Esterase 2-oligodeoxynucleotide conjugates as enzyme reporter for electrochemical detection of DNA and identification of bacterial species [Elektronische Ressource] / by Yiran Wang

Esterase 2-oligodeoxynucleotide conjugates as enzyme
reporter for electrochemical detection of DNA and
identification of bacterial species






A Thesis Submitted for the Degree of
Doktor der Naturwissenschaften

-Dr. Rer. Nat.-

der Fakultät für Biologie, Chemie und Geowissenschaften der

Universität Bayreuth

by
Yiran Wang


From

Zhejiang
P. R. China

Bayreuth, 2006



Die vorliegende Arbeit wurde in der Zeit von August 2003 bis November 2006 am Lehrstuhl
für Biochemie der Universität Bayreuth unter der Leitung von Herrn Prof. Dr. Mathias Sprinzl
angefertigt.


Vollständiger Abdruck der von der Fakultät Biologie, Chemie und Geowissenschaften der
Universität Bayreuth genehmigten Dissertation zu Erlangung des Grades eines Doktors der
Naturwissenschaften
- Dr. rer. nat.-











Promotionsgesuch eingericht am: 29. November 2006
Tag des Promotionskolloquiums: 8. Februar 2007



Erster Gutachter: Prof. Dr. Mathias Sprinzl
Zweiter Gutachter: Prof. Dr. Gerhard Krauss
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Table of Contents

