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Tailoring Peptide Nucleic Acids to Electrostatically Mimic TAILORING PEPTIDE NUCLEIC ACIDS TO ELECTROSTATICALLY MIMIC NATIVE NUCLEIC ACIDS AND TO EXECUTE SINGLE-NUCLEOTIDE TEMPLATED POLYMERIZATION by Narahenpitage Tilani Sandamala De Costa A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry The University of Utah December 2014 Copyright © Narahenpitage Tilani Sandamala De Costa 2014 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Narahenpitage Tilani Sandamala De Costa has been approved by the following supervisory committee members: Jennifer M. Heemstra , Chair 08/18/14 Date Approved Gary E. Keck Member 08/18/14 Date Approved Cynthia J. Burrows , Member 08/18/14 Date Approved Peter F. Flynn Member 08/18/14 Date Approved Thomas E. Cheatham, , Member 08/18/14 Date Approved and by Cynthia J. Burrows , Chair of the Department of Chemistry and by David B. Kieda, Dean of The Graduate School. ABSTRACT Peptide nucleic acid (PNA) is a nucleic acid mimic that shows tremendous potential for use in therapeutic and biosensing applications due to its high binding affinity for DNA and RNA and its excellent biostability. The therapeutic potential of PNA is hindered, however, by poor cellular uptake, solubility, and bioavailability. Although various approaches have been taken to overcome these critical limitations and realize the full potential of PNA, more efficient solutions are still desired. We hypothesize that negatively charged PNA analogues would electrostatically mimic DNA and RNA, thus overcoming the limitations mentioned above. This dissertation is mainly focused on our initial studies to investigate the tolerance of the PNA structure to the addition of negatively charged side chains. We explored the effect of ionic strength on binding affinity for modified PNAs having either negatively charged side chains or positively charged side chains (Chapter 2). We observed that as ionic strength is increased, negatively charged PNA increases in affinity for DNA and RNA, whereas positively charged PNA decreases in affinity for DNA and RNA. The point at which these trends intersect hovers near physiological salt concentration. In a simulated physiological buffer, negatively charged PNA shows slightly higher affinity for RNA whereas positively charged PNA shows slightly higher affinity for DNA. Intrigued by the effect of side chain structure and electrostatics on binding affinity, we were also curious to explore the mismatch and orientation selectivity of these y-substituted PNAs (Chapter 3). We observed that positively charged side chains provide higher selectivity in DNA binding, while negatively charged side chains provide higher selectivity in RNA binding. Our results provide insight into the impact of side chain structure and electrostatics on the binding affinity and selectivity with DNA and RNA under physiological conditions. Since PNA can be negatively charged without sacrificing binding affinity and selectivity, we anticipate that these molecules will show promise as therapeutics that take advantage of both the inherent benefits of PNA and the multitude of charge-based delivery technologies currently being developed for DNA and RNA. PNA also shows promise for use in synthetic biology applications, but the evolution of abiotic polymers such as PNA requires methods for sequence encoding and amplification. Chapter 4 describes our efforts to synthesize a modified PNA monomer that is designed to polymerize using dynamic reaction conditions. DNA-based micelles have the potential to be used as stimuli-responsive materials due to their ability to undergo programmable assembly and disassembly. Chapter 5 outlines our synthesis of a potential multivalent micellar scaffold. iv To the most wonderful people in my life, my parents, sister, and husband for their unconditional love and support TABLE OF CONTENTS ABSTRACT......................................................................................................................... iii LIST OF ABBREVIATIONS................................................................................................ ix ACKNOWLEDGEMENTS.................................................................................................. xiii CHAPTER 1. PEPTIDE NUCLEIC ACID: A VERSATILE NUCLEIC ACID ANALOGUE.............................................................................................................. 1 Nucleic Acid Analogues............................................................................... 4 Peptide Nucleic Acid (PNA)........................................................................ 8 Structures of PNA in complex with PNA and nucleic acids..........10 PNA backbone modifications...........................................................11 PNA nucleobase modifications.......................................................12 Applications of PNA......................................................................... 13 Cellular delivery of PNA.................................................................. 14 Negatively charged PNA: an attempt to enhance cellular delivery............................................................................................. 17 Objectives of this Dissertation................................................................. 20 References.................................................................................................. 22 2. TAILORING PEPTIDE NUCLEIC ACIDS TO ELECTROSTATICALLY MIMIC DNA AND RNA............................................................................................ 29 Introduction................................................................................................. 29 Results and Discussion.............................................................................. 31 Monomer synthesis..........................................................................31 Structure and sequence of modified PNA strands.........................33 The effect of ionic strength on duplex stability for DNA, RNA, and PNA.................................................................................. 33 Duplex stability of charged PNA with DNA at varying salt concentrations............................................................................35 Duplex stability of charged PNA with RNA at varying salt concentrations............................................................................39 Duplex stability of charged PNA with DNA and RNA under physiological salt conditions................................................. 40 Thermodynamic data analysis....................................................... 40 Conclusion.................................................................................................. 43 Experimental Section.................................................................................. 44 General techniques......................................................................... 44 Oligomer synthesis.......................................................................... 45 UV melting studies.......................................................................... 46 Buffer preparation............................................................................46 Thermodynamic analysis................................................................ 46 Procedures and characterization.................................................. 47 References.................................................................................................. 51 3. DIFFERENTIAL DNA AND RNA SEQUENCE DISCRIMINATION BY PNA HAVING CHARGED SIDE CHAINS.............................................................. 55 Introduction................................................................................................. 55 Results and Discussion............................................................................... 56 Structure and sequence of modified PNA strands.........................56 The effect of side chain structure on mismatch and antiparallel versus parallel discrimination.......................................58 Thermodynamic data analysis....................................................... 61 The effect of varying the position of the mismatch site relative to the side chains on mismatch discrimination.................64 Structural analysis by circular dichroism (CD) spectroscopy................................................................................... 65 Conclusion................................................................................................... 65 Experimental Section.................................................................................. 69 General techniques......................................................................... 69 UV melting studies.......................................................................... 70 Thermodynamic analysis............................................................... 70 Circular dichroism (CD) analysis................................................... 71 References.................................................................................................. 72 4. TOWARD DNA-TEMPLATED PNA POLYMERIZATION USING DYNAMIC COVALENT CHEMISTRY.................................................................... 75 Introduction................................................................................................... 75 Results and Discussion.............................................................................
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