Selective visualization of DNA G-quadruplex structures in live cells with 1-methylquinolinium-based molecular probes: The importance of indolyl moiety position towards specificity

Article abstract of DOI:10.1016/j.dyepig.2017.04.038

A class of small 1-methylquinolinium-based molecular fluorescent probes with high specificity in DNA G-quadruplex structures imaging in live cells was developed through the structural inclusion with an indolyl moiety. The study reveals that the scaffold of 1-methylquinolinium and indole ring is a distinctive combination for molecular design of new probes with effective and sensitive fluorescent signal switch-on functionality, particularly for identifying telomeric DNA, recognizing G-quadruplexes in promoters, and imaging or visualization of DNA G-quadruplex from other DNA structures. In addition, the experimental results demonstrate a rarely investigated factor on the importance of the indolyl moiety and its position when conjugating with a 1-methylquinolinium scaffold though a ethylene bridge in molecular probe design and synthesis; and these factors are determined as the crucial root leading to the excellent fluorescent signal switch-on property for the specific discrimination of DNA G-quadruplexes against other nucleic acids in live human prostate cancer cells (PC-3?cells) experiments. The characteristics of the new probes were comprehensively investigated with fluorescence titration, native PAGE experiments, and CD analysis to validate the selectivity, sensitivity, and stability while interacting with DNA G-quadruplex structures.

Full text of DOI:10.1016/j.dyepig.2017.04.038

Accepted Manuscript
Selective visualization of DNA G-quadruplex structures in live cells with 1-
methylquinolinium-based molecular probes: The importance of indolyl moiety position
towards specificity
Yu-Jing Lu, Xiao-Lu Guo, Miao-Han Xu, Wei-Wu Chen, Wing-Leung Wong, Kun
Zhang, Cheuk-Fai Chow
PII:
S0143-7208(17)30501-6
10.1016/j.dyepig.2017.04.038
DYPI 5935
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 11 March 2017
Revised Date: 3 April 2017
Accepted Date: 19 April 2017
Please cite this article as: Lu Y-J, Guo X-L, Xu M-H, Chen W-W, Wong W-L, Zhang K, Chow C-F,
Selective visualization of DNA G-quadruplex structures in live cells with 1-methylquinolinium-based
molecular probes: The importance of indolyl moiety position towards specificity, Dyes and Pigments
(2017), doi: 10.1016/j.dyepig.2017.04.038.
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ACCEPTED MANUSCRIPT
Selective Visualization of DNA G-quadruplex Structures in Live Cells with
1-Methylquinolinium-based Molecular Probes: The Importance of Indolyl Moiety
Position towards Specificity
Yu-Jing Lu,1,* Xiao-Lu Guo,1 Miao-Han Xu,1 Wei-Wu Chen,1 Wing-Leung Wong,3,4,*
Kun Zhang,1,2,* Cheuk-Fai Chow4
1
Institute of Natural Medicine and Green Chemistry, School of Chemical Engineering and
Light Industry, Guangdong University of Technology, Guangzhou 510006, P. R. China.
2 School of Chemical and Environment Engineering, Wuyi University, Jiangmen 529020, P. R.
China.
3
Research and Development Office, The Education University of Hong Kong, 10 Lo Ping
Road, Tai Po, Hong Kong SAR, P. R. China.
4
Centre for Education in Environmental Sustainability, The Education University of Hong
Kong, 10 Lo Ping Road, Tai Po, Hong Kong SAR, P. R. China.
* Corresponding authors:
E-mail: luyj@gdut.edu.cn; kzhang@gdut.edu.cn; wingleung@ied.edu.hk
1

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Graphical Abstract:
PAGE Staining & Light-up Signal
Live Cell Imaging
Highlights:
-
-
-
New turn-on fluorescent probe with high specificity towards DNA G-quadruplex struc-
tures;
Simple and small molecular probe integrated from natural product fragments of quinolin-
ium salt and indolyl ring;
Stable imaging signal in live cells staining;
2

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Abstract
A class of small 1-methylquinolinium-based molecular fluorescent probes with high specific-
ity in DNA G-quadruplex structures imaging in live cells was developed through the structur-
al inclusion with an indolyl moiety. The study reveals that the scaffolds of
1-methylquinolinium and indole ring is a distinctive combination for molecular design of new
probes with effective and sensitive fluorescent signal switch-on functionality, particularly for
identifying telomeric DNA, recognizing G-quadruplexes in promoters, and imaging or visu-
alization of DNA G-quadruplex from other DNA structures. In addition, the experimental re-
sults demonstrate a rarely investigated factor on the importance of the indolyl moiety and its
position when conjugating with a 1-methylquinolinium scaffold though a ethylene bridge in
molecular probe design and synthesis; and these factors are determined as the crucial root
leading to the excellent fluorescent signal switch-on property for the specific discrimination
of DNA G-quadruplexes against other nucleic acids in live human prostate cancer cells (PC-3
cells) experiments. The characteristics of the new probes were comprehensively investigated
with fluorescence titration, native PAGE experiments and CD analysis to validate the selec-
tivity, sensitivity, and stability while interacting with DNA G-quadruplex structures.
Keywords: DNA G-quadruplex; PC-3 human prostate cancer cells; Cell imaging; Indole ring,
1-Methylquinolinium derivative.
