Influence of substituent modifications on the binding of 2-amino-1,8-naphthyridines to cytosine opposite an AP site in DNA duplexes: thermodynamic characterization.

Abstract

Here, we report on a significant effect of substitutions on the binding affinity of a series of 2-amino-1,8-naphthyridines, i.e., 2-amino-1,8-naphthyridine (AND), 2-amino-7-methyl-1,8-naphthyridine (AMND), 2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND) and 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), all of which can bind to cytosine opposite an AP site in DNA duplexes. Fluorescence titration experiments show that the binding affinity for cytosine is effectively enhanced by the introduction of methyl groups to the naphthyridine ring, and the 1:1 binding constant (10(6) M(-1)) follows in the order of AND (0.30) < AMND (2.7) < ADMND (6.1) < ATMND (19) in solutions containing 110 mM Na(+) (pH 7.0, at 20 degrees C). The thermodynamic parameters obtained by isothermal titration calorimetry experiments indicate that the introduction of methyl groups effectively reduces the loss of binding entropy, which is indeed responsible for the increase in the binding affinity. The heat capacity change (DeltaC(p)), as determined from temperature dependence of the binding enthalpy, is found to be significantly different between AND (-161 cal/mol K) and ATMND (-217 cal/mol K). The hydrophobic contribution appears to be a key force to explain the observed effect of substitutions on the binding affinity when the observed binding free energy (DeltaG(obs)) is dissected into its component terms.

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Published online 9 January 2009Nucleic Acids Research, 2009, Vol. 37, No. 51411–1422
doi:10.1093/nar/gkn1079
Influence of substituent modifications on the
binding of 2-amino-1,8-naphthyridines to cytosine
opposite an AP site in DNA duplexes:
thermodynamic characterization
Yusuke Sato1, Seiichi Nishizawa1, Keitaro Yoshimoto2, Takehiro Seino1,
Toshiki Ichihashi1, Kotaro Morita1and Norio Teramae1,*
1Department of Chemistry, Graduate School of Science, Tohoku University, and CREST, Japan Science and
Technology Agency (JST), Aoba-ku, Sendai 980-8578 and2Department of Materials Science, Graduate School
of Pure and Applied Sciences, University of Tsukuba, 1-1-1-Ten-noudai, Tsukuba, Ibaraki 305-8573, Japan
Received September 1, 2008; Revised December 22, 2008; Accepted December 23, 2008
ABSTRACT
Here, we report on a significant effect of substitu-
tions on the binding affinity of a series of 2-amino-
1,8-naphthyridines, i.e., 2-amino-1,8-naphthyridine
(AND), 2-amino-7-methyl-1,8-naphthyridine (AMND),
2-amino-5,7-dimethyl-1,8-naphthyridine
and2-amino-5,6,7-trimethyl-1,8-naphthyridine
(ATMND), all of which can bind to cytosine opposite
an AP site in DNA duplexes. Fluorescence titration
experiments show that the binding affinity for cyto-
sine is effectively enhanced by the introduction
of methyl groups to the naphthyridine ring, and the
1:1 binding constant (106M?1) follows in the order of
AND (0.30) < AMND (2.7) < ADMND (6.1) < ATMND (19)
in solutions containing 110mM Na+(pH 7.0, at 208C).
The thermodynamic parameters obtained by isother-
mal titration calorimetry experiments indicate that
the introduction of methyl groups effectively reduces
the loss of binding entropy, which is indeed respon-
sible for the increase in the binding affinity. The
heat capacity change ("Cp), as determined from
temperature dependence of the binding enthalpy,
is found to be significantly different between AND
(?161cal/mol K) and ATMND (?217cal/mol K). The
hydrophobic contribution appears to be a key force
to explain the observed effect of substitutions on the
binding affinity when the observed binding free
energy ("Gobs) is dissected into its component
terms.
(ADMND)
INTRODUCTION
The chemistry of DNA-binding drugs and/or small
ligands is of ongoing interest due to their promising func-
tions and biological activities, including their anticancer
properties and ability to regulate gene expression (1–4).
