Hemphill, James B
(2015)
Applications of Optical Control of Oligonucleotide and Protein Function.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Optical regulation using light as an external trigger was applied to the control of biological processes with high spatio-temporal resolution. Photoremovable caging groups were site-specifically incorporated onto oligonucleotides and proteins to optically regulate their function in biological environments, typically for the photochemical control of gene expression. These caging group modifications enabled both OFF → ON and ON → OFF optochemical switches for important chemical biology tools. Oligonucleotides containing caging group modifications were synthesized to regulate nucleic acid function with light. Specifically, photocaged triplex-forming oligonucleotides were developed to optochemically control transcription in cell culture. Light-activated antagomirs were designed for the optical inhibition of miR-21 and miR-122 function in the regulation of endogenous microRNA activity. This technology was then applied to the study of miR-22 and miR-124 function in cortical neuron migration during cerebral corticogenesis. Splice-switching oligonucleotides were engineered to optically control mRNA splicing pathways in both human cells and zebrafish. The optical control of plasmid-based gene expression was demonstrated with a caged promoter, and applied to the photochemical activation of transcription in a live animal model. The caging of oligonucleotides was also applied to DNA computation in the production of optically controlled logic gates and amplification cycles, providing spatio-temporal control over hybridization cascades to add new functionality to DNA computation modules. These studies in DNA computation led to the development of novel biosensors for logic gate-based detection of specific micro RNA signatures in live cells. In addition, proteins were optically controlled through the site-specific installation of caging groups on amino acid side chains that are essential for protein function using unnatural amino acid mutagenesis in mammalian cells with an expanded genetic code. A caged lysine analogue was incorporated into T7 RNA polymerase to photochemically regulate transcription in the development of a light-activated synthetic gene network and light-triggered RNA interference. A light-activated Cas9 endonuclease was engineered through the installation of a caged lysine analogue to optically control CRISPR/Cas9 editing of both exogenous and endogenous genes. Lastly, a system for the incorporation of unnatural amino acids in zebrafish was studied in efforts to produce the first vertebrate species with an expanded genetic code.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
21 September 2015 |
| Date Type: |
Publication |
| Defense Date: |
29 July 2015 |
| Approval Date: |
21 September 2015 |
| Submission Date: |
9 July 2015 |
| Access Restriction: |
3 year -- Restrict access to University of Pittsburgh for a period of 3 years. |
| Number of Pages: |
302 |
| 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: |
optical
optochemical
photochemical
caged
oligonucleotide
protein |
| Date Deposited: |
21 Sep 2015 12:57 |
| Last Modified: |
21 Sep 2018 05:15 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/25604 |
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