viii
TABLE OF CONTENTS
CHAPTER
1
TITLE
PAGE
TITLE
i
SUPERVISOR’S DECLARATION
ii
DECLARATION
iii
DEDICATION
iv
ACKNOWLEDGEMENTS
v
ABSTRACT
vi
ABSTRAK
vii
TABLE OF CONTENTS
viii
LIST OF TABLES
xi
LIST OF FIGURES
xii
LIST OF ABBREVIATIONS
xv
LIST OF APPENDICES
xix
INTRODUCTION
1.1
Background of Study
1
1.2
Problem Statement
2
1.3
Research Objectives
3
1.4
Research Scope
3
1.5
Research Significance
4
ix
2
LITERATURE REVIEW
2.1
Influenza A Viruses
5
2.1.1
5
Evolutionary Process of Influenza A Viruses
2.1.2 Influenza A Virus: Structure and Function
2.2
2.1.3 Influenza A H1N1 2009
11
NS1 Protein
12
2.2.1
NS1A RNA Binding Domain
16
2.2.2
Effects of NS1A Gene Variation on
Structure and Function
2.3
2.4
3
17
Directed Evolution
18
2.3.1 Error-prone PCR
19
Bioinformatics Application
21
2.4.1 Protein Modeling
21
2.4.2
2.5
6
2.4.1.1 Comparative Protein Modeling
23
Protein Model Validation Tools
26
Nucleic Acid Aptamers
27
2.5.1
Advantage of Aptamers over Antibodies
28
2.5.2
Aptamers as Antiviral Drugs
29
MATERIALS AND METHODS
3.1
Experimental Design
31
3.2
Preparation of Luria-Bertani (LB) Broth and Agar
31
3.3
Culturing of Recombinant E. coli and Plasmid Extraction
32
3.4
Random Mutagenesis using Error-prone PCR
33
3.5
Separation of Bands using Gel Electrophoresis
34
3.6
Cloning and Transformation
35
3.6.1 Cloning of Mutated Genes into yT&A Vector
35
3.6.2
3.6.3
3.7
Transformation of Recombinant Plasmids into
E. coli DH5α
36
Screening for Clones with the Desired Inserts
37
Analysis of Mutants using Bioinformatics Tools
38
3.7.1
Sequence Analysis
38
3.7.2
Comparative Protein Modeling
39
x
4
3.7.3 Protein Validation
39
3.7.4
40
Molecular Docking
RESULTS AND DISCUSSION
4.1
Error-prone PCR
4.1.1
Error prone PCR with Various Concentrations of
MgCl2
4.1.2
43
Error-prone PCR with Increased Number of Cycles 45
4.2
Cloning and Colony Screening
46
4.3
Multiple Sequence Alignment
50
4.4
Comparative Protein Modeling of NS1 Protein and Structure
4.5
4.6
5
42
Error-prone PCR with Various Concentrations of
MnCl2
4.1.3
41
Analysis
57
Model Quality Validation
59
4.5.1
60
ERRAT
4.5.2 PROCHECK
62
4.5.3 Verify3D
65
4.5.4 ProSA-web
68
Structural Alignment between Wild-type NS1 Protein and
Mutants
71
4.7
Protein Side Chain Interactions
74
4.8
Prediction of Hot Spot Mutations
76
4.9
RNA Modeling and Validation
80
4.10
Molecular Docking Analysis
87
CONCLUSIONS AND FUTURE WORK
5.1
Conclusion
95
5.2
Future Works
96
REFERENCES
97
APPENDICES
APPENDIX A
111
xi
LIST OF TABLES
TABLE NO.
2.1
TITLE
PAGE
A brief summary on influenza A viral proteins and functions
(reviewed by O’Donnell and Subbarao, 2011)
8
2.2
NS1 protein amino acid functions.
