Studies on characterization, telomerase inhibitory properties and G-quadruplex binding of η6-arene ruthenium complexes with 1,10-phenanthroline-derived ligands

Article abstract of DOI:10.1039/c1dt11676b

Two arene ruthenium complexes [Ru(η⁶-C₆H ₆)(p-MOPIP)Cl]⁺1 and [Ru(η⁶-C ₆H₆)(p-CFPIP)Cl]⁺2, where p-MOPIP = 2-(4-methoxyphenyl)-imidazo[4,5f][1,10] phenanthroline and p-CFPIP = 2-(4-trifluoromethylphenyl)-imidazo[4,5f][1,10] phenanthroline, were prepared and the interactions of these compounds with DNA oligomers 5′-G3(T2AG3)3- 3′(HTG21) have been studied by UV-vis and circular dichroism (CD) spectroscopy, gel mobility shift assay, fluorescence resonance energy transfer (FRET) melting assay, polymerase chain reaction (PCR) stop assay and telomeric repeat amplification protocol (TRAP) assay. The results show that both complexes can induce the stabilization of quadruplex DNA but complex 1 is a better G-quadruplex binder than complex 2. The two ruthenium complexes tested led to an inhibition of the enzyme telomerase and complex 1 was the significantly better inhibitor. A novel visual method has been developed for making a distinction between G-quadruplex DNA and double DNA by our Ru complexes binding hemin to form the hemin-G-quadruplex DNAzyme. Furthermore, in vitro cytotoxicity studies showed complex 1 exhibited quite potent antitumor activities and the greatest inhibitory selectivity against cancer cell lines.

Full text of DOI:10.1039/c1dt11676b

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PAPER
Studies on characterization, telomerase inhibitory properties and
G-quadruplex binding of g6-arene ruthenium complexes with
1,10-phenanthroline-derived ligands†
Dongdong Sun, Rong Zhang, Fang Yuan, Du Liu, Yanhui Zhou and Jie Liu*
Received 5th September 2011, Accepted 26th October 2011
DOI: 10.1039/c1dt11676b
6
6
Two arene ruthenium complexes [Ru(h -C6H6)(p-MOPIP)Cl]+ 1 and [Ru(h -C6H6)(p-CFPIP)Cl]+ 2,
where p-MOPIP = 2-(4-methoxyphenyl)-imidazo[4,5f][1,10] phenanthroline and p-CFPIP =
2-(4-trifluoromethylphenyl)-imidazo[4,5f][1,10] phenanthroline, were prepared and the interactions of
these compounds with DNA oligomers 5¢-G3(T2AG3)3-3¢(HTG21) have been studied by UV-vis and
circular dichroism (CD) spectroscopy, gel mobility shift assay, fluorescence resonance energy transfer
(FRET) melting assay, polymerase chain reaction (PCR) stop assay and telomeric repeat amplification
protocol (TRAP) assay. The results show that both complexes can induce the stabilization of
quadruplex DNA but complex 1 is a better G-quadruplex binder than complex 2. The two ruthenium
complexes tested led to an inhibition of the enzyme telomerase and complex 1 was the significantly
better inhibitor. A novel visual method has been developed for making a distinction between
G-quadruplex DNA and double DNA by our Ru complexes binding hemin to form the
hemin-G-quadruplex DNAzyme. Furthermore, in vitro cytotoxicity studies showed complex 1 exhibited
quite potent antitumor activities and the greatest inhibitory selectivity against cancer cell lines.
reported.17–22 The metal can play a major structural role in orga-
Introduction
nizing the ligand(s) into an optimal structure for G-quadruplex
DNA interaction. Also, the electropositive metal can in principle
be positioned at the center of the guanine quartet, increasing
electrostatic stabilization by substituting the cationic charge of
the potassium or sodium that would normally occupy this site.
RuII complexes have prominent DNA-binding properties. For
example, the complex [Ru(bpy)2(dppz)]2+ (dppz = dipyrido [3,2-
a:2¢,3¢-c] phenazine) has been known as a DNA “light switch”.
