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Advancements in Firefly Luciferase-Based Assays and
Pyrosequencing Technology
Jonas Eriksson
© Jonas Eriksson
Jonas Eriksson
Department of Biotechnology
Royal Institute of Technology, KTH
AlbaNova University Center
SE-106 91 Stockholm
Sweden.
Printed at Universitetsservice US AB
KTH, 100 44 Stockholm
Sweden
ISBN 91-7283-618-0
Jonas Eriksson (2004): Advancements in Firefly Luciferase-Based Assays and
Pyrosequencing Technology. Doctoral dissertation from the Department of Biotechnology,
Royal Institute of Technology, KTH, AlbaNova University Center, Stockholm, Sweden.
ISBN 91-7283-618-0
Abstract
Pyrosequencing is a new DNA sequencing method relying on the sequencing-by-synthesis
principle and bioluminometric detection of nucleotide incorporation events. The objective of
this thesis was improvement of the Pyrosequencing method by increasing the thermal
stability of firefly luciferase, and by introducing an alternative DNA polymerase and a new
nucleotide analog. Furthermore, the development of a new bioluminescent assay is described
for the detection of inorganic pyrophosphatase activity.
The wild-type North American firefly (Photinus pyralis) luciferase is a heat-sensitive
enzyme, the catalytic activity of which is rapidly lost at temperatures over 30°C. Two
strategies for increasing the thermostability of the enzyme are presented and discussed. In the
first strategy, the solution thermodynamics of the system is affected by osmolytes in such a
way that heat-mediated inactivation of the enzyme is prevented. In the second strategy, the
enzyme is thermostabilized by mutagenesis. Both stabilizing strategies can be utilized to
allow bioluminometric assays to be performed at higher temperatures. For instance, both
DNA polymerase and ATP sulfurylase activity could be analyzed at 37°C.
The osmolyte strategy was successfully employed for increasing the reaction temperature
for the Pyrosequencing method. By increasing the reaction temperature to 37°C unspecific
signals from primer-dimers and 3’-end loops were reduced. Furthermore, sequencing of a
challenging template at 37°C, which previously yielded poor, non-interpretable sequence
signals at lower temperatures was now possible.
Introduction of a new adenosine nucleotide analog, 7-deaza-2’-deoxyadenosine-5’triphosphate (c7dATP) reduced the inhibitory effect on apyrase observed with the currently
used analog, 2’-deoxyadenosine-5’-O-(1-thiotriphosphate) (dATPαS).
Sequencing of homopolymeric T-regions has previously been difficult with the
exonuclease-deficient form of the DNA polymerase I large (Klenow) fragment. By using the
DNA polymerase from bacteriophage T7, known as Sequenase, templates with
homopolymeric T-regions were successfully sequenced. Furthermore, it was found that the
strand displacement activity for both polymerases was strongly assisted if the displaced
strand had a 5’-overhang. In contrast, the strand displacement activity for both polymerases
was inhibited without an overhang, resulting in reduced sequencing performance in double
stranded regions.
A firefly bioluminescent assay for the real-time detection of inorganic pyrophosphatase in
the hydrolytic direction was also developed. The assay is versatile and has a linear response
in the range between 8 and 500 mU.
Key words: bioluminescence, osmolytes, glycine betaine, thermostability, firefly luciferase,
inorganic pyrophosphatase, inorganic pyrophosphate, Pyrosequencing technology, secondary
DNA-structures, Sequenase, Klenow-polymerase, reaction rates, temperature, c7dATP,
dATPαS.
© Jonas Eriksson
A small step in science but a gigantic leap for me.
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
This thesis is based on the following papers and are referred to in the text by their
Roman numerals:
I.
Eriksson, J., Nordström, T., Nyrén, P. (2003) Method enabling firefly
luciferase-based bioluminometric assays at elevated temperature. Anal.
Biochem. 314: 158-161.
II.
Eriksson, J., Gharizadeh, B., Nordström, T., Nyrén, P. (2004)
PyrosequencingTM technology at elevated temperature. Electrophoresis, 25:
20-27.
III.
Eriksson, J., Gharizadeh, B., Nourizad, N., Nyrén, P. 7’-deaza-2’deoxyadenosine-5’-triphosphate as an alternative nucleotide for the
Pyrosequencing technology. Submitted.
IV.
Gharizadeh, B., Eriksson, J., Nourizad, N., Nyrén, P. Improvements in
Pyrosequencing technology by employing Sequenase polymerase. Submitted.
V.
Eriksson, J., Karamohamed, S., Nyrén, P. (2001) Method for real-time
detection of inorganic pyrophosphatase activity. Anal. Biochem. 293:67-70.
i
Jonas Eriksson
Abbreviations
ATP
adenosine triphosphate
ADP
adenosine diphosphate
AMP
adenosine monophosphate
APS
adenosine 5’-phosphosulfate
CCD
charge-coupled device
cDNA
complementary DNA
dATP
deoxyadenosine 5’-triphosphate
dCTP
deoxycytidine 5’-triphosphate
dGTP
deoxyguanosine 5’-triphosphate
dTTP
deoxythymidine 5’-triphosphate
dNTP
deoxynucleoside 5’-triphosphate
dNDP
deoxynucleoside 5’-diphosphate
dNMP
deoxynucleoside 5’-monophosphate
dATPαS deoxyadenosine alfa-thio 5’-triphosphate
PMT
photomultiplier tube
DNA
deoxyribonucleic acid
RNA
ribonucleic acid
EDTA
ethylenediamine tetra-acetic acid
HPV
human papillomavirus
KM
Michaelis-Menten constant
PCR
polymerase chain reaction
PPi
inorganic pyrophosphate
PPase
inorganic pyrophosphatase
SBH
sequencing-by-hybridization
SBS
sequencing-by-synthesis
SNP
single nucleotide polymorphism
ss
single-stranded
SSB
single-stranded DNA-binding protein
TA
Tris-acetate
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Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
Table of contents
Page
1. Introduction
1
2. Bioluminescence
2.1. Luciferases
2.2. Firefly luciferase
2.2.1. Firefly luciferase in biotechnology
2.2.1.1. Imaging and reporter gene
2.2.1.2. Biomass assays
2.2.1.3. Monitoring of enzymes and molecular substances
2.2.1.4. Immunoassays
3. DNA
2
2
3
5
5
8
8
10
11
11
11
3.1. Chemical structure of DNA
3.2. DNA amplification
4. DNA sequencing
4.1 Principles of DNA sequencing
4.1.1. The dideoxy method
4.1.2. Sequencing-by-hybridization
4.1.3. Sequencing-by-synthesis
4.2 Pyrosequencing technology
5. Present investigation
5.1. Bioluminometric assays at elevated temperatures
5.1.1. Increasing the thermostability of firefly luciferase using
osmolytes (I)
5.1.2. Pyrosequencing technology at elevated temperatures (II)
5.1.3. Coupled enzymatic assays at elevated temperatures (I)
5.2. Nucleotide analogs in Pyrosequencing technology (III)
5.3. DNA polymerases in Pyrosequencing technology (IV)
5.4. PPi and PPase (V)
13
13
14
14
15
15
18
18
19
22
29
34
39
44
6. Concluding remarks and future prospects
47
Acknowledgements
49
References
51
Original papers
iii
Jonas Eriksson
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Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
1. Introduction
One fundamental requirement, and perhaps the most important for life to exist on our
planet, is the ability to reproduce and give rise to offspring of one’s own kind. Ever
since Gregor J. Mendel proposed a solution in 1865 to the heritage enigma, research
has focused on understanding the requirements of life from a chemical and biological
perspective. Perhaps one of the most important moments in history was when Watson
and Crick presented the structure of DNA in 1953 (Watson et al., 1953). At the same
time, in a seemingly unrelated field, work was initiated which aimed to understand
the chemistry of bioluminescence. The scientists William McElroy, Emil White and
Howard Seliger performed their pioneering work at John Hopkins University on
crude extracts from North American fireflies. They described the importance of
adenosine triphosphate (ATP) in the light reaction catalyzed by firefly luciferase. It
became evident that firefly luciferase could be used as a tool to detect ATP and study
systems where ATP is being formed or consumed.
In the decades that followed, new tools in modern biochemistry made it possible
to understand some of the basic mechanisms involved in the process where the
information in the DNA is processed into a functional protein; today known as the
central dogma. However, at the time there was no practical and simple method
available to sequence DNA until Sanger presented the dideoxy method in 1977
(Sanger et al., 1977). The recent availability of the sequence of the firefly luciferase
gene, as well as the possibility to produce recombinant firefly luciferase, has opened
for many applications for this enzyme, such as reporter gene for detection of protein
expression, ATP detection, in vivo imaging of living higher organisms, and, recently,
DNA sequencing.
Today, several DNA sequencing methods are available. The most used method is
the dideoxy method by Sanger. Other methods include chemical degradation
techniques,
sequencing-by-hybridization,
single-molecule-detection,
mass
spectrometry and Pyrosequencing technology. The Pyrosequencing technology
combines the sequencing-by-synthesis principle with a coupled bioluminescence
assay. This thesis covers resent advancements in firefly luciferase bioluminescence-
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Jonas Eriksson
based assays with an emphasis on high-temperature reaction conditions and the
consequence for the Pyrosequencing method.
2. Bioluminescence
Bioluminescence is, by definition, enzyme catalyzed light emission. Bioluminescent
organisms are found throughout the biosphere. The biochemical and biological
diversity of bioluminescent systems both on land and in the sea suggests that the
ability to generate light arose from many separate origins during evolution (Hastings,
1983). 90 % of the animals in the mesopelagic zone of the ocean (depth of 200-1000
m) are bioluminescent. Some bioluminescent organisms that can be found in the
ocean are bacteria (such as Vibrio harveyi), dinoflagellates (unicellular algae such as
the phytoplankton Pyrocystis fusiformis), crustaceans (such as the copepod Gaussia
princeps and euphausiids (krill)), and various types of jellies and some fishes
(Harbour Branch Oceanographic Institute, URL:http://www.biolum.org, last visited
2004-01-24). Many of the land living bioluminescent higher organisms can be found
in the order Coleopera (beetles) in the families of Elateridae (click beetles),
Phengodidae (rail road worms) and Lampyridae (fireflies). ‘Firefly’ is the common
name for the nocturnal luminous insects of which there are over 2000 species
inhabiting the tropical and temperate regions (Lloyd, 1978).
2.1. Luciferases
The enzymes responsible for bioluminescence are known as luciferases. All
luciferases catalyze oxidation of the substrate luciferin into an electronically exited
state and light is emitted upon the return to the ground state. The quantum efficiency
(QE), the percentage of the molecular energy used to generate photons (light), can be
high and bioluminescence is for that reason sometimes referred to as “cold light”. The
firefly luciferase uses D-luciferin and adenosine triphosphate (ATP) as substrates to
generate light with a QE close to 90 %. In contrast, the bacterial luciferase uses flavin
mononucleotide (FMNH2) and an aldehyde as substrates to generate light with QE of
between 10-30 %. Other luciferase-luciferin systems include colenterazine, varguline
and the dinoflagellate luciferin.
