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RNA Kissing-Loop Interactions Supported by Bifacial Peptide Nucleic Acid

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RNA kissing-loop interactions supported by bifacial peptide nucleic acid DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Christopher Amedeo DeSantis Graduate Program in Chemistry The Ohio State University 2017 Dissertation Committee: Professor Dennis Bong, Advisor Professor Venkat Gopalan Professor Jovica Badjic Copyrighted by Christopher Amedeo DeSantis 2017 Abstract The dissertation to follow is an account of research efforts to utilize bifacial peptide nucleic acids (bPNA) triplexes to support kissing-loop complexes. A kissing-loop complex is composed of two nucleic acid stem-loops hybridized via Watson-Crick base pairing between their loop regions. For our studies, we employed two kissing-loop complexes in which a bPNA binding site, composed of ten sequential U-U mismatches, was inserted into the duplex stem of the interacting hairpins. The first system was a tRNA in which the anticodon stem was mutated with a 10-mer bPNA binding site and the loop region extended to nine nucleotides to facilitate a six-nucleotide loop-loop duplex during heterodimerization with a complementary tRNA mutant. The second system, based on the ColE1 plasmid replication control kissing-loop complex, involves two small hairpins which, after kissing complex formation, bind the protein Rop. The hairpins (RNAI-1 and RNAII-1) were mutated with a 10-mer bPNA binding site and have complementary seven- nucleotide loops. In the first part if this work, kissing-loop and Rop complex formation was analyzed in titration binding assays and native gel electrophoresis. tRNA duplex-triplex kissing complexes show a 5-fold decrease in affinity while omission of bPNA decreases affinity by a further 3-fold. Triplex-triplex tRNA mutant binding is 9-fold weaker compared to duplex kissing-loops and also show greater instability when bPNA is omitted. Triplex- duplex mutants in the ColE1 system show similar affinity to the wild type system with Rop ii present. Triplex-triplex kissing-loops bind 4-fold tighter than the wildtype system without the aid of Rop. Furthermore, Rop is able to bind to duplex-triplex and triplex-triplex kissing-loops; the first time a bPNA triplex has been installed at a protein-nucleic acid binding site without abolishing binding. In the latter part of this work, fluorescently-labeled bPNA were used to monitor kissing-loop formation in vitro. Triplex-triplex kissing-loop formation was monitored by FRET using Cy3- and Cy5-labeled bPNA triplexes. Competitive displacement of the U10 mutant tRNA by duplex tRNA in the triplex-triplex kissing-loop was observed via a decreased FRET signal. In the ColE1 system, Rop-catalyzed competitive displacement of a wild type hairpin by a U10 mutant in a WT-U10 kissing complex was monitored via increased FRET signal. In a separate study, fluorescence turn-on probes to detect the presence of a hairpin were developed. The fluorogen thiazole orange was conjugated to bPNA by several designed linkers and a series of synthetic RNA probes with a nine- nucleotide loop were tested for maximum fluorescence turn-on. A peptide with a 2-(2-(2- aminoethoxy)ethoxy)acetic acid linker combined with a probe in which the 1, 2 and 9 positions of the loop was mutated with propyl linkers provided 2.3-fold turn-on effect. In summary, we have shown that hairpins mutated with a bPNA triplex in the stem region are able to form kissing-loop complexes. Furthermore, proteins which interact with kissing-loops are still able to bind complexes with the triplex mutation. Using fluorescently labeled bPNA, we have proposed new technology to site selectively label and monitor RNA-RNA interactions. iii To my family. iv Acknowledgments First and foremost, I would like to thank my family. They were a constant source of support and love when I needed it most throughout this experience. Their sacrifices made moving all the way to Ohio for school possible and endurable. Their visits provided a welcomed reprieve and delicious Italian treats while reminding me what was most important in life. I love you Laura, Claudia, Mom, and Dad. Secondly, I would like to thank my friends Kendra Dewese, Laura Dzugan, Amanda Natter, Mike Davala, Johnny Wysocki, Dan Dilts, Emma Wei, and Nao Oda for reminding me to have fun. Their encouragement meant the world to me and lightened my heart on the toughest of days. Here is to many more home cooked meals together. I owe a debt of gratitude to Dr. Dennis Bong for the years of support as my advisor. Your constant encouragement and problem-solving discussions helped me when the projects stalled and