The complex can intercalate between the duplex DNA base
pairs and stabilize the DNA.23–25 Rickling et al. have studied the
action of the dinuclear [(tap)2Ru(tpac)Ru(tap)2]4+ complex (tap =
1,4,5,8-tetraazaphenanthrene, tpac = tetrapyridoacridine) with the
d(TTAGGG)4 sequence. They found that the complex would be
particularly interesting to damage these sequences by intramolec-
ular photobridging of two or more G bases by using the metallic
[(tap)2Ru(tpac)Ru(tap)2]4+. Indeed, this photoinduced bridging
process would “freeze” the folded G-quadruplex conformation.26
Arene ruthenium complexes have also been successfully used to
stabilise telomeric quadruplex DNA, such as cationic octanuclear
Telomeres are specific nucleoprotein structures essential for stabil-
ity and complete replication of chromosomes.1,2 A key function of
telomeres is to protect the termini of chromosomes from recom-
bination and end-to-end fusion. Human telomeres are essential
structures composed of telomeric DNA and telomere-binding
proteins located at the ends of every human chromosome, and are
composed of a repeated double-stranded [TTAGGGCCCTAA]n
sequence, except in the 3¢-terminal region which consists of
a single-stranded tandem [TTAGGG] repeated sequence over
several hundred bases.3–6 In normal somatic cells, telomeres are
shortened by 50–200 bases after each round of cell division, while
reversal of this degradation by a specialized enzyme called telom-
eraseincreases cellular replicativecapacity, leadingtouncontrolled
proliferation. However, telomerase is overexpressed in most tumor
cells and telomerase activity is reported in 80–90% of tumors.6–10
Thus, the inhibition of telomerase activity by stabiliz-
ing G-quadruplex formation and detection of G-quadruplex
DNA are important targets for developing new anticancer
chemotherapy.11–16 A number of small molecules have been re-
ported to efficiently stabilize G-quadruplex DNA, and recently
some metal complexes as G-quadruplex DNA stabilizers were
metalla-boxes [Ru8(arene)8(m-tpp-H2)2(m-C6H2O4)4]8+ (tpp-H2
=
5,10,15,20-tetra(4-pyridyl)porphyrin, arene = C6H5Me and p-
iPrC6H4Me). The octacationic arene ruthenium metalla-boxes
have shown promising quadruplex DNA stabilization and pos-
sessed a high degree of selectivity for quadruplex over duplex.27,28
In this paper, we studied the interaction of two complexes
Department of Chemistry, Jinan University, Guangzhou, 510632, PR China.
E-mail: tliuliu@jnu.edu.cn; Fax: +86-20-8522-1263
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1dt11676b
6
6
[Ru(h -C6H6)(p-MOPIP)Cl]+ 1 and [Ru(h -C6H6)(p-CFPIP)Cl]+
1734 | Dalton Trans., 2012, 41, 1734–1741
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Fig. 1 Synthetic route to ruthenium(II) complexes.
2 with G-quadruplexes. The synthetic route and structures of 1
and 2 are shown in Fig. 1.
d6 as a solvent and SiMe4 as an internal standard at 300 MHz
at room temperature. Electrospray ionization mass spectrometry
(ESI-MS) was recorded on a LQC system (Finnigan MAT,
USA) using CH3CN as a mobile phase. UV-Visible (UV-Vis)
and emission spectra were recorded on Perkin-Elmer Lambda-
850 spectrophotometer. Circular dichroism (CD) spectra were
recorded on a Jasco J-810 spectropolarimeter.
Experimental
Reagents and materials
All reagents and solvents were purchased commercially and used
without further purification unless specifically noted, and Ultra-
pure MilliQ water (18.2 mX) was used in all experiments. DNA
oligomers 5¢-G3(T2AG3)3-3¢ (HTG21) and the complementary
cytosine rich strand 5¢-C3(TA2C3)3-3¢((ssDNA) were purchased
from Shanghai Sangon Biological Engineering Technology &
Services (Shanghai, China) and used without further purification.
Calf thymus (CT) DNA (highly polymerized) purchased from
Sigma, was stored at 4 ◦C. Concentration of 5¢-G3(T2AG3)3-
3¢(HTG21) and 5¢-C3(TA2C3)3-3¢((ssDNA) was determined by
measuring the absorbance at 260 nm after melting. Single-strand
extinction coefficients were calculated from mononucleotide data
using a nearest-neighbour approximation.29 The formation of
intramolecular G-quadruplexes was carried out as follows: the
oligonucleotide samples, dissolved in different buffers, were heated
to 90 ◦C for 5 min, gently cooled to room temperature, and then
incubated at 4 ◦C overnight. Buffer A:10 mM Tris-HCl, pH = 7.4;
Buffer B:10 mM Tris-HCl, 100 mM NaCl, pH = 7.4; Buffer C:10
mM Tris-HCl, 100 mM KCl, pH = 7.4. Solutions of CT DNA in
the buffer 5 mM Tris HCl/50 mM NaCl in water gave a ratio of
UV absorbance at 260 and 280 nm, A260/A280, of 1.9,30 indicating
that the DNA was sufficiently free of protein. Concentrated stock
solutions of DNA (10 mM) were prepared in buffer and sonicated
for 25 cycles, where each cycle lasted 30 s with 1 min intervals.
The concentration of DNA in nucleotide phosphate (NP) was
determined by UV absorbance at 260 nm after 1 : 100 dilutions.
The extinction coefficient, e260 nm, was taken as 6600 M-1 cm-1.
Stock solutions were stored at 4 ◦C and used after no more than
4 days. Concentrated stock solutions of metal complexes were
stored at 1 or 2 mM in DMSO, further dilution being made in
the corresponding buffer to the required concentrations for all the
experiments.