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Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
2.2. Firefly luciferase
The firefly luciferase (EC 1. 13. 12. 7) is classified as a monooxygenase
(oxidoreductase). The cloning and sequencing of luciferases from over 17 beetle
species have reveled that these luciferases are closely related to a large family of
nonbioluminescent proteins including acyl-CoA sythetase (Suzuki et al., 1990) and 4chlorobenzoate dehalogenase (Babbitt et al., 1992). In addition, a comparison of the
mechanic features between firefly luciferases, fatty acyl-CoA synthetases and
aminoacyl-tRNA synthetases show many similarities (McElroy et al., 1967).
The gene for the North American firefly (Photinus pyralis) luciferase has been
cloned (de Wet et al., 1985) and sequenced (de Wet et al., 1987), and the structure has
been determined (Conti et al., 1996). The enzyme is a monomer and consists of 550
amino acids with a size of approximately 62 kDa. According to crystallographic data
the N-terminal domain makes up the major part of the enzyme comprising residues 1436 and the C-terminal, residues 437-550, is a small separate domain.
The light production by firefly luciferase is a complicated multi-step process not
fully understood in all details. However, the process is generally considered to
involve four main steps as summarized in Eq. 1-4.
E + MgATP + L → E-L-AMP + PPi
(Eq. 1)
E-L-AMP + O2 → E-oxyLΨ-AMP + CO2
(Eq. 2)
E-oxyLΨ-AMP → E-oxyL-AMP + hν
(Eq. 3)
E-oxyL-AMP → E + oxyL + MgAMP
(Eq. 4)
In the first step firefly luciferase (E) bind D-luciferin (L) and MgATP to form an
enzyme-adenylate-luciferyl complex (E-L-AMP) with the immediate release of PPi
(Eq. 1). In the second step L-AMP is oxidized into an electronically exited state
oxyluciferin (oxyLΨ-AMP) (Eq. 2). In the third step the exited-state product, still
bound to the enzyme, emits yellow-green light (hν) upon the return to the ground
state (Eq. 3). The final step involves the release of AMP and oxyL from the enzyme
(Eq. 4). Although the North American firefly P. pyralis and the Japanese firefly
Luciola crusiata generate light at the same wavelength of 562 nm there are
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Jonas Eriksson
differences in the emitted light wavelength between firefly species. For example, L.
mingrelica emits light at 570 nm whereas L. lateralis emits light at 552 nm.
Different luciferases utilize different luciferins and the luciferin used is, in part,
responsible for the wavelength of the generated light. The sea pansy Renilla
luciferase, for example, uses coelenterazine to generate light at 490 nm.
Furthermore, different forms of the exited state can contribute with light of different
wavelength. For example, the firefly luciferase product oxyluciferin has been
suggested to exist in two different forms; the enolate and the keto form responsible
for yellow-green light and red light, respectively (White et al., 1980). According to
recent research (Branchini et al., 1999; Tisi et al., 2002) the amino acids that are in
close proximity to the substrate in the suggested active site can in part control the
relation between of the two forms and thereby the bioluminescence color.
Specifically, the Thr343 plays an important role in this context (Branchini et al.,
1999).
The residues involved in the active site are suggested to be on the surfaces facing
each other across a cleft formed between β-sheet A and β-sheet B on the N-terminal
domain. The active site has been suggested to involve everything between 5 up to 12
amino acids (Branchini et al., 1998; Branchini et al., 2003; Ugarova et al., 1998).
Firefly luciferase shows two distinctly different kinetic patterns. At ATP
concentration over 1 µM an initial flash of light is observed that rapidly decays to a
relatively constant light emission. However, only constant light emission is observed
at ATP concentrations below 1 µM. Constant light emission is preferred for real-time
firefly luciferase based assays. Low amounts of PPi and L-luciferin (Lundin, 1982;
Lundin, 1993) and CoA (Airth et al., 1958; Ford et al., 1995) have been used for
further stabilization of the firefly luciferase reaction.
To explain the kinetics of firefly luciferase a two binding site model has been
suggested (DeLuca et al., 1984; Denburg et al., 1969; Lee et al., 1970; Steghens et al.,
1998). According to the simplest interpretation of the model one high affinity ATP
site is suggested to be responsible for the observed flash kinetics and a second low
affinity site responsible for the constant light emission (DeLuca et al., 1984).
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Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
However, so far no evidence has been presented for the localization of the suggested
two sites on the firefly luciferase enzyme.
2.2.1. Firefly luciferase in biotechnology
Bioluminescence from firefly luciferase has been applied in many different areas. In
the discussion below an attempt is made to cover the most important works in
different areas. The field of firefly bioluminescence has been divided into the
following subclasses: (1) imaging and reporter gene, (2) biomass assays, (3)
monitoring of enzymes and molecular substances, and (4) immunoassays.
2.2.1.1. Imaging and reporter gene
Visualization of firefly luciferase expression in eukaryotic and prokaryotic cells,
animal tissues, and in transgenic plants and animals has been made possible by
several advancements in modern biotechnology and by improved imaging
technologies. The introduction of recombinant DNA constructs with various
promoter-luciferase gene combinations and fusion gene products have extended the
application range and increased the usage specificity for recombinant firefly
luciferase. Furthermore, efficient and precise delivery of transgenic DNA and
exogenous substrates has enabled previously non-reachable areas to be studied with
bioluminescence. In addition, highly sensitive imaging technologies have been
developed.
The gene coding for green fluorescence protein (GFP) has been used for imaging.
GFP are proteins utilizing a mechanism to generate light that differs from that used by
luciferases. It has been discovered that many species such as the hydromedusa
Aequorea (Shimomura et al., 1975) and the anthozoan sea pansy Renilla reniformis
(Hart et al., 1979) have proteins that adsorb energy from photons generated by their
luciferase-luciferin systems. By doing so the GFP glows with green light with an
emission peak at 508 nm (Yang et al., 1996). The GFP is comprised of 238 amino
acids and can easily be engineered to emit light with other colors; blue and yellow
versions of the protein exist. The major advantage of using GFP as a reporter gene is
that only UV-light is needed to generate fluorescence and no substrate is necessary.
5
Jonas Eriksson
The GFP gene of A. victoria has been cloned and sequenced (Prasher et al., 1992).
Since Chalfie et al. showed that GFP could be expressed as a functional transgene
(Chalfie et al., 1994) GFP has been expressed in many systems such as bacteria
(Chalfie et al., 1994), yeast (Kahana et al., 1995), slime mold (Moores et al., 1996),
plants (Casper et al., 1996; Epel et al., 1996), drosophila (Wang et al., 1994),
zebrafish (Amsterdam et al., 1996) and in mammalian cells (De Giorgi et al., 1996;
Ludin et al., 1996).
Many of the available reporter genes, such as β galactosidase, chloramphenicolacetyl-transferase (CAT), β-glukuronidase (GUS), and human placental secreted
alkaline phosphatase (SEAP) are dependent on end-point assays and long incubation
times. The bacterial luciferase gene (luxCDABE) can be used as an imaging reporter
gene. The luxCDABE codes for five proteins. The 40 kDa α-subunit (A) and the 35
kDa β-subunit (B) makes up the active heterodimer form of the enzyme. The C, D
and E forms the fatty-acid-reductase complex needed for the synthesis of one of the
bacterial luciferase substrates. As we mentioned earlier the QE for bacterial luciferase
is low. In addition, the α- and β-subunits have to be assambled into an active form
before any activity could be assayed.
Firefly luciferase as a reporter gene has the benefits of high QE (90 %). In
addition, the expression can be followed directly as the firefly luciferase is expressed
in an active form. Furthermore, recent developments in reagent composition have
made the generated light highly stable and less dependent on fast read-out. Although
alternative luciferase-luciferin systems exits, such as vuc (vargulin), aeq (aequorin)
and ruc (Renilla luciferase) they are often used in specific cases where their unique
individual characteristics are required. Eucaryotic luciferase is by standard
conventions denoted luc. Important works using the luc (here referred to the firefly
luciferase) gene are listed in table 1.
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Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
Table 1. Examples of firefly luciferase constructs used for imaging (extract from (Greer et al., 2002)).
Application field
Construct/vector
Target/process
Reference
pCMV.luc, pCol.luc
MAP kinase signaling
i
pSP-Luc
PRL promotor activation
ii
pLPK-LucFF, pINS-LucFF, pβGK4-Luc,
Glucose-activated insulin
iii
AdCMVcLuc, pPPI-Luc
secretion and activation of
Target organism/
Cell lines
Cells and cellculture
phosphatidylinositol 3-kinase
LTR-luc in pGL3
Induction of SV40 promoter-
iv
luciferase expression
pcLuc (cytosolic, nin-targeted), pmLuc
Subcellular compartmentalization
(plasma membrane targeted)
of ATP
v
Transgenic plants
Arabidopsis
RD29A-luc
Monitoring of stress response
vi
pathways including induction of
the endogenous RD29A gene.
cab2-luc
Cab2-activity was inferred by
∆rbcs1b-luc rbcs2b-rbcs3b
Meiotic crossing
Zebra fish (Brachydanio rerio)
Luc
Expression, spatial distribution
Mouse
(β-actin promotor)-luc,
Luciferase mRNA,
xi
(HSP70.1-promotor)-luc
Transgenic selection
xii
(ho-1)-luc
Tissue oxygenation
xiii
(CMV-luc, (c-fos)-luc)
Promotor-enhancer activity in
xiv
vii
visualizing luc expression
viii
Transgenic animals
ix, x
brain cells
Transgenic viruses
Baculo virus
polyhedrin-enhancer-luc
Plaque assay
xv
Herpes/pseudorabies virus
Luc
Infection in mammalian cells
xvi
Adeno virus
Stain AdCMVLuc and Ad5LucRGD
Transduction efficiencies
xvii
Vaccina virus
Luc
Infection in African green
xviii
monkey BCS-40 cells
In vivo imaging of living rats
Transfected cell lines: Human
AdSV40/Luc, AdHIV/Luc, rLNC/Luc,
Monitoring of tumors in various
hepatocellular carcinoma
pLN/Luc
tissues
xix
(HepG2) and human prostate
adenocarcinoma (PC3.38)
i (Rutter et al., 1995), ii (Takasuka et al., 1998), iii (da Silva Xavier et al., 2000), iv (Contag et al., 1997), v (Kennedy et al.,
1999), vi (Ishitani et al., 1997), vii (Millar et al., 1992), viii (Jelesko et al., 1999), ix (Tamiya et al., 1990), x (Mayerhofer et al.,
1995), xi (Matsumoto et al., 1994), xii (Menck et al., 1998), xiii (Zhang et al., 1999), xiv (Sigworth et al., 2001), xv
(Langridge et al., 1994), xvi (Mettenleiter et al., 1996), xvii (Kratzer et al., 2001), xviii (Rodriguez et al., 1988), xix
(Honigman et al., 2001)
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Jonas Eriksson
2.2.1.2. Biomass assays
The simplicity and sensitivity of bioluminometric assays have made ATP detection
by firefly luciferase an attractive approach for detection and enumeration of cells. For
hygiene monitoring the sum of extra and intra cellular ATP is measured, while for
biomass assays only the intracellular ATP is measured. Therefore, the extraction of
intra cellular ATP is critical for obtaining precise results for both applications.
Extraction of ATP from cells can be achieved using acids, organic solvents or boiling.
