Development of a two-photon carbazole derivative probe for fluorescent visualization of G-quadruplex DNA in cells

Article abstract of DOI:10.1016/j.dyepig.2019.107749

G-quadruplex (G4) sequences are considered to play important roles in gene regulation, therefore the development of selective and sensitive probes for G4 DNAs is important for studying the functions of G-rich gene sequences, as well as designing of novel and effective anticancer drugs. Herein, a carbazole derivative (CZ-BT) was synthesized and characterized by a simple process for G4 DNA detection. CZ-BT was preferentially bound with G4 DNA compared with other types of nucleic acids according to the fluorescence assays. The fluorescence intensity and fluorescence lifetime of CZ-BT significantly increased after the interaction with G4 DNA. Molecular docking calculation proved the π-π stacking binding mode between CZ-BT and G4 DNA. Furthermore, the specificity of CZ-BT on G4 DNA was well demonstrated with the contrast of pyridostatin (PDS, a classical G-quadruplex ligand) using two-photon confocal fluorescent imaging technique and cell cycle experiment in cells. We believe that this study would give some favorable factors on developing of highly effective fluorescent probes for G4 DNA applications.

Full text of DOI:10.1016/j.dyepig.2019.107749

Accepted Manuscript
Development of a two-photon carbazole derivative probe for fluorescent visualization
of G-quadruplex DNA in cells
Fengli Gao, Shuhua Cao, Wen Sun, Saran Long, Jiangli Fan, Xiaojun Peng
PII:
S0143-7208(19)31202-1
DOI:
https://doi.org/10.1016/j.dyepig.2019.107749
Article Number: 107749
Reference:
DYPI 107749
To appear in: Dyes and Pigments
Received Date: 24 May 2019
Revised Date: 23 July 2019
Accepted Date: 23 July 2019
Please cite this article as: Gao F, Cao S, Sun W, Long S, Fan J, Peng X, Development of a two-
photon carbazole derivative probe for fluorescent visualization of G-quadruplex DNA in cells, Dyes and
Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107749.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
The molecule structure and docking calculation with G4 DNA of two-photon
carbazole based fluorescent probe (CZ-BT) which is specific on G4 DNA in cells.

ACCEPTED MANUSCRIPT
1
2
3
Development of a two-photon carbazole derivative probe for fluorescent
visualization of G-quadruplex DNA in cells
a
b
ac
ac
ac
ac
Fengli Gao,‡ Shuhua Cao,‡ Wen Sun, Saran Long,* Jiangli Fan,* and Xiaojun Peng,
4
5
a. State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, High-Tech district, Dalian
116024, China
6
7
8
9
0
b. College of chemistry, chemical and environmental engineering, Weifang University, No. 5147 Dongfeng Street, Weifang,
261061, China.
c. Shenzhen Research Institute, Dalian University of Technology, Nanshan District, Shenzhen, 518057, P.R. China.
E-mail: fanjl@dlut.edu.cn, srlong@dlut.edu.cn
1
1
‡ These authors contributed equally to this work.
1
2
3
4
5
6
7
8
9
0
1
2
3
Abstract: G-quadruplex (G4) sequences are considered to play important roles in gene
regulation, therefore the development of selective and sensitive probes for G4 DNAs is
important for studying the functions of G-rich gene sequences, as well as designing of novel
and effective anticancer drugs. Herein, a carbazole derivative (CZ-BT) was synthesized and
characterized by a simple process for G4 DNA detection. CZ-BT was preferentially bound
with G4 DNA compared with other types of nucleic acids according to the fluorescence
assays. The fluorescence intensity and fluorescence lifetime of CZ-BT significantly increased
after the interaction with G4 DNA. Molecular docking calculation proved the π-π stacking
binding mode between CZ-BT and G4 DNA. Furthermore, the specificity of CZ-BT on G4
DNA was well demonstrated with the contrast of pyridostatin (PDS, a classical G-quadruplex
ligand) using two-photon confocal fluorescent imaging technique and cell cycle experiment in
cells. We believe that this study would give some favorable factors on developing of highly
effective fluorescent probes for G4 DNA applications.
1
1
1
1
1
1
1
1
2
2
2
2
2
2
4
5
Keywords: two-photon; carbazole derivative; fluorescent probe; G-quadruplex DNA;
pyridostatin
2
6
1. Introduction
2
2
2
7
8
9
G-quadruplex DNAs (G4 DNAs) are noncanonical secondary DNA structures formed by
self-assembly of guanine rich nucleic acid sequences at the conditions of specific ionic
strength and pH values [1-2]. Owing to the direction of strands or parts of a strand that form