Synthesis of ligands and complexes
The following were obtained from the stated chemical suppliers:
RutheniumIII chloride hydrate (Alfa Aesar), 1,10-phenanthroline,
4-(trifluoromethyl)benzaldehyde,
4-methoxybenzaldehyde
and cyclohexa-1,4-diene (Sigma). The compounds of 1,10-
phenanthroline-5,6-dione,31 [Ru(h -C6H6)Cl2]Cl2,32,33 p-MOPIP
6
and p-CFPIP34 were prepared and characterized according to
methods in the literature.
Synthesis of [Ru(g6-C6H6)(p-MOPIP)Cl]+(1)
1 was synthesized in a similar manner to the complex [(arene)Ru
(N « N)Cl]+(arene = C6H6, N « N = phen).35 Two equivalents
(0.30 mmol) of the p-MOPIP ligand were added to a suspension
6
of [Ru(h -C6H6)Cl2]Cl2 (0.15 mmol) in dichloromethane (40 mL).
The mixture was stirred for 4 h at room temperature, during
this time the colour changed from orange to ash-coloured. After
evaporation to dryness, the residue was dissolved in water and the
solution filtered and evaporated to dryness to give the product.
The crude product was purified by column chromatography on a
neutral alumina column with acetonitrile and toluene (2 : 1, v/v)
as eluent. The mainly gray band was collected. The solvent was
removed under reduced pressure and a gray powder was obtained.
Yield, 60%. 1H NMR (300 MHz, [D6]DMSO, d (ppm)): 6.3 (6H,
s), 9.8 (2H, d), 9.2 (2H, m), 8.4 (2H, m), 8.0 (m, 2H), 7.4 (d, 2H).
ESI-MS (in MeOH, m/z): 541.13 [M]+. RuC26H20N4OCl: C, 57.64;
H, 3.67; N, 10.38; Found: C, 57.67; H, 3.69; N, 10.35.
Synthesis of [Ru(g6-C6H6)(p-CFPIP)Cl]+(2)
The complex was synthesized in the same way as has been
6
described for [Ru(h -C6H6)(p-MOPIP)Cl]+ with p-MOPIP (0.058
1
g, 0.17 mmol) in place of p-CFPIP. Yield, 53%. H NMR (300
Physical measurements
MHz, [D6]DMSO, d (ppm)): 6.3 (6H, s), 9.8 (2H, d), 9.2 (2H, m),
8.6 (2H, m), 8.1 (m, 2H), 7.9 (d, 2H). ESI-MS (in MeOH, m/z):
579.00 [M - H]+. RuC26H17N4F3Cl: C, 53.85; H, 2.95; N, 9.68;
Found: C, 53.88; H, 2.93; N, 9.67.
Elemental analyses (C, H, and N) were carried out with a Perkin-
Elmer 240 C elemental analyzer. 1H NMR spectra were recorded
on a Varian Mercury-plus 300 NMR spectrometer with DMSO-
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Absorption spectra studies
real time PCR detection system, using a total reaction volume
of 25 mL, with 1 mM of labeled oligonucleotide and different
concentrations of complexes in Tris-HCl buffer (10 mM, pH 7.4)
containing 60 mM KCl. A constant temperature was maintained
for 30 s prior to each reading to ensure a stable value. Final analysis
of the data was carried out using Origin 7.5 (OriginLab Corp.).
Absorption spectra titrations were carried out at room tempera-
ture to determine the binding affinity between DNA and complex.
Initially, 3 mL solutions of the blank buffer and the ruthenium
complex sample (10 mM) were placed in the reference and sample
cuvettes (1.0 cm path length), respectively, and then the first
spectrum was recorded in the range of 200–600 nm. During the
titration, an aliquot (1–10 mL) of buffered DNA solution was
added to each cuvette to eliminate the absorbance of DNA itself,
and the solutions were mixed by repeated inversion. Complex–
DNA solutions were incubated for 5 min before absorption spectra
were recorded. The titration processes were repeated until there
was no change in the spectra for four titrations at least, indicating
binding saturation had been achieved. The changes in the metal
complex concentration due to dilution at the end of each titration
were negligible.
PCR stop assay
The oligonucleotide HTG21 (5¢-G3(T2AG3)3-3¢) and
the corresponding complementary sequence (HTG21rev,
ATCGCT2CTCGTC3TA2C2) were used here. The reactions
were performed in 1¥ PCR buffer, containing 10 pmol of each
oligonucleotide, 0.16 mM dNTP, 2.5 U Taq polymerase, and
different concentrations of complexes. Reaction mixtures were
incubated in a thermocycler with the following cycling conditions:
94 ◦C for 3 min, followed by 30 cycles of 94 ◦C for 30 s, 58 ◦C for
30 s, and 72 ◦C for 30 s. PCR products were then analysed on 15%
nondenaturing polyacrylamide gels in 1¥ TBE and silver stained.
