G-quadruplex DNA fluorescence sensing by a bis-amine-substituted styrylquinolinium dye

Article abstract of DOI:10.1016/j.dyepig.2017.05.045

Searching for specific G-quadruplex DNA probes is important for study of the function of G-rich gene sequence, as well as design of novel effective anticancer drugs. In this paper, a novel bis-amine-substituted styrylquinolinium dye (BSAQ) was designed and synthesized to enhance the performance for the application as a G-quadruplex DNA probe. The studies on BSAQ with different DNA forms showed that it could be used as a colorimetric and red-emitting ?uorescent probe for G-quadruplex DNA. The limits of detection of BASQ with various G-quadruplex DNAs were found to below 1 nM. CD spectroscopy analysis revealed that BSAQ did not induce the G-rich sequence folding into G-quadruplex structure. These results of this study gave some crucial factors on developing of effective probes for G-quadrupex DNA applications.

Full text of DOI:10.1016/j.dyepig.2017.05.045

Accepted Manuscript
G-quadruplex DNA fluorescence sensing by a bis-amine-substituted styrylquinolinium
dye
Ming-Qi Wang, Yuan Wu, Zi-Yu Wang, Qiu-Yun Chen, Fu-Yan Xiao, Yu-Chi Jiang,
Amy Sang
PII:
S0143-7208(17)30573-9
10.1016/j.dyepig.2017.05.045
DYPI 6007
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 21 March 2017
Revised Date: 13 May 2017
Accepted Date: 23 May 2017
Please cite this article as: Wang M-Q, Wu Y, Wang Z-Y, Chen Q-Y, Xiao F-Y, Jiang Y-C, Sang A, G-
quadruplex DNA fluorescence sensing by a bis-amine-substituted styrylquinolinium dye, Dyes and
Pigments (2017), doi: 10.1016/j.dyepig.2017.05.045.
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ACCEPTED MANUSCRIPT

G-quadruplex DNA fluorescence sensing by a bis-amine-substituted
ACCEPTED MANUSCRIPT
styrylquinolinium dye
a*
a
a
b,**
a
a
a
Ming-Qi Wang , Yuan Wu , Zi-Yu Wang , Qiu-Yun Chen , Fu-Yan Xiao , Yu-Chi, Jiang , Amy Sang
a
School of Pharmacy, Jiangsu University, Zhenjiang, 212013, P. R. China
b
School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, 212013, P. R. China
Abstract: Searching for specific G-quadruplex DNA probes is important for study of the function of G-rich gene sequence,
as well as design of novel effective anticancer drugs. In this paper, a novel bis-amine-substituted styrylquinolinium dye
(BSAQ) was designed and synthesized to enhance the performance for the application as a G-quadruplex DNA probe. The
studies on BSAQ with different DNA forms showed that it could be used as a colorimetric and red-emitting fluorescent
probe for G-quadruplex DNA. The limits of detection of BASQ with various G-quadruplex DNAs were found to below 1
nM. CD spectroscopy analysis revealed that BSAQ did not induce the G-rich sequence folding into G-quadruplex structure.
These results of this study gave some crucial factors on developing of effective probes for G-quadrupex DNA applications.
Key words: G-quadruplex DNA; probe; Bis-amine-substituted; Styrylquinolinium dye
1
. Introduction
DNAs play essential roles in the storage and transfer of genetic information, which is essential for the living organisms
[1]. Guanine-rich DNA sequences can form non-canonical structures known as G-quadruplexes have attracted intense
scrutiny [2-4]. The basic unit of a G-quadruplex structure is the guanine tetrad that is derived from the association of four
guanines into a cyclic Hoogsteen hydrogen bonding planar arrangement. Stacking of several G-tetrads leads to form a
G-quadruplex motif, which can be stabilized by chelation of monovalent cations [5,6]. Computational analyses of the
human DNA has revealed an abundance of putative G-quadruplex-forming sequences in the human genome. For example,
G-quadruplex-forming sequences are found at the ends of the telomeres, ribosomal DNA, as well as in the important regions
of oncogene promoters [7-9]. Accumulating evidence has linked G-quadruplex structures to a number of biological
processes in vivo, including DNA replication, transcription, and genomic maintenance [10-13]. However, the precise
function and mechanism of G-quadruplex formation in mammalian cells remains poorly defined. The research of
G-quadruplex is still at an early stage. Therefore, the development of rapid and simple approaches for detection of
G-quadruplex DNA structures has attracted significant attention in recent years.
Notably, visualizing G-quadruplex DNA using small-molecule probes is an extremely active area and significant progress
has been made towards the development of colorimetric or fluorescent probes [14-26]. Such as the widely studied dyes
based on cyanine, thiazole, porphyrin, pyrene and carbazole. Most of them focused on the improvement of the probes’

