A novel fluorescent sensor for triplex DNA

Article abstract of DOI:10.1016/j.bmcl.2004.11.002

A novel triplex DNA fluorescent sensor which takes 4-aminonaphthalimide as a reporting group and a triplex-select intercalator as a recognizing group has been synthesized. The results show that the fluorescence of the sensor increases greatly upon addition of T?AT triplex, but the response to single strand DNA and duplex DNA is weak in PIPES 20 buffer (pH 7.0). A triplex DNA fluorescent sensor based on PET is described. The sensor takes 4-aminonaphthalimide as a reporting group and a triplex-select intercalator as a recognizing group. The results show that it is a selective sensor for T?AT triplex DNA in compared with duplex and ssDNA by fluorescence enhancement in PIPES 20 buffer.

Full text of DOI:10.1016/j.bmcl.2004.11.002

Bioorganic & Medicinal Chemistry Letters 15 (2005) 255–257
A novel fluorescent sensor for triplex DNA
Erhu Lu, Xiaojun Peng,* Fengling Song and Jiangli Fan
State Key Laboratory of Fine Chemicals, Dalian University of Technology, 158 Zhongshan Road, Dalian 116012, China
Received 19 September 2004; revised 31 October 2004; accepted 1 November 2004
Available online 26 November 2004
Abstract—A triplex DNA fluorescent sensor based on PET is described. The sensor takes 4-aminonaphthalimide as a reporting
group and a triplex-select intercalator as a recognizing group. The results show that it is a selective sensor for TÆAT triplex
DNA in compared with duplex and ssDNA by fluorescence enhancement in PIPES 20buffer.
Ó 2004 Elsevier Ltd. All rights reserved.
At present, many advances are being made in the under-
standing of the formation and the recognition of DNA
triple helices which should help the development of the
great potential in a variety of analytical and therapeutic
applications of triple helix forming oligonucleotides
(TFOs).1–6 Recognition and detection of triple helix
are of special importance for useful therapeutic agents
as modulators of gene expression in the ꢀantigeneꢁ thera-
peutic strategy.7–10 Therefore, there is a need to devel-
op new selective and sensitive methods for triple helix.
Of all detected signals, fluorescence is the favored sig-
naling technology for molecular diagnoses on account
of its simplicity and high sensitivity. Fluorescence assay
such as molecular beacon that is covalently linked in tri-
plex has been used to study the structure and thermody-
namic characterization of the triplex DNA.11,12 On the
other hand, there is few fluorescence sensor which is
selective for triplex DNA in comparison with other
structural forms of DNA, such as single and double
stranded DNA. These observations prompted us to de-
velop a new fluorescent biosensor for triplex structure
DNA.
In this paper, we synthesized a new triplex-selective flu-
orescent sensor 3 (Scheme 1). The 4-aminonaphtha-
limide was chosen as fluorescent-monitoring unit
which has been used in many studies such as sensors,
special interactions with some biomolecules (DNA,
enzymes, etc.) because of its outstanding spectroscopic
properties.13–18 Ethylene group as a spacer linked with
4-aminonaphthalimide could be used to build highly
sensitive photoinduced electron transfer (PET) sen-
sors.19–21 2-(2-Naphthyl)quinoline moiety, whose deriv-
atives are proved highly selective triplex intercalators
O
NH2
CH2
CH2
HN
N
CH3
CH2
CH2
NH
O
Cl
NH
(a)
(b)
N
N
N
3
2
1
Scheme 1. Reaction conditions: (a) SnCl4, ethylenediamine, 135°C, 4h, 85%; (b) C6H5N, N-methyl-4-bromo-1,8-naphthalimide, reflux 10h, then
chromatography separation (CH2Cl2:MeOH: 20:1), 70%.
Keywords: Biosensor; Triplex DNA; Fluorescent.
*
Corresponding author. Tel.: +86 411 88993899; fax: +86 411 84542668; e-mail: pengxj@dlut.edu.cn
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.002