Gel mobility shift assay
Oligonucleotides at a concentration of 10 mM were annealed by
heating in a 10 mM Tris/1 mM EDTA buffer containing 100 mM
KCl (pH 8.0) to 95 ◦C for 10 min followed by cooling to room
temperature. A stock solution (2 mL) of the metal complex was
added. The reaction mixture was incubated at room temperature
for 1 h and loaded onto a native 12% acrylamide vertical gel (1/19
bisacrylamide) in Tris borate EDTA (TBE) buffer, supplemented
with 20 mM KCl. The reaction was terminated by addition of
8 mL of gel loading buffer (30% glycerol, 0.1% bromophenol blue,
0.1% xylene cyanol), and the subsequent solution (10 mL) was
analyzed on a 12% native PAGE (the gel was prerun for 30 min).
Electrophoresis was performed at 4 ◦C in TBE buffer (pH 8.3)
containing 20 mM KCl for 15 h. The gels were dried and visualized
with a Phosphor Imager.
TRAP assay
Telomerase extract was prepared from HeLa cells. The TRAP
assay was performed using a modified procedure.36–38 PCR
was performed at a final reaction volume of 50 mL, composed
of a 45 mL reaction mix containing 20 mM Tris-HCl (pH
8.0), 50 mM deoxynucleotide triphosphates, 1.5 mM MgCl2,
63 mM KCl, 1 mM EGTA, 0.005% Tween 20, 20 mg mL-1
BSA, 3.5 pmol of primer HTG21 (5¢-G3(T2AG3)3-3¢), 18 pmol
of primer TS (5¢-A2TC2GTCGAGCAGAGT2-3¢), 22.5 pmol
of primer CXext (5¢-GTGC3T2AC3T2AC3T2AC3TA2-3¢),
7.5 pmol of primer NT (5¢-ATCGCT2CTCG2C2TTT4-
3¢),
0.01
amol
of
TSNT
internal
control
(5¢-
A2TC2GTCGAGCAGAGT2AA4AG2C2GAGA2GCGAT-3¢),
2.5 U of Taq DNA polymerase, and 100 ng of telomerase.
Compounds or distilled water were added under a volume of
5 mL. PCR was performed in an Eppendorf Master cycler
equipped with a hot lid and incubated for 30 min at 30 ◦C,
followed by 92 ◦C for 30 s, 52 ◦C for 30 s, and 72 ◦C for 30 s for
30 cycles. After amplification, 8 mL of loading buffer (containing
5 ¥ Tris-Borate-EDTA buffer (TBE buffer), 0.2% bromophenol
blue, and 0.2% xylene cyanol) was added to the reaction. A 15 mL
aliquot was loaded onto a 16% non-denaturing acrylamide gel
(19 : 1) in 1¥ TBE buffer and electrophoresed at 200 V for 1 h.
Gels were fixed and then stained with AgNO3.
Circular dichroism measurements
Circular dichroism (CD) spectra were measured on a Jasco J-810
spectropolarimeter. The CD titration procedure is described as
follows: 1.0 mL RuII (1 mM) complex was added sequentially to
solutions containing G-quadruplex DNA (2.0 mM). All solutions
were mixed thoroughly and allowed to equilibrate for 5 min before
data collection. The titration process was repeated several times
until no change was observed. It indicated that binding saturation
was achieved. For each sample, the spectrum was scanned at
least three times and accumulated over the wavelength range
of 200–350 nm at a temperature of 25 ◦C. The instrument was
flushed continuously with pure evaporated nitrogen throughout
the experiment. The scan of the buffer alone was subtracted from
the average scan for each sample.
Preparation of Ru complex-promoted G-quadruplex–hemin
DNAzyme
An equal volume of Ru complexes solution (in water) was added
to the DNA solutions (20 mM DNA, 10 mM Tris-HCl, 100 mM
EDTA, pH = 8.00), allowing the DNA strands to form the G-
quadruplex structure in 40 min. Then an equal volume of hemin
(in DMSO) was dissolved in the above G-quadruplex solutions
and kept for 2 h at room temperature to form the DNAzymes.
Subsequently, 180 mL of 296 mM TMB-1.76 mM H2O2 solution
was added as the substrate of above 20 mL peroxidatic DNAzyme
system. The mixture was kept for 1.5 h at room temperature,
different colors were observed with the naked eye and the
photograph of the mixture was taken with a digital camera.
Fluorescence resonance energy transfer (FRET) studies
The fluorescent labeled oligonucleotide, F21T (5¢- FAM-
G3[T2AG3]3-TAMRA-3¢, FAM: 6-carboxyfluorescein, TAMRA:
6-carboxy-tetramethylrhodamine) used as the FRET probes
were diluted in Tris-HCl buffer (10 mM, pH 7.4) containing
60 mM KCl and then annealed by heating to 92 ◦C for 5
min, followed by cooling slowly to room temperature overnight.
