Molecular Engineering of Thiazole Orange Dye: Change of Fluorescent Signaling from Universal to Specific upon Binding with Nucleic Acids in Bioassay

Article abstract of DOI:10.1021/acschembio.5b00987

The universal fluorescent staining property of thiazole orange (TO) dye was adapted in order to be specific for G-quadruplex DNA structures, through the introduction of a styrene-like substituent at the ortho-position of the TO scaffold. This extraordinary outcome was determined from experimental studies and further explored through molecular docking studies. The molecular docking studies help understand how such a small substituent leads to remarkable fluorescent signal discrimination between G-quadruplex DNA and other types of nucleic acids. The results reveal that the modified dyes bind to the G-quadruplex or duplex DNA in a similar fashion as TO, but exhibit either enhanced or quenched fluorescent signal, which is determined by the spatial length and orientation of the substituent and has never been known. The new fluorescent dye modified with a p-(dimethylamino)styryl substituent offers 10-fold more selectivity toward telomeric G-quadruplexes than double-stranded DNA substrates. In addition, native PAGE experiments, FRET, CD analysis, and live cell imaging were also studied and demonstrated the potential applications of this class of thiazole-orange-based fluorescent probes in bioassays and cell imaging.

Full text of DOI:10.1021/acschembio.5b00987

Articles
pubs.acs.org/acschemicalbiology
Molecular Engineering of Thiazole Orange Dye: Change of
Fluorescent Signaling from Universal to Specific upon Binding with
Nucleic Acids in Bioassay
,
†
†
§
†
†
†
Yu-Jing Lu,* Qiang Deng, Jin-Qiang Hou, Dong-Ping Hu, Zheng-Ya Wang, Kun Zhang,
Leonard G. Luyt,§ Wing-Leung Wong,* and Cheuk-Fai Chow
,∥
,‡
‡
†Institute of Natural Medicine and Green Chemistry, School of Chemical Engineering and Light Industry, Guangdong University of
Technology, Guangzhou 510006, Peoples’ Republic of China
‡
Department of Science and Environmental Studies, Centre for Education in Environmental Sustainability, The Hong Kong Institute
of Education, 10 Lo Ping Road, Tai Po, Hong Kong SAR, Peoples’ Republic of China
§
London Regional Cancer Program, 790 Commissioners Road East, London, Ontario N6A 4L6, Canada
∥
Departments of Oncology, Chemistry, Medical Imaging, The University of Western Ontario, London, Ontario N6A 3K7, Canada
*
S Supporting Information
ABSTRACT: The universal fluorescent staining property of thiazole
orange (TO) dye was adapted in order to be specific for G-quadruplex
DNA structures, through the introduction of a styrene-like substituent
at the ortho-position of the TO scaffold. This extraordinary outcome
was determined from experimental studies and further explored
through molecular docking studies. The molecular docking studies
help understand how such a small substituent leads to remarkable
fluorescent signal discrimination between G-quadruplex DNA and
other types of nucleic acids. The results reveal that the modified dyes
bind to the G-quadruplex or duplex DNA in a similar fashion as TO,
but exhibit either enhanced or quenched fluorescent signal, which is
determined by the spatial length and orientation of the substituent and
has never been known. The new fluorescent dye modified with a p-
(
dimethylamino)styryl substituent offers 10-fold more selectivity
toward telomeric G-quadruplexes than double-stranded DNA substrates. In addition, native PAGE experiments, FRET, CD
analysis, and live cell imaging were also studied and demonstrated the potential applications of this class of thiazole-orange-based
fluorescent probes in bioassays and cell imaging.
he development of highly sensitive and selective probes to
qualitatively and quantitatively detect nucleic acids is
for research in this area being eventual translation to the clinic.
Since G-rich telomeric DNA sequences synthesized by
telomerase have been shown to form G-quadruplexes in
T
1
−4
extremely important for a wide range of investigations,
such
11,15,16
as the active research areas of chemical biology, biochemistry,
and clinical diagnosis. The prototypical nucleic acid DNA, apart
from forming primarily double helical structures, is able to
adopt higher-ordered and functionally useful structures. G-
quadruplex is one such distinctive structure, which forms a
unique four-stranded structure containing guanine-rich nucleic
vivo,
dous interest and research effort.
telomeric G-quadruplexes therefore attract tremen-
12,18
Moreover, G-quad-
ruplexes were demonstrated as versatile building blocks that
19,20
offer substantial advantages on fluorescence sensors.
Small molecule based fluorescent probes have been identified
21
for in vitro or in vivo analysis of G-quadruplex DNA, such as
5
,6
acids sequences. G-quadruplexes can be divided into three
primary topologies: parallel, antiparallel, and hybrid-type
22
triphenylmethane dyes, cyanovinyl-pyridinium triphenyl-
23
24
7
amine, and triarylimidazole (IZCM-1). Among the reported
studies, thiazole orange (TO) is one of the most widely used
fluorescent probes in nucleic acid staining due to its high
structures. These unique sequences can be found in some
important genomic regions, such as telomere, rDNA, promoter
regions of some oncogenes, and the untranslated regions of
25−27
8
−10
fluorescence quantum yield.
However, TO is a universal
mRNA.
In recent years, G-quadruplex structures have
nucleic acid fluorescent dye which has no ability to differentiate
received attention because their unique structural features are
believed to be able to provide biological significance in
telomere maintenance, transcription regulation, and antitumor
Received: November 30, 2015
Accepted: January 11, 2016
1
1−18
chemotherapy.
