Harnessing the Dual Nature of Peptide Nucleic Acids Público

Abstract

Peptide nucleic acid is a unique synthetic nucleic acid analogue wherein the canonical negatively charged phosphate backbone has been replaced by a neutral pseudopeptide backbone. This peptide-like backbone imparts significant properties such as stronger affinity and specificity for base pairing with DNA and RNA as well as increased hydrolase resistance and stability in complex environments. These properties have made PNA a prime candidate to improve hybridization-based applications such as antisense interactions, nucleic acid detection, and gene editing by taking advantage of the nucleic acid nature. However, the ability to incorporate complex amino acid functional groups within the PNA backbone remains a less explored avenue for utilizing PNA as a peptide mimic. This thesis aims to highlight the dual nature of PNA, to be useful as both a nucleic acid and peptide mimic. In Chapter 1, I provide a literature review to introduce the concepts of PNA with amino acid functionality and applications therein. In Chapter 2, I describe a “bilingual” PNA wherein a complex amphiphilic amino acid sequence embedded in the backbone drives self-assembly into micellar architectures similar to peptide amphiphiles. The nucleic acid code can then be accessed sequence-specifically, resulting in disassembly and highlighting the first example of a PNA acting as both nucleic acid and peptide. In Chapter 3, I explore the use of forced-intercalation PNA probes (FIT-PNA) for detection of an adenosine to inosine modification in nucleic acids. I show the synthesis of a novel malachite green PNA monomer and the fluorescence properties of three individual FIT-PNAs when hybridized to RNA and DNA targets. I determine that the surrogate base dye thiazole orange is a viable candidate for future studies. In Chapter 4, I evaluate the effect of ionic strength on PNA:DNA hybridization and determine that the association rate is significantly affected whereas the dissociation rate is less affected. I then describe the use of a small molecule, glyoxal, to thermoreversibly cage and decage the Franklin-Watson-Crick face to disrupt and restore duplex formation, respectfully. Finally, in Chapter 5 I discuss the practical implications of the presented studies and consider avenues for future directions and applications. 

Table of Contents

ABSTRACT .................................................................................................................................iv

ACKNOWLEDGEMENTS............................................................................................................ vi

LIST OF TABLES AND FIGURES ..............................................................................................xi

1. PEPTIDE NUCLEIC ACIDS HARNESS DUAL INFORMATION CODES IN A SINGLE MOLECULE .................................................................................................................................. 1

1.1 Abstract..............................................................................................................................1

1.2 Introduction .......................................................................................................................2

1.3 Conjugation of PNA ..........................................................................................................3

1.3.1 PNA conjugated to peptides .....................................................................................4

1.3.2 Amphiphilic PNA conjugates ....................................................................................5

1.4 Functional modifications to the PNA backbone ............................................................8

1.4.1 Synthesis and properties of backbone modified PNA............................................8

1.4.2 Hybridization properties of PNA.............................................................................10

1.4.3 Applications of backbone modified PNA...............................................................12

1.5 Encoding Amino Acid Information in the PNA Backbone...........................................14

1.6 Summary and Objectives of this Dissertation..............................................................15

1.7 Conclusions.....................................................................................................................18

1.8 References.......................................................................................................................19

2. BILINGUAL PEPTIDE NUCLEIC ACIDS: ENCODING THE LANGUAGES OF NUCLEIC ACIDS AND PROTEINS IN A SINGLE SELF-ASSEMBLING BIOPOLYMER..........................33

2.1 Abstract............................................................................................................................33

2.2 Introduction .....................................................................................................................34

2.3 Results and Discussion..................................................................................................36

2.3.1 Design and Synthesis of Amphiphilic PNA ...........................................................36

2.3.2 Characterization of Amphiphilic PNA Assembly...................................................40

2.3.3 Stimuli-Responsive Switching of Amphiphilic PNA Assembly ...........................43

2.4 Conclusion.......................................................................................................................48

2.5 Materials and Methods ...................................................................................................49

References ............................................................................................................................. 58

3. FORCED INTERCALATION PEPTIDE NUCLEIC ACID PROBES FOR THE DETECTION OF AN ADENOSINE-TO-INOSINE MODIFICATION .................................................................65

3.1 Abstract............................................................................................................................65

3.2 Introduction .....................................................................................................................66

3.3 Results and Discussion..................................................................................................68

3.3.1 Design and Synthesis of Fluorogenic PNA Monomers ........................................68

3.3.2 Design and Synthesis of FIT-PNA Probes .............................................................70

3.3.3 Characterization of FIT-PNA Probes Binding to Target Transcripts...................71

3.4 Conclusions.....................................................................................................................77

3.5 Materials and Methods ...................................................................................................78

3.6 References.......................................................................................................................83

4. SINGLE-MOLECULE INVESTIGATION AND THERMOREVERSIBLE CONTROL OF PNA:DNA HYBRIDIZATION ......................................................................................................91

4.1 Abstract............................................................................................................................91

4.2 Introduction .....................................................................................................................92

4.3 Results and Discussion..................................................................................................94

4.3.1 Effect of Ionic Strength and Temperature on PNA:DNA Hybridization Kinetics 95

4.3.2 Comparison of PNA:DNA and DNA:DNA Hybridization Kinetics ........................97

4.3.3 Thermoreversible Control of PNA:DNA Hybridization........................................100

4.4 Conclusions...................................................................................................................102

4.5 Materials and Methods .................................................................................................104

4.6 References.....................................................................................................................109

5. CONCLUSIONS AND FUTURE PERSPECTIVES...............................................................115

5.1 Expanding the Repertoire of Bilingual PNA ...............................................................115

5.2 Applications of Bilingual PNA Amphiphiles...............................................................116

5.3 Utilization of FIT-PNA Probes for A-to-I Detection.....................................................117

5.4 Single-Molecule Exploration of PNA:DNA Hybridization ..........................................118

5.5 References.....................................................................................................................119

APPENDIX A: SPECTRAL AND OMITTED DATA OF CHAPTER 2 ......................................122

APPENDIX B: TABULAR, SPECTRAL, AND OMITTED DATA OF CHAPTER 3..................141

APPENDIX C: TABULAR, SPECTRAL, AND OMITTED DATA OF CHAPTER 4..................155

APPENDIX D: OPTIMIZATION OF PNA PURIFICATION AND SYNTHESIS.........................164 

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Nanotechnology Chemistry, Organic Chemistry, Biochemistry
Palabra Clave	Hybridization Self-Assembly Biosensing Stimuli-Responsive Peptide Nucleic Acid
Committee Chair / Thesis Advisor	Jen Heemstra, Emory University
Committee Members	David Lynn, Emory University Bill Wuest, Emory University

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