Published online 9 January 2009Nucleic Acids Research, 2009, Vol. 37, No. 51411–1422
Influence of substituent modifications on the
binding of 2-amino-1,8-naphthyridines to cytosine
opposite an AP site in DNA duplexes:
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
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),
(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
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
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: firstname.lastname@example.org
? 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.
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-
(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
(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-
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.
MATERIALS AND METHODS
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.
Nucleic Acids Research, 2009, Vol. 37,No. 5
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-
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
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:
D0¼ ½D? þ f L0K11½D?g=f1 þ K11½D?g
Together, Equations (1) and (2) describe the system.
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
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):
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.
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:
Nucleic Acids Research,2009, Vol.37, No. 51413
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
Fluorescence spectra of the resulting solutions (50ml)
were then measured at 58C with a JASCO FP-6500 spec-
trofluorophotometer equipped with a thermoelectrically
3mm?3mm); the slits for the excitation and emission
monochromators were 5 and 5nm, respectively.
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
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,
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.
Nucleic Acids Research, 2009, Vol. 37,No. 5
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
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
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, pH 7.0,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
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
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
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-
purinesremains unchangedfromAND to
?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
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
nonpolyelectrolyte contribution arises from all other
molecular interactions. As is shown in Supplementary
Figure S3, the binding constant K11 increases with
decreasing the salt concentration, and a linear relationship
is obtained between log K11and log [Na+] for all ligand–
cytosine interactions (cf. also Supplementary Table S1).
The slope (SK) of the linear plot was used to evaluate
?Gpein 110mM Na+using ?Gpe=(?SK)RTln [Na+],
followed by calculation of ?Gt(=?Gobs??Gpe).
Thermodynamic parameters thus obtained for the
1,8-naphthyridine-cytosine interactions are summarized
in Table 1. While the binding reaction is enthalpy-driven
in all cases, the effect of methyl groups can be clearly seen
from the comparison of binding enthalpy and entropy.
The most favorable gain in binding enthalpy is obtained
for AND (?Hobs=?20.5kcal/mol), but the loss of bind-
ing entropy is also significant (T?Sobs=?13.2kcal/mol).
This results in the weakest binding free energy for AND–
cytosine interactions (?Gobs=?7.3kcal/mol). By intro-
ducing methyl groups to the naphthyridine ring, the
enthalpy term becomes destabilized, and this is highly
compensated for by less negative values of entropy that
result in a more favorable binding free energy. In case
of AMND (?Gobs=?8.6kcal/mol), the gain in binding
enthalpy is somewhat reduced (?Hobs=?19.8kcal/mol),
whereas the loss of binding entropy is effectively decreased
(T?Sobs=?11.2kcal/mol). This leads to the increased
binding affinity of AMND (??Gobs=?1.3kcal/mol) as
compared to AND. Such an effect is more significant for
ADMND (?Gobs=?9.1kcal/mol, ?Hobs=?16.7kcal/
mol, T?Sobs=?7.6kcal/mol), and the loss of binding
entropy reaches the minimum for ATMND (?Gobs=
?3.0kcal/mol). These results clearly indicate that the
introduction of methyl groups effectively reduces the loss
of binding entropy, which is responsible for the increase
in the binding affinity of 1,8-naphthyridine–cytosine
It has been well recognized that, for DNA–ligand inter-
actions, the favorable binding entropy term may be due to
the release of structured water from DNA and/or ligand
into bulk solvent, and/or due to the release of condensed
counterions from DNA. The latter effect has been
reasonably observed for positively charged ligands such
Figure 4. Fluorescence titration curves for the binding of (A) AND (5.0mM), (B) AMND (1.0mM), (C) ADMND (1.0mM) and (D) ATMND
(0.5mM) to 21-meric AP site-containing DNA duplexes [50-GCA GCT CCC GXG GTC TCC TCG-30/30-CGT CGA GGG CNC CAG AGG
AGC-50, X=AP site (dSpacer), N=G, C, A or T]. Sample solutions were buffered to pH 7.0 with 10mM sodium cacodylate, containing
100mM NaCl and 1.0mM EDTA. Excitation wavelength, 350nm; temperature, 208C. Analysis: AND, 392nm; AMND, 400nm; ADMND,
400nm; ATMND, 403nm.
