Published online 9 January 2004
Nucleic Acids Research, 2004, Vol. 32, No. 1 278-286
© 2004 Oxford University Press
Detection of guanine–adenine mismatches by surface plasmon resonance sensor carrying naphthyridine–azaquinolone hybrid on the surface
Shinya Hagihara1,
Hiroyuki Kumasawa1,
Yuki Goto1,
Gosuke Hayashi1,
Akio Kobori2,
Isao Saito1 and
Kazuhiko Nakatani*,1,2
1 Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, Kyoto, Japan and
2 PRESTO, Japan Science and Technology Agency (JST), Kyoto 615-8510, Japan
*To whom correspondence should be addressed. Tel: +81 75 383 2756; Fax: +81 75 383 2759; Email: nakatani{at}sbchem.kyoto-u.ac.jp
Received July 18, 2003; Revised October 15, 2003;; Accepted November 15, 2003
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ABSTRACT
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We have discovered a new molecule naphthyridine–azaquinolone
hybrid (
Npt–Azq) that strongly stabilized the guanine-adenine
(G-A) mismatch in duplex DNA. In the presence of
Npt–Azq,
the melting temperature (
Tm) of 5'-d(CTA AC
G GAA TG)-3'/3'-d(GAT
TG
A CTT AC)-5' containing a single G-A mismatch increased by
15.4°C, whereas fully matched duplex increased its
Tm only
by 2.2°C.
Npt–Azq was immobilized on the sensor surface
for the surface plasmon resonance (SPR) assay to examine SPR
detection of duplexes containing a G-A mismatch. Distinct SPR
signals were observed when 27mer DNA containing a G-A mismatch
was analyzed by the
Npt–Azq immobilized sensor surfaces,
whereas the signal of the fully matched duplex was
6-fold weaker
in intensity. The SPR signals for the G-A mismatch were proportional
to the concentration of DNA in a range up to 1 µM, confirming
that the SPR signal is in fact due to the binding of the G-A
mismatch to
Npt–Azq immobilized on the surface. Examination
of all 16 G-A mismatches regarding the flanking sequence revealed
that the sensor surface reported here is applicable to eight
flanking sequences, covering 50% of all possible G-A mismatches.
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INTRODUCTION
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As a follow-on to the complete sequencing of the human genome,
typing of single nucleotide polymorphisms (SNPs) in an array
of disease-related genes is expected to be an indispensable
technique for realizing personalized medicine. A number of methods
have been developed for SNP typing (
1–
3), but much study
is still needed to design new typing methods that are simple
in operation, rapid and accurate in analysis, and low in cost.
One of the challenges we have focused on is the reduction and
eventual redundancy of labeled oligonucleotides for analysis.
The expense of fluorescently labeling oligonucleotides and PCR
products limits their application in large-scale typing by many
currently available methods. We have reported a conceptually
novel method of SNP typing that detects mismatch-containing
duplexes with a small molecular ligand immobilized on a gold
surface (
4). Hybridization of two sets of duplex DNAs that differ
from each other by a single nucleotide produces a DNA heteroduplex
containing a single mismatched site. Mismatch-containing duplexes
can be separated from homoduplexes by either gel electrophoresis
(
5,
6), chemical and enzymatic cleavages at the mismatched site
(
7–
9), or selective capture with mismatch-binding proteins
(
10,
11). While these heteroduplex analyses applied to low-throughput
screening are essentially free from oligonucleotide labeling,
new technologies for high-throughput analyses are yet to be
established. A small molecular ligand that selectively binds
to a mismatched site could replace mismatch-binding proteins
and bring an innovation to heteroduplex analyses (
5,
12–
20).
The ligand naphthyridine dimer (
Npt–Npt) strongly and
selectively binds to guanine–guanine mismatches in duplex
DNA (
5,
12) (Fig.
1). The binding constant to a G-G mismatch
in the 5'-CGG-3'/3'-GGC-5' sequence is 1.9
x 10
7 M
–1.
Npt–Npt consisting of two 2-amino-1,8-naphthyridine (
Npt)
chromophores, and a linker connecting the chromophores is designed
so that each
Npt produces three hydrogen bonds to each one of
the guanines in the G-G mismatch, and the resultant naphthyridine–guanine
pair is stabilized by stacking with the flanking base pairs.
