THE DEVELOPMENT OF ACIDIC PROTEIN APTAMERS USING CAPILLARY ELECTROPHORESIS METHODS AND THEIR USE IN SURFACE PLASMON RESONANCE JON ASHLEY (MChem, PGCE, NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTORATE OF CHEMISTRY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2013 I I Acknowledgements We acknowledge financial support from the National University of Singapore, National Research Foundation and Economic Development Board (SPORE, COY-15-EWI-RCFSA/N197-1) and Ministry of Education (R-143-000-441-112) I would like to thank the Singaporean government for allowing me to come to undertake my doctorate degree at the National University of Singapore I would also like to acknowledge my supervisor Professor Sam Fong Yau Li for his support and guidance during my time here I also wish to thank my fellow research group members for all their help and advice In particular I would like to thank Dr Grace Birungi and Dr Junie Tok for training me on the use of capillary electrophoresis, Dr Zuo Xing Bing for teaching me PCR, associate professor Christoph Winkler for training and advice on agarose gels, Kaili Ji for help with the cloning and general discussions on aptamers, and the proteomic centre for the use of the BIAcore T3000 SPR I would like to acknowledge the work done by the students I mentored, Dong Jia on the hybridized-SELEX using magnetic beads, Lim Wee Siang on the non-SELEX of hemoglobin aptamers and Lin weili for her work on the development of aptamers for β-lactoglobulin A using CE-SELEX I’d also like to thank my friends and family for their patience and support and my girlfriend Hyojae Park for her love and support II Table of Contents Acknowledgements II Outline VI List of Tables VIII List of Figures X List of Abbreviations XX Literature review .1 1.1 Aptamers 1.2 A comparison of different types of aptamer 1.3 Uses of aptamers 1.3.1 1.4 Bioanalytical uses of aptamers Selection of Aptamers 10 1.4.1 Partitioning methods 11 1.4.2 Determination of binding affinities KD and specificity 21 1.5 Objectives and Scope of the dissertation 25 Methodology 27 2.1 Methods and materials 27 2.1.1 Selection of aptamers using CE-SELEX, Non-SELEX and Hybridised-SELEX 27 2.1.2 Development of an aptamer based SPR biosensor 28 2.2 The CE-SELEX procedure for leptin aptamers 30 2.2.1 2.3 Validation of leptin clone sequences 32 The Non-SELEX of catalase and hemoglobin aptamers 33 2.3.1 Optimization of the Non-SELEX procedure 33 2.3.2 The Non-SELEX procedure for bovine catalase aptamers 34 2.3.3 Bulk affinity determination by NECEEM and validation of catalase aptamer clone sequences 35 2.3.4 The Non-SELEX procedure for hemoglobin aptamers 37 2.3.5 Bulk affinity analysis using ACE and validation of hemoglobin clone sequences 37 2.4 Hybridised-SELEX Procedure 39 III 2.4.1 Bulk affinity determination by NECEEM and validation of cholesterol esterase aptamer clone sequences 41 2.5 Development of aptamer based SPR biosensor 42 2.5.1 2.5.2 Optimization of the catalase biosensor 44 2.5.3 Preparation of the chip surface and optimization of the sensor 43 Real sample analysis 46 CE-SELEX of leptin aptamers and Implications for clone validation 47 3.1 Aim 47 3.2 Results and Discussion 49 3.2.1 The CE-SELEX procedure for leptin aptamers 49 3.2.2 Validation of leptin clone sequences 55 3.3 Summary 62 The Non-SELEX of bovine catalase and human hemoglobin aptamers 63 4.1 Aim 63 4.2 Results and Discussion 66 4.2.1 Optimization of the Non-SELEX procedure using catalase 66 4.2.2 The Non-SELEX of catalase aptamers 71 4.2.3 Validation of catalase aptamer clone sequences 78 4.2.4 The Non-SELEX of hemoglobin aptamers 83 4.2.5 Validation of hemoglobin aptamers clone sequences 86 4.3 Summary 91 Hybridised-SELEX of cholesterol esterase 94 5.1 Aim 94 5.2 Results and discussion 97 5.2.1 The Hybridised -SELEX procedure 97 5.2.2 Validation of cholesterol esterase clone sequences 101 5.3 Summary 108 The development of a aptamer based SPR sensor for the detection of catalase in milk samples 109 6.1 Aim 109 6.1.1 Preparation of chips sensor – SensiQ 110 IV 6.1.2 Preparation of chips sensors – BIAcore 113 6.1.3 Optimization of the aptamer based biosensor 116 6.1.4 Real sample analysis 124 6.2 Summary 128 Conclusion and future work 129 7.1 Conclusion 129 7.2 Future work 132 References 134 a Appendix of chapter 146 b Appendix of chapter 165 c Appendix of chapter 182 d Appendix of chapter 193 List of Publications 194 V Outline Aptamers are ssDNA or ssRNA which show affinity towards a wide range of biomolecules and small molecules We can screen for aptamers by incubating the target with a library of random oligonucleotides, separating binding oligonucleotides, amplifying them by Polymerase chain reaction (PCR) and regenerating the oligonucleotides by strand separation This is known as systematic evolution of ligands by exponential enrichment (SELEX) Traditionally scientists have used affinity chromatography or nitrocellulose membrane filters to select these aptamers Selection of aptamers can take a long time to finish due to the number of rounds needed to achieve an enriched library, typically >10 rounds A number of post SELEX modifications have appeared in the literature that decrease the time required for selection CE-SELEX and non-SELEX are capillary based methods that take advantage of the higher efficiency of separation and can reduce the number of rounds to