6,7-Dimethyllumazine as a potential ligand for selective recognition of adenine opposite an abasic site in DNA duplexes

Article abstract of DOI:10.1039/b816876h

6,7-Dimethyllumazine more selectively binds to adenine (A) base opposite the abasic site in DNA duplexes (5′-TCC AGX GCA AC-3′/3′-AGG TCN CGT TG-5′, X = AP site (Spacer C3), N = A, T, C and G) than the other three nucleobases with a dissociation constant K_d of ca. 1.0 μM; substituted methyl groups enhance the binding affinity to A and the selectivity for A over T, compared to the parent molecule, lumazine. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b816876h

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6,7-Dimethyllumazine as a potential ligand for selective recognition of
adenine opposite an abasic site in DNA duplexesw
a
a
Zhiqiang Ye,z Burki Rajendar,z Dai Qing,a Seiichi Nishizawaab and Norio Teramae*ab
Received (in Cambridge, UK) 26th September 2008, Accepted 20th October 2008
First published as an Advance Article on the web 12th November 2008
DOI: 10.1039/b816876h
6,7-Dimethyllumazine more selectively binds to adenine (A) base
opposite the abasic site in DNA duplexes (50-TCC AGX GCA
developing an efficient A-selective ligand still remains as a task
to be pursued, since alloxazine cannot sufficiently discriminate
A (the 1 : 1 binding constant, K11 = 1.21 ꢁ 106 Mꢀ1) from T
(K11 = 0.72 ꢁ 106 Mꢀ1).9 Accordingly, it is highly desired to
develop a new ligand to attain the selective detection of SNPs
related to adenine. Several strategies such as introducing
substituents into nucleotides and/or ligands have been proposed
to improve the binding affinity of ligands and stabilization of
DNA duplexes.7b,11 As one such approach, introduction of alkyl
groups into the nucleobase and/or ligands in a DNA strand has
been known to increase the stacking interaction11 and the binding
ability of ligands with a target nucleobase.7b
ꢀ
AC-30/30-AGG TCN CGT TG-50, X = AP site (Spacer C3),
ꢀ
ꢀ
N = A, T, C and G) than the other three nucleobases with a
ꢀ
dissociation constant Kd of ca. 1.0 mM; substituted methyl
groups enhance the binding affinity to A and the selectivity for
A over T, compared to the parent molecule, lumazine.
Single nucleotide polymorphisms (SNPs) are extremely important
as a genetic marker for the identification of disease genes and
detection of genetic mutations.1,2 Thus, simple and quick detec-
tion of SNPs is a key issue in today’s chemical biology, biotech-
nology, medicine and pharmacogenomics.3 Consequently,
considerable research efforts have been made to develop high-
throughput analysis of SNPs based on short oligo-nucleotides
labelled by fluorescent molecules.4 Nakatani et al.5 have recently
synthesized dimeric naphthyridine derivatives that specifically
bind to the guanine–guanine,5a cytosine–cytosine,5b and
guanine–adenine5c mismatches, and they succeeded in developing
a non-gel-based scanning method to detect the ligand-bound
SNPs,5a using a surface plasmon resonance (SPR) assay. On the
other hand, we have also proposed a new strategy of ligand-based
fluorescence assay for SNP typing (Fig. 1, left), and have
discovered a series of small fluorescence ligands that can bind
to a nucleobase opposite an abasic site (AP site) in DNA
duplexes.6–9 In contrast to typical DNA–drug binding ligands
(groove binders or intercalators), the AP site in DNA duplexes
provides a unique hydrophobic binding pocket which allows the
pseudo-base pairing of ligands with intrahelical nucleobases along
the Watson–Crick edge,10 where the ligand is stacked with two
nucleobases flanking the AP site. In combination with AP site-
containing DNA duplexes, we have reported a series of ligands
such as 2-amino-7-methyl-1,8-naphthyridine (AMND),6 2-amino-
6,7-dimethyl-4-hydroxypteridine,7 amiloride,8 and alloxazine9
with high affinities for cytosine (C), guanine (G), thymine (T) and
adenine (A), respectively. Among these base-selective ligands,
Here we report on new fluorescent ligands with a high
binding affinity for adenine, such as 6,7-dimethyllumazine.
