A triphenylamine derivative as a naked-eye and light-up fluorescent probe for G-quadruplex DNA

Article abstract of DOI:10.1016/j.tetlet.2016.09.100

G-quadruplex (G4) DNAs have attracted considerable interest because of their important biological functions and medical applications. Searching for highly specific binding molecules is important for the basic research of G4 DNA, as well as the design of novel anticancer drugs. Previous, we have developed a quinolone-substituted triphenylamine probe (TPA-2b) with selectivity to G4 DNAs. Herein, we further designed and synthesized a hydroxyethyl functionalized derivative (TPA-3) and investigated the interactions with G4 DNAs and living cells. TPA-3 was found to express significant fluorescence enhancement upon its interaction with G4 DNAs while shows almost no response to non-quadruplex DNAs. The distinction can even be easily distinguished by the naked eye under UV light. The spectral analysis showed TPA-3 bound to G4 DNAs mainly through intercalative binding mode. CD study results indicated TPA-3 did not disturb the conformation of G4 structure. Cellular uptake assay suggested that TPA-3 could pass through membrane and enter living cells. Our results suggested the side chain of core structure could change the binding affinity to G4 DNAs, as well as the interaction with living cells. Thus, this study gives some clues to design new G4 DNA probes with high selectivity, sensitivity, and biological imaging applications.

Full text of DOI:10.1016/j.tetlet.2016.09.100

Accepted Manuscript
A triphenylamine derivative as a naked-eye and light-up fluorescent probe for
G-quadruplex DNA
Ming-Qi Wang, Lie-Xiong Gao, Yi-Fan Yang, Xian-Ning Xiong, Zhao-Yi
Zheng, Shuo Li, Yuan Wu, Lu-Lu Ma
PII:
S0040-4039(16)31293-X
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.09.100
TETL 48170
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 August 2016
23 September 2016
30 September 2016
Please cite this article as: Wang, M-Q., Gao, L-X., Yang, Y-F., Xiong, X-N., Zheng, Z-Y., Li, S., Wu, Y., Ma, L-
L., A triphenylamine derivative as a naked-eye and light-up fluorescent probe for G-quadruplex DNA, Tetrahedron
Letters (2016), doi: http://dx.doi.org/10.1016/j.tetlet.2016.09.100
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A triphenylamine derivative as a naked-eye and light-up fluorescent probe
for G-quadruplex DNA
Ming-Qi Wanga, Lie-Xiong Gaoa, Yi-Fan Yanga, Xian-Ning Xionga, Zhao-Yi Zhenga, Shuo Lib,* Yuan Wua, Lu-Lu Maa
aSchool of Pharmacy, Jiangsu University, Zhenjiang, 212013, P. R. China
bCollege of Chemistry and Chemical Engineering, Chongqing University of Technology, Chongqing 400054, PR China
Abstract: G-quadruplex (G4) DNAs have attracted considerable interest because of their important biological functions
and medical applications. Searching for highly specific binding molecules is important for the basic research of G4 DNA, as
well as the design of novel anticancer drugs. Previous, we have developed a quinolone-substituted triphenylamine probe
(TPA-2b) with selectivity to G4 DNAs. Herein, we further designed and synthesized a hydroxyethyl functionalized
derivative (TPA-3) and investigated the interactions with G4 DNAs and living cells. TPA-3 was found to express
significant fluorescence enhancement upon its interaction with G4 DNAs while show almost no response to non-quadruplex
DNAs. The distinction can even easily be distinguished by the naked eye under UV light. The spectral analysis showed
TPA-3 bound to G4 DNAs mainly through intercalative binding mode. CD studies results indicated TPA-3 did not disturb
the conformation of G4 structure. Cellular uptake assay suggested that TPA-3 could pass through membrane and enter
living cells. Our results suggested the side chain of core structure could change the binding affinity to G4 DNAs, as well as
the interaction with living cells. Thus, this study gives some clues to design new G4 DNA probes with high selectivity,
sensitivity and biological imaging applications.
Keywords: G-quadruplex DNA; Fluorescent probe; Naked eye; Bioimaging.
Among various DNA structures, G-quadruplexes (G4s) have received intense research attention in recent years with
respect to their biological and medical applications.1, 2 G4 DNAs are unique structures that formed from the folding of
guanine rich DNA sequences by Hoogsteen hydrogen base-pairing.3 Analysis of the human DNA reveals that a variety of
G-rich sequences have been found in some pivotal genomic regions, such as telemetric DNA and promoter regions of some
oncogenes.4, 5 Consequently, numerous biochemical and structural analyses of G4s have been established in vitro.6-8 During
the past decades, the existence of G4 structures in mammalian cells remains controversial. To address these fundamental
questions, powerful tools for characterizing the G-quadruplexes in vivo are desperately needed.
Recently Balasubramanian et al. reported the direct evidence of endogenous G4 DNA formation using a small-molecule
ligand.9 More and more evidences show that G4 DNAs have important roles in various cellular pathways including
replication, recombination, transcription and translation, which are highly associated with various human diseases.10-12
However, the research on the role of G4 DNAs in vivo is still at an early stage.13,14 There is a need to know the formation
and location of G4 DNAs in cells at the appropriate time to understand their functions. Therefore, targeting the G4s using

