Exploration of Bilingual Peptide Nucleic Acids Public
Argueta-Gonzalez, Hector (Summer 2022)
Published
Abstract
Peptide nucleic acid (PNA) is an unnatural nucleic acid mimetic that contains a neutral pseudopeptide backbone, replacing the negatively charged phosphate backbone found in DNA and RNA. The change in backbone imparts changes such as having higher affinity and specificity for nucleic acid hybridization and increases stability. Due to these properties, PNA has a great potential for use in various applications such as nucleic acid detection and antisense interactions. As well, due to its modifiable backbone, amino acid residues could be installed at the γ-position to direct for assembly. Previous work has been done in our laboratory to develop a bilingual PNA biopolymer that could incorporate both nucleic acid and amino acid coding to develop a selfassembling oligomer that could undergo stimuli-responsive disassembly upon the recognition of a target sequence. However, this system has yet to be further explored to understand the coding interactions and explore its potential applications. This thesis aims to highlight the elucidation and potential applications of bilingual PNA systems. In chapter 1, I describe PNA as a nucleic acid, its previous applications, and potential for target recognition systems. In chapter 2, I describe a toehold-mediated displacement system development to induce stimuli-responsive assembly in bilingual PNA systems upon the addition of a releasing strand. In chapter 3, I elucidate the nucleic acid and amino acid influences on the bilingual PNA systems to further its potential in nanotechnology and biomedical applications. In chapter 4, I explore the use of triplex forced intercalation PNA probes for the detection of Adenosine to Inosine (A-to-I) edits. In chapter 5, I describe the application of a PNA as a capture strand for the development of a PNA based biosensor. In chapter 6, I discuss the testing and optimization of a coenzyme A (CoA) biosensor. In chapter 7, I describe the synthesis of a photocaged aspartic acid for use in bilingual PNA biopolymers to introduce spatial/temporal control. In chapter 8, I sought to synthesize a novel Nile Red (NR) PNA monomer to further expand PNA detection applications. Finally, I summarize how the presented studies could be utilized for future directions and applications.
Table of Contents
Chapter 1: Introduction ........................................................................................................... 2 1.1 Nucleic Acid Biosensors…………………………………………………….………………2 1.2 DNA-polymer conjugate assemblies……………………………………….………………2 1.3 Peptide Nucleic Acids ………………………………………….……………………………3 1.4 Modified Peptide Nucleic Acids………………………………………………….…………4 1.5 γ-Modified Peptide Nucleic Acid………………………………….………………………...6 1.6 Developing a Dual Encoded Biopolymer………………………………….……………….7 1.7 Summary and Conclusion of this Dissertation………………………………….…………8 1.8 References…………………………………….………………………………….…......…12 Chapter 2: Stimuli-Responsive Assembly of Bilingual Peptide Nucleic Acids...................17 2.1 Abstract……………………………………………………………………….……………..17 2.2 Introduction………………………………………………………………..….…………….18 2.3 Results and Discussion ……………………………………….………….……………….21 2.4 Conclusions…………………………………………………………………….….…..……31 2.4 Experimental Procedure…..………………………………………………….….……..…32 2.4 References…………………………………………………………………….….……...…36 Chapter 3: Elucidating the Structural Influence of Bilingual PNA Biopolymers.................40 3.1 Abstract……………………………………………………………………….……………..40 3.2 Introduction………………………………………………………………..….…………….41 3.3 Results and Discussion ……………………………………….………….……………….43 4 3.4 Conclusions…………………………………………………………………….….………..52 3.5 Experimental Procedure…..………………………………………………….….……..…52 3.6 References…………………………………………………………………….….……...…56 Chapter 4: Synthesis and development of a Triplex Forced Intercalation Peptide Nucleic Acid Probe for the Detection of Adenosine-to-Inosine Modification in Hairpin RNA*……..…………….61 4.1 Abstract……………………………………………………………………….……………..61 4.2 Introduction ………………………………………………………………..….…………….61 4.3 Results and Discussion ……………………………………….………….……………….64 4.4 Conclusions…………………………………………………………………….….………..67 4.5 Experimental Procedure …..………………………………………………….….……..…68 4.6 References…………………………………………………………………….….……...…70 Chapter 5: Development of a Structure-switching DNA:PNA Biosensor for Ochratoxin A………75 5.1 Abstract……………………………………………………………………….……………..75 5.2 Introduction ………………………………………………………………..….…………….75 5.3 Results and Discussion ……………………………………….………….……………….79 5.4 Conclusions…………………………………………………………………….….………..84 5.5 Experimental Procedure…..………………………………………………….….……..…84 5.6 References…………………………………………………………………….….……...…87 Chapter 6: Optimization of a CoA Structure-Switching Biosensor*…………………………………89 6.1 Abstract……………………………………………………………………….……………..89 5 6.2 Introduction ………………………………………………………………..….…………….89 6.3 Results and Discussion ……………………………………….………….……………….92 6.4 Conclusions…………………………………………………………………….….………..96 6.5 Experimental Procedure …..………………………………………………….….……..…96 6.6 References…………………………………………………………………….….……...…98 Chapter 7: Conclusions and Future Perspectives………………………………………………......101 7.1 Nucleic Acids and Bilingual Biopolymers ………………………………….……………101 7.2 Stimuli-Responsive Assembly of Bilingual Peptide Nucleic Acids.7.3 Nucleic Acids and Bilingual Biopolymers ……………………………………………..….…………….…….…….