ABSTRACT GEORGIANNA, WESLEIGH FOWLER EDWARDS. Development of Novel Methodologies for the Photoregulation of Biological Processes. (Under the direction of Dr. Alexander Deiters.) The flow of genetic information can be affected at various stages, ultimately leading to the up- or down-regulation of gene expression. Essential to elucidating gene function is first gaining precise external control over biological processes. Many techniques currently employed to investigate gene function may interfere with normal downstream processes or harm the organism of study, and their applications often lack specificity. Photochemical biology provides an alternative approach, in which a light-removable protecting group (termed “caging group”) installed on a protein or nucleic acid inhibits its nascent function; non-damaging light irradiation removes the caging group and restores activity. Additionally, light-responsive moieties such as diazobenzenes can be harnessed as reversible photoregulated groups for controlling biological macromolecules. Since light-irradiation can be precisely regulated, decaging can be achieved with a high level of spatial and temporal control. The research described herein reports the development of novel tools to achieve spatio-temporal control of biological processes through the photoregulation of proteins and nucleic acids, both in vitro and in vivo. Specifically, our approaches involve (1) modulating enzymatic activity by directly installing a caging group on the protein of interest, (2) utilizing photocaged nucleotides to regulate the polymerase chain reaction, (3) investigating the effects of microwave irradiation on nucleic acids, and (4) controlling protein dimerization through the use of a photoregulated chemical inducer of dimerization (CID). Development of Novel Methodologies for the Photoregulation of Biological Processes by Wesleigh Fowler Edwards Georgianna A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Chemistry Raleigh, North Carolina 2010 APPROVED BY: Alexander Deiters, PhD Jonathan Lindsey, PhD Committee Chair David Muddiman, PhD Lin He, PhD Ralph Dean, PhD DEDICATION This work is dedicated to My Mom and Dad, for their unconditional love and guidance, Steve Buchau, an exemplary teacher and scientist who put me on the right path, Asha Balakrishnan, for sharing piano lessons, tennis balls, and this amazing lifelong friendship, The wonderful friends and fellow graduate students I’ve met along the way, And to my husband, Ryan Georgianna, for his friendship, understanding and love, and for reminding me of what matters most in life. ii BIOGRAPHY The author, Wesleigh F. Edwards Georgianna, was born December 23, 1982 to parents Bill and Lisa Edwards in Virginia Beach, VA. She grew up at the beach and enjoyed playing tennis and volunteering at Sentara Leigh Hospital in the Emergency and Surgical Recovery Rooms during the summers. After graduating from Norfolk Academy in 2001, she attended Virginia Polytechnic Institute and State University, and worked in the lab of Dr. Timothy Long to investigate the use of poly(lactide)s as vascular sutures. Additionally, she was an Associate Justice with the Virginia Tech Undergraduate Honor System, and earned her Emergency Medical Technician license. She graduated from Virginia Tech in 2005, receiving a B.S. in Biochemistry with a concentration in Biotechnology. Wesleigh then entered the Chemistry graduate program at North Carolina State University under the direction of Dr. Alexander Deiters, where she undertook five years of doctoral research in biological chemistry. Wesleigh has accepted a position as a project leader at the Duke Translational Research Institute, where she works with other researchers to transition basic medical science discoveries into important clinical therapeutics. iii ACKNOWLEDGEMENTS I express my sincerest appreciation to everyone who has made this work possible. I would particularly like to acknowledge my advisor, Prof. Dr. Alex Deiters, for his research supervision and financial support during my graduate studies. I would also like to thank the former and current members of the Deiters Lab, especially Andrew, Yan, Jeane, Colleen, Jie, Hank, Jesse, and Harry for their humor and advice. Thank you also to my advisory committee for their assistance in the completion of my doctoral research. Additionally, thank you to those who have particularly contributed to my graduate studies through their guidance and indispensible fellowship. Doug, there are not enough words to tell you how grateful I am for your help over the last five years, and more importantly, your friendship. Virginia, thank you for your research counsel and for always being there to listen, both in graduate