DEOXYRIBOZYMES TO HYDROLYZE DNA PHOSPHODIESTER BONDS BY YING XIAO DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2014 Urbana, Illinois Doctoral Committee: Professor Scott K. Silverman, Chair Associate Professor Ryan C. Bailey Professor Paul J. Hergenrother Professor Satish K. Nair Abstract In nature, the main role of double-stranded DNA is storage of genetic information. However, once a DNA oligonucleotide is freed from its confining complementary strand, it can fold into complex three-dimensional structures that support catalytic activities. These catalytic single-stranded DNA oligonucleotides are called deoxyribozymes. Up to now, there are no known deoxyribozymes in nature, and the only method to identify them is in vitro selection. Since the identification of the first deoxyribozyme in 1994, many deoxyribozymes have been found to catalyze various chemical reactions, of which the substrates can be either oligonucleotides or small molecules. Our lab has recently reported the first DNA-hydrolyzing deoxyribozyme, 10MD5, which catalyzes the sequence-specific hydrolysis of a DNA phosphodiester bond. The 1 12 DNA hydrolysis by 10MD5 proceeds with kobs = 2.7 h and rate enhancement of 10 over the uncatalyzed P–O bond hydrolysis. However, 10MD5 has a sharp pH optimum near 7.5, with greatly reduced yield and rate when the pH is changed only by 0.1 units in either direction. Therefore, 10MD5 was optimized via reselection, leading to variants with broader pH tolerance. The reselection experiments and the follow-up characterization were described in Chapter 2. An artificial phylogeny constructed with sequences of the reselected variants suggested three mutations, T16R, G19Y and C30T, are strongly correlated with the broader pH tolerance. The reselected variants with broader pH tolerance were also found to suffer from relaxed site specificity, which could only be restored by expanding the “recognition site” beyond ATG^T (as in the parent 10MD5) to TATG^TT. These findings reflected functional compromises of the initial family of DNA-hydrolyzing deoxyribozymes. Additionally, a reselected variant, 9NL27, ii showed unique Zn2+-only hydrolysis activity, even though it differed from 10MD5 by only five out of 40 nucleotides. Evaluation of the five mutations in 9NL27 showed that only two nucleotide mutations in the 10MD5 sequence, T16A and G19C, were sufficient to convert the heterobimetallic 10MD5 deoxyribozyme into a monometallic deoxyribozyme that required Zn2+ alone. Systematic selection experiments were performed to establish broad generality of ssDNA-hydrolyzing deoxyribozymes and to identify a complete set of deoxyribozymes that can collectively cleave any arbitrarily chosen single-stranded DNA substrate at any predetermined site. These selection experiments were described in Chapter 3. Comprehensive selection experiments were performed, including in some cases a key selection pressure to cleave the substrate at a predetermined site. These efforts led to identification of numerous new DNA-hydrolyzing deoxyribozymes, many of which require merely two particular nucleotides at the cleavage site (e.g. X^G, X= A, T, C, or G) while retaining WatsonCrick sequence generality beyond those nucleotides along with useful cleavage rates. These results established experimentally that broadly sequence-tolerant and site-specific deoxyribozymes were readily identified for hydrolysis of single-stranded DNA. However, further selection experiments did not lead to identification of many new sequence-general deoxyribozymes due to various problems. The efforts toward the complete set of ssDNA-hydrolyzing deoxyribozymes were stopped because of the limited application of ssDNA-hydrolyzing deoxyribozymes alone. Finally, current efforts to achieve double-stranded DNA hydrolysis were described in Chapter 4. A targeted selection approach was designed to identify dsDNA-hydrolyzing deoxyribozymes, in which the deoxyribozyme pool would be conjugated to a dsDNA- iii binding protein, dHax3-TALE. The protein would recognize and bind to a specific region in the double-stranded DNA substrate, while the deoxyribozyme would catalyze the site- specific hydrolysis of the substrate. The ligation and capture steps were validated, and the selection conditions were determined. However, the key challenge of the selection approach was the requirement of a site-specific and efficient bio-conjugation method to link the deoxyribozyme pool with the TALE protein. Four conjugation strategies were designed, based on disulfide exchange reaction, FBDP-tyrosine reaction, PTAD-tyrosine reaction, or N-terminal threonine oxidation. After comprehensive assays, the strategies based on PTAD-tyrosine reaction