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
0
1
2
3
4
5
the tetrads, the structures of G4 DNAs are displayed as parallel, antiparallel, and mixed types
[3]. Researches reveal that G4 DNAs distribute in the regions which are closely related to the
gene functions, such as telomere and promoter regions [4-6]. They play an important role in
maintaining the stability of chromosome and are closely related to cancer initiation and
progression [7-11]. Therefore, it is of great biological significance for tracking and detecting
of G4 DNAs.
3
3
3
3
4
4
4
4
4
4
6
7
8
9
0
1
2
3
4
5
Up to now, several analytical tools are employed for the research of G4 DNAs, such as
electrochemistry [12-14], monoclonal antibodies [7,15], fluorescent probes [16-19], and so on.
Electrochemistry method is beneficial to miniaturization in terms of the instrumentation,
while the instable electrochemical signals lead to poor reproducibility. Monoclonal antibodies
have been implemented for G4 DNAs detection by means of secondary, labelled antibodies.
However, a number of issues were raised, including artefacts caused by chromatin fixation
and induced G4 formation [20]. In recent years, although some fluorescent small probes for
G4 DNAs have been developed due to their advantages of high sensitivity and low cost, some
of them with the short emission wavelength, poor selectivity and lack of relevant cell
experiments restricted their further applications [21-23].
4
4
4
4
5
5
5
5
5
5
5
5
6
7
8
9
0
1
2
3
4
5
6
7
Due to the excitation source of long wavelength (>700 nm), the two-photon microscopy
can effectively avoid the interference of autofluorescence and has widely been used for
imaging cells and tissues [24-26]. While the correlative research of two-photon fluorescent
probes on G4 DNAs are less [27-28]. Herein, with excellent optical properties, a novel
two-photon carbazole derivative probe (CZ-BT) for G4 DNA was designed and synthesized
(Scheme 1). In this probe, the conjugated complex of carbazole-benzothiazole possessed an
extended delocalized π-electron system which could interact with G4 DNA by π-π stacking.
The diethyl amine group was introduced to increase the hydrophilic and positive charged
benzothiazole was more easily to contact with DNA. Molecular docking calculation validated
the π-π stacking binding mode between CZ-BT and G4 DNA. The two-photon confocal
fluorescent images and cell cycle experiment of CZ-BT and PDS fully proved the specificity
on G4 DNA.

ACCEPTED MANUSCRIPT
5
5
8
9
2. Experimental section
2.1. Reagents and materials
6
6
6
6
6
6
6
6
6
6
7
7
0
1
2
3
4
5
6
7
8
9
0
1
Ultrapure water used in the whole experiment was from Milli-Q systems, other reagents
and solvents were of analytical grade without further purification. Tris-HCl (10 mM, pH=7.4,
60 mM KCl) buffer solution was used for the whole solution test. Silica column
chromatography was performed using silica gel (200-300 mesh, Qingdao Ocean Chemicals).
The G-quadruplet binding ligand pyridostatin (PDS) was purchased from Sigma-Aldrich.
Hoechst 33342, DNase and RNase were purchased from Thermo Fisher Scientific. MCF-7
and COS7 cells were obtained from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai,
China). The absolute fluorescent quantum yields of CZ-BT in various conditions were
recorded by Quanturus-QY (Hamamatsu C11347-11, Japan). MCF-7 and COS7 cells
fluorescent images were recorded on confocal laser fluorescent scanning microscope
(Olympus, FV1000, Japan). Cell cycle experiment was recorded on Acoustic focusing
cytometer of Thermo Fisher Scientific (Attune NxT).
7
7
2
3
2.2 DNA synthesis and purification
Table 1. Sequences of oligonucleotides used in the present study.
Name
dA21
dT21
ss26
Sequence
Structure
5`-d(AAAAAAAAAAAAAAAAAAAAA)-3`
5`-d(TTTTTTTTTTTTTTTTTTTTT)-3`
5`-d(ATACGATGCTTCACGGTGCTATCTG)-3` Single stranded
Single stranded
Single stranded
ssDNA
5`-d(GGATGTGAGTGTGAGTGTGAGG)-3`
Single stranded
Duplex
Duplex
Poly(A-T)9 5`-d(ATATATATATATATATAT)-3`
Poly(G-C)9 5`-d(GCGCGCGCGCGCGCGCGC)-3`
Hum24
Telo21
Oxy12
Pu22
5`-(TTAGGGTTAGGGTTAGGGTTAGGG)-3`
5`-d(GGGTTAGGGTTAGGGTTAGGG)-3`
5`-d(GGGGTTTTGGGG)-3`
G4 DNA
G4 DNA
G4 DNA
G4 DNA
5`-d(TGAGGGTGGGTAGGGTGGGTAA)-3`
7
7
7
4
5
6
All oligonucleotides used in this work were synthesized and HPLC purified by TaKaRa
Biotechnology Co., Ltd. (Dalian, China), and the sequences were listed in Table 1. The
original DNA sequences were dissolved in TE buffer and stored at -20°C for further use. To

ACCEPTED MANUSCRIPT
7
7
7
7
8
9
obtain DNA formation information, oligonucleotides were pre-treated in Tris-HCl buffer (10
mM, pH=7.4, containing 60 mM KCl) by raising the temperature to 95°C and keeping for 5
min, and then gradually cooling down to room temperature.
8
0
2.3. Synthetic procedures of CZ-BT
8
1
8
8
8
2
3
4
Scheme 1. Synthesis route of the G4 DNA probe CZ-BT: (a) acetone, KOH, rt, 24 h, 80%; (b) THF,
Na CO , N , reflux, 24 h, 65%; (c) DMF, POCl , 100, 4 h, 43%; (d) ethanol, piperidine, N , reflux, 10 h,
2
3
2
3
2
24%.
8
5
9-(4-bromobutyl)-9H-carbazole (1)
8
8
8
8
9
9
9
9
6
7
8
9
0
1
2
3
To a solution of carbazole (499 mg, 3 mmol) in 20 mL of acetone, 0.33 mL of
1,4-dibromobutane (3 mmol, 603 mg) and a catalytic amount of KOH were added. After
stirring for 24 hours at room temperature, the solvent was removed under reduced pressure.
Then the residue was purified by silica gel column chromatography (petroleum ether:
1
dichloromethane = 4:1, v/v) to afford the final compound of white solid (725 mg, 80%). H
NMR (400 MHz, CDCl ) δ 8.10 (s, 1H), 7.43 (dd, J = 24.4, 6.6 Hz, 2H), 4.34 (d, J = 5.7 Hz,
3
+
1H), 3.36 (d, J = 5.3 Hz, 1H), 2.24 – 1.78 (m, 2H); TOF HRMS: m/z calcd for C H NBr
1
6
16
+
M : 303.0549, found: 304.0552.
9
4
N, N-Diethyl-9H-carbazole-9-butanamine (2)
9
9
9
5
6
7
A reaction mixture which containing 9-(bromobutyl)-9H-carbazole (3.02g, 10 mmol),
diethylamine (1.0 mL, 10mmol) and a catalytic amount of Na CO in tetrahydrofuran (20 mL)
2
3
was refluxed for 24 hours within the protection of nitrogen. After cooling down to room