school and beyond. Thank you also to Elke, Rania, and Jennifer, with whom I served on PLU and whose camaraderie I very much enjoyed. I would also like to thank my Mom and Dad for the love and support I have received from them; I am blessed to have parents who wholeheartedly encouraged me from the beginning. Lastly, I would especially like to thank my husband, Ryan, who has kept me motivated these last five years, and has been there for me with research advice, understanding, laughter and companionship, and love. iv TABLE OF CONTENTS LIST OF TABLES………………………………………………………………………………. viii LIST OF FIGURES……………………………………………………………………………..... ix CHAPTER 1: INTRODUCTION TO PHOTOCHEMICAL BIOLOGY………………………… 1 CHAPTER 2: PHOTOCHEMICAL REGULATION OF THE POLYMERASE CHAIN REACTION……………………………………………………………………………………… 22 2.1 Introduction to the polymerase chain reaction………………………………………… 22 2.2 Hybridization studies with photocaged DNA…………………………………………. 23 2.3 Light-activation of PCR……………………………………………………………….. 26 2.4 Light-deactivation of PCR…………………………………………………………….. 29 2.5 Concurrent up- and down-regulation of PCR…………………………………………. 32 2.6 Summary and outlook…………………………………………………………………. 32 2.7 Experimental methods…………………………….……………………………………34 CHAPTER 3: THE EFFECT OF MICROWAVE IRRADIATION ON OLIGONUCLEOTIDE HYBRIDIZATION………………………………………………………………………………. 36 3.1 Introduction to DNA hybridization and microwave irradiation………………………. 36 3.2 Development of a fluorescence hybridization assay…………………………………... 37 3.3 Microwave irradiation of DNA……………………………………………………….. 40 3.4 Summary and outlook…………………………………………………………………. 48 3.5 Experimental methods………………………………………………………………….49 v CHAPTER 4: LIGHT-REGULATION OF PROTEIN FUNCTION THROUGH PHOTOCAGING………………………………………………………………………………… 52 4.1 Lysozyme………………………………………………………………………………. 52 4.1.1 Introduction to lysozyme……………………………………………………………... 52 4.1.2 PhotoPEGylation of lysozyme………………………………………………………... 54 4.1.3 In vitro activity assays of lysozyme function………………………………………… 58 4.1.4 Summary and outlook………………………………………………………………… 61 4.1.5 Experimental methods………………………………………………………………... 62 4.2 Cre Recombinase……………………………………………………………………... 64 4.2.1 Introduction to DNA recombination and Cre recombinase…………………………... 64 4.2.2 Expression and purification of wild type Cre………………………………………… 71 4.2.3 In vitro non-specific caging of lysine residues……………………………………...... 74 4.2.4 Unnatural amino acid mutagenesis …………………………………………………... 76 4.2.5 Expression and purification of CreONBY……………………………………………. 80 4.2.6 In vitro light-activation……………………………………………………………...... 82 4.2.7 In vivo light-activation to demonstrate spatiotemporal control of gene function…….. 83 4.2.8 Expression of TAT-modified Cre…………………………………………………...... 88 4.2.9 Summary and outlook………………………………………………………………… 90 4.2.10 Experimental methods………………………………………………………………... 91 4.3 Photochemical control over FimE-mediated DNA recombination in E. coli………… 99 4.3.1 Introduction to FimE………………………………………………………………..... 99 4.3.2 FimE expression and in vivo assays………………………………………………… 104 4.3.3 Summary and outlook……………………………………………………………….. 105 vi 4.3.4 Experimental methods………………………………………………………………. 106 4.4 Photochemical control over Flpe-mediated DNA recombination in E. coli………… 108 4.4.1 Introduction to Flpe recombinase………………………………………………….... 108 4.4.2 Expression of wild type Flpe………………………………………………………... 110 4.4.3 The Flpe bacterial assay……………………………………………………………... 110 4.4.4 Summary and outlook……………………………………………………………….. 113 4.4.5 Experimental methods………………………………………………………………. 114 CHAPTER 5: LIGHT-REGULATION OF PROTEIN DIMERIZATION…………………….. 119 5.1 Introduction to natural and synthetic protein dimerizers……………………………… 119 5.2 Expression and purification of DHFR and In Vitro Dimerization Assays……………. 128 5.3 Expression and purification of GFP-DHFR and In Vitro Fluorescent Dimerization Assays…………………………………………………………………………………. 132 5.4 In Vivo Dimerization Assays………………………………………………………….. 138 5.5 Summary and outlook…………………………………………………………………. 139 5.6 Experimental methods……………………………………………………………….... 140 CHAPTER 6: THE HALO-TAG AS A PROTEIN-BASED CAGING GROUP……………… 148 6.1 Introduction to the HaloTag protein…………………………………………………... 148 6.2 Expression and purification of HaloTag………………………………………………. 150 6.3 Summary and outlook…………………………………………………………………. 152 6.4 Experimental methods………………………………………………………………… 152 APPENDIX 1: RECIPES………………………………………………………………………. 155 REFERENCES…………………………………………………………………………………. 157 vii LIST OF TABLES Table 2.1. Melting temperatures of non-caged (P1) and caged (P2-P7) oligonucleotides before and after irradiation……………………...……………………………………… 25 Table 3.1. Sequences and melting temperature of DNA oligomers……………………. 38 Table 4.2. Percentage of red