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Evelyn Linardy The development of nuclease-mediated signal amplification technology for analyte detection Evelyn Linardy BSc (Molecular Biotechnology Hons) School of Biotechnology and Biomolecular Sciences Faculty of Science Submitted for the degree of Doctor of Philosophy (PhD) 2014 i Originality Statement ‗I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgment is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project‘s design and conception or in style, presentation and linguistic expression is acknowledged.‘ Signed Date 18th November 2014 Prelude ii Table of Contents Thesis dissertation sheet Copyright and authenticity statement Originality statement i Publications arising from work in this thesis xiii Acknowledgements xiv Abbreviations and Symbols xv List of Figures xx List of Tables xxvii Abstract xxix Chapter 1: Nucleases and their applications in nucleic acid detection chemistries 1 1.1 Nucleases 1 1.1.1 Restriction endonucleases 3 1.1.2 Nucleic acid endonucleases 8 1.2 Application of nucleases in signal amplification 11 1.2.1 Signal amplification employing sequence independent exonucleases and endonucleases 13 1.2.2 Signal amplification employing nicking endonucleases 15 1.2.3 Signal amplification employing RNA-cleaving DNAzymes 23 1.2.4 Comparison of signal amplification methods employing Prelude iii nucleases 27 1.3 Project aim 30 Chapter 2: Exonuclease III-mediated signal amplification 31 2.1 Introduction 31 2.2 Materials 34 2.2.1 Reagents, equipment and software 34 2.2.2 Oligonucleotides 35 2.2.2.1 Hairpin signal amplification components 35 2.2.2.2 3WJ signal amplification components 36 2.3 Methods 36 2.3.1 Exo III-mediated hairpin signal amplification strategy 37 2.3.1.1 Target dependent hydrolysis of hairpin probe 37 2.3.1.2 Target titration 37 2.3.1.3 Investigation into the extent of hairpin hydrolysis 37 2.3.2 Exo III-mediated 3WJ signal amplification strategy 38 2.4 Results 38 2.4.1 Exo III-mediated hairpin signal amplification strategy 38 2.4.1.1 Hairpin probe designs 38 2.4.1.2 Target dependent hydrolysis of the hairpin probe 39 2.4.1.3 Investigation into the extent of hairpin hydrolysis 41 2.4.2 Exo III-mediated 3WJ signal amplification 43 2.4.2.1 Hairpin probe designs 43 2.4.2.2 Screening the 3WJ designs for target dependent hydrolysis 45 2.5 Discussion 47 2.5.1 Exo III-mediated hairpin signal amplification strategy 47 Prelude iv 2.5.2 Exo III-mediated 3WJ signal amplification strategy 51 2.6 Conclusion 51 Chapter 3: Properties of restriction endonucleases for the development of EzyAmp signal amplification cascades 53 3.1 Introduction 53 3.2 Materials 58 3.2.1 Reagents, equipment and software 58 3.2.2 Oligonucleotides 58 3.2.2.1 Screening REases for their potential to specifically cleave CESA complexes which contain nicks within the enzymes‘ restriction sites 59 3.2.2.2 Investigation of the influence of the nick position within the MnlI restriction site 60 3.2.2.3 Impact of additional modifications to MnlI restriction sites containing nicks 61 3.2.2.4 Investigation of the influence of the nick position within the TspDTI restriction sites 62 3.2.2.5 Impact of additional modifications to TspDTI restriction sites containing nicks 63 3.3 Methods 64 3.3.1 Screening REases for their potential to specifically cleave CESA complexes which contain nicks within the enzymes‘ restriction sites 64 3.3.2 Investigation of the influence of the nick position or additional modifications within the MnlI restriction site 66 3.3.3 Investigation of the influence of the nick position or additional modifications within the TspDTI restriction sites 67 Prelude v 3.4 Results 68 3.4.1 Screening REases for their potential to specifically cleave CESA complexes which contain nicks within the enzymes‘ restriction sites 68 3.4.2 Screening MnlI activity in the presence of modifications at the restriction site 70 3.4.2.1 Investigation of the influence of the nick position within the MnlI restriction sites 70 3.4.2.2 Impact of additional modifications to MnlI restriction sites containing nicks 73 3.4.3 Screening TspDTI activity in the presence of modifications at restriction site 75 3.4.3.1 Investigation of the influence of the nick position within the TspDTI restriction site 75 3.4.3.2 Impact of additional modifications to TspDTI restriction sites containing nicks 77 3.5 Discussion 79 3.5.1 Screening REases for their potential to specifically cleave CESA complexes which contain nicks within the enzymes‘ restriction sites 79 3.5.2 Screening MnlI and TspDTI