The most accurate methods use trichloroacetic acids (TCA) (Lundin et al., 1975) or
cationic detergents, such as benzalkonium chlorid (BAC) (Ånséhn et al., 1979).
However, both TCA and BAC interfere with subsequent luciferase-luciferin assays.
To neutralize the effect of BAC on the luciferase reaction BSA (Ånséhn et al., 1979),
cyclodextrins (Lundin, 1994) and medium-chain fatty acids (Martin et al., 1996) have
been used. An interesting strategy is the use of a mutated firefly luciferase with
improved BAC resistance (Hattori et al., 2002).
Although actual typing of pathogenic bacteria is not possible by the
bioluminometric method, the speed of the assay makes it an attractive method for
determination of bacterial contamination. In this context firefly bioluminescence has
been used for biomass and hygiene monitoring in the medical environment and in the
food industry.
In the medical environment ATP bioluminescence is used for the study of
bacteriuria (Thore et al., 1975) and the effect of oral antiseptics on salivary biomass
(Gallez et al., 2000). In the food industry ATP bioluminescence is used for control of
the microbiological quality of milk (Niza-Riberio et al., 2000; Olsson et al., 1986)
and for estimation of total plate counts of surface micro flora on vegetables (Ukuku et
al., 2001).
2.2.1.3. Monitoring of enzymes and molecular substances
In principle, all processes where ATP is being formed or consumed can be monitored
using firefly luciferase based assays. The luciferase reaction is ideal to couple with
other enzymatic processes. Table 2 summarizes examples of firefly luciferase
bioluminescence based assays for enzymes and substrates. For example, ADP can be
8
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
assayed if the firefly luciferase reaction is coupled with pyruvate kinase (Holmsen et
al, 1972) and AMP by the additional enzyme adenylate kinase (Goswami et al, 1984).
Table 2. Summary of selected firefly bioluminescence based coupled assays.
Category
Enzymes
Enzyme/substrate
Reference
phosphoenolpyruvate
kinase
glycerol kinase
nucleoside diphosphate
kinase
creatine kinase
(Wimmer, 1988)
(Hellmer et al., 1989)
(Karamohamed et al., 1999a)
RT polymerase
DNA polymerase
ATPase
ATP sulfurylase
apyrase
(Lundin, 1978; Lundin et al.,
1982)
(Karamohamed et al., 1998)
(Nyrén, 1987)
(Hanocq-Quertier et al., 1988)
(Karamohamed et al., 1999b)
(Karamohamed et al., 2001)
AMP
ADP
ATP
cAMP
PPi
glucose
(Goswami et al., 1984)
(Holmsen et al., 1972)
(Aflalo et al., 1987)
(Ebadi, 1972)
(Nyrén et al., 1985)
(Idahl et al., 1986)
Substrates
Another example of a coupled enzymatic reaction is the DNA quantification
assay developed and commercialized by Promega Corporation. The assay consists of
a
set
of
coupled
reactions
consisting
of
pyrophosphorylation
and
transphosphorylation. The pyrophosphorylation reaction is the reverse of the DNA
polymerization reaction and is catalyzed by T4 DNA polymerase. In the presence of
pyrophosphate and dsDNA, dNTPs are released from the 3’ termini of the DNA
strands. The transphosphorylation reaction is catalyzed by nucleoside diphosphate
kinase. In this reaction, the terminal phosphate of the dNTP is transferred to ADP to
form ATP. Thus, the ATP formed and the light generated by firefly luciferase is
proportional to the amount of dsDNA added to the reaction. The assay is sensitive
and yields linear responses between 0.02-1 ng DNA.
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Jonas Eriksson
2.2.1.4. Immunoassays
Protein blotting is now an established technique in research and clinical laboratory
diagnosis. Blotted proteins are detected using labeled antibodies. Alkaline
phosphatase labels were not amendable to a firefly luciferase bioluminescent assay
until Miska and Geiger synthesized D-luciferin-O-phosphate (Geiger et al., 1987;
Miska et al., 1987). The alkaline phosphatase label cleaves the phosphate group to
produce D-luciferin, which, unlike the O-phosphate derivative, reacts with firefly
luciferase and ATP to produce light (Fig. 1). The technique can detect 5 pg of blotted
rabbit immunoglobin G, which represents a 100-fold improvement over radioactive or
other nonradioactive labels (Hauber et al., 1987). In addition, a protein A-firefly
luciferase fusion has been engineered that binds to the Fc region of IgG. The fusion
protein has been used for analysis of human IgG (Kobatake et al., 1993).
-Ag:Ab-Alkaline phosphatase
D-Luciferin-O-phosphate
30 min incubation
D-Luciferin
Firefly luciferase
ATP, Mg 2+
Light
Figure 1. An immunoassay, based on bioluminescence, using D-luciferin-O-phosphate substrate.
(After (Kricka, 1988). Reproduced by permission from the author.)
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Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
3. DNA
Deoxyribonucleic acid (DNA) molecules are the libraries in living cells where the
information required for building a cell and an organism are stored. This chapter
briefly describes the chemical structure of DNA. In addition, the principle of DNA
amplification is explained.
3.1. Chemical structure of DNA
DNA is a linear polymer composed of single chemical units called nucleotides. The
number of nucleotides in a cellular DNA molecule can exceed a hundred million. A
nucleotide has three parts: a phosphate group, a deoxyribose and an organic base. The
bases are adenine, guanine, cytosine and thymine; abbreviated A, G, C and T,
respectively. When the nucleotides polymerize to form DNA, the hydroxyl group
attached to the 3’ carbon of the sugar group of one nucleotide forms an ester bond to
the phosphate attached to the 5’ carbon of another nucleotide. This dictates the
extremely important property of the orientation of the polynucleotide strand. DNA
consists of two associated polynucleotide strands forming a double helix. The sugarphosphate backbones are on the outside of the double helix and the bases project into
the interior. The orientation of the two strands is antiparallel and complementary to
each other. The strands are held together by the cooperative energy of many hydrogen
bonds in addition to hydrophobic interactions. The opposite strands are held in
precise register by a regular base pairing between the two strands: A is paired with T
by two hydrogen bonds and G is paired with C by three hydrogen bonds.
3.2. DNA amplification
In the early 1980’s, the young scientist Kary B. Mullis, while working for the Cetus
Corporation, developed a method for amplifying specific DNA sequences that he
named polymerase chain reaction (PCR) (Mullis et al., 1986; Saiki et al., 1985).
According to the basic principle a target DNA is defined flanked by non-target DNA.
The PCR is then carried out by adding the following components to a solution
containing the target DNA: a pair of primers that hybridize with the flanking
11
Jonas Eriksson
sequences of the DNA target, all four deoxyribonucleoside triphosphates (dNTPs)
and a heat-stable DNA polymerase. A PCR cycle consists of three steps:
1. Strand separation. Heating the solution to 95°C for 10-20 s separates the two
strands of the target DNA.
2. Hybridization of primers. The solution is rapidly cooled to 45-55°C to allow each
primer (20-30 nucleotides long) to hybridize to the DNA target. One primer
hybridizes to the 3’-end of one strand of the target, and the other primer
hybridizes to the 3’-end of the complementary target strand. Parent DNA
duplexes are prevented by supplying primers in large excess.
3. DNA synthesis. The solution is heated to the optimal temperature of the DNA
polymerase. The polymerase elongates both primers in the direction of the target
sequence (5’ to 3’).
These three steps constitute one cycle of the PCR and can be carried out repetitively
by changing the temperature of the reaction mixture. After n cycles the target DNA is
theoretically amplified 2n times. Consequently, after 30 cycles the target DNA is
amplified a billion-fold.
Today, some 10 years after Kary B. Mullis was awarded the Nobel Prize for
Chemistry in 1993, the PCR has become an integral part in obtaining and compilation
of large volumes of genetic data. Recent advances in the technical platform around
the PCR method have focused on maximizing the time and cost efficiency, especially
through decreased reaction volumes and increasing the number of simultaneous
amplifications performed. For example, in a recent report a novel PCR platform was
presented, the PicoTiterPlateTM, where 300 000 discrete PCR reactions can be
performed in reaction volumes (for each reaction) as low as 39.5 pL (Leamon et al.,
2003).
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Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
4. DNA sequencing
The knowledge of the DNA sequence, the genetic code, is a very important element
towards the understanding of the organization framework life relies on. Specifically,
identification of protein-coding regions within the DNA sequence followed by
computer-assisted similarity searches in DNA and protein data bases, can lead to
important insights about the function and structure of a gene and its product. In
addition, DNA sequence information is essential for performing site directed
mutagenesis for functional studies of enzymes and proteins. In this context, the work
to map the human genome was initiated (Human Genome Project (HUGO)) and was
recently finished (Venter et al., 2001; Waterston et al., 2002). The laborious work to
find the function of the genes has just begun.
The HUGO initiative has created a market for conventional and new DNA
sequencing techniques. Here follows a brief description of a few DNA sequencing
techniques with specific attention on the Pyrosequencing technology.
4.1 Principles of DNA sequencing
The DNA sequencing methods that so far have been developed are based on two
fundamental different strategies namely direct or indirect sequencing. Direct
sequencing techniques involve a variety of synthesis, degradation, and/or separation
techniques, and includes two techniques (described below); the traditional dideoxy
method by Sanger (Sanger et al., 1977) and the Pyrosequencing method (Nyrén,
2001; Ronaghi et al., 1998b). Among the indirect sequencing methods sequencingby-hybridization dominate (Drmanac et al., 1989).
Regardless of what method is used DNA has to be processed before it can be
sequenced. First the DNA must be extracted from the cell, which is easily achieved
by modern biotechnology tools. However, the DNA amount in a cell is to small to be
used directly as a source for sequencing. In addition, the region of interest has to be
defined prior to sequencing. For the purpose of amplification of a specific DNA
region PCR is often utilized.
In most DNA sequencing methods the DNA template must be processed under or
after the amplification step simply for visualization, detection or capture reasons. In
13
Jonas Eriksson
the original gel based dideoxy method by Sanger (see description of the method
below) the terminated fragments were labeled by modified radioactive nucleotides. In
the Pyrosequencing method one of the primers in the PCR is biotinylated to allow
preparation of single-stranded DNA after capturing of the DNA on streptavidincoated magnetic beads.
4.1.1. The dideoxy method
In 1977, Sanger and co-workers developed an elegant DNA sequencing method that
has become known as the dideoxy or enzymatic method (Sanger et al., 1977). The
method capitalizes on the ability of the DNA polymerase to use 2’,3’dideoxynucleoside triphosphates (ddNTP). Four reactions are set up including primed
ssDNA template, DNA polymerase, dNTP and one of the four ddNTPs in each
reaction. When a ddNMP is incorporated at the 3’ end of the growing primer chain,
chain elongation is terminated at G, A, T or C due to the lack of a free 3’-hydroxyl
group. Each of the four elongation reactions contains a population of extended primer
chains, all of which have a fixed 5’ end determined by the annealed primer and a
variable 3’ end terminating at a specific nucleotide position. The chains can be
visualized after electrophoretic separation on a high-resolution denaturing
polyacrylamide gel.
4.1.2. Sequencing-by-hybridization
The sequencing-by-hybridization method (Drmanac et al., 1989) uses a set of
oligonucleotides immobilized on a solid phase in an array format to search for
complementary sequences on a longer target DNA molecule. The labeled target DNA
is exposed to the array where hybridization occurs only in the positions on the array
where the oligonucleotide has a sequence complementary to the target DNA. The
resulting hybridization pattern on the array represents a nested set of fragments that
can be used to determine the sequence of the target DNA.