Taking into account the structure of previously reported
ligands as a clue for binding to adenine, we have found that
lumazine and its derivative, 6,7-dimethyllumazine, possess
suitable hydrogen-bonding sites with high complementarity
for adenine (Fig. 1, middle), while fluorescence properties of
lumazine and its derivatives have been examined upon incor-
poration into DNA.12 Introduction of methyl groups to
lumazine at the 6,7-positions is a key idea to improve the
selectivity for adenine base; it is highly likely that introducing
an electron donating group neighboring the hetero atoms (N-8
position of lumazine) will enhance the electron density at the
binding site of 6,7-dimethyllumazine to adenine base. There-
fore, the high selectivity in complexation, with accompanying
significant fluorescence quenching, is obtained exclusively with
the adenine nucleobase in aqueous media.
First, we used fluorescence measurements to examine the
binding behavior of lumazine and 6,7-dimethyllumazine
with each target base opposite the AP site in DNA
duplexes (50-TCCA GXG CAAC-30/30-AGGT CNC GTTG-50,
ꢀ
ꢀ
a Department of Chemistry, Graduate School of Science, Tohoku
University, Aoba-ku, Sendai, 980-8578, Japan.
E-mail: teramae@mail.tains.tohoku.ac.jp; Fax: +81 22 7956552;
Tel: +81 22 7956549
b CREST, Japan Science and Technology Agency (JST), Aoba-ku,
Sendai, 980-8578, Japan
w Electronic supplementary information (ESI) available: Synthesis of
6,7-dimethyllumazine (Scheme S1), absorption and fluorescence data
(Fig. S1–S5), salt dependence (Fig. S6 and Table S1), ITC data
(Fig. S7 and S8), DNA sequence effect on fluorescence (Fig. S9). See
DOI: 10.1039/b816876h
Fig. 1 Schematic illustration of the ligand-based fluorescence detec-
tion of single-nucleotide polymorphisms in combination with an AP
site-containing probe DNA (left). Possible binding modes for adenine
by lumazine or 6,7-dimethyllumazine (middle) and alloxazine (right).
z The First authorship of this article is equally shared by Burki
Rajendar and Zhiqiang Ye.
y Present address: Department of Chemistry, University of Alabama
at Birmingham, 901 14th Street South, Birmingham, AL 35294, USA.
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6588 | Chem. Commun., 2008, 6588–6590

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magnitude higher than that of lumazine with adenine. Simi-
larly, the binding constants of lumazine and 6,7-dimethyl-
lumazine with thymine are 0.03 ꢁ 106 Mꢀ1 and 0.07 ꢁ 106 Mꢀ1
,
respectively. As expected, it is clear that the difference in the
binding constants of adenine against thymine is achieved with a
large discrimination ratio using 6,7-dimethyllumazine. Inter-
estingly, the binding affinity of 6,7-dimethyllumazine is further
strengthened by the use of 23-mer duplex (50-TCTGC GTCCA
GXG CAACG CACAC-30/30-AGACG CAGGT CAC GTTGC
ꢀ
ꢀ
GTGTG-50, X = Spacer C3, ESI, Fig. S4w) containing adenine
ꢀ
opposite the AP site (K11 = 1.0 ꢁ106 Mꢀ1). In contrast with
lumazine and the previously reported ligand, alloxazine9 (ESI
Fig. S5w), 6,7-dimethyllumazine has a striking ability to recognize
A over T, C and G.
As shown in Fig. 3, the binding affinity and the selectivity of
6,7-dimethyllumazine for adenine exhibit significant improve-
ment in comparison to lumazine (Fig. 2) due to the increased
stacking interactions between 6,7-dimethyllumazine and the
bases flanking the AP site by introducing two methyl groups to
lumazine. In the case of alloxazine and its derivative (lumi-
chrome: 7,8-dimethylalloxazine),9 the base-selectivity was
changed from adenine to thymine by adding methyl groups
at 7 and 8 positions of alloxazine. On the other hand, the base-
selectivity is kept for adenine and the binding affinity is
enhanced for 6,7-dimethyllumazine compared to lumazine.
