Zn(II)-DPA Coordinative fluorescent probe for enhancing G4 DNA binding

Article abstract of DOI:10.1016/j.dyepig.2021.109707

Novel dipicolylamino functionalized styryl-carbazole derivative (YCJ) was designed and synthesized. This derivative in combination with Zn(II) has exhibited large fluorescence intensity enhancement and prominent red-shift in absorption spectra with G4 DNA. Systematical analysis indicats that YCJ-Zn(II) complex shows much higher binding affinity and spectral response to G4 DNA than our previously reported styryl-carbazole scaffold (E1) due to the incorporationc of a Zn(II)-DPA moiety which could decrease the carbazole core electron density and consequently enhance the ability to display π-π stacking interaction with G4 DNA. Spectroscopic and molecular docking studies have unraveled YCJ-Zn(II) complex can stack both 3′ and 5′-ends and an associated with partial loop/groove interactions. The application of this Zn(II) complex as a fluorescent agent for living cell imaging was also demonstrated. The conjugation of the Zn-DPA moiety results in good cell permeability, endogenous DNA labeling, which is suitable for monitoring of nucleus activities.

Full text of DOI:10.1016/j.dyepig.2021.109707

Dyes and Pigments 195 (2021) 109707
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Zn(II)-DPA Coordinative fluorescent probe for enhancing G4 DNA binding
a
a
a
a
a
Quan-Qi Yu , Xue-Xian Lang , Juan-Juan Gao , Hong-Yao Li , Yi-Tong Bai ,
b
,
**
a,*
Hai-Jiao Wang
, Ming-Qi Wang
a
School of Pharmacy, Jiangsu University, Zhenjiang, 212013, PR China
b
The Key Laboratory of Biomedical Material, School of Life Science and Technology, Xinxiang Medical University, Xinxiang, 453003, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Novel dipicolylamino functionalized styryl-carbazole derivative (YCJ) was designed and synthesized. This de-
rivative in combination with Zn(II) has exhibited large fluorescence intensity enhancement and prominent red-
shift in absorption spectra with G4 DNA. Systematical analysis indicats that YCJ-Zn(II) complex shows much
higher binding affinity and spectral response to G4 DNA than our previously reported styryl-carbazole scaffold
Zn(II)-DPA
G4 DNA
Carbazole derivate
Fluorescent probe
Cell imaging
(
E1) due to the incorporationc of a Zn(II)-DPA moiety which could decrease the carbazole core electron density
and consequently enhance the ability to display stacking interaction with G4 DNA. Spectroscopic and mo-
π π
-
′
′
lecular docking studies have unraveled YCJ-Zn(II) complex can stack both 3 and 5 -ends and an associated with
partial loop/groove interactions. The application of this Zn(II) complex as a fluorescent agent for living cell
imaging was also demonstrated. The conjugation of the Zn-DPA moiety results in good cell permeability,
endogenous DNA labeling, which is suitable for monitoring of nucleus activities.
1
. Introduction
Guanine rich DNA sequences fold into tetra-stranded helical assem-
Collectively, G4s could be regarded as a vital biomarker for cancer
diagnosis and treatment.
Considering the wide range of biological processes associated with
DNA G4s, the requirements for selectively detecting G4 structures are
strong motivators for the development tools. Fluorescence imaging has
been attracting much more attention in recent years, due to the ad-
vantages of fluorescence including simply, high sensitivity, non-
destructivity, in situ examination, and intracellular detection [13].
Various G4 DNA fluorescent probes have been developed rely on a large
enhancement in emission intensity upon binding G4, compared to
binding other DNA forms (e.g., duplex, single-stranded DNA). So far,
some related review papers have discussed [14–18]. Most probes
designed to interact with G4s were initially based on organic com-
pounds, but to a far lesser extent, on metal complexes. It has been shown
that metal complexes are attractive G4 DNA binders due to their ver-
satile structural and electronic properties [19–22]. Metal complexes
provide the opportunity to access a much wider chemical space than
their purely organic counterparts due to the several geometries (e.g.,
linear, bent, trigonal planar, square planar, tetrahedral, trigonal bipyr-
amidal, octahedral) that coordinated metal ions have [23]. In addition,
metal ions can confer unique electronic properties to the system to
render the resulting metal complexes with interesting optical, catalytic
blies called DNA G-quadruplexes (G4s) [1]. The core structures in the
G4s are formed by stacked planar G-tetrads, which are held together via
Hoogsteen hydrogen bonds [2]. A series of bio-informatical studies
initially suggested that over 700 000 putative G4-forming sequences are
abundantly present across the human genome, especially located in the
chromosomes and transcriptomes [3–5]. While it is commonly accepted
that G4s are involved in a number of essential biological regulatory
processes, the exact roles that G4s play in biology are still under sig-
nificant scrutiny, even providing explicit evidence that G4 structures
actually form in native cellular DNA is a formidable challenge [6,7].
Recently, with widespread application in immunofluorescent staining,
several G4 structure-specific antibodies such as HF1, BG4, hf2, and 1H6
have been developed to visualize G4s in cells [8–11]. Among these,
immunostaining with BG4 in human cells showed the presence of G4
structures in all cell cycle phases, with maximum foci in S phase, which
corroborates the replication-dependent formation of G4 [9]. Meanwhile,
by developing a quantitative, comparative G4-ChIP-seq methodology,
researchers discovered that G4 DNA regions are highly associated with
critical drivers of triple-negative breast cancer models [12].