Fluorescence melting curves were determined with a Bio-Rad iQ5
1736 | Dalton Trans., 2012, 41, 1734–1741
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Cell culture
Cells were cultured in RPMI 1640 medium supplemented with
10% heat inactivated fetal bovine serum, 100 mg mL-1 penicillin,
and 100 mg mL-1 streptomycin. Cells were maintained at 37 ◦C in
a 5% CO2 incubator, and the media were changed twice weekly.
MTT assay
Cell viability was determined by measuring the ability of cells to
transform MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetra-
zolium bromide) to a purple formazan dye.39 Cells were grown in
a RPMI 1640 medium supplemented with 10% fetal calf serum,
100 mg mL-1 pen◦icillin and 100 mg mL-1 streptomycin. They were
incubated at 37 C in a humidified incubator with 5% CO2 and
95% air. Cells at the exponential growth stage were diluted to 5 ¥
103 cells mL-1 with RPMI 1640, and then seeded in 96-well culture
clusters (Costar) at a volume of 100 mL per cell, and incubated for
24 h at 37 ◦C in 5% CO2. Then the cells were treated with various
concentrations of complexes, including 5, 10, 25, 50, 75, 100, 150
and 200 mmol L-1, the media control and the drug-free control
were set at the same time. After incubation of cells for up to 48 h,
100 mL of MTT (5 mg mL-1) solution was added in each cell. After
a further period of incubation (4 h at 37 ◦C in 5% CO2), each cell
was added in 100 mL cell lysate. After 12 h at 37 ◦C, plates were read
on a microplate-reader at a wavelength of 570 nm (the absorbance
of the complexes at this wavelength can be neglected40–42). The
percentage growth inhibitory rate of treated cells was calculated
by (Atested - Amedia control)/(Adrug-free control - Amedia control) ¥ 100%, where A
is the mean value calculated using the data from three replicate
tests. The IC50 values were determined by plotting the percentage
viability versus concentration on a logarithmic graph and reading
off the concentration at which 50% of cells were viable relative to
the control.
Fig. 2 Absorption spectra of complexes 1 (A) and 2 (B) in buffer with
increasing amounts of G-quadruplex. [Ru] = 10.0 mM, [DNA] = 0–
12 mM from top to bottom. Arrows refer to the change in absorbance
upon increasing the concentration of DNA.
Gel mobility shift assay
Results and discussion
By employing a native PAGE assay, we examined the ability
of the complexes to assemble intermolecular G-quadruplexes
from the oligonucleotide HTG21, which contains four repeats of
the human telomeric sequence and hence has the potential to
form both parallel and antiparallel G-quadruplex structures, in
dimeric (D) and tetrameric (T) forms (Fig. 3).46–48 For example,
Hurley et al. have reported the DNA oligomer d(TTAGGG)4
(HT4) was incubated with increasing concentrations of the three
porphyrins; we observed an increased amount of dimers (D) in
the presence of TMPyP3, TMPyP4 and only tetrameric (T) in the
presence of TMPyP3. Che et al. has also proven the ability of
the complexes to assemble intermolecular G-quadruplexes from
the oligonucleotide which contains two tandem human telomeric
sequences and can associate into antiparallel and parallel G-
quadruplexes, in dimeric (D) and tetrameric (T) forms. Under
the buffer conditions used in the experiment (10 mM Tris, 1 mM
EDTA, pH 8.0) and in the absence of complexes, gel mobility
shift assays revealed that there was no formation of G-quadruplex
structure and only the band corresponding to the monomer (M)
could be observed (Fig. 4). When HTG21 was incubated with
increasing concentrations of complexes, we observed an increased
amount of dimers (D) in the presence of 1 and 2, in particular for
treatment of HTG21 oligonucleotide with complexes 1 and 2 at
concentrations up to 30 mM, the appearance of new bands with
The binding affinity by absorption spectra
Absorption spectra titrations were performed to determine the
binding affinity of complexes to HTG21. The changes in the
spectral profiles during titration were shown in Fig. 2 (CT-
DNA in Fig. S1, ESI†). The hypochromisms (H%), defined as
H% = 100%·(Afree - Abound)/Afree, of MLCT bands of complexes
1 and 2, were calculated as about 19.2% and 18.5%, respectively.
When CT-DNA was added to the same buffered solution, the
hypochromisms (H%) was 12.5% and 23.6%, respectively. In order
to compare the DNA-binding affinities of the two complexes
quantitatively, their intrinsic DNA-binding constants Kb were
obtained by monitoring the changes of the MLCT absorbance
of both complexes according to Eq. (1)43–45 (see ESI†). The
intrinsic binding constants Kb (HTG21) of complexes 1 and 2 were
3.87 ¥ 105 M-1 and 2.14 ¥ 105 M-1, respectively, from the decay
of the absorbance (Table S1, ESI†). The binding constant Kb
of complex 1 is larger than that of complex 2. However, when
compared to CT-DNA, the Kb order of complex 1 and complex 2
was reversed (Fig. S1, ESI†). It indicated that complex 1 bound to
the HTG21 more tightly than did complex 2. Upon comparison
of 1 and 2, compound 1 appeared to be more selective for HTG21
than CT-DNA.
