Nucleic acid-based fluorescent probes
Abstract/Contents
- Abstract
- This thesis describes the development of novel nucleic acid-based fluorescent probes. Chapters 2 and 3 describe the rational design of fluorogenic probes that measure the activity of specific DNA-targeting enzymes. These probes rely on fluorophores that are quenched in the initial probe state, and demonstrate a 40- to 250-fold increase in fluorescence following reaction of the probe with its target enzyme. The probes function both in vitro with recombinant enzyme and in more biologically relevant samples such as bacterial cell culture and cell lysate. One of the probes was applied in a high-throughput screen for enzyme inhibitors, and was used to identify a potential inhibitor. These probes are expected to be useful for screening the effects of polymorphisms, mutations, post-translational modifications, and competitive inhibitors of enzyme activity. Chapter 4 describes a method to identify the sequence of unnatural nucleic acids on single beads. Combinatorial one-bead-one-compound libraries are commonly used to identify library members with a desired property. Identification of the library member on a selected bead is essential to this approach. A method for determining the sequence of nucleic acids (including unnatural nucleic acids, which cannot be identified by traditional polymerase-based methods) was demonstrated on natural DNA and on oligodeoxyfluorosides (ODFs). This method has the potential to improve the way our lab discovers ODFs by decreasing library synthesis time and by decreasing bead identification time. In addition to using this method to sequence beads from our one-bead-one-compound combinatorial ODF libraries, we anticipate that this method can be expanded to sequence anything synthesized in a step-wise fashion where the mass difference between each intermediate can give the identity of the intermediates (e.g. backbone or sugar modifications such as LNA, PNA, DNA, RNA; or non-canonical nucleotides). Chapter 5 describes work towards selecting an RNA molecule that binds a fluorophore and catalyzes the release of a fluorescent quencher to covalently attach the fluorophore to itself. Such a tool will allow us to monitor the fluorescently tagged RNA spatiotemporally in live cells.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2016 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Edwards, Sarah |
|---|---|
| Associated with | Stanford University, Department of Biophysics. |
| Primary advisor | Kool, Eric T |
| Thesis advisor | Kool, Eric T |
| Thesis advisor | Smolke, Christina D |
| Thesis advisor | Wandless, Thomas |
| Advisor | Smolke, Christina D |
| Advisor | Wandless, Thomas |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Sarah Edwards. |
|---|---|
| Note | Submitted to the Department of Biophysics. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2016. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2016 by Sarah Katherine Edwards
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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