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Bifacial Peptide Nucleic Acid (Bpna) As a Regulator of Nucleic Acid Function

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Bifacial Peptide Nucleic Acid (bPNA) as a Regulator of Nucleic Acid Function Dissertation Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Xin Xia, B.S. Graduate Program in Chemistry The Ohio State University 2015 Dissertation Committee: Dr. Dennis Bong, Advisor Dr. Karin Musier-Forsyth Dr. Kotaro Nakanishi Copyright by Xin Xia 2015 Abstract This dissertation contains research summaries regarding the characterizations and applications of a novel bifacial peptide nucleic acid (bPNA) triplex system. The main content is divided into two parts: part one (chapter 2) presents systematic studies on effective in vitro inhibition of transcription, reverse-transcription and exonuclease function via the formation of synthetic bPNA-nucleic acid triplex structures; part two (chapters 3 and 4) demonstrates that bPNA triplex hybrid functionally substitutes for native duplex structures that are crucial for proper functions of aptamers and ribozyme, further a two- way communication system describing RNA-templated oxidative coupling of bPNA fragments leads to the emergence of ribozyme cleavage is discussed in details. In part one, we speculate that the thermodynamically stable synthetic bPNA-nucleic acid triplex can be utilized to generate template distortions that are inhibitory to nucleic acid based enzymatic reactions. Three enzymatic systems were investigated: T7 RNA polymerase, Exonuclease T, and AMV reverse transcriptase. bPNA hybridization kinetics and inhibitory efficacies on each system will be discussed in detail in chapter 2. In part two, study on bPNA-nucleic acid triplex system was expanded to investigate the structure-function relation of nucleic acid. In chapter 3, three biologically active nucleic acids folds were selected: IgE DNA aptamer, spinach RNA aptamer and minimal type I hammerhead ribozyme. Replacement of a duplex stem with unstructured oligo-T/U strands, which are bPNA binding sites, imposed structure-function loss in all three nucleic ii acids folds. Functional rescue was observed upon bPNA-driven refolding of oligo-T/U strands into triplex hybrid system. Further, in chapter 4, we demonstrated a 2-way communication between the abiotic bPNA hybridization site and the native ribozyme cleavage site, where the ribozyme-templated bPNA ligation in turn restore the ribozyme self-cleavage activity. In summation, we will demonstrate to you that bPNA triplex stem structure is compatible with biological processes and presented to be competitive inhibitor for DNA/RNA specific enzymes; further, bPNA triplex stem is biologically similar to native stem structures, not only the bPNA hybridization but also the nucleic acid templated bPNA ligation can restore native nucleic acid activities, demonstrate readout and transformation of non-native macromolecules through an abiotic template interface in DNA/RNA template topologies that are not accessible via native base-pairing. iii To my parents and my fiancé iv Acknowledgement I hold great appreciation to Dr. Bong, as he is an intelligent, patient and responsible mentor to me. He took me to into his lab on my third year, and since then he has been constantly helping me to not only build up knowledge, but also confidence. His persistence in research originality is highly admirable, and it has inspired me and will keep on inspiring me far beyond the graduate school. I also want to express my appreciation to Dr. Karin Musier-Forsyth and Dr. Kotaro Nakanishi for serving on my graduation committee, providing guidance and constructive input. Thank Dr. Thomas Magliery for his generosity in sharing his lab equipment constantly throughout my graduate study. Also, thank Dr. Kurt Fredrick for a productive collaboration. Finally, I want to thank my father Ming Xia and mother Ningning Zhang for their endless support and unconditional love throughout these years; thank my fiancé Zhun Zhou for holding my hands during all the ups and downs, studying and researching together; last but not the least, thank to my two fluffy friends Pangpang and Niuniu for necessary distractions. v Vita 2001-2004 ..................................................................Jinling High School, Nanjing, China 2004-2008 .................................... B.S., China Pharmaceutical University, Nanjing, China 2008-2009 ......................................Q.A., Simcere Pharmaceutical Group, Nanjing, China 2009-2015 ................ Graduate Research/Teaching Assistant, The Ohio State University Publications during Ph.D. program at the Ohio State University Zhou, Z., Xia, X. and Bong, D.* (2015) “Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function.” J. Am. Chem. Soc., DOI: 10.1021/jacs.5b05481 Piao, X., Xia, X., Mao, J. and Bong, D.