*
Corresponding author.
* Corresponding author.
E-mail addresses: taiyiyibo@126.com, taiyiyibo@126.com (H.-J. Wang), wmq3415@163.com, wmq3415@163.com (M.-Q. Wang).
*
https://doi.org/10.1016/j.dyepig.2021.109707
Received 25 May 2021; Received in revised form 3 July 2021; Accepted 5 August 2021
Available online 8 August 2021
0
143-7208/© 2021 Elsevier Ltd. All rights reserved.

Q.-Q. Yu et al.
Dyes and Pigments 195 (2021) 109707
◦
or redox properties which in turn can be exploited for imaging or
therapeutic applications [24,25]. Several metal complexes are routinely
used in the clinic as either therapeutic or imaging agents, with the most
prominent example being that of cisplatin and its derivatives for the
treatment of various cancers [26,27].
mixture and stirred at 50 C for 5 h. After cooling, the organic phase was
extracted with ethyl acetate (20 mL × 3) and purified by flash column
chromatography with PE/EA = 40:1 elution to afford brown oily liquid
1
1b (1.6 g, yield 47%). H NMR (400 MHz, CDCl
3
) δ 8.12 (d, J = 7.7 Hz,
2H), 7.55–7.45 (m, 4H), 7.3–7.22 (m, 2H), 4.52 (t, J = 6.5 Hz, 2H), 3.40
(t, J = 6.1 Hz, 2H), 2.47–2.44 (m, 2H).
′
Dipicolylamine (2,2 -dipyridylmethylamine, DPA) is one of the most
studied tridentate metal-binding units in coordination chemistry
[
28–32]. Recently, Zn-DPA complexes attracted considerable attention
2.2.2. Preparation of 9-(3-bromopropyl)-9H-carbazole-3-carbaldehyde
(1c)
in biological analysis and cell imaging, through electrically binding the
anionic molecular targets [33–35]. Previously, we have developed a
fluorescent probe (termed as E1) with excellent selectivity to G4s, which
possesses a pyridinium side group substituted carbazole planar core
through an ethylene bridge (Fig. 1) [36]. However, E1 has shown to be a
switch-on probe for G4s, with moderate fluorescence enhancements and
weak cellular potency. Thus, we speculated that by combining the ef-
fects of the positively charged Zn-DPA moiety, which can be involved in
electrostatic interactions with electronegative regions, and aromatic
Phosphorus oxychloride (1.5 mL, 16 mmol) was mixed and dissolved
in anhydrous DMF (5 mL) stirred at ice bath for 1 h. Intermediate 1b
(0.77 g, 2.7 mmol) dissolved in 1,2-dichloroethane was added into the
reaction mixture at refluxed temperature for 12 h. After cooling to room
temperature, the crude product was treated with saturated NaHCO
3
aqueous solution to reach the pH of 7.4 and extracted with dichloro-
methane (20 mL × 3). Then the combined organic phase was dried over
anhydrous sodium sulfate and organic solvent was removed by rotary
evaporation. The product was purified using flash silica gel column with
PE/EA = 40:1 elution to afford faint yellow oily liquid 1c (0.565 g, yield
68%).
rings designed to interact through -stacking, stronger G4 binder may be
π
obtained. Taking into account these highlights, herein, we proposed to
link a DPA subunit together with carbazole-pyridinium scaffold to
design a new class of G4 DNA ligand YCJ. Compared with precursor E1,
this ligand in combination with Zn(II) has exhibited the improved
binding affinity to G4 DNA and enhanced cellular uptake. The effect of
Zn-coordination was discussed in detail.
2.2.3. Preparation of N-(pyridin-2-ylmethyl)ethanamine (2c)
2-pyridinecarboxaldehyde (0.5 g, 4.6 mmol) was added to a stirred
solution of 2-picolylamine (0.5 g, 4.6 mmol) dissolved in methanol (50
mL) and stirred for 10 h under ambient temperature. The addition of
sodium borohydride (0.7 g, 18.5 mmol) turned the reaction mixture
from dark brown to pale yellow and stirred for another 2 h. All the
volatiles were removed under reduced pressure and diluted hydrochlo-
ric acid was dropwise added to neutralize the alkalinity of excess sodium
2
. Experimental methods
.1. Materials and instrumentation
A Bruker AM400 NMR spectrometer was used to test the H NMR and
2
1
borohydride. After extraction by CH
2
Cl
2
(20 mL × 3), the combined
1
3
organic phase was dried over anhydrous sodium sulfate and the crude
product was purified by aluminum trioxide column chromatography
using DCM/MeOH = 20:1 as the eluent to obtain pure compound 2c
3 6
C NMR spectra of synthetic compounds, in CDCl or DMSO‑d with
TMS as an internal standard. High-resolution mass spectra (HRMS) were
recorded on a Shimazu LCMS-IT-TOF instrument with an ESI detector.
All chemicals were purchased from commercial sources and all the
solvents were of analytical reagents used without further purification.
All oligonucleotides listed in Table S1 were purchased from Sangon
Biotechnology Co., Ltd. (Shanghai, China), and G4 structures were pre-
folded in 10 mM Tris-HCl buffer (containing 60 mM KCl, pH 7.4) unless
(
0.18 g, yield 20%). 1H NMR (400 MHz, CDCl
3
) δ 8.53 (d, J = 8.4 Hz,
2
1
H), 7.62 (t, J = 21.5, 10.8 Hz, 2H), 7.35 (d, J = 7.8 Hz, 2H), 7.14 (t, J =
5.7, 8.0 Hz, 2H), 3.98 (s, 4H), 2.84 (s, 1H).