The proposed binding of
Npt–Npt to the G-G mismatch has
been verified by the 2D- NOESY spectrum of the complex (
12).
In addition to
Npt–Npt, ligands that selectively and strongly
bind to G-A, G-T and A-A mismatches are needed to accomplish
SNP typing by a mismatch binding ligand. Taking into account
the structure of
Npt–Npt as a clue for the molecular design
of ligands binding to a G-A mismatch, we have discovered naphthyridine–azaquinolone
hybrids (
Npt–Azq) where one
Npt chromophore in
Npt–Npt is replaced by a 8-azaquinolone chromophore (
Azq) having complementary
hydrogen-bonding surfaces to adenine (Fig.
2). Herein, we report
the remarkable stabilization of G-A mismatch DNA by
Npt–Azq and the first synthesis of a surface plasmon resonance (SPR)
sensor detecting a G-A mismatch by immobilization of
Npt–Azq on a gold surface.
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MATERIALS AND METHODS
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Chemistry
In order to know the structure–activity relationship for
the binding of
Npt–Azq hybrid to G-A mismatches, hybrid
molecules consisted of chromophores with different hydrogen-bonding
groups were synthesized. These chromophores include
Npt,
Azq,
quinoline (
Q) and 2-aminoquinoline (
AQ). Synthesis of the hybrid
molecules consisting of
Npt with
Azq and other heterocycles
is straightforward using
N-(
tert-butoxycarbonyl)imino-3,3'-bis(pentafluorophenyl
propionate) (
12), in which two carboxyl groups are activated
as a pentafluorophenyl ester. Mono-substitution of the pentafluoroester
with 2-amino-7-methylnaphthyridine produces intermediate amide
Npt-OC
6F
5 that subsequently reacts with the heterocyclic amines
to give hybrid ligands (Fig.
3). A similar procedure was used
for the synthesis of
Azq-based hybrid ligands (Fig.
4). Details
of the synthetic procedure is described in the Supplementary
Material.
Synthesis of Npt–Azq
To a solution of
N-(
tert-butoxycarbonyl)imino-3,3'-bis(pentafluorophenyl
propionate) (1.5 g, 2.53 mmol) in dry DMF (5 ml) was added 2-amino-7-methyl-1,8-naphthyridine
(180 mg, 1.14 mmol) and diisopropylethylamine (163 mg, 1.26
mmol). The reaction mixture was stirred at room temperature
for 15 h. The solvent was evaporated to dryness and the residue
was purified by column chromatography on silica gel to give
Npt-OC
6F
5 (1.29 g, 90%) as pale yellow solids:
1H-NMR (CDCl
3,
400 MHz)
= 9.01 (br, 1H), 8.44 (d, 1H,
J = 8.8 Hz), 8.12 (d,
1H,
J = 8.8 Hz), 7.99 (d, 1H,
J = 8.0 Hz), 7.26 (d, 1H,
J =
8.0 Hz), 3.66 (m, 4H), 2.90 (m, 2H), 2.74 (m, 2H), 2.73 (s,
3H), 1.42 (s, 9H),
13C-NMR (CDCl
3, 100 MHz)
= 163.6, 155.3,
154.6, 153.4, 142.6, 140.1, 139.5, 138.5, 136.6, 121.9, 118.8,
114.5, 80.9, 44.9, 44.4, 37.6, 37.2, 36.7, 33.4, 32.7, 28.6,
25.8, FABMS (NBA),
m/
e 569 [(M + H)
+], HRMS calc. for C
26H
26O
5N
4F
5 [(M + H)
+] 569.1821, found 569.1827.
