A fluorescent guanosine dinucleoside as a selective switch-on sensor for c-myc G-quadruplex DNA with potent anticancer activities

Article abstract of DOI:10.1002/chem.201302107

Like likes like! A novel fluorescent C₂-symmetric guanosine-based dinucleoside has been engineered by chemical ligation of two guanosine units with a biocompatible dansyl tag. The nucleoside exhibits high selectivity for c-myc G-quadruplex DNA through fluorescence enhancement over duplex DNA and other promoter G-quadruplexes (see scheme). It stains the nucleus preferentially, arrests the cell cycle at the G2/M phase, inhibits cell growth, and induces apoptosis in A375 cancer cells. Copyright

Full text of DOI:10.1002/chem.201302107

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DOI: 10.1002/chem.201302107
A Fluorescent Guanosine Dinucleoside as a Selective Switch-On Sensor
for c-myc G-Quadruplex DNA with Potent Anticancer Activities
Y. Pavan Kumar,[a] Sudipta Bhowmik,[a] Rabindra Nath Das,[b] Irene Bessi,[c]
Sushovan Paladhi,[a] Rita Ghosh,[d] Harald Schwalbe,[c] and Jyotirmayee Dash*[a, b]
Dedicated to Professor Krishna N. Ganesh on the ocasion of his 60th birthday
Development of fluorescent chemical probes that can rec-
ognize biomacromolecules with high specificity and interfere
with cellular processes is an emerging trend in chemical bi-
ology.[1,2] Within this context, G-quadruplex DNA has re-
ceived considerable attention as a prospective target for de-
signing anticancer drugs.[3–4] G-Quadruplex structures are
widespread in the genome, found at the end of telomeres, in
the promoter regions proto-oncogenes, and in the untrans-
lated regions of mRNAs. These structures are believed to
play key roles in the human genome, such as telomere main-
tenance and gene regulation.[4] While several classes of mol-
ecules have been reported for stabilization of G-quadruplex
DNA, only a few fluorescent chemosensors for the selective
detection of G-quadruplex motifs have been developed.[5,6]
In addition, there is current interest in developing fluores-
cent probes with multiple signals that can find applications
in analytical and computational devices.[1–2,7] Recently we
have shown that it is possible to create biomolecular logic-
gate systems based on the interaction of fluorescent molecu-
lar probes with the G-quadruplex DNA.[5f]
DNA. However, the G-quartets (self-assembled Hoogsteen-
type hydrogen-bonded macrocycles of four guanine bases)
are common to all quadruplexes, making discrimination be-
tween the quadruplexes challenging.
Previously we have reported synthesis of G-quadruplex
binding ligand[9] using “click-chemistry”[9c] and fabrication of
complex nanoarchitectures using supramolecular self-assem-
bly of guanosine derivatives.[10] Among the five natural nu-
cleobases, guanosine and its derivatives have received con-
siderable interest in supramolecular chemistry and nano-
technology.[11] Taking inspiration from the natural self-or-
ganization of nucleosides, we envisioned designing a flexible
ligand by linking a biocompatible fluorescent tag between
two guanosine units using CuI-catalyzed 1,3-dipolar azide–
alkyne cycloaddition.[12,13] We hypothesized that the guano-
sine units can interact with the G-quartet of the quadruplex
by means of hydrogen bonding and the flexible linker can
tether in the groove region of quadruplex sequences. Since
quadruplex sequences vary in the groove and loop regions,
the ligand may show selectivity for a particular quadruplex
sequence, for which the groove region would be maximally
occupied. Further the ligand based on a guanosine nucleo-
side can be incorporated into DNA and the attached fluoro-
phore would enable visualization of the nucleus in living
cells.[14]
Monchaud and co-workers have reported the design and
synthesis of two molecules containing guanine bases and
their possible self-assembly to form artificial G-quartets as a
nature-inspired strategy to interact with the G-quadruplex.[8]
These ligands show selectivity for qudruplexes over duplex
Based on our design principle, we have synthesized a gua-
nosine azide 1 from the natural nucleobase guanosine in
three steps and a biocompartible fluorescent dansyl probe 2
(Scheme 1 and Scheme S1 in the Supporting Information).
The C2-symmetric dinucleoside 3 (DDG) was prepared in
high yields by employing a double click reaction of 1 with
the dansyl dialkyne 2 in the presence of Na ascorbate and
CuSO4·5H2O in tBuOH/H2O (1:1).
