Thesis
Binding of DNA/DNA duplexes to single tethered HIV RT molecules
Washington State University
Master of Science (MS), Washington State University
2011
Handle:
https://hdl.handle.net/2376/103727
Abstract
Single molecule spectroscopy has been used to study the binding of primertemplate DNA complexes to tethered HIV-1 reverse transcriptase (HIV RT) molecules. In this study, the 5‟ end of primer DNA was fluorescently tagged with a green quantum dot and then annealed to a longer strand of template DNA. The rates of association and dissociation were observed in terms of „on‟ and „off‟ times of the DNA duplex with a single tethered reverse transcriptase molecule. In this work, HIV RT was successfully immobilized onto an optical surface and the binding „on‟ and „off‟ times for quantum dotprimer / template complexes at concentrations of 50 pM, 75 pM, and 100 pM were measured at 25° C. The histogram for the „on‟ times of each concentration is a direct measure of the dissociation rate of the QD-primer / template / HIV RT complex. The histogram for the „off‟ times is a direct measure of the association rate of the complex. While the dissociation and association rates were determined, many factors needed verification, including the functionality of HIV RT, as well as surface homogeneity. The surface was characterized by a number of studies involving the direct observation of immobilized streptavidin and tethered HIV RT molecules as a function of increasing amounts of biotinylated BSA laid on the surface, as well as checking for non-specific binding events using labeled / unlabeled streptavidin and HIV RT antibodies. Finally, in an effort to observe colocalized binding events, a simultaneous two color single molecules microscope was developed. Red quantum dots were used as a label for the DNA and Alexa® Fluor 555 were used as a label for the HIV RT. Unfortunately, blinking, blueing, and bleaching of the red quantum dots proved to be a limit in their use for single molecule detection and led to day to day inconsistencies in these particular results. Fortunately, we were able to identify these chemical processes and devise an alternate plan for future experiments.
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Details
- Title
- Binding of DNA/DNA duplexes to single tethered HIV RT molecules
- Creators
- Lillian Pearl Wong
- Contributors
- James A. Brozik (Degree Supervisor)
- Awarding Institution
- Washington State University
- Academic Unit
- Chemistry, Department of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University; Pullman, Wash. :
- Identifiers
- 99900525135801842
- Language
- English
- Resource Type
- Thesis