Nucleic Acids Research, 2009, Vol. 37,No. 5
as ethidium (25), and even for uncharged ligands such as
actinomycin (39) or chartreusin (42), the binding has been
shown to accompany the release of counterions from
DNA. This is generally attributed to lengthening and
unwinding of the DNA duplex, both of which increase
the phosphate spacing along the helix axis. This results
in a decrease in the charge density of the duplex, thereby
releasing condensed counterions from DNA. In the pres-
ent case, 2-amino-1,8-naphthyridines have a positive
charge due to the protonation at the N1 moiety when
binding to cytosine (cf. Figure 7), and the chemical mod-
ification of the naphthyridine ring seems cause some
additional effects on the binding-induced release of coun-
terions. As summarized in Table 1, on increasing the
number of methyl groups from AND to ATMND, the
slope (SK) of the linear plot (SK), which is equivalent to
the number of counterions released from DNA upon
ligand binding, increases somewhat, providing a more
favorable gain from the polyelectrolyte contribution
(?Gpe). In the case of AND, the binding is accompanied
by the release of 1.1 counterions, which corresponds to the
favorable gain of ?1.4kcal/mol from ?Gpe. In the case of
ATMND, 1.4 counterions are released upon binding, and
the value of ?Gpeincreases to ?1.8kcal/mol. It is there-
fore likely that the effect of methyl groups is ascribed par-
tially to the increased release of condensed counterions
from DNA, which provides a favorable entropy contribu-
tion to the overall binding free energy (?Gobs).
The effect of methyl groups is however more evident for
the non-polyelectrolyte contribution (?Gt). Again, as
summarized in Table 1, ?Gt is indeed fundamental in
the stabilization of the binding events, and ?Gtclearly
increases as the number of methyl groups increases. As
compared to AND (?Gt=?5.9kcal/mol), the favorable
gain from ?Gtis increased by as much as ?2.1kcal/mol
for ATMND, which is roughly comparable to the value
for increased gain in the overall binding free energy
(??Gobs=?2.5kcal/mol). Thus, the effect of methyl
groups on the binding affinity is mainly ascribed to the
increased gain from the nonpolyelectrolyte contribution
?Gt, so as to provide a favorable entropic term, probably
due to the release of structured water from DNA and/or
the ligand itself into bulk solvents.
According to the literatures (24,25,42–44), the nonpo-
lyelectrolyte (?Gt) contribution is further dissected into
four contributions that drive the binding process, and
thus the observed binding free energy (?Gobs) is totally
composed of at least five contributions:
?Gobs¼ ?Gpeþ ?Grþtþ ?Ghydþ ?Gconfþ ?Gint
where ?Gr+tis the free energy cost resulting from losses
in rotational and translational degrees of freedom upon
complex formation, ?Ghyd is the free energy for the
hydrophobic transfer of the ligand from aqueous solution
into the DNA binding site, ?Gconfis the contribution due
to conformational transitions in DNA and the ligand, and
?Gint is the contribution from intermolecular DNA–
ligand contacts within the binding site. Among these con-
tributions consisting of ?Gobs, it has been well shown that
the hydrophobic contribution (?Ghyd) is a key parameter
which is related to the change in surface area that is
exposed to solvent upon complex formation, and it is
also possible to correlate changes in solvent-accessible sur-
face area (?SASA) with the heat capacity changes (?Cp).
This relationship has been successfully shown to hold for a
typical DNA–ligand interaction, e.g. the Hoechst 33258
binding to DNA duplex, where the negative change in
heat capacity was observed due to the removal of nonpo-
lar surface from bulk solvent upon complexation, and the
experimentally determined value for ?Cpwas in excellent
agreement with the value computed using ?SASA
obtained by two crystal structures (24). We therefore esti-
mated the values of ?Ghydfrom the heat capacity change
(?Cp), for which temperature dependence of the binding
enthalpy was examined.