G-quadruplex binders have great potential
for development as anticancer drugs with the expected outcome
©
XXXX American Chemical Society
A
DOI: 10.1021/acschembio.5b00987
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Articles
a
Scheme 1. Synthesis Route to 4a−4d and Their Molecular Structures
a
Reagents and conditions: (i) NaHCO , methanol, room temp., 1 h; (ii) reaction with aromatic aldehydes, 135 °C, reflux 3 h.
3
G-quadruplex DNA from other DNA species. A recent
approach is to develop a new generation of G-quadruplex
selective fluorescent probes by the integration of a G-
quadruplex selective binder into a TO molecule. Successful
2. RESULTS AND DISCUSSION
.1. Synthesis of New Fluorescent Dyes. Intermediate 1
4-chloro-1,2-dimethylquinolin-1-ium iodide) was obtained by
2
(
the reaction of 4-chloro-2-methylquinoline with iodomethane.
This was followed by reaction with 2,3-dimethylbenzo[d]-
thiazol-3-ium iodide (2) to give compound 3 ((Z)-1,2-
dimethyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)
28,29
30
31
examples include benzofuroquinolinium,
ISTO, TOPI,
3
2
33
PyroTASQ, and PDC-TO, demonstrating that the struc-
tural modification on the basis of the TO framework is able to
provide direct and remarkable G-quadruplex specificity and/or
fluorescence discrimination. Recently, a styryl-substituted TO
35
quinolin-1-ium iodide) as the key intermediate. A series of the
targeted thiazole orange based fluorescent dyes 4a−4d
(Scheme 1) with different styryl substituents introduced at its
ortho-position was synthesized by the reaction of 3 with a
variety of aromatic aldehydes including p-dimethylaminoben-
zaldehyde (4a), indole-2-carboxaldehyde (4b), p-
34
molecule was developed as a RNA fluorescent dye. Currently,
how a small substituent such as a styryl group allied to a TO
molecule can induce signaling specificity is unclear and has not
been discussed in the literature. We believe that understanding
the fundamentals on the relationship of interaction and
signaling between the dye and DNA at the molecular level is
very important for structure-based probe design either for
biosensing or for drug target applications. In the present study,
we report on a series of new fluorescent dyes, designed on the
basis of a TO framework by systematically introducing various
styrene-like substituents at its ortho-position, and investigate
the influence on the binding property and fluorescence
discrimination with various duplex and G-quadruplex-DNA
substrates through both experimental and modeling studies.
These TO derived dyes are able to bind with nucleic acids, and
surprisingly, excellent fluorescent signal discrimination is found
to be determined by the spatial length and orientation of a
small substituent of the TO molecule. In addition, the
photophysical properties of the dyes, including fluorescence
quantum yields, equilibrium binding constants, the detection
limit toward G-quadruplex-DNA, and their potential applica-
tions in live cell staining and imaging, were explored.
(
dimethylamino)cinnamaldehyde (4c), and p-methylbenzalde-
hyde (4d). The compounds were obtained with high isolated
yields (83−90%). The purity of the compounds was confirmed
to be above 95% by using HPLC analysis.
2.2. Fluorescence Discrimination of the Dyes toward
Different DNA Substrates and the Comparison of Their
Binding Kinetics. As indicated from the screening results
shown in Figure 1, the parent TO dye provides no selectivity,
and fluorescence is increased by both dsDNA and G4-DNA,
while the four styryl modified dyes show impressive fluorescent
signal discrimination that is specific to G-quadruplex DNA. For
the modified TO compounds, both ssDNA and dsDNA
substrates give very weak fluorescent signal induction. The
binding behavior and specificity of 4a−4d toward a number of
nucleic acids were evaluated. The experiments were conducted
using fluorescence titration with 20 targeted nucleic acids
including single-stranded DNA (ssDNA), double-stranded
34
DNA (dsDNA), G-quadruplex DNA (G4-DNA), and RNA.
Generally, as shown in Figure 1, the fluorescence intensity of
the TO dyes after modification with a styryl substituent are
B
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Figure 1. Fluorescence intensities at 630 nm (λex = 475 nm) of 4a−4d and TO with different nucleic acids in Tris−HCl buffer containing 60 mM
KCl. Single-stranded DNA: da21, dt21. Duplex DNA: 4a4t, 4at, ds12, and ds26. Telomere G-quadruplex DNA: htg22, telo21, 4telo, human 12,
oxy12, and oxy28. Promoter G-quadruplex DNA: bcl2, ckit-1, ckit-2, Pu27, Pu18, RET, and VEGF. Concentration of dye was 5 μM, and DNA
concentration was 10 μM. The mixture solutions were 10 μM dt21, 10 μM ds26, 10 μM telo21, and 10 μM bcl2. The fluorescence intensity is the
average of three repeated tests.
preferentially retained with G-quadruplexes’ DNA over dsDNA
and ssDNA (Figure S6). However, their fluorescence intensities
induced upon interaction were found to be quite varied. It
seems that for performance of DNA substrate discrimination in
terms of induced fluorescence signal changes, compound 4a is
the best, and the remaining are in the following order: 4b > 4c
dyes show preference to bind with G-quadruplex DNA over
single-stranded DNA, duplex DNA, and RNA. As shown in
Figure 2, 4a exhibits much better performance than its
analogues 4b−4d. We believe this is a consequence of the
effect of the substituent at the 2 position of TO. The binding
isotherm of 4a with telo21 quadruplex (up to 8 μM) was
recorded in the presence of a large excess of duplex DNA
(ds26, 100 μM; Figure 2, 4a). It seems that the interference
from ds26 is very minor; although ds26 leads to a slight
increase in the fluorescence signal, the addition of G-
quadruplex DNA (telo21, 6 μM) for binding with the dye
results in a remarkable induction of fluorescence intensity,
more than 6 fold, compared to duplex DNA ds26 at 100 μM.
