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Complete 3D Description of Dynamic Behaviour of Enzyme Mimics: Role of Various Structural Elements in Catalysis and Interactions with Bio-Target

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Complete 3D Description of Dynamic Behaviour of Enzyme Mimics: Role of Various Structural Elements in Catalysis and Interactions with Bio-Target Complete 3D description of dynamic behaviour of enzyme mimics: role of various structural elements in catalysis and interactions with bio-target A thesis submitted to the University of Manchester for the degree of Doctor in Philosophy in the Faculty of Biology, Medicine and Health 2018 Linda T. Trivoluzzi The University of Manchester School of Health Sciences Division of Pharmacy and Optometry Table of Contents List of Figures……………………………………………………………………………..….……. 7 List of Tables……………………………………………………………………………………..... 20 List of Schemes……………………………………………………..…………………….….…….. 21 List of abbreviations……………………………………………………………………….…….… 22 Abstract………………………………………………………………………………….….……… 25 Declaration……………………………………………………………………….……….…..……. 26 Copyright Statement……………………………………………………………….….………....… 27 Acknowledgements…………………………………………………………………….………..…. 28 Publications…………………………………………………………………………………………. 29 Chapter 1: Introduction 1.1 Low molecular weight drugs: decline in drug discovery process………………… 30 1.2 RNA targeting………………………………………………………………………..……… 33 1.2.1 Rationale, possible impact and advantages of RNA targeting………………………..…… 33 1.2.2 MicroRNAs as attractive therapeutic targets……………………………………………… 37 1.3 Spontaneous, enzymatic and chemical catalysis of RNA cleavage…………..….. 38 1.3.1 RNA chemical stability……………………………………………………………………. 38 1.3.2 Spontaneous RNA transesterification……………………………………………………… 39 1.3.3 Base catalysis……………………………………………………………………………… 40 1.3.4 Metal ion catalysis……………………………………………………………………….… 41 1.3.5 RNA cleaving enzymes……………………………………………………………………. 42 1.3.5.1 Ribonucleases……………………………………………………………….……..…. 42 1.3.5.2 Ribozymes…………………………………………………………………….…..….. 44 1.4 Oligonucleotide-based chemical ribonucleases………………………………………. 44 1.4.1 Metal-dependent artificial ribonucleases………………………………………….…..…… 47 1.4.2 Metal-independent artificial ribonucleases……………………………………….….…….. 51 1.4.3 Drawbacks of existing ribonucleases……………………………………………...………. 54 1.4.4 Peptidyl oligonucleotide conjugates…………………………………………………..…… 54 1.4.5 Mechanism of RNA cleavage by peptidyl-oligonucleotide conjugates…………………… 60 1.4.5.1 Gaps in knowledge……………………………………………………………….…... 63 1.5 Aims and objectives of the project………………………………………………………. 63 1.6 Structure and dynamics of macromolecules by NMR………………………………. 66 1.6.1 Introduction to NMR methods…………………………………………………………….. 66 1.6.2 Common NMR Isotopes………………………………………………………………..….. 69 1.6.3 Chemical shifts………………………………………………………………………..…… 70 1.6.4 Spin-Spin or J-coupling…………………………………………………………….…….... 71 1.6.5 Relaxation in NMR Spectroscopy…………………………………………………….…… 74 1.6.6 Fourier Transform in NMR………………………………………………….…………….. 75 1.6.7 Multidimensional NMR………………………………………………………………..….. 76 2 1.6.8 NMR techniques……………………………………………………………………..…….. 79 1.6.8.1 Water suppression………………………………………………………………….… 79 1.6.9 COSY: Correlation Spectroscopy…………………………………………………………. 