* (2015) “Peptide ligation and RNA cleavage via an abiotic interface.” J. Am. Chem. Soc., 137, 3751-3754. Xia, X., Piao, X., and Bong, D.* (2014) "Bifacial PNA as an allosteric switch for aptamer and ribozyme function." J. Am. Chem. Soc., 136, 7265-7268. Xia, X., Piao, X., Fredrick, K, and Bong, D., (2014) “Bifacial PNA Complexation Inhibits Enzymatic Access to DNA and RNA.” ChemBioChem. 15, 31-36. Piao, X., Xia, X., and Bong, D.* (2013) “Bifacial Peptide Nucleic Acid Directs Cooperative Folding and Assembly of Binary, Ternary, and Quaternary DNA Complexes.” Biochemistry, 52, 6313-6323. Saibal Bandyopadhyay, Xin Xia, Andre Matseiyeu, Georgeta Mihai, Sanjay Rajagopalan and Dennis Bong.* (2012) “Z-Group Ketone Chain Transfer Agents for RAFT Polymer Nanoparticle Modification via Hydrazone Conjugation.” Macromolecules, 45, 6766-6773. Field of Study Major field: Chemistry vi Table of Contents Abstract ........................................................................................................................... ii Acknowledgement ........................................................................................................... v Vita ................................................................................................................................. vi Table of Contents .......................................................................................................... vii List of Figures .............................................................................................................. xvii List of Tables ................................................................................................................ xxi CHAPTER 1 Nucleic Acids Structures and Targeting Strategies ................................................... 1 1.1 Native Nucleic Acids Structures ............................................................................ 2 1.1.1 Duplexes ........................................................................................................ 2 1.1.2 G-Quadruplex ................................................................................................ 4 1.1.3 RNA Folds ..................................................................................................... 6 1.2 Nucleic Acids Triplex ............................................................................................ 7 1.2.1 Structural Characters of Triplex ...................................................................... 7 1.3.2 Nucleobase Modifications .............................................................................10 1.3.3 Sugar Modifications ......................................................................................12 1.3.4 Backbone Modifications ................................................................................13 1.4 Peptide Nucleic Acid (PNA) as DNA Mimic ..........................................................14 1.4.1 PNA Structure and Recognition Mechanism .................................................14 vii 1.4.2 Antigene Therapeutics of PNA ......................................................................17 1.4.3 Antisense Regulation of PNA ........................................................................20 1.4.4 PNAs as Detection Probes and Biosensors ..................................................21 1.4.5 Chemical Modifications of PNA .....................................................................21 1.5 Bifacial Nucleobase Analogues ............................................................................23 1.5.1 Bifacial Nucleobase Displaying PNA for Triplex Formation ...........................24 1.5.2 Melamine Derivatives Recognize T-T/U-U Mismatch ....................................25 1.6 PNA With Native Peptide Backbone ....................................................................26 1.7 Melamine Displayed Bifacial Peptide Nucleic Acid (bPNA) ..................................27 1.8 References for Chapter 1 .....................................................................................31 CHAPTER 2 bPNA Triplex Inhibits Enzymatic Access to DNA and RNA ......................................46 2.1 Introduction ..........................................................................................................47 2.2 Results and Discussion ........................................................................................50 2.2.1 Triplexes Inhibit in vitro T7 RNA Transcription. .............................................52 2.2.2 Triplexes are Resistant Towards Exonuclease T Digestion ...........................56 2.2.3 Triplexes Inhibit AMV Reverse Transcription .................................................59
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