Development of template-mediated reactive probes for the fluorescence detection of nucleic acids in cells
Abstract/Contents
- Abstract
- Templated fluorescence activation is an elegant nucleic acid detection technique, which relies on a target-activated chemical reaction between two oligonucleotide probes, eliciting a fluorescence readout. This method reports on genetic markers in solution phase and in cells. The research described in this thesis was aimed to expand the reaction scope of templated fluorescence activation and to develop probes, which overcome shortcomings of previous designs. Examples of DNA template-mediated fluorogenic reactions developed during this project include an organomercury-induced cyclization of Rhodamine thiosemicarbazide and the deprotection of a 7-azidomethoxycoumarin profluorophore by a Staudinger reaction. The most promising probe design (Q-STAR probes) is based on the reductive cleavage of an [alpha]-azidoether quencher release linker conjugated to a fluorophored-labeled DNA. A triphenylphosphine DNA-probe rapidly activates Q-STAR probes in the presence of the matched DNA target strand, reporting its presence by a strong fluorescence turn-on signal. Q-STAR probes are inert to aqueous conditions and cellular components, properties that were suboptimal for previous probe designs. Q-STAR probes report the target with single nucleotide specificity and enable an amplified detection signal by harnessing the target as a catalyst for the templated reaction. Q-STAR probes efficiently detect the presence of rRNAs in bacteria and mammalian cells. The probes are responsive to single nucleotide differences, which allows discriminating bacteria species by genetic variations using fluorescence microscopy or flow cytometry. Similarly, rRNAs in mammalian cells generate a strong fluorescence turn-on signal for Q-STAR probes. Templated fluorescence activation schemes bear considerable promise for applications in clinical diagnostics and molecular biology. The probe designs described in this thesis, in particular Q-STAR probes, constitute a major advancement in the field and will help achieve these goals.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2010 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Franzini, Raphael Marcel | |
|---|---|---|
| Associated with | Stanford University, Department of Chemistry | |
| Primary advisor | Kool, Eric T | |
| Thesis advisor | Kool, Eric T | |
| Thesis advisor | Du Bois, Justin | |
| Thesis advisor | Huestis, Wray | |
| Advisor | Du Bois, Justin | |
| Advisor | Huestis, Wray |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Raphael Marcel Franzini. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2010. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2010 by Raphael Marcel Franzini
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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