To a solution of Npt-OC6F5 (300 mg, 0.53 mmol) in dry DMF (2 ml) was added 7-(aminomethyl)hydro-8-azaquinolin-2-one (93 mg, 0.53 mmol) and diisopropylethylamine (77 mg, 0.6 mmol). The reaction mixture was stirred at room temperature for 15 h. The solvent was evaporated to dryness and the residue was purified by column chromatography on silica gel to give N-Boc-Npt–Azq (227 mg, 77%) as pale yellow solids: 1H-NMR (CDCl3, 400 MHz) = 11.29 (br, 1H), 9.08 (br, 1H), 8.38 (d, 1H, J = 8.8 Hz), 8.06 (d, 1H, J = 8.0 Hz), 7.95 (d, 1H, J = 8.0 Hz), 7.81 (d, 1H, J = 8.0 Hz), 7.64 (d, 1H, J = 9.6 Hz), 7.27 (d, 1H, J = 8.0 Hz), 7.22 (d, 1H, J = 8.0 Hz), 6.64 (d, 1H, J = 9.6 Hz), 4.63 (d, 2H, J = 6.0 Hz), 3.58 (t, 2H, J = 6.8 Hz), 3.57 (t, 2H, J = 6.8 Hz), 2.71 (t, 2H, J = 6.8 Hz), 2.70 (s, 1H), 2.54 (t, 2H, J = 6.8 Hz), 1.36 (s, 9H), 13C-NMR (CDCl3, 100 MHz) = 171.7, 163.9, 163.4, 159.8, 155.7, 154.5, 153.6, 149.3, 139.3, 139.2, 137.4, 136.9, 123.1, 121.9, 118.8, 118.2, 114.6, 114.0, 80.5, 44.8, 41.8, 37.5, 28.6, 25.7, FABMS (NBA), m/e 560 [(M + H)+], HRMS calc. for C29H34O5N7 [(M + H)+] 560.2621, found 560.2618.
To a solution of N-Boc-Npt–Azq (62 mg, 0.11 mmol) in CHCl3 (3 ml) was added ethyl acetate containing 4 M HCl (2 ml) at room temperature and the reaction mixture was stirred at room temperature for 0.5 h. The solvent was evaporated to dryness to give hydrochloride of Npt–Azq (quantitative yield) as white solids: 1H-NMR (CDCl3, 400 MHz) = 11.45 (br, 1H), 8.67 (t, 1H, J = 5.6 Hz), 8.31 (d, 1H, J = 8.8 Hz), 7.98 (d, 1H, J = 8.8 Hz), 7.88 (d, 1H, J = 8.0 Hz), 7.65 (d, 1H, J = 7.6 Hz), 7.53 (d, 1H, J = 9.6 Hz), 7.15 (d, 1H, J = 7.6 Hz), 7.14 (t, 1H, J = 8.0 Hz), 6.58 (d, 1H, J = 9.6 Hz), 4.65 (d, 2H, J = 5.6 Hz), 3.08 (t, 2H, J = 6.0 Hz), 3.06 (t, 2H, J = 6.0 Hz), 2.65 (t, 2H, J = 6.0 Hz), 2.58 (t, 2H, J = 6.0 Hz), 2.57 (s, 3H), 13C NMR (CD3OD, 100 MHz) = 171.8, 171,3, 165.0, 160.2, 159.7, 157.0, 148.5, 147.3, 146.4, 140.3, 140.2, 138.8, 122.5, 120.9, 120.2, 117.6, 117.0, 114.8, 44.1, 43.9, 43.1, 32.5, 30.5, 19.7, FABMS (NBA), m/e 460 [(M + H)+], HRMS calc. for C24H26O3N7 [(M + H)+] 460.2095, found 460.2097.
Synthesis of Npt–Azq having an aminoalkyl linker for immobilization onto SPR sensor surface
SPR sensors having Npt–Azq on their surface were synthesized to examine SPR detection of the G-A mismatch in a flow system (Fig. 5). Npt–Azq was immobilized on a dextran matrix coated gold surface (CM5 chip, BIAcore) through a bivalent linker of N-Boc-aminoaldehyde. First, Npt–Azq was tethered by a reductive amination to the linker, which was efficiently prepared from Boc-protected 4-aminobutanoic acid and 3-aminopropionaldehyde diethylacetal (Supplementary Material). Deprotection of a Boc group produced a primary amine, which was subsequently immobilized on the sensor surface by a coupling between the amino group and an activated carboxyl group on the CM5 chip using a standard method with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS).