256
E. Lu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 255–257
for TÆAT triplex,22–24 was used as triplex receptor. The
results showed that 3 displayed a highly selective re-
sponse of fluorescence enhancement in the presence of
TÆAT triplex in PIPES 20(pH 7.0) buffer.
14
12
10
8
Compound 3 was obtained in two steps. Compound 1,
prepared according to literature,25 was used as a starting
material. A nucleophilic displacement of chloride from 1
by the reaction with ethylenediamine in the presence of
a catalytic amount of SnCl4 furnished 2. N-Methyl-4-
bromo-1,8-naphthalimide was readily prepared from 4-
bromo-1,8-naphthalimide and 33% aq methylamine by
stirring at room temperature for 2.5h. Then it was
refluxed with 2 in pyridine solution to afford 3, which
was characterized by 1H NMR spectroscopy, and
high resolution mass spectrometry.26 The kmax(ab) and
kmax(em) of 3 are 457nm and 538nm, respectively, in
PIPES 20buffer (10mM piperazine- N,N0-bis(2-ethane-
sulfonic acid), 1mM EDTA, and 200mM NaCl ad-
justed to pH7.0). The fluorescent emission spectrum
was recorded on a spectrometer with excitation at
457nm.
TAT
6
4
2
0
500
550
600
650
Wavelength (nm)
Figure 1. Emission spectra (excitation at 457nm) of 3 (1lM) in the
presence of various concentrations of TÆAT ranging from 0, 1, 5, 10,
20, 40, 60, 80, 100lM. These spectra were measured in pH7.0PIPES
20buffer.
2-(2-Naphthyl)quinoline derivatives are highly selective
triplex intercalators for TÆAT triplex which is widely
used in studies of ligand–triplex interactions, and is
one of the best characterized triple helix structures.24
Thus, TÆAT was chosen as our target triplex structure.
Calf thymus, purchased from Sigma, was chosen as a
mixed sequence duplex at hand for comparison purpose.
Nucleic acid concentration was expressed in terms of the
monomeric unit for each polymer, that is, nucleotides
for single strands, base pairs for duplex forms and tri-
plets for the triplex.
6
5
4
3
2
1
0
The nucleic acids oligomers, dA19 and dT19, were pur-
chased from Takara, and dissolved in PIPES 20buffer.
The concentration of dA19 and dT19 were determined
spectrophotometrically according to the reported extinc-
tion coefficients (L/mol of base)/cm, at 260nm and
25°C) for dA19 (9000) and dT19 (8200), respectively.27
TÆAT was prepared by mixing 1:2 molar ratio of dA19
and dT19, and heating the mixture in a water bath to
90°C then slowly cooling to 5°C to form the final sam-
ple before use according to literatures.22,27 AT was
prepared by mixing 1:1 molar ratio of dA19 and dT19
with the same method. All of the fluorescence experi-
ments were carried out with the samples in PIPES 20
buffer.
none
dA19
CT DNA
nucleic acid sample
AT
TAT
Figure 2. Fluorescence response of 1lM 3 to nucleic acid samples
(50lM) in PIPES 20buffer (pH7.0). The temperature was 5 °C.
To clarify the selectivity of fluorescence enhancement of
3 toward TÆAT triplex, several kinds of DNA with differ-
ent structures were studied. As shown in Figure 2, the
addition of ssDNA (dA19, 50lM) to the solution of 3
has little increase in fluorescence emission. Addition of
AT (50lM) or calf thymus (50lM) duplex shows a
modest increase in fluorescence emission. However, the
significant fluorescence enhancement was observed upon
adding 50lM TÆAT, suggesting that compound 3 is a
triplex-selective DNA sensor.
To ensure that the TÆAT remains in its triplex structure,
we investigated the fluorescent response of 3 to DNA
triplex at 5°C. As expected, 3 showed a very clear fluo-
rescence enhancement by the addition of TÆAT triplex.
When the intercalation of 2-(2-naphthyl)quinoline moi-
ety into the bases of TÆAT triplex, hydrogen bond be-
tween the electron pair of the N atom of amino-
quinoline and the triplex bases or p stacking between
the naphthylquinoline and triplex bases or both might
cause the decrease of PET to the fluorophore, which re-
sults in the fluorescence enhancement. There was about
6-fold fluorescence enhancement, when 1 was 1.0lM
and TÆAT was 60lM (Fig. 1).
Fluorescence recovery is often occurred when a PET
sensor combines with a proton. As shown in Figure 3,
the fluorescence intensity is high in the low pH, this is
due to the protonation of N atom of amino-quinoline
and quinoline. Therefore, the normal fluorescent
quenching pathway by PET is blocked. The pKa of 3

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2
4
6
pH
8
10
Figure 3. Effect of pH on the fluorescence intensity of 1lM 3
(excitation at 457nm, emission from 470to 680nm) at 25 °C.
is about 5.4 obtained from the fluorescence titration
curve (Fig. 3). The fluorescence intensity of 3 decreases
along with the increase of pH value, but when
pH > 6.5, the fluorescence intensity changes very little
in PIPES 20buffer. This pH range might be helpful to
avoid the interference of possible pH change induced
by biological stimulation.28
In summary, a simple fluorescent biosensor 3 for recog-
nition of TÆAT was designed and synthesized, and it dis-
plays selectivity for TÆAT triplex by fluorescence
enhancement when pH > 6.5.
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A. N.; Kiselyov, A. S.; Lin, S. Y.; Tanious, F. A.; Wilson,
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Acknowledgements
This work was supported by ꢀ973 programꢁ of the Min-
istry of Science and Technology of China and National
Science Foundation of China (project 20128005,
20376010 and 20472012).
26. mp = 233–234°C; 1H NMR (DMSO-d6,400MHz) d 3.22–
3.24 (d, J = 6.0Hz, 2H), 3.34 (s, 3H), 3.53–3.54 (d,
J = 4.2Hz, 2H), 7.03 (s, 1H), 7.08–7.10 (d, J = 7.6Hz,
1H), 7.36–7.38, (d, J = 8.0Hz, 1H), 7.41–7.43 (m, 1H),
7.45–7.46 (m, 1H), 7.48–7.50(m, 1H), 7.52–7.55 (m, 1H),
7.60–7.64, (t, J = 7.6Hz, 1H), 7.91–7.93 (t, J = 8.0Hz,
1H), 7.96–7.98 (d, J = 8.0Hz, 1H), 8.00–8.04, (t,
J = 8.4Hz, 1H), 8.16–8.17, (m, 2H), 8.32–8.34, (m, 2H),
8.38–8.40, (d, J = 8.4Hz, 1H), 8.63, (s, 1H) ppm; HRMS
(ESI) calcd for [M + H]+ 523.2134, found 523.2131.
27. Hopkins, H. P.; Hamilton, D. D.; Wilson, W. D.; Zon, G.
J. Phys. Chem. 1993, 97, 6555.
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