1. Introduction
The specific identification and imaging of higher-order DNA and RNA G-quadruplex (G4)
structures formed from guanine-rich sequences in the human genome1-4 from any other nu-
cleic acids is currently a hot research topic because these unique bio-molecules have been
known to play many substantial roles in the regulation of various biological processes, in-
cluding telomere maintenance, transcription regulation of oncogenes, and anti-tumor chemo-
therapy.5-12 It is thus extremely important to develop highly specific and sensitive molecular
fluorescent probes for qualitative and quantitative detection and discrimination from various
nucleic acids in-situ.13-16 In recent years, the G4-forming oligonucleotide sequences have
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been identified in promoter regions of various genes such as c-kit, c-myc, bcl-2, VEGF on-
cogenic proteins.17-26 More importantly, it has been demonstrated that using a G4-specific
chelating ligand to target the quadruplex motif in the promoter of proto-oncogenes is able to
decrease effectively the transcriptional activity of these proto-oncogene and helps to inhibit
aberrant cell proliferation.27 However, the effective and sensitive probe or ligand with a sim-
ple molecular structure for the purpose is still rare, and also the understanding of its function-
al mechanism on the specific interaction of G4-DNA and followed to regulate the biological
activities is a challenging task.
Currently, on the basis of the topology known in G4-DNA structures (parallel, hybrid, and
anti-parallel forms), a variety of G4-DNA binding probes and ligands has been reported.28-35
Some small molecule-based fluorescent probes were demonstrated for in vitro or in vivo
analysis of G4-DNA,36 such as triphenylmethane dyes,37 cyanovinyl-pyridinium triphenyla-
mine,38 and triarylimidazole (IZCM-1),39 bisquinolinium,40 bisindole,41 bisbenzimidazole de-
rivatives,42 tetracationic porphyrin derivatives,43 phthalocyanine derivatives,44 and thiazole
orange analogues45,46. Among these probes, 1-methylquinolinium is one of the most fre-
quently applied scaffolds for ligand modification probably due to its merit property in fluo-
rescence signaling like thiazole orange. With respect to our experience on the molecular de-
sign for specific G4-DNA and RNA interaction study and live cell imaging applications, the
selection of a small piece of fragment for molecular adaptation and its position to conjugate
to 1-methylquinolinium scaffold is the crucial factor.47 Interestingly, a simple combination of
a plain indolyl ring with the typical 1-methylquinolinium scaffold is rarely found in literature.
In the present study, we intended to incorporate a plain indolyl moiety to
1-methylquinolinium scaffold as a specific G4-DNA fluorescent probe for interaction studies
and live cell staining and imaging because the plain indolyl group is found in a large number
of important naturally occurring compounds (such as tryptophan, 5-hydroxytryptamine, 5-HT)
and is associated with a lot of biological activities and biomedical importance (such as yo-
himbine48: a drug for male impotency; and delavirdine49: an anti-HIV drug). By combining
the structural features of 1-methylquinolinium scaffold and a plain indole ring, the simple
probes developed potentially renders a new class of specific G4-DNA with bioactive func-
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tions, and also it engages and inspires biochemists for the design of special ligand or mole-
cule to understand and to solve biological problems in biochemistry that could lead to im-
portant clinical applications.
In the present study, we reported a series of new fluorescent probe which is designed on the
combination of a 1-methylquinolinium scaffold and a plain indole ring by systematically in-
troducing at different position (Scheme 1). The results show that both the specificity and the
turn-on fluorescent signal enhancement towards G4-DNA of the molecular probes exhibited
remarkable differentiation with respect to its structural position of the indole ring. The char-
acteristics of the probes were comprehensively investigated with fluorescence titration, native
PAGE experiments and CD analysis in order to validate the selectivity, sensitivity, and stabil-
ity while interacting with various G4-DNA structures.
2. Experimental Sections
2.1. Materials
Melting point (m. p.) measurement was performed using a SRS Opti Mel automated melting
point instrument without correction. Mass spectra (MS) were recorded on Bruker amazon SL
mass spectrometer with an ESI or ACPI mass selective detector and the high resolution mass
1
spectra (HRMS) were recorded with Shimadzu LCMS-IT-TOF. H and 13C NMR spectra
were recorded in DMSO using TMS as the reference with a Bruker BioSpin GmbH spec-
trometer at 400 MHz and 100 MHz, respectively. All chemicals were purchased from com-
mercial sources unless otherwise specified. All the solvents were of analytical reagent grade
and were used without further purification. All oligonucleotides used in this work were syn-
thesized and purified by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China), and
their sequences were listed in Table 1.
2.2. Synthesis and characterization
1,2-dimethylquinolin-1-ium iodide (1a) To a solution of 2-methylquinoline (0.5 g, 3.5 mmol)
in sulfolane (10 mL) iodomethane (0.42 mL, 6.74 mmol) was added. The reaction mixture
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was stirred at 60 °C for 8 h in a closed pressure bottle. After cooled to room temperature, to
the reaction mixture anhydrous ether was added to precipitate the products. The solids were
collected by suction filtration and washed with anhydrous diethyl ether and then were dried in
1
vacuum to give compound 1a (0.85 g, 85%): H NMR (400 MHz, DMSO-d6) δ 9.11 (d, J =
8.5 Hz, 1H), 8.60 (d, J = 9.0 Hz, 1H), 8.38 (t, J = 15.7 Hz, 1H), 8.23 (t, J = 7.5 Hz, 1H), 8.13
(d, J = 8.5 Hz, 1H), 7.99 (t, J = 7.5 Hz, 1H), 4.46 (s, 3H), 3.09 (s, 3H). ESI-MS m/z: 158.2
[M+H]+.
1,4-dimethylquinolin-1-ium iodide (1b) To a solution of 4-methylquinoline (0.5 g, 3.5 mmol)
in sulfolane (10 mL) iodomethane (0.42mL, 6.74mmol) was added. The reaction mixture was
stirred at 60 °C for 8 h in a closed pressure bottle. After cooled to room temperature, to the
reaction mixture anhydrous ether was added to precipitate the products. The solids were col-
lected by suction filtration and washed with anhydrous diethyl ether and then were dried in
1
vacuum to give compound 1b (0.91g, 91 %): H NMR (400 MHz, DMSO-d6) δ 9.38 (d, J =
6.0 Hz, 1H), 8.53 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 8.9 Hz, 1H), 8.27 (t, J = 7.9 Hz, 1H), 8.07
(t, J = 6.3 Hz, 2H), 4.59 (s, 3H), 3.00 (s, 3H). ESI-MS m/z: 158.2 [M+H]+.