2
.2.4. Preparation of 9-(3-(bis(pyridin-2-ylmethyl)amino)propyl)-9H-
carbazole-3-carbaldehyde (3)
65 mg (1.8 mmol) compound 1c and 355 mg (1.8 mmol) compound
otherwise noted and prepared as 100
μ
M stock solution, then their to-
5
pological structures were confirmed by CD studies (Fig. S1).
2
c were dissolved in 50 mL acetonitrile, the resultant mixture was
◦
refluxed at 80 C for 12 h after the addition of anhydrous potassium
carbonate (493 mg, 3.6 mmol). After cooling, small amount of water was
added into the solution to dissolve potassium carbonate and the crude
product was obtained by extraction with dichloromethane. With the
completion of dryness over anhydrous sodium sulfate, pure compound 3
2
2
.2. Synthesis and characterization of synthesized compounds
.2.1. Preparation of 9-(3-bromopropyl)-9H-carbazole (1b)
Under nitrogen atmosphere, sodium hydride (0.57 g, 23 mmol) was
added to the mixed solution of carbazole (2 g, 12 mmol), DMF (2 mL)
and THF (30 mL) stirred for 15 min at room temperature. Then 1,3-
dibromopropane (3.6 g, 17.8 mmol) was blended into the reaction
(
464 mg, 60%) was afforded with aluminum trioxide column chroma-
1
tography using DCM/MeOH = 30:1 as the eluent. H NMR (400 MHz,
Fig. 1. Molecular structures of E1, YCJ and its Zn(II) complex.
2

Q.-Q. Yu et al.
Dyes and Pigments 195 (2021) 109707
CDCl
3
) δ 10.05 (s, 1H), 8.58–8.48 (m, 3H), 8.10 (d, J = 7.7 Hz, 1H), 7.93
covalent and non-covalent interactions between ligand and acceptor
1XAV nucleic acid.
(
d, J = 8.5, 1.5 Hz, 1H), 7.65–7.55 (m, 2H), 7.53–7.39 (m, 4H),
7
4
2
.32–7.25 (m, 2H), 7.17–7.10 (m, 2H), 6.95 (d, J = 11.2 Hz, 4.6 Hz, 1H),
.37–4.24 (m, 2H), 3.83 (s, 4H), 2.69 (t, J = 6.9 Hz, 2H), 2.14–2.01 (m,
2.6. Cell staining experiments
H).
4
T1 cells were grown in DMEM medium with fetal bovine serum
2
9
.2.5. Preparation of 2-(2-(9-(3-(bis(pyridin-2-ylmethyl)amino)propyl)-
H-carbazol-3-yl)vinyl)-1-methylpyridin-1-ium (YCJ)
(10%). For the localization study, cells were cultured in confocal dishes
◦
at 37 C for 24 h first. After the cells were washed twice with PBS, 20
μ
M
A solution of 3 (464 mg, 1.1 mmol) in absolute ethyl alcohol (40 mL)
in situ generated YCJ-Zn(II) ensemble was added. After the cells were
◦
was refluxed with 1,2-dimethylpyridin-1-ium iodide (251 mg, 1.06
mmol) along with a few drops of piperidine. After 4 h, the combined
organic phase was concentrated under reduced pressure and pure
cultured at 37 C for 30 min, the medium was removed, and the cells
◦
were further incubated with 1.0
μ
g/mL DAPI for 30 min at 37 C. After
the cells were washed three times with PBS, the images were processed.
product YCJ (110 mg, 20%) was obtained by recrystallization from
1
DCM/MeOH. H NMR (400 MHz, DMSO‑d
6
) δ 8.85 (d, J = 6.1 Hz, 1H),
2.7. DNase and RNase digest test
8
1
.70 (s, 1H), 8.54 (d, J = 8.0 Hz, 1H), 8.50–8.40 (m, 3H), 8.26–8.16 (m,
H), 8.14 (s, 1H), 7.94 (dd, J = 8.7, 1.2 Hz, 1H), 7.81 (dd, J = 10.1, 3.7
4T1 cells were cultured in confocal dishes for 24 h. Then, the cells
were fixed by precooled methanol for 15 min. After being washed twice
with PBS, the cells were treated with Triton X-100 (1%) for 2 min. After
being washed twice with PBS, the pretreated cells were incubated with
Hz, 1H), 7.76–7.64 (m, 3H), 7.64–7.54 (m, 2H), 7.46 (t, J = 8.4 Hz, 3H),
7
6
.32–7.17 (m, 3H), 4.41 (d, J = 12.1 Hz, 5H), 3.72 (s, 4H), 2.56 (t, J =
.7 Hz, 2H), 2.00 (d, J = 13.5, 6.8 Hz, 2H) HRMS: (positive mode, m/z)
+
◦
calculated 524.2809, found 524.2814 for [M ꢀ I] .
YCJ-Zn(II) ensemble or DAPI, respectively for 1 h at 37 C. After the
cells were washed three times with PBS, one dish was used as a blank,
2
.3. Spectrophotometric and spectrofluorimetric titrations
and the other dishes were respectively treated with DNase (RNase-free,
◦
3
0
μg/mL) or RNase (30
μg/mL) at 37 C for 2 h. Cells were rinsed by
UV–vis absorption studies were performed on UV-8000 spectropho-
PBS three times before imaging.
