Ratiometric fluorescent signaling of small molecule, environmentally sensitive dye conjugates for detecting single-base mutations in DNA

Article abstract of DOI:10.1002/chem.201200219

Bind it! A ratiometric fluorescent probe based on conjugation of a DNA-binding ligand with an environmentally sensitive dye to probe hydrophobic grooves of the DNA helix was developed. The resulting conjugate ATMND-DBD can bind to an orphan nucleobase opposite an abasic (AP) site with high selectivity and affinity for pyrimidine bases (see figure). Copyright

Full text of DOI:10.1002/chem.201200219

COMMUNICATION
DOI: 10.1002/chem.201200219
Ratiometric Fluorescent Signaling of Small Molecule Environmentally
Sensitive Dye Conjugate for Detecting Single-Base Mutations in DNA
Chun-xia Wang, Yusuke Sato, Megumi Kudo, Seiichi Nishizawa, and Norio Teramae*[a]
Since the report on Ca2+ indicators by Tsien and co-work-
ers,[1] ratiometric fluorescent probes have come to be recog-
nized as a promising tool for biosensing and bioimaging
studies.[2] Significant efforts have been devoted to develop
this class of probes for a wide range of analytes including
metal ions,[3] inorganic and organic anions,[4] enzyme activi-
ties,[5] and nucleic acids.[6–8] A typical way to produce the ra-
tiometric signal is by using double fluorescent dyes, in which
techniques based on fluorescence resonance energy transfer
(FRET) are widely used.[2] For example, for the analysis of
single-nucleotide polymorphism (SNP) in genomic DNAs,
two-fluorophore-labeled hybridization probes have been de-
veloped based on the FRET strategy,[6,7] in which pairs of
Alexa 488-Cy5,[6] 6-carboxyfluorescein-coumarin,[7a] car-
boxy-x-rhodamine-Cy3,[7b] and Cy3-Cy5[7c] molecules were
utilized. Another major approach to produce the ratiometric
signal is based on the formation of excimers,[6,8] and ratio-
metric fluorescent detection of SNP has been demonstrated
by using pyrene-[6,8a,b] or perylene-bisimide[8c]-modified hy-
bridization probes, and bispyrene diamines.[8d]
(Figure 1), was used simultaneously with an AP-containing
DNA probe as following two steps: 1) a target single-strand-
ed DNA is hybridized with the AP-containing DNA probe
Herein, we report on a different way to design ratiometric
fluorescent probes for SNP genotyping. In contrast to most
approaches based on the interaction between two fluores-
cent dyes in hybridization probes,[6–8] we focused on the
unique structure of the helical duplex to produce the ratio-
metric signaling. For this purpose, an environmentally sensi-
tive fluorescent dye to probe hydrophobic grooves of the
DNA helix was used,[9,10] in which the dye is conjugated
with a fluorescent ligand developed by our group to recog-
nize an orphan nucleobase opposite an abasic (AP) site in
the DNA duplex.[11] Herein, a benzofurazan derivative, 4-
(N,N-dimethylaminosulphonyl)-1,2,3-benzoxadiazole
Figure 1. A) Schematic illustration of the binding of ATMND–DBD to
the target nucleobase opposite an AP site in a DNA duplex. B) One of
the possible binding modes of ATMND–DBD with thymine in the DNA
duplex obtained with software MacroModel (Ver. 9.0, AMBER* force
field, GB/SA solvent model). Thymine is shown in green, ATMND
moiety in blue, and DBD moiety in orange. Pseudo-base pairing with
thymine is also shown. In this binding mode, DBD moiety protrudes into
the minor groove of the DNA duplex.
to place the AP site opposite a nucleobase to be detected;
2) the ligand (ATMND moiety) selectively binds to the
orphan (target) nucleobase in the duplex through comple-
mentary hydrogen bonding, and the DBD moiety becomes
located at the surface of the minor or major groove of the
duplex (cf. also Figure S1 in the Supporting Information).