Preparation of a sensor chip carrying Npt–Azq on its surface
SPR measurements were performed with a BIAcore 2000 system (BIAcore,
Uppsala, Sweden). Immobilization of
Npt–Azq on a sensor
chip CM5 (carboxymethylated dextran surface, BIAcore) was carried
out using amine coupling kit (BIAcore) in a continuous flow
of HBS-N buffer (10 mM HEPES, pH 7.4) containing NaCl (150 mM)
at a flow rate of 10 µl/min. A solution (70 µl)
of NHS (0.05 M) and EDCI (0.2 M) was injected using the QUICKINJECT
command to activate the carboxymethylated dextran surface of
a CM5 sensor chip. A solution (70 µl) of
Npt–Azq having an aminoalkyl linker (2 mM in borate buffer, pH 9.2)
was injected using the QUICKINJECT command on the activated
surface. Residual activated surface was completely blocked by
injection of a solution (20 µl) of ethanolamine hydrochloride
(1.0 M, pH 8.5). Non-covalently bound material was removed by
washing with 5 µl of 50 mM NaOH to produce the sensor
chip carrying
Npt–Azq on its surface. The amount of
Npt–Azq immobilized on the surface was modulated by the reaction period
of the immobilization, and was monitored as an increase of SPR
signal [resonance unit (RU)] after the deactivation of the unreacted
NHS-esters and a conditioning of the surface. Three sensor surfaces
carrying
Npt–Azq for 527, 722 and 951 RU were obtained
by the immobilization for 1, 3 and 7 min with a 2 mM solution
of
Npt–Azq in a borate buffer (pH 9.2).
Measurements of thermal denaturation profiles of mismatch-containing duplexes
Thermal denaturation profiles of the duplexes 5'-d(CTAA vGw AATG)-3'/3'-d(GATT xyz TTAC)-5' where G-y mismatches were flanked by v-x and w-z base pairs (4.5 or 5.0 µM for each strand) were measured in a sodium cacodylate buffer (10 mM, pH 7.0) containing NaCl (100 mM) using a Shimadzu UV-2550 UV-Vis spectrometer linked to a Peltier temperature controller. The absorbance of the sample was monitored at 260 nm from 4 to 70°C with a heating rate of 1°C/min in the absence and presence of a hybrid ligand. The measurements were carried out at the ligand concentration of 50 or 200 µM. The melting temperature of these duplexes was determined as the temperature crossing the melting curve and the median of two straight lines drawn for the single and duplex region in the melting curve.
General procedure for SPR binding experiments using synthetic oligonucleotides
All measurements were carried out at 25°C in a continuous flow of a buffer (10 mM HEPES, pH 7.4) containing NaCl (1 M) at a flow rate of 30 µl/min. A 1 µM solution of 27mer duplexes 5'-d(GTT ACA GAA TCT VGW AAG CCT AAT ACG)-3'/3'-d(CAA TGT CTT AGA XYZ TTC GGA TTA TGC)-5' containing G-Y mismatches flanked by V-X and W-Z base pairs in the buffer were injected for 180 s for analyzing the association to the sensor surface. The buffer was subsequently injected for another 180 s for analyzing the dissociation of the bound oligomer form the surface. After each analysis, all binding materials were removed by washing with 30 µl of NaOH solution (50 mM). Immediately after washing, this system can be used for the next assay.
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RESULTS AND DISCUSSION
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UV melting temperature analyses
The bindings of
Npt–Azq and other hybrid ligands to G-
y mismatches were examined by measuring the melting temperature
(
Tm) of mismatch-containing duplexes (5.0 µM) in the presence
of the ligand. The difference of the melting temperature (
Tm)
in the absence and presence of the ligands is summarized in
Table
1. A large
Tm of 26.2°C was obtained for the 11mer
duplex containing the G-G mismatch flanked by two G-C base pairs
(
vGw/xyz =
cGg/gGc) in the presence of
Npt–Npt. In order
to distinguish the 11mer duplexes used for the
Tm measurements
from the 27mer duplexes used for SPR studies, the flanking sequences
to the mismatch of 11mer were described with lowercase letters,
whereas uppercase letters were used of the flanking sequences
of 27mer. Under these conditions,
Npt–Npt also increased
the
Tm of the G-A mismatch (
cGg/gAc) by 12.8°C, whereas
only a modest
Tm of 4.3°C was observed for the matched duplex.