…….102 7.3 Elucidating the Structural Influence of Bilingual PNA Biopolymers ……………………………………………………………………………….………….……………….102 7.4 Synthesis and development of a Triplex Forced Intercalation Peptide Nucleic Acid Probe for the Detection of Adenosine-to-Inosine Modification in Hairpin RNA*…………...……..103 7.5 Development of a Structure-switching DNA:PNA Biosensor for Ochratoxin A……...103 7.6 Optimization of a CoA Structure-Switching Biosensor*………………………………..104 Appendix A: Omitted Data from Chapter 2………………………………………..……………...105 Appendix B: Omitted Data from Chapter 3..........................................................................119 Appendix C: Omitted Data from Chapter 4..........................................................................135 Appendix D: Omitted Data from Chapter 5..........................................................................145 Appendix E: Omitted Data from Chapter 6..........................................................................155 6 List of Tables and Figures Figure 1.1: Structure-switch aptamer biosensor ................................................................... 2 Figure 1.2: DPC conjugate monomer structure and possible reorganization structures…...3 Figure 1.3: Chemical structures of DNA, RNA and PNA………………………………………......4 Figure 1.4: Toehold-mediated Displacement inducing assembly......................................... 9 Figure 1.5: Elucidation of dual coding in Bilingual PNA Biopolymer………………………….9 Figure 1.6: A-to-I FIT PNA Probe fluorogenic response………………………………………....10 Figure 1.7: PNA:DNA SS Biosensor………………………..……………………………………....11 Figure 1.8: Novel SS SELEX method for targeting CoA………………………………………....11 Figure 2.1: PNA self-assembly and toehold-mediated disassembly. ...................................................................................................…………………………………19 Table 2.1: Oligonucleotide sequences used for stimuli-responsive assembly system. ....21 Figure 2.2: Hybridization of MS-D, MS-R, and MS-D-1m to PNA-C1-FAM. ..........................22 Figure 2.3: Fluorescence-based toehold mediated release of control PNA........................24 Figure 2.4: Fluorescence-based toehold mediated release of bilingual PNA. ....................26 Figure 2.5: Normalized size distribution of PNA assemblies...............................................27 Figure 2.6: CD spectroscopy of toehold mediated release of PNA-A1-FAM.......................28 Figure 2.7: TEM images of assembled, disassembled, and reassembled……………………29 Figure 3.1: Assembly variation via changes to bilingual PNA coding. ...............................43 Table 3.1: Oligonucleotide sequences used for bilingual PNA assembly and disassembly .................................................................................................................................................44 Figure 3.2: DLS and CMC of bilingual PNA library. ..............................................................46 7 Table 3.2: Calculated CMC values of bilingual PNA library. ................................................47 Table 3.3: DLS sizes of bilingual PNA library........................................................................47 Figure 3.3: TEM images of bilingual PNA library assembled...............................................49 Table 3.4: Average TEM of bilingual PNA library..................................................................50 Figure 3.4: CD spectroscopy of bilingual PNA library binding DNA………………………….50 Figure 3.5: TEM images of bilingual PNA library disassembled..........................................52 Scheme 4.1: Synthesis of M and P Monomers......................................................................64 Table 4.1: RNA sequences and tFIT PNA probe....................................................................65 Figure 4.1: Fluorescence measurements of tFIT PNA probe. ..............................................66 Table 5.1: PNA and DNA sequences.....................................................................................79 Figure 5.1: % Signal Quenching and % Displacement of unmodified PNA:DNA OTA biosensor system. ..................................................................................................................80 Figure 5.2: % Signal Quenching and % Displacement of unmodified DNA:DNA OTA biosensor system. ..................................................................................................................81 Figure 5.3: % Signal Quenching and % Displacement of modified PNA:DNA biosensor systems ...................................................................................................................................83 Figure 5.4: Assembly formation apriori to the OTA induced displacement........................84 Table 6.1: CoA candidate sequences. ...................................................................................92 Figure 6.1: CoA Candidate Sequence Biosensor optimizations..........................................93 Figure 6.2: CoA Candidate Sequence Biosensor displacements ........................................94 Figure 6.3: CoA Candidate Sequence #6 Binding studies. ..................................................95 8 Figure A1. Melting temperature measurements for MS-D duplexes..................................106 Figure A2. Melting temperature measurements for MS-R duplexes..................................107 Figure A3. Melting temperature measurements for PNA-C1-FAM duplexes.....................108 