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Fig. 3 The quadruplex: tetrameric, dimeric and monomeric G-quadru-
plex composed of three G-quartets.
Fig. 5 CD spectra of HTG21 acquired at 20 ◦C in the absence or presence
of different concentrations of 1 (A) and 2 (B). All experiments were carried
out in 10 mM Tris-HCl, pH 7.4. The HTG21 concentration was 2 mM.
to increase, the CD spectrum of this new DNA conformation
would appear similar to the antiparallel G-quadruplexes described
in previous studies, where the major positive band was usually
observed around 295 nm with a negative band at 265 nm and a
smaller positive band at 246 nm. However, a strange phenomenon
was found in the CD spectrum. A major positive band at 295 nm
started to wane and a new positive band gradually appeared. The
addition of complex 2 did not induce obvious changes in CD
spectra. This interesting phenomenon occurs probably because
HTG21 DNA is a quadruplex, there are also several different
folding motifs possible to create a quadruplex as alluded to in Fig.
3, and another point is closely related to the different ligands. The
structures of G-quadruplex were also investigated in the Na+ or K+
buffer solution. Upon addition of complexes 1 and 2 to HTG21 in
Na+ or K+ buffer solution, the CD spectrum exhibited a maxima–
minima pattern, similar but not identical to the spectrum in Na+
or K+ without addition of the two complexes (Fig. S3 and S4,
ESI†), which implied that the conformation of the G-quadruplex
was stabilized by Na+ or K+, and 1 or 2 could not change
the conformation of the G-quadruplex at high ionic strength.
For ssDNA, when complex 2 was added, the bands at about
280 nm and 250 nm significantly decreased and reached saturation.
However, upon addition of complex 1, no obvious spectral changes
were observed under the same conditions (in Fig. S5, ESI†). Upon
interacting complex 2 with CT DNA ([DNA]/[Ru] = 1 : 1), we find
the maximum at 275 nm with a slightly higher intensity and a new
negative band at about 290 nm, suggesting the conformation of
CT DNA has been changed. In contrast to 2, the CD spectrum of
DNA in the presence of complex 1 showed no change under the
same experimental conditions (in Fig. S6, ESI†). These data clearly
suggest that both complexes, especially 1, strongly and selectively
Fig. 4 Effect of complex 1 (a) and 2 (b) on the assembly of the HTG21
structure illustrated by native PAGE analysis. Ruthenium complexes at the
indicated concentrations were incubated with HTG21 (10 mM) at 20 ◦C in
a buffer containing 10 mM Tris, 1 mM EDTA, pH 8.0. Major bands were
identified as monomer (M), dimer (D).
reduced mobility, corresponding to the D G-quadruplex structures
were most conspicuous. These bands were also observed when
oligonucleotide HTG21 was incubated in K+ containing Tris buffer
(10 mM Tris, 1 mM EDTA, 100 mM KCl, pH 8.0) (Fig. S2, ESI†).
Compared to in the absence of K+ buffer, the D G-quadruplex
structures bands were not obvious until the concentration of 1
and 2 reached 35 mM. The results showed that in the presence
of potassium ions or not, complexes 1 and 2 were able to induce
HTG21 oligonucleotide to form D G-quadruplex structures.
Circular dichroic spectral studies
Circular dichroism (CD) spectroscopy was used to characterize the
solution conformations of HTG21. Without any metal cations, the
CD spectra of HTG21 at room temperature exhibited a negative
band centered at 235 nm, a major positive band at 257 nm,
which probably corresponded to the signal of the random coil
HTG21 (characterized by a positive peak at 257 nm).49,50 Upon
addition of complex 1 to HTG21 aqueous solution (Fig. 5), a
significant change in the CD spectrum was observed. With the
increase of 1 from 1 mM to 3 mM, the maximum at 257 nm
was gradually suppressed and shifted to 249 nm, while a major
negative band at about 270 and a major positive band at 295 nm
started to appear. We thought that as the concentration continued
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Table 1 ꢀTm values at various concentrations of 1 and 2, F21T (1 mM)
DTm (◦C)
Complexes (mM)
1
2
1
2.5
5
15
22
27
9
17
21
interact with G-quadruplex DNA over ssDNA and CT-DNA. The
results reveal that the different ligands of complexes 1 and 2 are
related to the mode of DNA binding, and 2 can bind more strongly
to CT DNA than complex 1.
Thermodynamic stabilization of the telomeric G-quadruplex by
complexes 1 and 2
FRET (fluorescence resonance energy transfer) can be used as a
convenient method to monitor the 3¢-to-5¢-end distance.51,52 In this
paper, we used a FRET melting assay to investigate the binding
abilities of 1 and 2 to G-quadruplex DNA F21T (sequence: FAM-
G3[T2AG3]3-TAMRA, mimicking the human telomeric repeat).53
As shown in Fig. 6, in the absence of any RuII complex, the DNA
melting temperature (Tm) of F21T in Tris/KCl buffer was 48 ◦C.