1,2,4-trimethylquinolin-1-ium iodide (1c) To a solution of 2,4-dimethylquinoline (0.5 g, 3.2
mmol) in sulfolane (10 mL) iodomethane (0.71 mL, 11.2 mmol) was added. The reaction
mixture was stirred at 70 °C for 8 h in a closed pressure bottle. After cooled to room temper-
ature, to the reaction mixture anhydrous ether was added to precipitate the products. The sol-
ids were collected by suction filtration and washed with anhydrous diethyl ether and then
were dried in vacuum to give compound 1c (0.73 g, 76.8 %): 1H NMR (400 MHz, DMSO-d6)
δ 8.51 (d, J = 9.0 Hz, 1H), 8.45 – 8.36 (m, 1H), 8.23 – 8.09 (m, 1H), 8.00 (s, 1H), 7.94 (t, J =
7.6 Hz, 1H), 4.35 (s, 3H), 2.99 (s, 3H), 2.88 (s, 3H). ESI-MS m/z: 172.2 [M+H]+.
2.3. General procedures for synthesis of target compounds 2a-c and 3a-c.
Compound 1 (0.71 mmol) and 1H-indole-3-carbaldehyde or 1H-indole-2-carbaldehyde (1.42
mmol) were added to a flask with butanol (7 mL) and followed adding 0.5 ml
4-methylpiperidine. The reaction solution was refluxed at 120 degrees Celsius for 3 h. After
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cooling to room temperature, the mixture was poured into ethyl acetate (10 mL) to precipitate
the products formed. The collected precipitates were purified by using column chromatog-
raphy with dichloromethane/methanol (v/v = 20:1) as the eluent to obtain the pure com-
pounds. The characterizations of each compound were listed as follows:
(E)-2-(2-(1H-indol-2-yl) vinyl)-1-methylquinolin-1-ium iodide (2a): Purple solid; yield
76%;1H NMR (400 MHz, DMSO-d6) δ 11.94 (s, 1H), 9.29 (d, J = 6.5 Hz, 1H), 8.90 (d, J =
8.4 Hz, 1H), 8.49 (d, J = 6.5 Hz, 1H), 8.45 (d, J = 8.9 Hz, 1H), 8.35 – 8.22 (m, 3H), 8.12 (t, J
= 7.7 Hz, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H),
7.08 (dd, J = 14.8, 7.1 Hz, 2H), 4.52 (s, 3H). 13C NMR (101 MHz, DMSO) δ 153.99 (s),
146.98 (s), 139.22 (s), 138.96 (s), 138.03 – 138.01 (m), 135.00 (s), 133.54 (s), 128.99 (s),
126.62 (s), 125.92 (s), 125.82 (s), 123.56 (s), 121.95 (s), 120.78 (s), 119.41 (s), 115.14 (s),
113.82 (s), 113.18 (s), 113.06 (s), 44.39 (s). Melting point: 256–258 °C, HRMS m/z: calcd for
C20H17N2 [M − I] + 285.1386, found 285.1378.
(E)-4-(2-(1H-indol-2-yl)vinyl)-1-methylquinolin-1-ium iodide (2b): Purple solid; yield
80%;1H NMR (400 MHz, DMSO-d6) δ 11.99 (s, 1H), 9.00 (d, J = 9.0 Hz, 1H), 8.60 (d, J =
9.1 Hz, 1H), 8.52 (d, J = 9.0 Hz, 1H), 8.34 (dd, J = 11.3, 6.5 Hz, 2H), 8.21 – 8.14 (m, 1H),
7.93 (t, J = 7.5 Hz, 1H), 7.83 (d, J = 15.7 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.2
Hz, 1H), 7.31 (t, J = 7.3 Hz, 1H), 7.14 (s, 1H), 7.10 (t, J = 7.4 Hz, 1H), 4.54 (s, 3H). 13C
NMR (101 MHz, DMSO) δ 152.43 – 152.33 (m), 148.12 (s), 139.28 (s), 139.20 (s), 136.13
(s), 135.37 (s), 133.31 (s), 129.62 (s), 128.55 (s), 128.55 (s), 126.62 – 126.52 (m), 126.12 (s),
125.34 (s), 121.98 (s), 120.60 (s), 119.91 (s), 117.72 (s), 115.61 (s), 111.99 (s), 110.86 (s),
45.00 (s). Melting point: 259–262 °C, HRMS m/z: calcd for C20H17N2 [M − I] + 285.1386,
found 285.1378.
2,4-bis((E)-2-(1H-indol-2-yl)vinyl)-1-methylquinolin-1-ium iodide (2c): Purple solid; yield
69%;1H NMR (400 MHz, DMSO-d6) δ 12.01 (s, 2H), 12.01 (s, 2H), 8.79 (d, J = 8.2 Hz, 1H),
8.67 (s, 1H), 8.57 (dd, J = 22.0, 15.7 Hz, 2H), 8.38 (d, J = 8.9 Hz, 1H), 8.31 – 8.23 (m, 4H),
8.09 (t, J = 7.7 Hz, 1H), 7.94 (d, J = 15.8 Hz, 1H), 7.86 (t, J = 7.6 Hz, 1H), 7.58 – 7.51 (m,
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3H), 7.29 (ddd, J = 9.5, 6.1, 3.1 Hz, 4H), 4.39 (s, 3H). 13C NMR (101 MHz, DMSO) δ 154.32
(s), 149.41 – 149.03 (m), 140.27 (s), 139.34 (s), 139.01 (s), 136.37 (s), 136.02 (s), 136.02 (s),
135.73 (s), 134.83 (s), 132.24 (s), 128.63 (s), 128.58 (d, J = 10.5 Hz), 128.52 (s), 125.82 (s),
125.62 (s), 125.56 (d, J = 13.9 Hz), 125.49 (s), 125.04 (s), 122.18 (s), 122.18 (s), 121.87 (s),
121.87 (s), 120.71 (s), 120.71 (s), 120.51 (s), 120.51 (s), 120.10 (s), 120.10 (s), 118.09 –
118.03 (m), 117.24 (s), 117.24 (s), 114.47 (s), 114.47 (s), 112.12 (s), 112.02 (d, J = 19.9 Hz),
111.92 (s), 111.55 (s), 111.55 (s), 109.95 (s), 40.54 (d, J = 21.0 Hz), 40.22 (s), 40.01 (s),
39.80 (s), 39.60 (s), 39.39 (s). Melting point: 267–269 °C, HRMS m/z: calcd for C30H24N3
[M − I] + 426.1962; found 426.1960.