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Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
4.1.3. Sequencing-by-synthesis
The sequencing-by-synthesis (SBS) method is based on the detection of nucleotide
incorporation during a primer-directed polymerase extension with the four
nucleotides being added cyclically in a specific order (Melamede, 1985). The
incorporation can be detected directly or indirectly. For the direct detection SBS
approach, a strategy called base addition sequencing scheme (BASS) has been
described where labeled nucleotides are used. Many groups have described different
strategies based on BASS (Canard et al., 1994; Cheesman, 1994; Metzker et al.,
1994; Rosenthal, 1989; Tsien et al., 1991). However, Metzker et al. showed that the
polymerase has low efficiency for incorporation of these modified nucleotides,
thereby limiting the number of identified bases to only a few bases. The
Pyrosequencing method, described in more detail in the next section is an example of
a SBS based technique where an indirect detection is employed.
4.2. Pyrosequencing technology
The Pyrosequencing technology is an enzymatic based indirect sequencing-bysynthesis DNA sequencing method with detection based on firefly bioluminescence
(Nyrén, 2001; Ronaghi et al., 1998b). Single-stranded DNA (ssDNA) with an
oligonucleotide hybridized adjacent to the sequence of interest is used as a template
for the DNA polymerase. The four different deoxynucleotides are added iteratively
and DNA polymerase catalyzes the incorporation of the deoxynucleotide into the
DNA-strand if it is complementary to the base in the template strand.
Each
incorporation event is accompanied by release of inorganic pyrophosphate (PPi) in a
quantity equimolar to the amount of incorporated nucleotide. ATP sulfurylase
quantitatively converts the PPi to ATP in the presence of adenosine 5’-phosphosulfate
(APS). Firefly luciferase is applied to the reaction enabling monitoring of the
produced ATP in real time. Removal of nucleotides and ATP is achieved in two
different ways: (i) the solid-phase approach with washing steps between each
nucleotide addition (Ronaghi et al., 1996) and (ii) the liquid-phase approach with
enzymatic degradation (Nyrén, 2001; Ronaghi et al., 1998b). Figure 2 shows illustrate
the Pyrosequencing method using the liquid-phase approach. The liquid-phase
15
Jonas Eriksson
Pyrosequencing method has been commercialized and a company established
(Biotage AB (former Pyrosequencing AB), Uppsala, Sweden). Automated systems
and customer friendly ready-to-use kits are now available and the method has been
successfully applied in several fields.
Iterative additions
dATP
dCTP
dTTP
dGTP
Apyrase
dATP
*
dNTP
ATP
dNDP
ADP
Pi
dNMP
AMP
Pi
DNA polymerase
... G A T G G C T A G G G A C T G C G T C A T G G T T A A A T T G T T A T A C A C G A A G A T G T G
3'
... C T A C C G A T C C C T G
PPi
APS + PP i
5'
*
ATP sulfurylase
2-
ATP + SO 4
Firefly
luciferase
D-luciferin + MgATP + O 2
oxyluciferin + AMP + CO2 + hυ + PP i
Computer assisted
collection and
interpretation of data
by a software
Light detector
(CCD or PMT)
Sequence result is presented in a Pyrogram
Nucleotide addition order
(A C G T)
Figure 2. The liquid-phase Pyrosequencing method is a non-electrophoretic real-time DNA
sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide
incorporation into target DNA. The four different nucleotides are added iteratively to a four-enzyme
mixture. The inorganic pyrophosphate (PPi) released in the DNA polymerase-catalyzed reaction is
quantitatively converted to ATP by ATP sulfurylase, which provides the energy to firefly luciferase to
oxidize luciferin and generate light (hν). The light is detected by a photon-detection-device and
monitored in real-time by a computer program. Finally, apyrase catalyzes degradation of nucleotides
that are not incorporated and the system will be ready for the next nucleotide addition.
16
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
The Pyrosequencing method has opened new possibilities for performing
sequence-based DNA analysis. The technique has been successful for both
confirmatory sequencing and de novo sequencing. Pyrosequencing has been
employed for many different applications such as genotyping (Ahmadian et al.,
2000a; Alderborn et al., 2000; Milan et al., 2000; Nordström et al., 2000), mutation
detection (Ahmadian et al., 2000b; Garcia et al., 2000), tag sequencing of selected
cDNA library (Nordström et al., 2001), allele frequence quantification (Gruber et al.,
2002; Neve et al., 2002; Wasson et al., 2002), multiplex analysis (Pourmand et al.,
2002), monotoring resistance to HIV inhibitors (O'Meara et al., 2001), forensic
identification (Andreasson et al., 2002), evolutionary studies (Kaessmann et al.,
2002), bacteria typing (Gharizadeh et al., 2003b; Monstein et al., 2001; Unnerstad et
al., 2001), virus typing (Gharizadeh et al., 2001; Gharizadeh et al., 2003a; Gharizadeh
et al., 2003b; Pourmand et al., 2002), fungal typing (unpublished data), sequencing of
disease-associated genes (Garcia et al., 2000), analysis of DNA methylation profiles
(Uhlmann et al., 2002), and sequencing of difficult secondary structure (Ronaghi et
al., 1999).
The 454 Life Sciences Company currently develops an interesting application of
the solid-phase approach of the Pyrosequencing method. The 454 Life Sciences
approach uses a high-density, microfluidic PCR platform, PicoTiter PlateTM, which
allows amplification of several houndred of thousands DNA samples simultaneously.
The PicoTiter PlateTM is used in the sequencing process where the amplified DNA is
immobilized in each well and the reagents are supplied by microfluidics. A highsensitive, high-resolution CCD device allows detection of each well. The 454 Life
Sciences Technology was recently reporting success in sequencing of the entire
adenovirus genome.
17
Jonas Eriksson
5. Present investigation
The aim of the research presented in this thesis was to improve the Pyrosequencing
technology. Questions related to bioluminescence, DNA sequencing,
and
thermostability were addressed. The specific objectives of this work were (i) to find a
strategy to extend the temperature range of the native firefly luciferase, (ii) evaluate
the use of higher temperature on the Pyrosquencing technology, (iii) find and evaluate
a new adenine nucleotide analog for the Pyrosequencing technology, (iv) evaluate a
faster and more processive polymerase for the Pyroseqencing technology and (v)
develop a new bioluminometric method for monitoring of inorganic pyrophosphatase
activity in real-time. In the following text these objectives are described and
summerized.
5.1. Bioluminometric assays at elevated temperatures
In this section the thermostability of firefly luciferase is addressed. A new approach
(activity stability) was used to measure the effect of temperature on the firefly
luciferase activity in real-time. The thermostabilizing effect of osmolytes was studied
with the new approach (paper I). In addition, the osmolyte strategy was applied on the
Pyrosequencing technology and the effect of an increased reaction temperature on
secondary DNA-structures was evaluated (paper II). Finally, two different strategies
were used to enable firefly bioluminescence assays of enzymes at higher temperatures
(paper I).
The interest in storage stability of the firefly luciferase can be partially explained
by the interest for commercialization of easy-to-use customer ready kits and reagents.
In a normal storage stability experiment the firefly luciferase is exposed to different
conditions, e.g. pH, temperature or additives. After an incubation period the
remaining activity (in % related to a control) is assayed under optimal conditions and
temperature. The storage stability approach was successfully used for finding
thermostable firefly luciferase mutants (Kajiyama et al., 1993; Kajiyama et al., 1994;
White et al., 1996). However, the storage stability approach does not give any
information about the activity of the enzyme at higher temperatures. Therefore, a
different approach was presented in paper I based on activity stability. In the activity
18
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
stability approach the activity of the firefly luciferase sample after exposion to
different conditions is always related to the initial activity of the same sample.
In figure 3 the activity stability approach was compared with the more traditional
stability approach. Although the results obtained by the two methods differed to some
degree (Fig. 3) both methods were comparable for describing the general trend of
thermal inactivation of luciferase from the wt North American firefly (P. pyralis). In
paper I the activity stability approach was used when the effect of glycine betaine was
analyzed and the storage stability approach for the effect of proline and trehalose.
Figure 3. Stability of wt North American firefly (P. pyralis) luciferase at 37°C monitored by two
strategies; activity stability approach (closed circles) and storage stability approach (open circles).
5.1.1. Increasing the thermostability of firefly luciferase using osmolytes (I)
In nature organisms living under high osmotic pressure, or at conditions where the
temperature fluctuates, can raise the osmotic pressure in the cytoplasm and protect
proteins against denaturation by accumulation of specific substances. These
substances are known as osmolytes. The major classes of stabilizing organic
osmolytes are (I) sugars and polyhydric alcohols (polyols), (II) amino acids and
amino acid derivatives, and (III) methylated ammonium and sulfonium compounds.
Proline and betaine have been shown in vitro to protect several different enzymes
19
Jonas Eriksson
against the inactivating effect of heat (Paleg et al., 1981). Trehalose has been reported
to thermostabilize reverse transcriptase (Carninci et al., 1998), firefly luciferase
(Singer et al., 1998), inorganic pyrophosphatase (Sola-Penna et al., 1996) and βgalactosidase (Mazzobre et al., 1999).
Figure 4. Effect of glycine betaine on the wt North American firefly (P. pyralis) luciferase reaction at
37°C. The activity was assayed at 37°C by the activity stability approach at different time intervals in
the presence of different concentrations of glycine betaine. The stabilizing effect was strongly
dependent on the glycine betaine concentration.
In paper I the effect of osmolytes on the thermostability of the luciferase from the
North American firefly (P. pyralis) was studied. One osmolyte from each of the
above-described classes (glycine betaine, proline, and trehalose) were selected; all
well known from the literature to protect proteins against heat denaturation. A first
goal was to stabilize the firefly luciferase activity higher temperatures. At 37°C, and
in the absence of osmolytes, the activity continuously decreased (Fig 4). The
strongest stabilizing effect was obtained in the presence of glycine betaine. The
stabilizing effect was concentration dependent (Fig. 4). In the presence of 1.6 M
glycine betaine the firefly luciferase retained most of its activity for more than 60 min
at 37°C (90 % of the activity was left). At higher temperatures, the effect of glycine
betaine was less pronounced, althought nearly 85% of the activity was left after 30
min at 40°C and 40% at 42°C (Fig. 5).
20
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
Figure 5. Temperature effect on the North American firefly (P. pyralis) luciferase activity. The
activity was assayed by the activity stability approach at different temperatures in the presence of 1.6
M glycine betaine. At temperatures over 40°C the stabilizing effect of glycine betaine was less
pronounced.
It is worth noting that at 22°C and in the presence of 1.6 M glycine betaine the
firefly luciferase activity was inhibited by 35% and at 2.0 M by 50%. For this reason
higher concentrations were not tested.