Upon treatment of the F21T (1 mM) with concentration ratios
[Ru1]/[DNA] = 1 : 1, 2.5 : 1 and 5 : 1, the highest Tm deviation [DTm
(change in DNA melting temperature) = 27 ◦C] was found with 1.
When compared to 1, complex 2 (DTm = 9, 17, 21 ◦C, respectively)
is not a very effective quadruplex-DNA stabilizer (Table 1). This
is consistent with the results of the absorption titration studies
showing that 1 has the higher K value [3.87 ¥ 105 M-1]. The results
indicated that both complexes can stabilize G-quadruplex DNA
and that complex 1 affected the stability of the G-quadruplex more
than complex 2. The difference may originate from the different
DNA-binding affinity. The mechanism is not yet clear, however the
ligand of the RuII complex plays a key role in stabilization. It was
found that, in relation to binding abilities, the electron withdrawing
group trifluoromethyl was inferior to the electron donor methoxy,
consistent with circular dichroism spectral studies.
Fig. 6 (A) FRET-melting curves obtained with F21T (1 mM) alone (ꢀ)
and with 1 = 1.0 mM to 5 mM. (B) FRET-melting curves obtained with
F21T (1 mM) alone (ꢀ) and with 2 = 1.0 mM to 5 mM.
Fig. 7 Effect of complexes 1 (0–5.0 mM, left) and 2 (0–7.5 mM, right) on
the hybridization of HTG21 in the PCR-stop assay.
TRAP assay
On the basis of the obtained results, it appeared to be of interest
to compare the ability of the two arene ruthenium complexes to
inhibit the enzyme telomerase. For this, a modified version of
the telomeric repeat amplification protocol (TRAP) assay was
performed (Fig. 8). In this experiment, solutions of complexes 1
and 2 were added to a telomerase reaction mixture containing
extracts from HeLa cells, which express high levels of telomerase
activity. Fig. 8 showed the in vitro inhibitory effect of complex
1 towards telomerase studied in a dose-dependent manner and
the number of bands clearly decrease with respect to the control,
at drug concentrations in the range of 1–16 mM. In contrast,
no complete inhibition was observed in the presence of 2, even
at 16 mM (Fig. 8). The two ruthenium complexes tested led to
an inhibition of the enzyme telomerase, but there were great
differences in the extent of inhibition. The results clearly revealed
that the telomerase inhibitory properties of 1 were significantly
greater, which is in accordance with the experimental data from
the thermodynamic stability study and the PCR stop assay.
Inhibition of amplification of HTG21 by Ru complexes 1 and 2
In order to further evaluate the ability of complexes 1 and 2
to stabilize G-quadruplex DNA, a polymerase chain reaction
(PCR) stop assay was used to ascertain whether complexes bound
to the test oligomer (5¢-G3(T2AG3)3-3¢) and stabilized the G-
quadruplex structure.54–56 In the presence of complexes 1 or 2,
the template sequence HTG21 was induced into a G-quadruplex
structure that blocked the hybridization with a complementary
primer sequence. In that case, 5¢ to 3¢ primer extension by DNA
Taq polymerase was arrested and the final double-stranded DNA
PCR product could not be detected.55,57 The inhibitory effect of 1
was clearly enhanced as the concentration increased from 0.2 to
5.0 mM, with no PCR product detected at 5.0 mM (Fig. 7, left).
However, even if the concentration of 2 was allowed to exceed
7.5 mM, 2 could not completely inhibit the appearance of the
PCR product, which further indicates that complex 1 is a better
G-quadruplex binder.
Visual detection of G-quadruplex structures by Ru complex
Although certain kinds of G-rich sequences have been demon-
strated to form G-quadruplex structures readily at physiological
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Dalton Trans., 2012, 41, 1734–1741 | 1739
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and cisplatin. The tested cancer cells, especially the HeLa cells,
were susceptible to the complexes. It is particularly interesting
that the antiproliferative activities of complex 1 were higher than
those of 2, as evidenced by the lower IC50 values. Notably, complex
1 exhibited a broad spectrum of inhibition on human cancer cells,
with IC50 values ranging from 8.70 to 35.72 mM, indicating the
higher cytotoxic effects of complex 1 on cancer cells. It is also
worth noting that 1 shows a distinct preference for HeLa cells
(approximately as potent as cisplatin towards this cell line). In
addition, we also detected the antiproliferative activity of the RuII
complexes on the mouse embryonic fibroblast cell line NIH/3T3.
Generally, this series of complexes inhibited the growth of cancer
cell lines better than normal cells, suggesting the RuII complexes
were much less toxic towards normal cells, with IC50 values of
48.37 mM and greater than 100 mM, which were significantly
higher than those of cisplatin (19.72 mM). The results showed
that complex 1 exhibited quite potent antitumor activities and the
greatest inhibitory selectivity against cancer cell lines.
