relaxation Assay

Inhibition of Homo sapiens topoisomerase I activity by CPO was evaluated by plasmid relaxation assay. Serially diluted CPO from 100 μM to 0.1 nM was incubated with the enzyme (PROSPEC, Ness-Ziona, Israel) for 2 hours and the reaction was carried on with the presence of supercoiled pBR322 plasmid DNA (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 0.5 hours at 37 °C. The reaction was stopped by sodium dodecyl sulfate solution plus proteinase K, and agarose gel electrophoresis was performed to visualize the bands. The inhibition was quantified by measuring the intensity of supercoiled DNA band followed by calculation of EC₅₀ values in Prism7.