A triphenylamine-based colorimetric and fluorescent probe with donor-bridge-acceptor structure for detection of G-quadruplex DNA

Article abstract of DOI:10.1016/j.bmcl.2015.11.007

In this Letter, three triphenylamine-based dyes (TPA-1, TPA-2a and TPA-2b) with donor-bridge-acceptor (D-π-A) structure were designed and synthesized for the purpose of G-quadruplexes recognition. In aqueous conditions, the interactions of the dyes with G-quadruplexes were studied with the aim to establish the influence of the geometry of the dyes on their binding and probing properties. Results indicate that TPA-2b displays significant selective colorimetric and fluorescent changes upon binding of G-quadruplex DNA. More importantly, its distinct color change enables visual detection and differentiation of G-quadruplexes from single and duplex DNA structures. CD titration date reveals that TPA-2b could induce and stabilize the formation of G-quadruplex structure. All these remarkable properties of TPA-2b suggest that it should have promising application in the field of G-quadruplexes research.

Full text of DOI:10.1016/j.bmcl.2015.11.007

Accepted Manuscript
A triphenylamine-based colorimetric and fluorescent probe with donor–bridge-
acceptor structure for detection of G-quadruplex DNA
Ming-Qi Wang, Wen-Xiang Zhu, Zhuan-Zhuan Song, Shuo Li, Yong-Zhao
Zhang
PII:
S0960-894X(15)30216-X
http://dx.doi.org/10.1016/j.bmcl.2015.11.007
BMCL 23263
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 September 2015
16 October 2015
4 November 2015
Please cite this article as: Wang, M-Q., Zhu, W-X., Song, Z-Z., Li, S., Zhang, Y-Z., A triphenylamine-based
colorimetric and fluorescent probe with donor–bridge-acceptor structure for detection of G-quadruplex DNA,
Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.11.007
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A triphenylamine-based colorimetric and fluorescent probe with
donor–bridge-acceptor structure for detection of G-quadruplex DNA
Ming-Qi Wanga,*, Wen-Xiang Zhua, Zhuan-Zhuan Songa, Shuo Lib,*, Yong-Zhao Zhanga
aSchool of Pharmacy, Jiangsu University, Zhenjiang, 212013, P. R. China. Fax: +86 511 85032801;
E-mail: wmq3415@163.com
bSchool of Chemical Engineering, Chongqing University of Technology, Chongqing 400054, P. R. China.
E-mail: lishuo@cqut.edu.cn
Abstract: In this paper, three triphenylamine-based dyes (TPA-1, TPA-2a and TPA-2b) with
donor–bridge-acceptor (D–π–A) structure were designed and synthesized for the purpose of
G-quadruplexes recognition. In aqueous conditions, the interactions of the dyes with
G-quadruplexes were studied with the aim to establish the influence of the geometry of the
dyes on their binding and probing properties. Results indicate that TPA-2b displays significant
selective colorimetric and fluorescent changes upon binding of G-quadruplex DNA. More
importantly, its distinct color change enables visual detection and differentiation of
G-quadruplexes from single and duplex DNA structures. CD titration date reveals that TPA-2b
could induce and stabilize the formation of G-quadruplex structure. All these remarkable
properties of TPA-2b suggest that it should have promising application in the field of
G-quadruplexes research.
Keywords: G-quadruplex, Triphenylamine, Donor–bridge-acceptor, Visual detection
Single-stranded nucleic acids containing repetitive guanine-rich bases are known to adopt
non-canonical four-stranded secondary structures called G-quadruplexes (G4s) which are
composed of several planar stacks of guanine quartets stabilized by hydrogen bonds and metal
cations (usually K+ or Na+).1 During the past two decades, G4 structures have been well
characterized by nuclear magnetic resonance and X-ray crystallography and they can exhibit
diversity topological conformations, depending on the primary sequence, strand orientation and
numbers of strands involved.2 In spite of being well studied in vitro, the evidence for the

