title: A high-throughput-compatible, fluorescence anisotropy-based assay for ATP-dependent supercoiled DNA relaxation by human topoisomerase IIα.
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.
authors: Bioscience Department, Infection Innovative Medicines, AstraZeneca R&D Boston, Waltham, MA 02451 USA. adam.shapiro@astrazeneca.com
authors: Adam B
authors: AB
authors: Shapiro
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D000951:Antigens, Neoplasm / Q000737:chemistry*; D000970:Antineoplastic Agents / Q000737:chemistry; D004250:DNA Topoisomerases, Type II / Q000737:chemistry*; D004278:DNA, Superhelical / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D005047:Etoposide / Q000737:chemistry; D005454:Fluorescence Polarization; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D010957:Plasmids; D059005:Topoisomerase II Inhibitors / Q000737:chemistry
chemical_list: D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D059005:Topoisomerase II Inhibitors; D005047:Etoposide; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II