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Applications of Optical Control of Oligonucleotide and Protein Function

Hemphill, James B (2015) Applications of Optical Control of Oligonucleotide and Protein Function. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Hemphill, James Bjbh55@pitt.eduJBH55
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairDeiters, Alexanderdeiters@pitt.eduDEITERS
Committee MemberHorne, Sethhorne@pitt.eduHORNE
Committee MemberIslam, KabirulKai27@pitt.eduKAI27
Committee MemberTsang, Michaeltsang@pitt.eduTSANG
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


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