presence of G4 structures in vivo remains mainly controversial, but is currently supported by a
growing wealth of biochemical and molecular genetics data.3-5 Because of their abundance in
functional genomic regions, such as telomere, promoter regions of important oncogenes and
the untranslated regions of mRNAs, they have possible implication in a range of biological
functions, including telomere maintenance, transcription and translation regulation, the DNA
damage response, genome rearrangements and anti-tumor chemotherapy.6-9 In this context, the
recognition of G4s with small-molecule probes has emerged as a strategy to design: chemical
tools to detect these biological processes and potential anticancer drugs.10, 11
In recently years, fluorescence-based detection method has received great attention due to its
simple operation, high selectivity and sensitivity. Therefore, great efforts have been made to
develop small-molecule-based fluorescent probes for G4s.12-19 One of the common limitations
is that most of them have only displayed one type of output, which are prone to be disturbed in
quantitative detection by many factors, thus it would highly desire to develop probes
containing different outputs. Such probes will take advantage of different methods for more
reliable measurements and versatile applications. Recently, colorimetric detection with visual
readouts has been attracted much interest because it is rapid, facile and even allow
instrument-free.20, 21 To this end, design a probe for detection G4s with colorimetric and
fluorescent properties is a promising strategy.
Triphenylamine-based dyes have been widely used in opto- and electro-active materials,
fluorescence probes and cell imaging for their good electron donating and transporting
capability.22-24 Moreover, the triphenylamine moiety usually plays the role of donor in various
systems and can form a donor–acceptor (D-A) system to increase the fluorescence. Some
triphenylamine derivatives have demonstrated the ability of interaction with AT regions, but
rarely reported to have the optical response to G4s.22a, 25
Inspired by those studies, we attempted to screen and discover new dual probes for G4
structures based on triphenylamine dyes with push-pull architectures (Scheme 1). TPA-1 and
TPA-2a were designed by linking an indolium hemicyanine and a N-methyl-quinoline unit to
the triphenylamine skeleton via a methine chain respectively, whereas TPA-2b possesses an
additional functional amine side chain compared to TPA-2a. The N-methylated heterocycles
were chosen because of their electron-accepting as well as DNA binding properties.26 The

rotation of the methine-bridge would impact the push–pull effects between the donor and
acceptor moieties, and also may be restricted after binding to the specific DNA structure, and
give rise to a different spectroscopy response, which could be utilized to distinguish the DNA
structures. The amino-side group could be protonated at physiological pH, which may further
increases solubility in aqueous solution.
Scheme 1. Chemical structures of triarylamine-based dyes.
The desired dyes were synthesized in our laboratories. TPA-1 and TPA-2a were prepared
from commercially available materials using simple methylation and condensation procedures
(Scheme S1). TPA-2b, an amine-side chain derivative of TPA-2a, was obtained through a
facile three-component one-pot reaction (Scheme S1). All of the new compounds were
characterized by 1H NMR, 13C NMR and MS (see the Supplementary Information).
With the dyes in hand, their photophysical properties were first elucidated in different
solvents. In buffered solution of relatively high ionic strength (10 mM Tris-HCl, 60 mM KCl,
pH 7.4), all the dyes displayed broad absorption bands and blue shifted with respected to the
bands observed in organic solvents (Figures S1-S3, Table S2 for the detailed date). The spectral
shifts may be attributed to specific solute-solute and solute-solvent interactions or
charge-transfer interactions. It also has been found that the three dyes were almost
non-fluorescent in buffered solutions, with very weak and broad fluorescence bands in the long
wavelength region between 400 and 580 nm and thus display remarkable Stockes shifts of
about 100 nm for dyes TPA-1 and TPA-2a and about 90 nm for TPA-2b. Taken together, these
spectral features (i.e., broad, long-wavelength absorption bands, large Stokes shifts) are
characteristic of donor–acceptor systems exhibiting internal charge transfer upon excitation. In
this regard, the absorption maxima of the dyes TPA-1 and TPA-2a are redshifted with respect

