Contents
CONTENTS
Certificate --------------------------------------------------------------------------- i
Acknowledgement -------------------------------------------------------------- ii-iv
Abbreviations ---------------------------------------------------------------------- v-vii
Aim and 0 bj ectives ------------------------------------------------------------------ viii
CHAPTER 1: REVIEW OF LITERATURE -------------------------------------1-35
1.1 Irttro<i11ction--------------------------------------------------------------------------- 1-3
1.2 ~i\ vvorl<i---------------------------------------------------------------------------- 4
1.3 ~i\ aptamers------------------------------------------------------------------------ 4-6
1.4 Designing combinatorial library of DNJ\ an<i ~}\ ---------------------------- 6
1.4.1 DNJ\ combinatorial library Clesign ----------------------------------------- 6
1.4.2 ~}\combinatorial library synthesis -------------------------------------- 8
1.5 The SELEX process ----------------------------------------------------------------- 9-10
1.6 Properties of therape11tics ~i\ aptamers ---------------------------------------- 10
1.6.1 Efficiency an<i specificity---------------------------------------------------- 10
1.6.2 Pharmacokinetics an<i n11clease resistance -------------------------------- 10
1.6.3 Clearance----------------------------------------------------------------------- 10
1.6.4 Toxicity an<i imm11nogenicity ----------------------------------------------- 11
1.6.5 1\Clministration----------------------------------------------------------------- 11
1.6.6 Scalability, cost an<i stability------------------------------------------------ 12
1. 7 i\pplications of aptamers ----------------------------------------------------------- 12
1. 7.1 i\ptazyrnes --------------------------------------------------------------------- 12
1.7.2 Protein targete<i inhibitors --------------------------------------------------- 12
1. 7.3 Target i<ientification an<i vali<iation ---------------------------------------- 13
1. 7.4 Therape11tics ------------------------------------------------------------------- 13
1. 7.5 i\ptamers in cancer me<iicines ---------------------------------------------- 13
1. 7.6 Reg11lation of gene expression ---------------------------------------------- 13
1.7. 7 Combating infectio11s agents ------------------------------------------------ 13
1. 7.8 i\nti-i<iiotype approach------------------------------------------------------- 13
1. 7.9 i\ffinity p11rification me<ii11m------------------------------------------------ 14
1. 7.10 Diagnostics an<i biosensors ------------------------------------------------ 14
1. 7.11 Irttramers ---------------------------------------------------------------------- 15
1. 7.12 Molec11lar svvitch assays---------------------------------------------------- 15
1. 7.13 Biochips----------------------------------------------------------------------- 15
1.8 ~i\ secon<iary strllctme ----------------------------------------------------------- 16-17
1.9 ~i\ strllctme pre<iiction----------------------------------------------------------- 18-20
1.10 Tertiary strllctme of ~i\ --------------------------------------------------------- 20-22
1.11 i\Clvancement of aptamer research an<i technology --------------------------- 22-24
1.12 Gl11tathione -------------------------------------------------------------------------- 24-25
1.12.1 Gl11tathione an<i cancer----------------------------------------------------- 25-26
1.12.2 Gl11tathione an<i apoptosis -------------------------------------------------- 27-29
1.12.3 Gl11tathione Clepletion an<i cancer therapy ------------------------------- 29-31
1.12.4 Gl11tathione homeostasis---------------------------------------------------- 31
1.12 5 Gl11tathione an<i gl11tathione-S-transferases (GSTs) -------------------- 31
1.12.6 Gl11tathione Clepletion an<i Bcl-2 ------------------------------------------ 32-33
Contents
1.12.7 Glutathione and telomerase activity-------------------------------------- 33-35
CHAPTER 2: MATERIALS AND METHODS
2.1 Materials -------------------------------------------------------- 36-48
2.1.1 Chemicals---------------------------------------------------------------------- 36
2.1.2 Media stock solutions, reagents and buffers ------------------------------ 37-40
2.1.3 Generation of nucleic acid library------------------------------------------ 40
2.1.3.1 DNA end labeling------------------------------------------------------ 40
2.1.3.2 PCR reaction------------------------------------------------------------ 40
2.1.4 Primers used for SELEX ----------------------------------------------------- 41
2.1.5 Primers used for expression ------------------------------------------------- 41
2.1.6 In vitro transcription---------------------------------------------------------- 41
2.1. 7 Column and beads ------------------------------------------------------------ 41
2.1. 7.1 Columns ----------------------------------------------------------------- 41
2.1. 7.2 Bead---------------------------------------------------------------------- 42
2.1.8 R1r PCR-------------------------------------------------------------------------42