The ability of DDG to discriminate nucleic acid sequen-
ces was investigated using fluorescence spectroscopy
(Figure 1). The emission spectra of the guanosine–dansyl
conjugate DDG was characterized by a twofold intense
peak at 430 nm and a minor peak at 553 nm, when excited
at 350 nm (quantum yield, F=0.25). Guanosine azide 1 is
essentially non-fluorescent; however, the emission bands of
DDG at 430 and 553 nm were assigned to the guanosine
and the dansyl group, respectively, owing to the excitation
energy transfer from the guanosine to the dansyl chromo-
[a] Y. P. Kumar, Dr. S. Bhowmik, S. Paladhi, Prof. Dr. J. Dash
Department of Organic Chemistry
Indian Association for the Cultivation of Science Jadavpur
Kolkata-700032 (India)
E-mail: ocjd@iacs.res.in
[b] R. N. Das, Prof. Dr. J. Dash
Department of Chemical Sciences
Indian Institute of Science Education and Research Kolkata
Mohanpur, West Bengal 741252 (India)
[c] I. Bessi, Prof. Dr. H. Schwalbe
Institute of Organic Chemistry and Chemical Biology
Goethe University Frankfurt and
Center for Biomolecular Magnetic Resonance
Max-von-Laue Strasse 7, 60438 Frankfurt am Main (Germany)
[d] R. Ghosh
Department of Biochemistry and Biophysics
University of Kalyani, Kalyani-741235, West Bengal (India)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201302107.
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Scheme 1. Modular synthesis of a fluorescent guanosine dinucleoside 3 (DDG).
duplex DNA (Figure 1d).
negligible fluorescence
A
en-
hancement of DDG was ob-
served for duplex DNA at both
the guanosine and dansyl
bands. By comparing the
change in ratio of the fold in-
crease between guanosine and
dansyl emission of DDG upon
titration with DNA sequences,
it was observed that in all cases
the guanosine band was in-
creased from one- to fourfold
compared to the dansyl band.
The ratiometric variation of
DDG was dependent upon the
nucleic acid sequences and rec-
ognition occurred at very low
concentration of DNA. Inter-
estingly, even in the presence of
100 nm c-myc, the emission in-
tensity of DDG was increased
fivefold at 430 nm (Figure 1d).
It is important to point out that
such nanomolar sensitivity is
highly impressive for the devel-
opment of biomarker for the
quadruplex DNA. To the best
Figure 1. Fluorimetric titration spectra of DDG (500 nm) with a) c-myc quadruplex DNA (0–500 nm) and b)
with duplex DNA (0–1500 nm). c) Fluorescence intensity profiles of DDG (500 nm) with quadruplex sequences
~
&
*
(0–500 nm) at 430 nm ( =c-myc, =c-kit1, =c-kit2). d) Fold increase of fluorescence intensity for DDG
(500 nm) upon titration with DNA sequences (100 nm).
phore.[15,16] This indicates that DDG, which contains biologi-
cally active non-natural and natural components, such as
dansyl sulphonamide, triazole and guanosine, exhibits dis-
tinct optical properties and may lead to enhance biological
activities.
When the fluorimetric titration of DDG (500 nm) was car-
ried out at pH 7.4 with increasing concentration of quadru-
plex sequences (0–500 nm), the fluorescence intensity of
both dansyl and guanosine band was enhanced in a dose-de-
pendent manner from which the dissociation binding con-
stants (Kd) were calculated (Figure 1 and Figures S2 and S3
and Table S1 in the Supporting Information).[17] In the emis-
sion spectra, the addition of c-myc resulted higher selectivi-
ty, with a 6-fold enhancement of guanosine band and 2.5-
fold enhancement for the dansyl band. It shows high selec-
tivity for c-myc over c-kit2 and c-kit1 quadruplexes and
of our knowledge, none of the previously reported probes
can detect nanomolar DNA with high selectivity.[5] DDG
showed both excellent affinity for c-myc quadruplex (Kd =
20.8 nm) as well as substantial specificity (44.5-fold) over
duplex DNA (Kd =925.7 nm) at 430 nm. DDG also exhibited
3.8-fold selectivity for c-myc over c-kit2 (Kd =79.4 nm) and
7.6-fold selectivity for c-myc over c-kit1 (Kd =158.8 nm).
Based on the high specificity of the guansoine–dansyl con-
jugate for c-myc quadruplex DNA, we performed excited-
state lifetime measurements[18] of DDG in the absence and
presence of c-myc quadruplex DNA (Figure 2). The results
reveal that DDG has two forms in the excited state, a major
component (95.5%) with a lifetime of 454 ps and a minor
form with a higher lifetime (1.7 ns). Upon addition of
1.0 equivalents of c-myc quadruplex, an enhancement in the
excited state lifetime of both the species was observed.
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G-Quadruplex Detection
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Figure 2. Fluorescence decay profiles (lex =340 nm) of a) the excitation
lamp profile, b) DDG (500 nm), and c) DDG and c-myc (500 nm).
