SELECTION OF APTAMERS FOR SIGNAL TRANSDUCTION PROTEINS AND DEVELOPMENT OF HIGHLY SENSITIVE APTAMER PROBES TOK JUNIE (B.Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements Acknowledgements It is my pleasure to thank those who made this thesis possible. First and foremost, I am grateful to Professor Sam Li, who has given me much flexibility for my research and allowed me the room to work in my way. He has also supported me throughout my publications and thesis with his patience and knowledge. His friendly disposition and open-mindedness has made the route to my PhD degree more manageable. I am thankful to my collaborators from the Institute of Molecular and Cell Biology (IMCB). Professor Thomas Leung who has given me many valuable advices and guided me with great patience; and Miss Jesyin Lai who has worked hard with me over the years, withstanding the failures we faced in our experiments. I also enjoyed working with the other people in their lab who have helped me in one way or another. I am indebted to many of my labmates who supported me during the course of my research. I would like to show my gratitude to the following people: Dr Yu Lijun and Dr Zuo Xinbing for sharing their expertise with me through invaluable suggestions and providing me with technical assistance; Dr Xu Yan for her contribution in this project; Dr Liu Mahe and Ms Helen Yek for their technical support; Dr Feng Huatao for helping me in the liaising for the use of instrument at Bioprocessing Technology Institute (BTI); Dr Law Wai Siang, Miss Lau Hiu Fung and Miss Elaine Tay i Acknowledgements for helping me to orientate in the lab when I first joined and engaged in many discussions when I am faced with problems; the rest in the lab who have made the laboratory a pleasant working environment. I would also like to acknowledge my past UROPS and Honours students, Miss Yu Lijie, Miss Jasmine Goh, Miss Chan Shu Ann, Miss You Kailun and Miss Joyce Ko for their contribution to my research. They have injected much fun into my work and I truly enjoyed working with them. I would like to specially mention a few important colleagues and friends in the National University of Singapore (NUS) for their constant support and endless encouragement. Mdm Irene Teo who has taken good care of me like a mother over the past few years; Mdm Frances Lim for the technical help provided; Mr Soh Ying Teck and Mr Wong Chee Leong who willingly provided me with a good pair of listening ears whenever I met with any obstacles in my research. My most heartfelt appreciation goes to my family for their unconditional love and emotional support. They have given me the strength and optimism that I needed to see through my project. Finally, I dedicate this thesis to my husband, Gay Yong, who stood by me over these years, maintaining his faith in my capabilities through the encouragement and motivation he has given me during my pursuit for the PhD degree. ii List of Abbreviations List of Abbreviations 6-His Hexahistadine APS Ammonium persulphate ATP Adenosine triphosphate Cdc42-GDP Cell division cycle 42 – guanosine diphosphate Cdc42-GTP Cell division cycle 42 – guanosine triiphosphate CE Capillary electrophoresis CZE Capillary zone electrophoresis DABCYL 4-((4-(dimethylamino)phenyl)azo)benzoic acid DAPI 4'-6-Diamidino-2-phenylindole DI Deionized DMEM Dulbecco’s modified Eagle medium DPBS Dulbecco’s phosphate-buffered saline DNA Deoxyribonucleic acid DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid EOF Electroosmotic flow FAM Carboxyfluorescein GST Glutathione-S-transferase HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High-performance liquid chromatography HRP Horseradish peroxidase iii List of Abbreviations id Inside diameter Ig Immunoglobulin Im Imidazole IPTG Isopropyl-β-D-thiogalactopyranoside KCE Kinetic capillary electrophoresis LB Luria-Bertani LIF Laser-induced fluorescence LMP Low melting point MAB Molecular aptamer beacon MB Molecular beacon MES 2-(N-morpholino)ethanesulfonic acid MRCK Myotonic dystrophy kinase-related Cdc42-binding kinase MRCKα-KD MRCKα kinase domain MW Molecular weight NECEEM