15
4.1
NS1 variant library with change in chemical properties
56
4.2
Total energy of wild-type NS1A and mutant protein models
59
4.3
Validation of models using PROCHECK
64
4.4
RMSD calculations of mutant proteins
73
4.5
Mutability of wt NS1A protein residues obtained from error-prone
PCR
4.6
79
Secondary and tertiary of RNA aptamers predicted from
sequence
82
4.7
Validation of RNA aptamers via Molprobity
86
4.8
Docking energy or free energy of binding (kJ/mol)
87
4.9
List of hydrogen bonds between proteins and aptamers
88
4.10
Protein-RNA aptamer docking based on lowest free energy
conformation
93
xii
LIST OF FIGURES
FIGURE NO.
2.1
TITLE
PAGE
Structure of influenza A virus with 8 RNA segments that code
for viral proteins (Vincent et al., 2008)
7
2.2
Evolutional history of 2009 A (H1N1) virus (Khanna et al., 2009) 12
2.3
Diagram of NS1 protein structure and interactions with other
biological molecules (Hale et al., 2008)
14
2.4
Schematic diagram of error-prone PCR (Fujii et al., 2004)
21
2.5
Schematic diagram of steps involved for comparative protein
modeling (Sanchez et al., 2000)
2.6
24
Relationship between level of sequence identity in comparative
modeling and various applications in computational biology
(Sanchez et al., 2000)
25
2.7
Schematic diagram of SELEX process (Lee et al., 2010)
28
3.1
Map of yT&A cloning vector (Yeastern Biotech)
35
3.2
Multiple cloning sites in sequence of yT&A cloning
vector (Yeastern Biotech)
36
4.1
Effects of varying concentrations of MgCl2 on PCR products
42
4.2
PCR products with addition of MnCl2 ranging from 1µM to
20µM
43
xiii
4.3
PCR products with addition of MnCl2 ranging from 10 µM to
40 µM
4.4
44
PCR products with addition of MnCl2 ranging from 60 µM to
150 µM
44
4.5
DNA bands after error prone PCR of 80 cycles
45
4.6
Blue-white colonies on LB agar containing X-gal and IPTG after
TA cloning and transformation into E. coli DH5α
4.7
47
Screening of transformed colonies via colony PCR and products
resolved using gel electrophoresis
4.8
Screening of transformed colonies via colony PCR and products
resolved using gel electrophoresis
4.9
49
Screening of transformed colonies via colony PCR and products
resolved using gel electrophoresis
4.12
49
Screening of transformed colonies via colony PCR and products
resolved using gel electrophoresis
4.11
48
Screening of transformed colonies via colony PCR and products
resolved using gel electrophoresis
4.10
48
50
Nucleotide sequence of NS1A mutants aligned with the wild-type
NS1A gene for sequence comparison and identification of mutation
sites using Jalview program
4.13
51
Amino acid sequence of the mutant NS1A proteins aligned with the
amino acid sequence of NS1A protein for mutation identification
using Jalview program
4.14
54
Cartoon representation wild-type NS1 protein model
from influenza A virus (A/California/04/2009(H1N1)) viewed in
4.15
PyMOL
58
ERRAT plot of each protein with overall quality factor
61
xiv
4.16
Ramachandran plots generated via PROCHECK for different
protein structures
63
4.17
3D profile window plots of structures
66
4.18
Protein quality scores generated through ProSA web server
69
4.19
Structural alignment of all mutants against wt-NS1A protein
based on all Cα atoms with arrows pointing to mutation sites
4.20
Difference between wt NS1A and mutant proteins based on amino
acid side chain hydrogen bond interactions
4.21
72
74
The amino acid sequence of the wt NS1A protein with each
residue represented by mutability colour scale with 1 (lowest)
represented in blue to 9 (highest) represented in red
4.22
77
The ‘hot spots’ of wt-NS1A protein predicted via Hotspot Wizard
server prepared in PyMOL. The spheres in magenta indicate
4.23
‘hot spot’ residues.