The finding reveals that the telo21 quadruplex is able to
displace the bound ds26 very effectively and induces a much
stronger fluorescent adduct complex with 4a in situ (Figure S7).
The in vitro results prove that the new TO dyes are able to
show high G-quadruplex binding ability in the competitive
biological assay conditions.
>
4d. Both 4c and 4d show poor differential ability for
discriminating between dsDNA and G-quadruplex DNA. From
the screening tests, it is interesting that TO showed a very
stable response for DNA substrates in the same types of DNA,
while 4a gave a fluctuation of response toward various G4-DNA
substrates; for instance, the fluorescent intensities for telo 21
and oxy 28 were largest in Telomere G4-DNA and less
intensive signals for promoter G4-DNA. The observation may
imply that the introduced styrene-like substituents at the ortho-
position of TO show significant influence on binding
preference due to the difference of binding orientation in the
G4-DNA pockets, which can be further elaborated in the
modeling study.
The binding kinetics of the dyes with different concen-
trations of DNA substrates including single-stranded DNA
da21), duplex-stranded DNA (ds26), G-quadruplex DNA
telo21), and RNA were performed for comparison (Figure 2).
The compounds 4a−4d were designed with different
substituents at the ortho-position of the TO structure,
including 4-(dimethylamino)styryl, 2-vinyl-indole, 4-dimethyla-
mino-phenyl-buta-1,3-dienyl, and 4-methylstyryl, to allow for a
comparative study. These modifications may result in an
improved understanding of the induction of the fluorescence
signal and changes in the binding specificity of these modified
(
(
The concentration of dyes was kept constant at 5 μM in a
Tris−HCl buffer containing 60 mM KCl. The fluorescence
signal was measured at 630 nm (λ = 475 nm). In general, the
ex
C
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Figure 2. Binding kinetics of the dyes with different concentrations of DNA substrates: single-stranded DNAs da21, duplex-stranded DNAs ds26, G-
quadruplex DNA telo21 and RNA. The concentration of dyes was 5 μM in Tris−HCl buffer containing 60 mM KCl. The fluorescence signal was
measured at 630 nm (λ = 475 nm) at 25 °C.
ex
TO dyes toward G-quadruplex DNA rather than duplex DNAs.
The TO with a 4-(dimethylamino)styryl substituent (4a and
Table 1. Spectroscopic Data of 4a−d and Their Binding
Properties with telo21
4
c) demonstrated better selectivity in terms of induced
a
Ex
Em
(λmax
nm)
c
d
fluorescent signal intensity; however, with a longer distance
of the styryl substituent away from the TO structure (4c), a
reduction in the induced fluorescence signal was found (Figures
absorbance
(λmax, nm)
(λmax
nm)
,
,
LOD
K
(×
d
−1
b
5
ligand
Φf
(nM)
10 M )
4a
4b
4c
473
465
474
535
475
474
478
540
630
584
535
620
0.170
0.120
0.103
0.054
2.55
3.08
5.86
8.52
9.65
7.71
5.38
2.33
1
and 2). The reasons leading to the reduction of fluorescence
can be further explained by modeling studies of the substituent
effects, as impacted by molecular interactions and orientation
between the dye and the DNA substrate.
4
d
a
Experiments were performed in a 10 mM Tris-HCl buffer at pH 7.4.
E is the maximum excitation wavelength; E is the maximum emission
wavelength. Relative fluorescence quantum yield of probes upon the
addition of 50 μM telo21; standard of the relative fluorescence
quantum yield is fluorescein (Φ = 0.85, methanol with 1% NaOH).
Limit of detection for telo21. Equilibrium binding constant between
2
.3. Spectroscopic Property, Binding Affinity, and
x
m
b
Stabilization Studies of the Dyes with G-Quadruplex
DNA. The spectroscopic properties of 4a−4d are shown in
Table 1 and Table S2. The dyes structurally possess a similar
conjugation scaffold, and thus the absorption maxima of the
compounds in a Tris-HCl (10 mM, pH 7.4) buffer were found
in the range of 465−535 nm, and the emission maxima of the
dyes upon binding with telo21 were found in the range of 535−
f
c
d
the compound and telo21 at 25 °C.
trend may imply that the introduced substituents possibly lead
to quite different interaction orientation and environment and
that these could be the key sources of the resultant specificity.
630 nm. This shift of absorption and emission maxima among
the dyes is probably due to the effects of the differing
substituents on the TO. Interestingly, there is an obvious trend
observed in the relative fluorescence quantum yield (Φ ), limit
of detection (LOD), and equilibrium binding constant (K ) of
We believed that the K values are most probably depending on
d
the binding orientation and also the extra interactions with the
introduced side groups. In addition, the position of the side
groups, inside or projected out of the G-quartet pocket,
f
d
the dyes when interacting with telo21: 4a > 4b > 4c > 4d. This
influences the Φ value. The docking study in the latter part
f
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Figure 3. (A) A typical fluorescence titration spectrum of dye 4a with telo21 in a Tris-HCl buffer (10 mM, pH 7.4). The final equivalent amount of
telo21 added was 2 with respect to 4a. (B) Fitted curve and linear relationship of 4a with 0−1500 nM telo21. F represents the fluorescence intensity
0
without telo21, and F represents the fluorescence intensity after adding telo21.