Table 2 summaries the binding enthalpy (?Hobs) for
two typical ligands, AND and ATMND, as determined
by ITC measurements at different temperatures. For both
ligands, the value for ?Hobsbecomes less negative as the
Figure 5. ITC data obtained at 208C for the addition of ATMND
aliquots (each 15ml of 175mM) into the solution containing DNA
duplex [1.43ml of 20mM, 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]. Sample solutions were buffered to pH
7.0 with 10mM sodium cacodylate, containing 100mM NaCl and
1.0mM EDTA. The data were best fitted to a model that assumes
a single set of identical binding sites, giving the binding enthalpy
(?Hobs) of ?12.8kcal/mol with a binding stoichiometry (n) of 1.1.
Nucleic Acids Research,2009, Vol.37, No. 51417
temperature is lowered, and ?Cpis determined from the
slope of a plot of ?H versus temperature (Figure 6). The
obtained ?Cpis ?161cal/mol K for AND, and ?217cal/
mol K for ATMND. These values are then used to esti-
mate hydrophobic contribution (?Ghyd) using Record’s
relationship, ?Ghyd=80 (?10)??Cp(38), giving ?12.8
(?1.6) kcal/mol and ?17.3 (?2.2) kcal/mol for AND and
ATMND, respectively. The observed gain from the hydro-
phobic contribution is significantly different between
AND and ATMND (??Ghyd=?4.5kcal/mol), indicat-
ing that ?Ghydis effectively modulated by the introduc-
tion of methyl groups to the naphthyridine ring. This
result seems to be very consistent with the observed
increase in the binding affinity, due to the reduction of
the loss of binding entropy.
Further parsing of the binding free energy (?Gobs) was
done by consideration of a value for the loss of translation
and rotational freedoms (?Gr+t=T?Sr+t) upon bimole-
cularcomplex formationfor ligand–DNA binding
reactions. While there is some controversy about the
value of ?Sr+t (45,46), Spolar and Record (47) have
empirically derived a value of ?Sr+t=50 (?10)cal/mol
K, which seems the most appropriate value to use, as has
been extensively discussed in literatures (24,25,31). By
using this value, we estimate ?Gr+tto be 14.7kcal/mol
at 208C, with about 20% uncertainly. The remaining two
contributions from unfavorable ?Gconf and favorable
?Gint are considered together in this work, and are
obtained by subtracting the sum of the other three con-
from the experimental ?Gobs.
The energetic profiles thus determined for the binding
of AND and ATMND are summarized in Table 3. For
both ligands, the contribution from the hydrophobic
transfer process (?Ghyd) is very large as compared to
the other favorable contributions (?Gpe, and ?Gconf+
?Gint), and appears to be a key force to explain the
observed effect of methyl groups on the binding affinity.
In the case of AND, the favorable gain mainly comes
from ?Ghyd (–12.8kcal/mol), and further from ?Gpe
(?1.4kcal/mol) and ?Gconf+?Gint(?7.8kcal/mol). The
entropic cost of ?Gr+t(14.7kcal/mol) is overcome by the
sum of these favorable contributions. In contrast, in case
(?17.3kcal/mol) is of sufficient magnitude to overcome
the unfavorable contribution from ?Gr+t (14.7kcal/
mol), so that the other favorable gains from ?Gpe
(?1.8kcal/mol) and ?Gconf+?Gint(?5.4kcal/mol) effec-
tively contribute to the overall binding free energy. It is
therefore highly likely that a major driving force for the
1,8-naphthyridine binding is the contribution from the
hydrophobic transfer process (?Ghyd), which is indeed
highly dependent on the chemical modification of the
naphthyridine ring with methyl groups.