(E)-2-(2-(1H-indol-3-yl) vinyl)-1-methylquinolin-1-ium iodide (3a): Purple solid; yield
78%;1H NMR (400 MHz, DMSO-d6) δ 12.31 (s, 1H), 8.81 (d, J = 9.1 Hz, 1H), 8.64 (d, J =
15.5 Hz, 1H), 8.60 (d, J = 9.2 Hz, 1H), 8.44 (d, J = 8.9 Hz, 1H), 8.40 (d, J = 2.8 Hz, 1H),
8.31 – 8.17 (m, 2H), 8.08 (t, J = 7.7 Hz, 1H), 7.84 (t, J = 7.5 Hz, 1H), 7.64 – 7.48 (m, 2H),
7.31 (dd, J = 5.9, 3.0 Hz, 2H), 4.46 (s, 3H). 13C NMR (101 MHz, DMSO) δ 156.06 (s),
156.06 (s), 143.74 (s), 139.65 (d, J = 16.5 Hz), 137.30 (s), 135.54 (s), 135.15 (s), 130.47 (s),
129.17 (s), 128.46 (s), 127.95 (s), 125.99 (s), 122.30 (s), 120.80 (d, J = 5.4 Hz), 119.61 (s),
116.65 (s), 112.55 (s), 112.19 (s). Melting point: 253–254 °C HRMS m/z: calcd for C20H17N2
[M − I] + 285.1386; found 285.1384.
(E)-4-(2-(1H-indol-3-yl)vinyl)-1-methylquinolin-1-ium iodide (3b): Purple solid; yield
84%;1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 9.11 (d, J = 6.7 Hz, 1H), 8.97 (d, J =
8.6 Hz, 1H), 8.57 (d, J = 15.6 Hz, 1H), 8.42 (d, J = 6.7 Hz, 1H), 8.34 (d, J = 6.6 Hz, 2H),
8.26 (dd, J = 6.1, 2.6 Hz, 1H), 8.24 – 8.17 (m, 1H), 8.01 (dd, J = 15.6, 9.0 Hz, 2H), 7.62 –
7.50 (m, 1H), 7.29 (dd, J = 5.9, 3.0 Hz, 2H), 4.44 (s, 3H). 13C NMR (101 MHz, DMSO) δ
157.14 (s), 143.28 (s), 142.15 (s), 139.58 (s), 137.99 (s), 135.02 (s), 134.53 (s), 130.26 (s),
128.34 (s), 127.11 (s), 125.68 (s), 123.88 (s), 122.38 (s), 120.81 (s), 120.36 (s), 119.21 (s),
115.00 (s), 113.31 (s), 111.95 (s). Melting point: 255–257 °C HRMS m/z: calcd for C20H17N2
[M − I] + 285.1386, found 258.1378.
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2,4-bis((E)-2-(1H-indol-3-yl)vinyl)-1-methylquinolin-1-ium iodide (3c): Purple solid; yield
71%;1H NMR (400 MHz, DMSO-d6) δ 12.05 (s, 1H), 8.77 (d, J = 8.2 Hz, 1H), 8.63 (d, J =
14.3 Hz, 1H), 8.59 – 8.49 (m, 1H), 8.36 (d, J = 8.8 Hz, 1H), 8.30 (s, 1H), 8.29 – 8.18 (m, 2H),
8.08 (t, J = 7.7 Hz, 1H), 7.94 (s, 1H), 7.86 (dd, J = 16.7, 9.0 Hz, 1H), 7.58 – 7.48 (m, 2H),
7.32 – 7.25 (m, 2H), 4.37 (s, 2H). 13C NMR (101 MHz, DMSO) δ 155.19 (s), 150.03 (s),
140.73 (s), 140.13 (s), 138.00 (s), 137.80 (s), 136.71 (s), 134.22 (s), 133.74 (s), 131.93 (s),
127.75 (s), 126.28 (s), 125.82 (s), 125.58 (s), 124.71 (s), 123.61 (s), 123.29 (s), 121.98 (s),
121.56 (s), 120.91 (s), 120.84 (s), 119.46 (s), 114.84 (s), 114.66 (s), 113.90 (s), 113.20 (s),
113.08 (s), 112.99 (s), 112.75 (s), 39.11 (s). Melting point: 266–269 °C, HRMS m/z: calcd for
C30H24N3 [M − I] + 426.1962; found 426.1960.
2.4. Measurements and methodology
2.4.1. UV-Vis and fluorescence assays
UV-Vis spectra were obtained with a Lambda 25 Spectrophotometer (Perkin Elmer). Fluo-
rescence spectra were recorded on a LS-45 fluorescence spectrometer (Perkin Elmer). With
the data obtained through fluorimetric titrations, the binding constants were analyzed accord-
ing to the independent site model50 by nonlinear fitting with Equation (1).51 The parameters P
and M, Q and N were found via the Levenberg-Marquardt fitting routine in the Origin 9
software, where as a varied to obtain a better fit. Kd = Ka-1.