Indeed, the binding of ATMND–DBD is highly selective to
pyrimidine (C, T) over purine (G, A) bases, and a clear ra-
tiometric response was obtained based on quenching of the
ATMND emission (403 nm) and enhancing of the DBD
emission (615 nm). Although the sensing ability depends on
the DNA sequence to some extent, the limit of detection
(LOD) reaches to nanomolar level, and the ratiometric re-
sponse to 500 nm samples can be judged even with the
naked eye. Therefore, our approach offers a convenient and
(DBD),[12] was utilized as the environmentally sensitive fluo-
rescent dye, which is linked through an alkyl spacer to 2-
amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND).[11f] The
resulting conjugate with an ethylene linker, ATMND–DBD
[a] C.-x. Wang, Dr. Y. Sato, M. Kudo, Dr. S. Nishizawa,
Prof. Dr. N. Teramae
Department of Chemistry, Graduate School of Science
Tohoku University, Aoba-ku, Sendai 980-8578 (Japan)
Fax : (+81)22-795-6552
E-mail: teramae@m.tohoku.ac.jp
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201200219.
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label-free method for detecting SNP as has been reported
previously,[11] and this study further offers a basis for the ad-
vanced design of ratiometric fluorescent probes for detect-
ing DNAs, especially for SNP genotyping.
ATMND–DBD was synthesized in five steps from 2,6-di-
aminopyridine (Scheme 1 and the Supporting Informa-
tion).[13] The introduction of the ethylene linker (1,2-diami-
noethane) to the naphthyridine ring was done according to
Scheme 1. Synthesis of ATMND–DBD. Reagents and conditions: a) 3-
methylpentane-2,4-dione, H3PO4, 48%; b) NaNO2, H2SO4; c) POCl3;
d) 1,2-diaminoethane, 31% (three steps); e) DBD-F, Et3N, DMF, 36%.
Figure 2. A) Fluorescence responses of ATMND–DBD (2.0 mm) to target
nucleobases (N=G, C, A, or T) in 21-meric AP-site-containing DNA du-
plexes (2.0 mm) in solutions buffered to pH 7.0 (10 mm sodium cacody-
late) and containing NaCl (100 mm) and EDTA (1.0 mm, water/ethanol
97.2:2.8). Excitation: ATMND moiety, at 376 nm; DBD moiety, at
451 nm; T=208C. Duplex: 5’-GCAGCTCCC GXG GTCTCCTCG-3’/3’-
CGTCGAGGG CNC CAGAGGAGC-5’, X=AP site; SpacerC3 (a tri-
methylene residue), N=G, C, A, or T. B) Fluorescence titration for the
binding of ATMND–DBD (1.0 mm) to thymine (N=T) in the 21-meric
AP-site-containing DNA duplex (0–5.0 mm). Other conditions are the
same as those for Figure 2A. Inset: nonlinear regression analysis of the
fluorescent intensity ratio at 585 nm based on a 1:1 binding isotherm
model. F and F0 denote the intensities of ATMND–DBD in the presence
and absence of duplexes, respectively.
the literature,[13b] and then the DBD moiety was attached.
For comparison, two conjugates with different lengths of the
linker (see the Supporting Information: C3, trimethylene;
C4, tetramethylene) were also prepared similar to
ATMND–DBD.