3. Results and discussion
3.1. Chemistry
tometer (Metash, China) using matched quartz cuvettes of 10 mm path
length in 10 mM Tris-HCl (60 mM KCl) buffer. Fluorescence titration
studies were performed on RF-5301PC fluorescence spectrophotometer
(
Shimazu, Japan) with 10 mm quartz cuvettes in which fluorescence was
measured in 10 mM Tris-HCl (60 mM KCl) buffer and the spectra were
recorded at 2 nm excitation and emission slit widths unless otherwise
specified. The fluorescence excitation wavelength was set as 419 nm and
emission was ranged from 420 to 700 nm. For the titration test both in
spectrophotometry and spectrofluorimetry, the concentration of stock
solution was constant while different DNA solutions were added step by
step until no change was observed in the spectra, indicating that binding
saturation was achieved.
With the aim to increase the binding affinity and the biocompati-
bility of probe E1, we decorated carbazole-pyridinium scaffold with DPA
substituent via different alkyl linker to afford ligand YCJ. As was out-
lined in Scheme 1, in the first step, the commercially available carbazole
was reacted with 1,3-dibromopropane under inorganic basic condition
to afford 9-(3-bromopropyl)-9H-carbazole (1b). The intermediate 1b
was then treated with phosphonium oxychloride (POCl ) in DMF under
3
heating condition to obtain 9-(3-bromopropyl)-9H-carbazole-3-carbal-
2
.4. CD spectra measurement
dehyde (1c). In the second step, treating pyridine-2-carboxaldehyde
with 2-aminopyridine and sodium borohydride led to the formation of
′
CD spectra titration was performed by using a JASCO J-815 CD
2,2 -dipicolylamine (DPA), which was subsequent reacted with com-
spectrometer continuously flushed with pure evaporated nitrogen
pound 1c to give the intermediate 3. Finally, knoevenagel condensation
of intermediate 3 with pyridinium in the presence of organic base in
anhydrous ethanol led to afford desired ligand YCJ as a yellow solid.
Experimental data including 1H NMR, C NMR and high-resolution
mass spectrometry supported their structures (see the Supplementary
Information).
throughout the experiment. The concentration of c-myc DNA was fixed
at 2
μ
M while the incremental amount of in situ generated YCJ-Zn(II)
13
ensemble from 2
μ
M to 20 M was added into the Tris-HCl buffer con-
μ
taining c-myc DNA. The spectra were accumulated over the wavelength
range of 230–320 nm with a bandwidth of 1.0 nm and 60 nm/min
scanning speed. After each addition of compound, the mixed solution
was vibrated adequately and equilibrated at least 2 min before scan. The
data of Tris-HCl buffer as a background baseline had been subtracted
from each sample at the beginning of titration.
3.2. Fluorescence response towards c-myc G4 DNA
Previously, when the probe E1 was used to interact with c-myc G4
DNA, the observed fluorescence enhancement was only about 8-fold,
that is, the fluorescence response is relatively low toward G4 DNA. In
order to investigate the ability of this new ligand in the fluorescence
detection of G4 DNA, the fluorescence response of YCJ toward c-myc G4
DNA in the presence of Zn(II) was first studied. The ligand YCJ exhibits a
weak fluorescence signal in buffer solution with λmax = 544 nm upon
excitation at 419 nm. When combined with Zn(II), a little decrease was
observed in the fluorescence (Fig. S2). The job plot shows the formation
of a 1:1 bonding mode between YCJ and Zn (II) (Fig. S3). To get further
2
.5. Molecular docking
Docking analysis was carried out by using the Autodock 1.5.6
modeling tool. The crystal structure of c-myc G4 (PDB ID:1XAV) was
downloaded from RCSB PDB nucleic acid database. And all of the water
molecules and metal ions were extracted from the maternal structure of
nucleic acid 1XAV and all of the hydrogen atoms (including polar and
nonpolar hydrogen atoms) were added to define the correct configura-
tions and tautomeric states. The ligand was converted from a two-
dimensional structure into three-dimensional structure using Chem-
Draw 3D with minimized energy. The grid box dimension was deter-
mined by visual inspection to encompass the whole G 4 structure but
also left additional space for maximum flexibility in ligand orientation.
All of the possible docking sites recommended by docking-scoring
analysis were displayed by PyMOL 1.7.6 software for visualizing
insight into the binding mode of YCJ and Zn2 , the H NMR analysis of
+
1
+
ligand YCJ in DMSO‑d
recorded and the result was shown in Fig. S4. The protons at pyridine
6
in the presence of 1.0 equiv. of Zn2 was
2
+
were all broadened and downfield shifted upon addition of Zn
.
Meanwhile, the singlet peak of methylene protons in DPA moiety at
3.72 ppm was shifted downfield to 4.13 ppm. Accordingly, the structure
of YCJ-Zn2 was proposed in Fig. S4, in which Zn may coordinate with
+
2+
3

Q.-Q. Yu et al.
Dyes and Pigments 195 (2021) 109707
◦
Scheme 1. Synthetic routes for the preparation of ligand YCJ. Reagents and conditions: a.1,3-dibromopropane, NaH, DMF, THF, rt, 5 h; b. DMF, POCl
NaBH , MeOH, rt, 10 h; d.K CO , acetonitrile, reflux,12h; e. Piperidine, EtOH, reflux, 12 h.