Besides the anthracycline antibiotics doxorubicin and dau-
norubicin, many DNA-binding molecules have been
developed as effective pharmaceutical agents, especially
in cancer chemotherapy (5–7). Another class of DNA-
binding molecules is useful stain agents for nucleic acids,
and typical of such molecules are ethidium and Hoechst
33258 (8–9). Of particular interest to us is a class of
ligands applicable to gene analysis (10,11), especially
single-nucleotide polymorphism (SNP) typing (12).
We have recently found a series of aromatic ligands that
can selectively bind to a nucleobase opposite an abasic site
(AP site) in DNA duplexes, and we have proposed a new
strategy of ligand-based fluorescence assay for SNP typing
(Figure 1A) (13–23). In contrast to typical DNA binding
such as intercalation or groove binding (24,25), it is
characteristic of ligands to bind to non-Watson–Crick
base-pairing sites in DNA duplexes, where the binding
is selectively promoted by a pseudo-base pairing along
the Watson–Crick edge of the intrahelical target nucleo-
bases (cf. Figure 7). Successful examples of this class of
ligands are the mismatch-binding molecules developed by
Nakatani and co-workers, and a surface plasmon reso-
nance (SPR) assay has been proposed based on these
molecules for the detection of mismatched base pairs in
heteroduplexes (26–28). In our approach, on the other
hand, we have paid attention to the AP site as a binding
Present address:
Kotaro Morita, Department of Chemistry, Faculty of Science, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan
*To whom correspondence should be addressed. Tel: +81 22 795 6549; Fax: +81 22 795 6552; Email: teramae@mail.tains.tohoku.ac.jp
? 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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cavity for effective ligand–nucleobase interactions: while
naturally occurring AP site is one of the most common
forms of DNA damages (29), we have incorporated
a chemically stable AP site in a probe DNA so as to orient
the AP site toward a target nucleobase (Figure 1A). Such
AP sites in the duplex provide a unique binding pocket
that allows a direct contact of ligands to nucleobases
via hydrogen bonding (cf. Figure 7), where the ligand is
further stacked with two nucleobases flanking the AP site.
Useful affinity and selectivity for target nucleobases
opposite the AP site has been indeed obtained by various
kinds of heterocyclic planar compounds, including cyto-
sine selective2-amino-7-methyl-1,8-naphthyridine
(AMND, Figure 1B) (13,14), guanine selective 2-amino-
6,7-dimethyl-4-hydroxypteridine (15), adenine selective
alloxazine (16) and thymine selective amiloride (17), ribo-
flavin (18). All of these ligands show a complexation-
induced fluorescence signaling, and SNP genotype of
samples can be clearly distinguished by combining ligands
with selectivity for respective target nucleobases.
Simple substituent modification of the parent ring was
found to be quite effective for tuning the binding affinity
and selectivity when designing such AP site-binding
ligands. In the case of pteridines capable of selectively
binding to guanine, for example, the binding affinity
of the parent ligand, 2-amino-4-hydroxypteridine, was
0.62?106M?1(in 110mM Na+, pH 7.0, at 58C) when
binding to guanine in an 11-meric AP site-containing
DNA duplex(50-TCCAGXGCAAC-30/30-AGGTCG
CGTTG-50, X=APsite
(15,19). In contrast, the pteridine modified with two
methyl groups, 2-amino-6,7-dimethyl-4-hydroxypteridine,
showed an affinity with one order of magnitude higher
(6.2?106M?1) than that of the parent ligand (15).