All the osmolytes that were tested stabilized the firefly luciferase reaction at
37°C. Although both proline and trehalose protected the luciferase to a similar extent
as glycine betaine, there were some disadvantages to using them. For instance,
proline had a strong inhibiting effect on the luciferase activity. In the presence of 1.4
M proline the firefly luciferase was inhibited by 70% at 22°C. For trehalose, on the
other hand, the problem was viscosity. Trehalose is more than 5 times as viscous as
equimolar proline and glycine betaine (Diamant et al., 2001). At a final concentration
of 0.6 M trehalose about 70% of the firefly luciferase activity was left after 60 min at
37°C. At this high trehalose concentration the solution is 50% saturated and very
viscous. In addition, the sticky nature of the aerosol produced from stirring of this
solution negatively affected the performance of the mixing system.
To find a general theory that can explain the observed effect of the osmolytes on
the thermostability of the firefly luciferase the thermodynamics of the system has to
be scrutinized rather than the individual substances. In 1985, Arkawa and Timasheff
21
Jonas Eriksson
(Arakawa et al., 1985) proposed a theory for how osmolytes from different classes
can stabilize proteins. Their theory was based on measurement of the effectiveness of
various osmolytes, including glycine betaine and proline, to protect lysozyme from
guanidine hydrochlorid mediated denaturation. The fundamental discovery by
Timasheff and colleagues was that osmolytes are preferentially excluded from the
immediate hydration shell around a protein in contrast to solutes favoring
denaturation, e.g. guanidine hydrochlorid, urea or sodium dodecyl sulfate. The later
substances work in an opposite way by direct interaction with the surface of the
protein and thereby excluding the water. If a solute is excluded from the water
immediately adjacent to the protein surface, a local decrease in system entropy takes
place, as the solute will be distributed less randomly throughout the entire aqueous
phase. Thermodynamically, this is an unfavorable situation. However, if the protein is
unfolded, e.g. by increased temperature, and thereby exposes even more surface area,
the situation would be even worse from a thermodynamic perspective. A bad situation
thermodynamically would be made even worse. Therefore, solutes that are excluded
preferentially from water near the protein surface favor the compact, folded state of
the protein.
5.1.2. Pyrosequencing technology at elevated temperatures (II)
Formation of different types of secondary structures is a well-known problem in
DNA and RNA research. Self-complementary regions with high GC content can form
hairpin structures both for RNA and DNA. Processive DNA synthesis by reverse
transcriptase is frequently interrupted by secondary structures (pause sites), causing
difficulties in full-length cDNA synthesis (DeStefano et al., 1992; Klarmann et al.,
1993; Kuo et al., 1997; Malboeuf et al., 2001). Templates with DNA hairpins are both
difficult to amplify by PCR (Jung et al., 2002) and to sequence by the Sanger method
(band compressions) (Barr et al., 1986; Jung et al., 2002; Mizusawa et al., 1986).
Primer-dimer is another type of secondary structure that reduces the performance of
PCR (Brownie et al., 1997).
To reduce problems associated with secondary structures several strategies have
been developed. The observation that tetraalkylammonium ions binds to AT rich
22
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
DNA (Shapiro et al., 1969) and reduce the melting temperature of DNA (Melchior et
al., 1973) has stimulated the search for other substances with similar features. For
example, betaine was found to eliminate the base pair composition dependence of
DNA melting (Rees et al., 1993). Betaine has therefore been applied to reduce
secondary DNA structures in PCR in a number of studies (Henke et al., 1997;
McDowell et al., 1998; Weissensteiner et al., 1996). In addition, the combination of
glycine betaine and trehalose improved reversed transcription, resulting in longer
cDNA products (Spiess et al., 2002). Dimetyl sulfoxid (Winship, 1989), glycerol
(Bachmann et al., 1990), polyethylene glycol (Pomp et al., 1991) and formamide
(Weissensteiner et al., 1996) have also been found to yield significant improvements
in PCR. A different strategy to reduce problems due to secondary structures is to use
higher temperature, which has been made possible by the introduction of
thermostable enzymes (successfully used in cycle sequencing (Innis et al., 1988) and
PCR).
The Pyrosequencing method has traditionally been performed at 28°C, mainly
because of the low thermostablility of the firefly luciferase. The lower temperature
increases the probability of unspecific hybridization and secondary structure
formation. The consequence for the Pyrosequencing technology is unspecific signals
not related to the sequencing signals. Three different kinds of DNA related structures
have been identified to cause these problems and are illustrated in figure 6. Singlestranded DNA (ssDNA) can form loop structures by self-hybridization. Primers can
make unspecific binding to each other and form primer-dimers. Loop structures and
primer-dimers can function as templates for DNA polymerase if there is a match in
the 3’-end. In addition, ssDNA can form hairpin structures that can be a hindrance for
the DNA polymerase and terminate further polymerization.
primer-dimer
loop structure
3'
5'
5'
hairpin structure
3'
5'
5'
3'
3'
Figure 6. Illustration of secondary DNA structures identified to cause problems for the
Pyrosequencing technology.
23
Jonas Eriksson
In paper II the osmolyte strategy was employed to increase the temperature for
the Pyrosequencing reaction. A self-hybridizing single-stranded template with an
annealed sequencing primer was analyzed at 28°C. Signals were obtained from
specific priming of the sequence primer and from unspecific priming of the free 3’end of the ssDNA (Fig. 7 (A)). At 37°, the signals from the unspecific priming
decreased and the signals from the sequence primer dominated (Fig. 7(B)). The
sequence primer still bound strongly at 37°C, whereas the weaker interaction of selfhybridization was decreased. Further, it was proven, simply by using the abovedescribed template without any sequence primer, that the background signals
observed at 28°C were indeed from unspecific self-hybridization of free 3’-ends (Fig.
7(C)). In addition, by analyzing the background signals and the sequence of the
single-stranded template it was shown that the unspecific self-hybridization of the 3’end occurred at a specific site on the DNA (paper IV, Fig. 5). However, a more
common situation is when unspecific signals are generated for every dNTP addition.
A possible explanation for the later observation is that the 3’-end can make unspecific
binding at multiple sites and that the observed signals are an average from all these
sites.
24
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
A
B
C
D
Figure 7. Effect of increasing the temperature from 28°C to 37°C on a PCR generated 222-base-long
self-looping template. Analysis was performed in the presence of a sequencing primer, at 28°C (A) and
37°C (B), and in the absence of a sequencing primer at 28°C (C) and 37°C (D). The arrows in A
indicate signals originating from the loop (see C). At 37°C the signals from the loop is clearly reduced
as indicated by the arrows in B. The low signals observed in D indicate destabilization of the loop
structure at the higher temperature. The arrows in C and D indicate the expected height of a signal
from incorporation of one correct base if the sequencing primer used in A and B would have been
present. The correct read sequence is indicated above trace (B) and the order of nucleotide addition is
indicated under the traces.
A 320 base-pair long PCR generated fragment from the human glutathione
peroxidase gene was used for analysis of the effect of increased temperature on
primer-dimers. The fragment was sequenced and the sequencing primer formed
primer-dimers that contributed to unspcific signals (Fig. 8(A)). Increasing the
temperature destabilized the primer-dimers, and at 37°C only very low background
signals were observed and the correct sequence could be easily read (Fig. 8(B)).
25
Jonas Eriksson
A
B
Figure 8. Effect of increasing the temperature from 22°C to 37°C on a single-stranded template in the
presence of a primer-dimer. Analysis, using Pyrosequencing technology, was performed on a PCRgenerated 320-base-long template in the presence of 5 pmol sequencing primer at 22°C (A) and 37°C
(B). The unspecific signals that were due to the primer-dimer, indicated by arrows, were reduced at the
higher temperature. The correct read sequence is indicated above trace B.
In the process of finding the origin of disturbing background signals it is
important to have a proper evaluating tool. For this reason a method was developed to
predict contribution of signals from primer-dimers to the real sequencing signals. In
the new method a synthetic DNA template was sequenced and used as an internal
reference. With the assumption that the analyzed primers do not bind to the reference
DNA, the only signals apart from the reference signals are signals from the primerdimers. In this way the contribution from any primer that generates background
signals can be easily observed and quantified.
In an earlier study another type of problematic template was found. The template
had a region that consisted of three C followed by seven G. The observed sequence
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Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
signals in the seven-G-region were very low with no improvement by increasing the
temperature (Fig. 9(B)). By analysis of the template with a software (Oligo 4.0) we
found that 21 different possible hairpin structures (19 with a steam length of 2 bp and
two with 3 bp) could be formed. The structure of the hairpin with the lowest ∆G (-1.7
kcal/mol) is illustrated in figure 9(A). As sequence data was not improved by
increasing the temperature a different strategy was employed. An attempt was made
to weaken the structure by using 7’-deaza-dGTP instead of the natural dGTP during
the PCR. This strategy has previously been reported to improve both the PCR in GCrich regions (Liu et al., 1998) and the sequencing of GC-rich regions using the Sanger
dideoxy method (Fernandez-Rachubinski et al., 1990). By combining the use of 7’deaza-dGTP in the PCR and increased temperature (37°C) during the sequencing
procedure the sequence could be read beyond the homopolymeric region (Fig. 9(C)).
27
Jonas Eriksson
A
B
28oC
C
37oC
Figure 9. Effect of a nucleotide change and increased temperature on the sequence data obtained on a
131-base long template with a hairpin structure. The 131-base long DNA sequence was analyzed
(Oligo 4) for possible hairpin structures. The hairpin structure with the lowest ∆G (-1.7 kcal mol-1) and
the highest Tm (56°C) is illustrated (A). The template in (B) was generated by PCR using the natural
dGTP and sequenced at 28°C. In (C) the natural dGTP was replaced by 7’-deaza-dGTP in the PCR and
sequenced at 37°C. At 28°C the sequence could not be read beyond the 7C due to minus shift
(indicated by vertical arrows in B). In (C) the vertical arrows indicated the same positions as in (B) but
with reduced shift. The correct read sequence is indicated above trace (C). The order of nucleotide
addition is indicated under the traces.
The thermodynamic stability of double-stranded nucleic acids depends on the
base composition/sequence (Breslauer et al., 1986; Freier et al., 1986; Lesnik et al.,
1995) and effects of polyelectrolytes in the surrounding solution (Le Bret et al., 1984;
Manning, 1978; Record et al., 1978). In the context of the latter relationship glycine
betaine has been reported to isostabilize DNA at concentrations over 5 M (Rees et al.,
1993). However, in the experiments described above and in paper II we could not
observe any effect of glycine betaine itself on the stability of the primer-dimers or the
loop structures. Neither did glycine betaine affect the hairpin structure with the
28
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
natural dGTP. Although, a less stable hairpin was observed in the presence of glycine
betaine when the natural dGTP was replaced by 7’-deaza-dGTP, indicating an
isostabilization effect.
5.1.3. Coupled enzymatic assays at elevated temperatures (I)
To be able to correctly estimate any temperature dependent difference in the firefly
luciferase activity by the activity stability method used in paper I the following
parameters were considered. Firstly, the pH-optimum for the firefly luciferase (7.75)
did not change when the temperature was increased from 22°C to 37°C. Therefore,
for all concentrations of glycine betaine used and at all temperatures tested the pH
was held at 7.75.