ACCEPTED MANUSCRIPT
9
9
8
9
temperature, the mixture was filtered, and the filtrate was extracted with ethyl acetate, dried
over Na SO , concentrated. The residue was purified with a silica gel column and was eluted
2
4
1
1
1
1
1
1
00
01
02
03
04
with dichloromethane and CH OH (50:1, v/v) to afford 1.91 g (65%) of the compound. H
3
NMR (400 MHz, MeOD) δ 8.07 (d, J = 7.8 Hz, 1H), 7.46 (dt, J = 14.5, 7.8 Hz, 2H), 7.18 (t, J
= 7.4 Hz, 1H), 4.42 (t, J = 6.9 Hz, 1H), 2.56 – 2.35 (m, 3H), 1.96 – 1.80 (m, 1H), 1.61 – 1.42
+
2
+
(m, 1H), 0.95 (t, J = 7.2 Hz, 3H). TOF HRMS: m/z calculated for C H N M : 294.2155,
2
0
26
found: 295.2161.
1
05
9-(4-(diethylamino) butyl)-9H-carbazole-3-carbaldehyde (3)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
06
07
08
09
10
11
12
13
14
15
16
17
18
19
To a solution of N, N-Diethyl-9H-carbazole-9-butanamine (1.61 g, 5 mmol) in DMF (5
mL), POCl (0.9 mL, 20 mmol) was added into the solution in ice-water bath under the
3
protection of nitrogen. The mixture was stirred vigorously for half an hour, then the
temperature was risen to 100°C and continued for 4 hours. The nigger-brown reaction mixture
was poured into a solution of sodium acetate (20% mass fraction) to neutralize the acidity and
then extracted with ethyl acetate. The solvent was dried with anhydrous sodium sulfate
overnight and evaporated on a rotary evaporation. The crude product was purified on a silica
gel column using dichloromethane and CH OH (40:1, v/v) as eluent, yielding 693 mg (43%)
3
1
of the desired product as light yellow power. H NMR (400 MHz, MeOD) δ 10.00 (s, 1H),
8.63 (d, J = 1.4 Hz, 1H), 8.18 (d, J = 7.4 Hz, 1H), 8.00 (dd, J = 8.6, 1.5 Hz, 1H), 7.66 (dd, J =
17.4, 8.4 Hz, 2H), 7.55 (q, J = 8.6 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 5.49 (s, 1H), 4.51 (t, J =
6.9 Hz, 2H), 3.06 (ddd, J = 16.6, 11.5, 6.4 Hz, 6H), 1.97 (dt, J = 14.6, 7.1 Hz, 2H), 1.76 (ddd,
+
J = 20.9, 10.2, 6.3 Hz, 3H), 1.21 (t, J = 7.3 Hz, 6H). TOF MS: m/z calculated for C H N O
2
1
26
2
+
M : 323.70, found: 323.78.
1
1
20
21
(E)-2-(2-(9-(4-(diethylamino)butyl)-9H-carbazol-3-yl)vinyl)-3-ethylbenzo[d]thiazol-3-ium
(CZ-BT)
1
1
1
22
23
24
To a 50 mL round bottom flask, 2-methyl-3- ethyl benzothiazole iodide (305 mg, 1 mmol)
and (E)-2-(2-(9-(4- (diethylamino) butyl) -9H- carbazole -3-yl) vinyl)-3- ethylbenzo [d]
thiazol -3-ium (322 mg, 1 mmol) were dissolved in ethanol (15 mL), then several drops of