3
, 80 C; c.
4
2
3
DPA nitrogen atoms. The observed spectral behavior of YCJ-Zn(II)
complex is particularly interesting from the perspective of an ideal
fluorescence probe that is almost non-fluorescence in free-state and
strongly intensity in the analyte bound-state. On the basis of this result,
quantitative analysis of the in situ generated YCJ-Zn(II) ensemble to-
ward c-myc G4 DNA was investigated by fluorescent titration in Tris-HCl
buffer (pH 7.4, containing 60 mM KCl). As shown in Fig. 2A, when c-myc
G4 DNA was gradually added, there was a large fluorescence enhance-
ment at λ = 544 nm. Fig. 2B compares the fluorescence responses of E1
and YCJ-Zn(II) complex with c-myc G4 DNA, which demonstrates this
complex gives much better performance than its precursor E1. Addi-
tionally, according to the titration curve, a linear correction between
Combining with the study on E1, the strong fluorescence enhance-
ment of YCJ-Zn(II) complex originates from the restriction of rotation
around the vinylic-bridge upon its G4 binding, which is also illustrated
by the emission enhancement in a viscous medium (Fig. S5). Commonly,
fluorescent molecular rotors usually show longer fluorescence lifetimes
in a restricted environment. Fluorescence intensity decay measurements
were then conducted to demonstrate the binding of YCJ-Zn(II) complex
with c-myc G4 DNA (Fig. 2D). The normalized fluorescence decay data of
free YCJ-Zn(II) complex and c-myc G4 DNA bound YCJ-Zn(II) complex
has been shown in a scatter plot and typical decay profiles along with
autocorrelation diagrams are shown in Fig. 2D. The decays were fitted
with a three-exponential function. The major decay component (34%)
had a lifetime of 2.1 ns for G4 DNA bound YCJ-Zn(II) complex, and the
other two decays (2.3 ns and 7.2 ns) had an amplitude of 58%. The
average fluorescence lifetime of 4.71 ns was calculated from the above
three exponentials, which was 4 times the 1.10 ns of free YCJ-Zn(II)
complex. This result indicates that the strong interaction between
YCJ-Zn(II) complex and c-myc G4 DNA hinders the molecular rotation
and increases its lifetime.
fluorescence intensity of YCJ-Zn(II) complex and the concentration of c-
2
myc G4 DNA in the range of 0.2–1.6
μ
M was observed with R = 0.991
(
Fig. 2C). The limit of detection (LOD) was calculated to be as low as 68
nM according to the IUPAC-based method [37]. The LOD value of
YCJ-Zn(II) complex for c-myc G4 DNA in solution is significantly
improved relative to ligand E1 (LOD = 160 nM). The above fluorescence
measurement demonstrates that the YCJ-Zn(II) complex displays greatly
enhanced fluorescence performance over ligand E1 in the detection of
c-myc G4 DNA. It has initially validated our proposed idea.
Fig. 2. (A) Fluorescence titration spectra of
2
+
YCJ (1
μ
M) + 1 eq Zn with the addition of
c-myc from 0.2 to 1.6
μ
M in 10 mM Tris-HCl
buffer, 60 mM KCl, pH 7.4. (B) Intensity
ratio change of E1 and YCJ-Zn(II) complex
(
1
μ
M) with the addition of c-myc from 0.2
to 1.8 M in buffered solution. (C) The linear
μ
fit equation for calculating LOD value be-
tween YCJ-Zn(II) complex and c-myc G4
DNA at 545 nm. (D) Fluorescence lifetime
decay of YCJ-Zn(II) complex with/without
c-myc in 10 mM Tris-HCl buffer, 60 mM KCl,
pH 7.4.
4

Q.-Q. Yu et al.
Dyes and Pigments 195 (2021) 109707
3
.3. Selectivity study of YCJ-Zn(II) complex towards G4 DNAs against
shift and hypochromic effect, it can be assumed that YCJ-Zn(II) complex
may be bound through strong stacking interactions to the c-myc G4 DNA
structure. Coordination of Zn(II) to aromatic ligand may decrease the
ligand’s electron density and consequently enhance the ability to display
other DNA forms
We then studied the fluorescence responses of YCJ in combination
with Zn(II) towards other G4s (22AG, EAD, G3T3, HT, Htg-21, Hum24,
π
- stacking interactions with G4 DNA, which resulted in larger extent of
π
Pu27, VEGF, ODN, CM22), double-stranded DNAs (dsDNA26, (A-T)
2
,
bathochromism.
(
A-T) , (A-T) , (G-C) ), and single-stranded DNA (ss26) (Fig. S6). The
5
9
9
titration curves manifest that the fluorescence enhancements at 544 nm
are increased to different extents and depend on the types of DNA.