In contrast to
Npt–Npt, substitution of one
Npt chromophore
to
Azq in
Npt–Azq showed a striking difference in the
spectrum for mismatch stabilization.
Npt–Azq strongly
stabilizes
cGg/gAc as indicated by the
Tm of 15.4°C, exceeding
the
Tm of
Npt–Npt by 2.6°C. Since the non-specific
binding to a fully matched duplex is weaker for
Npt–Azq (2.2°C) than
Npt–Npt (4.3°C),
Npt–Azq is
currently the strongest ligand described that stabilizes the
G-A mismatch.
To gain insight into a role of
Azq chromophore for the binding
to the G-A mismatch, the
Azq and
Npt chromophores in
Npt–Azq were replaced by either quinoline (
Q) or aminoquinoline (
AQ)
chromophores. A dramatic decrease of the
Tm from 15.4 to 1.6°C
was observed by replacing
Azq chromophore in
Npt–Azq with
Q in
Npt–Q. The hydrogen-bonding donor of N-H in
Azq was
replaced by a non-hydrogen bonding group of C-H. Aminoquinoline–azaquinolone
(
AQ–Azq) obtained by a substitution of
Npt in
Npt–Azq with
AQ completely lost the binding to both G-A and G-G mismatches
(Fig.
6). Furthermore, the azaquinolone dimer (
Azq–Azq),
the single chromophore
Npt and
Azq, or the 1:1 mixture of
Npt and
Azq did not stabilize the G-A mismatch. These results clearly
indicated that a covalent attachment of
Npt and
Azq chromophores
is essential for the stabilization of the G-A mismatch. These
remarkable effects of the substitution of the
Npt and
Azq chromophores
in
Npt–Azq on the stabilization of the G-A mismatch suggests
the significance of the hydrogen bonding of
Azq to adenine and
Npt to guanine (Fig.
2). It has been recently shown that
Azq is superior to thymine in the recognition of adenine in duplex
and triplex structure (
21). These observations are well consistent
to our results.
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Figure 6. Schematic representation of the effect of structural modification of chromophores in Npt–Azq on the increased melting temperature (Tm) of the G-A mismatch.
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Evaluation of the binding of Npt–Azq to G-A mismatches
Having found that
Npt–Azq strongly stabilizes the G-A
mismatch, and that the hydrogen bonding groups in the two chromophores
are essential, the binding of the ligand was studied in detail.
First, the UV absorption of the
Npt–Azq (20 µM)
was measured in the presence of different concentrations of
the 11mer G-A mismatch duplex (
cGg/gAc) (0–50 µM).
In the absence of DNA, the ligand showed an absorption maximum
at 320 nm and a shoulder at 333 nm. At increasing
cGg/gAc concentrations,
these absorptions decreased in intensity with a concomitant
red shift of the peak from 320 to 324 nm (Fig.
7). An isosbestic
point was observed at 340 nm for the spectral change, indicating
that UV absorbance of
Npt–Azq linearly changes from the
free state to the bound state to
cGg/gAc. With the data points
at 320 nm, the fraction of the total ligand bound against the
molar fraction of
cGg/gAc ([
cGg/gAc] / [
Npt–Azq]) was
plotted (Fig.
8). The bound fraction of
Npt–Azq rapidly
increased with increasing
cGg/gAc concentration and saturated
in the presence of approximately one molar equivalent of DNA.
In contrast, the bound fraction of
Npt–Npt to the G-A
mismatch steadily increased with increasing
cGg/gAc concentration
and reached saturation with two molar equivalents of DNA. This
suggests that the binding of
Npt–Azq to the G-A mismatch
is stronger than that of
Npt–Npt. CD spectra of
cGg/gAc in the presence of
Npt–Azq showed an increase of the ellipticity
at 275 nm in addition to strong and weak CD signals induced
at 320 and 345 nm, respectively (Fig.
9). Distinct induced CD
signals indicated that
Npt–Azq was under the influence
of the chiral environment of duplex DNA in the complex.
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Figure 7. UV absorption spectra of Npt–Azq (20 µM) in the presence of different concentrations of the G-A mismatch duplex cGg/gAc (0–50 µM). The experiments were conducted in a sodium cacodylate buffer (10 mM, pH 7.0) containing NaCl (100 mM) at 15°C. The concentrations of DNA are 0, 1, 2.5, 5, 7.5, 10, 15, 20, 25 and 37.5 µM.
