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I Bifacial PNA in Nucleic Acid Folding, Peptide Ligation and in Vitro

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I Bifacial PNA in Nucleic Acid Folding, Peptide Ligation and in Vitro Bifacial PNA in Nucleic Acid Folding, Peptide Ligation and in vitro Selection Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Xijun Piao, B.S. Graduate Program in Chemistry The Ohio State University 2016 Dissertation Committee: Dr. Dennis Bong, Advisor Dr. Jovica Badjic Dr. Jonathan R. Parquette i Copyright by Xijun Piao 2016 ii Abstract This dissertation summarizes three research projects regarding the fundamental studies and applications of bifacial Peptide Nucleic Acid (bPNA) – nucleic acid hybrid triplex system. Chapter 2 describes the syntheses of a series of bPNAs with different length and the cooperative folding of thymine-rich deoxyoligonucleotides by bPNA into binary, ternary and quaternary complexes. Chapter 3 demonstrates that template effect exists in abiotic bPNA – nucleic acid triplex system in that nucleic acid templates acyl group transfer, bPNA ligation and extension. Chapter 4 presents the application of bPNA – nucleic acid triplex as a replacement of native duplex in constructing a reversibly-structured RNA library for in vitro selection against stapled highly helical HIV-1 Rev peptides and also explores the concept of target-directed evolution of RNA library. In Chapter 2, we demonstrate melamine-displaying bPNA recognizes thymine-rich DNA in predictable and multifaceted ways that allow binding affinity, structure stability and stoichiometry to be tuned through simple bPNA length modification and matching with DNA length. The longer recognition interface results in the increase of melting temperature and enthalpy change for dissociation, as well as decrease of dissociation constant. 10mer bPNA can tolerate a high number of “mismatches” on thymine tracts and still yields triplex formation whose melting temperature correlates directly with thymine content. Interestingly, when a DNA host has more T − T sites than melamine sites on bPNA, two or three bPNAs can bind to a single DNA, resulting in ternary and quaternary complexes that have higher thermal stability than the binary (1:1) bPNA − DNA complex, suggestive of cooperative multisite binding. In contrast, when two bPNAs of different lengths bind to ii the same DNA host, a ternary complex is formed with two melting transitions, corresponding to independent melting of each bPNA component from the complex. This set of data serve as the foundation for our future research. In Chapter 3, we demonstrate DNA- and RNA-templated chemical transformation of bifacial peptide nucleic acid (bPNA) fragments directed by an abiotic thymine/uracil – melamine – thymine/uracil triplex hybrid interface. Watson-Crick base-pairing is widely studied in nucleic acid-templated chemistry, however very few reports are about non- Watson-Crick recognition. Triplex hybridization of reactive bPNA fragments with DNA template is shown to catalyze acryl group transfer, bPNA native chemical ligation and length-controlled bPNA extension. Gratifyingly, RNA-templated oxidative coupling of bPNA fragments is found to result in the emergence of engineered ribozyme cleavage. These data demonstrate that nucleic acid template effect exists in abiotic melamine- thymine/uracil bifacial recognition and establish a connection between engineered and native reaction sites. In Chapter 4, we report the use of bPNA – nucleic acid triplex hybridization to construct a reversibly-structured RNA library for in vitro selection against side chain-stapled HIV-1 Rev peptides. Bifacial PNA – nucleic acid triplex stem structurally replaces native duplex stem evolved from random region and forces the library into a stem-loop structure, leaving the precious random nucleotides for the evolution of binding motifs instead of non-binding duplex stem. This bPNA-assisted reversibly-structured RNA library also facilitates the problematic reverse transcription of selected RNA aptamers by loosening the extensive secondary structures. Although many peptide targets have been used for in vitro selection, a stapled peptide is not seen and known to enhance helicity and binding affinity. In vitro selection against stapled original Rev peptide yields RNA aptamers showing the conserved core element presented in native Rev Responsive Element (RRE) RNA. A key iii asparagine on the stapled Rev peptide is mutated to two synthetic triazine-displaying amino acids that direct the evolution of random oligonucleotides based on likely hydrogen- bonding preference. This ongoing project integrates engineered bPNA – nucleic acid triplex stem into in vitro selection and may solve the problem of reverse transcription of in vitro selection. Further single mutations on stapled peptide targets