Notably, G4s could significantly enhance the fluorescence signal of YCJ-
Zn(II) complex (12- to 20-fold). Only weak fluorescence enhancements
are observed upon binding to single-stranded and double-stranded DNAs
3.5. The effect of YCJ- Zn(II) complex on the conformation and stability
of c-myc G4 DNA
To gain insights into the binding event, highlighting the effect of the
complex on the conformation of c-myc G4 DNA structure, circular di-
chroism (CD) experiment was conducted. The CD spectrum of the pre-
folded c-myc alone in a buffer containing 60 mM KCl and 10 mM Tris-
HCl at pH 7.4 shows the characteristic signature reported in the litera-
ture for this sequence, displaying a maximum at 260 nm and a negative
at 245 nm, corresponding to the parallel G4 structure [41]. After addi-
tion of YCJ-Zn(II) complex, a slight dropping of the intensity of the
positive band at 260 nm, an increase of the negative band at 245 nm was
observed (Fig. 5). Since the G4 structure might be affected by metal ions,
a CD titration of pure Zn(II) into the solution of c-myc G4 DNA under the
same conditions was also conducted. As seen in Fig. S7, it had almost no
effect on the CD intensity, indicating that Zn(II) would not affect the
c-myc G4 DNA structure. In general, addition of YCJ-Zn(II) complex
does not affect the positions of peaks, suggesting that the G4 structure
retained the parallel topology. A gradual decrease in the intensity of CD
signal suggests more or less perturbation in the perfect stacking between
tetrads in the G4 DNA by YCJ-Zn(II) complex. This finding is also
consistent with the UV–vis signal observed in the absorption titration
experiment.
(
less than 5-fold). The results are summarized in Fig. 3. Thus, these re-
sults show that YCJ-Zn(II) complex preferentially recognizes G4 DNA in
vitro.
3
.4. Binding characteristics
Electronic absorption spectroscopy is a sensitive method for deter-
mining the binding characteristics of small organic molecules and/or
their metal complexes with G4 DNA. Changes observed in the electronic
absorption spectrum reflect the interaction between the molecules of a
particular compound/complex and DNA, and inferences may be drawn
regarding the mechanism of this interaction from wavelength shifts of
the corresponding absorption bands. In our experiments, we investi-
gated the effect of adding c-myc G4 DNA to a solution of YCJ-Zn(II)
complex. For comparison, the absorption spectrum of E1 in the presence
of c-myc G4 DNA was also collected. In the case of E1, as shown in
Fig. 4A, hypochromicity of 54.4% and small bathochromic shift of 5 nm
are observed. Addition of the c-myc G4 DNA to the YCJ-Zn(II) complex
has led to a 33.9% decrease in intensity and a large red shift of 32 nm in
the absorption spectra as shown in Fig. 4B. Generally, bathochromism
3
.6. Study on the binding mechanism of YCJ-Zn(II) complex with c-myc
G4 DNA
can be explained on the basis of the coupling of an antibonding
of the intercalating ligand and the -orbital of the DNA base pairs, which
thus lowers the * transition energy, and in turn results in a shift to
longer wavelengths [38]. Given that the coupled orbital is not fully
π* orbital
π
The above experimental results show that the probe YCJ-Zn(II)
π
→ π
complex is giving more effective fluorescent signal discrimination to G4
DNA. The enhanced fluorescence signal may also reveal that the mo-
lecular rotating ability may be effectively restricted because the mo-
lecular size of the complex is small enough to fit into the G4-binding
π
filled with electrons, there is a decrease in the values of the spectral
transition probabilities and this is reflected in the spectrum as a decrease
in absorbance (hypochromism) [39,40]. In other words, hypochromism
is indicative of intercalative binding whereas the bathrochromic shift is
an indication of strong stacking interaction between the ligand chro-
mophore and bases of DNA. Comparing the data on the bathochromic
pocket. The favorable adduct may probably form through the
π-π
stacking interactions between the planner carbazole and G4-structure.
Next, the binding stoichiometry of YCJ-Zn(II)/c-myc complex in solu-
tion was assessed by using Job’s plot method, in which the maximum
fluorescence intensity peaked at about 0.67 fraction of YCJ-Zn(II),
indicated that a 2:1 complex was formed (Fig. S8). In order to
comprehensively understand the nature of interaction and binding mode
between YCJ-Zn(II) complex and G4 DNA, an elementary and rapid
technique, fluorescent intercalator displacement (FID) assay, was
exploited in this study. This assay is based on the displacement of an
“
on/of” fluorescence probe YCJ-Zn(II) complex from G4 DNA matrices
by increasing amounts of a putative G4 binder hemin which considered
′
′
as binding to G4 DNA through both 5 and 3 end-stacking modes by
forming a 2:1 (hemin/G4) complex [42]. If hemin has the same binding
site as YCJ-Zn(II) complex, will compete with YCJ-Zn(II) complex for
G4 DNA binding, resulting in the large decrease of the emission. As
illustrated in Fig. S9, the displacement with hemin induced almost fully
quenched in the fluorescence intensity of YCJ-Zn(II)/c-myc complex.
The result clearly indicates that YCJ-Zn(II) complex binds to G4 DNA
with the same site as hemin.
To verify whether the binding site of YCJ-Zn(II) complex to c-myc G4
′
′
DNA was at the 3 or 5 terminal, we monitored the impact of YCJ-Zn(II)
complex binding on the modified c-myc G4 DNA with amino-
′
′
methylcoumarin (AMCA) substituted at position of 3 and 5 -terminus of
the c-myc sequence. Theoretically, at which end YCJ-Zn(II) complex
bound, the AMCA fluorescence will be quenched Fig. 6. It was found that
Fig. 3. Distribution for the values of F/F
tested oligonucleotides at the saturated concentration in 10 mM Tris-HCl buffer,
0 mM KCl, pH 7.4.
0
of YCJ-Zn(II) complex for all the
′
′
6
the fluorescence intensities of 5 and 3 -labeled c-myc G4 DNA were
5

Q.-Q. Yu et al.