selectivity for G-quadruplex DNA. However, an ideal G-quadruplex probe should display several key features, including
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strong photostability and chemical stability, an intense emission enhancement in the presence of G-quadruplexes, excellent
discrimination for G-quadruplex DNA over other forms of DNA, and low detection limit.
Ionic styryl dyes represent an important class of function dyes that have a number of favorable properties. They are
fluorescent, have high photostability and synthetically easily available [27,28]. Some of them exhibited remarkably high
affinities toward double-stranded DNA along with significant changes of their photo-physical properties [29,30]. Recently,
our group has designed and synthesized several triphenylamine derivatives that exhibited good selectivity for G-quadruplex
DNAs [31]. These derivatives contain an amine side chain, which is considered crucial for G-quadruplex DNA sensing. In
order to enhance the selectivity and sensitivity toward G-quadruplex DNAs of the ionic styryl dyes, we attempted to
assemble styrylquinolinium and amine side group in a fusion scaffold. Herein, we reported a new bis-amine-substituted
styrylquinolinium dye (BASQ), which was synthesized by a simple and rapid process. The detailed interactions of BASQ
with G-quadruplex DNAs were investigated.
2
. Experimental methods
2
.1. Oligonucleotides
All oligonucleotides (HPLC purified) were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China) and the
sequences were listed in Table S1. Oligonucleotides were dissolved in 10 mM Tris-HCl buffer (containing 60 mM KCl, pH
o
7
.4). Prior to use, all oligonucleotides were pre-treated by heating at 95 C for 5 min, followed by gradual cooling to room
temperature and kept at this temperature for 30 min. Binding experiments were performed in the same buffers as used to
dissolve the oligonucleotides.
2
.2. Synthesis and characterization
1
13
H NMR and C NMR spectra were measured on a 400 MHz spectrometer using TMS as internal standard in CDCl or
3
DMSO-d . Mass spectra (MS) were recorded on a Shimazu LCMS-2010A instrument with an ESI detector.
6
4
-Fluorobenzaldehyde, N-methyl piperazine and 4-hydroxy-2-methylquinoline were purchased from Beijing InnoChem
Science & Technology Co., Ltd. All common solvents and chemical reagents were used as received without further
purification. Stock solutions of BASQ (5 mM) were prepared in DMSO.
2
.1.1. Synthesis and characterization of intermediate 1
-Fluorobenzaldehyde (400 mg, 3.22 mmol), N-methyl piperazine (322 mg, 3.22 mmol) and K CO (469 mg, 3.40 mmol)
4
2
3
were stirred in dry DMF at 130 °C under nitrogen atmosphere. After 12 h, the reaction was quenched by addition of water.
The resulting mixture was extracted with EtOAc (3 × 50 mL), washed with brine, and dried over anhydrous Na SO . The
2
4
1
resulting residue was purified by flash chromatography (PE/EtOAc 1:4). Yield 65%, white solid. H NMR (400 MHz,