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
25
26
27
28
29
30
31
32
33
34
piperidine was added. The reaction mixture was refluxed for 10 hours under the protection of
nitrogen. After cooling down to room temperature, the precipitate was filtered and purified by
silica gel column chromatography (dichloromethane: methanol = 18:1, v/v) to afford the brick
1
red powder (146 mg, 24%). H NMR (400 MHz, DMSO-d ) δ 8.98 (s, 1H), 8.46 – 8.37 (m,
6
1H), 8.33 – 8.18 (m, 1H), 8.03 (d, J = 15.5 Hz, 1H), 7.86 (dd, J = 18.3, 9.0 Hz, 1H), 7.75 (t, J
= 7.6 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.34 (t, J = 7.4 Hz, 1H), 5.00 (d, J = 7.0 Hz, 1H), 4.53
(s, 1H), 3.07 (d, J = 6.6 Hz, 3H), 1.46 (dd, J = 36.5, 29.4 Hz, 2H), 1.16 (dd, J = 17.8, 10.8 Hz,
1
3
4H); C NMR (100 MHz, DMSO-d ) δ 8.98, 8.44, 8.42, 8.40, 8.27, 8.25, 8.24, 8.05, 8.01,
6
7.89, 7.87, 7.85, 7.82, 7.77, 7.75, 7.58, 7.56, 7.54, 7.36, 7.34, 7.33, 1.52, 1.50, 1.48, 1.17, 1.15,
+
+
1.13. TOF HRMS: m/z calculated for C H N S M : 482.2620, found: 482.2625.
3
1
36
3
1
35
2.4. UV-vis and fluorescence spectroscopic studies
1
1
1
1
1
1
1
1
1
36
37
38
39
40
41
42
43
44
CZ-BT was dissolved in dimethyl sulfoxide (DMSO) to prepare a concentration of 1.0 mM
as the stock solution for further use. The UV-vis and fluorescence spectra were obtained on an
Agilent Cary 60 UV−vis spectrophotometer (G6860A) and Agilent Cary Eclipse fluorescence
spectrophotometer at room temperature. The absorption and emission spectra were performed
by fixed CZ-BT concentration (10.0 µM) and titrated with increasing oligonucleotides
concentrations using a 1.0 cm length quartz curette. Before recording the spectrum, each
oligonucleotide was added into the solution gently, and then stirred and allowed to equilibrate
for at least 1.0 min. Each experiment was carried out for three times in replicate
determination.
1
45
2.5. The two-photon properties of CZ-BT probe
1
1
1
1
1
1
46
47
48
49
50
51
It is difficult to measure the two-photon fluorescence collection efficiency of molecules
directly. Therefore, for comparing the two-photon induced fluorescence of unknown sample
and standard sample with known two-photon absorption cross section, the two-photon
absorption cross section of target molecule was obtained. The two-photon confocal
fluorescence microcopy was used as the test platform, and the sample was tested by infrared
pulse laser. In this work, the fluorescein of NaOH aqueous solution (pH=11) was chosen as

ACCEPTED MANUSCRIPT
1
1
52
53
reference [29], the two-photon absorption cross section of CZ-BT was calculated according
to the following formula:
1
54
δ = δ (S Φ ψ C ) / (S Φ ψ C )
s
r
s
r
r
r
r
s
s
s
1
1
1
1
1
1
55
56
57
58
59
60
The subscript of s and r represented sample and reference; δ was two-photon absorption cross
section; S indicated the fluorescence signal intensity recorded by charge coupled device
(CCD), that was the integral value of fluorescence intensity; Φ expressed the fluorescence
quantum yield; ψ showed the fluorescence collection efficiency of experimental device (ψr
=ψ ), C was the concentration of solution. In order to minimize the experimental error,
s
repeated three times for average.
1
1
1
1
61
62
63
64
For the double logarithmic plot of two-photon induced fluorescence integral intensity
against the average laser power, whether the signal measured was a simple two-photon
induced fluorescence could be confirmed. If the slope of the fitting line was close to 2, then
the signal was proved as two-photon absorption.
1
65
2.6. The lifetime measurements of CZ-BT
1
1
1
1
1
66
67
68
69
70
The lifetime experiments were carried out by the time-correlated single photon counting
(TCSPC) technique (PicoQuant PicoHarp 300) at room temperature. Using deconvolution/fit
program (PicoQuant FluFit), the time resolution was reached down to 10 ps. The second
harmonic of a titanium sapphire laser (Mai Tai DeepSee) at 400 nm (150 fs, 80 MHz) was
selected as excitation source.
1
71
2.7. Molecular modelling study of CZ-BT and G4 DNA
1
1
1
1
1
72
73
74
75
76
The crystal structure of human telomeric G4 DNA (Telo21, PDB: 4DA3) was used
as a receptor to perform docking study on CZ-BT. The 3D structures of CZ-BT were
sketched using chem3D. Autodock Tools (ver. 1.5.6) was used to convert the structure
files to pdbqt format. Docking calculation was performed using the AUTODOCK vina
program. The dimensions of the active site box were chosen to be large enough to

ACCEPTED MANUSCRIPT
1
1
77
78
encompass the entire G4 structures. An exhaustiveness of 100 was used and other
parameters were left as default.
1
79
2.8. Circular dichroism (CD) spectroscopy test
1
1
1
1
1
1
1
80
81
82
83
84
85
86
CD studies were performed on the Chirascan circular dichroism spectrophotometer
(Jasco J-810, Japan). The circular dichroism spectra of Telo21 and other DNAs were
recorded with and without the addition of CZ-BT (20 µM) in Tris-HCl buffer solution
(10 mM, 60 mM KCl, pH=7.4) at room temperature using a cuvette with a 1 mm path
length over a wavelength range of 220-320 nm with 1 nm bandwidth and the step size
of 1 nm. The CD spectra were obtained by taking the average of repeat three times.
The final analyses of the data were carried out using Origin 8.0 (Origin Lab Corp).
1
87
2.9. Cell digestion and laser confocal imaging
1
1
1
1
1
1
1
1
1
1
1
1
88
89
90
91
92
93
94
95
96
97
98
99
MCF-7 and COS7 cells were cultured in high glucose DMEM supplemented with
10% FBS and penicillin/streptomycin from Gibco in CO incubator at 37°C. The cells
2
were seeded into a confocal glass bottom dish (35.0 mm dish with 20.0 mm bottom
well) with 2.0 mL of culture medium in the incubator until the cell density was
approximately 50% and then washed with PBS for three times and fixed with ice
ethanol. The cell dishes were stored in the refrigerator at minus twenty degree for ten
minutes, and washed with PBS for 3 times, DNase and RNase were added into
different experimental groups for digestive enzyme and incubated for several hours and
then CZ-BT (2.0 µM) was added and incubated for 30 min. The fluorescent images
were recorded by confocal laser fluorescent scanning microscope under the two-photon
excitation wavelength of 820 nm and the emission spectra was recorded within the
range of 580-620 nm.
2
00
2.10. The impact of CZ-BT on cell cycle
2
2
01
02
Plant cells: The cells in exponential phase of growth were digested and transferred
6
into a 6-well flatbottomed plate with the density of 1×10 per milliliter; Add drugs:

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
03
04
05
06
07
08
09
10
11
12
13
14
15
16
After 24 hours cultivation, the cell density was reached up to almost 50%. Two
milliliters of fresh medium was added into each well of the plate, after removing the
original medium out and washed for three times by PBS. Different concentrations of
CZ-BT and PDS were added and then incubated at 37°C in incubator for another 24
hours. Collect cells: Transferred the original medium into centrifuge tubes, then the
cells were digested by pancreatic enzyme and collected into the previous tubes; Fix
cells: The cells were collected with centrifugation under the speed of 1500 rpm for
5min, then washed for two times by PBS. Two milliliters of precooled ethanol (70%)
was added into the cell suspension under low vortex, then immobilized for 12 hours at
4°C; PI staining: 0.5 ml of PI staining solution was added into every cell sample
separately, overhanging cell precipitation slowly and incubated far from light in 37°C
for 30 min; Flow cytometer detection: Red fluorescence was detected using an acoustic
focusing cytometer, then the effects of CZ-BT and PDS on the cell phases were
determined on microplate reader spectrophotometer (Thermo Fisher Scientific).
2
2
17
18
3. Results and discussion
3.1. Synthesis of CZ-BT
2
2
2
2
2
2
2
2
19
20
21
22
23
24
25
26
Compound
the yield of 80%; the ethylated compound
of bromine with diethyl amino in 65% yield; the C-3 aldehyde reaction of
phosphorus oxychloride in dimethyl formamide gave the compound (43%); the
condensation reaction of compound and ethylated benzothiazole afforded the desired
compound CZ-BT (24%, Scheme 1.). CZ-BT and its intermediate products were
1
was obtained by the reaction of carbazole and 1,4-dibromobutane with
was prepared by following the substitution
with
2
2
3
3
1
13
purified by silica gel column chromatography and confirmed by H NMR, C NMR,
and HRMS spectrometry (see the Supplementary Information).
2
2
27
28
3.2. The spectra studies of CZ-BT upon interaction with G4 DNAs
The absorbance increased linearly with the increasing concentration of CZ-BT

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
29
30
31
32
33
34
35
36
37
indicating its good solubility in Tris-HCl buffer solution (Fig. S1). CZ-BT shows a
very low intrinsic fluorescence in Tris-HCl (ΦF-free=0.01), which is of prime
importance for a fluorescent probe for G4 DNA detection. Upon binding with DNAs
(ssDNA: dA21, dT21, ss26 and ssDNA; dsDNA: Poly(A-T) and Poly(G-C) ; G4
9
9
DNA: Telo21, Hum24, Oxy12 and Pu22), the fluorescence intensity of CZ-BT was
significantly enhanced (ΦF-DNA =0.109, Table S1) with the gradual addition of Telo21
with good selectivity (Fig. 1B and Fig. S2). A good linear relationship between
fluorescent intensity and the concentration of Telo21 was presented in Figure 1D with
the linear correlation coefficient of 0.991 and the detection limit of 15.6 nM.
2
38
2
2
2
2
2
39
40
41
42
43
Fig. 1. The absorbance spectra (A) and fluorescence intensity histogram (B) of CZ-BT (10.0 µM) in the
absence and presence of DNAs in Tris-HCl buffer solution (10 mM, 60 mM KCl, pH=7.4); (C) The
fluorescence spectra of CZ-BT (10.0 µM) upon titration with Telo21 (0-15.0 µM), λ =460 nm; (D) The
ex
linear relationship between the fluorescent intensity of CZ-BT and the concentrations of Telo21 (0-3.0 µM),
n=3.
2
2
2
2
44
45
46
47
CZ-BT in Tris-HCl buffer solution presented a freedom state, so there was no
rotational restriction around the methine bridge which connect the carbazole and
benzothiazole, therefore CZ-BT exhibited negligible fluorescence in low viscosity
buffer. When the rotation of methine bridge was restrained after binding with G4 DNA