molecule tools has emerged as an alternative strategy for functional exploration of G4s and drug discovery.15-17
Fluorescent probes are attractive molecular tools for both analytical sensing and bioimaging because of their high
sensitivity, fast response time, and technical simplicity.18,19 Great efforts have been made to develop fluorescence probes for
the detection of G4 DNAs, and many of them have been reported.20-25 In previous work, we have reported two dyes TPA-2a
and TPA-2b by incorporating a triphenylamine fluorophore into the quinolinium framework (Fig.1).26 Results showed that
TPA-2b with an amine-side chain had excellent selectivity to G4 DNAs over other DNA forms in vitro. However, TPA-2b
expressed only about 10-fold increase in fluorescence intensity upon binding to G4 DNA. And it cannot penetrate living
cells with an intact membrane, thus the further application in cellular detection was restricted. The direct intracellular
detection of G4 DNAs remains a formidable challenge because a limited number of fluorescent G4 probes can penetrate
into living cells and their actual targets in cells are not fully known.13 Inspired by these phenomena, we have great interest
in developing a new fluorescence probe based on TPA-2b for G4 DNA aiming not only to induce a remarkable fluorescence
signal output but also to improve its biocompatibility. Additionally, this will provide deep insights for the structure-based
rational design of G4 DNA probes.
In the present study, a new triphenylamine derivative TPA-3 was designed on the basis of a TPA-2b framework by
introducing a hydroxyethyl group. The influence on the G4 DNAs binding properties, fluorescence discrimination with
other DNA species, and its potential application on live cells staining and imaging were investigated through experimental
studies.
Figure1. Structures of TPA-2a and 2b and the hydroxyethyl group tethered derivative TPA-3.
The desired compound TPA-3 was synthesized according to the method reported by our previous paper.26 As shown in
Scheme S1, TPA-3 was prepared by condensing 4-formyltriphenylamine, 1, 2-dimethyl-4-chloroquinolin-1-ium iodode and
1
N-(2-Hydroxyethyl)piperazine in one-pot. The new compound was characterized by H NMR, 13C NMR and MS (see the
Supplementary Information).
First, the fluorescence properties of TPA-3 to different DNA forms, including G4s with different conformations, duplex
and single-stranded DNAs, were investigated via fluorescence titration assays. As shown in Fig. 2A, free TPA-3 displayed a
weak emission at 550 nm in buffer solution (10 mM Tris-HCl, 60 mM KCl, pH 7.4) when excited at 470 nm. With the
gradual addition of G4 DNA 22AG, the fluorescence intensity around 615 nm was remarkably increased. This light-up
fluorescence effect was also observed when TPA-3 was treated with other G4 DNAs, including Ckit1, C-myc, CM22, Scr1,