Figure A4. Melting temperature measurements for PNA-A1-FAM duplexes.....................109 Table A1. Calculated melting temperature measurements. ...............................................110 Figure A5. % Displacement of PNA-C1-FAM:MS-D System. ..............................................110 Figure A6. Structure, HPLC, and ESI-TOF of PNA-C1-FAM................................................111 Figure A7. Structure, HPLC, and ESI-TOF of PNA-A1-FAM................................................112 Table A2. PNA Sequence, expected mass, and found mass .............................................113 Figure A8. TEM images of PNA-A1-FAM assembled….......................................................114 Figure A9. TEM images of PNA-A1-FAM disassembled via MS-D. ....................................115 Figure A10. TEM images of PNA-A1-FAM reassembled via RS-D. ....................................116 Figure A11. CD spectroscopy of PNA-A1-FAM, MS-D, RS-D..............................................117 Figure A12. % Signal quenching of PNA-A1-FAM:MS-D . ..................................................117 Figure A13. DLS of PNA-C1-FAM and PNA-A1-FAM assembled, disassembled, and reassembled .........................................................................................................................118 Figure B1. Melting temperature measurements for PNA:DNA duplexes...........................120 Table B1. Calculated melting temperature measurements. ...............................................121 Figure B2. TEM images of PNA control sequences assembled.........................................122 Figure B3. CD spectroscopy of PNA control sequences and DNA. ..................................123 Figure B4. DLS of PNA sequences. .....................................................................................124 9 Table B2. DLS sizes of PNA sequences. .............................................................................125 Figure B5. CMC studies of PNA sequences........................................................................126 Figure B7. Structure, HPLC, and ESI-TOF of PNA-A-B………………………………………...127 Figure B8. Structure, HPLC, and ESI-TOF of PNA-N-A.......................................................128 Figure B9. Structure, HPLC, and ESI-TOF of PNA-10-A. ....................................................129 Figure B10. Structure, HPLC, and ESI-TOF of PNA-8-A. ....................................................130 Figure B11. Structure, HPLC, and ESI-TOF of PNA-N-C.....................................................131 Figure B12. Structure, HPLC, and ESI-TOF of PNA-10-C. ..................................................132 Figure B13. Structure, HPLC, and ESI-TOF of PNA-8-C. ....................................................133 Table B3. PNA sequence, expected mass, and found mass..............................................134 Figure C1: Structure, HPLC, and ESI-TOF of TO tFIT PNA Probe......................................136 Figure C2: ESI-TOF of Fmoc-M-Bz Monomer......................................................................137 Figure C3: ESI-TOF of Fmoc-P-Bz Monomer.......................................................................138 Figure D1: % Signal Quenching of PNA-1, PNA-2, and PNA-3...........................................140 Figure D2: PNA-CM-3 biosensor testing. ............................................................................141 Table D1: FAM-labeled PNA-CM sequences and BHQ-1-labeled OTA aptamer sequence. ...............................................................................................................................................142 Figure D4: % Signal Quenching and % Displacement via OTA recognition using PNA-CM4:DNA biosensor...................................................................................................................143 Figure D5: % Signal Quenching and % Displacement via OTA recognition using PNA-CM5:DNA biosensor...................................................................................................................144 Figure D6: % Signal Quenching of PNA-CM-5:DNA............................................................145 10 Figure D7: Structure, HPLC, and ESI-TOF of PNA-1...........................................................146 Figure D8: Structure, HPLC, and ESI-TOF of PNA-2...........................................................147 Figure D9: Structure, HPLC, and ESI-TOF of PNA-3...........................................................148 Figure D11: Structure, HPLC, and ESI-TOF of PNA-CM-1 ..................................................149 Figure D12: Structure, HPLC, and ESI-TOF of PNA-CM-2. .................................................150 Figure D13: Structure, HPLC, and ESI-TOF of PNA-CM-3. .................................................151 Figure D14: Structure, HPLC, and ESI-TOF of PNA-CM-4 ..................................................152 Figure D15: Structure, HPLC, and ESI-TOF of PNA-CM-5. .................................................153 Figure D16: Structure, HPLC, and ESI-TOF of PNA-CM-6. .................................................154 Figure E1: Gel purification of candidate sequences ..........................................................156 Figure E2: Sequence secondary structures via Nupack ....................................................157
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