81 1.6.10 DQF-COSY: Double-Quantum Filtered Correlation Spectroscopy……………………… 82 1.6.11 TOCSY: Total Correlation Spectroscopy…………………………………………….….. 83 1.6.12 NOESY: Nuclear Overhauser Effect Spectroscopy………………………………..…….. 84 1.6.13 HSQC: Heteronuclear Single-Quantum Correlation……………………………….……. 87 1.6.13.1 CT-HSQC: Constant-time Heteronuclear Single-Quantum Correlation…………..…… 88 1.6.14 2D HSQC-NOESY……………………………………………………………………..… 88 1.6.15 2D ω1-X-filtered NOESY……………………………………………………………..…. 89 1.6.16 3D HNHA………………………………………………………………………….…….. 91 1.7 Combination of NMR and Molecular Modelling for structural analysis……….. 92 1.7.1 Molecular Modelling……………………………………………………………………… 92 1.7.1.1 Molecular Mechanics……………………………………………………………..…. 93 1.7.1.2 Energy Minimisation and Geometry Optimization…………………………….……. 93 1.7.1.3 Molecular Dynamics……………………………………………………………..….. 94 1.7.1.4 Restrained Molecular Dynamics…………………………………………………….. 96 1.7.1.5 Simulated Annealing and NMR assisted folding…………………………….……… 96 1.7.2 Analysis…………………………………………………………………………………… 98 1.7.2.1 Geometrical Analysis………………………………………………………………… 98 1.7.2.2 RMSD………………………………………………………………………….……. 99 1.7.2.3 Cluster Analysis……………………………………………………………………… 100 1.8 References………………………………………………………………………….………… 101 Chapter 2: Materials and methods 2.1 Source of materials………………………………………………………………………… 115 2.2 Mass Spectrometry…………………………………………………………….…………… 115 2.3 Synthesis of peptidyl-oligonucleotide conjugates…………………………………… 116 2.3.1 DNA oligonucleotide……………………………………………………………..……….. 117 2.3.2 Solid phase peptide synthesis with Fmoc Chemistry………………………………..……. 117 2.3.3 Peptidyl-oligonucleotide conjugate synthesis………………………………………….…. 120 2.3.4 Synthesis of the selectively (13C, 15N) labelled conjugates……………………..………… 122 2.4 Conjugate quantification…………………………………………………………….……. 123 2.5 Ribonuclease activity assay………………………………………….…………………… 123 2.6 Nuclear Magnetic Resonance Spectroscopy…………………………………..………. 124 2.6.1 Spectrometers……………………………………………………………………………… 124 2.6.2 NMR Sample preparation…………………………………………………………………. 124 2.6.3 Data acquisition…………………………………………………………………….……… 125 2.6.3.1 NMR spectra acquired for assignment of the unlabelled conjugate…………………. 125 2.6.3.2 NMR spectra acquired for assignment of the labelled conjugates……..…………….. 127 2.6.3.3 NMR spectra acquired at different temperature and pH………………………..……. 127 2.6.4 Data processing and analysis………………………………………………………………. 128 3 2.7 NMR assignment methods and constraint generation…………………….…………. 128 2.7.1 Distance restraints…………………………………………………………..……………… 130 2.7.1.1 Generation of restraint file……………………………………………………………. 130 2.8 Restrained molecular dynamics (rMD)………………………………..……………….. 132 2.9 MD simulations………………………………………………………………………….….. 134 2.10 References………………………………………………………………………….……….. 136 Chapter 3: Solution structure and dynamics of the model conjugate by NMR spectroscopy 3.1 Introduction……………………………………………………………………….………….. 139 3.2 Design of the Peptidyl-oligonucleotide conjugate……………………..….…………. 141 3.3 Design of the Peptidyl-oligonucleotide labelled conjugate………………………… 141 3.4 Synthesis of the unlabelled and site-specifically 13C- and 15N-labelled peptidyl- oligonucleotide conjugates……………………………………………..…….. 142 3.4.1 Solid phase synthesis of the peptide NH2-[Leu-Arg]4-Gly-CONH2 and site-specifically 13C- and 15N-labelled peptides……………………………………………………………. 