CDCl ) δ 9.78 (s, 1H), 7.76 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 3.46-3.37 (m, 4H), 2.59-2.54 (m, 4H), 2.36 (s, 3H).
3
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+
LC-MS: (positive mode, m/z) calculated 205.1431, found 205.1321 for [M+H] .
2
.1.2. Synthesis and characterization of BASQ
A solution of intermediate 1 (320 mg, 1.59 mmol), 1,2-dimethyl-4-chloroquinolin-1-ium iodode (3, 507 mg, 1.59 mmol)
and N-methyl piperazine (200 mg, 2.0 mmol) in anhydrous ethanol (30 mL) was refluxed for 24 h under nitrogen with
stirring. After cooling to room temperature, the solvent was evaporated under reduced pressure. The residue was purified by
column chromatography on silica gel eluting with CH Cl /CH OH (20:1, v/v) to afford BASQ (0.35 g, 38.7 %) as a red
2
2
3
1
solid. H NMR (400MHz, DMSO-d ) δ: 8.24 (d, J = 8.28 Hz, 1H), 8.09 (d, J = 7.84 Hz, 1H), 7.99-7.90 (m, 2H), 7.78 (d, J =
6
7
2
1
4
.32 Hz, 2H), 7.72 (t, J=6.6 Hz, 1H), 7.47 (s, 1H), 7.43 (s, 1H), 7.01 (d, J = 7.08 Hz, 2H), 4.19 (s, 3H), 3.75 (br, 8H),
1
3
.79-2.73 (m, 8H), 2.48 (s, 3H), 2.37 (s, 3H); C NMR (100 MHz, DMSO-d ) δ: 158.86, 154.35, 151.67, 143.76, 140.54,
6
33.53, 130.35, 126.31, 126.14, 125.15, 119.62, 119.11, 115.02, 114.31, 104.86, 53.89, 53.42, 53.36, 51.12, 45.69, 45.04,
+
4.24; LC-MS: (positive mode, m/z) calculated 442.2965, found 442.2955 for [M-I] .
2
2
.3. Measurements and methodology
.3.1. UV-vis spectral studies
The UV-vis spectra were recorded on an UV-2550 spectrophotometer using a 1 cm path length quartz cuvette at room
temperature. For the titration experiments, small aliquots of a stock solution of the DNAs were added to the solution
containing BASQ at a fixed concentration (8 ꢀM) in aqueous buffer (10 mM Tris-HCl, pH 7.4, 60 mM KCl). After each
DNA addition, the solution was incubated for 1 min before absorption spectra recorded.
2
.3.2. Fluorimetric titrations
Fluorescence spectra were measured on a Shimadzu RF-5301PCS spectrofluorophotometer in a 10 mm quartz cell at
room temperature. The concentration of BASQ was fixed at 2 ꢀM and the titration process was similar to absorption spectra
titration experiment. Both excitation and emission slits were set at 10 nm. The fluorescence measurement was obtained at an
excitation wavelength of 470 nm. The date from the fluorimetric titrations were analyzed according to the independent-site
model by nonlinear fitting to equation (1) [32], in which F is the fluorescence intensity of BSAQ in the absence of
0
G-quadruplex DNA, n is the putative number of BASQ molecules binding to a given DNA matrix, Q is the fluorescence
enhancement upon saturation, A = 1/[K C ] and x = nC /C . The parameters Q and A were found by
a
dye
DNA dye
Levenberg-Marquardt fitting routine in Origin 8.5 software.
ꢀ
ꢂ − 1
2
ꢉ
=
1 +
ꢃꢄ + 1 + ꢅ − ꢆꢇꢄ + 1 + ꢅꢈ − 4ꢅꢊꢋꢋꢋꢋꢋꢋꢋꢋꢋꢋꢋꢋꢋꢋꢇ1ꢈ
ꢀꢁ
2
.3.3. FID assay
The experiment was carried out in 10 mM Tris-HCl buffer (containing 60 mM KCl, pH 7.4) by adding different
concentrations of BASQ to the TO-bound Htg-21 solution. The concentrations of TO and Htg-21 were set at 0.5 and
.1 ꢀM, respectively. BSAQ was added until no change was observed in the fluorescence intensity indicating the
0