Dyes and Pigments 195 (2021) 109707
Fig. 4. UV–vis spectra for titrations of c-myc stepwise to (A) E1 and (B) YCJ-Zn(II) complex (5
recorded at 2 min intervals.
μ
M) in 10 mM Tris-HCl buffer, 60 mM KCl, pH 7.4. The spectra were
additional binding sites, several mutated c-myc G4 DNA sequences with
extension or shortening the length of different loops were prepared. CD
spectroscopy confirms that these mutated c-myc DNA sequences do not
disturb the parallel G4 conformation (Fig. S10). The changes of their
fluorescence emission spectra and fluorescence intensity were observed
and recorded (Table 1). The elongation or shortening of the loop near
5
’ end has almost no effect on fluorescence intensity, while other
loops have a little effect on the enhancement of fluorescence that
compared with original c-myc G4 DNA. The above results suggest
that YCJ-Zn(II) complex targets the c-myc G4 DNA mainly by end-
stacking mode, and partially by groove/loop mode.
Molecular docking is proven to be an important technique, useful to
predict the interaction of small ligands with biological macromolecules.
Ligand YCJ and its Zn(II) complex were introduced in the docking
analysis to further clarify detail DNA interactions toward c-myc G4 DNA
(
PDB:1XAV) using Autodock Vina software [43]. The best favorable
binding poses were selected from the docking results as depicted in
Figs. 7 and S11, and their binding energies were calculated (Table 2). It
Table 1
Relative fluorescence intensities of YCJ-Zn(II) complex (1
presence of c-myc G4s (2 M) with different flanking loops in 10 mM Tris-
HCl buffer, 60 mM KCl, pH 7.4.
μ
M) in the
Fig. 5. CD titration spectra of c-myc G4 DNA (2
μM) with 0–10 equiv YCJ-Zn
μ
(
II) complex in 10 mM Tris-HCl buffer, 60 mM KCl, pH 7.4.
′
′
Name
Sequence (5 -3 )
Relative FI
significantly disturbed upon the addition of YCJ-Zn(II) complex, indi-
cating that YCJ-Zn(II) complex had close contact with these regions. In
c-myc
TTGA GGG T GGG TA GGG T GGG TAAA
TTGA GGG T AAA GGG TA GGG T GGG TAAA
TTGA GGG T GGG TAATT GGG T GGG TAAA
TTGA GGG T GGG TA GGG TAAT GGG TAAA
TTGA GGG T GGG T GGG T GGG TAAA
TTGA GGG T GGG TA GGG TA GGG TAAA
1000
1000
704
′
′
other words, both 3 and 5 -ends of G4 DNA may be the binding sites for
YCJ-Zn(II) complex.
c-myc-A
c-myc-B
c-myc-C
c-myc-D
c-myc-E
658
Because loops are distributed on the side faces of parallel c-myc G4
DNA, the side arms on the carbazole core may approach the groove and
loop regions and interact with them. To further gain insight into the
641
816
Fig. 6. (A) Schematic representation of AMCA (aminomethylcoumarin) modified G-quartet regulated by YCJ-Zn(II) complex. (B) The ratio (F/F
0
) of fluorescence
′
intensity of 5’/3 AMCA-labeled c-myc G4 (0.2
μ
M) with the addition of YCJ-Zn(II) complex in 10 mM Tris-HCl buffer, 60 mM KCl, pH 7.4. λex = 353 nm and λem
=
4
48 nm.
6

Q.-Q. Yu et al.
Dyes and Pigments 195 (2021) 109707
has been previously shown that G4 binders contain a planar
π
-aromatic
Table 2
Estimated free energy of binding (ΔG, kcal⋅molꢀ ) for G4 DNA.
1
surface can stack on the surface of both terminal G-quartet planes. Here,
′
′
both of them were found to stack in the centers of 3 and 5 -terminals.
′
′
Compound
5 terminus
3 terminus
′
When the ligand YCJ binds to at 5 -terminal, the two benzene rings of
YCJ5
ꢀ 6.1
ꢀ 9.0
ꢀ 7.8
ꢀ 9.2
carbazole have
π
-
π
stacking interactions with DG-10 and DG-15
stacking also occurred between the vinyl group
YCJ5-Zn(II) complex
respectively, and CH-
π
in the pendant chain and DG-10 base. In addition, one of the pyridine
rings of DPA moiety was encapsulated by a hydrophobic cavity
composed of DG-9, DG-10, DA-12, and DG-14. After DPA moiety
chelated with zinc ion, it had the propensity to be closer to the central
ion channel that is negatively charged in the center of G-tetrad due to
suggesting that YCJ-Zn(II) complex interacted with endogenous DNA in
cells. The nucleolus is an organelle where rDNA undergoes transcription
to rRNA, and G-rich double strands can separate into single-strand DNA
which may transitorily fold into G4s during transcription and replica-
tion. Although YCJ-Zn(II) complex exhibited selectivity towards G4
DNAs in vitro experiments, the selectivity in vivo was insufficient. The
co-staining experiment results revealed that the abundant presence of
duplex DNA in the nucleus is expected to play important role in the
fluorescence enhancement of YCJ-Zn(II) complex due to non-specific
binding to duplex DNA. Therefore, the strong green fluorescence of
YCJ-Zn(II) complex was induced within almost the whole nucleus. The
clear staining images demonstrated that YCJ-Zn(II) complex was suit-
able for monitoring of nucleus activities.
electrostatic interactions, and divalent zinc ion exhibited cation-
π
interaction with DG-15. Besides, a hydrophobic cavity composed of
DG-10, DA-12, DG-9, DG-14, and DG-13 wrapped the pyridine group
connected by the vinyl bond. It is noteworthy that the lower binding free
energy of YCJ-Zn(II) complex also suggests more favorable binding in-
teractions with G4 DNA than its metal-free ligand due to the expected
′
electrostatic interactions. Similar results were found with the 3 -termi-
nal binding process. They indicated that introducing metal ions in the
ligand could create some interesting differences in the DNA binding
properties, therefore, such structural information of the complex is
important for designing new G4 probes with enhanced binding activity.
