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Applications of Optical Control of Oligonucleotide and Protein Function Applications of Optical Control of Oligonucleotide and Protein Function by James Bride Hemphill B.S. Molecular Genetics, University of Vermont, 2007 Submitted to the Graduate Faculty of the Dietrich School of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2015 UNIVERSITY OF PITTSBURGH Dietrich School of Arts and Sciences This dissertation was presented by James Bride Hemphill It was defended on July 29th 2015 and approved by W. Seth Horne, Assistant Professor, Department of Chemistry Kabirul Islam, Assistant Professor, Department of Chemistry Michael Tsang, Associate Professor, Department of Developmental Biology Dissertation Advisor: Alexander Deiters, Professor, Department of Chemistry ii Copyright © by James Bride Hemphill 2015 iii Applications of Optical Control of Oligonucleotide and Protein Function James Bride Hemphill, PhD University of Pittsburgh, 2015 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 iv 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. v Table of Contents 1.0 Introduction to Caged Oligonucleotides and Proteins .............................................. 1 1.1 Optical Control of Oligonucleotide Function ..................................................... 4 1.1.1 Synthesis of Modified Oligonucleotides. ...................................................... 18 1.2 Optical Control of Protein Function ................................................................. 22 2.0 Application of Caged Oligonucleotides in the Regulation of Gene Function ....... 41 2.1 Optical Regulation of Transcription through Caged Triplex-Forming Oligonucleotides .................................................................................................. 41 2.2 Optical Control of MicroRNA Function using Caged Antagomirs................ 45 2.3 Optical Control of microRNAs Controlling Polarization of Migrating Cortical Neurons ................................................................................................. 50 2.4 Optical Control of Gene Expression through Site-Specific Promoter Caging ..........................................................................................................................54 2.4.1 Methods and Materials ................................................................................. 72 2.5 Optical Control of Alternative Splicing through Caged Splice-Switching Oligonucleotides .................................................................................................. 77 2.5.1 Methods and Materials ................................................................................. 98 3.0 DNA Computation: Photochemical Regulation and Application in Live Cells.. 101 3.1 DNA Computation: A Photochemically Controlled AND Gate ................... 112 vi 3.2 DNA Computation in Mammalian Cells: AND Gates ................................... 119 3.2.1 Methods and Materials ............................................................................... 137 3.3 DNA Computation in Mammalian Cells: OR Gate, OR/AND Circuit, and Release of a Splice-Switching Oligonucleotide ............................................... 140 3.3.1 Methods and Materials ............................................................................... 156 3.4 Optically Controlled Signal Amplification for DNA Computation .............. 159 3.5 DNA Computation in Mammalian Cells: Signal Amplification of miRNA. 170 3.5.1 Methods and Materials ............................................................................... 177 4.0 Application of Caged Proteins in the Regulation of Gene Function ................... 179 4.1 Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells ........ 179 4.1.1 Methods and Materials ............................................................................... 193 4.2 Optical Control of CRISPR/Cas9 Gene Editing ............................................ 198 4.2.1 Methods and Materials ............................................................................... 211 4.3 Expression of Caged Proteins in Live Zebrafish ............................................ 218 4.3.1 Methods and Materials ............................................................................... 226 5.0 Expanded Methods and Materials .......................................................................... 227 5.1 Sequences of Synthesized Oligonucleotides .................................................... 227 5.2 Oligonucleotide Synthesis ................................................................................. 233 5.3 General Molecular Biology Techniques .......................................................... 237 5.3.1 Polymerase Chain Reaction (PCR) ............................................................ 237 5.3.2 Site-Directed Mutagenesis (SDM) .............................................................. 238 5.3.3 Restriction Enzyme Digestion..................................................................... 239 vii 5.3.4 Plasmid Ligation. ......................................................................................... 239 5.3.5 Plasmid E. coli Transformation ................................................................. 240 5.3.6 Plasmid Purification .................................................................................... 242 5.3.7 Plasmid Analysis .......................................................................................... 242 5.4 General Cell Culture Techniques .................................................................... 243 5.4.1 Cell Growth and Maintenance ................................................................... 243 5.4.2 Cell Transfection.......................................................................................... 244 5.4.3 Cell Irradiations ........................................................................................... 246 5.4.4 Cell Fixing .................................................................................................... 246 5.4.5 Cell Imaging ................................................................................................. 247 5.4.6 RNA Isolation ............................................................................................... 248 5.4.7 RNA Analysis by Quantitative Real-Time PCR (qRT-PCR) .................. 248 5.4.8 Protein Isolation from Mammalian Cells .................................................. 249 5.4.9 Protein Analysis ........................................................................................... 250 5.5 General Zebrafish Techniques......................................................................... 250 5.5.1 Zebrafish Mating ......................................................................................... 250 5.5.2 Zebrafish Injection ...................................................................................... 251 5.5.3 Zebrafish Imaging ......................................................................................
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