Figure 2A shows the fluorescence responses of ATMND–
DBD (2.0 mm) upon addition of 21-meric AP-site-containing
DNA duplexes (2.0 mm; 5’-GCA GCT CCC GXG GTC
TCC TCG-3’/3’-CGT CGA GGG CNC CAG AGG AGC-5’,
X=AP site; SpacerC3 (a trimethylene residue), N=G, C,
A, or T) at 208C in solutions buffered to pH 7.0. ATMND
moiety exhibited the emission band with a maximum at
420 nm when excited at 376 nm (the absorption maximum
wavelength of ATMND moiety), and the emission is
quenched in the presence of DNA duplexes. As has been
observed for parent ATMND,[11f] the responses are signifi-
cant for pyrimidine bases (N=T and C), whereas moderate
responses were observed for purine bases (N=G and A). It
is highly likely that ATMND moiety binds to the AP site of
the DNA duplex, and the binding is promoted selectively to
pyrimidine bases by three-point hydrogen bonding (Fig-
ure 1B).[11f]
The fluorescence response of DBD moiety was clearly ob-
served and is also highly selective to pyrimidine bases over
purine bases (Figure 2A, right). In the absence of DNA du-
plexes, the weak emission with a maximum at 615 nm was
observed when excited at 451 nm (the maximum absorption
wavelength of DBD moiety), and the binding to thymine
(N=T) or cytosine (N=C) resulted in the enhancement of
fluorescence intensities. The binding to these two nucleobas-
es was also accompanied by a 30 nm blueshift of the emis-
sion-maximum wavelength, indicating a change in the micro-
environment of the DBD moiety. A hypothetical binding of
DBD moiety to the AP site is exclusive, because a control
ligand, a DBD derivative that lacks ATMND moiety,
showed no fluorescence responses to 21-meric AP-site-con-
taining DNA duplexes (Figure S2 in the Supporting Infor-
mation). Therefore, it is feasible that the observed response
of DBD moiety is due to its being in the hydrophobic micro-
environment of a surrounding DNA molecule, namely, the
major or minor groove of the duplex DNA, when ATMND–
DBD binds to the AP site (Figure 1B and Figure S1 in the
Supporting Information).
The binding affinity of ATMND–DBD was then quantita-
tively determined by fluorescence-titration experiments, for
which the responses of DBD moiety were utilized. As can
be seen from the typical response to thymine (Figure 2B),
the response is concentration dependent, which is well fitted
by a nonlinear least-square regression analysis based on
a 1:1 binding isotherm (Figure 2B, inset). ATMND–DBD
showed the strongest binding affinity for thymine with a 1:1
binding constant K11 of 6.7
ty for cytosine (K11 =2.2
ACHTUNGTRENNUNG
U
to the affinity for thymine, although it is two orders of mag-
nitude lower for guanine and adenine (K11/106 mÀ1: G, 0.05;
A, 0.06). We note that the binding selectivity and affinity of
ATMND–DBD are comparable to those of the parent
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Ratiometric Fluorescent Signaling of Small Molecule
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ligand, ATMND,[11f] indicating that conjugation with the
DBD moiety has a small influence on the original binding
properties of the pyrimidine-selective ATMND.
the Supporting Information). However, the response selec-
tivity between cytosine and thymine depends on the flanking
nucleobases. Although the response is selective to thymine
for GXG, the cytosine-selective response is obtained for
TXT and AXA, and the response is comparable for CXC.
More importantly, the response of DBD moiety becomes
larger than that for GXG, probably due to the difference in
the microenvironment surrounding the DBD moiety. As for
the response to cytosine (2.0 mm), the largest increase in the
intensity by 61-fold is observed for AXA with a maximum
at 565 nm, and the response follows the order of AXA>
TXT (35-fold, 566 nm)>CXC (12-fold, 579 nm)>GXG
(3.6-fold, 585 nm). The response to thymine is the largest
also for AXA (50-fold, 563 nm), and follows the order of
AXA>TXT (17-fold, 567 nm)>CXC (15-fold, 579 nm)>
GXG (7.5-fold, 581 nm). Therefore, as compared with pyri-
midine detection demonstrated for GXG (Figure 3A), sensi-
tivity would be better for the other three sequences. Indeed,
for the analysis of AXA/TNT (Figures 3B and S3 in the
Supporting Information), the LOD for cytosine and thymine
was estimated to be 1.4 and 2.1 nm, respectively, and the ra-
tiometric responses to 500 nm samples can be judged even
with the naked eye—the change in the emission color is
clearly seen for pyrimidine bases under UV irradiation (Fig-
ure 3C).