ACCEPTED MANUSCRIPT
2
2
2
48
49
50
in the high viscosity glycerol solvent, the fluorescence was recovered. This
interpretative statement was further supported by the correlation between fluorescence
quantum yield and solvent viscosity (Fig. S3) [30].
2
2
2
2
2
2
2
51
52
53
54
55
56
57
The two-photon properties of CZ-BT was confirmed by a power dependence
experiment. Fig. S4A showed the double logarithmic plot of two-photon induced
fluorescence integral intensity against the average laser power, a linear regression equation
(Log (F -Fbackground) = 1.95 Log W + 6.07) with a slope of 1.95 was obtained, which indicated
s
an obvious two-photon absorption process. The maximum two-photon absorption cross
section of CZ-BT was 21.0 GM at the presence of G4 DNA in Tris-HCl buffer solution when
excited at 820 nm (Fig. S4B).
2
58
3.3. The impact of G4 DNA on lifetime of CZ-BT
2
59
2
2
2
60
61
62
Fig. 2. (A) Lifetimes of CZ-BT (10.0 µM) and (B) Lifetimes of CZ-BT (10.0 µM) mixed with G4 DNA
(5.0 µM) in Tris-HCl buffer solution. The detection wavelength was 580 nm. Fitting results were obtained
by a deconvolution of the instrument response function (blue line).
2
2
2
2
2
2
2
2
2
63
64
65
66
67
68
69
70
71
In order to further confirm the interaction between CZ-BT and G4 DNA (Telo21),
the lifetimes of CZ-BT in the absence and presence of G4 DNA were recorded using
TCSPC technique excited at 400 nm. Free CZ-BT presented a fluorescence lifetime
about 1.43 ns and a short lifetime about 142 ps owing to the fast twisting process of
C=C double bond between carbazole and benzothiazole (Fig. 2A). As shown in Fig. 2B
and Table S2, the proportion of twisting process ( 1
to 4.67%) after the interaction with G4 DNA, meanwhile, besides the fluorescence
lifetime of free CZ-BT τ2), there was a new longer lifetime (τ3) which was assigned to
the fluorescence lifetime of CZ-BT fixed by G4 DNA [31]. The results above
τ ) significantly decreased (from 96.9%
(

ACCEPTED MANUSCRIPT
2
2
2
2
72
73
74
75
suggested that the fast twisting process of C=C double bond could be inhibited and the
fluorescence lifetime of CZ-BT would become longer in the presence of G4 DNA,
those are the reasons why the fluorescence intensity of CZ-BT increased after the
interaction with G4 DNA.
2
76
3.4. Binding mode between CZ-BT and G4 DNA
2
77
2
2
78
79
Fig. 3. Top view of the interaction between CZ-BT and the charge on Telo21 (PDB id 4DA3) (A) or not
(B); (C) Circular dichroism spectrum of Telo21 (20.0 µM) and with the addition of CZ-BT (20.0 µM).
2
2
2
2
2
2
2
2
2
2
2
2
2
80
81
82
83
84
85
86
87
88
89
90
91
92
The molecular docking study was performed using the AUTODOCK 4.2 program to
explore the interaction between CZ-BT and G4 DNA. Computation result showed that
CZ-BT was able to stack onto the Telo21 (PDB id 4DA3) [32] via π-π stacking
interaction. Whether there is charge on G4 (Fig. 3A) or not (Fig. 3B), this π-π stacking
mainly embodied on the interaction between the aromatic nucleus of CZ-BT and
purine ring of DG3 in G4. In addition, the secondary structure change of G4 DNA after
the interaction with CZ-BT was recorded by circular dichroism (CD). As shown in Fig
3C, the CD spectrum of Telo21 structure alone was of the typical antiparallel G4 DNA,
with the positive band (272 nm) and negative band (247 nm) [33]. The addition of
CZ-BT to G4 DNA solution only made the peaks increased, while other DNAs were
not changed obviously (Fig. S5). The results indicated that CZ-BT did not change the
conformation transition of G4 DNA structure. This was also consistent with the
fluorescent signal enhancements from the fluorescence titration experiments.
2
2
93
94
3.5. Application of CZ-BT for the specificity on G4 DNA in cells
The applications of CZ-BT as a selective probe for the detecting and imaging of G4

ACCEPTED MANUSCRIPT
2
2
2
2
2
3
3
3
3
95
96
97
98
99
00
01
02
03
DNA in cells were further investigated using two-photon confocal laser scanning
microscopy. As shown in Fig. 4 (and Fig. S6), CZ-BT was able to induce a strong
fluorescence response in the regions of nucleus and mainly located in nucleoli where
rDNA (ribosomal DNA) undergoes transcription. It has been reported that G-rich
rDNA may also adopt temporal quadruplex conformations [34]. The fluorescence
signal of CZ-BT in nucleus was almost disappeared after DNase treatment, while
nothing has changed after RNase treatment in compared with control, which indicated
that CZ-BT could selectively interact with G4 DNA in cells and generated strong
fluorescence.
3
04
3
3
05
06
Fig. 4. Digestion experiments for two-photon fluorescence images of fixed COS7 and MCF-7 cells with
CZ-BT (2.0 µM), λ =820 nm, collection wavelength: 600±20 nm, scale bar: 20 µm.
ex
3
07

ACCEPTED MANUSCRIPT
3
3
3
08
09
10
Fig. 5. Two-photon CZ-BT (2.0 µM) fluorescence images and the corresponding normalization intensity of
fixed MCF-7 and COS7 cells with the addition of PDS (0, 1.0, 2.0 µM). λ = 820 nm, collection
ex
wavelength: 600±20 nm scale bar: 20 µm.
3
3
3
3
3
3
3
3
11
12
13
14
15
16
17
18
As a classical G-quadruplex ligand, PDS could promote growth arrest in cells by
inducing replication and transcription dependent DNA damage [35]. Therefore, PDS
was introduced as the contrast reagent with CZ-BT for the endogenous cellular G4
DNA target. As shown in Fig. 5 and Fig. S7, strong fluorescence was presented in the
nucleus after the incubation of CZ-BT, while the fluorescence intensity was
dramatically decreased along with the addition increasing of PDS. This can be
attributed to the preferential occupation of G4 DNA binding sites by PDS. Therefore,
the specificity of CZ-BT on the G4 DNA in cells was verified.
3
19
3.6. The cell cycle experiment of CZ-BT
3
20
3
3
3
21
22
23
Fig. 6. Effects of CZ-BT on cell cycle distribution of MCF-7 cells. The cells were treated with control (A),
CZ-BT (B), and PDS (C) for 24h, (D) showed the cell cycle percentage distribution. The concentration of
CZ-BT and PDS was 10.0 µM.
3
3
3
3
24
25
26
27
Cell cycle refers to the whole process of a cell from the beginning to the end of the division
and each phase preserves different function, respectively [36]. G2 phase is the late period of
DNA synthesis, therefore the vast majority of G4 DNAs are accumulated in this phase during
the whole cell cycle and the cell growth would be stagnated at G2 stage after the incubated

