Lau Pui Man Irene. / Thesis submitted in: July 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 162-172). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.ii / Abstract --- p.v / Abstract in Chinese --- p.vii / List of Figures --- p.ix / List of Tables --- p.xii / Contents --- p.xiii / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.1.1. --- Therapeutic uses of nucleic acids --- p.2 / Chapter 1.1.1.1 --- Antisense oligonucleotides --- p.2 / Chapter 1.1.1.2 --- RNA interference --- p.4 / Chapter 1.1.1.3 --- Aptamer --- p.6 / Chapter 1.2 --- Selection of Aptamer --- p.7 / Chapter 1.2.1 --- SELEX 'Systematic Evolution of Ligands by Exponential enrichment' --- p.7 / Chapter 1.2.1.1 --- In vitro selection --- p.8 / Chapter 1.2.1.2 --- Amplification --- p.8 / Chapter 1.2.1.3 --- Monoclonal Aptamer --- p.10 / Chapter 1.2.2 --- Photo-SELEX --- p.10 / Chapter 1.3 --- Examples of target molecules of aptamers --- p.12 / Chapter 1.4 --- Applications of aptamer --- p.14 / Chapter 1.4.1 --- Detection of Aptamer --- p.14 / Chapter 1.4.2 --- Examples of diagnostic use Contents --- p.15 / Chapter 1.4.2.1 --- Aptamer against theophylline with high specificity --- p.15 / Chapter 1.4.2.2 --- Aptamer chip --- p.16 / Chapter 1.4.3 --- Examples of therapeutic use --- p.18 / Chapter 1.4.3.1 --- Vascular endothelial growth factor (VEGF) --- p.18 / Chapter 1.4.3.2 --- Aptamer as a reversible antagonists of coagulation factor IXa is another example to show the potential case of aptamers as therapeutic agents --- p.20 / Chapter 1.4.4 --- Problem faced by aptamer --- p.21 / Chapter 1.4.4.1 --- Stability --- p.21 / Chapter 1.4.4.2 --- Clearance from blood --- p.22 / Chapter 1.5 --- Comparison between aptamer and antibody --- p.24 / Chapter 1.5.1 --- General comparison between aptamer and antibody --- p.24 / Chapter 1.5.1.1 --- Diversity --- p.24 / Chapter 1.5.2 --- Specificity --- p.26 / Chapter 1.5.3 --- Disadvantages of antibody --- p.26 / Chapter 1.5.4 --- Advantages of aptamer --- p.27 / Chapter 1.6 --- Project Objectives --- p.29 / Chapter Chapter 2. --- Materials and Methods --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Chemicals --- p.32 / Chapter 2.1.2 --- Buffers --- p.36 / Chapter 2.1.2.1 --- Buffers commonly used --- p.37 / Chapter 2.1.2.2 --- Reagents for molecular work --- p.37 / Chapter 2.1.3 --- Bacterial Culture --- p.38 / Chapter 2.1.4 --- Culture of cell --- p.38 / Chapter 2.1.4.1 --- "TNF-α Sensitive Cell Line, L929" --- p.38 / Chapter 2.1.4.2 --- Medium for cell culture --- p.38 / Chapter 2.1.5 --- Reagent for Western blotting --- p.39 / Chapter 2.1.5.1 --- Protein extraction --- p.39 / Chapter 2.1.5.2 --- SDS-PAGE --- p.40 / Chapter 2.1.5.3 --- Electro-blotting --- p.41 / Chapter 2.2 --- Methods --- p.42 / Chapter 2.2.1 --- Conjugation of protein to solid support --- p.42 / Chapter 2.2.1.1 --- Conjugation of protein on PVDF membrane --- p.42 / Chapter 2.2.4.2 --- Conjugation of protein on Sepharose --- p.42 / Chapter 2.2.4.3 --- Conjugation of protein on magnetic bead --- p.42 / Chapter 2.2.2 --- SELEX --- p.43 / Chapter 2.2.2.1 --- Selection --- p.43 / Chapter 2.2.2.2 --- Photo-selection --- p.44 / Chapter 2.2.2.3 --- PCR --- p.45 / Chapter 2.2.3 --- Separation of oligonucleotides --- p.46 / Chapter 2.2.3.1 --- Separate short length double-stranded oligonucleotides by using polyacrylamide gel --- p.46 / Chapter 2.2.3.2 --- Separate short length single-stranded oligonucleotides by using denaturing polyacrylamide gel --- p.47 / Chapter 2.2.3.3 --- Extract the DNA from polyacrylamide gel --- p.48 / Chapter 2.2.3.4 --- Estimate the amount of DNA in solution after extraction --- p.49 / Chapter 2.2.3.5 --- Agarose Gel Electrophoresis --- p.49 / Chapter 2.2.4 --- Cloning of selected polyclonal aptamer --- p.50 / Chapter 2.2.4.1 --- Restriction cutting --- p.50 / Chapter 2.2.4.2 --- Ligation --- p.50 / Chapter 2.2.4.3 --- Preparation of the competent cells --- p.50 / Chapter 2.2.4.4 --- Transformation of plasmid into competent cell --- p.51 / Chapter 2.2.4.5 --- Plasmid extraction from bacterial culture --- p.51 / Chapter 2.2.5 --- Cell culture --- p.52 / Chapter 2.2.5.1 --- Cell culture of L929 --- p.52 / Chapter 2.2.5.2 --- Preservation of cells --- p.52 / Chapter 2.2.5.3 --- Treatment with TNF-α --- p.53 / Chapter 2.2.5.4 --- Fixation of cells --- p.53 / Chapter 2.2.6 --- Western blotting analysis --- p.54 / Chapter 2.2.6.1 --- Preparation of proteins from cells --- p.54 / Chapter 2.2.6.2 --- SDS polyacrylamide gel electrophoresis (SDS-PAGE) --- p.54 / Chapter 2.2.6.3 --- Electroblotting of protein --- p.55 / Chapter 2.2.6.4 --- Probing antibodies or aptamers for proteins --- p.55 / Chapter 2.2.6.5 --- Enhanced chemiluminescence (ECL) Assay --- p.56 / Chapter Chapter 3. --- Results --- p.57 / Chapter 3.1 --- Selection of aptamer against cytochrome c dotted on membrane with counter selection against BSA on membrane --- p.58 / Chapter 3.1.1 --- Selection process --- p.58 / Chapter 3.1.1.1 --- PCR cycles --- p.59 / Chapter 3.1.1.2 --- Polyclonal aptamer --- p.61 / Chapter 3.1.1.3 --- Monoclonal aptamer Contents --- p.63 / Chapter 3.1.2 --- Binding test of cy-1 to cy-4 to cytochrome c --- p.65 / Chapter 3.1.3 --- Binding of cy-3 to the cytochrome c dotted on PVDF membrane --- p.67 / Chapter 3.1.4 --- Test the binding of cy-3 with cytochrome c by ELISA --- p.68 / Chapter 3.1.5 --- Competitive binding between monoclonal aptamer cy-3 and anti-cytochrome c antibody --- p.70 / Chapter 3.1.6 --- Western blotting of pure cytochrome c by cy-3 --- p.71 / Chapter 3.1.7 --- Western blotting of pure cytochrome c from different species --- p.73 / Chapter 3.1.8 --- Cell lysate SDS-PAGE labeled with cy-3 --- p.75 / Chapter 3.1.9 --- Cell lysate labeled with cy-1 to cy-9 after SDS-PAGE --- p.77 / Chapter 3.2 --- Selection of cytochrome c-specific aptamer with counter selection against cytosolic protein --- p.79 / Chapter 3.2.1 --- Selection of aptamer against cytochrome c with counter selection against cytosolic cell lysate --- p.79 / Chapter 3.2.2 --- Selection of aptamer against cytochrome c by fixed cell followed by cytochrome c elution --- p.82 / Chapter 3.2.3 --- Selection of aptamer from cytochrome c band --- p.84 / Chapter 3.3 --- Primers Testing --- p.86 / Chapter 3.3.1 --- Cell lysate labeled with primers after SDS-PAGE --- p.86 / Chapter 3.3.2 --- Cell lysate labeled with cy-3 without primers --- p.87 / Chapter 3.3.3 --- Test the effect of sense oligonucleotide --- p.89 / Chapter 3.3.4 --- Sequence of monoclonal aptamer --- p.90 / Chapter 3.3.5 --- Cell lysate labeled with aptamers without primer ends --- p.92 / Chapter 3.3.6 --- Test of the aptamers after mutations --- p.93 / Chapter 3.3.7 --- Test for other