Ckit3, HTG-21, HRAS, G3T3, which caused 20- to 50-fold fluorescence enhancement. However, the addition of
single-strand DNA (ss26, ss22) and duplex DNA (Ct-DNA, ds26, Polyd(A-T)9 and Polyd(G-C)9) under the same conditions
induced negligible changes in the fluorescence intensity (Figs. 2B and S1). The distinction between G4 DNA and ssDNA,
dsDNA can easily be distinguished by naked eye under UV light (Fig. 2C). The finding indicated that TPA-3 exhibited
excellent selectivity for G4 DNA over single- and double-strand DNA. As we previously reported, the fluorescence
emission of TPA-2b (Fig. 1) was nearly 10-fold increase could be observed with the addition of G4 DNAs. Thus, TPA-3
might be better than TPA-2b.
Figure 2. (A) Fluorescence spectrum of TPA-3 (2 μM) in the absence or presence of 2 μM G4s, duplex and single-stranded DNAs. (B) The
fluorescence enhancement of 2 μM TPA-3 at 615 nm versus the concentration of [DNA]. (c) Visual detection of G4 DNA in the presence of
TPA-3 under the irradiation of 470 nm excitation light.
The sensitivity of TPA-3 as a “light-up” fluorescence G4 DNA probe was investigated by its fluorimetric titration with
22AG. The concentration-response curve to 22AG (0.2-0.8 μM) showed a good linear relationship with R2 = 0.993 (Fig. S2).
The limit of detection (LOD) was estimated to be 34 nM according to the equation 3σ/s (The σ value represents the standard
deviation for multiple measurements of blank solution. The s value is the slope derived from the linear range of the
fluorescence titrations).27 It is also noteworthy that the LOD value of TPA-3 for G4 DNA in solution is significant improved
relative to TPA-2b.26 Therefore, the different side chains on the core structure may cause different interaction with G4
DNAs, resulting in the different binding affinity. And the additional hydroxyethyl chain of TPA-3 was found to be good in
the terms of induced fluorescence intensity toward G4 DNAs. Based on the G4 ligands reported previously, we have known
that the introduction of side chain would increase the affinity of ligand for G4 DNA because the side chain may dock onto
the groove of G4 DNA and interact with the G4 DNA by forming intermolecular hydrogen bond or electrostatic
interaction.28
In order to examine whether the significant fluorescence enhancement of TPA-3 was due to its interactions with G4
secondary structure or just simply G-rich sequence, we mixed 22AG and its complementary sequence in a ratio of 1:1 to

form a G-rich duplex structure. Following a similar titration protocol (Fig. S3), the fluorescence intensity of the G-rich
duplex structure was found to be very low compared to G4 DNA 22AG, which demonstrated that TPA-3 indeed interacted
with G4 secondary structure instead of simple G-rich sequence.
Besides, although most of G-rich sequences are prone to form monomeric G4s, a small number of them tend to form
higher-order G4 structures consisting of consecutive G4 units.29 In addition, it has been reported that there are some
diseases are associated with expansion repeats of G-rich sequences capable of forming multimeric G4s.30 Thus, the
recognition ability of TPA-3 toward multimeric G4 DNA was studied. As shown in Fig. S4, addition of multimeric G4 DNA
Hum45 increased the fluorescence of TPA-3 steadily, implying TPA-3 also has the potential to be used for probing
multimeric G4 DNA. Collectively, these results demonstrated the promising potential of TPA-3 to serve as a selective and
sensitive probe toward G4 DNAs.
Encouraged by the strong fluorescence emission enhancement of TPA-3 with G4 DNAs, it was therefore interesting to
understand the details of their interactions. On this basis, we then conducted spectrophotometric titrations of TPA-3 with
different DNAs. As shown in Fig.3, after adding 22AG G4 DNA, the absorption band of TPA-3 at approximately 447 nm
gradually decreased, and a large red-shift in the maximum was observed, which led to a new peak at approximately 493 nm.
In addition, the titration spectra exhibited a well-resolved isosbestic point (at 484 nm) revealed the existence of one major
binding mode, whereas remarkable hypochromic and bathochromic changes are indicative of an intercalative binding
mode.31 Specifically, the quinoline subunit would interact with G-quartet plane by π-π stacking. Furthermore, we
investigated the interaction between TPA-3 and other DNAs. The changes in the spectral profiles during titration were
shown in Fig. S5. Addition of G4 DNAs with different conformations also led to significant hypochromisms and red shifts,
which meant TPA-3 could bind with these G4 DNAs through intercalation. While, in the case of single-stranded DNA (ss26)
and double-stranded DNAs (ds26, ct-DNA), the bands showed much smaller redshift (Figs. 3B and S5), suggesting that
TPA-3 had much stronger interaction with G4s than with other DNA forms, which was consistent with the results of
fluorescence spectra.
Figure 3. (A) Absorbance spectra of TPA-3 in buffer (10 mM Tris–HCl, pH 7.4, 60 mM KCl) in the presence of increasing amounts of 22AG.
TPA-3 = 8 μM; [22AG] = 0-2 μM from top to bottom. Arrows indicate the change in absorbance upon increasing the 22AG concentrations. (B)
Absorbance response of TPA-3 to DNAs, the y-axis corresponds to the shift in the absorbance maximum (λmax).

