Development of techniques for live cell RNA imaging
Abstract/Contents
- Abstract
- The development of live cell RNA imaging techniques will lead to the unraveling of many important biological processes. To achieve this goal, there have been three different strategies developed. They are the development of small molecule probes, nucleic acid probes, and green fluorescent protein (GFP) probes. In the following thesis, the pros and cons of each approach are discussed, followed by a proposal to resolve the limitations. In the small molecule case, a probe was developed that utilized a quenched sulforhodamine dye. It was designed so that its structure can be rationally modified from the initial lead compound. An aptamer sequence that activates the sulforhodamine probe with micro molar affinity was found by in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX), followed by fluorescence screening in E.coli. The rational modification of the structure of the initial sulforhodamine probe resulted in an overall 33-fold increase in binding affinity compared to the initial lead compound. Instead of the chemical modification of the lead compound, the small molecule's cell permeability and binding affinity to the target could be improved by linking to cell penetrating peptides (CPP). A CPP is a short peptide sequence composed of poly arginine amino acids which shows excellent cell uptake and affinity to RNA. However, the use of the CPP-linked dye in live cell imaging has been limited by strong signals in the endosome region. An attempt was made to overcome this difficulty by linking a quencher molecule to the dye-CPP via a disulfide bond, which only breaks when it enters the cytosol. For the nucleic acid probe, the major problem was its low cell permeability and low signal-to-background ratio due to the low copy number of mRNA targets within the cell. We made mutant Hammerhead ribozymes and embedded them in a non-coding region of the GFP expression vector that can be transfected to mammalian cells. This modified Hammerhead ribozyme acts as a logic gate, and the signal is amplified by the expression of GFP in the presence of the target mRNA. In vitro and in vivo results are discussed. Finally, a fragmented GFP system, the fluorescence of which could be recovered by binding to a specific RNA tag, was developed. The major problem for the GFP-mediated RNA imaging system was the low signal-to-background ratio from the GFP probe that is not bound to the RNA tag. To find the non-fluorescent GFP, the GFP was truncated from the C-terminus such that it loses its fluorescence with minimum loss of amino acids. An RNA sequence that has high affinity to this GFP was found by in vitro SELEX. The subsequent E.coli screening found an RNA sequence that reactivates the fluorescence of the GFP probe.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2011 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Lee, Jungjoon Kempthorne | |
|---|---|---|
| Associated with | Stanford University, Department of Chemistry | |
| Primary advisor | Rao, Jianghong | |
| Thesis advisor | Rao, Jianghong | |
| Thesis advisor | Huestis, Wray | |
| Thesis advisor | Moerner, W. E. (William Esco), 1953- | |
| Advisor | Huestis, Wray | |
| Advisor | Moerner, W. E. (William Esco), 1953- |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Jungjoon Kempthorne Lee. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Ph.D. Stanford University 2011 |
| Location | electronic resource |
Access conditions
- Copyright
- © 2011 by Jungjoon Kempthorne Lee
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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