F-F0/F0 = 1+(Q-1)/2{N+1+X-[(X+1+N)24X]1/2} …………Eq. (1)
Where F0 is the fluorescence intensity of 2a-c and 3a-c in the absence of DNA, Fmax is the
fluorescence intensity upon saturation of DNA, Q = Fmax(F0)-1, N = (KaCdye)-1, and X =
nCDNA(Cdye)-1.
The limit of detection (LOD) was calculated on the basis of the equation: LOD = K Sb/m. The
K value is in accordance with the International Union of Pure and Applied Chemistry (IU-
PAC) and is generally taken to be 3; Sb represents the standard deviation of the blank multiple
measurements (n = 20) and m is the slope of the calibration curve, which represents the sen-
sitivity of this method.52
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2.4.2. Measurement of fluorescent quantum yields of the new compounds
The fluorescent quantum yields of the new compounds were measured in the presence of
DNA with fluorescein (Ф=0.85) in 1 % NaOH ethanol solution as a standard.53 Values of ab-
sorbance and fluorescence were recorded after adding the new compound with a series of in-
creasing concentrations into a fixed concentration DNA solution. Quantum yields can be cal-
culated according to Equation (2):54
Фx=ФST (Gradx / GradST) (η2x/η2ST) …………. Eq. (2)
Where the subscripts ST and X denote the standard and test respectively; Ф is the fluores-
cence quantum yield; Grad is the gradient from the plot of integrated fluorescence intensity
versus absorbance, and is the refractive index of the solvent.
2.5. Polyacrylamide gel electrophoresis (PAGE) experiment conditions
Gel electrophoresis was performed with 30 % (29:1) polyacrylamide gels. The select ssDNA,
dsDNA, and quadruplex-forming oligonucleotides (5ꢀM, detailed sequences listed in Table 1
were run in 1x TBE buffer (80 mM Tris-borate, 2mM EDTA, pH=8.3), by using the follow-
ing parameters: temperature = 20 °C, voltage = 100 V, time = 3 h. The gel was post-stained
with a solution of the new compound (5ꢀM) in 1 x TBE.
2.6. Circular dichroism (CD) experiment conditions
The CD spectra were obtained using a Chirascan spectrophotometer (Applied Photophysics)
and these experiments were carried out on DNA samples in quartz cuvette with 4mm path
length. The final concentration of pu27 was 1 ꢀM and the concentrations of the new probes
were from 1 to 5-fold increment. Both the DNA and the new probes were dissolved in 10 mM
Tris-HCl, 60 mM KCl, pH = 7.4. The quartz cuvette with 4 mm path length was employed for
the spectra recorded over a wavelength range of 230 nm to 330 nm, with the bandwidth 1 nm,
step size 1 nm. The CD spectra were obtained by taking the average of at least three repeated
trials. Final analyses of the data were carried out using Origin 9.0 (Origin Lab Corp).
2.7. Cell culture conditions and fluorescence microscopy imaging study
Fluorescence microscopy images were obtained on Olympus IX71 inverted fluorescence by
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PC3 cells (human prostate cancer cells, ATCC® CRL-1435). Before staining, the cells grown
on a six-well cell culture cluster were fixed by precooled 99 % methanol (-4 °C) for 1 min
and then was washed twice with phosphate-buffered saline (PBS) for 5 min. In Fluorescence
microscopy images, the fixed PC3 cells were stained by 5 µM 3c for 20 min at ambient tem-
perature and washed by PBS for 5 minutes twice. Then a reference probe
6-diamidino-2-phenylindole (DAPI, 5 mg/mL) was used to stain nucleus for 5 min for locali-
zation, and then also was washed twice by PBS for 5 min. PC3 cells were both incubated in
PBS during the fluorescence microscopy experiments.
2.8. DNase and RNase digestion tests
The cell membrane permeabilization was done by immersing cells in 1 % Triton X-100 for 2
min. Then rinsed twice with PBS, then 100 mL of probe-PBS solution (1 mM) was added in-
to three adjacent wells. Cells were incubated with the probe in PBS for 15 min in ambient
temperature and then were rinsed twice by clean PBS. For control experiments, a total of 100
mL clean PBS, 30 mg/ml DNase (Sigma), or 25 mg/ml DNase-Free RNase (GE) was added
into the three adjacent wells and then was incubated at 37 °C in 5 % CO2 for 2 h. Cells were
rinsed twice by clean PBS and then was used for imaging. For each probe tested, the fluores-
cent imaging pictures were obtained by using an equal exposure time for control, DNase, and
RNase experiments.
3. Results and Discussion
3.1. Synthesis and the spectroscopic properties of the fluorescent probes
A series of new fluorescent probes with an indolyl ring fused to 1-methylquinolinium were
readily obtained in high yields by a straightforward two-step synthesis under mild conditions
(Scheme 1). The key intermediate (1,2-dimethylquinolin-1-ium iodide hydrochloride) was
prepared by the reaction of 2-methylquinoline derivatives with iodide. Further reaction of the
intermediate with a suitable starting of indolyl carbaldehyde, six analogs of the
1-methylquinolinium-based fluorescent probes conjugated with an indole-ring at different
position were synthesized for the investigation of binding specificity towards G4-DNA struc-
11

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tures and switch-on signaling effects. The incorporation of positively charged
1-methylquinolinium fragment is able to enhance the compound solubility in buffer solu-
1
tions.55 The new compounds obtained were confirmed unambiguously with H NMR, 13C
NMR, and HRMS.
The spectroscopic properties of the compounds (2a-b and 3a-c) were examined in a Tris-HCl
buffer (10 mM, pH = 7.4) as show in Table 2. In general, the absorption maxima of the
compounds in buffer solution are found in the range of 465 - 531 nm while the emission
maxima are in the range of 556 - 617 nm which the emission from orange to red region is
suitable for cell imaging applications. To our surprise, although these probes have a similar
structure, which composed of 1-methylquinolinium scaffold and an indole-ring, the connec-
tion with an ethylene bridge at different position for the two components results remarkable
difference in both strokes shift and fluorescence quantum yield. This may imply that the
combination of 1-methylquinolinium scaffold and an indole-ring has a special structural con-
formation affecting its electronic effects of the conjugated rotatable flat-ring systems. The
observation has not been disclosed in literature. The conformation effects would be further
demonstrated through the specificity of the probe on the fluorescent signaling of discrimina-
tion of G4-DNA structures from other nucleic acids.