However, the decay profiles show that the minor form be-
comes the major component (75%) with a higher lifetime of
6.4 ns (Table S2, in the Supporting Information). These re-
sults indicate that DDG binds to c-myc G-quadruplex DNA
and forms a stable complex with higher fluorescence as well
as higher lifetime.
The interaction of DDG with G-quadruplex DNA was
further evaluated by Fçrster resonance energy transfer
(FRET) melting analysis[19] using dual-labeled DNA sequen-
ces (Figure S4 in the Supporting Information). FRET melt-
ing analysis has been used to determine the G-quadruplex
stabilization and selectivity of a ligand towards a particular
G-quadruplex target. We have evaluated the melting of four
DNA sequences with G-rich DNA sequences found in the
promoter of c-myc, two G-quadruplexes found in the pro-
moter of c-kit (c-kit1 and c-kit2), and a self-complementary
duplex DNA sequence. FRET melting studies showed good
agreement with the fluorescence binding titrations (Table S3
in the Supporting Information). The stabilization potential
for c-myc saturates at 1 mm concentration of DDG (DTm =
12.6 K at 1 mm, that is, a Tm of 938C). DDG also showed
good stabilization for c-kit quadruplex sequences with a
DTm of 17.5 K for c-kit1 and DTm of 7.4 K for c-kit2 at a
DDG concentration of 1 mm, whereas no detectable duplex
stabilization (DTm =0.58C) was observed for the DDG at
this concentration.
Figure 3. NMR spectra (600 MHz) of a) the imino region and b) the aro-
matic region of 200 mm c-myc in the presence of DDG at different
DDG:c-myc DNA molar ratio. c) Overlay of imino region of 1H, 1H-
NOESY of the complex (grey: c-myc:DDG=1:2, 300 mm c-myc,
800 MHz) with the DNA alone (black: 1 mm c-myc, 950 MHz); Titration
was performed at 298 K, in buffer containing 25 mm tris·HCl at pH 7.4
and 100 mm KCl, H2O/[D6]DMSO 80:20. d) Map of chemical shift pertur-
bations detected by NMR spectrosopcy on the c-myc model (PDB:
1XAV); adenosine and guanosine perturbed residues in black.
from, confirming that G13 must be involved in the interac-
tion with DDG, in line with the NMR data from titration
experiments. Our initial NMR analysis therefore shows that
DDG interacts with both the external G-quartets and with
the groove defined by G8-G9-G10 and G13-G14-G15 (Fig-
ure 3d).
Human malignant melanoma A375 cells were used as a
model to investigate the cell membrane permeability of
DDG (Figure 4). The cells were incubated with DDG
(2.5 mm) for 30 min at 378C and examined by fluorescence
microscopy. It was found that DDG, which contains the gua-
nosine nucleoside unit and hydrophobic heteroaromatic
core, is cell-membrane permeable and displays blue fluores-
cence (Figure 4b). The images indicated that DDG binds to
the nucleus preferentially, but it also stains other parts of
the cell. This result was further confirmed by co-staining
DDG with the nucleus marker propidium iodide (PI; Fig-
ure 4c).
To shed light on the binding mode, the titration of DDG
with c-myc DNA was monitored by 1D 1H NMR spectra
(Figure 3). In the region of imino proton signals, NMR reso-
nances from G13, G14/G4 (overlapped signals) residues re-
vealed significant line broadening. Also in the aromatic
region, changes in chemical shifts could not be quantified
for all residues due to signal overlap. Among the monitored
aromatic protons, residues G2, A12, G13, and A22 showed
the most pronounced chemical-shift perturbations. Further,
1
1
in the H, H-NOESY spectrum (Figure 3c), the cross-peak
of imino signal of G6 to G5 and of G6* to G5* in the
second minor conformation of c-myc[20] is shifted, suggesting
that G6 is interacting with the ligand. Furthermore, the
cross-peaks of imino signal of G13 to G8, G9 and G14 are
much weaker in the presence of the ligand than in the free
The antiproliferative activities of the dansyl–guanosine
conjugate DDG was then determined on the A375 cell line.
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Figure 4. Fluorescence microscope images of A375 cells. a) Bright-field
transmission image of cells after incubation with DDG (2.5 mm) at 378C
for 30 min. b) Fluorescence image of a). c) Fluorescence image of DDG
stained (same as a) fixed with 4% paraformaldehyde and stained with PI
(1.0 mm) for 30 min.
The cytotoxic effect of DDG on A375 cells was determined
with varying concentrations (0, 0.5, 1, 10, 25, 50 mm) for 24 h
by MTT assay (MTT=3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide).[21] The IC50 value of DDG was
found to be 5 mm (Figure S5 in the Supporting Information).