The enhancement in the binding affinity can be ascribed to
the increased hydrophobicity and polarizability of methyl
groups on the ligand. In addition, the presence of two methyl
groups (i.e., electron-donating groups) neighboring the bind-
ing site, the electron density at the N-8 position of the
lumazine ring significantly increases, hence 6,7-dimethyl-
lumazine prefers adenine-recognition (at N-1 and N-8 positions)
more strongly than lumazine.
Fig. 2 Fluorescence spectra of lumazine in the absence and presence
of AP site-containing DNA duplexes (50-TCCA GXG CAAC-30/30-
ꢀ
AGGT CNC GTTG-50, X = AP site, N = G, C, A, T). [DNA duplex] =
ꢀ
ꢀ
ꢀ
10.0 mM, [lumazine] = 10.0 mM in a solution buffered to pH 7.0
(10 mM sodium cacodylate containing 100 mM NaCl and 1.0 mM
EDTA). Excitation wavelength = 335 nm, 5 1C.
X = Spacer C3, N = G, C, A, T) by fluorescence measurements.
ꢀ
ꢀ
As is shown in Fig. 2, lumazine exhibits significant quenching of
its fluorescence upon binding with the target adenine base, and
the quenching efficiency is in the order of G o C o T o A,
while almost no quenching is observed in the presence of
normal duplexes (fully matched DNA) containing no AP sites.
The fluorescence response of 6,7-dimethyllumazine upon bind-
ing with the target nucleobase shows higher quenching than
lumazine in the order of G o C o T oo A as shown in
Fig. 3, and it can be seen that highly selective detection of A
over T, C and G is attained by using 6,7-dimethyllumazine as a
ligand. From the fluorescence titration analysis (ESI,
Further, the binding constants of lumazine and 6,7-dimethyl-
lumazine were also determined at different salt concentrations
using fluorescence measurements. As is shown in ESI Fig. S6,w
the binding constants for lumazine and 6,7-dimethyllumazine
decrease slightly as the salt concentration increases. These results
indicate that the binding of lumazine or 6,7-dimethyllumazine to
DNA is accompanied by rather less release of counterions. From
the double logarithmic linear fit to binding constants under
different salt concentration, a slope of ꢀ0.061 was estimated,
by which an apparent charge of 6,7-dimethyllumazine was
obtained (Z = +0.07) for the binding to adenine in AP
site-containing DNA duplexes according to M. T. Record
et al.13 From the dependence of the binding constant on salt
concentration, the binding free energies of polyelectrolyte
contribution (DGpe) and non-polyelectrolyte contribution (DGt)
were calculated based on the polyelectrolyte theory by Record
et al.13 The polyelectrolyte contribution to the binding free
energy change (DGobs) is small for the 6,7-dimethyllumazine
binding (DGpe = ꢀ0.074 kcal molꢀ1) and the lumazine binding
(DGpe = ꢀ0.19 kcal molꢀ1) as listed in Table 1. Whereas, DGt of
the 6,7-dimethyllumazine binding and the lumazine binding are
ꢀ7.46 kcal molꢀ1 and ꢀ6.08 kcal molꢀ1, respectively. The values
of DGt reveal that the non-polyelectrolyte component of binding
is fundamental for the stabilization of the complex between
lumazine or 6,7-dimethyllumazine complex and DNA.
Moreover, it also indicates that the increase in the binding
Fig. S2w), K11 for lumazine with adenine is 0.085 ꢁ 106 Mꢀ1
,
and for 6,7-dimethyllumazine with adenine, it is 0.83 ꢁ
106 Mꢀ1 (see ESI Fig. S3w) which is about one order
Fig. 3 Fluorescence spectra of 6,7-dimethyllumazine in the absence and
presence of AP site-containing DNA duplexes (50-TCCA GXG CAAC-
ꢀ
30/30-AGGT CNC GTTG-50, X = AP site, N = G, C, A, T). [DNA
ꢀ
ꢀ
ꢀ
duplex] = 5.0 mM, [6,7-dimethyllumazine] = 5.0 mM in a solution
buffered to pH 7.0 (10 mM sodium cacodylate containing 100 mM NaCl
and 1.0 mM EDTA). Excitation wavelength = 350.5 nm, 5 1C.