143 3.4.2 Peptide Characterization (Mass spectrometry)……………………………………………. 144 3.4.3 Coupling reaction between the oligonucleotide 5’-d(pTCAATC)-3’ and peptide NH2-[Leu-Arg]4- Gly- CONH2…………………………………………………….……… 145 3.4.4 Conjugate Characterization (Mass spectrometry)…………………………………………. 146 3.5 Cleavage of 5’-[32P]-microRNA-24 by the peptidyl-oligonucleotide conjugate ……………………………………………………………………………………. 147 3.6 NMR spectroscopy and structure determination of the conjugate………………... 151 3.6.1 Preliminary NMR studies on the conjugate………………………………………..……… 151 3.6.2 Optimisation of solution conditions for NMR experiments………………………………. 153 3.6.3 NMR strategies for nucleic acid resonance assignments………………………………..… 158 3.6.3.1 Identification of base protons and sugar proton spin systems…………………….….. 158 3.6.3.2 Assignment of the exchangeable proton resonances…………………………….…….160 3.6.3.3 Sequential backbone assignment……………………………………………..………. 161 3.6.3.4 Resonance assignments from 31P correlated experiments……………………………. 167 3.6.3.5 Nucleic acid chemical shifts………………………………………………………….. 168 3.6.4 NMR strategies for conjugate peptide resonance assignments……………………………. 170 3.6.4.1 Backbone resonance assignment……………………………………………………... 170 3.6.4.2 Sequential backbone assignment……………………………………….……….……. 173 3.6.4.3 Side chain assignments…………………………….…………………………………. 174 3.6.4.4 Peptide medium and long-range assignments………………………………………... 179 3.6.5 Identification of intramolecular NOEs…………………………………………………….. 180 3.6.6 Chemical shift temperature dependence………………………………………………..…. 183 3.6.7 Assignment of the peptide protons assisted with isotopic labelling………………………. 185 3.6.8 Solution structure of the conjugate………………………………………………………… 195 3.6.8.1 Experimental restraints……………………………………………………………….. 195 4 3.6.8.2 Simulated Annealing…………………………………………………………………. 199 3.6.8.3 Structural analysis……………………………………………………….…………… 200 3.7 Molecular modelling ……………………………………………………….……………… 207 3.8 References………………………………………………………………………..…….…….. 215 Chapter 4: miRNases: Novel peptide-oligonucleotide bioconjugates that silence miR-21 in lymphosarcoma cells 4.0. DECLARATION……………………………………………………….………………….. 218 4.1 Abstract……………………………………………………………………..………………… 219 4.2 Introduction………………………………………………………………………………….. 220 4.3 Material and Methods……………………………………………….……...……………… 222 4.3.1 Instrumentation……………………………………………………………………………. 222 4.3.2 Oligonucleotides…………………………………………………………...……………… 222 4.3.3 Peptide synthesis and purification…………………………………………………………. 223 4.3.3.1 Acetyl-(LeuArg)2-Gly-(LeuArg)2-Gly-COOH………………………...…………… 223 4.3.4 Synthesis of the conjugates………………………………………………….………….…. 223 4.3.5 Conjugate purification…………………………………………………….………………. 224 4.3.6 Characterisation of conjugates………………………………………………..…………… 224 4.3.6.1 Conjugate 5’-16………………………………………………………...…………….. 224 4.3.6.2 Conjugate 5’-h-6/14………………………………………………………...………… 224 4.3.6.3 Conjugate 5’-h-9/14………………………………………………………………….. 225 4.3.6.4 Conjugate 5’-h-6/16………………………………………………….………………. 225 4.3.6.5 Conjugate 5’-h-9/16………………………………………………..………………… 225 4.3.6.6 Conjugate 5’-luc-h-9/14…………………………………………………...…………. 226 4.3.7 5’-RNA labelling………………………………………………………...………………… 226 4.3.8 Gel-retardation assay………………………………………………………...……………. 226 4.3.9 Ribonuclease activity assay……………………………………………………………….
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