Part I, unnatural thymidine analogs and shape mimics as substrates for human thymidine kinases; Part II, fluorescent size-expanded DNA analogs as efficient substrates for a template-independent DNA polymerase
Abstract/Contents
- Abstract
- This thesis is composed of two separate studies involving unnatural nucleoside (DNA) analogs in two different types of enzymes: human thymidine kinases 1 and 2 and terminal deoxynucleotidyl transferase (TdT). The ability of two types of unnatural DNA analogs, nonpolar nucleoside analogs and expanded nucleoside analogs, to act as efficient substrates in enzymes will be described. Nonpolar nucleoside analogs lacking the ability to hydrogen bond were synthesized to systematically vary in size and shape, and then used to probe the ability of two types of human thymidine kinases (TK1 and TK2) to recognize and phosphorylate these analogs. The results establish that nucleoside recognition mechanisms for these two classes of thymidine kinase are very different. On the basis of this data, nonpolar nucleosides are likely to be active in the nucleotide salvage pathway in human cells, suggesting new designs for bioactive molecules. Another class of nucleoside analogs, expanded nucleoside (xDNA) analogs, maintain the ability to hydrogen-bond to their respective natural bases, but have enhanced pi-stacking due to their larger size, allowing the molecule to have greater stability in a DNA duplex, and unique fluorescent properties. It was found that terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase, can accept multiple xDNA nucleotide analogs as substrates with efficiencies close to that of natural nucleotides. In addition, the expanded adenine (xA) and cytosine (xC) analogs show a visible and spectral change in fluorescence when TdT incorporates multiple analogs. The ease of enzymatic synthesis of these analogs and their inherent fluorescence suggest their use in nucleic acid labeling and hybridization studies. The comparable efficiencies which nonpolar nucleoside analogs and xDNA nucleotide analogs have to natural bases in thymidine kinases and TdT give new information about the steric and electronic requirements of these enzymes, and will be useful for potential therapeutic and biotechnological applications.
Description
| Alternative title | Unnatural thymidine analogs and shape mimics as substrates for human thymidine kinases |
|---|---|
| Alternative title | Fluorescent size-expanded DNA analogs as efficient substrates for a template-independent DNA Polymerase |
| Type of resource | text |
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2011 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Jarchow-Choy, Sarah King |
|---|---|
| Associated with | Stanford University, Department of Chemistry |
| Primary advisor | Kool, Eric T |
| Thesis advisor | Kool, Eric T |
| Thesis advisor | Chen, James Kenneth |
| Thesis advisor | Waymouth, Robert M |
| Advisor | Chen, James Kenneth |
| Advisor | Waymouth, Robert M |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Sarah King Jarchow-Choy. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2011. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2011 by Sarah King Jarchow-Choy
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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