Abstract
We report a mechanism based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells and retained DNA is detected by Picogreen fluorescence. Compounds that result in an increase in Picogreen staining represent potential topoisomerase interfacial poisons while those that reduce fluorescence report catalytic inhibitors; therefore, the solid phase assay represents a „bimodal‟ readout that reveals mechanisms of action. The method has been demonstrated to work with known interfacial poisons and catalytic inhibitors. In addition to specific topoisomerase targeting drugs, the method also weakly detects other relevant anticancer agents, such as potent DNA alkylating and intercalating compounds; therefore, topoisomerase I HTS represents an excellent tool for searching and identifying novel genotoxic agents. This method is rapid, robust, economical and scalable for large library screens.
Notes
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Graduation Date
2011
Semester
Fall
Advisor
Muller, Mark T.
Degree
Master of Science (M.S.)
College
College of Medicine
Department
Molecular Biology and Microbiology
Degree Program
Molecular and Microbiology
Format
application/pdf
Identifier
CFE0004473
URL
http://purl.fcla.edu/fcla/etd/CFE0004473
Language
English
Release Date
June 2013
Length of Campus-only Access
1 year
Access Status
Masters Thesis (Open Access)
Subjects
Dissertations, Academic -- Sciences, Sciences -- Dissertations, Academic
STARS Citation
Cyril, Vidusha, "A Solid Phase Assay For Topoisomerase I Interfacial Poisons And Catalytic Inhibitors" (2011). Electronic Theses and Dissertations. 1836.
https://stars.library.ucf.edu/etd/1836