Figure 4. (A) Stabilization of G-quadruplex F21T and duplex-stranded F10T (0.4 μM) by compounds 4a−4d (2.0 μM) in 10 mM Tris−HCl, 60
mM KCl, pH = 7.4. (B) Normalized FRET melting curves of G-quadruplex F21T (0.4 μM) at different concentrations of compound 4a (0.2−2.0
μM) in 10 mM Tris−HCl, 60 mM KCl, pH = 7.4.
provides more information on the trends. By comparing the
effects of substituents of 4-(dimethylamino)styryl (4a) and 4-
methylstyryl (4c) on TO framework, 4a shows 3 times higher
fluorescence signal and the concentration of telo21 was
obtained (Figure 3B: the inserted graph, R = 0.9933). The
results suggest that the dye is a useful and selective quadruplex
DNA sensor for quantitative determination and bioanalysis.
The analytical parameters for limit of detection (LOD) and
2
fluorescence quantum yield and a K value with a much lower
d
LOD value than that of 4c. These results are also found to be in
accord with the fluorescent signal enhancement (induced signal
intensity: Figures 1 and 2) due to the substituent on the TO
molecule. The mechanism for the fluorescent signal induction
is possibly due to the suppression of free rotation of the
thiazole fragment and the vibrational motions of the molecule
when it is bound inside the pocket of the DNA substrate.
Human telomeric sequence telo21 (5′-GGGTTAGGGT-
TAGGGTTAGGG-3′) is known to form different types of G-
equilibrium binding constant (K ) for 4a−4d were determined
d
and summarized in Table 1. Among the new compounds
examined, LOD values vary from 2.55 nM to 8.52 nM, and 4a
exhibits the best performance with the lowest LOD (2.55 nM)
5
−1
and highest K (9.65 × 10 M ). Both data indicate the
d
compound has very high G-quadruplex DNA binding
specificity.
To further understand the binding properties of the dyes on
the stabilization ability for the G-quadruplex structure,
fluorescence resonance energy transfer (FRET) measurement
was carried out, since FRET is a quantitative, rapid, and
convenient method to identify promising G-quadruplex ligands
3
6
quadruplex structures under certain incubation conditions.
For example, antiparallel G-quadruplexes are formed in Na
+
solution and hybrid-type G-quadruplexes are formed in K+
solution. From the fluorescence titration studies with the
addition of various concentrations of telo21 into 4a−4d
solutions buffered with Tris-HCl, a progressive increase in
fluorescence intensity (Figure 1) was observed. The induced
signal (fluorescence intensity) illustrates the effectiveness of the
interaction taking place for the dyes with telo21. The results are
also directly related to how significantly these substituents have
effects and influence on the molecular interaction, which leads
to remarkable discrimination ability toward various DNA
substrates. This can be further elaborated by comparison to
docking results described in the next section.
3
7−39
and binders.
Figure 4 shows the ΔT values of F21T and
m
F10T with different concentration of 4a−4d in a 60 mM
potassium solution. The ΔT of F21T increased from 5 to 30
m
°C with the compounds at 2.0 μM (Figure 4A). The ΔT of
m
F10T increased from 0.2 to 0.8 °C with the compounds at 2.0
μM (Table S4). The large ΔT (F21T) values compared with
m
ΔT (F10T) values of the dyes are generally observed in the
m
experiments, because the compounds are able to bind tightly
with G-quadruplex DNA, and thus the G-quadruplex structure
is further stabilized to give higher ΔT values. Among the four
m
A typical fluorescence titration experiment of 4a with G-
quadruplex DNA is shown in Figure 3. The background
fluorescence signal at 630 nm is very weak. A significant
emission is induced upon the addition of telo21, and a plateau
is reached at 6 μM. A good linear relationship for the induced
new ligands tested, the difference in ΔT (F21T) values found
m
are very distinct and in the order of 4a > 4b > 4c > 4d. This
further establishes the impact and critical role that the
substituent on the TO molecule plays in the interaction with
the G-quadruplex.
E
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Figure 5. CD spectra of the dyes 4a−4d bound to telo21 (5 μM) in 10 mM Tris−HCl, 60 mM KCl, pH = 7.4.
Figure 6. Top view (A, B) and side view (C, D) of the structures of Telo21 G-quadruplex in complex with TO (A, C) and 4a (B, D) from molecular
docking studies.
Circular dichroism (CD) spectroscopy is a useful technique
to study nucleic acid conformation and is widely used to study
the G-quadruplex conversion induced by G-quadruplex binders.
In the absence of TO compounds, the CD spectrum of telo21
was typical of an antiparallel G-quadruplex, with the positive
is consistent with the observation obtained from fluorescence
titration experiments.
2.4. Modeling Study to Investigate the Influence of
Styryl Substituents on the Fluorescent Discrimination
Ability for G-Quadruplex DNA. The above experimental
results show that the modified dyes are giving very good
fluorescent signal discrimination to G-quadruplex DNA, which
is not the same as their parent TO dye being nonselective for
dsDNA and G-quadruplex DNA (Figures 1 and S6). In other
words, the substituent on the TO molecule may either weaken
the interaction with dsDNA/ssDNA or may result in the loss of
fluorescence even if strong binding happens. To better
understand how the structural changes lead to fluorescent
discrimination on nucleic acid binding, molecular docking
studies were performed to elucidate the interaction difference
7
,40
peak at 290 nm and a negative peak at 265 nm.