The observed nature of the thermodynamic profile,
i.e., the large magnitude of negative binding enthalpies,
is similar to the profile for typical intercalators such
as ethidium (?G=?6.7kcal/mol, ?H=?9.0kcal/mol,
T?S=?2.3kcal/mol, in 0.2M NaCl, pH 7.0, at 258C)
?9.0kcal/mol, T?S=?1.1kcal/mol, in 0.2M NaCl, pH
7.0, at 208C) (31). This is consistent with the intercalation-
like binding mode proposed for the 1,8-naphthyridine-
cytosine interaction in AP site-containing DNA duplexes
Table 2. Temperature dependence of the observed binding enthalpy (?Hob) and calculated heat capacity change (?Cp) for the 1,8-naphthyridine
binding to cytosine in the 21-meric AP site-containing DNA duplex
a?Hobswas directly determined by ITC experiments. Errors are the standard deviations obtained from at least three independent measurements at
each temperature (?14 times;?23 times). Sample solutions were buffered to pH 7.0 with 10mM sodium cacodylate, containing 100mM NaCl and
1.0mM EDTA. DNA duplex: 50-GCA GCT CCC GXG GTC TCC TCG-30/30-CGT CGA GGG CCC CAG AGG AGC-50, X=AP site (dSpacer),
bHeat capacity change calculated from the slope d(?H)/dT obtained by linear least squares fit (cf. Figure 6, r=0.9906 for AND, r=0.9999
Figure 6. Temperature dependence of the binding enthalpy for 2-
ATMND. Errors are the standard deviations obtained from at least
three independent measurements. The linear least squares fit to the
data yielded the heat capacity change, ?Cp, of –161cal/mol K for
AND (r=0.9906), and –217cal/mol K for ATMND (r=0.9999),
respectively. See Table 2 for further details.
Nucleic Acids Research, 2009, Vol. 37,No. 5
(cf. Figure 1). However, it is of interest to note that the
values of ?Cpobtained for 1,8-naphthyridines, especially
for ATMND (?217cal/mol K), are indeed larger than the
values of such typical intercalators. In the case of ethidium
consisting of a hetrocyclic phenanthridine, the value
of ?Cphas been estimated to be ?139cal/mol K (25),
and even for much larger anthracycline antibiotics,
daunorubicin, the ?Cp value has been estimated to
be ?160cal/mol K (31). The ?Cpvalues estimated for
1,8-naphthyridines therefore appear to be somewhat
large when considering their relatively smaller molecular
size as compared to those of these intercalators. This inter-
esting result may be ascribed to a unique local conforma-
tion of AP site-containing DNA duplexes.
As has been reviewed in the literature (48), the existing
NMR data show that the local duplex structure strongly
depends on the type of adjacent base pairs, the AP residue,
and the orphan base, i.e., the base opposite the AP site.
While the orphan purine bases, being largely hydrophobic,
always stack inside the helix, the position of orphan pyr-
imidine residues shows more variability (49). When the
THF abasic site analog is used, an orphan cytosine residue
adopts extrahelical conformations, and tends to be solvent
exposed when it is flanked by cytosine residues that have
weak stacking ability. In the present study, the THF ana-
logue is used for the AP site, and the target cytosine is
flanked by cytosine residues, indicating that the target
cytosine base is located outside the helix and exposed to
solvent. For the binding of 2-amino-1,8-naphthyridines,
it is therefore likely that the DNA duplex must undergo
a conformational transition to form the cavity suitable
for ligand binding, where the target cytosine stacks
inside the helix, and this is followed by the transfer of
1,8-naphthyridines from solution into the binding cavity
(AP site). This kind of binding events seem to accompany
a considerable change in ?SASA, providing the relatively
large values of ?Cpas compared to those of typical DNA
1,8-naphthyridine-cytosine binding, molecular interac-
tions (?Gint), arising generally from specific hydrogen
bonds, van der Waals contacts and other interactions,
are effective contributors to the observed binding free
energy (Table 3). Although the sums of values of ?Gint
and ?Gconf are estimated in the present study, the
sign andmagnitude of
to notethat,for the
the valuesobtained for
?5.4kcal/mol) clearly indicate that the molecular interac-
tion (?Gint) is overcoming the unfavorable contribution
due to conformational transitions in the DNA and the
ligand (?Gconf), and it is a more effective driving force
than the polyelectrolyte contribution (?Gpe: AND,
?1.4kcal/mol; ATMND, ?1.8kcal/mol). This is similar
to intercalators such as ethidium (25), but distinct from
groove binders such as Hoechst 33258 (24), where molec-
ular interactions (?Gint) were found to contribute little to
the observed binding free energy, and both the hydropho-
bic (?Ghyd) and polyelectrolyte (?Gpe) contributions were
found to be the primary driving forces for association.
