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Thermodynamics and Kinetics of DNA Nanostructure Assembly Development of an Artificial Genetic System Capable of Darwinian Evolution by Hanyang Yu A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Approved April 2013 by the Graduate Supervisory Committee: John C. Chaput, Chair Julian J.-L. Chen Hao Yan ARIZONA STATE UNIVERSITY May 2013 ABSTRACT The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules simultaneously for a pre-defined chemical property, and enrich for molecules with the particular property. DNA and RNA sequences with versatile functions have been identified by in vitro selection experiments, but many basic questions remain to be answered about how these molecules achieve their functions. This dissertation first focuses on addressing a fundamental question regarding the molecular recognition properties of in vitro selected DNA sequences, namely whether negatively charged DNA sequences can be evolved to bind alkaline proteins with high specificity. We showed that DNA binders could be made, through carefully designed stringent in vitro selection, to discriminate different alkaline proteins. The focus of this dissertation is then shifted to in vitro evolution of an artificial genetic polymer called threose nucleic acid (TNA). TNA has been considered a potential RNA progenitor during early evolution of life on Earth. However, further experimental evidence to support TNA as a primordial genetic material is lacking. In this dissertation we demonstrated the capacity of TNA to form stable tertiary structure with specific ligand binding property, which suggests a possible role of TNA as a pre-RNA genetic polymer. Additionally, we discussed the challenges in in vitro evolution for TNA enzymes and developed the necessary methodology for future TNA enzyme evolution. i ACKNOWLEDGEMENTS First and foremost, I would like to thank my wife Zhe for her love, support and patience. Earning a Ph.D. degree in a foreign country would be an unimaginably difficult mission for me to accomplish if she was not by my side encouraging and spurring me on along the way. As a scientific researcher herself, she initiated numerous stimulating discussions with me and I benefited a lot from her sparkling ideas. I am especially indebted to her for her devoting more time to taking good care of our daughter. I am also thankful for my daughter Audrey, who brought innumerable little moments of fun and happiness to my life and also made me become a more independent and responsible person. I am grateful to my parents for their understanding and support. They provided everything I need to finish my study in China and encouraged me to study abroad even though knowing they would only be able to see me very infrequently thereafter. After Audrey was born, they traveled a long way from China to an unfamiliar country to see us and took over the chief responsibility of babysitting Audrey, so that I could focus more on my research. I owe them too much for their dedication and sacrifice. I want to express gratitude to my parents-in-law. They have been supporting Zhe and me in many ways. Their help of looking after Audrey was crucial to release our anxiety and stress as new parents. I want to acknowledge my mentor, Dr. John C. Chaput. He inspired me to explore a thrilling field of science full of challenges, and provided a privileged opportunity to work on exciting research projects. He taught me how to design experiments seamlessly, how to present results passionately and how to write articles accurately. Under his guidance, I was able to grow from a student to a qualified researcher armed with critical ii thinking and solid experimentation skills. He broadened my knowledge by encouraging me to attend seminars, conferences and to converse with world renowned scientists. I am also grateful to the members of my committee, Dr. Julian Chen and Dr. Hao Yan, for their time and advices. They challenged me to think more deeply about the significance of my projects and gave valuable suggestions on how to present research to a broad audience. I also want to thank all the former and present graduate students, postdoctoral fellows, and lab researchers who I have interacted with along the way. Their advice and support have been instrumental to my accomplishments. I want to especially thank my collaborators Dr. Noureddine Fahmi, Dr. Su Zhang, Bing Jiang and Matthew R. Dunn. Without their contributions, my research projects would not be as successful and complete as it is today. I would like to thank Department of Chemistry and Biochemistry at ASU for accepting me into the Biochemistry PhD program and offering the opportunity to meet excellent faculty, talented students, brilliant researchers and friendly staff. I would also like to thank the Biodesign Institute and the Center for Evolutionary Medicine and Informatics for creating a collaborative and inspirational working environment. I would like to thank the Graduate & Professional Student Association at ASU for sponsoring me to go to an international conference and to share my research findings with the scientific community. iii TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES .............................................................................................................x CHAPTER 1. IN VITRO EVOLUTION OF NATURAL AND ARTIFICIAL NUCLEIC ACIDS ....................................................................................................................1 1.1. Abstract .........................................................................................................1 1.2. In Vitro Selection of Natural Nucleic Acids .................................................2 1.2.1 In Vitro Selection of Nucleic Acid Aptamers ...................................3 1.2.2 In Vitro Selection of Nucleic Acid Enzymes ....................................6 1.3. In Vitro Selection of Modified Nucleic Acids ..............................................8 1.4. Threose Nucleic Acid ..................................................................................10 1.4.1 Initial Synthesis of TNA ..................................................................10 1.4.1.1 -D-Hexo-pyranosyl Oligonucleotide System ......................12 1.4.1.2 Pento-pyranosyl Oligonucleotide System .............................13 1.4.1.3 -L-Tetro-furanosyl Oligonucleotide System .......................14 1.4.2 Synthesis of TNA Phosphoramidites and Triphosphates ................18 1.4.3 Structure of TNA Duplex ................................................................20 1.4.4 Towards Evolution of TNA Polymers .............................................26 1.5. Projects ........................................................................................................33 1.6. References ...................................................................................................34 iv CHAPTER Page 2. APTAMERS CAN DISCRIMINATE ALKALINE PROTEINS WITH HIGH SPECIFICITY ......................................................................................................37 2.1. Abstract .......................................................................................................37 2.2. Introduction .................................................................................................38 2.3. Results and Discussion ................................................................................41 2.3.1. Selection for Single-Stranded DNAs that Bind to Histone H4 .......41 2.3.2. Affinity and Specificity of the DNA Aptamers ...............................45 2.3.3. Salt Effects ......................................................................................47 2.3.4. Directed Evolution ..........................................................................48 2.3.5. Studies of Site-Directed Mutations .................................................50 2.3.6. Implications of Aptamer Selection ..................................................55 2.4. Conclusion ...................................................................................................58 2.5. Experimental Design ...................................................................................59 2.5.1. General ............................................................................................59 2.5.2. In Vitro Selection ............................................................................59 2.5.3. Capillary Electrophoresis ................................................................60 2.5.4. Directed Evolution ..........................................................................60 2.5.5. DNA Sequencing and Analysis .......................................................61 2.5.6. Dot Blot Binding Assay ..................................................................62 2.5.7. Structural Probing by Hydroxyl Radical Footprinting ....................63 2.6. References ...................................................................................................63 v CHAPTER Page 3. DARWINIAN EVOLUTION OF AN ALTERNATIVE GENETIC SYSTEM PROVIDES SUPPORT FOR TNA AS AN RNA PROGENITOR .....................67 3.1. Abstract .......................................................................................................67
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