binding saturation has been achieved. The fluorescence spectra were measured using excitation wavelength at 504 nm
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and the emission range was set between 515 and 700 nm.
2
.3.4 Circular dichroism (CD)
CD spectra (230-400 nm) were performed on a JASCO-J815 circular dichroism spectrophotometer using a 10 mm path
-1
length quartz cuvette. The scanning speed of the instrument was set to 500 nm min . The strand concentration of
oligonucleotide Htg-21 used for measurement was 5 ꢀM. The buffer condition is 10 mM Tris-HCl, pH 7.4 in the
presence/absence of 60 mM KCl. Final analysis of the data was carried out using Origin 8.5.
3
. Results and discussion
3
.1 Synthesisi of the bis-amine-substituted styrylquinolinium dye BASQ
The desired dye, BASQ, was synthesized following the procedure reported previously by our group [31]. As shown in
Scheme 1, intermediate 1 was obtained by the reaction of p-fluorobenzaldehyde with N-methyl piperazine. This compound
was followed by reaction with 1,2-dimethyl-4-chloroquinolin-1-ium iodode, N-methyl piperazine in one-pot to give the final
1
13
dye BASQ. The structure of this new dye was characterized by H NMR, C NMR and MS (see the Supplementary
Information).
<
Scheme 1. Synthetic route for the synthesis of BASQ.>
3
.2 Fluorescence spectroscopic studies of BASQ upon interaction with nucleic acids
To investigate the interactions of BASQ with G-quadruplex DNAs, fluorescence titrations in 10 mM Tris-HCl buffer at
pH 7.4 were first performed. The fluorescence emission band of BASQ (2 ꢀM) at 514 nm is very weak at room temperature
Fig.1). Upon gradual addition of human telomeric G-quadruplex DNA Htg-21, an emission peak at approximately 587 nm
(
appeared and was significantly enhanced and eventually reached to ~240 fold at the concentration as low as 0.68 ꢀM. We
speculated that the interaction between BASQ and Htg-21 may restrict the rotation of the methine-bridge of BASQ, as
shown by the emission enhancement in a viscous medium (Fig. S1), therefore enhancing the radiative pathway relaxation
and fluorescence emission [33]. This fluorescent turn-on properties were also observed when treated with other
G-quadruplex DNAs (22AG, CM22, C-myc, G3T3, Hras and Ckit1) (Fig.1B and Fig. S2). The changes in fluorescence
intensities could even be detected by the naked eye under UV light (Fig. S3). It is worth pointing out that the specific red
emission of BASQ with G-quadruplex DNA is very attractive for cellular imaging experiments, as low photo-damage, and
minimal fluorescence background can be achieved [34]. In contrast, much smaller changes were observed with
single-stranded DNA (ss26), double-stranded DNA (ctDNA) and the protein BSA. These results suggest that BASQ exhibits
a selectively fluorescence response toward G-quadruplex DNAs.

