High / Medium-Throughput Topoisomerase Assay
A high-throughput microtitre plate-based assay was developed in the laboratory of Professor A. Maxwell of the John Innes Centre along with staff from Inspiralis. It is based upon the fact that negatively-supercoiled plasmids form intermolecular triplex DNA more readily than relaxed plasmids do.
This assay overcomes some of the problems of gel-based assays, which are time consuming and are therefore inherently low-throughput. Inspiralis are able to offer this assay as a means of screening compounds for inhibitory activity against all DNA topoisomerases. The assay is adaptable to other enzymes that change DNA supercoiling such as restriction enzymes. Some of the results of a comparison of the assay with the conventional gel-based assay are shown below.
The assay is particularly useful for screening of compounds and may also be used for determination of IC50 values. In the examples given below, the determination of IC50values for the inhibition of supercoiling by E. coli DNA gyrase and relaxation by E. coli DNA topo IV and Human topo I and II by the plate- and gel-based assays are compared. Similar inhibition profiles and IC50 values were obtained in both assays.