G4 DNA has been recognized as a potential target for anti-cancer drug design. Normal human physiological pH is ~7.4,
while tumors generally exhibit acidic microenvironments with pH 6.2–6.9.32 Considering the practical applications, the G4
recognition fluorescence behavior of TPA-3 was investigated and compared in the range pH 6.0~7.4 (Fig. 4). Though a
change of pH value in this range had a little effect on the G4 recognition, TPA-3 had a significant fluorescence
enhancement to all tested mediums. The titrations resulted in absorption changes in different pH values also displayed
similar trends (Fig. S6). The finding shows that detection of G4 DNA is indeed possible under these conditions.
Figure 4. Fluorescence intensity enhancement of TPA-3 at 612 nm plotted against the concentration of 22AG in different pH solutions.
Cationic species especially K+ and Na+ ions also play a very important role in the G4s formation. It has been reported
that the structure of G4 formed in the presence of K+ is more stable than that in the presence of Na+.33 To explore this, the
fluorescence titrations assays were performed with K+ or Na+ (Fig. S7). The titrations of TPA-3 with 22AG in solution
containing 60 mM NaCl displayed a relatively small emission enhancement and with a nominal bathochromic shift in the
absorption band (Fig. S7). Evidently, TPA-3 interacted stronger with 22AG/K+ than that of Na+, showing a binding
specificity to the K+-induced G4 of 22AG. The structural changes in 22AG caused by TPA-3 were further studied using
circular dichroism (CD) measurements. CD has been widely used as a powerful tool to monitor the conformation of DNA
sequence due to its outstanding sensitivity. In the presence of K+, 22AG sequence has formed a hybrid-type of quadruplex
DNA containing parallel and anti-parallel structure, with two positive peaks at 265 and 290 nm, and a negative band near
235 nm. While in the presence of Na+, 22AG has formed an anti-parallel structure with a positive band around 295 nm and a
negative band around 260 nm. Upon additions of TPA-3, very little change of the CD spectrum was observed in the solution
of K+ and no apparent spectral change in that of Na+(Fig. 5A and B), which implied that the conformation of G4 was
stabilized by alkali metal ions, and TPA-3 did not disturb the conformation of G4 at high ionic strength.

