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Directed Molecular Evolution of Novel DNA Aptamers Raised Against an Antibiotic Resistant Escherichia Coli

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Directed Molecular Evolution of Novel DNA Aptamers Raised Against an Antibiotic Resistant Escherichia Coli Directed Molecular Evolution of Novel DNA Aptamers Raised Against an Antibiotic Resistant Escherichia coli. Robert Michael Nicholas Gowland This thesis is submitted in partial fulfilment of the requirements for the award of the degree of Doctor of Philosophy of the University of Portsmouth. Submission date: June 2015 Whilst registered as a candidate for the above degree I have not been registered for any other research award. The results and conclusions embodied in this thesis are my own and have not been submitted for any other academic award. Signature: Rob Gowland Date: 16.06.2015 Word Count: 55,848 i Abstract Antimicrobial resistance presents a serious, worldwide threat to public health. New tools are required to combat the evolving problem. Here we report the systematic evolution of nucleic acid ligands to live Escherichia coli cells. Two SELEX methods were used to generate aptamers to the HB101: pAT153 strain of antibiotic resistant bacterial cells. These different Cell-SELEX methods were compared to analyse how DNA adapts to selective evolutionary pressure. The first method was performed using asymmetric PCR as the mechanism of single strand regeneration, with target cells taken from bacterial colonies. The second method used a novel strand protection exonuclease method for single strand regeneration and target cells taken from live cultures. Sequences evolved during both methods were analysed and stored in plasmid libraries. These methods produced a combined total of fifty-eight putative DNA ligands (aptamers). These sequences were analysed using alignment and structural prediction software and six candidate molecules were taken for further analysis. Evolved sequences were synthesised and target cell binding assays were performed using fluorescence laser-scanning confocal microscopy to visualise binding. A number of control sequences were also analysed. These included scrambled versions of each aptamer sequence, complementary oligonucleotides to deform binding structures and positive control sequences of published cell binding aptamers. Five of the six novel sequences exhibited cell specific localisation. Two of these sequences, named Oligo 3.1 and Oligo 4.4 (one from each SELEX Method) demonstrated apparent ligand binding affinities app app in the low-micromolar range (KD Oligo 3.1= 3.35 μM. KD Oligo 4.4 = 6.69 μM). ii Contents Declaration i Abstract ii Table of Contents iii Abbreviations and Units viii Index of Figures x Index of Tables xii Chapter 1: Introduction 1 1.1. Nucleic Acid Chemistry 2 1.1.1. Nucleobases 2 1.1.2. Nucleosides and Sugars 4 1.1.3. Nucleotides and Backbones 6 1.1.4. Synthetic Nucleic Acid Analogues 8 1.2. Intermolecular Nucleic Acid Interactions 10 1.2.1. Base Pairings 10 1.2.2. Duplex Nucleic Acid Structures 12 1.2.3. Triplex Nucleic Acid Structures 14 1.2.4. Quadruplex Nucleic Acid Structures 16 1.2.5. Pentaplex Nucleic Acid Structures 18 1.2.6. Intermolecular Functional RNAs 18 1.3. Intramolecular Nucleic Acid