Dissertation
Physical Investigations of DNA/DNA and RNA/DNA Duplexes Transiently Bound to Tethered HIV RT and DNA Assembled Gold Nanoparticles
Doctor of Philosophy (PhD), Washington State University
01/2013
Handle:
https://hdl.handle.net/2376/111380
Abstract
The focus of this dissertation is within the biological and analytical auspices of physical chemistry. Two individual systems were investigated. The first system relied upon single molecule total internal reflectance fluorescence (TIRF) microscopy to study the kinetics associated with the binding of DNA/DNA duplexes to human immunodeficiency virus reverse transcriptase (HIVRT) in a tethered system, as well as the limit of detection of various RNA/DNA duplexes utilizing RNA from West Nile Virus (WNV) and a similar tethered system. The second technique used was scanning transmission electron microscopy (STEM) to study the drying effect as well as the native, in situ liquid state of DNA assembled gold nanoparticles (AuNPs) with two DNA binding conformations.
The DNA/DNA duplex TIRF studies yielded binding rates of 162 ± 4 bases per second, more than 50% faster than previous studies which measured the polymerization rate to be 106 ± 6 bases per second. Reduced early release rates were also observed in this novel tethered system. The activity of the polymerase and binding affinity of the DNA to the HIVRT was increased demonstrating increased performance for detection modalities in the tethered system. The RNA/DNA duplex TIRF studies utilized the tethered system and limit of detection, dissociation rates, and surface coverage for the WNV RNA were obtained. With three different parts of the WNV genome represented with three different RNA lengths, it was shown that RNA length affects RNA/DNA duplex formation.
Application of the in situ liquid cell STEM technique showed significant differences between the inter-particle distances seen for dry and in situ samples. The drying effect falsely indicated certain grouping arrangements and changed the impression of the size of groups formed. It was also shown that both hybridized and bridged DNA controlled AuNP assemblies formed at room temperature within 72 hours with average distances as predicted based on the DNA lengths. These experiments, the first of their kind, are the foundation for continued progress within targeted drug delivery research and other downstream applications of AuNP assemblies. Further fluorescence correlation spectroscopy studies are planned to investigate assembly size through diffusion in solution, focusing on DNA and RNA detection schemes.
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Details
- Title
- Physical Investigations of DNA/DNA and RNA/DNA Duplexes Transiently Bound to Tethered HIV RT and DNA Assembled Gold Nanoparticles
- Creators
- Angela Rose Rudolph
- Contributors
- James A Brozik (Advisor)Susan M Brozik (Committee Member)Lisa M Gloss (Committee Member)Jeffrey P Jones (Committee Member)Jeanne L McHale (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Chemistry, Department of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 166
- Identifiers
- 99900581536801842
- Language
- English
- Resource Type
- Dissertation