to the TPA-2b, which gives evidence for more pronounced charge-transfer interactions in
TPA-1 and TPA-2a.
The interactions of the dyes with G4 DNA were studied using absorption spectra titrations.
Electronic absorbance spectroscopy is one of the most useful ways to investigate the
interactions of compounds with DNA.27 Compound binding to DNA usually results in
hypochromism and red shift. The extent of the hypochromism and red shift parallels the
binding affinity.28 Titrations of dyes TPA-1, TPA-2a and TPA-2b in the absence and presence
of Ckit1 as well as representative G4 DNA were obtained at room temperature. The changes in
the spectral profiles were shown in Figure 1 and Supplementary Figures S4 and S5. With the
increase in concentration of Ckit1, the intensity of TPA-2b at 450 nm gradually decreased, and
a notable redshift of the maximum was observed with an isosbestic point at 485 nm, which led
to a new peak at appromximately 495 nm. The hypochromism (H%), as defined by H% = 100%
(Afree-Abound)/Afree, of band at 480 nm of TPA-2b were 41.3%. As compared to TPA-2b,
addition of Ckit1 led to a 20 nm red shift and 31.2% hypochromism for TPA-2a and only 11.9%
hypochromism for TPA-1. The exact date was shown in Table S3. In order to further compare
quantitatively the binding affinities of the three dyes, the intrinsic binding constants Kb of
TPA-1, TPA-2a and TPA-2b to Ckit1 were measured to be 0.12 × 106, 5.47 × 106 and 14.15 ×
106 M-1, respectively, using the equation from Eq.(1) (see supplementary information). The
larger binding constant Kb of TPA-2b, indicating TPA-2b binds to Ckit1 more tightly than the
other two dyes. As the three dyes containing the same triphenylamine skeleton, this may be
caused the quinolinium moiety may also interact with G-quartet plane by π-π stacking or with
the phosphate backbone via electrostatic interaction.29 In addition, introduction of amine side
chain would also increase the stabilizing ability of TPA-2b to Ckit1. This might be related to
that introduction of the amine side chain into the quinolinium ring led to binding mode of
ligands with G4 DNA change to a certain extent. Interesting, the specific red-shift of TPA-2b
gave rise to a marked and vivid color change from pale yellow to pink under daylight, which
was valuable for the naked-eye detection of Ckit1 (Figure 1C-D).

Figure 1. (A) Absorbance spectra of TPA-2b in buffer (10 mM Tris-HCl, pH 7.4, 60 mM KCl) in the
presence of increasing amounts of Ckit1. TPA-2b = 8 μM, [Ckit1] = 0-2.5 μM from top to bottom. Arrows
indicate the change in absorbance upon increasing the Ckit1 concentrations. (B). Corresponding
half-reciprocal plot of TPA-2b (R = 0.9978). (C) Photograph of 8 μM TPA-2b (a) and 8 μM TPA-2b with 3
μM Ckit1 mixture (b) in buffer taken under daylight. (D) Color changes of TPA-2b (8 μM) upon addition of
Ckit1 (0-4 μM) under daylight.
The detailed fluorescence properties of the dyes with G4 DNA were explored by using
fluorescence titration assays. As shown in Figure 2A, TPA-2b alone in buffer displayed weak
fluorescence emission. With the gradual addition of the G4 DNA Ckit1, an emission peak at
approximately 627 nm was significantly enhanced and accompanied by a 75 nm red shift,
indicating the interaction occurred with G4 DNA. The change of (F/F0-1) versus Ckit1
concentration can be satisfactorily fitted to the Boltzmann function (Figure 2B). This
fluorescence enhancement might be caused by conformational changes in the excited state of
TPA-2b, most likely by the rotation restriction around the vinyl-bridge that separates the
triphenylamine and N-methylated quinolinium motieties, as clearly shown by the emission
enhancement in a viscous medium (Figure S7).20b It can be seen that there was a good linear
relationship between the concentration of Ckit1 (0.4-1.6 μM). and the fluorescence intensity.
The limit of detection was estimated to be 0.158 μM according to the standard method
(Supplementary information, Figure S8). However, TPA-1 and TPA-2a did not show any
significant fluorescence enhancement after adding Ckit1 (Supplementary Figure S6). Taken