and N-terminal threonine oxidation showed high site- specificity and promising reaction yields. iv To My Parents v Acknowledgments It is my pleasure to express my gratitude to people who have been helping me throughout my Ph.D. studies. First of all, I would like to thank my research advisor, Prof. Scott K. Silverman for his constant support and guidance. He is a great mentor who teaches me how to become a good researcher. I am really grateful for the opportunity he gave me to work in the lab and the trust he has in me to pursue my own research ideas. It has been a really great experience interacting with Scott, both scientifically and personally. Additionally, I am grateful to the past members of the Silverman group. I would like to thank Dr. Madhav Chandra for training me in the lab, answering all my questions, and helping me out when I faced difficulties. I also thank Amit and Adrienne for their advice and help about my Ph.D. courses, assignments, and research projects. I would like to thank all current members of Silverman group, Victor, Ben, Jagdeesh, Spurti, Jimmy, Shannon, Puzhou, Cong, and Peter for their friendship, support and helpful discussions. Especially, I thank Victor for his help on my job applications, Ben for all the arguments that lead to better understanding of our research and great scientific ideas, Jagdeesh for all the help to synthesize FBDP reagent and all the discussions about processing proteins, and Jimmy and Shannon for dragging me to the gym to keep me fit. I am also grateful to the talented undergraduate researchers I have worked with, especially Emily, Nora, Rebecca, Jay, Alex, Darren, and Tania. It has been great fun to work with them, and I am very proud of them! Moreover, I am very grateful to Nancy from van der Donk group for teaching me everything about protein, answering all my silly questions, and just being a great friend. I vi am also very grateful to Kyle from Mitchell group for teaching me about fluorescence anisotropy and help me to use their microplate reader. Furthermore, I am very thankful to my thesis committee members, Prof. Paul J. Hergenrother, Prof. Ryan C. Bailey, and Prof. Satish K. Nair for their suggestions during the committee meetings, as well as advice and support for my current and future career. Last but not least, I would like to thank my parents for their support, motivation and unconditional love throughout these years. I would also thank all my dear friends for years of friendship, encouragement and support. vii Table of Contents List of figures .......................................................................................................................... xi List of tables ........................................................................................................................ xviii Chapter 1: Introduction to Enzymes—Natural and Artificial ........................................... 1 1.1 Natural Enzymes .......................................................................................................... 1 1.1.1 Protein Enzymes.................................................................................................. 2 1.1.1.1 Structural Components of Protein ............................................................. 2 1.1.1.2 Structure, Function, and Mechanism of Protein Enzymes ....................... 5 1.1.1.3 Applications of Protein Enzymes ............................................................ 10 1.1.2 Ribozymes ......................................................................................................... 10 1.1.2.1 Ribozymes and Their Biological Functions ............................................ 10 1.1.2.2 Structural Components of Ribozymes .................................................... 11 1.1.2.3 Ribozyme Catalyzed Transesterification Reaction ................................. 13 1.1.2.4 Ribozyme Catalyzed Hydrolysis Reaction ............................................. 17 1.1.2.5 Ribozyme Catalyzed Peptidyl Transfer Reaction ................................... 20 1.2 Artificial Enzymes ..................................................................................................... 22 1.2.1 Engineered/Designed Protein Enzymes .......................................................... 23 1.2.1.1 Enzyme Engineering via Directed Evolution ......................................... 23 1.2.1.2 In Vitro Selection from a Protein Library ............................................... 27 1.2.1.3 Designing Protein Enzymes .................................................................... 30 1.2.2 Artificial Nucleic Acid Enzymes ...................................................................... 32 1.2.2.1