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Figure 8. Plot of the fraction of total ligand bound against the molar fraction of the G-A mismatch duplex cGg/gAc to the concentration of Npt–Azq (filled circles) and Npt–Npt (open circles), respectively. The data were obtained from experiments shown in Figure 7.
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Figure 9. CD spectra of cGg/gAc (4.5 µM) measured in 10 mM sodium cacodylate buffer (pH 7.0) and 100 mM NaCl at 25°C in the absence (gray) and presence (black) of 20 µM Npt–Azq.
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The effect of the concentration of
Npt–Azq on the
Tm of
cGg/gAc was examined by measuring the melting curve at different
drug concentrations. Increasing the concentration of
Npt–Azq,
the
Tm of the duplex increased and reached a plateau (Fig.
10).
At 50 µM
Npt–Azq, UV-melting profiles were obtained
for all G-A mismatches with regard to the sequence flanking
to the mismatch (
vGw/xAz). The
Tm of all G-A mismatches except
for
tGt/aAa in the absence and presence of
Npt–Azq are
summarized in Figure
11. The
Tm of
tGt/aAa was too low to measure
under the conditions. The largest
Tm was recorded for the sequence
of
tGg/aAc. The sequence
cGg/gAc we have examined in detail
had the second largest
Tm value. While we have anticipated that
the binding of
Npt–Azq would be favorable for the G-A
mismatches flanking to G-C base pairs due to the increased stacking
stabilization of the complex, there seems no obvious rationalization
for the sequences stabilized by the drug. The melting curves
of the sequences showing the top five
Tm values are shown in
Figure
12. The energy gains of these five G-A mismatches in
the presence of 50 µM
Npt–Azq were estimated by
curve fitting of the melting profiles. The melting profile of
the G-A mismatch in the absence and presence of
Npt–Azq were fitted to a two-state model with a non-linear least-squares
program by using Sigma Plots (version 2001) (
22). The energy
gains obtained by these simulations are summarized in Table
2. The largest energy gain of –6.0 kcal/mol (277.15 K)
was obtained for
cGg/gAc, whereas the sequence
tGg/aAc, which
recorded the highest
Tm value, was ranked third. As we reported
earlier, the magnitude of
Tm is not consistent with the energy
gain by the drug binding especially when the duplex showed a
low melting temperature (
23).
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Figure 10. Concentration dependency of Tm of cGg/gAc (4.5 µM). The melting temperature of cGg/gAc (4.5 µM) was measured in the presence of 0, 5, 10, 15, 25, 50, 100 and 200 µM Npt–Azq in 10 mM sodium cacodylate (pH 7.0) and 100 mM NaCl.
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Figure 11. Tm values for G-A mismatches (4.5 µM) of different flanking sequence in the presence of Npt–Azq (50 µM) (black) and melting temperatures of G-A mismatches in the absence of Npt–Azq (white).
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Figure 12. Melting curves of G-A mismatch duplexes (4.5 µM) in the absence (dots) and presence (filled circles) of Npt–Azq (50 µM) in 10 mM sodium cacodylate (pH 7.0) and 100 mM NaCl. tGg/aAc, green; cGg/gAc, blue; gGa/cAt, red; tGc/aAg, orange; tGa/aAt, black.
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SPR analyses
Having discovered that
Npt–Azq strongly stabilizes the
G-A mismatch DNA, the binding of G-containing mismatches to
the SPR sensor where
Npt–Azq was immobilized on the surfaces
was investigated. SPR analyses of a 1 µM solution of 27mer
duplexes 5'-d(GTT ACA GAA TCT
VGW AAG CCT AAT ACG)-3'/3'-d(CAA
TGT CTT AGA
XYZ TTC GGA TTA TGC)-5' containing a G-Y mismatch
flanked by G-C base pairs (5'-
VGW-3'
/3'-
XYZ-5' = CGG/GYC, Y
= A, G or C) were performed with the sensor carrying
Npt–Azq for 951 RU on the surface. To suppress a non-specific absorption
of DNA to the sensor surface, binding experiments were carried
out under high salt conditions (1 M NaCl and 10 mM HEPES buffer,
pH 7.4). A sensorgram obtained for the G-A mismatch (
CGG/GAC)
showed strong SPR signals, of which intensity reached to 193
RU, after 180 s of the association time (Fig.