Secondly, the optimal concentration of
D-luciferin
has been
reported to be 100 µg/ml (Lundin et al., 1976) (confirmed by us). However, at 22°C
in the presence of 1.6 M glycine betaine the optimal concentration was found to be
500 µg/ml. Therefore, when the dependence of temperature on the firefly luciferase
activity was estimated both 100 µg/ml and 500 µg/ml of D-luciferin were used. A
20% increase of the luciferase activity was observed by increasing the temperature
from 22°C to 37°C in the presence of 100 µg/ml D-luciferin (paper I). In contrast, the
activity increased by 66% in the presence of 500 µg/ml D-luciferin. It is worth noting
that at 37°C in the presence of 1.6 M glycine betaine the firefly luciferase activity
increased by 30% if the D-luciferin concentration was increased from 100 µg/ml to
500 µg/ml.
Most methods for measuring enzymatic activity rely upon the detection of
molecules being formed or consumed by the enzyme. In contrast, the normal way of
measuring firefly luciferase activity is by quantifying the generated light by detecting
the photons with a photomultiplier tube (PMT), e.g. luminometer. The sensitivity of a
PMT varies for light of different wavelength. One parameter not examined in paper I
is the possibility of a temperature dependent wavelength change. For example, the
performance of the broad range PMT in the LKB 1251 luminometer is reduced by
20% by a change in wavelength from 550 to 600 nm (Kyösti Kinnunen, personal
communication).
29
Jonas Eriksson
If an enzyme-catalyzed reaction is studied over a range of temperatures, the
overall rate passes through a maximum. The temperature at which the rate is maximal
is known as the optimum temperature. Two independent processes affect the optimal
temperature, the catalyzed reaction itself and the thermal inactivation of the enzyme.
At lower temperatures inactivation is very slow and has no appreciable effect on the
rate of the catalyzed reaction; the overall rate therefore increases with rise in
temperature, as with ordinary chemical reactions. The temperature dependence for
many reactions fall somewhere in the range spanned by the hydrolysis of methyl
ethanolate (the rate coefficient at 35°C is 1.82 times that at 25°C) and the hydrolysis
of sucrose (the factor is 4.13) (Atkins, 1986). At temperatures over the optimal
temperature the concentration of active enzyme falls during the course of the reaction
due to thermal inactivation. This is the case for firefly luciferase at 37°C (Fig. 4).
One strategy to increase the thermostability of firefly luciferase is, as shown
above, the use of osmolytes. In the presence of glycine betaine the thermal
inactivation of firefly luciferase is reduced and only 10 % of the activity is lost over
60 min at 37°C (Fig. 4). A different strategy to thermostabilize firefly luciferase is
protein engineering. Several firefly luciferase mutants with increased thermostablility
have been reported. Some of them are summarized in table 3. For example, the Luc5-pos mutant retained more then 80% of the activity after 60 min at 42°C (Nourizad
et al, work in preparation).
Table 3. Thermostable firefly luciferase mutants.
Mutant
name
Lucigen
Luc-T
Source of the
wt
P. pyralis
L. crusiata
Mutation position
Reference
Glu354Arg, Asp357Tyr
Thr217Ile
Ultra Glow
Photuris
pennsylvanica
P. pyralis
43 positions
(Kajiyama et al.,
1993)
(McElroy et al.,
1993)
-
Luc-5-pos
Thr214Ala, Ile232Ala,
Phe295Leu, Glu354Lys,
Leu550Val
Lucigen, Luc-T and Ultra Glow have all been reported to display increased
storage stability against heat. However, it was found that the Ultra Glow luciferase
30
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
had a 10-times longer rise time compared to the wt P. pyralis luciferase. The Ultra
Glow luciferase has a nearly 100-fold lower KM for ATP (0.7 µM) (Rita Hannah,
personal communication) than what has been reported for the P. pyralis firefly
luciferase (Branchini et al., 1998), which might explain the observed lower catalytic
activity. In a Pyrosequencing reaction it is important that all ATP produced in each
polymerization step reacts and generates light with firefly luciferase. If the firefly
luciferase is to slow the apyrase has more time for degradation of the ATP and a
lower signal will be recorded.
Coupled bioluminometric assays can now be perform at elevated temperatures
using different strategies. In paper I the osmolyte strategy was used for studying of
the ATP sulfurylase and DNA polymerase catalyzed reactions at 37°C. These assays
were also successfully performed using a thermostable firefly luciferase. In figure 10,
ATP sulfurylase was assayed by the osmolyte strategy and DNA polymerase by using
a thermostable firefly luciferase. The activity of both enzymes were increased by over
100%, regardless of what strategy used, when the temperature was increased from
22°C to 37°C. It is worth noting that no loss in activity (assayed at 22°C) with or
without glycine betaine could be observed after 1-hour storage at 37°C for either ATP
sulfurylase or DNA polymerase.
31
Jonas Eriksson
B
A
22 o C
37 C
22 o C
o
1 min
37 C
o
1 min
Light
ATP sulf
40 pmol
ATP
ATP sulf
20 pmol
ATP
1 min
1 min
Figure 10. Coupled bioluminometric assays performed at different temperatures. A firefly luciferase
based assay was used to monitor ATP sulfurylase activity at 22°C and 37°C, respectively, in the
presence of 1.6 M glycine betaine (A). The initial rates at 22°C and 37°C were 18 and 35 pmols
ATP/min, respectively. In (B) DNA polymerase activity was recorded at 22°C and 37°C by using the
thermostable firefly luciferase mutant Luc-5-pos. The initial rate at 22°C and 37°C were 1.6 and 4.6
pmols PPi/min, respectively.
The Pyrosequencing reaction was successfully performed at 37°C by using the
osmolyte strategy (glycine betaine) (paper II). The increase in temperature was
advantageous and improved the sequence result in several ways (described in section
5.1.2.). An additional advantage with higher temperature is increased reaction rate for
individual enzymes, which has been observed for ATP sulfurylase and DNA
polymerase.
The observed advantages with the osmolyte strategy encouraged us to study the
possibility to use a thermostable firefly luciferase in the Pyrosequencing technology.
By applying a thermostable firefly luciferase the disadvantages of using osmolytes,
such as inhibition and increased viscosity could be circumvented (described in 5.1.1.).
Initially, two firefly luciferase mutants, Lucigen and Luc-T, were selected to replace
the wt firefly luciferase in the Pyrosequencing reaction. The selection was based on
activity stability measurements at 37°C and the time needed to reach peak light
emission. The Lucigen and Luc-T mutants showed high activity stability at 37°C and
the time to reach maximal activity was comparable to the wt firefly luciferase for both
mutants. However, by using one of the selected mutants for sequencing of a short 17-
32
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
base-long template at 37°C we found that the base line of the signals increased over
time (Fig. 11(A)). The result indicates that apyrase has low thermal stability at 37°C.
To estimate the activity loss a simple bioluminometric assay was used. The assay was
performed with Lucigen firefly luciferase and apyrase in the absence of glycine
betaine. We found that 50% of the apyrase activity was lost after 60 minutes at 37°C.
In contrast, a strong increase in the thermal stability of apyrase was observed in the
presence of glycine betaine (Fig. 11(B)).
37o C
- glycine betaine
A
A G T C A G T C A G T C A G T C A G T C A G T C A G T C A G T C A G T C A G T C A
B
C
T
G
A
C
G
2
A
2
2
T C AG
C
37o C
+ glycine betaine
A G T C A G T C A G T C A G T C A G T C A G T C A G T C A G T C A G T C A G T C A
Figure 11. Pyrosequencing technology with a thermostable firefly luciferase. The Pyrosequencing
reaction was performed on a short 17-base-long synthetic template at 37°C in the absence (A) and in
the presence (B) of 1.6 M glycine betaine. The peak height is maintained due to the use of the
thermostable Lucigen firefly luciferase mutant. Note the increased base line of the peaks in the absence
of glycine betaine (A) indicating that apyrase has low thermal stability at 37°C. In the presence of 1.6
M glycine betaine a strong increase in the thermal stability of apyrase was observed (B). The order of
nucleotide addition is indicated on the bottom of the traces, and the read sequence on top of trace (B).
An interesting observation was made with the Japanese firefly luciferase mutant
(Luc-T). When Luc-T was used in the Pyrosequencing reaction at 37°C the luciferase
activity increased dramatically with increasing glycine betaine concentrations.
33
Jonas Eriksson
Similar results have been observed with a different Japanese firefly luciferase mutant
(Luc-H) (Maria Murby, personal communication) but not with the Lucigen mutant.
The stimulation in activity was only valid for the Japanese firefly mutants from L.
crusiata while the North American firefly P. pyralis mutants were unaffected. It has
been reported that firefly luciferase can aggregate at higher temperatures (Hannah et
al., 1998; Lundovskikh et al., 1998).The aggregation leads to inactivation, which can
be observed as reduced activity. Perhaps the L. crusiata mutants more easily form
aggregates at higher temperatures than the P. pyralis mutants, and that the
aggregation might be prevented by the presence of glycine betaine.
Until a more thermostable apyrase has been found the osmolyte strategy is the
only practical procedure for performing the Pyrosequencing reaction at higher
temperatures.
5.2. Nucleotide analogs in Pyrosequencing technology (III)
A number of modified nucleosides and nucleotides have been synthesized in many
different laboratories all over the world for over 40 years and been used for numerous
applications. One field where modified nucleosides are used is in medicine. For
example, 6-mercaptopurine can be used clinically as a cancer and malaria drug due to
the interference with nucleic acid synthesis. A different application is modification of
natural nucleotides, e.g. labeling. For nucleotides used in the Sanger DNA sequencing
method the label can be radioactive, e.g. dATPα[35S], or fluorecent. In addition,
dideoxy nucleotides are used for termination of DNA fragments.
If a modified nucleotide is used for DNA sequencing by the SBS strategy the
positions where modifications can be made are limited. Firstly, the modification
should not affect the substrate specificity for the DNA polymerase. For example,
introduction of methyl and ethyl labels at the 4’-position in the 2’-deoxyribose part do
not affect the 1-base incorporation efficiency by Klenow polymerase (Summerer et
al., 2001), but once incorporated further DNA synthesis is reduced 2000-fold
(Summerer et al., 2002). For the purines the N3 position is proposed to have an
important role for the fidelity of DNA polymerases (Eom et al., 1996). In addition,
substitution by fluorescin-15 (Augustin et al., 2001) or 2-(4-imidazolyl)ethylamino in
34
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
position 8 showed low incorporation efficiency by polymerase (Perrin et al., 1999).
Secondly, the base pairing rule and steric factors limits the available positions further.
In the base part of the purines, position 1 and 6 are important for base pairing and
position 2 is important for the DNA structure. 2-aminoadenine substitutions for
adenine introduce changes in the minor groove of DNA and create an additional
hydrogen bond in the base pair with thymine (Chollet et al., 1988).
A third kind of limitation is set by the Pyrosequencing technology where a
modified adenine nucleotide has to be used due to the fact that the natural dATP is a
substrate to firefly luciferase and gives light. Modifications in the phosphate part are
only possible on the α-phosphate. The remaining two phosphates must be unchanged
due to their destiny to be cleaved off during polymerization, as PPi, and converted to
ATP. Analogs containing boron instead of oxygen on the α-phosphate have been
described (Porter et al., 1997). The currently used analog (dATPαS) has sulphur at
this position. This substitution makes the dATPαS less then 0.05% as effective as
substrate for the luciferase compared to dATP (Ronaghi et al., 1996). Still, the
requirements for the Pyrosequencing technology on the performance of incorporation
and extension rates of dATPαS with Klenow polymerase were matched. Analogs
with bromo- and hydroxy-group replacements in position 8 are not substrates for
firefly luciferase, but have low incorporation efficiency and consequently could not
be used for the Pyrosequencing technology (unpublished observations). The adenine
nucleotide studied in paper III (c7dATP) was base modified in position 7; carbon
replaced nitrogen.