together with the results of absorption and fluorescence spectral experiments, it can be
concluded that TPA-2b has great potential as a colorimetric and fluorescent G4 DNA probe.
Therefore, TPA-2b was chosen as the most promising probe for further detailed investigation.
Figure 2. (A) Fluorescence spectra of TPA-2b (2 μM) upon titration with prefolded Ckit1 (0-2 μM) in 10
mM Tris-HCl buffer (pH 7.4) and 60 mM KCl. (B) Plots of F/F0-1 at 627 nm versus Ckit1 concentration. λex
= 462 nm.
The interactions with different DNA oligomers, including G4, single- and double-stranded
DNAs under identical salt conditions were conducted to examine the selectivity. Changes in
absorption spectra were shown in Figure 3 and Figure S9, the interactions with G4s, including
Ckit1, CM22, HTG-21, were characterized by the clearly enhanced the absorbance at 495nm
and formation of new bands redshifted about 45 nm. In the case of single-stranded DNA (ss26)
and double-stranded DNAs (ds26, polyd(A-T)9 and polyd(G-C)9), the new bands showed slight
decrease and much smaller redshift (by 6-26 nm) (Figures 3A-B and S9). Of note, the red-shift
of the absorption bands point out to differences in the TPA-2b binding affinities in single-,
duplex and G4 DNA. Moreover, TPA-2b also exhibited excellent selectivity for G4s over
single- and double-stranded DNAs (Figures 3C-D and S10) by fluorescence measurements,
which was consistent with the absorption titration studies. The fluorescence quantum yield
values of TPA-2b with different nucleic were summarized in Table S4. Meanwhile, as shown
in Figure 3E, the incubation of G4s with TPA-2b caused significant color change under
daylight. And other DNAs did not cause noticeable color change, suggesting that G4s could be

easily detected and differentiated from other forms of DNA with just the naked eyes.
Figure 3. (A)The absorbance enhancement of TPA-2b (8 μM) at 495 nm with different amounts of DNAs.
(B) Absorbance response of TPA-2b to DNAs, the y-axis corresponds to the shift in the absorbance
maximum (λmax). (C) The fluorescence intensity enhancement of TPA-2b (2 μM) with different amounts of
DNAs. (D) Distribution of the values of the F/F0 for TPA-2b with DNAs. λex = 462 nm. (E) Photograph of
the aqueous solutions of TPA-2b/DNA mixtures taken under daylight. Conditions: 10 mM Tris-HCl buffer
(pH 7.4, 60 mM KCl).
It is known that cationic species play an important role in determining the structure of G4s.1
Therefore, the fluorescence measurements were also performed with different cationic species
(K+ or Na+), or without any metals ions (Figure 4). The binding affinity of TPA-2b to the K+
was found to be stronger than that of Na+, indicating the higher fluorescence enhancement in
the buffer solution with KCl (14-fold) than that with NaCl (9-fold). In other words, G4
structure formed in the presence of K+ is stable than that in the presence of Na+. Interestingly,

in the absence of any metal ions, the TPA-2b-G4 system shows a fluorescence turn-on up to
32-fold, which is larger in the case of K+ or Na+. Besides, after adding Ckit1 without prefolding,
the absorption spectra showed 50 nm red-shift and approximately 43% hypochromism (Figure
S11, Table S5), which is similar to the result of absorption spectra in the presence of K+. These
might indicate that the positively charged quinolinium group and amine side chain in TPA-2b
increased the solubility of the dye in the aqueous buffer and strengthened its interaction with
the negatively charged Ckit1 via electrostatic interaction. When the positively charged K+ or
Na+ ions added, the TPA-2b was partially detached and driven into the aqueous buffer,
probably due to its unfavorable docking on the G-quadruplex surface.
Figure 4. Fluorescence intensity enhancement (F/F0) of TPA-2b (2 μM) plotted against the Ckit1 in 10 mM
Tris-HCl buffer (pH 7.4) in the presence of 60 mM KCl, 60 mM NaCl and absence of any metal ions. λex =
462 nm.
To validate the assumption, the structural changes in Ckit1 caused by TPA-2b were studied
using Circular dichroism (CD) measurements. CD spectroscopy is an extremely useful
technique to study nucleic acids conformation, so it is widely used to study the G4 conversion
induced by probes.14 In the presence of 60 mM K+ ions and absence of any molecules, the CD
spectrum of Ckit1 was of the typical parallel G-quadruplex, with the positive peak at 264 nm
and a negative peak at 240 nm.30 After addition of TPA-2b to the solution, the characteristic
peaks of the parallel G4 had no obvious change, which the positive peak at 264 nm enhanced