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
28
29
30
31
32
33
34
with PDS [37]. Cells were treated with control, CZ-BT, and PDS for 24 h respectively, and
then were analyzed by flow cytometry to determine the influence on cell cycle (Fig. 6 and Fig.
S8). In comparison with the control group (cells were treated with PBS), the G2 phase
population of MCF-7 cells showed a remarkable increasement (10.32% to 17.07%) after the
treatment with CZ-BT (10.0 µM), which is similar with that of PDS. Therefore, the selective
interaction on G4 DNAs of two-photon probe CZ-BT with low cytotoxicity (Fig. S9) was
fully proved.
3
35
4. Conclusion
3
3
3
3
3
3
3
3
36
37
38
39
40
41
42
43
In conclusion,
a
novel two-photon fluorescent probe CZ-BT of
carbazole-benzothiazole derivative for G4 DNA in vitro and vivo was designed and
synthesized. UV-vis, fluorescence emission and lifetime showed the good specificity
of CZ-BT on G4 DNA, and the molecular docking calculation proved that the binding
mode between CZ-BT and G4 DNA was π-π stacking. More importantly, the
two-photon confocal fluorescent images and cell cycle experiment demonstrated the
specificity of CZ-BT on G4 DNA in cells. All these results suggested that CZ-BT
could have promising applications in G4 DNA research.
3
3
44
45
Conflicts of interest
There are no conflicts of interest to declare.
3
46
Acknowledgements
3
3
3
47
48
49
This work was financially supported by the National Natural Science Foundation of
China (21421005, 21576037, 21703025 and 21878039) and the NSFC-Liaoning
United Fund (U1608222)
3
50
References
3
3
51
52
1 Gellert M, Lipsett MN, Davies DR. Helix formation by guanylic acid. Proc Natl Acad Sci USA
1962;48:2013-2018.

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
2 Bochman ML, Paeschke K, Zakian VA. DNA secondary structures: stability and function of
G-quadruplex structures. Nat Rev Genet 2012;13:770-780.
3 Burge S, Parkinson GN, Hazel P, Todd AK Neidle S. Quadruplex DNA: sequence, topology and
structure. Nucleic Acids Res 2006;34:5402-5415.
4 Bidzinska J, Cimino-Reale G, Zaffaroni N, Folini M. G-Quadruplex structures in the human
genome as novel therapeutic targets. molecules 2013;18:12368-12395.
5 Dexheimer TS, Sun D, Hurley LH. Deconvoluting the structural and drug-recognition complexity
of the G-Quadruplex-forming region upstream of the bcl-2 P1 promoter. J Am Chem Soc
2006;128:5404-5415.
6 Blackburn EH. Structure and function of telomeres. Nature 1991;350:569-573.
7 Biffi G, Tannahill D, McCafferty J, Balasubramanian S. Quantitative visualization of DNA
G-quadruplex structures in human cells. Nat Chem 2013;5:182-186.
8 Rhodes D, Lipps HJ. G-quadruplexes and their regulatory roles in biology. Nucleic Acids Res
2015;43:8627-8637.
9 Ou TM, Lu YJ, Tan JH, Huang ZS, Wong KY, Gu LQ. G-quadruplexes: targets in anticancer drug
design. Chem Med Chem 2008;3:690-713.
10 Rezler EM, Seenisamy J, Bashyam S, Kim MY, White E, Wilson WD, Hurley LH. Telomestatin
and diseleno sapphyrin bind selectively to two different forms of the human telomeric
G-quadruplex structure. J Am Chem Soc 2005;127:9439-9447.
11 Chambers VS, Marsico G, Boutell JM, Antonio MD, Smith GP, Balasubramanian S.
High-throughput sequencing of DNA G-quadruplex structures in the human genome. Nat
Biotechnol 2015;33:877-881.
12 Wu ZS, Chen CR, Shen GL, Yu RQ. Reversible electronic nanoswitch based on DNA
G-quadruplex conformation: a platform for single-step, reagentless potassium detection.
Biomaterials 2008;29:2689-2696.
13 Ge BX, Huang YC, Sen D, Yu HZ. Arobust electronic switch made of immobilized
duplex/quadruplex DNA. Angew Chem Int Ed 2010;49:9965-9967.
14 Zhang J, Chen JH, Chen RC, Chen GN, Fu FF. An electrochemical biosensor for ultratrace terbium
3
+
based on Tb promoted conformational change of human telomeric G-quadruplex. Biosens
Bioelectron 2009;25:378-382.
15 Henderson A, Wu YL, Huang YC, Chavez EA, Platt J, Johnson FB, Brosh RM, Sen D, Lansdorp
PM. Detection of G-quadruplex DNA in mammalian cells. Nucleic Acids Res 2014;42:860-869.
16 Kim HN, Lee EH, Xu ZC, Kim HE, Lee HS, Lee JH, Yoon J. A pyrene-imidazolium derivative
that selectively recognizes G-quadruplex DNA. Biomaterials 2012;33:2282-2288.
17 Yang P, Cian AD, Teulade-Fichou MP, Mergny JL, Monchaud D. Eangineering
bisquinolinium/thiazole orange conjugates for fluorescent sensing of G-quadruplex DNA. Angew
Chem Int Edit 2009;48:2188-2191.
18 Seo YJ, Lee IJ, Yi JW, Kim BH. Probing the stable G-quadruplex transition using quencher-free
end-stacking ethynyl pyrene-adenosine. Chem Commun 2007;27:2817-2819.
19 Vummidi BR, Alzeer J, Luedtke NW. Fluorescent probes for G-quadruplex structures.
ChemBioChem 2013;14:540-558.
20 Schnell U, Dijk F, Sjollema KA, Giepmans BNG, Immunolabeling artifacts and the need for
live-cell imaging. Nat Methods 2012;9:152-158.