13). The SPR signal
for the G-G mismatch (
CGG/GGC) was also distinct, but much lower
in intensity (114 RU) than that obtained for
CGG/GAC. In marked
contrast, only a weak SPR signal (31 RU) was observed for the
G-C match DNA (
CGG/GCC). These results are consistent with those
of UV-melting studies in the presence of
Npt–Azq showing
a higher
Tm for
cGg/gAc than for
cGg/gGc and almost negligible
stabilization for the G-C match DNA. The distinct differences
in the intensity of the SPR signal between the G-A mismatch
and G-C match DNA clearly indicate a unique character of the
Npt–Azq immobilized sensor surface.
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Figure 13. Sensorgrams for the binding of duplexes containing G-Y mismatches to the Npt–Azq immobilized SPR sensor surface. Aliquots of 90 µl of the duplexes (1.0 µM in 10 mM HEPES buffer pH 7.4, 1 M NaCl) containing G-A, G-G mismatch and G-C matches were injected for 180 s to measure the association to the sensor surface.
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Concentration dependency for the SPR responses
To further evaluate the fidelity of the novel sensor surfaces
detecting the G-A mismatches, the concentration dependency of
the SPR signal for the binding of the G-A mismatch to the sensor
surface was investigated. The duplex
CGG/GAC containing a G-A
mismatch of different concentrations (0.13–1.0 µM)
was analyzed by three sensor surfaces carrying
Npt–Azq for 951, 722 and 527 RU. The SPR responses after the association
time of 180 s were plotted against the concentration of the
G-A mismatch DNA (Fig.
14). The SPR signals produced by each
sensor surface clearly showed a linear correlation to the concentration
of
CGG/GAC, with a correlation coefficient of 0.99. A linear
correlation between SPR intensity and DNA concentrations validated
that the observed SPR signals were definitely due to a specific
interaction between
CGG/GAC and
Npt–Azq on the sensor
surface. Furthermore, it was suggested that the sensor surface
is not only effective to detect the G-A mismatch, but also applicable
to quantify G-A mismatch DNA at the concentration range up to
1 µM (
24). It is worth noting that the SPR responses of
three sensor surfaces were not proportional to the amount of
Npt–Azq immobilized on the surface. This is most likely
due to the different surfaces of the three sensors produced
by independent immobilization processes for the three CM5 chips
of research grade. Thus, calibration of the sensor surface is
necessary for quantitative applications.
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Figure 14. Concentration dependency for the SPR responses for 27mer duplex (CGG/GAC) containing a G-A mismatch. CGG/GAC at concentrations of 0.13, 0.25, 0.5 and 1.0 µM were analyzed by three sensors carrying Npt–Azq for 951 (filled circles), 722 (open circles) and 527 RU (squares) on the surface. Responses after 180 s of the association time were plotted against the DNA concentration.
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Effect of the flanking sequence to the G-A mismatch on the SPR responses
To know the scope and limitation of the novel G-A mismatch detecting
sensor, the effect of the sequence flanking to the mismatch
on the binding to the
Npt–Azq immobilized sensor surface
was examined. In heteroduplex analyses, it is important to differentiate
the mismatch-containing duplex from the fully matched duplex.
Thus, the SPR signal of a mismatched duplex relative to that
of the fully matched duplex was investigated. DNA duplexes containing
G-A mismatches in the sequence of 5'-
VGW-3'/3'-
XAZ-5' were analyzed
by SPR using an
Npt–Azq immobilized sensor surface. SPR
intensities of 16 G-A mismatches were reported by the relative
intensity to the highest signal of the fully matched duplex.
G-A mismatches are largely divided into three groups with regard
to the affinity to the surface. The first group of G-A mismatches
showed a strong response to the
Npt–Azq immobilized surfaces.
Among 16 duplexes, the strongest SPR signal was observed for
TGG/AAC (Fig.