With the
addition of 4a−4d (15 μM) to telo21 (5 μM), the characteristic
peaks of the antiparallel G-quadruplex had an obvious change,
with the positive peak at 290 nm gradually increasing while the
negative peak at 265 nm gradually decreased as shown in Figure
5. The results indicate that the dyes have no influence on the
conformation of G-quadruplexes. However, a slight enhance-
ment of the positive peak observed may be attributed to the
stabilization of G-quadruplexes by the dye. Also, the results
suggest that the dyes are able to bind to G-quadruplexes, which
F
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between the dye and G-quadruplex DNA as well as duplex
respect to our docking results, the TO molecule is almost
completely embedded in the 5′-CpT-3′ binding site of the
dsDNA. Interestingly, 4a also intercalates in the 5′-CpT-3′
binding site in a very close orientation to TO; however, its
substituent p-(dimethylamino)styryl at the ortho-position of
TO completely projects out of the binding pocket without any
interaction with duplex DNA (Figure 7B). The substituent is
likely to interact with solvent molecules and thus leads to
negative effects on both binding affinity and fluorescence signal
due to its loosely bound manner as compared to the situation
when bound with G-quadruplex (Figure 6B,C). The docking
results indicate that the dye does have interactions with the
dsDNA and also explains why the dyes induce a weak
fluorescence signal in the study with dsDNA substrates. Similar
results were also observed for 4b−4d.
DNA. Previous studies reported that TO prefers to stack on the
4
1,42
G-quartet,
which is in accord with our docking results
shown in Figure 6A,C.
As compared to TO, 4a with a p-(dimethylamino)styryl
substituent also stacks on the G-quartet in a very similar
fashion, except that the styrene substitution contributes
additionally to the stacking interaction with the G-quadruplex
(Figure 6B,D) and its dimethylamine group is very close to the
side of the G-quartet pocket. Previously, a NMR spectroscopy
study suggested that a homodimeric thiazole orange dye (TO)
acted as a duplex DNA intercalator with the TO chromophore
43
stacking in the 5′-CpT-3′ binding site (Figure 7A). With
The comparison of the binding modes of the dyes on the G-
quartet binding pocket of the telo21 G-quadruplex also gives
meaningful information on how the substituents disturb the
binding affinity. Figure 8 shows that the TO moiety is located
in the center part of the G-quartet binding pocket, and the
substituents with similar structure (p-(dimethylamino)styryl
(
4a), p-dimethylamino-phenyl-buta-1,3-dienyl (4c), and p-
methylstyryl (4d)) are lying in almost the same manner.
However, in 4b the 2-vinyl-indole substituent is located in quite
a different position, probably due to the bulky indole moiety.
The amine group on the substituents appears to be beneficial
for the interaction compared to 4-methylstyryl (4d). However,
the performance of 4a was found to be better than 4c, because
the substituent on 4c is extended by two methylene units,
resulting in the p-(dimethylamino)styryl moiety being located
outside of the G-quartet binding pocket of telo21. In general,
the binding modes found by the docking studies are able to
explain the experimental observations on the intensity of the
binding properties.
Figure 7. (A) A duplex DNA in complex with homodimeric thiazole
orange dye (TO) from NMR spectroscopy studies, which shows that
the TO chromophore is stacking in the 5′-CpT-3′ binding site. (B)
The same duplex DNA in complex with 4a from molecular docking
studies.
Figure 8. Comparison of binding modes of the dyes on the G-quartet binding pocket of telo21 G-quadruplex: (A) 4a, (B) 4b, (C) 4c, and (D) 4d.
The interpolated charge of G-quadruplex was computed by DS viewer 3.5.
G
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.5. The Application of the Dye As a Fluorescent
Articles
2
PAGE gels with a 5 μM solution of 4a is able to selectively
visualize the bands corresponding to G-quadruplexes including
htg2, oxy28, telo21, bcl-2, and bcl-1. In contrast, almost no
fluorescence was observed in the bands containing duplex-
stranded DNA (ds26) and single-stranded DNA (dt21). The
results highlight the potential of using 4a as a G-quadruplex-
selective fluorescent staining agent.
2.6. Application of the Dye in Live Cell Staining and
Imaging. The application of the dye as a selective staining
agent for the detection and imaging of G-quadruplexes in
human prostate cancer cells PC3 was investigated. Fluorescence
microscopy of the treated PC3 cells showed that both 4a and
DAPI are able to induce a strong fluorescence response in
certain regions of the nucleus (Figure 10). The stained region
corresponds to the nucleoli where rDNA undergoes tran-
scription. It has been reported that guanine-rich rDNA may
Staining Agent in Native Polyacrylamide Gel Electro-
phoresis (PAGE). The significant and selective fluorescence
enhancement of the dyes when bound with quadruplex DNA
structures led us to investigate the feasibility of using the dyes
as fluorescent stains in native polyacrylamide gel electro-
phoresis (PAGE). Quadruplex DNA is poorly visualized in
native gel electrophoresis by common fluorescent stains such as
ethidium bromide, GelRed, or SYBR Safe. The experiments
were performed using 4a, since it has the best performance
between the TO analogues. Figure 9 shows that poststaining of
44,45
also adopt temporal quadruplex conformations.
