Sparano, Brian Andrew
(2007)
The Development of Fluorescent Sensors for Specific RNA Using Chemistry and Combinatorial Biology.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Here we describe the development of small molecule-based fluorescent sensors for RNA. The sensors described here and the new paradigm they represent are a convergence of chemical and biological methods. The resulting sensor system consists of two components: a rationally designed small-molecule fluorescent sensor and a reporter RNA sequence that binds to and enhances its fluorescence, isolated with combinatorial biology. First, we rationally designed and synthesized a series of modular fluorescent chemosensors which would be quenched in solution and fluorescent only when bound to their target. These sensors consist of a fluorophore (2',7'-dichlorofluorescein, DCF) conjugated to a pair of quencher units (aniline derivatives in this case) which quench the fluorescence of DCF by the PET (photoinduced electron transfer) mechanism. NMR and fluorescent spectroscopic analyses of these DCF derivatives revealed important correlations between their conformations, the PET, and fluorescent intensity in addition to insight on their solvatochromism and pH dependence. These studies allowed us to choose a candidate sensor for the next step: using combinatorial biology to isolate a reporter sequence. In order to isolate RNA that would enhance the fluorescence of our sensor, RNA that bound to the aniline-based quencher was isolated via in vitro selection (a.k.a SELEX or systematic evolution of ligands by exponential enrichment). These RNA sequences (aptamers) were then screened for their ability to switch on the fluorescence of the sensor. One of these aptamers was found to enhance the fluorescence intensity of the DCF-aniline conjugate in a concentration-dependent manner, leading to further study of that aptamer with mutation studies and additional in vitro selection experiments. To demonstrate the power and generality of this approach, a parallel in vitro selection was performed with aptamers from this selection having similar activities. These results show that one can develop fluorescence-inducing reporter RNA and morph it into remotely related sequences without prior structural insight into RNA-ligand binding through the rational design of fluorescent chemosensors and subsequent isolation of RNA from combinatorial libraries.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
| Title | Member | Email Address | Pitt Username | ORCID |
|---|
| Committee Chair | Koide, Kazunori | koide@pitt.edu | KOIDE | | | Committee Member | Day, Billy W | bday@pitt.edu | BDAY | | | Committee Member | Schafmeister, Chris | | | | | Committee Member | Weber, Stephen | sweber@pitt.edu | SWEBER | |
|
| Date: |
27 September 2007 |
| Date Type: |
Completion |
| Defense Date: |
9 August 2007 |
| Approval Date: |
27 September 2007 |
| Submission Date: |
6 August 2007 |
| Access Restriction: |
5 year -- Restrict access to University of Pittsburgh for a period of 5 years. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
fluorescence spectroscopy; fluorescent sensors; in vitro selection; RNA |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-08062007-141424/, etd-08062007-141424 |
| Date Deposited: |
10 Nov 2011 19:57 |
| Last Modified: |
15 Nov 2016 13:48 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/8931 |
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