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<
Fig.1. (A) Fluorescence spectra of 2 µM BASQ titrated with Htg-21 in 10 mM Tris-HCl buffer, 60 mM KCl, pH 7.4; Arrow: concentrations of
Htg-21 ranged from 0 to 0.68 µM. (B) Distribution of the values of fluorescence intensity at 587 nm for BASQ (2 µM) with all the tested samples
0.68 µM). Error bars represent the standard deviations of the results from three independent experiments.>
(
3
.3 UV-vis spectroscopic studies of BASQ upon interaction with nucleic acids
The absorption properties of BASQ with nucleic acids were explored by UV-vis spectroscopic titrations. Electronic
absorbance spectroscopy is one of the most useful tools to investigate the interactions of dyes with DNA. As shown in Fig.
A, upon addition of Htg-21, the intensity of 377 nm gradually decreased, and a large redshift of the maximum was
2
observed with an obvious isosbestic point at 433 nm, which finally led to a new peak at about 456 nm. Other tested
G-quadruplex DNAs could also clearly enhance the absorbance of BASQ at 456 nm (Fig. S4). It has been reported that
intercalative mode of binding between compound and DNA usually results in hypochromism and red shift due to the strong
stacking interactions [35]. The large red shift and hypochromism between BASQ and G-quadruplexes parallels intercalative
binding mode. In general, quinolinium moiety may interact with G-quartet plane by π-π stacking. The effects of duplex and
single-stranded DNAs on the absorption bands of BASQ were much weaker than the G-quadruplex DNAs, which only
caused a hypochromic effect at 433 nm. Thus, the value of A456 nm/A377 nm could be used as a criterion to discriminate
between G-quadruplexes and other DNA forms. At the concentration of 2.4 ꢀM DNA, The values of A456 nm/A377 nm of all
tested G-quadruplexes were greater than 1.0. In contrast, the values of other DNAs were less than 0.75 (Fig. 2B).
Accordingly, the changes in absorption spectra were in good agreement with that of fluorescence titration experiments. The
above results indicate that BASQ could also be used as a colorimetric probe to distinguish G-quadruplexes from other DNA
forms.
<
Fig.2. (A) Absorption spectra of 8 µM BASQ titrated with Htg-21 in 10 mM Tris-HCl buffer, pH 7.4, 60 mM KCl; Arrows: concentrations of
Htg-21 ranged from 0 to 2 µM. (B) Plots of A456 nm/A377 nm of 8 µM BASQ against the DNA concentrations.>
3
.4. The detection limits of BASQ with G-quadruplex DNAs
For an ideal G-quadruplex DNA probe, the selectivity and sensitivity are both important criteria. Therefore, we measured
the detection limits (LODs) of BASQ toward DNA G-quadruplexes by means of spectrofluorimetric titrations. The LOD
values were calculated according to the equation 3σ/k [36]. The σ value represents the standard deviation for multiple
measurements of blank solution. The k value is the slope derived from the linear range of the BASQ fluorescence titration
curve with different G-quadruplex DNAs. The linear ranges of the fluorescence titration curves for BASQ with most of
G-quadruplex DNAs ranged from 0-400 nM. Thus, the k values were obtained by fitting these curves and the corresponding
values of the LODs were shown in Table 1 and Fig. S5. Clearly, the LODs of BASQ for different G-quadruplex DNAs in

solution were below 1 nM, which meant it was able to detect G-quadruplex DNAs in the nanomolar range. It is also
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noteworthy that the LOD values of BASQ are significant improved in comparison with mono-amine-substituted
quinolinium dye reported previously [31], which has a detection limit of 158 nM. Accordingly, the improving detection
limits of BASQ for G-quadruplex DNA revealed the importance of the amine chain. The date underline that BASQ shows
high sensitivity toward G-quadruplex DNAs.
<
Table 1 The detection limits and linear ranges of BASQ (2 µM) for different G-quadruplex DNAs in solution.>
3
.5. Binding properties of BASQ with G-quadruplex DNAs
In an effort to understand the details of binding process, we evaluated the binding stoichiometries and intrinsic constants
(
K ) between BASQ and individual G-quadruplexes. The fluorescence titration curves were further fitted to an
a
independent-site model (Experimental methods). The results are summarized in Table 2. Analyses of the relationship
between the fluorescence intensity of BASQ and the concentrations of DNA G-quadruplexes afforded binding constants in
6
-1
the range from 0.10-3.36 × 10 M . However, the binding affinities and fluorescence enhancements are not directly
correlated, due to the different microenvironments in the binding sites, which avoid the nonradiative relaxation process to
different extents. The results were further confirmed by Job plot experiments (Fig. S6). To gain further insight into the
binding mode between BASQ and G-quadruplex, the dye affinity for Htg-21 was tested by an indirect FID (fluorescence
intercalator displacement) approach [37]. This assay measures the decrease in fluorescence intensity upon the displacement
of thiazole-orange (TO) from the G-quadruplex-TO complex by the dye. TO is highly fluorescent upon interaction with
G-quadruplex DNA mainly by an π-π stacking mode whereas totally quenched when free in solution. Efficiency TO
displacement would thus be an indication of an same binding mode of our dye. As shown in Fig S7, BASQ could partially
displace (41%) TO from Htg-21, indicating the fact that quinolinium moiety of BASQ can only undergo partial stacking
interactions with G-quadruplex DNA.
<
Table 2. Binding stoichiometries and intrinsic binding constants (K ) of BASQ in the presence of different G-quadruplex DNAs.>
a
3
.6. Effect of cationic species of Htg-21 on the interactions with BASQ
Since the G-rich sequence is prone to fold into G-quadruplex structure in the presence of metal ions. And cationic ion
plays an important role in determining the secondary structures [5,6]. To explore this, the fluorescence behaviors of BASQ
+
with Htg-21 in the different concentrations of K ion were investigated. The finding showed that fluorescence intensities of
+
BASQ strongly depended on the concentrations of K ion, with maximal value occurring near 5 mM. Above this