As for molecularinteractions
are crucial for the selective binding to nucleobases in
AP site-containing DNA duplexes. As suggested by
Nakatani et al. (28,50) and revealed by15N NMR experi-
ments (51), 1,8-naphthridine (AMND, pKb=6.8) exists
as a tautomeric mixture of N1 and N8 protonated form
in acidic solutions, and the N1 protonated form of
1,8-naphthridine binds to cytosine, so that a fully comple-
mentary base-pairing is formed via three-point hydrogen
bonds (Figure 7). The N8 protonated form seems respon-
sible for the binding to thymine, and this would also allow
a fully complementary base-pairing based on three-point
hydrogen bonds (Figure 7). These binding modes explain
the observed binding selectivity for pyrimidines over pur-
ines in AP site-containing DNA duplexes (cf. Figure 4). In
the case of ATMND, while the cytosine/thymine selectiv-
ity is only moderate, the binding affinity for cytosine is
indeed two-order of magnitude higher than those for ade-
nine and guanine (Kd/nM: C, 53?6.0; T, 111?4.1, A,
5800, G, 6000). Similarly, the other three 2-amino-
1,8-naphthyridines show the selectivity for pyrimidines
over purines, irrespective of the number of methyl
groups attached to the naphthyridine ring. Thus, the
1,8-naphthyridine–DNA interaction does involve specific
hydrogen bonds, and is not simply promoted by the trans-
fer of ligands from solution into the hydrophobic binding
site (AP site), which is another distinct feature, differing
from typical DNA intercalation (25).
It should be here noted that the introduction of methyl
groups does not contribute to the improvement of
the binding selectivity between cytosine and thymine,
Figure 7. Proposed binding modes of 2-amino-1,8-naphthyridines with
cytosine or thymine opposite the AP site in DNA duplexes.
Table 3. Energetic profiles for the 1,8-naphthyridine binding to cyto-
sine in the 21-meric AP site-containing DNA duplexa
aThe estimated contributions to the observed free energy (?Gobs) from
the five sources discussed in the text are given: Binding to the 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)
in 100mM NaCl, 1.0mM EDTA, 10mM sodium cacodylate, pH 7.0
Nucleic Acids Research,2009, Vol.37, No. 51419
another important issue from a practical point of view.
While ATMND shows some preference for cytosine
over thymine, the thermodynamic parameters for the
with parameters for the binding to cytosine. As sum-
marized in Supplementary Table S2, the binding to
?Hobs=?13.6kcal/mol, T?Sobs=?4.3kcal/mol), and
the values of binding enthalpy and entropy are com-
interaction (?Gobs=?9.8kcal/mol, ?Hobs=?12.8kcal/
mol, T?Sobs=?3.0kcal/mol). The polyelectrolyte (?Gpe)
contribution is also effective for the thymine binding
(?Gpe=?1.8kcal/mol), which is consistent with the pro-
posed binding mode by the positively charged (proto-
nated) 1,8-naphthyridine (cf. Figure 7). Thus, a different
approach rather than the methylation strategy should be
required in order to develop 1,8-naphthyridine-based
ligands with the improved binding selectivity between
cytosine and thymine.