In the case of alloxazines, substitutions at the 7- and
8-position of the ring have been found to significantly
affect the binding selectivity (16). While alloxazine
showed a useful affinity and selectivity for adenine
(1.2?106M?1, in 110mM Na+, pH 7.0, at 58C),
7,8-dimethylalloxazine (lumichrome) showed a clear selec-
tivity for thymine over other nucleobases (K11 for T:
1.6?107M?1). These results clearly indicate that the
ligand–nucleobase interaction at the AP site is strikingly
sensitive to substitutions of the parent ring, and the subtle
difference in the ligand structure is crucial to getting desir-
able binding properties. However, little is known about
the details of such a substituent effect on the binding
events, and therefore the detailed thermodynamic and/or
structural characterization would provide significant
knowledge for the rational design of this class of ligands
(30–33). This would also offer a valuable insight into the
molecular basis of interactions for the further develop-
ment of various kinds of DNA-binding molecules.
In this work, we report on the effect of substitutions
in 2-amino-1,8-naphthyridines on the binding to nucleo-
bases in AP site-containing DNA duplexes, for which a
series of 2-amino-1,8-naphthyridines (Figure 1B) was
prepared, including 2-amino-1,8-naphthyridine (AND),
2-amino-7-methyl-1,8-naphthyridine (AMND), 2-amino-
5,7-dimethyl-1,8-naphthyridine
2-amino-5,6,7-trimethyl-1,8-naphthyridine
We examined their binding characteristics by melting tem-
perature (Tm) measurements, fluorescence spectroscopy
and isothermal titration calorimetry (ITC). Interestingly,
with increasing number of methyl groups attached
to the naphthyridine ring, the binding affinity of
1,8-naphthyridines clearly increases for pyrimidine bases
opposite the AP site in DNA duplexes: ATMND having
three methyl groups does bind to cytosine with a 1:1 bind-
ing constant of 1.9?107M?1(in 110mM Na+, pH 7.0, at
208C), which is indeed two order of magnitude higher than
that of the parent AND (0.030?107M?1). The binding
affinity for thymine is also enhanced effectively from
AND (0.012?107M?1) to ATMND (0.91?107M?1).
Such a significant effect of substitutions in 2-amino-1,
8-naphthyridines is discussed based on the examination
of thermodynamic parameters.
(Spacer-C3), G=target)
(ADMND)and
(ATMND).
MATERIALS AND METHODS
Materials
A series of 2-amino-1,8-naphthyridines examined here
were purchased from Specs, Akos, Maybridge and
Enamine for AND, AMND, ADMND and ATMND,
respectively. All of the DNAs used in the present study
were custom synthesized and HPLC-purified (>97%) by
Figure 1. (A) Schematic illustration of the ligand binding to nucleotides
opposite an AP site in a DNA duplex. For the detection of SNPs, an
AP site-containing probe DNA is hybridized with a target DNA so as
to place the AP site towards a target nucleotide, by which a hydro-
phobic binding pocket is provided for aromatic ligands to bind to
target nucleotide. (B) Structures of the series of 2-amino-1,8-naphthy-
ridines examined in this work.
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Nihon Gene Research Laboratories Inc. (Sendai, Japan).
For the synthesis of abasic (AP) site-containing DNAs,
a tetrahydrofuranyl residue (dSpacer) was utilized. The
concentration of DNA was determined from the molar
extinction coefficient at 260nm (34). Water was deionized
(?18.0MV cm specific resistance) by an Elix 5 UV Water
Purification System and a Milli-Q Synthesis A10 system
(Millipore Corp., Bedford, MA, USA). The other reagents
were commercially available analytical grade and were
used without further purification.
Unless otherwise mentioned, all measurements were
performed in 10mM sodium cacodylate buffer solutions
(pH 7.0) containing 100mM NaCl and 1.0mM EDTA.
Before measurements, the sample solutions were annealed
as follows: heated at 758C for 10min, and gradually
cooled to 58C (38C/min), after which the solution temper-
ature was raised again to 208C (18C/min).
Melting temperature measurements
Absorbance of DNA was measured at 260nm as a
function of temperature using an UV-vis spectrophot-
ometerModel UV-2450
Japan) equipped with a thermoelectrically temperature-
controlled micro-multicell holder (8 cells; optical path
length=1mm). The temperature ranged from 28C to
928C with a heating rate of 1.08C/min. The resulting
absorbance versus temperature curve was analyzed by a
differential method to determine Tmvalues.