Nonequilibrium capillary electrophoresis of equilibrium mixtures od Outside diameter PAK P21-activated kinase PBS Phosphate-buffered saline PCR Polymerase chain reaction PIPES piperazine-N,N′-bis(2-ethanesulfonic acid) PMSF Phenylmethanesulphonylfluoride PVA Poly(vinyl alcohol) iv List of Abbreviations PVDF Polyvinylidene difluoride RNA Ribonucleic acid SDS Sodium dodecyl sulphate SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SELEX Systematic evolution of ligands by exponential enrichment ssDNA Single-stranded DNA TAMRA Carboxytetramethylrhodamine TAE Tris-actate-EDTA TE Tris-EDTA TEMED N,N,N’,N’-tetramethylethylenediamine Tris Tris(hydroxymethyl)aminomethane v Table of Contents Table of Contents Acknowledgements .i List of Abbreviations .iii Summary .x List of Tables xii List of Figures xiii List of Symbols .xvii Chapter 1.1 Introduction .1 Selection of aptamers . 1.1.1 Systematic Evolution of Ligands by EXponential enrichment (SELEX) …………………………………………………………………1 1.1.2 Capillary Electrophoresis as a tool for SELEX (CE-SELEX) …………………………………………………………………4 1.1.3 Basic principles of capillary electrophoresis 1.1.4 Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM) 12 1.1.5 1.2 Non-SELEX . 19 Applications of aptamers 21 Chapter Selection of aptamers for signal transduction proteins by capillary electrophoresis 25 2.1 Introduction 25 2.1.1 Signal transduction protein as targets of interest 25 vi Table of Contents 2.2 Materials and Methods . 27 2.2.1 Materials . 27 2.2.2 Buffers . 28 2.2.3 Equipment . 29 2.2.4 Procedure for non-SELEX . 30 2.3 Results and Discussion 38 2.3.1 Optimization of non-SELEX conditions . 38 2.3.2 Optimization of run buffer 40 2.3.3 Optimization of selection buffer and protein concentration ………………………………………………………………50 2.3.4 Sample injection size 52 2.3.5 Optimization of PCR conditions . 53 2.3.6 Affinity studies for enriched aptamer pools . 56 2.3.7 Sequence and binding analysis of aptamers . 65 2.4 Summary 76 Chapter Development of aptamer probes for cellular imaging of protein localization .79 3.1 Introduction 79 3.1.1 Aptamers for cellular imaging 79 3.1.2 Principles of molecular beacon . 82 3.1.3 Aptamer probe designs . 83 3.1.4 MRCKα as protein target for probe design 88 3.2 Materials and Methods . 89 vii Table of Contents 3.2.1 Materials . 89 3.2.2 Design of molecular aptamer beacons 91 3.2.3 Selectivity studies for MRCKα molecular aptamer beacon ………………………………………………………………91 3.2.4 Quenching efficiency . 92 3.2.5 HeLa cell culture . 92 3.2.6 Fixed cell imaging of normal expression of MRCKα 93 3.2.7 Fixed cell imaging of overexpressed MRCKα and P35A ………………………………………………………………93 3.3 Results and Discussion 94 3.3.1 MRCKα-binding aptamer beacon design 94 3.3.2 Selectivity studies of MRCKα molecular aptamer beacon …………………………………………………………… 100 3.3.3 Detection of MRCKα localization in cells using fluorescently labeled aptamer . 104 3.4 Summary 109 Chapter Conclusion and Future Work .111 Bibliography 116 Appendix I .134 A1.1 Protein Methodology 134 A1.1.1 Transformation and protein expression in competent cells …………………………………………………………… 134 A1.1.2 Protein purification and ion-exchange 135 viii Table of Contents A1.1.3 Cdc42-GTP exchange reaction . 138 A1.1.4 Activation of PAK with Cdc42wt-GTP and ATP 139 A1.1.5 Testing kinase activity of MRCK 139 A1.1.6 Protein analysis by SDS-PAGE . 140 A1.1.7 Coomassie blue staining . 141 A1.1.8 Protein detection with Western blot 141 A1.2 DNA methodology 142 A1.2.1 Analysis of PCR products 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Y., Bioconjugate Chem. 2005, 16, 1433-1441. 133 [...]