Table of Contents .................................................................................................II
Abbreviations ..................................................................................................... VI
1. Introduction.......................................................................................................1
1.1 Electrochemical detection of nucleic acids........................................................................1
1.1.1 Introduction of electrochemical nucleic acid biosensor..............................................1
1.1.2 Electrochemical biosensing of DNA hybridization ....................................................3
1.1.2.1 Sensor structure of fully integrated electrical DNA chip.....................................3
1.1.2.2 Capture immobilization........................................................................................4
1.1.2.3 DNA amplification...............................................................................................5
1.1.2.4 The hybridization event7
1.1.2.5 Electrochemical transduction of DNA hybridization...........................................7
1.1.2.5.1 Direct oxidization of nucleobases .................................................................8
1.1.2.5.2 Indirect oxidization of nucleobases...............................................................8
1.1.2.5.3 DNA-mediate charge transport .....................................................................9
1.1.2.5.4 Conductivity-based detection........................................................................9
1.1.2.5.5 Enzyme amplified transduction...................................................................10
1.2 Esterase 2 and its potential as a reporter enzyme.............................................................12
1.2.1 Structure and function of esterases ...........................................................................12
1.2.2 Esterase 2 from Alicyclobacillus acidocaldarius......................................................13
1.2.3 Mechanism of EST2 catalysis...................................................................................15
1.2.4 Trifluoromethyl ketones inhibit active-serine esterases............................................17
1.2.5 Affinity purification of esterase by trifluoromethyl ketones ligand..........................18
1.2.6 EST2 as a reporter enzyme .......................................................................................19
1.3 Hybridization behavior ....................................................................................................19
1.3.1 Properties of solution-phase hybridization................................................................19
1.3.2 Properties of solid-phase hybridization.....................................................................21
1.3.2.1 Thermodynamics and kinetics of solid-phase hybridization..............................21
1.3.2.2 Capture surface density......................................................................................22
1.3.2.3 Impact of capture layer structure........................................................................22
1.3.2.4 Impact of mismatches on solid-phase hybridization..........................................23
1.4 Bacterial species identification through detection of 16S rRNA.....................................23
1.5 Molecular beacon.............................................................................................................26
1.6 Statement of objectives ....................................................................................................28
2. Materials and Methods...................................................................................29
2.1 Materials...........................................................................................................................29
2.1.1 Instruments................................................................................................................29
2.1.2 Materials....................................................................................................................29
2.1.3 Chromatographic materials .......................................................................................29
2.1.4 Chemicals, enzymes and proteins .............................................................................29
2.1.4.1 Chemicals...........................................................................................................29
2.1.4.2 Enzymes and proteins ........................................................................................30
2.1.5 Bacterial strains.........................................................................................................30
2.1.6 Plasmids30
2.1.7 Oligodeoxynucleotides..............................................................................................31
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Table of Contents
2.1.7.1 Oligodeoxynucleotides for construction of mutant............................................31
2.1.7.2 Oligodeoxynucleotides for detection of DNA ...................................................31
2.1.7.3 Oligodeoxynucleotides for bacteria species identification ................................32
2.1.7.4 Oligodeoxynucleotides for molecular beacon....................................................32
2.1.8 Bacterial media .........................................................................................................32
2.1.9 Buffers and solutions ................................................................................................32
2.2 Methods............................................................................................................................34
2.2.1 Standard methods......................................................................................................34
2.2.1.1 Spectrophotometer determination of protein and nucleic acids.........................34
2.2.1.2 Bradford protein assay .......................................................................................34
2.2.1.3 Culture of bacteria..............................................................................................34
2.2.1.4 Gel electrophoresis.............................................................................................35
2.2.1.4.1 Agarose gel electrophoresis ........................................................................35
2.2.1.4.2 SDS-polyacrylamide gel electrophoresis ....................................................35
2.2.2 Isolation and purification of nucleic acids ................................................................36
2.2.2.1 DEPC treatment .................................................................................................36
2.2.2.2 Isolation of plasmid DNA ..................................................................................36
2.2.2.3 Purification of DNA fragments from agarose gels ............................................36
2.2.2.4 Acidified phenol method extraction of ribosomal RNA....................................36
2.2.2.5 Mini-preparation of ribosomal RNA..................................................................37
2.2.3 Recombinant DNA techniques37
2.2.3.1 Digestion of DNA with restriction endonucleases.............................................37
2.2.3.3 Cloning of PCR products ...................................................................................37
2.2.3.4 Ligation of DNA fragments ...............................................................................37
2.2.3.5 Site-directed mutagenesis of EST2 by overlap extension..................................38
2.2.4 Preparation and transformation of competent cells...................................................38
2.2.5 Normal PCR and asymmetry PCR............................................................................39
2.2.6 Protein purification ...................................................................................................39
2.2.6.1 Purification of A. acidocaldarius EST2 from E. coli Bl21(DE3)......................39
2.2.6.2 Preparation and purification of EST2-ODN conjugate......................................40
2.2.6.3 Preparation of EST2-streptavidin conjugate ......................................................40
2.2.7 Chemical synthesis....................................................................................................41
2.2.7.1 Preparation of trifluoromethyl ketone modified Sepharose...............................41
2.2.7.2 Synthesis of p-aminophenyl esters.....................................................................41
2.2.7.2.1 Preparation of p-am...........................................................41
2.2.7.2.2 EI-MS and NMR analysis of p-aminophenyl esters ...................................42
2.2.7.2.3 Analysis of purity and stability of p-aminophenyl esters............................43
2.2.8 SDS-PAGE gel esterase activity staining ................................................................43
2.2.9 Chip construction and instrumentation43
2.2.10 Esterase activity and kinetics spectrophotometer measurements44
2.2.10.1 Esterase activity assay by spectrophotometer..................................................44
2.2.10.2 Kinetic parameters measurement by spectrophotometer .................................45
2.2.11 Amperometric detection of EST2 ...........................................................................45
2.2.11.1 p-Aminophenol measurement ..........................................................................45
2.2.11.2 Determination of soluble esterase activity .......................................................45
2.2.11.3 Measurement of substrate specificity of immobilized esterase........................46
2.2.12 Pretreatment of electrodes and immobilization of capture ODN............................46
2.2.13 E-Chip detection of nucleic acids46
2.2.13.1 Low limit of detection......................................................................................46
2.2.13.2 Directly detection of mismatched capture ODN..............................................47
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Table of Contents
2.2.13.3 Detection of 49-mer ODN analyte ...................................................................47
2.2.13.4 Detection of a mismatch in a 510-nucleotide partial gene...............................47
2.2.13.5 Identification of bacterial species through 16S rRNA sequence .....................48
2.2.13.6 E-Chip EST2 activity assay .............................................................................48
2.2.14 Magnetic beads assisted preparation of ssDNA......................................................48
2.2.15 Modification of stem-loop structured ODN with 5’ thiol and 3’ biotin..................49
2.2.16 Construction and hybridization assay of stem-loop structured ODN .....................49
3. Results ..............................................................................................................52
3.1 Purification and biochemical properties of EST2, and synthesis and amperometric
characterization of its electrochemical substrate....................................................................52
3.1.1 Construction of EST2 mutant plasmid and its expression ........................................52
3.1.2 TFK-Sepharose purification of EST2E118C from E. coli Bl21(DE3) .....................53
3.1.3 Kinetic characterization of EST2E118C...................................................................54
3.1.4 Detergent effect and substrate specificity of EST2...................................................55
3.1.4.1 Effects of detergents to the activity of EST2 .....................................................55
3.1.4.2 Substrate specificity of EST2.............................................................................55
3.1.5 Synthesis and stability of p-aminophenyl esters .......................................................56
3.1.5.1 Synthesis of p-aminophenyl esters.....................................................................56
3.1.5.2 Analysis of the stability of p-aminophenyl esters..............................................57
3.1.6 Amperometric detection of EST258
3.1.6.1 Effect of various solvents to the activity of EST2 .............................................58
3.1.6.2 Substrate specificity of soluble EST2 ................................................................60
3.1.6.3 Substrate specificity of immobilized EST2........................................................60
3.1.7 Comparison of spectrophotometric and amperometric detection of EST2...............62
3.1.7.1 Calibration curve of p-nitrophenol and p-aminophenol.....................................62
3.1.7.2 Detection of EST2 by spectrophotometric and amperometric methods ............63
3.2 E-Chip based EST2-ODN conjugates detection of DNA ................................................65
3.2.1 Preparation and purification of EST2-A34 conjugates .............................................65
3.2.2 Preparation of EST2-streptavidin conjugates............................................................66
3.2.3 Sensitivity of the detection........................................................................................67
3.2.4 Selectivity of the detection69
3.2.4.1 Directly detection of mismatched capture ODN69
3.2.4.2 Detection of 49-mer ODNs analyte ...................................................................71
3.2.4.3 Detection of a mismatch in a single gene...........................................................72
3.3 E-Chip based bacterial species identification ..................................................................73
3.3.1 Comparison of 16S rRNA sequences of eight representative foodborne pathogens 73
3.3.2 Fragmentation of rRNA ............................................................................................75
3.3.3 Bacterial species identification based on the 16S rRNA sequences .........................76
3.4 Stem-loop structured ODN for oligodeoxynucleotide analyte detection78
4. Discussion.........................................................................................................79
4.1 Expression and purification of EST2 ...............................................................................79
4.2 Factors affectting EST2 specific activity .........................................................................80
4.3 Comparison of the spectrophotometric and amperometric methods for detection of
soluble EST2 ..........................................................................................................................81
4.4 Sensitivity of EST2-A34 conjugate for E-Chip detection of DNA..................................83
4.5 Capture ODN mismatch discrimination by the EST2-ODN conjugate and EST2-
streptavidin conjugate ............................................................................................................84
4.6 Discrimination of single nucleotide mismatches .............................................................87
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4.7 Bacterial species identification through 16S rRNA sequence .........................................89
4.8 Molecular beacon for oligodeoxynucleotide analyte detection .......................................90
5. Summary..........................................................................................................93
6. Zusammenfassung...........................................................................................94
7. Acknowledgement96
8. References ........................................................................................................97
9. Erklärung.......................................................................................................110
10. Curriculum Vitae ........................................................................................111
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Abbreviations
Abbreviations