slightly (Figure 5). In addition, we tested the effect of Na+ ions on conformation of
G-quadruplex. As shown in Figure S12, it was found that the spectrum was almost unchanged
on addition of different concentrations of TPA-2b. These results indicated the TPA-2b had no
influence on the conformation of G4 structure in monovalent cations. However, the slight
enhancement of the positive peak attribute to the stabilization of the G4 structure. On the other
hand, as shown in 5B, Ckit1 sequence adopted a parallel structure in the absence of monovalent
cations. Upon addition of TPA-2b, significant changes in ellipticity were observed. And the
positive band at 260 nm increased significantly and the negative band at 238 nm also increased.
The results indicated that TPA-2b could induce and stabilize the Ckit1 sequence to form
parallel structure of quadruplex in the absence of salt.
Figure 5. CD titrations spectra of TPA-2b for Ckit1 G4 DNA (4 μM) in 10 mM Tris-HCl buffer (pH 7.4) in
the presence(A) and absence (B) of 60 mM KCl. [TPA-2b]/μM: (1) 0; (2) 2; (3) 4; (4) 8.
Competition titrations were performed to further confirm the selective of TPA-2b binding to
G4 DNA, in which the ability of TPA-2b to retain an enhanced fluorescence intensity with the
addition of G4 DNA challenged by single-strand or duplex DNA. As shown in Figures 6 and
S9, in the presence of an excess of duplex DNA (ds26, 50 μM) or single-strand DNA (ss26, 50
μM), very slightly increased the intensity of TPA-2b (about 2 folds) was observed, however,
upon addition of an increasing amount of Ckit1 led to a significant enhancement of the

fluorescence intensity (about 10 folds). Similar results were found by using absorption spectra.
Although the absorbance of the dye was affected by the presence of dsDNA and ssDNA, the
addition of the Ckit1 led to much larger red-shifts of the absorption bands.(Figure S13) The
finding shows that detection of G4 DNA is indeed possible under these conditions. These
results demonstrate the high potential of TPA-2b to serve as quadruplex-sensitive probe in the
competitive biological environment.
Figure 6. Spectrofluorimetric titrations of Ckit1 (0-4 μM) to the dye TPA-2b in the presence of an excess of
dsDNA (50 μM) or ssDNA (50 μM). The concentration of TPA-2b was 2 μM in buffer. λex = 462 nm.
In conclusion, three new dyes with D-π-A structure were rationally designed and synthesized
for G4 recognition. Results show the TPA-2b can be used as a colorimetric and fluorescent
dual probe for specific detection of G4s, based on its significant absorbance red-shift and
fluorescence enhancement in the presence of G4s. Furthermore, the distinct color changes of
TPA-2b offered a convenient visualization method for discriminating G4s from other DNA
forms (including single- and double-strand DNAs). The CD studies showed that TPA-2b could
induce and stabilize G4 structure. Further studies on the exploration of biomedical applications
of this probe are ongoing in our laboratories.

Acknowledgements
This work was financially supported by the Senior Talent Cultivation Program of Jiangsu University (Nos.
15JDG068). This work was also supported by Natural Science Foundation Project of CQ CSTC
(cstc2013jcyjA50012), Scientific and Technological Research Program of Chongqing Municipal Education
Commission (Grant No. KJ130820), China Postdoctoral Science Foundation Funded Project (No.
2014M562326). We thank Analytical & Testing Center of Jiangsu University for NMR, MS and CD analysis.
Supplementary date
1
13
Experimental procedures, fluorescence and absorption spectra of the probe; Copies of H and C NMR,
MS spectra for new compounds.
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Graphical abstract
A colorimetric and fluorescent dual probe with D–π–A structure for G-quadruplexes was
successfully developed. Its significant and distinct changes in color enable the label-free and
visual detection of and differentiation of G-quadruplexes from single and duplex DNA
structures.