concentration, however, the maximum fluorescence intensity was found to show a regular decrease, perhaps because the
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high concentration of K disrupted the docking between the Htg-21 and BASQ. Based on the G-quadruplex ligands reported
+
previously, we have known that the positively charged or protonable amine side group would enhance electrostatic
interaction strength with the negatively charged DNA phosphate backbone. The strong dependence of the fluorescence
intensities on the ionic strength of the buffer indicates that the electrostatic interactions play an important role in the binding
process.
+
<
Fig.3. (A) Fluorescence intensities of BASQ (2 µM) at 587 nm with different amounts of Htg-21 in the various K buffers (1-100 mM). (B)
+
Fluorescence intensities of BASQ (2 µM) at 587 nm with Htg-21 (0.32 µM) in the 1-100 mM K buffers.>
3
.7. CD studies
To investigated whether BASQ has the G-quadruplex induced ability with Htg-21 which can not spontaneously fold into
+
G-quadruplex in the absence of metal ion K , the effects of BASQ on the conformation of G-quadruplex Htg-21 in the
+
absence and presence of K ion were studied by using circular dichroism (CD), which is an extremely useful technique to
study nucleic acids conformation. The typical CD spectrum of Htg-21 is a hybrid-type of quadruplex DNA containing
+
parallel and anti-parallel structure in the presence of K ion, with a large positive band at about 293 nm, a shoulder around
2
72 nm, a small positive band at 251 nm, and a minor negative band near 234 nm [6,38,39]. Upon gradual addition of
BASQ to this solution, a maximum band at 292 nm increased and the shoulder at 272 nm decreased and merged into the
band at 292 nm. Meanwhile, the small positive band at 251 nm decreased. These changes indicated that the dye BASQ did
not cause significant conformation transition of Htg-21 G-quadruplex structure. However, the enhancement of the positive
peak may be attributed to the stabilization of G-quadruplex structure by this dye. In order to explore whether BASQ can
induce the formation of G-quadruplex structure or not in the absence of salt, the CD experiment was carried out in the
+
K -free buffer. In this case, the Htg-21 alone displayed a characteristic CD band at 255 nm and 297 nm, conforming its
unfold state (Fig. 4B). As the concentration of BASQ increased, there were still two characteristic positive CD band at 255
nm and 297 nm, confirming its unfolded state, indicating that BASQ can not induce the structure exchange from unfolded
state to quadruplex state.
<
Fig. 4. CD titrations spectra of BASQ for Htg-21 (4 µM) in 10 mM Tris-HCl buffer (pH 7.4) in the presence (A) and absence (B) of 60 mM KCl.
The concentration of BASQ is from 0 to 8 µM. Arrows indicate the change direction of peaks.>
4
. Conclusions
In conclusion, a bis-amine-substituted styryl dye, BASQ, has been designed and synthesized as a G-quadruplex DNA