ꢂc
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Table 1 Thermodynamic parameters for the binding of lumazine or 6,7-dimethyllumazine to adenine in the 11-mer AP site-containing DNA
duplexa
DGobs
/
DGpe
/
DGt/
DHobs
/
TDSobs
/
Kobs/Mꢀ1
kcal molꢀ1
ꢀSK
0.153 (ꢃ0.012) ꢀ0.19 (ꢃ0.01)
kcal molꢀ1
kcal molꢀ1
kcal molꢀ1
kcal molꢀ1
Lumazine
6,7-Dimethyllumazine 0.83 (ꢃ0.02) ꢁ 106
0.085 (ꢃ0.002) ꢁ 106 ꢀ6.27 (ꢃ0.01)
ꢀ6.08 (ꢃ0.02) ꢀ9.5 (ꢃ0.26) ꢀ3.23 (ꢃ0.27)
ꢀ7.53 (ꢃ0.01)
0.061 (ꢃ0.0047) ꢀ0.074 (ꢃ0.006) ꢀ7.46 (ꢃ0.02) ꢀ14.0 (ꢃ0.08) ꢀ6.47 (ꢃ0.09)
Kobs (Mꢀ1), determined by fluorescence titration experiments, is the 1 : 1 binding constant in 110 mM Na+ at 5 1C ([sodium cacodylate] =
a
10 mM, [EDTA] = 1 mM, [NaCl] = 100 mM, pH 7.0). DGobs is the observed binding free energy calculated from DGobs = ꢀRT ln Kobs. SK is
the slope of the plot of log Kobs versus log aNa+. DGpe and DGt are the polyelectrolyte and non-polyelectrolyte contributions to the binding free
energy (DGpe = (ꢀSK)RT ln[Na+]) evaluated at 110 mM Na+. DHobs was determined by ITC at 5 1C. TDSobs was calculated from TDSobs
=
DHobs ꢀ DGobs. DNA duplex: 50-TCC AGX GCA AC-30/30-AGG TCA CGT TG-50, X = AP site; A = adenine. Errors: Kobs; the SD by three
ꢀ
ꢀ
ꢀ
ꢀ
independent repeated measurements, DHobs; fitting error.
constant for 6,7-dimethyllumazine is mainly due to a large non-
polyelectrolyte contribution.
be necessary to improve the dependence of fluorescence
responses on nucleobases flanking the AP site (ESI Fig. S9w),
the new ligand (6,7-dimethyllumazine) would be useful for the
detection of adenine-related SNPs, and the results presented here
will provide a rational basis for further studies on the design and
synthesis of new adenine-selective fluorescent ligands with the
improved binding and sensing properties.
For analyzing the energetics and stoichiometry of the inter-
action, the binding of 6,7-dimethyllumazine with adenine was
further characterized by isothermal titration calorimetry (ITC)
experiments. As shown in ESI Fig. S8,w the addition of the
duplex aliquots (200 mM) into the solution containing 6,7-
dimethyllumazine (20 mM) caused a large exothermic heat of
reaction, and the corrected binding isotherm was obtained
after the heat of dilution was subtracted. The resulting titra-
tion curve was able to be best fitted using a model that assumes
a single set of identical binding sites with a stoichiometry n of
1.1, suggesting a 1 : 1 binding. The thermodynamic parameters
of the interactions for the 6,7-dimethyllumazine-adenine and
lumazine-adenine are summarized in Table 1. As can be seen
there, the ligand-duplex interaction is enthalpy-driven for both
lumazine and 6,7-dimethyllumazine. Compared to the
enthalpy loss of lumazine (DHobs = ꢀ9.5 kcal molꢀ1), the
Notes and references
1 A. J. Schafer and J. R. Hawkings, Nat. Biotechnol., 1998, 16, 33.
2 F. S. Collins, M. S. Guyer and A. Chakravarti, Science, 1997, 278,
1580.
3 (a) A.-C. Syvanen, Nat. Rev. Genet., 2001, 2, 930;
(b) A. Chakravarti, Nature, 2001, 409, 822; (c) J. J. McCarthy
and R. Hilfiker, Nat. Biotechnol., 2000, 18, 209.