In figure 12 the substrate specificity for selected analogs with firefly luciferase
are shown. The firefly luciferase activity in the presence of dATP was 2.5-3% of the
activity obtained by an equivalent molar amount of ATP. Both dATPαS and c7dATP
showed much lower activity; 0.005% and 0.006%, respectively. The data indicate that
c7dATP has low substrate specificity for firefly luciferase and in the same low range
as dATPαS.
35
Jonas Eriksson
Figur 12. The structure and substrate specificity for adenine analogs with the firefly luciferase. For
each analog the substrate specificity is illustrated by an activity trace. The amount of analog added is
shown above each trace. The sensitivity of the recorder is indicated beside each trace.
It is important for the Pyrosequencing technology that the modified dATP analog
is efficiently degraded by apyrase otherwise traces of left over nucleotides cause nonsynchronized extension and reduced sequence quality. Problems can also occur if
apyrase is product inhibited by the analog. Although the initially used Sp/Rp mixture
of dATPαS and the currently used pure Sp-dATPαS solution are degraded by apyrase
an inhibitory effect can be observed after several additions. The inhibitory effect is
lower for the pure Sp-dATPαS solution than for the Sp/Rp-dATPαS mixture
36
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
(Gharizadeh et al., 2002). However, there is a need for a dATP analog with reduced
inhibitory effect on the apyrase.
In figure 13 the effect of dATPαS and c7dATP, respectively, on the apyrase
activity is shown. The experiments show the hydrolysis of ATP by apyrase in the
presence of dATPαS (Fig. 13(A)) and c7dATP (Fig. 13(B)). The observed initial
signal increase after each addition is due to the reaction between ATP and luciferase
and the following decrease is due to ATP degradation by apyrase. In the presence of
dATPαS a continuous decrease in apyrase activity could be observed as an increased
base line (Fig. 13(A)). However, when c7dATP was used at the same concentration as
dATPαS the inhibitory effect on apyrase was significantly reduced (Fig 13(B)).
A
B
Figure 13. The effect of different adenosine nucleotides on the apyrase activity. The reaction was
started by addition of 0.2 µl of a solution containing (A) 0.4 pmol ATP and 320 pmol dATPαS or (B)
0.4 pmol ATP and 320 pmol c7dATP. The addition was repeated 100 times with 1-min intervals. Only
the first 14 and the last 17 additions are shown. The reaction was detected by the firefly luciferase
system.
A single satisfactory explanation for the observed reduced inhibitory effect on
apyrase with c7dATP has not yet been found. One hypothesis suggested in paper III is
that there is a difference between the intermediate products dADPαS and c7dADP.
According to the hypothesis c7dADP is degraded by apyrase but dADPαS is not, or to
37
Jonas Eriksson
a lower degree, and thereby dADPαS is accumulated over time. If this is the case the
accumulated dADPαS might inhibit the apyrase reaction.
In paper III, an attempt was made to understand and reduce a phenomenon known
as the A-effect. The A-effect is defined as a 5-15% higher than expected peak-hight
after A incorporations in the Pyrosequencing reaction (Fig. 14). The effect is related
to the adenine nucleotide alone and is more pronounced at higher concentrations. No
similar effect has been observed with the guanine, thymine or cytosine nucleotides.
The origin is still unknown so in order to explain the A-effect a hypothesis was
suggested.
In addition to the light reaction the firefly luciferase has a side reaction that
catalyze the formation of di-nucleoside-tetra-phosphate (dNp4dN) (Guranowski et al.,
1990; Sillero et al., 1991). Deoxyadenosine triphosphate (dATP) is a better substrate
for the dNp4dN formation than dGTP, dCTP and dTTP (Ortiz et al., 1993). A
hypothesis is that the side reaction increases the turnover of the firefly luciferase
when adenine nucleotides are used. Consequently, the product is released faster and
therefore more firefly luciferase is available to react with ATP and produce light. If
the hypothesis is right an adenine analog more equal to dGTP, dCTP and dTTP as a
substrate for the side reaction should reduce the A-effect. The dATP analog c7dATP
was tested, but still the A-effect was observed (Fig. 14(B)). Perhaps there are other,
not yet tested, adenine nucleotide analogs that might be useful for solving the Aeffect problem.
38
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
B
A
Figure 14. Pyrograms illustrating the A-effect. Sequencing was performed on a 131-base-long PCRgenerated DNA fragment in the presence of dATPαS (A) or c7dATP (B). Note the higher signals
(arrows) for the adenine nucleotides. The nucleotides were added with 65 s intervals and the order is
indicated on the bottom of the traces. The read sequence is indicated above the traces.
The sequence quality and readlength obtained with c7dATP was equal to that
obtained with dATPαS. Several templates were successfully sequenced using c7dATP
and the results obtained were used for typing various bacterial species and several
clinically important Human Papilloma Viruses (HPV). In addition, both c7dATP and
dATPαS were stable for at least 3 months when stored in 2 mM solutions at 8°C
without reduced sequence performance.
5.3. DNA polymerases in Pyrosequencing technology (IV)
A DNA polymerase used in the Pyrosequencing reaction should fulfill a number of
characteristics if high quality data should be obtained. The DNA polymerase must be
3'-5' exonuclease-deficient in order to avoid degradation of the sequencing primer
annealed to the template. The processivity must be reasonable high to achieve full
incorporation of nucleotides in each step. Moreover, the DNA polymerase must be
able to use modified nucleotides (such as dATPαS and c7dATP) and to incorporate
such nucleotides with high efficiency, especially across homopolymeric T-regions.
Furthermore, it is a clear advantage if primer-dimers and loop-structures cannot be
used as substrates. It is also desirable that the polymerase has an efficient strand
displacement activity.
Since the exonuclease-deficient form of the DNA polymerase I large fragment,
also known as Klenow fragment and related here as Klenow polymerase, displays
many of the above described features it has traditionally been used in the
39
Jonas Eriksson
Pyrosequencing method. However, sequencing problems due to inefficient
incorporation of nucleotide dATPαS in homopolymeric T-regions and extension of
mispairs formed by unspecific hybridization events have occasionally been
encountered. For example, sequencing of templates containing regions with 4 to 5 T
with Klenow polymerase resulted in unsynchronized sequence signals. Further,
primer-dimers and 3’-end loops gave increased background signals (see section
5.1.2.). Therefore, in order to improve the performance of the Pyrosequencing
technology a modified T7 polymerase (exonuclease deficient T7 DNA polymerase),
Sequenase (Tabor et al., 1989), was tested.
The effect of Sequenase on sequencing performance on poly(T)-rich templates
was evaluated in parallel with Klenow DNA polymerase. Sequenase demonstrated far
better catalytic efficiency for all homopolymeric T-regions tested. Synchronized
extension was observed on homopolymeric templates containing regions with up to 8
T (Fig. 15(B)). It was also found that both Klenow and Sequenase incorporated all the
other nucleotides with good efficiency.
40
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
A
B
Figure 15. Sequencing performance by two different DNA polymerases. The Pyrosequencing reaction
was performed on a synthetic oligonucleotide, containing a homopolymeric region of eight T, in the
presence of (A) Klenow and (B) Sequenase. Note the low C and false A signal peaks obtained
(indicated by filled and dotted arrows, respectively) when Klenow was applied. When Sequenase was
used the sequence could easily be read. The C signal peak was more close to the expected level (filled
arrow) and the false A was reduced (dotted arrow). The read sequence is indicated above trace (B).
The most important factors responsible for the better result obtained with
Sequenase are processivity and nucleotide discrimination. A more processive DNA
polymerase is bound a longer time to the template during each binding event and can
therefore polymerize longer regions before it falls off. Sequenase is over 10-times
more processive than Klenow DNA polymerase (Bedford et al., 1997). However, the
most important factor is probably that Sequenase shows lower discrimination against
modified nucleotides than Klenow (Tabor et al., 1990). For this reason Sequenase has
been used in dATPα[35S] labeling of templates in Sanger DNA sequencing. The
adenine nucleotide analogs dATPαS and c7dATP were both more efficiently
incorporated into poly(T)-rich templates using Sequenase compared to Klenow in the
Pyrosequencing reaction.
41
Jonas Eriksson
It has been shown that Sequenase is more discriminating for 3'-mismatches than
Klenow polymerase (Nyrén et al., 1997). In addition, Sequenase require a longer stem
(15 bases) than Klenow polymerase (12 bases) if a loop-structure is used as primer
(Ronaghi et al., 1998a). In paper IV it was shown that Sequenase was reluctant to
extend some free 3'-ends formed by loop-structures and primer-dimers in contrast to
Klenow polymerase.
Under some conditions, DNA polymerase may stall when it reaches secondary
structures formed by the template itself (hairpin structures) or due to mispriming by
the sequence primer, i.e. DNA polymerase can not pass these DNA structures unless
the enzyme has good strand-displacement activity. To analyze if such structures may
create problems for Klenow polymerase and/or for Sequenase a model system was set
up. Two different oligonucleotides were designed, both binding ten bases in front of
the sequencing primer, one with a 5’-overhang (eight bases) and one without an
overhang. Both oligonucleotides were blocked (by a phosphate group) at the 3’-end to
prevent extension. In figure 16 the results from sequencing of the model template, in
the absence of oligonucleotide (Fig. 16(A)), in the presence of an oligonucleotide
without an overhang (Fig. 16(B)), and in the presence of an oligonucleotide with an
overhang (Fig. 16(C)) are shown. Similar results were obtained regardless of which
DNA polymerase that was used (only the Sequenase experiments are shown). Both
polymerases had problem to read through the dsDNA structure that lacked a 5’overhang. After extension of the primer five bases into the dsDNA structure a lower
signal was observed for the following C addition (the position is indicated by an
arrow in Fig. 16(B)). However, the difficulty in reading through the dsDNA region
was reduced when the dsDNA structure had a 5’-overhang (Fig. 16(C)). The later
result indicates that a 5’-overhang is needed for the polymerases (Klenow and
Sequenase) to exert their strand-displacement activity.
42
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
A
B
C
Figure 16. Analysis of strand-displacement activity. The Pyrosequencing reaction was performed with
13 units Sequenase on a primed 279-base-long PCR-generated fragment in the (A) absence and in the
(B and C) presence of oligonucleotides binding ten bases in front of the primer. The vertical dotted
arrows indicate starting position of the oligonucleotide-binding region. The vertical solid arrows
indicate the first signal, in trace B and C, that were lower than expected (A). One of the
oligonucleotides was perfectly matched to the template (B) and one had an eight-base-long 5’overhang (C). Sequenase had problem to read through the dsDNA structure lacking a 5’-overhang.
After extension of the primer five bases into the dsDNA structure a lower signal was observed for the
following C addition (indicated by solid arrows in trace B and C, respectively). The order of nucleotide
addition is indicated on the bottom and the read sequence on the top of the traces.