targeting probe. BASQ was found to express large red shifts of its absorption spectra and significant fluorescence
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enhancements upon binding with G-quadruplex DNAs, while showed insignificant changes upon interactions with
non-quadruplex structures. In further studies, BASQ exhibited highly sensitive fluorescence detection of G-quadruplex
DNAs with the LOD values below 1 nM in solution. The CD spectra titrations results revealed that BASQ could not convert
G-rich sequence into G-quadruplex structure. All these remarkable properties of BASQ suggested it should have promising
applications in the field of G-quadruplex DNA research, and provide useful information for future studies.
Authors Informations
*
*
Corresponding author.
* Corresponding author.
E-mail addresses: wmq3415@163.com (M.-Q. Wang), chenqy@ujs.edu.cn
Acknowledgements
This work was financially supported by the China Postdoctoral Foundation (Grant No. 2017M611704), Senior Talent
Cultivation Program of Jiangsu University (Nos. 15JDG068), National Natural Science Foundation of China (21571085).
Appendix A. Supplementary date
Supplementary date related to this article can be found at XXXXXX.
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Captions：
ACCEPTED MANUSCRIPT
Scheme 1 Synthetic route for the synthesis of BASQ.
Table 1 The detection limits and linear ranges of BASQ (2 ꢀM) for different G-quadruplex DNAs in solution.
Table 2 Binding stoichiometries and intrinsic binding constants (K ) of BASQ in the presence of different G-quadruplex
a
DNAs.
Fig.1. (A) Fluorescence spectra of 2 ꢀM BASQ titrated with Htg-21 in 10 mM Tris-HCl buffer, 60 mM KCl, pH 7.4; Arrow:
concentrations of Htg-21 ranged from 0 to 0.68 ꢀM. (B) Distribution of the values of fluorescence intensity at 587 nm for
BASQ (2 ꢀM) with all the tested samples (0.68 ꢀM). Error bars represent the standard deviations of the results from three
independent experiments.
Fig. 2. (A) Absorption spectra of 8 ꢀM BASQ titrated with Htg-21 in 10 mM Tris-HCl buffer, pH 7.4, 60 mM KCl; Arrows:
concentrations of Htg-21 ranged from 0 to 2 ꢀM. (B) Plots of A456 nm/A377 nm of 8 ꢀM BASQ against the DNA
concentrations.
+
Fig. 3. (A) Fluorescence intensities of BASQ (2 ꢀM) at 587 nm with different amounts of Htg-21 in the various K buffers
+
(
1-100 mM). (B) Fluorescence intensities of BASQ (2 ꢀM) at 587 nm with Htg-21 (0.32 ꢀM) in the 1-100 mM K buffers.
Fig. 4. CD titrations spectra of BASQ for Htg-21 (4 ꢀM) in 10 mM Tris-HCl buffer (pH 7.4) in the presence (A) and
absence (B) of 60 mM KCl. The concentration of BASQ is from 0 to 8 ꢀM. Arrows indicate the change direction of peaks.

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Scheme 1
DNA sample
Htg-21
LOD (nM)
Linear range (nM)
0.84
0.63
0.36
0.64
0.72
0.63
0.85
0-400
0-240
0-320
0-400
0-400
0-320
0-320
22AG
C-myc
CM22
Ckit1
G3T3
Hras
Table 1

6
-1
DNA sample
Stoichiometry (BASQ:DNA)
a
K (10 M )
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Htg-21
2:1
0.91±0.07
0.62±0.05
0.22±0.02
0.10±0.01
0.56±0.04
3.36±0.12
3.01±0.11
2
2AG
1:1
4:1
2:1
1:2
4:1
4:1
C-myc
CM22
Ckit1
G3T3
Hras
Table 2
Fig. 1

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Fig. 2
Fig. 3

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Fig. 4

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Highlights：
A bis-amine-substituted styrylquinolinium dye (BSAQ) was designed and synthesized.
BSAQ could be used as a colorimetric and red-emitting fluorescent probe for G-quadruplex
DNAs.
The limits of detection of BASQ with various G-quadruplex DNAs were found to below 1 nM.
The binding properties were demonstrated.