Structures 19 1.3.1. Stem-loops, Pseudoknots and Bulges 20 1.3.2. Intramolecular Triplex Structures 22 1.3.3. Intramolecular Quadruplex Structures 22 1.3.4. Riboswitches and Ribozymes 24 1.3.5. Intramolecular Deoxyribozymes 27 1.4. Molecular Evolution and Genetic Information Systems 29 1.4.1. Strategies for Molecular Evolution 31 1.4.2. The RNA World 34 1.4.3. Directed Molecular Evolution 35 1.5. SELEX 38 1.5.1. A Brief History of SELEX 38 1.5.2. DNA SELEX 41 iii 1.5.3. Cell SELEX 42 1.6. DNA Aptamers and Ligand-Binding 43 1.7. Aptamers vs. Antibodies 46 1.8. Antibiotic Resistance 48 1.9. The Gram Negative Bacterial Cell Wall 50 1.10. Purpose of this Work 53 Chapter 2: Materials and Methods 54 2.1. Chemicals & Reagents 54 2.2. Buffers 55 2.3. Enzymes 58 2.4. DNA Reagents 61 2.4.1. Plasmids 61 2.4.2. Primers and Nucleic Acid libraries 63 2.5. PCR 66 2.5.1. Symmetric PCR 66 2.5.2. Asymmetric PCR 67 2.5.3. Colony PCR 68 2.5.4. Radiolabelled PCR 69 2.6. Nucleic acid purification methods 69 2.6.1. Ethanol Precipitation 69 2.6.2. Qiagen PCR purification 70 2.6.3. Exonuclease I - Shrimp Alkaline Phosphatase PCR purification 70 2.6.4. GE Healthcare microspin column 70 2.7. Spectrophotometry 70 2.7.1. Nanodrop 2000 UV Spectrophotometry 71 2.7.2. Cell Culture Optical Density 71 2.8. Microbial Techniques 71 2.8.1. Media & Antibiotics 71 2.8.2. Strains of E.coli 72 iv 2.8.3. Culturing E.coli 72 2.8.4. Preparation of Competent Cells 72 2.8.5. Transformation of Plasmid DNA 73 2.8.6. Mini-preparation of Plasmid DNA 73 2.9. Systematic Evolution of Ligands by Exponential Enrichment 74 2.9.1. SELEX Method I 74 2.9.2. SELEX Method II 75 2.10. Gel Electrophoresis 76 2.10.1. Agarose Gel Electrophoresis 76 2.10.2. Native Polyacrylamide Gel Electrophoresis (N-PAGE) 78 2.10.3. Denaturing Polyacrylamide Gel Electrophoresis (D-PAGE) 79 2.10.4. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 79 2.11. Molecular Cloning 80 2.12. Phenol Chloroform Extraction 81 2.13. Restriction Endonuclease Digestion 81 2.14. λ Exonuclease Digestion 81 2.15. DNA Sequencing 82 2.16. Oligonucleotide Annealing 82 2.17. Bioinformatics 82 2.17.1. DNA Sequence Analysis and Alignment 82 2.17.2. mfold Structural Prediction 82 2.17.3. ifold Structural Prediction 82 2.18. Confocal Microscopy 83 2.18.1. Sample Preparation 83 2.18.2. Confocal Microscopy 83 2.18.3. Image Processing 83 2.19. Liquid Scintillation Counting 86 Chapter 3: Cell-SELEX using Bacterial Colonies and Asymmetric PCR 87 3.1. SELEX Overview & Rationale 87 3.2. SELEX Primers & Library 89 v 3.3. SELEX Preliminary Experiments 93 Preliminary Experiment I – Sampling the Initial Library 93 Preliminary Experiment II – Primer Walking Experiment 98 Preliminary Experiment III – ssDNA Production 100 Preliminary Experiment IV – Primer Specificity Experiments 105 Preliminary Experiment V – Target Cell Viability 105 3.4. SELEX Method I 108 3.5. Cloning and Sequencing of the enriched pool of molecules 115 3.6. SELEX Method I Conclusions 119 Chapter 4: Cell-SELEX using Bacterial Cell Cultures and Lambda Exonuclease 122 4.1. Introduction and Rationale 122 4.2. SELEX Method II Overview and Strategy 123 4.3. SELEX Preparation and Preliminary Experiments 124 Preliminary Experiment I – PCR Optimisation 124 Preliminary Experiment II – PCR Primer to PCR Template