Finally, ATMND was applied to the analysis of single-
base mutation present in 107-meric DNAs (K-ras gene,
codon 12) (54). After asymmetric PCR amplification
(52,53), the products were analyzed at 58C in a buffer
solution (pH 7.0, 100mM sodium cacodylate) containing
1.6mM EDTA, 50 nM ATMND and 5.0mM AP site-
containing 20-mer probe DNA. As shown in Figure 8A,
in the case of sense strand analysis, a significant fluores-
cence quenching of ATMND (excitation wavelength:
350nm, analysis: 403nm) is observed for the cytosine-
containing sequence (GCT: 79%), while the response is
relatively moderate for the thymine- or purine-containing
sequences (GTT: 40%; GAT: 18%; GGT: 21%). In the
case of antisense strand analysis (Figure 8B), a fluores-
cence response is observed effectively for pyrimidine-
containing sequences over purine-containing sequences
(ACC: 48%; ATC: 38%; AAC: 1%; AGC: 2%).
ATMND would be thus applicable to the detection of
cytosine (thymine)-related transversion such as C(T)>G
and C(T)>A, for which the simultaneous use of purine-
selective ligands would assure the analysis, as has been
previously demonstrated for G>A detection (16).
In summary, we have demonstrated a significant effect of
the methyl substitutions on the binding of a series of
2-amino-1,8-naphthyridines to pyrimidines in AP site-
containing DNA duplexes. Despite the relatively simple
modification, the binding affinity of 1,8-naphthyridines
clearly increased with increasing the number of methyl
groups of the naphthyridine ring, and ATMND having
three methyl groups showed the strongest binding affinity
of 1.9?107M?1and 0.91?107M?1, respectively for cyto-
sine and thymine (in 110mM Na+, pH 7.0, at 208C).
These value were nearly two order of magnitude higher
than those of the parent AND having no methyl groups
(0.030?107M?1for cytosine, 0.012?107M?1for thy-
mine). The obtained thermodynamic parameters for
1,8-naphthyridine-cytosine interactions (?Gobs, ?Hobs,
T?Sobs) indicated that, while the binding was enthalpy-
driven for all ligands, the introduction of methyl groups
effectively reduced the loss of binding entropy, which
was responsible for the increase in the binding affinity of
1,8-naphthyridine–cytosine interactions. From the analy-
sis based on the polyelectrolyte theory, we found that the
nonpolyelectrolyte contribution (?Gt) was fundamental in
the stabilization of the binding events, and ?Gtclearly
increased with increasing the number of methyl groups.
Interestingly, the value of the heat capacity change
(?Cp) was found to be significantly different between
AND and ATMND, and the estimated contribution
from the hydrophobic transfer process (?Ghyd) was
found to be effectively modulated by the introduction
of methyl groups to the naphthyridine ring. Indeed, the
obtained energetic profiles revealed that a major driving
force for the 1,8-naphthyridine binding was the contribu-
tion from the hydrophobic transfer process, which
appeared to be a key force to explain the observed effect
of methyl groups on the binding affinity.
As has been reported for some DNA-binding molecules,
thermodynamic characterization of the binding reactions
offers valuable insights into the major driving forces
involved in the complex formation, and the obtained
thermodynamic data, together with structural characteri-
zation, would be a rational basis for the further
Figure 8. Fluorescence detection of single-base mutation of PCR products present in (A) sense strand and (B) antisense strand of K-ras gene (107-
mer, codon 12). After PCR, the products were analyzed in solutions buffered to pH 7.0 with 100mM sodium cacodylate containing 1.6mM EDTA,
50nM ATMND and 5.0mM AP site-containing probe DNA. Excitation 350nm; detection 403nm. Temperature 58C.
Nucleic Acids Research, 2009, Vol. 37,No. 5
development of the chemistry of DNA-binding molecules.
In particular, the comprehensive examination of a series of
structurally related compounds might be an effective
approach for this direction. We strongly expect that the
results described here represent a piece of such data, espe-
cially for the further design of DNA-binding molecules
applicable to gene analysis. We are now undertaking fur-
ther studies on the design and synthesis of this class of
ligands including the thermodynamic and structural
Supplementary Data are available at NAR Online.
Research (B) (No. 20350032) and Exploratory Research
(No. 19655022), and for G-COE Project, from the
Ministry of Education, Culture, Sports, Science and
charge: CREST, JST.
Conflict of interest statement. None declared.
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Nucleic Acids Research, 2009, Vol. 37,No. 5