Moreover,
from the costaining experiments with DAPI (a duplex DNA
binder), a low fluorescence response was observed due to the
nonspecific binding of the dye to duplex DNA. However, we
cannot completely rule out the possibility that this weak signal
is coming from the binding of 4a with RNAs in the nucleus. For
further clarification, experiments were performed whereby, after
staining with 4a and DAPI, the cells were subsequently treated
with deoxyribouclease (DNase) or ribonuclease (RNase). The
enhanced fluorescence signals of 4a in nucleoli clearly
disappeared after DNase treatment, but not after RNase
treatment (Figure 10). Therefore, this supports the premise
that the dye enters the nucleus and interacts with the G-
quadruplexes, resulting in the strong fluorescence signal. The
results demonstrated that the new dyes have great potential for
live cell staining and imaging, for the purpose of monitoring G-
quadruplex DNA.
2
.7. Investigation of Photostability. Photostability is an
Figure 9. Gel electrophoresis (20% acrylamide in 1 × TBE) of htg22,
oxy28, telo21, bcl-2, ckit-2, dt21, and ds26 at a concentration 5.0 μM
stained with SYBR green (top) and 4a (bottom).
important factor when evaluating the usefulness of new
fluorescent probes for live cell imaging. Since 4a exhibited
good cell tolerability (IC50 was about 0.598 μM) at imaging
Figure 10. Fluorescence images of PC3 cells (fixed) stained with 5.0 μM of 4a for 15 min and 1.0 μg/mL DAPI for 5 min without and with DNase
or RNase treatment. 1000× magnification was utilized in the imaging. Scale bar is 10 μm.
H
DOI: 10.1021/acschembio.5b00987
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
Figure 11. Photostability demonstration: The time-lapse imaging of nucleolus DNA in PC3 cells with 4a in the range of 0−3 h. 1000× magnification
was utilized in the imaging. Scale bar is 10 μm.
concentration after 24 h of incubation, we next examined the
photostability of 4a in cell imaging. Figure 11 shows the
photostability of 4a over 3 h when using PC3 cells. After 3 h,
the fluorescence signal from 4a in the nucleoli of PC cells was
retained with sufficient intensity for clear visualization. The
results demonstrated that 4a is a robust dye for imaging of
nucleoli G-quadruplex DNA in live cells and is particularly
attractive for experiments requiring longer irradiation times.
Conclusions. In conclusion, a series of new fluorescent dyes
were synthesized by introducing different styryl moieties at the
ortho-position of thiazole orange. The compounds show very
good fluorescence discrimination toward G-quadruplex DNA
rather than dsDNA, exhibit high relative quantum yields upon
binding with G-quadruplexes, and give excellent G-quadruplex
NMR (400 MHz, DMSO-d
8
7
6
): δ 8.77 (d, J = 8.3 Hz, 1H), 8.18 (d, J =
.7 Hz, 1H), 8.02−7.96 (m, 2H), 7.74 (d, J = 8.2 Hz, 2H), 7.59 (t, J =
.7 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.34 (s, 1H), 6.85 (s, 1H), 4.07
+
(
s, 3H), 3.98 (s, 3H), 2.87 (s, 3H). ESI-MS: m/z 319.0 [M − I] .
General Procedures for Synthesis of Target Compounds
4
a−4d. A mixture of 3 (0.072 g, 0.16 mmol), 4-methylpiperidine (0.5
mL), and n-butanol (10 mL) was stirred at RT. The chosen aldehyde
0.32 mmol) was added into the mixture and refluxed for 3 h. After the
(
mixture was cooled and suction filtered, the solid was washed with n-
butanol. The precipitate was purified by using column chromatography
to afford the pure target compounds 4a−4d.
Synthesis of N-Methyl-2-((E)-4-(dimethylamino)styryl)-4-((Z)-(3-
methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium Iodide
1
(
4a). Purple solid, yield 90%. mp = 301−304 °C. H NMR (400 MHz,
DMSO-d ): δ 8.73 (d, J = 7.8 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 8.06−
8
(m, 3H), 7.64 (s, 1H), 7.61−7.55 (m, 2H), 7.42−7.35 (m, 3H), 6.87
(
6
.02 (m, 1H), 8.00−7.95 (m, 1H), 7.87 (d, J = 8.5 Hz, 2H), 7.76−7.68
stabilization as indicated by the larger ΔT values. A modeling
m
1
3
study on the interaction of the dyes with DNA illustrates the
influence of the introduced ancillary substituent at the ortho-
position of thiazole orange on the binding discrimination
between dsDNA and G-quadruplex DNA. In addition, the
potential application of the dyes as in vitro biochemical reagents
was successfully demonstrated by native PAGE experiments
and live cell staining and imaging. The results indicate that
these new dyes can be utilized for G-quadruplex specific
biomolecular sensing and cell imaging.
s, 1H), 4.13 (s, 3H), 3.97 (d, J = 3.7 Hz, 3H), 2.56 (s, 6H). C NMR
(100 MHz, DMSO-d ): δ 158.77, 153.24, 152.30, 147.53, 142.83,
6
141.10, 139.57, 133.43, 130.97, 128.40, 126.63, 125.57, 124.30, 124.08,
123.92, 123.38, 123.09, 118.93, 115.07, 112.69, 112.16, 108.03, 87.58,
+
+
38.28, 33.91. HRMS (ESI) m/z calcd for C H N S ([M − I] ):
450.1998. Found: 450.1986. HPLC retention time of 4a was 5.439
29 28
3
min.