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Scheme 1. General synthetic routes to 2a-3c and their molecular structures.
13

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By comparing fluorescent properties of the compounds, it seems that the position of the in-
dole ring to fuse with 1-methylquinolinium scaffold is very crucial. Obviously, the series of
2a-c that the indole ring connected at its position 2 (1H-indol-2-yl) is almost not emissive as
indicated by their small quantum yields (0.017-0.022, and 2c is not detectable), and also by
their weak fluorescence response upon binding with various DNA G-quadruplexes (Figure 1
and Table 2). Interestingly, when the 1H-indol-3-yl moiety connected at position 3, 3a-c are
able to exhibit about 10 times higher quantum yields (0.13-0.26). It is out of our expectation
that the series of 3a-c shows strong switch-on fluorescent signal specifically towards DNA
G-quadruplex structures (hras, htg22, oxy28, telo21, 4telo, human12, bcl2, ckit2, pu22, pu27,
ckit1, vegh), while the fluorescence response for binding with ssDNA (da21) and dsDNA
(ds12, ds26, 4at, and 4a4t) was found very weak.56-58 In addition, 3a-c displays different de-
grees of fluorescence response when binding with RNA. Nonetheless, with the preliminary
results from Figure 1, 3c gives the best performance on the switch-on fluorescent signal and
is suitable for the study of binding property with various nucleic acid substrates and for the
visualization of G4-DNA in live cells.
800
800
2a
dsDNA Telomere G4DNA Promoter G4DNA
RNA ssDNA
Telomere G4DNA Promoter G4DNA
ssDNA dsDNA
RNA
3a
600
600
400
200
0
400
200
0
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800
3b800
RNA ssDNA dsDNA
Telomere G4DNA
Promoter G4DNA
Telomere G4DNA Promoter G4DNA
RNA ssDNA dsDNA
2b
600
400
200
0
600
400
200
0
800
800
Promoter G4DNA
RNA ssDNA dsDNA Telomere G4DNA
Promoter G4DNA
ssDNA dsDNA Telomere G4DNA
RNA
2c
3c
600
400
200
0
600
400
200
0
Figure 1. Fluorescence intensity of the probes (2a-c and 3a-c) with RNA and different DNA.
Single-stranded DNA: da21; duplex DNA: 4a4t, 4at, ds12, ds26; G-quadruplex DNA: htg22,
pu27, hras and vegf. The concentration of the probes was 5 ꢀM in a Tris–HCl buffer contain-
ing 60 mM KCl.
Table 1. Sequences of oligonucleotides used in the present study
Abbrevia- Sequence
tion
Structure/origin
da21
4a4t
AAAAAAAAAAAAAAAAAAAAA
ssDNA
AAAATTTT
dsDNA
4at
ATATATATATAT
dsDNA
ds12
ds26
htg22
oxy28
GCGCAATTGCGC
dsDNA
CAATCGGATCGAATTCGATCCGATTG
AGGGTTAGGGTTAGGGTTAGGG
dsDNA
Telomere G-quadruplex
Telomere G-quadruplex
GGGGTTTTGGGGTTTTGGGGTTTGGGG
15

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4telo
GGGTTAGGGTTAGGGTTAGGGTTAGGGTTAG Telomere G-quadruplex
telo21
human12
bcl2
GGGTTAGGGTTAGGGTTAGGG
TTAGGGTTAGGG
Telomere G-quadruplex
Telomere G-quadruplex
Promoter G-quadruplex
Promoter G-quadruplex
Promoter-G-quadruplex
Promoter G-quadruplex
Promoter G-quadruplex
Duplex
GGGCGCGGGAGGAAGGGGGCGGG
TGAG3TG3TAG3TG3TAA
pu22
ckit1
AGGGAGGGCGCTGGGAGGAGGG
GGGCGGGCGCGAGGGAGGGG
GGGGCGGGCCGGGGGCGGGG
16S- and 23S-Ribosomal from E. coli
ckit2
vegf
RNA
Table 2. The spectroscopic properties of the fluorescent probes.a
Abs
Ex
Em
Stoke Фf c
shiftb
(λmax/nm) (λmax/nm) (λmax/nm)
485
492
477
466
465
531
495
502
487
476
475
541
598
566
N.D.d
556
595
617
103
64
0.022
0.017
2a
2b
2c
3a
3b
3c
N.D.d N.D.d
80
0.21
0.26
0.13
120
76
a Experiments were performed in 10 mM Tris-HCl buffer at pH = 7.4.
b Stokes shift.
c
Relative fluorescence quantum yield of probes upon addition of 10 mM telo21, standard of
the relative fluorescence quantum yield is fluorescein (Φf = 0.85, in CH3OH).
d Not determined.
16

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3.2. Specificity study of the probe towards DNA G-quadruplex against other nucleic ac-
ids
The selectivity of 3c to DNA G-quadruplexes was investigated by using fluorescence titra-
tions with various DNA oligomers including G-quadruplex, single-, double-stranded DNA
and RNA. As shown in Figure 3A, the enhanced fluorescence intensity of 3c was found to be
about 150-290 folds upon binding with different DNA G-quadruplex (telo21, htg22, ckit2,
pu22) at saturated concentration. In contrast, under same experimental conditions, when 3c
binds with duplex DNA (ds26), single-stranded DNA (da21), and RNA a much smaller (less
than 50-fold) fluorescence enhancement was observed generally. The results indicate that 3c
exhibited excellent switch-on fluorescent signal for the discrimination of G-quadruplexes
from the single-, double-stranded DNA, and RNA. To further confirm the structural im-
portance attributed from the position of the indolyl moiety, we also performed the same fluo-
rescence titration with 2c under the same experimental conditions and the results show almost
no fluorescence enhancement for the nucleic acids tested.