progress looked bleak. By working in your group, I have become a better scientist and for this I thank you. I would also like to thank Jie Mao. My constant companion throughout grad school who was always ready with a friendly smile and encouraging words. My teachers, Xijun Piao, Xin Xia, and Zhun Zhou for putting up with my incessant questions and teaching me a myriad of lab skills. I would also like to thank the other lab group members for their helpful discussions. Finally, I would like to thank Dr. Gopalan and Dr. Badjic. v Vita 2007................................................................Hamilton High School East 2011................................................................B.A. Chemistry, Rutgers, the State University of New Jersey 2011 to present ..............................................Graduate Research Associate, The Ohio State University Publications Mao, J., DeSantis, C., and Bong, D.* (2017) “Small molecule recognition triggers secondary and tertiary interactions in DNA folding and hammerhead ribozyme catalysis.” J. Am. Chem. Soc., DOI: 10.1021/jacs.7b05448 Fields of Study Major Field: Chemistry vi Table of Contents Abstract ............................................................................................................................... ii Acknowledgments............................................................................................................... v Vita ..................................................................................................................................... vi Publications ........................................................................................................................ vi Fields of Study ................................................................................................................... vi Table of Contents .............................................................................................................. vii List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii Chapter 1: Structure of nucleic acids and their mimics ..................................................... 1 1.1.1 SECONDARY STRUCTURES OF NUCLEIC ACIDS: bulges and loops. ........ 6 1.1.2 SECONDARY STRUCTURES OF NUCLEIC ACIDS: junctions ...................... 8 1.1.3 SECONDARY STRUCTURES OF NUCLEIC ACIDS: G-quadruplex .............. 9 1.2 COMPONENTS OF TERTIARY STRUCTURE OF NUCLEIC ACIDS ............... 9 1.2.1 TERTIARY STRUCTURE OF NUCLEIC ACIDS: coaxial stacking. ........... 10 vii 1.2.2 COMPONENTS OF TERTIARY STRUCTURE OF NUCLEIC ACIDS: kissing-loops and pseudoknots .................................................................................. 11 1.2.3 COMPONENTS OF TERTIARY STRUCTURE OF NUCLEIC ACIDS: base triples. ........................................................................................................................ 12 1.2.4 COMPONENTS OF TERTIARY STRUCTURE OF NUCLEIC ACIDS: tetraloops ................................................................................................................... 13 1.2.5 COMPONENTS OF TERTIARY STRUCTURE OF NUCLEIC ACIDS: triplexes. .................................................................................................................... 14 1.3 MODIFICATIONS TO NUCLEIC ACIDS ............................................................ 16 1.3.1 MODIFICATIONS TO NUCLEIC ACIDS: natural modifications ................ 17 1.3.2 MODIFICATIONS TO NUCLEIC ACIDS: metal-coordinating bps. ............ 18 1.3.3 MODIFICATIONS TO NUCLEIC ACIDS: hydrogen bonding attenuation. 19 1.3.4 MODIFICATIONS TO NUCLEIC ACIDS: triplex promoting modifications. ................................................................................................................................... 20 1.3.5 MODIFICATIONS TO NUCLEIC ACIDS: triplex formation via Janus wedge. ........................................................................................................................ 21 1.4 MODIFICATIONS TO NUCLEIC ACID BACKBONES. .................................... 23 1.4.1 MODIFICATIONS TO NUCLEIC ACID BACKBONES: phosphodiester modifications. ............................................................................................................ 23 viii 1.4.2 MODIFICATIONS TO NUCLEIC ACID BACKBONES: ribose modifications. ............................................................................................................ 25 1.4.3 MODIFICATIONS TO NUCLEIC ACID BACKBONES: addressing backbone charge repulsion. ....................................................................................... 26 1.4.4 MODIFICATIONS TO NUCLEIC ACID BACKBONES: ribose substitutes.
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