Synthesis of 2-((E)-2-(1H-Indol-3-yl)vinyl)-1-methyl-4-((Z)-(3-
methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium Iodide
1
(
4b). Purple solid, yield 85%. mp = 298−302 °C. H NMR (400 MHz,
DMSO-d ): δ 11.97 (s, 1H), 8.62 (d, J = 8.4 Hz, 1H), 8.20 (s, 1H),
6
METHODS FOR LIGAND SYNTHESIS
Synthesis of 4-Chloro-1,2-dimethylquinolin-1-ium Iodide
1). To the solution of 4-chloro-2-methylquinoline (0.2 g, 1.12
mmol) in sulfolane (10 mL) was added iodomethane (0.42 mL, 6.74
mmol). The reaction mixture was stirred at 50 °C for 18 h and then
cooled. Anhydrous ether was added to the reaction mixture and the
solids collected by suction filtration, washed with anhydrous ether, and
8.09 (d, J = 7.3 Hz, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.98 (d, J = 7.8 Hz,
■
1
H), 7.88 (dd, J = 15.5, 5.5 Hz, 2H), 7.63 (t, J = 7.6 Hz, 1H), 7.51 (q,
J = 8.4 Hz, 4H), 7.33−7.23 (m, 4H), 6.70 (d, J = 8.5 Hz, 1H), 4.07 (s,
3H), 3.83 (s, 3H). 13C NMR (100 MHz, DMSO-d
): δ 158.38, 153.65,
(
6
147.23, 140.96, 139.40, 136.84, 133.30, 128.29, 126.47, 125.49, 125.13,
124.14, 123.92, 123.80, 123.38, 123.26, 121.71, 120.70, 118.78, 114.21,
113.98, 113.05, 112.54, 107.63, 87.39, 38.20, 33.84. HRMS (ESI) m/z
+
+
dried in vacuo to give compound 1 (0.36 g, 95.8%). mp = 245−247 °C.
calcd for C29H N S ([M − I] ): 446.1685. Found: 446.1674. HPLC
24 3
1
H NMR (400 MHz, DMSO-d ): δ 8.56 (d, J = 8.4 Hz, 1H), 8.46 (d, J
retention time of 4b was 3.608 min
6
=
(
−
8.3 Hz, 1H), 8.22 (t, J = 8.1 Hz, 1H), 8.01 (t, J = 7.9 Hz, 1H), 7.55
Synthesis of 2-((1E,3E)-4-(4-(Dimethylamino)phenyl)buta-1,3-
dien-1-yl)-1-methyl-4-((Z)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)-
s, J = 7.4 Hz, 1H), 4.20 (s, 3H), 2.68 (s, 3H). ESI-MS: m/z 192.1 [M
+
methyl)quinolin-1-ium Iodide (4c). Purple solid, yield 88%. mp =
I] .
1
2
1
89−293 °C. H NMR (400 MHz, DMSO-d ): δ 8.70 (d, J = 8.5 Hz,
Synthesis of 1,2-Dimethylbenzothiazol-1-ium Iodide (2). A
mixture of 2-methylbenzothiazole (0.25 g, 1.68 mmol), iodomethane
0.63 mL, 10.08 mmol), and anhydrous ethanol (10 mL) was stirred at
reflux temperature for 15 h. After cooling, the mixture was dried over
anhydrous ethanol and chloroform oscillating suction filtered. The
precipitate was washed with chloroform and with a small amount of
6
H), 8.12 (d, J = 8.9 Hz, 1H), 8.07 (d, J = 7.9 Hz, 1H), 7.95 (t, J = 7.8
Hz, 1H), 7.73−7.68 (m, 2H), 7.59 (dd, J = 13.5, 7.0 Hz, 3H), 7.49 (d,
J = 8.7 Hz, 2H), 7.39 (t, J = 7.6 Hz, 1H), 7.17 (dd, J = 24.7, 13.8 Hz,
(
3
(
H), 6.83−6.76 (m, 3H), 4.09 (d, J = 8.5 Hz, 3H), 3.96 (s, 3H), 3.00
d, J = 11.7 Hz, 6H). 13C NMR (100 MHz, DMSO-d ): δ 158.75,
6
1
1
1
52.13, 151.49, 147.13, 143.68, 142.22, 140.98, 139.42, 133.35, 129.38,
28.39, 126.59, 125.49, 124.30, 124.08, 124.00, 123.81, 123.62, 123.41,
20.93, 118.77, 112.78, 112.53, 107.66, 87.68, 37.99, 33.94. HRMS
ethanol and vacuum-dried to give compound 2 (0.448 g, 91.7%). mp =
1
2
1
32−235 °C. H NMR (400 MHz, DMSO-d ): δ 8.44 (d, J = 8.1 Hz,
6
H), 8.30 (d, J = 8.4 Hz, 1H), 7.90 (t, J = 7.8 Hz, 1H), 7.81 (t, J = 7.7
+
+
+
Hz, 1H), 4.20 (s, 3H), 3.17 (s, 3H). ESI-MS: m/z 164.4 [M − I] .
(ESI) m/z calcd for C31
476.2144. HPLC retention time of 4c was 4.889 min.
H
30
N
3
S
([M − I] ): 476.2154. Found:
Synthesis of (Z)-1,2-Dimethyl-4-((3-methylbenzo[d]thiazol-
2
(
(3H)-ylidene)methyl) Quinolin-1-ium Iodide (3). Compound 1
Synthesis of 1-Methyl-4-((Z)-(3-methylbenzo[d]thiazol-2(3H)-
0.5 g, 1.60 mmol), 2 (0.5 g, 1.72 mmol), and aqueous sodium
ylidene)methyl)-2-((E)-4-methylstyryl)quinolin-1-ium Iodide (4d).