demonstrate the concept of directed evolution of RNA library. iv Dedication Dedicated to my wife and parents v Acknowledgements I am very grateful to my PhD advisor, Dr. Dennis Bong. We have worked together for six years and he not only helped me enrich my knowledge in organic chemistry and biophysics, but also made me become a more confident and competent researcher. His patience, persistence and trust led us to complete our very challenging in vitro selection project, in which I learned nucleic acid chemistry from the very beginning and improved my problem- solving ability. His goal for research originality is highly admirable and everything I learned in his lab would become a great fortune for my future research. I would also like to thank Dr. Badjic and Dr. Parquette for serving my candidacy and defense committee, as well as writing reference letters in support of my postdoctoral researcher application. I also highly appreciate the helpful advice and discussions provided by friendly labmates, as well as productive collaborations. Finally, I am very grateful and indebted to my family. My parents, Mingtao Piao and Huiqing Gu, have been giving me unconditional love and tremendous support all the time throughout the years I was far away from home. My wife Ying Yu and my parents-in-law have been giving me endless love and indispensable trust since my marriage and I believe their love, understanding and support will make me become a better, more responsible and supportive husband to my family. vi Vita 2003 – 2006……………………………………. Anshan No.1 Middle School, Anshan, China 2006 – 2010………………………………………… B.S., Fudan University, Shanghai, China 2010 – 2016......……Graduate Teaching / Research Assistant, The Ohio State University Publications during PhD (1) Xijun Piao, Xin Xia, Jie Mao, and Dennis Bong*, “Peptide Ligation and RNA Cleavage via an Abiotic Template Interface” J. Am. Chem. Soc. 2015, 137, 3751-3754. (2) Xijun Piao, Xin Xia, and Dennis Bong*, “Bifacial Peptide Nucleic Acid Directs Cooperative Folding and Assembly of Binary, Ternary, and Quaternary DNA Complexes” Biochemistry, 2013, 52, 6313-6323. (3) Xin Xia, Xijun Piao, and Dennis Bong*, “Bifacial Peptide Nucleic Acid as an Allosteric Switch for Aptamer and Ribozyme Function” J. Am. Chem. Soc. 2014, 136, 7265-7268. (4) Xin Xia, Xijun Piao, Kurt Fredrick, and Dennis Bong*, “Bifacial PNA Complexation Inhibits Enzymatic Access to DNA and RNA” ChemBioChem, 2014, 15, 31-36. (5) Yingying Zeng, Yaowalak Pratumyot, Xijun Piao and Dennis Bong*, "Discrete assembly of synthetic peptide-DNA triplex structures from polyvalent melamine - thymine bifacial recognition" J. Am. Chem. Soc. 2012, 134, 832-835. Fields of Study Major field: Chemistry vii Table of Contents Abstract…………………………………………………………………………………………....ii Acknowledgement…………………………………………………………………………….…vi Vita………………………………………………………………………………………………..vii Table of Contents………………………………………………………………………….…….vii List of Figures…………………………………………………………………………………...xiv List of Tables………………………………………………………………………………......xviii Chapter 1. Structures, Recognition and Template Effect of Nucleic Acids ....................... 1 1.1 Nucleic acid structures ............................................................................................ 2 1.1.1 DNA duplex ....................................................................................................... 3 1.1.2 DNA triplex ........................................................................................................ 4 1.1.3 Nucleic acid G-quadruplex ................................................................................ 6 1.1.4 RNA folding ....................................................................................................... 7 1.2 Modifications on nucleic acid to enhance triplex formation...................................... 8 1.3 Peptide Nucleic Acid (PNA) – Native nucleobases on pseudopeptide backbone . 10 1.3.1 Chemical structure of PNA .............................................................................. 10 1.3.2 PNA-native nucleic acid duplex and triplex ..................................................... 11 1.3.3 Biological and medicinal applications of PNA ................................................. 12 1.4 Targeting nucleobase pairs with Janus-Wedge insertion strategy ........................ 12 1.4.1 Janus-Wedge Insertion of a single nucleobase pair by small molecules ........ 13 1.4.2 Single Janus-Wedge insertion on biopolymer scaffold ................................... 14 viii 1.4.3 Multivalent insertion of Janus-Wedge heterocycles into nucleobase pairs ..... 14 1.4.4 Triazine used to target nucleobase or nucleobase pairs and bifacial Peptide Nucleic Acid (bPNA) ...............................................................................................
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