... affinities of 1st -3rd enriched Cdc42-GTP aptamer pools 57 Table 2.3 Comparison of bulk affinities of 4th to 6th enriched Cdc42-GTP aptamer pools for Cdc42-GTP and Cdc42-GDP 59 Table 2.4 Bulk affinities of enriched MRCKα aptamer pools 62 Table 2.5 Bulk affinities of enriched PAK1 aptamer pools 63 Table 2.6 Sequences and affinities of the best five selected aptamers for the respective signal transduction. ..Summary Summary This thesis examines and addresses the challenges faced in the development of protein-specific probes by integration of various emerging interdisciplinary and enabling platforms The first part of the thesis presents the selection of aptamers for different signal transduction proteins, i.e Cell division cycle 42 (Cdc42), p21activated kinase (PAK) and Myotonic dystrophy kinase-related... according to their respective electrophoretic and electroosmotic flow mobilites; and illustration of the diffuse double layer on the capillary wall 11 Figure 1.4 NECEEM for selection and characterization of aptamers 14 Figure 1.5 Difference between traditional SELEX and non-SELEX selection of aptamers 19 Figure 2.1 Illustration of aptamer collection window determination when... specificity for MRCK in a cellular environment x Summary Lastly, this thesis concludes the research findings with a discussion on the challenges that arose and suggestions to overcome these problems were provided for consideration for future development of aptamer probes xi List of Tables List of Tables Table 2.1 Estimated bulk affinities of the naïve DNA library for respective signal transduction proteins. .. Bischromophoric labeled aptamer (d) Hairpin molecular aptamer beacon for single-stranded DNAbinding proteins (Note: a half stem sequence can be added to the 5’ end of aptamer to form the hairpin structure) 87 xv List of Figures Figure 3.3 Secondary structure of (a) MRCKα aptamer and (b) MRCKα molecular aptamer beacon predicted using mFold program under selection conditions of 50 mM tris, 50 mM... integrating into the structure of larger molecules such as proteins [13] Aptamers have the potential to change the field of affinity probes and replace antibodies as diagnostic, analytical [14, 15] and therapeutic reagents [16, 17] The ease and low cost of production; and the simplicity of chemical modifications and integration into different analytical schemes are clear advantages aptamers have over traditional... DNA·P DNA dissociated from DNA·P D N A Time of migration to the end of the capillary Figure 1.4 NECEEM for selection and characterization of aptamers The unique feature of NECEEM is its very low background: the amount of non -aptamers collected in the aptamer- collection window normalised by the amount of the library loaded is approximately 10-5, which is two orders of magnitude better than the lowest previously... characterised (i.e Kd and koff are determined), they can be used for quantitative analyses of the targets for which they are selected NECEEM-based determination of Kd and koff is fast, accurate, and has a wide and adjustable dynamic range The upper limit of Kd values depends on the highest concentration of P available, which allows measurement of Kd values for very low bulk affinities of naïve libraries... Electrophoresis of Equilibrium Mixtures (NECEEM) to identify the best binders The second part of the thesis describes the modification of a representative MRCK aptamer into a hairpin structure for the development of aptamer probes Solution studies on the selectivity of the aptamer probe were carried out to check the target specificity of the modified aptamer Preliminary experiments were also done using the MRCK aptamer. .. the aptamer can be introduced; enhancing the stability, affinity and specificity of the molecules, thus imparting greater resistance to denaturation and a much longer shelf life [2, 16, 18, 19] Despite the vast potential and significant effort in the development of aptamers over the past 20 years, the progress has been slow largely due to the limitations of conventional technologies used for aptamer development . SELECTION OF APTAMERS FOR SIGNAL TRANSDUCTION PROTEINS AND DEVELOPMENT OF HIGHLY SENSITIVE APTAMER PROBES TOK JUNIE (B.Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF. Affinity studies for enriched aptamer pools 56 2.3.7 Sequence and binding analysis of aptamers 65 2.4 Summary 76 Chapter 3 Development of aptamer probes for cellular imaging of protein localization. the development of protein-specific probes by integration of various emerging interdisciplinary and enabling platforms. The first part of the thesis presents the selection of aptamers for