APS ammonium persulfate
BSA bovine serum albumin
CFU colony-forming unit
CTAB cetyltrimethylammonium bromide
ddH O double distilled H O 2 2
DNA deoxyribonucleic acid
DTT dithiolthretiol
E-Chip Electrical Chip system
EDTA ethylenediaminetetraacetic acid
efts elongation factor Ts
-2g gram or Earth’s gravity (g=9.81 m*s )
HPLC high-pressure liquid chromatography
IPTG isopropyl thio- β-galactoside
kDa kilo daltons
Ki inhibition constant
3L liter (dm )
LB Luria-Bertani medium
3M molar concentration (mol/dm )
μ micro
MALDI-TOF MS matrix assisted laser desorption ionization time-of-flight mass
spectrometry
MB molecular beacon
MBTFP 3-(4-mercaptobutylthio)- 1,1,1-trifluoro-2-propanone
3mM millimolar concentration (mmol/dm )
nA nano Amper
NMR nuclear magnetic resonance
ODN oligodeoxynucleotide
PAGE polyacrylamide gel electrophoresis
pAP p-aminophenol
pAPB p-aminophenylbutyrate
pK negative logarithm of the dissociation constant K (-logK)
pKa e logarithm of the acid dissociation constant K (-logKa)
pNP p-nitrophenol
pNPB p-nitrophenylbutyrate
RNA ribonucleic acid
rpm revolutions per minute
rRNA ribosomal RNA
S 100 supernatant after ultracentrifugation at 100 Svedberg unit
SA streptavidin
SDS sodium dodecyl sulfate
SSPE saline-sodium phosphate-EDTA
sulfo-SMCC sulfosuccinimidyl 4-(Nmaleimidomethyl)cyclohexane-1-carboxylate
TBE Tris-borate-EDTA
TCEP tris (2-carboxyethyl) phosphine hydrochloride
TEMED N,N,N ′,N ′-Tetramethylethylene diamine
TFK trifluoromethyl ketone
VIAbbreviations
Tris tris-(hydroxymethyl)-aminoethane
U units
UV ultra violet
v/v volume per volume
v/w e per weight
X-Gal 5-bromo-4-chloro-3-indolyl- β-D-galactoside