(Shimadzu Corp.,Kyoto,
Determination of binding constants by fluorescence
titrations
To determine the 1:1 binding constant (K11), fluorescence
spectra of ligands were measured at 208C with a JASCO
FP-6500 spectrofluorophotometer (Japan Spectroscopic
Co. Ltd., Tokyo, Japan) equipped with a thermoelectri-
cally temperature-controlled cell holder (quartz cuvette,
3mm?3mm). Typically, in the case of ATMND titra-
tion, the ligand concentration was fixed at 500nM, and
the concentration of DNA duplex ranged from 0 to
2.5mM. The changes in fluorescence intensity at 403nm
(maximum wavelength) were monitored as a function of
duplex concentration. The resulting titration curve was
analyzed by nonlinear least-squares regression based on
a 1:1 binding isotherm (35):
F=F0¼ f1 þ kK11½D?g=f1 þ K11½D?g
where F and F0are the observed fluorescence intensities of
ligand in the presence and absence of DNA duplexes,
respectively, and k (=k11/kL) represents the ratio of pro-
portionality constants connecting the fluorescence intensi-
ties and concentrations of the species (1:1 complex: k11,
free ligand: kL). The free duplex concentration, [D], can be
related to known total concentrations of duplex (D0) and
ligand (L0), by the following equation:
1
D0¼ ½D? þ f L0K11½D?g=f1 þ K11½D?g
Together, Equations (1) and (2) describe the system.
2
Saltdependence ofthe binding constants
The effect of different NaCl concentrations on the 1:1
binding constants was examined at 208C (pH 7.0) by flu-
orescence titration experiments, as described above, and
analyzed according to the polyelectrolyte theory by
Record et al. (36) The observed salt dependence of the
bindingconstantsis explained
relationship:
bythefollowing
dlogK11=dlog½Naþ? ¼ ?Zc ¼ SK
where Z is the apparent charge on the ligand, and c is the
proportion of counterions associated with each DNA
phosphate group. The slope (SK) of the plot, which is
equivalent to the number of counterions released from
DNA upon ligand binding, was obtained from lines of
best linear least squares fit, and was used to evaluate the
polyelectrolyte contribution (?Gpe) to the observed bind-
ing free energy (?Gobs) using the relationship (36):
?Gpe¼ ð?SKÞRTln½Naþ?
The nonpolyelectrolyte contribution (?Gt) was then given
by the following equation (37):
?Gobs¼ ?Gpeþ ?Gt
Isothermal titration calorimetry
ITC experiments were carried out using a Microcal
VP-ITC calorimeter (Microcal Inc., Northampton, MA,
USA). The Origin software (Microcal) was used for data
acquisition and analysis. All solutions were degassed by
stirring under vacuum before use. Typically, the reference
cell contained deionized water, and a titration was done at
208C so that 15ml of ligand solution were added (a total of
17 injections) to 1.43ml of DNA solution in the sample
cell. The injection time was 30s, and the interval between
injections was 300s. In order to remove any air bubbles in
the tip of syringe, the initial injection was set as 5ml and
the resulting peak was neglected in the analysis. The peaks
produced during titration were converted into heat output
per injection by integration and correction for the cell
volume and sample concentration. The heats of dilution
for the addition of ligand into buffer solution were deter-
mined independently, and the net enthalpy for ligand–
DNA interactions was determined by subtraction of the
heats of dilution. The data thus obtained were best fitted
to a model that assumed a single set of identical binding
sites, giving binding enthalpies and stoichiometries.