biotinylated primers --- p.96 / Chapter 3.4 --- Elimination of non-specific binding --- p.98 / Chapter 3.4.1 --- Different types of cell lysate --- p.98 / Chapter 3.4.2 --- Heating effect on the non-specific binding --- p.99 / Chapter 3.4.3 --- Using milk as a blocking agent --- p.101 / Chapter 3.4.3.1 --- Milk blocked membrane --- p.101 / Chapter 3.4.3.2 --- Milk prevented the binding of aptamer to cytochrome c --- p.102 / Chapter 3.4.3.3 --- Cell lysate labeled with cy-3 after SDS-PAGE by using milk as blocking agent --- p.104 / Chapter 3.4.3.4 --- Aptamer selection against cytochrome c in the presence of milk --- p.105 / Chapter 3.4.4 --- Using DNA as a Blocking agent --- p.107 / Chapter 3.4.4.1 --- DNA blocked the non-specific binding --- p.107 / Chapter 3.4.4.2 --- Cell lysate labeled with cy-3 after SDS-PAGE by using DNA as blocking agent --- p.109 / Chapter 3.4.4.3 --- Selection against cytochrome c blocked by DNA --- p.110 / Chapter 3.4.4.4 --- "Labeling of cell lysate treated with DNase, RNase or both after SDS-PAGE" --- p.112 / Chapter 3.5 --- Photo-SELEX --- p.114 / Chapter 3.5.1 --- Selection process --- p.114 / Chapter 3.5.2 --- Cell lysate labeled with photo-aptamer --- p.116 / Chapter 3.5.3 --- Testing by immunoprecipitation --- p.118 / Chapter 3.6 --- Application --- p.120 / Chapter 3.6.1 --- Detection of the cytochrome c in cytosolic proteins after treatment of TNF-α --- p.120 / Chapter 3.6.2 --- Detection of the cytochrome c in total cell lysate after treatment of TNF-α --- p.123 / Chapter 3.6.3 --- Detection of cytochrome c in different cellular compartments after treatment of TNF-α --- p.125 / Chapter Chapter 4. --- Discussion --- p.130 / Chapter 4.1 --- General information --- p.131 / Chapter 4.1.1 --- The pool of oligonucleotide --- p.131 / Chapter 4.1.2 --- Design of oligonucleotides --- p.131 / Chapter 4.1.3 --- SELEX --- p.133 / Chapter 4.1.3.1 --- Buffer condition of selection --- p.133 / Chapter 4.1.3.2 --- Binding equilibrium --- p.134 / Chapter 4.1.3.3 --- Prevalence of matrix-binding species --- p.134 / Chapter 4.2 --- Selection --- p.135 / Chapter 4.2.1 --- Cycle numbers of PCR --- p.135 / Chapter 4.3 --- Assay of aptamers selected --- p.137 / Chapter 4.3.1 --- The use of biotin-streptavidin for recognition --- p.137 / Chapter 4.3.2 --- Polyclonal aptamers --- p.137 / Chapter 4.3.3 --- Monoclonal aptamer --- p.137 / Chapter 4.3.4 --- Cy-3 shows the highest affinity to cytochrome c --- p.138 / Chapter 4.3.5 --- The presence of non-specific binding --- p.138 / Chapter 4.4 --- Counter selection against cell lysate --- p.140 / Chapter 4.5 --- Primer testing --- p.143 / Chapter 4.6 --- Sequences and secondary structures of monoclonal aptamers --- p.145 / Chapter 4.7 --- Elimination of non-specific binding Contents --- p.147 / Chapter 4.7.1 --- Non-specific binding may be mediated by sequence-independent recognition --- p.147 / Chapter 4.7.2 --- Elimination of non-specific binding by milk --- p.147 / Chapter 4.7.3 --- Eliminate the non-specific binding by using DNA --- p.149 / Chapter 4.8 --- Photo-aptamer --- p.151 / Chapter 4.9 --- Application of the monoclonal aptamer cy-3 --- p.153 / Chapter 4.9.1 --- Aptamer can label cytochrome c as antibody does --- p.153 / Chapter 4.10 --- Conclusion I --- p.158 / Chapter 4.11 --- Conclusion II --- p.159 / Chapter Chapter 5 --- References --- p.161