In order to ensure the switch-on fluorescent signal is due to the specific binding of 3c with
G4-DNA structure, a competitive fluorescence titration was conducted in the presence of a
large excess of a duplex DNA (ds26, 100 µM) with telo21 quadruplex (Figure 3B). At the
high concentration of ds26, a weak background fluorescent signal of 3c was observed; how-
ever, when adding telo21 to the solution, it results a significant enhancement of fluorescent
signal and the intensity was kept increasing (up to 7 folds) as more telo21 was added. The
results revealed that ds26 originally bound with 3c was replaced by telo21 and thus generated
stronger fluorescent signal. These results demonstrated that 3c possesses excellent specificity
for discrimination of G4-DNA from other nucleic acids even in a more competitive biological
condition.
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da21
ds26
rna
A 600
B
300
250
200
150
100
50
ckit2
telo21
pu22
htg22
500
400
300
200
100
0
0
4
8
12
16
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Concentration/µM
telo21/µM
Figure 3. (A) Fluorescence intensities of 3c at 617 nm with different amounts of DNAs (λex
= 531 nm). DNA G-quadruplex: telo21, htg22, ckit-2, pu22; duplex DNA: ds26; single-stand
DNA: da21 The concentration of 3c was 5 µM in Tris–HCl buffer containing 60 mM KCl. (B)
Fluorescence titration of the telo21 quadruplex to the 3c (λex=531 nm) in the presence of an
excess of ds26 (100 µM). The concentration of 3c was 3 µM in Tris–HCl buffer containing
60 mM KCl.
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3.3. Utility of the probe as a stain for PAGE study
G4-DNA substrates are usually hard to be visualized in native gel electrophoresis (PAGE)
stained by common fluorescent strains such as ethidium bromide, CelRed and SYBR Safe.
From the above experimental results, 3c shows good binding ability and fluorescent
switch-on specificity towards G4-DNA and is not significantly interfered by other form of
nucleic acids such as dsDNA even under high concentration and RNA. We thus attempt to
explore the performance of 3c as a staining agent in PAGE. In the experiments, we selected a
number of common nucleic acids including htg22, oxy28, telo21, ckit-1, hras, da21, ds26,
and RNA for testing. As shown in Figure 4A, the post-staining of PAGE gels with 5 µM 3c in
Tris-HCl solution, clear staining bands are observed for G4-DNA substrates, while ds26 or
RNA only results very weak band, and almost no fluorescence was visualized in the band of
da21. This is also in accord with the observation from the visualization test in solution (Fig-
ure 4B). The results are found positive and indicate that 3c can be used as a good staining
probe specific for visualization of DNA G-quadruplex substrates.
Figure 4. (A) Gel electrophoresis (20 % acrylamide in 1 × TBE) of RNA, ds26, da21, hras,
ckit1, telo21, oxy28, htg22, at a concentration 5 ꢀM, stained with 3c for 30 min; (B) The flu-
orescence changes of 3c (ex=302 nm) with the presence of various nucleic acids:
G-quadruplex telo21, hras, ckit1, oxy28, htg22 double-stranded DNA ds26, single-stranded
DNA da21, and RNA in at 10 mM Tris-HCl, 60 mM KCl, pH = 7.4 buffer solution
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3.4. G4-DNA binding affinity, limit of detection (LOD), and interaction study
The binding property of the probes with DNA G-quadruplex and the intrinsic binding con-
stant were also studied by fluorescence titrations. A typical titration experiment using 3c to
titrate with telo21 quadruplex in Tris-HCl (10 mM, pH = 7.4) buffer was shown in Figure 5.
The experimental results suggested that 3c are the most useful and highly selective probe for
the determination of DNA quadruplex. The analytical parameters showed in Table 3 gave a
good linear relationship between the enhanced fluorescent intensity and the amounts of telo21
quadruplex present in the test solution over a wide range of concentrations (0.14–2.14 ꢀM).
Among the new probes examined, 3c exhibits the best performance with the lowest LOD
(13.8 nM). Surprisingly, the compound series of 2a-c show very weak interactions with te-
lo21 quadruplex because their equilibrium binding constants were found less than 0.1 x 105
M-1 while the series 3a-c were found having much stronger interactions with telo21 indicated
by their large equilibrium binding constants (from 2.5 x 105 M-1 to 7 x 105 M-1). The results
reveal that the position of the indolyl moiety conjugated to 1-methylquinolinium scaffold is a
crucial factor and obviously at position 3 of the indole ring moiety (1H-indol-3-yl) is the best.
A
500
B
250
telo21
400
300
200
100
0
200
150
100
50
B 160
140
120
100
80
R2=0.99465
2
3
4
5
telo 21 (µM)
0
600
650
700
750
800
0
3
6
9
12
15
Wavelength/nm
telo21(µM)
Figure 5. (A) Fluorescence titration spectra of 3c with concentrations of G-quadruplex in
Tris-HCl (10 mM, pH = 7.4) buffer. (B) Fitted curve of 3c with good linear relationship with
0-14.25 µM telo21. F0 represents the fluorescence intensity without telo21 and F represents
the fluorescence intensity after adding telo21.
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Table 3. Fluorescence titration results of the probes with telo21 quadruplex.a
F/F0
R shift Total
Lrd
LOD
Kd(x105 R2
(nm)b
DNA
(ꢀM)c
(nM)d
M-1)e
(ꢀM)
11.3
5
11.06
10.08
9.80
0.14~1.58 638
0.14~1.44 313
0.09
0.038
N.D.
3.95
7.01
2.52
0.90792
2a
2b
2c
3a
3b
3c
18.6
5
0.92155
N.D.
