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

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

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