6
Fig.
8
The influence of [Ru(h -C6H6)(p-MOPIP)Cl]+ (1) and
[Ru(h -C6H6)(p-CFPIP)Cl]+ (2) on the telomere activity of HeLa.
6
concentrations of Na+ and K+ in vitro,58–61 the existence of G-
quadruplex structures in vivo is still controversial.62,63 The lack of
direct evidence for this quadruplex structure in living cells is a
serious obstacle to determining its function. Thus, detecting G-
quadruplex structures has great significance for cell proliferation,
cancer research, and drug development. Here we report a facile
and visual approach to detect G-quadruplexes with the naked eye.
It is well known that most G-quadruplex DNA can be effectively
formed by K+, and G-quadruplexes have the ability to bind with
hemin to form peroxidase-like DNAzymes. It is proven that in the
presence of the DNAzymes, H2O2-mediated oxidation of TMB
(3,3¢,5,5¢-tetramethylbenzidine) could be sharply accelerated and
the color change is very sensitive and easy to identify. The design
is based on this principle. As shown in Fig. 9, in the presence
of complex 1 or 2, HTG21 can also fold into a G-quadruplex,
and such a quadruplex structure is able to bind hemin to form
the hemin–G-quadruplex DNAzyme that catalyzes the H2O2-
mediated oxidation of colorless TMB to the blue product, as well
as control K+. But for complex 1 with double strands of CT-DNA,
the solution remains colorless. The reason is obvious, because
CT-DNA cannot form the G-quadruplex structure. Furthermore,
under the same conditions, when using the ligands p-MOPIP (L1)
and p-CFPIP (L2) the solution does not change colour at all (Fig.
S7, ESI†).
Conclusions
6
In conclusion, two h -arene ruthenium with 1,10-phenanthroline-
derived ligands have been prepared and interacted with G-
quadruplex DNA. The results showed that complex 1 bound
to the DNA more tightly than did complex 2. Complex 1 is a
potent telomerase inhibitor and a very good G-quadruplex DNA
stabilizer that can increase the Tm value of G quadruplexes by
9–27 ◦C. Successful quadruplex DNA binders should not only in-
teract strongly with their target but also exhibit high selectivity for
quadruplex versus duplex DNA. In this work, more importantly,
complex 1 could significantly stabilize and induce intramolecular
G-quadruplex structural transitions and has made a significant
choice of G-quadruplex DNA. The visual observation of G-
quadruplexes has also been successfully used in investigating the
one-stranded telomeric and double-stranded calf thymus DNA.
In vitro cytotoxicity studies showed complex 1 exhibited quite
potent antitumor activities and the greatest inhibitory selectivity
against cancer cell lines. The electron-withdrawing or electron-
donating properties of substituents were determined to be critical
for interaction. Complex 1 was observed to have a greater ability
to interact with quadruplex DNA as it contains a ligand with a
pendant –OCH3 functional group, which may be involved in H-
bonding interactions with the guanine in the external tetrad of G-
quadruplex DNA. The PIP ligands might be partially inserted into
6
the plane of G-quadruplex DNA and the h -arene ligand might
Fig. 9 Characterization of the DNAzyme functions of HTG21 DNA and
CT-DNA in the presence of 500 nM K+, 500 nM 1 and 500 nM 2 in the
TMB–H2O2 system. Conditions: TMB, 266 mM in Tris-MES buffer (25
mM MES, pH = 5.10); H2O2, 794 mM; DNA, 500 nM; hemin, 500 nM.
interact with G-quadruplex DNA by p–p stacking. Although the
details of the binding modes of these complexes with the G-
quadruplex and the structure of the G-quadruplex are not clear
yet, in our research we can draw the following conclusions: (1)
6
h -arene ruthenium complexes exert a stabilization effect towards
the G-quadruplex structure and show good selectivity between
dsDNA and G-quadruplexes. The changes in microenvironment
(–OCH3, –CF3) in the complexes affects the binding capacity. (2)
Complexes can induce changes in G-quadruplex DNA conforma-
tion and the structures of complexes can also affect the changes
of G-quadruplex DNA. (3) The complexes have the ability to
stabilize G-quadruplex DNA and induce the transformation of
G-quadruplex DNA conformation, which closely relates to the
inhibition of telomerase activity and in vitro antitumor activity.
In vitro cytotoxicity
To explore the antitumor potential of the RuII complexes, HepG2
(human hepatocellular liver carcinoma), HeLa (human cervical
cancer), A549 (human lung carcinoma), SW620 (colorectal ade-
nocarcinoma) and NIH/3T3 (mouse embryonic fibroblast) cells
were treated with varying concentrations of RuII complexes for
48 h, and cell viability was determined by the MTT assay. Table
S2, ESI† shows the IC50 values of two arene ruthenium complexes
1740 | Dalton Trans., 2012, 41, 1734–1741
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We speculate telomerase is the target of the antitumor activity of
these complexes.
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©