Despite this potency, DDG exhibited lower cytotoxicity to-
wards human normal keratinocyte (HaCaT) cells with an
IC50 value at 69 mm. These results suggest that DDG possess
significant selectivity for cancer cells over normal cells. Next
we have investigated the ability of DDG to arrest the cell
cycle and induce apoptosis using A375 cells by fluorescence-
activated cell sorting (FACS).[22–24] The cell cycle analysis re-
vealed that DDG can disturb the cell cycle of the A375 cell
line after exposure for only 12 h, which is shown with the
decreased G0–G1 phase and an increased G2/M phase in a
dose-dependent manner (Figure S6 in the Supporting Infor-
mation). The treatment of DDG caused cell cycle arrest in
G2/M phase,[25] possibly due to repair of damaged DNA
phase.
Figure 5. Flow cytometric analysis of the change in mitochondrial poten-
tial. A375 cancer cells were incubated for 24 h in the presence of (0–
5 mm) of DDG using JC1-stained cells. Histograms a) 0 mm, b) 1.0 mm, c)
2.5 mm, and d) 5.0 mm indicate the percent of normal (M1) and apoptotic
cells (M2) upon treatment with DDG.
real time, which could potentially be developed as a bio-
marker for the quadruplex in the nanomolar concentration
range. Dinucleoside DDG induces cell cycle arrest at G2/M
phase, exhibits specific cytotoxicity to the human melanoma
A375 cells over normal human keratinocyte cells, and pro-
motes cell death by apoptosis.
Acknowledgements
To determine whether the observed cell death induced by
DDG was due to apoptosis, a biochemical marker of apop-
tosis, for example, mitochondrial membrane depolarization
(Ym), was monitored by flow cytometry after JC-1 stain-
ing.[22–24] As shown in Figure 5, untreated cells have intact
mitochondria and normal DYm; however, the loss of DYm
exhibits a dose-dependent increase after the cells were treat-
ed with DDG, as evidenced by the shift of fluorescence of
the JC-1 dye from red to green. DDG at the concentrations
of 1, 2.5 and 5 mm for 24 h increased DYm of cells from 8.36
to 39.06, 46.27, and 50.27%, respectively. These experiments
indicated that cancer cells treated by DDG lose DYm and
induce apoptosis in A375 cells through mitochondria-medi-
ated pathways. The viability of A375 cells treated with
DDG was further investigated by PI-flow cytometric analy-
sis.[24] The in vitro apoptosis analysis indicated that exposure
of A375 cells to different concentrations of DDG for 24 h
resulted in a dose-dependent increase in the proportion of
apoptotic cells (16.06 to 36.11%, Figure S7 in the Support-
ing Information).
This work was supported by the Department of Science and Technology
(DST), Department of Biotechnology (DBT) and Council of Science and
Industrial Research (CSIR), India. R.N.D. and S.P. thank CSIR India for
research fellowships. The authors thank Prof. Dr. Shankar Balasubrama-
nian, University of Cambridge, and Prof. Dr. Kankan Bhattacharyya,
IACS Kolkata, for helpful comments on the manuscript.
Keywords: cell cycles · click chemistry · fluorescence ·
nucleosides · G-quadruplex DNA
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Received: June 3, 2013
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J. Dash et al.
G-Quadruplex Detection
Y. P. Kumar, S. Bhowmik, R. N. Das,
I. Bessi, S. Paladhi, R. Ghosh,
H. Schwalbe, J. Dash* ....... &&&& — &&&&
A Fluorescent Guanosine Dinucleo-
side as a Selective Switch-On Sensor
for c-myc G-Quadruplex DNA with
Potent Anticancer Activities
Like likes like! A novel fluorescent
C2-symmetric guanosine-based dinu-
cleoside has been engineered by chem-
ical ligation of two guanosine units
with a biocompatible dansyl tag that
exhibits high selectivity for c-myc G-
quadruplex DNA through fluorescence
enhancement over duplex DNA and
other promoter G-quadruplexes (see
scheme). It stains the nucleus preferen-
tially, arrests the cell cycle at the G2/M
phase, inhibits cell growth, and induces
apoptosis in A375 cancer cells.
A fluorogenic guanosine-dansyl conjugate (depic-
ted)……that detects G-quadruplex DNA through
fluorescence enhancement has been developed by J.
Dash et al. and reported in their Communication on
page && ff. This dinucleoside interacts with both
the external G-quartets and in the groove regions of
c-myc G-quadruplex DNA. It stains the nucleus
preferentially and exhibit potent antiproliferative
activities against A375 human melanoma cell line.
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