ACCEPTED MANUSCRIPT
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
21 Wu SR, Wang LL, Zhang N, Liu Y, Zheng W, Chang A, Wang FY, Li SQ, Shangguan DH. A
bis(methylpiperazinylstyryl) phenanthroline as a fluorescent ligand for G-quadruplexes. Chem-Eur
J 2016;22:6037-6047.
22 Zuffo M, Ladame S, Doria F, Freccero M. Tuneable coumarin-NDI dyads as G-quadruplex specific
light-up probes. Sensor Actuat B-Chem 2017;245:780-788.
23 Wang MQ, Wu Y, Wang ZY, Chen QY, Xiao FY, Jiang YC, Sang A. G-quadruplex DNA
fluorescence sensing by a bis-amine-substituted styrylquinolinium dye. Dyes Pigments
2017;145:1-6.
24 Liu Q, Guo BD, Rao ZY, Zhang BH, Gong JR. Strong two-photon-induced fluorescence from
photostable, biocompatible nitrogen-doped graphene quantum dots for cellular and deep-tissue
imaging. Nano Letters 2013;13:2436-2441.
25 Lim CS, Masanta G, Kim HJ, Han JH, Kim HM, Cho BR. Ratiometric detection of mitochondrial
thiols with a two-photon fluorescent probe. J Am Chem Soc 2011;133:11132-11135.
26 Yu HB, Xiao Y, Jin LJ. A lysosome-targetable and two-photon fluorescent probe for monitoring
endogenous and exogenous nitric oxide in living cells. J Am Chem Soc 2012;134:17486-17489.
27 Sun W, Cui JX, Ma LL, Lu ZL, Gong B, He L, Wang RB. Analyst 2018;143:5799-5804.
28 Grande V, Shen CA, Deiana M, Dudek M, Olesiak-Banska J, Matczyszyn K, Wurthner F. Chem.
Sci 2018;9:8375-8381.
29 Xu C, Webb WW. Measurement of two-photon excitation cross sections of molecular fluorophores
with data from 690 to 1050 nm. J Opt Soc Am B 1996;13:481-491.
30 Granzhan A, Ihmels H, Viola G. 9-donor-substituted acridizinium salts: versatile
environment-sensitive fluorophores for the detection of biomacromolecules. J Am Chem Soc
2007;129:1254-1267.
31 Byrne CD, Mello AJ. Photophysics of ethidium bromide complexed to ct-DNA: a maximum
entropy study. Biophys Chem 1998;70:173-184.
32 Lu YJ, Deng Q, Hou JQ, Hu DP, Wang ZY, Zhang K, Luyt LG, Wong WL, Chow CF. Molecular
engineering of thiazole orange dye: change of fluorescent signalling from universal to specific
upon binding with nucleic acids in bioassay. ACS Chem Biol 2016;11:1019-1029.
33 Balagurumoorthy P, Brahmachari SK, Mohanty D, Bansal M, Sasisekharan V. Hairpin and parallel
quartet structures for telomeric sequences. Nucleic Acids Res 1992;20:4061-4067.
34 Drygin D, Siddiqui-Jain A, O`Brien S, Schwaebe M, Lin A, Bliesath J, Ho CB, Proffitt C, Trent K,
Whitten JP, Lim JKC, Hoff DV, Anderes K, Rice WG. Anticancer activity of CX-3543: a direct
inhibitor of rRNA biogenesis. Cancer Res 2009;69:7653-7661.
35 Muller S, Kumari S, Rodriguez R, Balasubramanian S. Small-molecule-mediated G-quadruplex
isolation from human cells. Nat Chem 2010;2:1095-1098.
36 Sherr CJ. Cancer cell cycles. Science 1996;274:1672-1677.
37 Koirala D, Dhakal S, Ashbridge B, Sannohe Y, Rodriguez R. A single-molecule platform for
investigation of interactions between G-quadruplexes and small-molecule ligands. Nat Chem
2011;3:782-787.

ACCEPTED MANUSCRIPT
Highlights
1
: A novel two-photon carbazole based fluorescent probe (CZ-BT) was designed and
synthesized for specific interaction on G4 DNA with excellent optical properties and
large conjugate plane.
2
: Compared with ssDNA and dsDNA, CZ-BT selectively impacted on G4 DNA with
excellent fluorescent emission.
3
: Combined with lifetime, molecular docking, circular dichroism, co-staining with
PDS (a classical commercial G-quadruplex ligand), nucleic digestion experiments and
cell cycle experiments showed CZ-BT was specifically impacted on G4 DNA in
cells.