2.1.8.1 Primers used for R1r-PCR --------------------------------------------- 42
2.1.9 Bacterial strains --------------------------------------------------------------- 42
2.1.1 0 Cell lines---------------------------------------------------------------------- 42
2 .1.11 Plasmids ---------------------------------------------------------------------- 4 3-44
2.1.11.1 Plasmid used for expression of GSH aptamers------------------- 44
2.1.12 GSH aptamers used for binding characterization----------------------- 44
2.1.13 GSH aptamers and truncated GSH aptamer used for SPR ------------ 45
2.1.14 GSH aptamers and truncated RNA aptamer used for CD------------- 45
2.1.15 Software used for structural characterization ofGSH aptamers------ 46
2.1.16 Binding and kinetic characterization------------------------------------- 47
2.1.17 Cell culture ------------------------------------------------------------------- 48
2.2 Methods ----------------------------------------------------------------- 49-75
2.2.1 Preparation of competent cells and their transformation---------------- 49
2.2.2 Long term storage of bacterial cultures------------------------------------ 49
2.2.3 Alkaline lysis (mini preparation) for plasmid isolation ----------------- 49
2.2.4 LiCl procedure for DNA isolation------------------------------------------ 50
2.2.5 Midi preparation for plasmid------------------------------------------------ 50
2.2.6 Genomic DNA isolation from cultured mammalian cells--------------- 51
2.2. 7 1rotal RNA isolation from mammalian cells ------------------------------ 51
2.2.8 Agarose gel electrophoresis ------------------------------------------------- 52
2.2.9 Extraction ofDNA from agarose gel: phenol freeze fraction method- 52
2.2.1 0 Urea PAGE------------------------------------------------------------------- 53
2.2.11 Phenol chloroform extraction---------------------------------------------- 53
2.2.12 Ethanol precipitation-------------------------------------------------------- 53
2.2.13 Measurement of DNA and RNA concentrations------------------------ 53
2.2.14 Generation of nucleic acid library ---------------------------------------- 54
2.2.14.1 Primer annealing reaction ------------------------------------------- 54
2.2.14.2 End Labeling of dsDNA ---------------------------------------------55
2.2.14.3 Klenow end filling reaction----------------------------------------- 55
2.2.14.4 PCR Reaction--------------------------------------------------------- 55
2.2.15 In- Vitro transcription-------------------------------------------------------- 56
2.2.15.1 Purification of RNA-------------------------------------------------- 56
2.2.15.2 Column purification and percentage incorporation-------------- 57
2.2.16 SELEX ------------------------------------------------------------------------ 57-60
Contents
2.2.16.1 Selection ofGSH aptamers (affinity chromatography---------- 58
2.2.16.2 Elution scheme-------------------------------------------------------- 59
2.2.16.3 RT-PCR ---------------------------------------------------------------- 59-60
2.2.17 Cloning and sequencing of GSH aptamers ------------------------------ 60
2.2.18 Automated DNA sequencing---------------------------------------------- 60
2.2.19 Sequence analysis and secondary structures prediction --------------- 61
2.2.20 Tertiary structure prediction ---------------------------------------------- 61
2.2.21 Docking Studies of GSH aptamers --------------------------------------- 62
2.2.22 Binding assays and in vitro kinetic characterization ------------------- 62
2.2.22.1 Effect of RNA concentration on binding-------------------------- 62-63
2.2.22.2 Time course of binding and elution-------------------------------- 63
2.2.22.3 Competition assay and specificity assay -------------------------- 63
2.2.22.4 In vitro kinetic characterization ------------------------------------ 63-64
2.2.22.5 Surface Plasmon resonance studies -------------------------------- 64-65
2.2.22.6 Circular dichorism (CD) studies------------------------------------ 65
2.2.23 BLAST------------------------------------------------------------------------ 65
2.2.24 Cloning of GSH aptamer in expression vectors------------------------- 66
2.2.24.1 PCR Reaction with U6 primers------------------------------------- 66
2.2.24.2 Restriction digestion of vector and insert ------------------------- 66-67
2.2.24.3 Ligation of insert into vector DNA -------------------------------- 67
2.2.24.4 Screening of clones--------------------------------------------------- 67-68
2.2.25 Cell culture--------------------------------------------------------------------- 68-70
2.2.25.1 --------------------------------------------------------------------------- 68
2.2.25.2 Splitting of culture cells -----------------------------------------,---- 68
2.2.25.3 Determining the viability of cells ---------------------------------- 69
2.2.25.4 Freezing human cells grown in monolayer culture -------------- 70
2.2.25.5 Thawing and recovering human cells------------------------------ 70
2.2.26 GSH aptamers transfection------------------------------------------------- 70
2.2.27 Analysis of transfection efficiencies-------------------------------------- 71