By keeping such binding properties, ATMND-DBD can
provide ratiometric fluorescence signaling to detect pyrimi-
dine nucleobases (Figures 3A and S3 in the Supporting In-
formation). We assessed the binding-induced change in the
ratio of the two emission intensities (F585/F420), that is, the
emission of DBD moiety at 585 nm (F585) and the emission
of ATMND moiety at 420 nm (F420). In the case of detection
of thymine in the 21-meric DNA duplex, a linear response
of the emission ratio (F585/F420) was obtained in the concen-
tration range from 0 to 500 nm. LOD was estimated to be
16 nm. A linear response was also obtained for cytosine with
LOD of 32 nm. In contrast, the responses are almost negligi-
ble for guanine and adenine in this concentration range.
Therefore, ATMND–DBD will provide analysis of poly-
merase-chain reaction (PCR)-amplification products with
a sufficient sensitivity and selectivity for pyrimidine bases
(see below).
The ratiometric response of ATMND–DBD can be effec-
tively obtained irrespective of the nucleobases flanking the
AP site (N’XN’, N’=G, C, A, or T), and the response is
highly specific to pyrimidine over purine bases (Figure S4 in
These useful ratiometric responses cannot be obtained by
conjugates with a longer linker between ATMND and the
DBD moiety (Figure S5 in the Supporting Information). In
the case of the trimethylene-linker-containing conjugate
(C3), the response of the DBD moiety becomes smaller
compared with that of ATMND–DBD with an ethylene
linker, and the resulting ratiometric response is only moder-
ate even in the presence of 2.0 mm DNA samples (F585/F420
:
T, 1.3Æ0.17; C, 0.8Æ0.01; A, 0.2Æ0.01; G, 0.2Æ0.01; DNA
free, 0.17Æ0.01). In the case of the conjugate with the
longer tetramethylene linker (C4), the ligand has almost no
responses attributable to the DBD moiety. Thus, the longer
linker in the conjugates resulted in the decreased ratiomet-
ric fluorescence response. In addition, the binding affinities
of these conjugates for thymine are one order of magnitude
lower than that of ATMND–DBD (Figure S6 in the Sup-
porting Information; K11/106 mÀ1: C3, 0.53Æ0.03; C4, 0.11Æ
0.02). Apparently, the binding and sensing properties strong-
ly depend on the spacer, and the choice of the linker length
is crucial for the design of this type of conjugates.
It was noted herein that two fluorescent moieties
(ATMND and DBD) can be excited simultaneously to give
the ratiometric response. This can be seen from the change
in the emission color (Figure 3C, excitation at 365 nm), and
also from the whole emission spectra upon excitation at
375 nm, Figure S7 in the Supporting Information). However,
as shown in Figure 2, the excitation at longer wavelength
(451 nm, absorption maximum of DBD moiety) makes the
response of DBD moiety more significant compared with
that obtained by the excitation at shorter wavelength
(375 nm). Thus, in the case of ATMND–DBD, doubled irra-
diation is better way to obtain more effective ratiometric re-
Figure 3. Ratiometric responses of ATMND–DBD (2.0 mm) to detect nu-
cleobases (N=G, C, A, or T) in 21-meric AP-site-containing DNA du-
plexes (5’-GCA GCT CCC N’XN’ GTC TCC TCG-3’/3’-CGT CGA
GGG N’NN’ CAG AGG AGC-5’, X=AP site; SpacerC3, N’=G or A):
A) GXG/CNC; B) AXA/TNT. Sample solutions containing NaCl
(100 mm) and EDTA (1.0 mm, water/ethanol 97.2:2.8) were buffered to
pH 7.0 (10 mm sodium cacodylate). Excitation: ATMND moiety, at
376 nm; DBD moiety, at 451 nm. Analysis: 420 (F420) or 421 (F421), 585
(F585), or 565 nm (F565). T=208C. LOD was estimated from LOD=3
(Sblank/slope). C) Visual detection of nucleobases (500 nm, AXA/TNT,
N=G, C, A, or T) by ATMND–DBD (500 nm). Samples were excited
with a UV lamp at 365 nm and photographed with a digital camera. T=
238C (RT). Other conditions are the same as those for Figure 3A and B.
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sponses and would be not always drawback to the analysis
of in vitro samples.