VIIIntroduction

1. Introduction

The progress of human genomic sequencing unravels genotype related diseases (Hudson,
2006). This brings a perspective for an individual therapy based on the DNA analysis. The
emerged “lab-on-chip” enables a fast, robotic and cost-effective way to fulfill the so-called
“point-of-care” tasks. Point-of-care diagnostic testing, or testing performed at the patient
bedside, allows physicians to diagnose patients more rapidly than traditional laboratory-based
testing. The capacity of current microarray technology allows processing massive data
accumulation based on large numbers of genes or sequences sampled, i.e. gene transcriptional
profiling, single-nucleotide polymorphism discovery, or portions of the genome resequencing
(Abdullah-Sayani et al., 2006). However, clinical diagnostics do not require massive data
accumulation simultaneously, but reliability, reproducibility and automated analysis
(Drummond et al., 2003; Abdullah-Sayani et al., 2006). To practically realize this purpose,
different disciplines, including molecular biology, electrical engineering, material science,
physics and chemistry, are needed to work together to reach the aim of nucleic acids
diagnostics on electrical chips, which possess characters of accurate, fast, robotic and
inexpensive for patient dianostics (Drummond et al., 2003; Nebling et al., 2004).

1.1 Electrochemical detection of nucleic acids
1.1.1 Introduction of electrochemical nucleic acid biosensor

In the 1990’s progress in genomics and particularly in the Human Genome Project greatly
stimulated interest in new methods capable of unraveling the genetic information stored in the
nucleotide sequence of DNA. Wide-scale genetic testing requires the development of easy-to-
use, fast, inexpensive, miniaturized analytical devices. Traditional methods for detecting DNA
hybridization, such as gel electrophoresis or membrane blots, are too slow, discontinuous and
labor intensive. This increases the demand for exploitation of a new method.
The development of microfabricated devices built on silicon, glass, or plastic supports is a
modern trend in biological techniques area in the last two decades, resulting in many start-up
companies serving the pharmaceutical, biotechnology, and diagnostics markets. However, the
idea of implementing such devices on microelectronic substrates has been introduced only
1Introduction
recently (Tartagni et al., 2004). Electrochemical biosensors are small devices linking specific
biochemical recognition properties for a selective analysis to report the diagnosis result by
means of electrical signal. And the analysis of complex DNA samples and acquisition of
sequence and expression information would require the integration of multiple biosensors into
arrays or chip form for parallel analysis (Service, 1998; Wang, 2000). Therefore, development
of DNA sensors and the construction of a fully electronic DNA chip with electrochemical
detection method has become a booming field. It is a great effort in biology, chemistry, and
engineering to utilize the advantages of miniaturization for cheaper, better, and faster sample
analysis. A number of terms, like electrical chip, electrochemical chip, electrochemical DNA
array, electrical arrays, microelectronic chips, electrical biochips and electrical microarray are
often being intermixed to describe this kind of parallel analysis device.
In brief, the common principle of such devices is the coupling of a biological recognition
element with a physical transducer (Fig. 1.1). Transducing elements include optical (Piunno et
al., 1995), electrochemical (Palecek et al., 2002), and microgravimetric (Zhou et al., 2001)
devices, but electrochemical transducers have received considerable more attention because of
its simpler, faster, and cheaper characters (Paeschke et al., 1996; Palecek et al., 2002; Gooding,
2002; Drummond et al., 2003). The first electrochemical DNA biosensor based on
hybridization was developed in 1993 (Millan and Mikkelsen, 1993). Since then, the progress of
semiconductor technology enables the construction of fully electrical chip, with high
integration at acceptable product costs. The advantage of a fully electrical chip is the intrinsic
high spatial resolution allowing highly parallel reaction and compact construction without the
common expensive optical components (Drummond et al., 2003; Hintsche et al., 2005).


Fig. 1.1. Steps involved in the detection of a specific DNA sequence using an electrochemical
DNA hybridization biosensor. Adapted from (Gooding, 2002).
2