Heat capacitymeasurements
ITC titration experiments were carried out at four tem-
peratures between 58C and 208C, and the binding enthal-
pies were determined as described above. From the
observed temperature dependence of the binding enthalpy,
the change in heat capacity, ?Cp, was determined accord-
ing to the relationship:
?Cp¼ dð?HÞ=dT
Nucleic Acids Research,2009, Vol.37, No. 51413

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The obtained value of ?Cpwas then used to estimate the
hydrophobic contribution (?Ghyd) according to the rela-
tionship of Record et al. (38):
?Ghyd¼ 80ð?10Þ ? ?Cp
Preparation andanalysis ofPCR products
Asymmetric PCR (52,53) amplification of 107-meric sense
or antisense strands of K-ras gene (codon 12) (54) was
done with a 20-meric forward primer (50-GACTGAATA
TAAACTTGTGG-30) and a 20-meric reverse primer (50-C
TATT GTTGG ATCAT ATTCG-30). The reaction solu-
tion (100ml) contained dNTPs (0.2mM each), 10? PCR
buffer (10ml; TaKaRa), forward primer (300pmol or
20pmol), reverse primer (20pmol or 300pmol), template
(0.5ng) and Taq (2.5 U; TaKaRa Hot Start Version).
PCR conditions: 948C for 5min, followed by 40 cycles
of 948C for 30s, 528C for 30s and 728C for 30s, and
then 728C for 7min and kept at 48C. The 107-meric
PCR product: sense strand, 50-GACTG AATAT AAAC
T TGTGG TAGTT GGAGC TGNTG GCGTA GGCA
A GAGTG CCTTG ACGAT ACAGC TAATT CAGAA
TCATT TTGTG GACGA ATATG ATCCA ACAAT
AG-30; antisense, 50-CTATT GTTGG ATCAT ATTCG
TCCAC AAAAT GATTC TGAAT TAGCT GTATC G
TCAA GGCAC TCTTG CCTAC GCCAN CAGCT CC
AAC TACCA CAAGT TTATA TTCAG TC-30(N=G,
C, A, or T).
After PCR amplification, an aliquot (40ml) from the
PCR product was buffered to pH 7.0 with 100mM
sodium cacodylate containing EDTA (1.6mM). Then,
ATMND (50nM), and a 20-meric AP site-containing
probe oligonucleotide (5.0mM) were added (for sense
strand analysis, 50-CCT ACG CCA XCA GCT CCA
AC-30; for antisense strand analysis, 50-GTT GGA GCT
GXT
GGCGTA GG-30,
Fluorescence spectra of the resulting solutions (50ml)
were then measured at 58C with a JASCO FP-6500 spec-
trofluorophotometer equipped with a thermoelectrically
temperature controlledcell
3mm?3mm); the slits for the excitation and emission
monochromators were 5 and 5nm, respectively.
X=APsite; dSpacer).
holder (quartz cuvette:
RESULTS AND DISCUSSION
First, we examined the binding of 2-amino-1,8-naphthyr-
idines to cytosine in an 11-meric AP site-containing DNA
duplex [50-TCC AGX GCA AC-30/30-AGG TCC CGT
TG-50, X=AP site (dSpacer), C=target] by Tmmeasure-
ments. As is shown in Figure 2, all melting curves of the
duplex give a sigmoidal shape typical for the thermal
denaturation of DNA duplexes. In the absence of ligands
(curve a), Tmvalue of the duplex is determined as 30.58C
from the first derivative of the melting curve. In the pres-
ence of ligands (curves b–e), an increase in Tmis clearly
observed, indicating that each ligand is incorporated into
the AP site by the binding to cytosine, which results in
an increase in the thermal stability of the DNA duplex.
The stabilization by ATMND is the most significant as
compared to that of the other three ligands, where the
Tm increases by as much as 20.68C (curve e), and the
?Tm follows in the order of ADMND (+17.48C)>
AMND (+13.68C)>AND (+10.88C). It is therefore
highly likely that the binding affinity of 2-amino-1,
8-naphthyridines strongly depends on the number of
methyl groupsattachedto
ATMND, having three methyl groups, shows the stron-
gest binding affinity among these ligands.