78
Docking of wt-NS1A protein to aptamer 2
90
xv
LIST OF SYMBOLS/ ABBREVIATIONS/ NOTATIONS/
TERMINALOGY
A
-
Adenine
Ampr
-
Ampicillin resistant
BLAST
-
Basic Local Alignment Search Tool
bp
-
Base pairs
C
-
Cytosine
CASP
-
Critical Assessment of Structure Prediction
CPSF30
-
30-kDa subunit of the cellular cleavage and polyadenylation
specificity factor
dH2O
-
Distilled water
DNA
-
Deoxyribonucleic acid
dNTPs
-
Deoxynucleotide triphosphates
dsRNA
-
Double stranded RNA
E. coli
-
Escherichia coli
ED
-
Effector domain
eIF4F
-
Translation initiation factor
ELISA
-
Enzyme-linked immunosorbent assay
EP-PCR
-
Error-prone polymerase chain reaction
EtBr
-
Ethidium bromide
g
-
Gram
G
-
Gravitational force
G
-
Guanine
G-factor
-
Goodness factor
GUI
-
Graphical User Interface
h
-
Hour
HA
-
Hemagglutinin
H
-
Histidine
xvi
IFN
-
Interferon
IPTG
-
Isopropyl-β-D-thiogalactoside
K
-
Kelvin
Kd
-
Dissociation constant
kDa
-
Kilo Dalton
kJ
-
Kilo Joule
L
-
Liter
LB
-
Luria-Bertani
m
-
Mille
MFE
-
Minimum free energy
ml
-
Milliliter
mg/ml
-
Milligram/milliliter
Mg2+
-
Magnesium ion
Mn2+
-
Manganese ion
MgCl2
-
Magnesium chloride
MnCl2
-
Manganese chloride
mmol/L; mM -
Milli molar
mRNA
-
Messenger RNA
NA
-
Neuraminidase
NaCl
-
Sodium chloride
NCBI
-
National Center for Biotechnology Information
NEP
-
Nuclear export protein
NES
-
Nuclear export signal
NLS
-
Nuclear localization sequence/signal
NoLS
-
Nucleolar localization signal
NMR
-
Nuclear magnetic resonance
ns
-
Nano second
No.
-
Number
NS1
-
Nonstructural protein 1
OAS
-
Oligo (A) synthetase
PABP
-
Poly (A)-binding protein
PCR
-
Polymerase chain reaction
PDB
-
Protein Data Bank
PI3K
-
Phosphatidylinositol 3-kinase
xvii
PKR
-
Protein kinase R
ProSA
-
Protein Structure Analysis
ps
-
Pico second
RBD
-
dsRNA-binding domain
RMSD
-
Root mean square deviation
RNA
-
Ribonucleic acids
RNP
-
Ribonucleoprotein
rpm
-
Rounds per minute
s
-
Seconds
SDS-PAGE
-
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SELEX
-
Systematic evolution of ligands by exponential enrichment
ssRNA
-
Single stranded RNA
T
-
Thymine
TAE
-
Tris-Acetate electrophoresis buffer
Taq
-
Thermus aquaticus
Trp
-
Tryptophan
µl
-
Microliter
µg/ml
-
Microgram/milliliter
µM
-
Micro molar
U
-
Uracil
UV
-
Ultraviolet
v
-
Volt
vRNP
-
Viral ribonucleoprotein
WHO
-
World Health Organization
wt
-
Wild-type
w/v
-
Weight/volume
X-gal
-
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
3D
-
Three-dimensional
Å
-
Angstrom
Α
-
Alpha
Β
-
Beta
°C
-
Degree Celsius
γ
-
Gamma
δ
-
Delta
xviii
ε
-
Epsilon
ζ
-
Zeta
η
-
Eta
Φ
-
Phi
Ψ
-
Psi
xix
LIST OF APPENDICES
APPENDIX
A
TITLE
Map of pET-32c(+) cloning vector (Novagen).
PAGE
118