4 R. T. Ranasinghe and T. Brown, Chem. Commun., 2005, 5487.
5 (a) K. Nakatani, S. Sando and I. Saito, Nat. Biotechnol., 2001, 19,
51; (b) A. Kobori, S. Horie, H. Suda, I. Saito and K. Nakatani,
J. Am. Chem. Soc., 2004, 126, 657; (c) S. Hagihara, H. Kumasawa,
Y. Goto, G. Hayashi, A. Kobori, I. Sato and K. Nakatani, Nucleic
Acids Res., 2004, 32, 278.
6 (a) K. Yoshimoto, S. Nishizawa, M. Minagawa and N. Teramae,
J. Am. Chem. Soc., 2003, 125, 8982; (b) S. Nishizawa,
K. Yoshimoto, T. Seino, C.-Y. Xu, M. Minagawa, H. Satake,
A. Tong and N. Teramae, Talanta, 2004, 63, 175.
7 (a) K. Yoshimoto, C.-Y. Xu, S. Nishizawa, T. Haga, H. Satake
and N. Teramae, Chem. Commun., 2003, 2960; (b) Q. Dai,
C.-Y. Xu, Y. Sato, K. Yoshimoto, S. Nishizawa and
N. Teramae, Anal. Sci., 2006, 22, 201.
8 (a) C. Zhao, Q. Dai, T. Seino, Y.-Y. Cui, S. Nishizawa and
N. Teramae, Chem. Commun., 2006, 1185; (b) B. Rajendar,
Y. Sato, S. Nishizawa and N. Teramae, Bioorg. Med. Chem. Lett.,
2007, 17, 3682; (c) N. B. Sankaran, S. Nishizawa, T. Seino,
K. Yoshimoto and N. Teramae, Angew. Chem., Int. Ed., 2006,
45, 1563.
9 B. Rajendar, S. Nishizawa and N. Teramae, Org. Biomol. Chem.,
2008, 6, 670.
10 J. Lhomme, J. F. Constant and M. Demeunynck, Biopolymers
(Nucleic Acid Sci.), 1999, 52, 65.
11 (a) S. Wang and E. T. Kool, Biochemistry, 1995, 34, 4125;
(b) R. A. Hutchins, J. M. Crenshaw, D. E. Graves and
W. A. Denny, Biochemistry, 2003, 42, 13754.
12 (a) M. E. Hawkins, W. Pfleiderer, F. M. Balis, D. Porter and
J. R. Knutson, Anal. Biochem., 1997, 244, 86; (b) M. E. Hawkins,
W. Pfleiderer, O. Jungmann and F. M. Balis, Anal. Biochem., 1997,
244, 86.
increase in enthalpy loss of 6,7-dimethyllumazine (DHobs
=
ꢀ14.0 kcal molꢀ1) can be ascribed primarily to the effect of the
introduced methyl groups. Despite the value of entropy loss
from lumazine to 6,7-dimethyllumazine being negative
(D(TDS) = ꢀ3.24 kcal molꢀ1), it is highly compensated by
the less negative value of enthalpy (DDHobs = ꢀ4.5 kcal molꢀ1),
that results in a more favorable free energy change (DDGobs
=
ꢀ1.26 kcal molꢀ1). Therefore the DNA binding constant
of 6,7-dimethyllumazine is 11-fold higher than that of luma-
zine. It is highly likely that the formation of hydrogen
bonds and stacking interactions are more effective in the
6,7-dimethyllumazine-duplex binding.
In summary,
a new class of site-recognition ligand,
6,7-dimethyllumazine, has been developed for fluorescence
detection of the adenine base by using the AP site as the field
of molecular recognition. 6,7-Dimethyllumazine was able to
selectively recognize adenine base with 1 : 1 binding affinity of
1.0 ꢁ 106
M
ꢀ1, accompanied by significant fluorescence
quenching. It was demonstrated that the hydrogen bonding
and the stacking interactions with flanking nucleotides played
an important role in the complexation of 6,7-dimethyl-
lumazine with adenine at the AP site, which indeed controlled
the binding stability and selectivity. While further efforts will
13 M. T. Record, C. F. Anderson and T. M. Lohman, Q. Rev.
Biophys., 1978, 11, 103.
ꢂc
This journal is The Royal Society of Chemistry 2008
6590 | Chem. Commun., 2008, 6588–6590