activity in the presence of modifications at restriction sites 80 3.5.2.1 Investigation of the influence of the nick position within the enzyme restriction site 80 3.5.2.2 Impact of additional modification to enzyme restriction sites containing nicks 82 3.6 Conclusion 84 Prelude vi Chapter 4: An EzyAmp signal amplification cascade mediated by MnlI 4.1 Introduction 86 4.2 Materials 92 4.2.1 Reagents, equipment and software 92 4.2.2 Oligonucleotides 92 4.2.2.1 MnlI EzyAmp signal amplification cascade components 92 4.2.2.1.1 PESA1 93 4.2.2.1.2 PESA2 94 4.2.2.1.3 Initiator DFs 94 4.2.2.2 MNAzyme components 95 4.2.2.2.1 Assembly facilitators (AF) 95 4.2.2.2.2 Partzymes 95 4.2.2.2.3 Substrates 97 4.2.2.3 Intermediate substrate blocker components 98 4.2.2.3.1 Substrate blockers 98 4.2.2.3.2 Hairpin substrate blockers 99 4.2.2.3.3 DNAzyme 99 4.3 Methods 100 4.3.1 MnlI-mediated EzyAmp signal amplification cascade 100 4.3.1.1 Demonstration of the feedback 101 4.3.1.2 MgCl2 titration 101 4.3.2 MNAzyme-initiated MnlI EzyAmp signal amplification cascade 101 4.3.3 Intermediate substrate blocker step 103 4.3.3.1 DNAzyme-initiated substrate blocker step 103 4.3.3.2 MNAzyme-initiated substrate and hairpin blocker step 103 Prelude vii 4.4 Results 104 4.4.1 MnlI-mediated EzyAmp signal amplification cascade 104 4.4.1.1 Demonstration of the feedback 104 4.4.1.2 MgCl2 titration 106 4.4.2 MNAzyme initiated MnlI EzyAmp feedback cascade 108 4.4.2.1 Demonstration of initiation by MNAzyme 108 4.4.2.2 MNAzyme substrate optimisation 113 4.4.2.3 MNAzyme substrate concentration optimisation 115 4.4.2.4 DF binding site optimisation 116 4.4.2.5 Buffer optimisation 123 4.4.2.6 Sensitivity and quantification investigation 124 4.4.3 Intermediate substrate blocker step 126 4.4.3.1 DNAzyme-initiated substrate blocker step 127 4.4.3.2 MNAzyme-initiated substrate blocker step 130 4.4.3.3 MNAzyme-initiated hairpin substrate blocker step 131 4.5 Discussion 132 4.5.1 MnlI-mediated EzyAmp signal amplification cascade 132 4.5.2 MNAzyme-initiated MnlI EzyAmp signal amplification cascade 135 4.5.3 Intermediate substrate blocker step 139 4.6 Conclusion 141 Chapter 5: An EzyAmp signal amplification cascade mediated by TspDTI 143 5.1 Introduction 143 5.2 Materials 149 5.2.1 Reagents, equipment and software 149 5.2.2 Oligonucleotides 149 Prelude viii 5.2.2.1 PESA1 150 5.2.2.2 PESA2 151 5.2.2.3 Stabilisers 151 5.2.2.4 Junction DFs 152 5.2.2.5 Targets (assembly facilitators) 153 5.3 Methods 153 5.4 Results 157 5.4.1 4WJ TspDTI EzyAmp signal amplification cascade containing two complexes 157 5.4.1.1 Preliminary Junction DF designs screening 157 5.4.1.2 Demonstration of two PESA‘s feedback cascade 164 5.4.1.3 Investigation into the effect of reaction temperature to 4WJ TspDTI feedback cascade containing two complexes 167 5.4.2 4WJ TspDTI unimolecular EzyAmp signal amplification cascade 169 5.4.2.1 Design and demonstration of feedback cascade 169 5.4.2.2 Planar Junction DF strategy 171 5.4.2.3 Investigation into the effect of buffer compositions to 4WJ TspDTI unimolecular feedback cascade 176 5.4.3 4WJ TspDTI EzyAmp self-feedback cascade 178 5.4.4 Comparison of the 4WJ TspDTI two complexes, unimolecular, and self-feedback cascade strategies 182 5.5 Discussion 183 5.5.1 4WJ TspDTI EzyAmp signal amplification cascade containing two complexes 183 5.5.2 4WJ TspDTI unimolecular EzyAmp signal amplification cascade 185 5.5.3 4WJ TspDTI EzyAmp self-feedback cascade 187 5.5.4 Comparison of the 4WJ TspDTI utilising two complexes, unimolecular, and self-feedback cascade strategies 188 Prelude ix 5.6 Conclusion 189 Chapter 6: Applications of the EzyAmp signal amplification cascades 6.1 Introduction 191 6.1.1 Ideal Point-of-Care tests 191 6.1.2 Detection of non-nucleic acid targets via aptamers 192 6.2 Materials 194 6.2.1 Reagents, equipment and software 194 6.2.2 Oligonucleotides 195 6.2.2.1 MNAzyme components 195 6.2.2.1.1 Assembly facilitator (AF) 195 6.2.2.1.2 Partzymes 195 6.2.2.1.3 Substrates 197 6.2.2.2 MnlI EzyAmp signal amplification cascade components 197 6.2.2.2.1 PESA1 198 6.2.2.2.2 PESA2 198 6.2.2.2.3 Initiator DF 198 6.2.2.3 4WJ TspDTI EzyAmp signal amplification cascade components 199 6.2.2.4 VEGF aptamer components 200 6.2.2.4.1 Aptamer 200 6.2.2.4.2 Initiator DF (VEGF aptamer complement) 200 6.3 Methods 201 6.3.1 Characterisation in a background of a complex genomic sample 201 6.3.1.1 Detection of endogeneous target using MNAzyme initiated MnlI EzyAmp signal amplification cascade 201 Prelude x 6.3.1.2 Testing
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