4. Conclusions
Metal complexes have been considered as an emerging class of
compounds targeting G4 DNA. To uncover the important structural
determinants contributing to the improvement of effective G4 DNA
probes, we have prepared a ligand YCJ based on the promising structure
of the previously discovered G4 DNA probe E1, which covalently linking
a metal ion chelator DPA and a carbazole-pyridinium scaffold. This
ligand in combination with Zn(II) has exhibited affinity toward G4 DNA
with promising fluorescence intensity enhancement and red-shift in
absorption spectra. To characterize in more detail the interaction be-
tween this complex and G4 DNA, comprehensive studies were carried
out with c-myc DNA as a representative example of a G4 structure. It is
proposed that the Zn-coordination could significantly enhance G4 DNA
binding affinity and cellular uptake. We expect that this research would
help to guide the development of high performance metal complex as G4
DNA probes with higher binding affinity, improved biocompatibility
and enhanced functional properties to monitor and study in live cells.
3
.7. Confocal fluorescence imaging
Following consideration of above properties of YCJ-Zn(II) complex,
we further studied its biological application in cell imaging Fig. 8 pre-
sented the fluorescence images of 4T1 cells stained with YCJ-Zn(II)
complex directly or after deoxyribouclease (DNase) and ribonuclease
(
RNase) treatments. The localization in cells was illustrated through co-
staining with the nucleus-targeted dye DAPI. Apparently, the DAPI dye
brightened up the whole nucleus with blue-emitting. The 4T1 cells after
treated with YCJ-Zn(II) complex showed intensive green fluorescence
signal in the nucleolus region. Compared with our previous reported
probe E1 that localized in the cytoplasm with weak fluorescence signal,
the results suggested that Zn(II)-DPA moiety could enhance the ability
for penetration through the nuclear membrane. To verify the binding
target of YCJ-Zn(II) complex in nucleoli, enzymes DNase and RNase
digest experiments were conducted. As shown in Fig. 8, the produced
green fluorescence signals of YCJ-Zn(II) complex in nucleus clearly
disappeared after DNase treatment but did not affect by RNase,
Fig. 7. Structure of ligand YCJ-Zn(II) com-
′
plex with G4 DNA at the 3 terminal (A–C)
′
and 5 terminal (D–F), derived from NMR
(
PDB:1XAV). (A)(D) Metal potassium ions
used to stabilize the G4 structure were rep-
resented by purple spheres. Guanine bases
that make up for G-tetrad and bases in the
loop region were shown in the stick model.
Carbon atoms on benzene ring and alkane
chain were displayed by cyan color stick,
nitrogen atoms in tv-red color and zinc ion
in pink sphere. Non-covalent interactions
including hydrogen bond,
π
-
π
stacking, CH-
π
and cation- interactions were shown in
π
dotted line. Figs were rendered using
PyMOL. (For interpretation of the references
to color in this figure legend, the reader is
referred to the Web version of this article.)
7

Q.-Q. Yu et al.
Dyes and Pigments 195 (2021) 109707
Fig. 8. Fluorescence images of 4T1 cells stained with 20
μ
M of YCJ-Zn(II) complex for 30 min and 1.0
μ
g/mL DAPI for 30 min without and with DNase or RNase
treatment. The cells were excited and imaged by using a confocal fluorescence microscope within the given range of wavelengths pass through 335–385 nm for DAPI
and 465–496 for YCJ-Zn (II) complex.
CRediT authorship contribution statement
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[
[
Quan-Qi Yu: Conceptualization, Methodology, Data curation. Xue-
Xian Lang: Methodology. Juan-Juan Gao: Methodology. Hong-Yao Li:
Data curation. Yi-Tong Bai: Data curation. Hai-Jiao Wang: Conceptu-
alization, Methodology. Ming-Qi Wang: Supervision, Conceptualiza-
tion, Writing – review & editing.
[
[
12] H ¨a nsel-Hertsch R, Simeone A, Shea A, Hui WWI, Zyner KG, Marsico G, Rueda OM,
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2
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imaging in colorectal surgery-an updated review. Expet Rev Med Dev 2020;17(12):
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[
[
14] Ma DL, Wang M, Lin S, Han QB, Leung CH. Recent development of G-quadruplex
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Acknowledgements
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16] Bhasikuttan AC, Mohanty J. Targeting G-quadruplex structures with extrinsic
fluorogenic dyes: promising fluorescence sensors. Chem Comm 2015;51:7581–97.
This work was financially supported by the National Natural Science
Foundation of China (21907042), Natural Science Foundation of
Jiangsu Province (BK20180857), The Key Scientific Research Project of
Colleges and Universities in Henan Province (20A150034).
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