Keywords: DNA recognition · fluorescent probes · ligand
design · noncovalent interactions · nucleobases
Finally, ATMND–DBD was applied to the analysis of the
single-base mutation present in 107-meric DNAs (K-ras
gene, sense trand, codon 12: GNT, N=G, C, A or T).[14]
After PCR amplification (see the Supporting Information),
an aliquot from the PCR product was analyzed in buffer so-
lution (pH 7.0) containing ATMND–DBD and 20-meric
AP-site-containing probe DNA (Figure S8 in the Supporting
Information). ATMND–DBD showed the all-or-none re-
sponses to pyrimidine over purine bases (F585/F420: T 1.0; C
1.5; A 0.6; G, 0.6; control 0.5) so as to distinguish between
the two types of nucleobases. ATMND–DBD would be
therefore applicable to the analysis of transversion, that is,
replacements of purine for pyrimidine bases (or vice versa),
such as A/C, C/G, A/T, and G/T SNPs. Herein, the analysis
requires no time-consuming steps, such as purification of
PCR products and careful control of temperature, and the
result is readily obtained after PCR.
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In summary, a ratiometric fluorescent signaling probe,
ATMND–DBD, for SNP genotyping was successfully devel-
oped. In contrast to conventional approaches based on
FRET or excimer formations in hybridization probes, the
design of small ligand-based probes with the environmental-
ly sensitive fluorescent dye was demonstrated. The ratiomet-
ric signal was indeed effective, and the strategy would be
sufficiently flexible for further design of this class of probes
to detect all possible mutations. Also, because the naphthyr-
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Experimental Section
Synthesis of ATMND–DBD: DBD-F (4-(N,N-Dimethylaminosulfonyl)-7-
fluoro-2,1,3-benzoxadiazole, 50.3 mg, 0.205 mmol) and triethylamine
(2 mL) were added to a solution of 3 (50.1 mg, 0.205 mmol) in DMF
(8 mL). The reaction mixture was stirred under nitrogen gas at RT for
24 h and then concentrated in vacuo. The crude product was purified by
FPLC (CHCl3/MeOH) on a NH2-modified silica gel with the gradient
elution system to give ATMND–DBD (33.6 mg, 0.0737 mmol, 36%).
1H NMR (500 MHz, CDCl3): d=2.36 (s, 3H), 2.52 (s, 3H), 2.71 (s, 3H),
2.84 (s, 6H), 3.77 (t, 2H, J=5.5 Hz), 4.06 (t, 2H, J=5.5 Hz), 6.26 (d, 1H,
J=8.0 Hz), 6.62 (d, 1H, J=9.0 Hz), 7.86 (d, 1H, J=8.0 Hz), 8.03 ppm (d,
1H, J=9.0 Hz); HRMS (ESI) for calcd for C21H26N7O3S: 456.1818
[M+H]+; found: 456.1812.
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Acknowledgements
This work was supported by Grant-in-Aid for Scientific Research (S),
No. 22225003, from the Ministry of Education, Culture, Sports, Science
and Technology, Japan. Y.S. acknowledges supports by Grant-in-Aid for
Young Scientists (Start-up), No. 22850001.
Received: January 20, 2012
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!

Ratiometric Fluorescent Signaling of Small Molecule
COMMUNICATION
Bind it! A ratiometric fluorescent
probe based on conjugation of a DNA-
binding ligand with an environmentally
sensitive dye to probe hydrophobic
grooves of the DNA helix was devel-
oped. The resulting conjugate
ATMND–DBD can bind to an orphan
nucleobase opposite an abasic (AP)
site with high selectivity and affinity
for pyrimidine bases (see figure).
Fluorescent Probes
C.-x. Wang, Y. Sato, M. Kudo,
S. Nishizawa, N. Teramae* . &&&& — &&&&
Ratiometric Fluorescent Signaling of
Small Molecule Environmentally
Sensitive Dye Conjugate for Detecting
Single-Base Mutations in DNA
Chem. Eur. J. 2012, 00, 0 – 0
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