1
bicarbonate solution (0.5 mol/L, 2 mL) were mixed with 10 mL of
methanol. The mixture was stirred at RT for about 1 h. To the
reaction solution, 4 mL of saturated KI solution was added. The
mixture was then stirred for about 15 min. The solid obtained was
collected by filtration, washed with water and acetone, and vacuum-
Purple solid, yield 83%. mp = 273−276 °C. H NMR (400 MHz,
DMSO-d ): δ 8.77 (d, J = 8.3 Hz, 1H), 8.19 (d, J = 8.8 Hz, 1H), 8.07
6
(d, J = 7.8 Hz, 1H), 8.00 (t, J = 7.9 Hz, 1H), 7.85 (d, J = 7.9 Hz, 2H),
7.75 (dd, J = 8.7, 5.6 Hz, 3H), 7.63 (dt, J = 15.6, 9.9 Hz, 3H), 7.43−
7.34 (m, 3H), 6.92 (s, 1H), 4.17 (s, 3H), 4.00 (d, J = 8.1 Hz, 3H), 2.41
1
13
dried to give compound 3 (0.49 g, 92.5%). mp = 268−270 °C. H
(s, 3H). C NMR (100 MHz, DMSO-d ): δ 159.88, 152.60, 148.26,
6
I
DOI: 10.1021/acschembio.5b00987
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
1
1
1
41.32, 141.05, 140.79, 139.52, 133.74, 132.99, 130.03, 128.95, 128.55,
26.97, 125.69, 124.69, 124.27, 124.01, 123.48, 121.10, 119.03, 113.14,
08.44, 88.27, 38.53, 34.12, 21.57. HRMS (ESI) m/z calcd for
(5) Parkinson, G. N., Lee, M. P., and Neidle, S. (2002) Crystal
structure of parallel quadruplexes from human telomeric DNA. Nature
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+
+
C H N S ([M − I] ): 421.1732. Found: 421.1721. HPLC retention
time of 4d was 3.384 min.
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2
8
25
2
Modeling Method. The crystal structure of telo21 G-quadruplex
46
DNA 5′-d[GGG(TTAGGG)3]-3′ (PDB id 4DA3) in complex with
naphthalene diimide ligands was used as the G-quadruplex target. The
NMR structure of duplex DNA 5′-d(CGCTAGCG)-3′ in complex
with homodimeric thiazole orange dye (TO) (PDB id: 108D) was
used for the modeling of intercalation interaction. Docking studies
were performed using the AUTODOCK 4.0 program. The
dimensions of the active site box were chosen to be large enough to
encompass the entire G-quadruplex DNA and the active site of duplex
DNA. All docking calculations were carried out using the Lamarckian
genetic algorithm (LGA). A maximum number of 2 500 000 energy
evaluation was used. Each docking experiment includes 200
independent runs.
(
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2
(
4
7
4
(
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(
ASSOCIATED CONTENT
■
telomeric G-overhang are more than simple telomerase inhibitors.
Curr. Med. Chem.: Anti-Cancer Agents 4, 439−443.
*
S
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.5b00987.
(12) Dunnick, W., Hertz, G. Z., Scappino, L., and Gritzmacher, C.
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Additional experimental details, Figures S1−S10, and
Tables S1−S3 (PDF)
(
in Escherichia coli global regulation. Genome Res. 16, 644−655.
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AUTHOR INFORMATION
Corresponding Authors
Tel.: +86-20-39322235. E-mail: luyj@gdut.edu.cn.
Tel.: +852-2948-8401. Fax: +852-2948-7676. E-mail:
wingleung@ied.edu.hk.
Notes
The authors declare no competing financial interest.
■
1
5908−15912.
15) Rezler, E. M., Bearss, D. J., and Hurley, L. H. (2002) Telomeres
and telomerases as drug targets. Curr. Opin. Pharmacol. 2, 415−423.
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2002) Molecular modelling and cytotoxicity of substituted
anthraquinones as inhibitors of human telomerase. Curr. Pharm. Des.
, 2491−2504.
17) Rezler, E. M., Seenisamy, J., Bashyam, S., Kim, M. Y., White, E.,
*
(
*
(
(
8
(
ACKNOWLEDGMENTS
We acknowledge the support received from the National
Nature Science Foundation of China (21102021 and
■
Wilson, W. D., and Hurley, L. H. (2005) Telomestatin and diseleno
sapphyrin bind selectively to two different forms of the human
telomeric G-quadruplex structure. J. Am. Chem. Soc. 127, 9439−9447.
8
1473082), Nature Science Foundation of Guangdong
(18) Ou, T. M., Lu, Y. J., Tan, J. H., Huang, Z. S., Wong, K. Y., and
Province, China (S2012010010200), and Science and Tech-
nology Program of Guangdong Province (2012B020306007).
The authors are also grateful to the support from Guangdong
Province Higher Education “Qianbaishi Engineering” project,
and Guangdong University of Technology “Pei Ying Yu Cai”
Project. We thank the Shared Hierarchical Academic Research
Computing Network (SHARCNET, www.sharcnet.ca) for
allocation of computer resources. J.-Q.H. thanks Prostate
Cancer Canada for fellowship support.
Gu, L. Q. (2008) G-quadruplexes: Targets in anticancer drug design.
ChemMedChem 3, 690−713.
(19) Fu, R., Li, T., and Park, H. G. (2009) An ultrasensitive
DNAzyme-based colorimetric strategy for nucleic acid detection.
Chem. Commun. 39, 5838−5840.
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luminescent switch-on molecular probe. Analyst 140, 5998−6004.
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DOI: 10.1021/acschembio.5b00987
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