Figure 5. CD titration spectra of TPA-3 for 22AG DNA (5 μM). (a) Solution buffered with 10 mM Tris (pH 7.4) and 60 mM KCl; (b) Solution
buffered with 10 mM Tris (pH 7.4) and 60 mM NaCl. [TPA-3]/ μM: 0-5 μM.
The experiments described above showed the TPA-3 bound to 22AG G4 DNA mainly through intercalative binding
mode. To gain further insight into this binding mode, a G4 fluorescent intercalator displacement (G4-FID) experiment was
performed. The G4-FID is a well-known method assay based on the loss of fluorescence of a G4-bound intercalator
(thiazole orange, TO) upon displacement of a G4-bind molecule.34 TO binds to G4s in a single-site manner of intercalation
with high fluorescence intensity. Molecules that compete with TO for G4 binding are also considered to bind through
intercalation and will result in fluorescence intensity decreased. The emission spectra of TO bound to 22AG in the absence
and presence of TPA-3 were shown in Fig. S8. Addition of TPA-3 to the G4-bound TO solution caused a great reduction in
the emission intensity, implying that the molecule could bind to G4 DNA mainly in an intercalative binding mode.
Moreover, the results obtained from photo-fluorimetric titrations indicated that TPA-3 interacted with 22AG in a 1:1
stoichiometry (Fig. S9). Thus, we deduced that one molecular of TPA-3 might simultaneously interact with one G-quartets
of 22AG. According to the 1:1 ratio obtained from the Job plot, the binding constant (Kb) for the TPA-3/22AG
complexation derived from the fluorescence titration data can be estimated to be 1.21×106 (R2 = 0.997, Fig. S10) by using
Benesi−Hildebrand plot.35
In addition, to examine the bioactivity of TPA-3, the cellular uptake and localization were investigated. The living cells
were incubated with TPA-3 (10 μM) for 2h, and then imaged by under a standard cell staining protocol. The confocal
imaging showed that TPA-3 could pass through membrane and enter living cells (Fig. 6). However, the co-staining with
DAPI (duplex DNA probe) showed that TPA-3 mainly located in cytoplasm. Thus, the fluorescence of TPA-3 upon binding
to G4s might be difficult to observe in cells. Fluorescence from the cytoplasm may be due to TPA-3 located in an
environment of low polarity. Our previous results have shown that TPA-2b with amino group could not enter living cells,
therefore, the results suggested the hydroxyethyl group of TPA-3 increased the cellular uptake.

Figure 6. Confocal images of Hep-G2 cells stained by TPA-3 (10 μM) (λex=470 nm) and DAPI (5 μg/mL) (λex =405 nm).
In summary, a hydroxyethyl-functionalized triphenylamine derivative TPA-3 has been designed and synthesized. The
interactions with G4 DNAs and living cells were investigated. Fluorescence spectra showed that TPA-3 could serve as a
naked-eye and light-up probe for G4 DNAs over ss-/ds-DNA. Absorption and G4-FID assays suggested that TPA-3 bound
to G4 DNA mainly through intercalation binding mode. Furthermore, CD studies showed TPA-3 did not disturb the
conformation of G4 structure. Confocal imaging showed TPA-3 could enter into cancer cells and mainly located in
cytoplasma. These results confirmed that the side chain of the ligand could affect the binding ability to G4 DNAs and
change the cellular uptake. These characteristics of TPA-3 provide some information that may be useful for the design of
new specific G4 DNA probes for diagnostic and biosensing applications.
Acknowledgements
This work was financially supported by the Senior Talent Cultivation Program of Jiangsu University (Nos. 15JDG068).
This research was also supported by Technical Research Program for Chongqing Education Commission (KJ1600916),
Chongqing Research Program of Basic Research and Frontier Technology (No.cstc2013jcyjA50012, cstc2016jcyjA0508),
China Postdoctoral Science Foundation funded project (2014M562326, 2016T90851).
Supplementary date
1
Experimental procedures, fluorescence and absorption spectra of the probe; Copies of H and 13C NMR, MS spectra for
new compounds.
Corresponding author.
E-mail addresses: lishuo@cqut.edu.cn (Shuo. Li).
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A triphenylamine derivative as a naked-eye and light-up fluorescent probe
for G-quadruplex DNA
Ming-Qi Wanga, Lie-Xiong Gaoa, Yi-Fan Yanga, Xian-Ning Xionga, Zhao-Yi Zhenga, Shuo Lib,* Yuan Wua, Lu-Lu Maa
aSchool of Pharmacy, Jiangsu University, Zhenjiang, 212013, P. R. China
bCollege of Chemistry and Chemical Engineering, Chongqing University of Technology, Chongqing 400054, PR China

Highlights：
A hydroxyethyl-functionalized triphenylamine derivative was developed.
Significant “light-up” effect was observed when this probe bound with G4 DNAs.
The G4-targeting probe displayed excellent selectivity, and sensitivity.
The cellular application in cancer cells was demonstrated.