15a). The SPR intensity was more than two times
stronger than the signal for
CGG/GAC, and 12-fold stronger than
that observed for the matched duplex. Besides these two, G-A
mismatches in
GGA/CAT,
TGC/AAG,
AGG/TAC and
AGA/TAT showed SPR
signals that are markedly stronger than the signal of a matched
duplex by >2-fold. The fitting of the response curve to a
1:1 Langmuir model with BIAevaluation software (version 3) provided
an estimate of the association constant (
Ka) of each G-A mismatch
to the
Npt–Azq immobilized surface. The
Ka obtained for
the G-A mismatches were 1.8
x 10
6 M
–1 for
TGG/AAC, 1.0
x 10
6 M
–1 for
CGG/GAC, 9.0
x 10
5 M
–1 for
GGA/CAT,
9.2
x 10
5 M
–1 for
AGG/TAC 7.5
x 10
5 M
–1 for
AGA/TAT.
The
Ka for the
TGC/AAG could not be estimated due to a very
slow dissociation of DNA from the surface. The second group
of G-A mismatches showed reduced relative intensities compared
with those in the first group (Fig.
15b). The SPR responses
of
AGT/TAA and
GGG/CAC are clearly distinguished from that of
matched duplex. The relative intensity of the SPR signal to
the mismatches is
1.5-fold. The third group of mismatches did
not show any noticeable differences in SPR intensity from that
of fully matched duplex. These analyses showed that the sensor
we reported here would be effective to detect G-A mismatches
in eight flanking sequences, covering 50% of all G-A mismatches.
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Figure 15. Relative SPR intensities of the binding of G-A mismatches to the Npt–Azq immobilized sensor surfaces. G-A mismatches (1 µM) were analyzed in HEPES buffer for 180 s of association to the sensor surface and then dissociation of the bound DNA from the surfaces. SPR intensities were reported as a relative intensity by setting the maximum intensity of the fully matched duplex to be 1.0. (a) G-A mismatches strongly bind to the sensor. TGG/AAC, yellow; CGG/GAC, black; TGC/AAG, green; GGA/CAT, blue; AGG/TAC, pink; AGA/TAT, aqua; matched duplex, red. (b) G-A mismatches with weak binding to the sensor. AGT/TAA, violet; GGG/CAC, aqua; matched duplex, red.
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A sequence-dependent binding of
Npt–Azq to the G-A mismatches
implies that
Npt–Azq not only binds to the G-A mismatches,
but also interacts to the bases flanking to the mismatch. SPR
analyses showed that the binding kinetics for the G-A mismatch
in
TGC/AAG are markedly different from those of other G-A mismatches.
The association of
TGC/AAG to the
Npt–Azq immobilized
surface and the dissociation from the surface was quite slow.
These observations are particularly important for the molecular
design of the improved version of
Npt–Azq that is aiming
to detect the rest of the eight G-A mismatches.
Conclusion
The new molecule Npt–Azq was discovered to be the ligand strongly stabilizing the G-A mismatch. The SPR sensor surface on which Npt–Azq was immobilized was the first sensor detecting the G-A mismatch in duplex DNA. The G-A mismatch is produced by a heteroduplex formation from a pair of duplex DNAs containing a G·C to T·A mutation. The G to T transversion is high in frequency because the oxidation of guanine leading to a formation of 8-oxoguanine eventually resulted in the mutation (25). Oxidation of guanine is known to be sensitively affected by the base 3' side to the guanine (26). The 5'-GG-3' is most easily oxidizable and the 5'-GA-3' is second in 5'-GX-3' sequences. The G in the G-A mismatches that are detectable by the SPR surface are mostly flanked by 3' side G and A, suggesting that the oxidation at those Gs is high in frequency. Thus, the Npt–Azq immobilized sensor surface reported here would be a useful and important tool for the discovery of a G to T mutation by detecting the G-A mismatches.
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SUPPLEMENTARY MATERIAL
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Supplementary Material is available at NAR Online.
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ACKNOWLEDGEMENTS
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|---|
This work was supported by Grant-in-Aid for Scientific Research
on Priority Areas (C) ‘Medical Genome Science’ from
the Ministry of Education, Culture, Sports, Science and Technology
of Japan.
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