N.D.f
96.6
N.D.
0.14~1.4
N.D.
4
5
4
16.2
0.14~2.31 71.24
0.14~2.04 6.0
0.14~2.14 13.8
0.99309
0.99646
0.99607
378.0
14.28
237.1
14.98
a The concentration of the probe used is 5 ꢀM; b Hypochromatic shift of probes; c Linear de-
tection range of telo21; d Limit of telo21 detection; e Binding constant of the probe and telo21;
f Not determined.
The G-quadruplex structural conversion or changes induced by G-quadruplex binder 3c were
investigated with circular dichroism (CD) spectroscopy. In the absence of any salt, the telo21
oligonucleotide was dissociated partially to single-stranded molecules with a negative band
centered at 238 nm, a major positive band at 257 nm, a minor negative band at 280 nm, and a
positive band near 298 nm (Figure 6A). The bands at 238 and 257 nm disappeared gradually
upon the addition of 3c, thereby revealing a major negative band at 266 nm and significantly
increasing the intensity of the band centered at 298 nm. These changes are consistent with the
induction of the guanine-rich DNA in forming the antiparallel G-quadruplex structure by 3c.
It is known that in the absence of binder molecules, the typical CD spectrum of telo21 is a
hybrid-type G-quadruplex59,60 in K+ buffer and is an anti-parallel G-quadruplex in Na+ buffer.
For the experiments conducted in a K+ buffer, the addition of 3c (concentration at 5 ꢀM, 15
ꢀM, 25 ꢀM) to a solution with 5 ꢀM telo21, the characteristic peaks of an anti-parallel
G-quadruplex was found to change with the positive peak at 290 nm and the negative peak at
265 nm (Figure 6B). However, 3c showed only slightly influence on the anti-parallel type of
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telo21 in Na+ buffer (Figure 6C). The results indicate that 3c binds with telo21 G-quadruplex
and also induces no sequence transform. This finding is also consistent with the switch-on
fluorescent signal observed in the fluorescence titration experiments.
telo21alone
8
telo21:3c=1:1
A
telo21:3c=1:3
telo21:3c=1:5
6
4
2
0
-2
-4
-6
240
260
280
300
320
Wavelength/nm
telo21alone
telo21:3c=1:1
telo21:3c=1:3
telo21:3c=1:5
10
8
B
6
4
2
0
-2
240
260
280
300
320
Wavelength/nm
telo21 alone
telo21:3c=1:1
telo21:3c=1:3
telo21:3c=1:5
10
8
C
6
4
2
0
-2
-4
-6
-8
240
260
280
300
320
Wavelength/nm
Figure 6. (A) CD spectra of 3c bound to telo21 (5 µM) in 10 mM Tris–HCl, pH 7.4. The
concentration of 3c is 5 µM 15 µM and 25 µM. (B) CD spectra of 3c bound to telo21 (5 µM)
in 10 mM Tris–HCl, pH 7.4, 60 mM KCl. The concentration of 3c is 5 µM 15 µM and 25 µM.
(C) CD spectra of 3c bound to telo21 (5 µM) in 10 mM Tris–HCl, pH 7.4, 60 mM NaCl. The
concentration of 3c is 5 µM 15 µM and 25 µM.
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3.5. Living cell staining and imaging
The binding ability and selectivity of the probe to distinguish DNA G-quadruplex in living
PC3 cells were investigated. With respect to the experimental results, 3c is the best candidate
for the study because it exhibits a number of good properties over its analogs, such as good
fluorescence quantum yield, very low LOD, high selectivity towards G4-DNA with strong
binding affinity. Fluorescence microscopic measurements of the treated PC3 cells were taken
place for comparison. As shown in Figure 7A, a strong fluorescence response of the probe
was induced within the nucleus in certain regions where probably are the nucleoli that rDNAs
undergo transcription because guanine-rich rDNA is known to adopt temporal quadruplex
conformations. In addition, a low signal response was observed due to non-specific binding to
duplex DNA, which is revealed by the co-staining experiments with the duplex DNA binder
DAPI.1 Nonetheless, the possibility of 3c binding with RNAs in the nucleus may also happen
and thus induce weak fluorescence signal enhancement. To further verify the sensing and
binding target of 3c in nucleoli, ribonuclease (RNase) and deoxy-ribouclease (DNase) digest
experiments Figure 7B and 7C were conducted.61 The enhanced fluorescence signals of 3c in
nucleoli clearly disappeared after DNase treatment, but not after RNase treatment. Therefore,
the probe could enter into the nucleus and interact with the G-quadruplex generating strong
fluorescence. The clear staining images demonstrated that 3c is suitable for living cells appli-
cations on monitoring of nucleus activities.
Conclusion
In conclusion, we demonstrated the structural importance of a simple molecular probe that
integrated from natural product fragments for selective visualization of DNA G-quadruplex
structures in live cells. One of the compounds exhibits significant application potential as a
specific staining and imaging probe for monitoring of DNA G-quadruplex activities particu-
larly suitable for those experiments in the nuclei of the cell. Also, the fragments adopted to
compose the probe with high specificity are from the natural products of quinoline and indole
give excellent cell tolerance for live cell experiments. This type of fluorescent probes is not
commonly found in commercial and could be utilized to establish useful and important visu-
23

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alization protocols for clinical research and medical applications and treatment.
Figure 7. Fluorescence images of PC3 cells stained with 5.0 µM 3c for 5 min and 1.0 µM
DAPI for 20 min without and with DNase or RNase treatment. The 400x magnification in
figure A, E and I, and 200x magnification in other figures. Scale bar is 10 ꢀm.
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Acknowledgments
We acknowledge the support received from the National Nature Science Foundation of China
(21102021 and 81473082), Nature Science Foundation of Guangdong Province, China
(S2012010010200), and Science and Technology 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 Technol-
ogy “Pei Ying Yu Cai” Project.
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