The examination by fluorescence titration experiments
clearly supports the above consideration. Figure 3 shows a
typical fluorescence response of 2-amino-5,6,7-trimethyl-
1,8-naphthyridine (ATMND, 500nM) to cytosine in a
21-meric AP site-containing DNA duplex [50-GCA GCT
CCC GXG GTC TCC TCG-30/30-CGT CGA GGG CCC
CAG AGG AGC-50, X=AP site (dSpacer), C=target
cytosine], as measured in solutions containing 110mM
Na+(pH 7.0, at 208C). While almost no response is
observed for a fully complementary duplex (500nM,
50-GCA GCT CCC GGG GTC TCC TCG-30/30-CGT
CGA GGG CCC CAG AGG AGC-50), ATMND shows
significant quenching in the presence of the AP site-
containing duplex, indicating that the binding event
is taking place at the AP site. The other three
1,8-naphthyridines (ADMND, AMND and AND) also
show fluorescence quenching upon binding to cytosine in
the AP site-containing duplex (Supplementary Figure S1).
For all ligands, the response is concentration-dependent,
which is well analyzed by nonlinear least-squares regres-
sion based on a 1:1 binding isotherm (inset of Figure 3,
themotherring, and
Figure 2. Thermal denaturation profiles of a 11-meric AP site-contain-
ing DNA duplex [50-TCC AGX GCA AC-30/30-AGG TCC CGT
TG-50, X=AP site (dSpacer), C=target cytosine]. (a) DNA alone,
and in the presence of (b) AND, (c) AMND, (d) ADMND and (e)
ATMND. [DNA duplex]=30mM, [ligand]=580mM, in 100mM
NaCl, 1.0mM EDTA and 10mM sodium cacodylate (pH 7.0).
Absorbance of DNA was measured at 260nm as a function of
temperature, which ranged from 28C to 928C with a heating rate of
1.08C/min. Optical path length=1mm.
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and Supplementary Figure S1). The 1:1 binding constants
K11for cytosine thus obtained are summarized in Table 1.
As compared to AND having no methyl groups, the
introduction of even one methyl group is effective for
increasing the binding affinity, and the resulting ligand,
AMND, shows an affinity for cytosine with one order of
magnitude higher (K11=2.7?106M?1) than that of
AND (K11=0.30?106M?1). The binding affinity is fur-
ther enhanced for ADMND having two methyl groups
(K11=6.1?106M?1),
19?106M?1for ATMND. As for the binding to other
three nucleobases (Figure 4), the binding affinity for thy-
mine is also enhanced effectively by the introduction of
methyl groups [K11/106M?1(n=3): AND: 0.12?0.01,
AMND: 0.98?0.09, ADMND: 2.4?0.2, ATMND:
9.1?0.3], and the binding selectivity for pyrimidines
andtheaffinity does reach
over
ATMND. Significantly, the magnitude of binding affinity
of ATMND for cytosine is stronger than that of typical
intercalators such as ethidium (0.1?106M?1, in 0.2M
NaCl, pH7.0,at258C)
(3.8?106M?1, in 0.1M NaCl, pH 7.0, at 108C) (39),
and is almost comparable to that of groove binders such
as distamycin (36?106M?1, in 30mM NaCl, pH 7.0, at
208C) (40).
In order to understand such a favorable effect of methyl
groups on the binding affinity, thermodynamic parameters
for DNA–ligand interactions were examined, focusing on
the binding to cytosine in the 21-meric AP site-containing
duplex. First, the binding enthalpy was determined by
ITC. Figure 5 shows typical ITC data for the binding
of 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND)
to cytosine in the AP site-containing DNA duplex
[50-GCA GCT CCC GXG GTC TCC TCG-30/30-CGT
CGA GGG CCC CAG AGG AGC-50, X=AP site
(dSpacer), C=target cytosine], obtained in solutions con-
taining 110mM Na+(pH 7.0, at 208C). For all ligands,
a large exothermic heat of reaction was observed upon
addition of the ligand aliquots into the DNA solution
(Supplementary Figure S2). After correction of the dilu-
tion heat, the resulting titration curve was best fitted using
a model that assumed a single set of identical binding sites,
giving the binding enthalpy (?Hobs). This value was then
used to calculate the binding entropy (T?Sobs) using
T?Sobs=?Hobs??Gobs, for
lnK11) values determined from fluorescence titration
experiments were utilized, since ITC cannot be used to
obtain an accurate value for K11under our experimental
conditions: this limitation of ITC has been highlighted in
literature (24,41).