2.2.28 Cell cycle analysis----------------------------------------------------------- 71
2.2.29 Measurement of ROS production----------------------------------------- 71
2.2.30 Hoechst 33258 nuclear staining------------------------------------------- 72
2.2.31 RT PCR ----------------------------------------------------------------------- 72
2.2.32 Apoptotic DNA ladder assay---------------------------------------------- 73
2.2.33 Caspase assay ---------------------------------------------------------------- 73-7 4
2.2.33.1 Quantification by flow cytometry ---------------------------------- 73
2.2.33.2 Detection by fluorescence microscopy---------------------------- 74
2.2.33.3 Analysis by fluorescence plate reader----------------------------- 74
2.2.34 Effect of caspase Inhibition and DTT ------------------------------------ 74
2.2.35 Cell viability ----------------------------------------------------------------- 74
2.2.36 Glutathione estimation------------------------------------------------------ 74-7 5
2.2.37 Effects ofGSH RNA aptamers in 293T cells and non-specific------ 75
RNAs control for transfection studies
CHAPTER3:
SELECTION AND CHARACTERIZATION OF GLUTATHIONE BINDING
RNA APTAMERS ----------------------------------------------------- 76-87
3.1 Generation of oligonucleotides library ------------------------------------------- 76-79
3.2 SELEX (Selection of GSH-binding RNA aptamers) --------------------------- 79-81
3.3 Cloning-------------------------------------------------------------------------------- 81-83
Contents
3.4 Nucleotide sequences of aptamers ------------------------------------------------ 83-87
CHAPTER4:
PHYLOGENETIC STUDIES AND STRUCTURAL CHARACTERIZATION
-------------------------------------------------------------- 88-117
4.1 Phylogenetic studies of glutathione-binding RNA aptamers ------------------ 88-90
4.2 Multiple sequence alignment of GSH aptamers -------------------------------- 90-110
4.2.1 Multiple alignment of class-I aptamers ------------------------------------ 90
4.2.2 Multiple alignment of class-II aptamers ----------------------------------- 90-91
4.2.3 Multiple alignment of class-III aptamers ---------------------------------- 91
4.2.4 Multiple alignment of class-IV aptamers ---------------------------------- 92
4.2.5 Multiple alignment of class-V aptamers ----------------------------------- 92-93
4.3 Secondary structures prediction of glutathione-binding RNA aptamers----- 93-97
4.4 Structural alignment and comparisons-------------------------------------------- 98-99
4.5 Tertiary structures prediction and docking--------------------------------------- 100-110
4.6 Homology of Aptamer Sequence with Natural Sequences -------------------- 111-117
CHAPTERS:
KINETICS OF APTAMERS-LIGAND INTERACTION -----------------118-128
5.1 Effect of RNA (GSH aptamers) concentrations on binding------------------- 118-119
5.2 Time course of binding and release----------------------------------------------- 119-120
5.3 Determination ofKd using isocratic affinity chromatography---------------- 120-121
5.4 Competition and specificity assays------------------------------------------------ 122-123
5.5 Surface plasmon resonance studies ----------------------------------------------- 124-127
5.6 Circular Dichorism ------------------------------------------------------------------ 12 7-128
CHAPTER6:
BIOLOGICAL EFFECTS OF GSH APTAMERS IN MCF CELL LINE .. 129-148
6.1 PCR amplification of GSH aptamers --------------------------------------------- 129-130
6.2 In vitro production of GSH aptamers --------------------------------------------- 130
6.3 Phase contrast images of MCF -7 -------------------------------------------------- 130-131
6.4 GSH RNA aptamers cause altered cell morphology
indication of apoptosis -----------------------------------------------------------------6.5 RT-PCR shows presence oftransfected RNA in cells-------------------------6.6 Cloning of GSH aptamers in pTZU6+27 ----------------------------------------6.6.1 PCR amplification for incorporation of restriction sites ---------------6.6.2 Cloning of GSH aptamers --------------------------------------------------6. 7 Flow cytometry ---------------------------------------------------------------------6.8 RNA aptamer induces generation of ROS --------------------------------------6.9 DNA fragmentation and nuclear staining ---------------------------------------6.10 Activation of caspase 3 following transfection with GSH aptamers-------6.11 Effects ofGSH aptamers and Non-specific RNAs on cell viability
in 293 T cells----------------------------------------------------------------------------6.12 Effect ofGSH aptamers on GSH levels-----------------------------------------
131
132
133-135
133
134-13 5
13 6-13 7
13 8-140
140-141
142-144
144-147
147-148
CHAPTER 7: DISCUSSION ---------------------------------------------149-154
Summary and Conclusions------------------------------------------------ 155-160
Future prospects ----------------------------------------------------- 161
References ----------------------------------------------------------------- 162-172

Supplemental Materials - Molecular Biology of the Cell