43
Jonas Eriksson
5.4. PPi and PPase (V)
The Pyrosequencing technology consists a major part of this thesis. From the
principle of the technology (see section 4.2) it can be easily realized that the only
inorganic pyrophosphate (PPi) that is allowed in the system is the PPi generated by
the DNA polymerase during the polymerization of the DNA template. All other
sources of PPi are considered to be contaminants. There are several sources for
contaminating PPi, such as the dNTP solutions and the DNA polymerase buffers.
Degradation of dNTP is a likely reason for PPi in the dNTP solutions, while
phosphate is the most probable source for the contamination of the DNA polymerase
buffers. In addition, PPi from the PCR can be a limiting factor for further
developments of DNA template preparation methods for the Pyrosequencing
technology.
According
to
early
procedures
high
amounts
of
inorganic
pyrophosphatase (PPase) was used for removal of contaminant PPi in the dNTP
solutions, followed by removal of the PPase by filtration. Although the method was
successful in PPi removal, a major drawback was that once the PPase had been
removed the PPi levels could increase again due to continuous dNTP degradation.
Therefore, it was argued that it should be beneficial to have small amounts of PPase
present in the dNTP solutions to ensure PPi-free conditions. In that case, careful
calibrations must be performed to avoid adding to much PPase to the Pyrosequencing
reaction.
PPase catalyze the hydrolysis of PPi to orthophosphate (Pi). Several assays have
been developed for detection of PPase activity. The enzymatic activity can be
followed either in the synthesis or hydrolysis direction. The most convenient assay in
the synthesis direction is the continuous bioluminometric method (Nyrén et al.,
1985), which utilizes ATP sulfulylase coupled to firefly luciferase. The method by
Nyrén et al. has the advantage of sensitivity and speed; the assay can be completed
within seconds. However, most PPase activity methods are based on colorimetric
estimation of liberated Pi. The most widely used method for this purpose is the
procedure of Fiske and Subbarow (Fiske et al., 1925) that relies on the formation of a
blue molybdenum complex, which forms upon reduction of an ammonium
molybdate-phosphate complex in strong acid solutions. Discontinuous enzymatic
44
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
methods have also been described. However, the method described by Kawasaki et al.
involves the laborious and intriguing work of immobilization of three different
enzymes (Kawasaki et al., 1989). Furthermore, the method by Hill et al. involved
centrifugation and the use of HPLC equipment (Hill et al., 1997). Only two methods
for continuous detection of the hydrolytic activity have been described (Baykov et al.,
1981; Shakhov et al., 1982). The main drawback with the method by Baykov et al.
was the gradual deposition of precipitate on the optical cuvett containing the reaction
solution, resulting in drift of the baseline with prolonged measurements. Furthermore,
the initial rate of PPase activity could not be measured due to the design of the
automatic analyzer. The method by Sharkhov et al. based on recording change in pH
was developed for a specific application and has not been proven to be generally
applicable, most probably due to a complicated preparation procedure and lack in
sensitivity. Many of the methods described above involve many steps, long (hour’s)
incubation times, advanced technical equipment and extensive use of hazardous
chemicals. Furthermore, the PPase activity is not followed directly. In addition, the
colorimetric methods suffer in the uncertainty of the low stability color complexes.
In paper V a bioluminometric assay is described for real-time detection of PPase
activity in the hydrolytic direction. The assay relies on the observation that the ATPinduced firefly luciferase reaction is inhibited in the presence of PPi. In the presence
of PPase the PPi is hydrolyzed and the bioluminoscence increases (Fig. 17). In an
optimized assay the initial rate increase of the luciferase reaction is proportional to
the amount of PPase added. The linear range for the assay is between 8-500 mU
PPase and an assay can be completed within minutes. In situations where ATPase
activity is common, such as in crude cell extract, the assay can still be employed
simply by replacing the ATP by adenine 5’-phosphosulfate (Fig. 17(B)).
45
Jonas Eriksson
Figure 17. Typical traces obtained from real-time monitoring of PPase activity. The assay was
performed in the presence of 25 µM PPi and (A) 0.2 µM ATP or (B) 500 µM APS. The reaction was
started by addition of 8 mU yeast PPase. The luminescence was measured with a tube luminometer.
The ATP and PPi additions were done with the luminometer in the opened position, whereas the PPase
addition was done in the closed position.
Apart from the general applications described in paper V the PPase method was
found to be particularly well suited for calibration of the PPase solutions used for
removal of contaminating PPi. Calibration ensures that identical amounts of PPase are
used every time. In addition, variations between PPase batches can easily be adjusted
for. Therfore, all work described in this thesis were the Pyrosequencing method was
used the dNTP solutions were treated with a carefully calibrated PPase amount.
Furthermore, in all cases where Sequenase polymerase was applied to the
Pyrosequencing reaction, mainly in paper IV, determination of the exact PPase
amount necessary for removal of the PPi originating from the phosphate buffer is
essential. Finally, the PPase assay was also found to be useful in connection with the
double-stranded DNA preparation technique (Nordström et al., 2002) where a specific
PPase amount was needed for optimal performance.
46
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
Concluding remarks and future prospects
In this work it was demonstrated for the first time that osmolytes could increase the
thermostability of firefly luciferase. The number of osmolytes described in this thesis
is only a fraction of what is available. Perhaps there are other osmolytes or substances
in the kingdoms of plants and fungi that could increase the thermostability even
further. An ideal osmolyte should also be able to mediate a thermostabilizing effect at
low concentrations to avoid the problems related to high viscosity.
The improved thermostability, in turn, allowed the Pyrosequencing method to be
performed at higher temperatures, which reduced unspecific signals resulting in
improved sequence results. Careful template and primer design, together with the use
of single-stranded DNA-binding protein, are no longer the only available tools for
reducing unspecific signals. Although the benefits of increased enzymatic reactions
rates for Pyrosequencing at higher temperature have not yet been fully investigated,
some advantages have been observed in sequencing a template with a strong hair-pin
structure.
We also demonstrated, using several thermostable firefly luciferase mutants,
monitoring of DNA polymerase and ATP sulfurylase at 37°C in the absence of any
osmolyte. Unfortunately, the nucleotide-degrading enzyme in the Pyrosequencing
method, apyrase, was found to be heat-sensitive and lost significant activity at 37°C.
Osmolytes improved the thermostability of apyrase in a similar manner to luciferase.
A thermostable apyrase must therefore be found if osmolytes could be discarded in
the application of high temperature DNA sequencing using the Pyrosequencing
technology. One possibility is to use mutagenesis on the apyrase or to find alternative
more thermostable nucleotide degrading enzymes.
DNA polymerase is a key enzyme in the performance of the Pyrosequencing
method. In this thesis, it was demonstrated how a highly processive and less
discriminatory polymerase could improve sequence results. Further improvement
could be envisioned with other polymerases, such as the chimerical Klenow
polymerase engineered to include the thioredoxin-binding domain of T7 Sequenase
(Bedford et al., 1997), T4 polymerase or Bst DNA polymerase.
47
Jonas Eriksson
For the first time since the α-phosphate modified dATPαS was presented, an
alternative exists for the Pyrosequencing technology in the possibility to use a base
modified nucleotide. It is very likely that the use of c7dATP, which has a lower
inhibitory effect on apyrase than dATPαS, will be an important factor in the quest to
reach even longer sequence read-length.
Hopefully the thermostabilization of firefly luciferase by osmolytes could lead to
new applications of firefly luciferase-based bioluminescence. It would also be
satisfying if the higher reaction temperature could be applied to Pyrosequencing
technology in a more general perspective and as a parameter to reach longer sequence
read-length. With the recent advancements in the Pyrosequencing technology now
available and with new technological platforms being developed based on the
Pyrosequencing method, the future for the Pyrosequencing technology looks very
promising.
48
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
Acknowledgement
Now is the time to pop out from behind the screen and announce the following: Hereby I
would like to thank the following people for making the research presented in his thesis
possible:
Pål Nyrén – My supervisor and scientific guide who introduced me to the field of
bioluminescence and Pyrosequencing technology. Thank you so very much for the
confidence you have shown to me over the years.
Mostafa Ronaghi – For making the move from Uppsala (BMC) to Stockholm (KTH)
possible and introducing me, in my absence, in such way that I got accepted as PhD-student.
Thanks to the Applied Enzymology group presently consisting of Baback Gharizadeh,
Nader Nourizad and Tommy Nordström for scientific collaboration and contributing to a
warm and friendly atmosphere in the writing room. Also thanks to some of the past members
of the group, especially Carlos Garcia for proving that good rhythms never get out of style
and Samer Karamohamed for teaching me the practical craftsmanship of proper handling of
luminometers as well as being a close friend.
Måns Ehrenberg – For great positive and constructive influence on me during my pre-KTH
years at BMC, Uppsala, as well as giving me encouragement to proceed into the world of
research and science. Also thanks to Michail Pavlov for your excellent scientific supervision
during my master thesis work on initiation factor 3.
Thanks to the staff at Pyrosequencing AB, especially Maria Murby, Peter Hagelid and
Björn Ekström.
Thanks to Fredik Viklund for computer assistance. Thanks to Harry Brumer for i)
convincing me to change my computer strategy to the better and ii) for critical reading of
parts of this thesis. Thanks to Eva Hedin for taking care of our common real estate interests.
Thanks for having the privilege to work with the people at the AlbaNova, floor 2, from year
2001 to present time consisting of the researchers and students in the groups headed by Karl
Hult, Tuula Teeri and Gunnel Dalhammar.
49
Jonas Eriksson
Thanks for having the privilege to work at the Department of Biotechnology, located at
Teknikringen 34, under the years 1998-2001, with researchers and students headed by
Mattias Uhlén, Per-Åke Nygren, Stefan Stål and Sofia Hober.
Thanks to the Helge Ax:son Johnsons stiftelse, SFBM (Svenska föreningen för biokemi och
molekylärbiologi), ESF (European Science Foundation) and KTHs fond för vetenskap och
högre forskning for financial support attending conferences and meetings.
Thanks to Kjell Backman for friendship and introducing me to the nightlife of Stockholm.
Also Anders Hagblom for getting me interested in live music scene of Stockholm and
introducing county music and new artists I never discovered otherwise. Erik Söderström for
all funny moments and your honesty.
Thanks to Simon Fredriksson for mentioning something about “light DNA sequencing” at
the Lucce-gasquen 1997 at Gästrike Hälsinge nation (GH), Uppsala. Also all members in
Hälsinge Bockar (GH), especially Magnus Holmquist for support on partial integration and
being “someone to lean on”. Gunnar Carlsson for explaining to me why Frank Zappa is
necessary for a happy living. Ola Sundgren for friendship under the compact living years.
Henrik Nilzén for introducing me to the founders of Flogsta funken, Offspring and the
Friday-night student-sub-world of Uppsala nation. My friends from Gävle: Lumpan,
Jansson, Lowie. Thanks to the great staff at Texaco (Preem) Eriksberg for unforgettable
memories from the years 1995-1998. Tord Nyström for friendship and all the trips to
Hultsfred.
Ove och Birgitta, my parents and my best friends, for your never-ending support in all the
things I do and have done. Without you, this “klass resa” would not have been possible.
Finally I would not have been even half the man I am today without you in my life, my love
and closest friend Johanna and to our son Måns.
50
Advancements in Firefly Luciferase-Based Assays and Pyrosequencing Technology
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