Salt dependence of binding constants was then exam-
ined according to the polyelectrolyte theory of Record
et al. (36), and the observed binding free energy
(?Gobs=?RT lnK11) was dissected into its polyelec-
trolyte(?Gpe) and
contributions (37):
purinesremainsunchanged from AND to
(25) or actinomycin
which ?Gobs
(=?RT
nonpolyelectrolyte(?Gt)
?Gobs¼ ?Gpeþ ?Gt
The polyelectrolyte contribution arises from a release of
counterions from DNA upon ligand binding, while the
Table 1. Thermodynamic parameters for the 1,8-naphthyridine binding to cytosine in the 21-meric AP site-containing DNA duplexa
K11
(M?1)
?Gobs
(kcal/mol)
SK
?Gpe
(kcal/mol)
?Gt
(kcal/mol)
?Hobs
(kcal/mol)
T?Sobs
(kcal/mol)
AND
AMND
ADMND
ATMND
3.0 (?0.2)?105
2.7 (?0.2)?106
6.1 (?0.5)?106
1.9 (?0.2)?107
?7.3 (?0.1)
?8.6 (?0.1)
?9.1 (?0.1)
?9.8 (?0.1)
?1.15
?1.31
?1.36
?1.42
?1.4
?1.7
?1.8
?1.8
?5.9
?6.9
?7.3
?8.0
?20.5?0.6
?19.8?0.4
?16.7?0.3
?12.8?0.7
?13.2 (?0.6)
?11.2 (?0.4)
?7.6 (?0.3)
?3.0 (?0.7)
aK11(M?1), determined by fluorescence titration experiments (cf. Figures 3 and 4), is the 1:1 binding constant in 110mM Na+at 208C ([sodium
cacodylate]=10mM, [EDTA]=1.0mM, [NaCl]=100mM, pH 7.0) with the standard deviations obtained from three independent experiments.
?Gobs is the observed binding free energy calculated from ?Gobs=?RTlnK11. SK is the slope of the plot of log K11 versus log [Na+]
(cf. Supplementary Figure S3): fitting error is within 0.02. ?Gpeand ?Gtare the polyelectrolyte and nonpolyelectrolyte contributions to the observed
binding free energy (?Gobs) evaluated at 110mM Na+(?Gobs=?Gpe+?Gt, ?Gpe=?SKRT ln [Na+]). ?Hobswas directly determined by ITC at
208C (cf. Figure 5): Errors are the standard deviations obtained from three independent measurements. T?Sobs was calculated from
T?Sobs=?Hobs??Gobs. DNA duplex: 50-GCA GCT CCC GXG GTC TCC TCG-30/30-CGT CGA GGG CCC CAG AGG AGC-50, X=AP
site (dSpacer), C=target cytosine.
Figure 3. Fluorescence reponses of ATMND (500nM) to 21-meric AP
site-containing DNA duplex [50-GCA GCT CCC GXG GTC TCC
TCG-30/30-CGT CGA GGG CCC CAG AGG AGC-50, X=AP site
(dSpacer), C=target cytosine], measured in solutions buffered to pH
7.0 (10mM sodium cacodylate) containing 100mM NaCl and 1.0mM
EDTA. Excitation wavelength, 350nm; temperature, 208C. Inset: non-
linear regression analysis of the changes in the fluorescence intensity
ratio at 403nm based on a 1:1 binding isotherm model. F and F0
denote the fluorescence intensities of ATMND in the presence and
absence of DNA duplexes, respectively.
Nucleic Acids Research,2009, Vol.37, No. 51415