Ratio Experiments 125 Preliminary Experiment III– PCR Primer to PCR Template Ratio Experiments II 129 4.4. λ Exonuclease Digestion of DNA 131 4.5. SELEX Method II 143 4.6. Cloning and Sequencing 146 4.7. SELEX Method II Conclusions 150 Chapter 5: Molecular Evolution of DNA during SELEX Methods I & II 151 5.1. Introduction 151 5.2. Aberrant SELEX Products from SELEX Method I 152 5.3. Aberrant SELEX Products from SELEX Method II 158 5.4. Development of Homogeneity in SELEX Method I and II 160 Chapter 6: Analysis of Evolved Sequences 161 6.1. Analysis of Individual Selected Sequences 161 6.2. Collective Alignment Analysis of Selected Sequences 167 vi 6.3. Selection of Sequences for Confocal Microscopy 173 Chapter 7: Binding Analysis of Radioactively and Fluorescently Labelled Oligonucleotides 179 7.1. Sample Preparation 179 7.2. Laser Scanning Confocal Microscopy Setup 181 7.3. Laser Scanning Confocal Microscopy 181 7.3.1. Negative Control Experiments 181 7.3.2. LSCM Analysis of Evolved Sequences from SELEX Method I 187 7.3.3. LSCM Analysis of Evolved Sequences from SELEX Method II 196 OLIGO 4.1 and OLIGO 4.1s 196 OLIGO 4.2 and OLIGO 4.2s 199 OLIGO 4.3 and OLIGO 4.3s 199 OLIGO 4.4 and OLIGO 4.4s 202 7.3.4. LSCM Analysis of Aptamer Sequences from the Literature 206 OLIGO J and OLIGO Js 206 OLIGO K and OLIGO Ks 206 7.3.5. LSCM Analysis of Evolved Sequences and Human Cells 206 7.3.6. LSCM Analysis of Evolved Sequences and Elongated E.coli cells 210 7.4. Quantitation of LSCM results 210 7.5. Radiolabelled Binding Assay 215 Chapter 8: Discussion 217 8.1. Novel ssDNA Sequences which Interact with Gram Negative Bacteria 217 8.2. Insights into Molecular Evolution of DNA 220 8.3. Development of SELEX Methods 222 8.4. Future Work 222 References Appendices vii Abbreviations 1D, 2D, 3D – One, Two or Three MONOLEX- One-step SELEX Dimensional NADH - Reduced β-Nicotinamide adenine 3’ – Three prime dinucleotide 5’ - Five prime NDP - Nucleotide Diphosphate 5mC – 5-methylcytosine NMR - Nuclear Magnetic Resonance A - Adenine N-PAGE – Native PAGE AMR – Anti Microbial Resistance NTP - Nucleotide Triphosphate APS - Ammonium Persulphate PAGE - Polyacrylamide Gel BLAST - Basic Local Alignment Search Electrophoresis Tool PBS - Phosphate Buffered Saline BSA - Bovine Serum Albumin PCR - Polymerase Chain Reaction C - Cytosine PDB - Protein Database CD – Circular Dichroism Pi - Inorganic Phophate CPM - counts per minute PNA – Peptide nucleic acid Cot – Copy over template Qβ - Bacteriophage Qβ CotC - Beta-globin co-transcriptional Rh - hydrodynamic radius cleavage rRNA - ribosomal RNA DNA - Deoxyribonucleic Acid RT - Reverse Transcriptase dNTP - Deoxynucleotide Triphosphate SDS - Sodium Dodecyl Sulphate D-PAGE – Denaturing PAGE SELEX - Systematic Evolution of Ligands dsDNA - double-stranded DNA by Exponential Enrichment dsRNA - double-stranded RNA ssDNA - single-stranded DNA DTT - Dithiothreitol ssRNA - single-stranded RNA E.coli – Escherichia coli T – Thymine EDTA – Ethylene diamine tetra acetic TAE – Tris Acetate EDTA acid. TBE – Tris Borate EDTA EtOH - Ethanol TEMED - Tetramethylethylenediamine FAM - Fluorescein amidite TFO - Triplex Forming Oligonucleotide G - Guanine Tris –Tris(hydroxymethyl)aminomethane GIS – Genetic Information System
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