Studies on synthesis, characterization, and G-quadruplex binding of Ru(II) complexes containing two dppz ligands

Article abstract of DOI:10.1016/j.jinorgbio.2010.10.005

In this work, the interaction between the guanine-rich single-strand oligomer AG₃(T₂AG₃)₃ quadruplex and two Ru(II) complexes, [Ru(L¹)(dppz)₂](PF₆) ₄ (1) and [Ru(L²)(dppz)₂](PF₆) ₄ (2) (L¹ = 5,5′-di(1-(trimethylammonio)methyl)-2, 2′-dipyridyl cation, L² = 5,5′-di(1-(triethylammonio) methyl)-2,2′-dipyridyl cation, dppz = dipyrido[3,2-a:2′,3′-c] phenazine), has been studied by UV-Visible, fluorescence, DNA melting, and circular dichroism in K⁺ buffer. The two complexes after binding to G-quadruplex have shown different DNA stability and fluorescence enhancement. The results show that both complexes can induce the stabilization of quadruplex DNA. ΔT_m values of complexes 1 and 2 at [Ru]/[DNA] ratio of 1:1 were 9.4 and 7.0, respectively. Binding stoichiometry along with the quadruplex was investigated through a luminescence-based Job plot. The major inflection points for complexes 1 and 2 were 0.49 and 0.46, respectively. The data were consistent with the binding mode at a [quadruplex]/[complex] ratio of 1:1. In addition, the conformation of G-quadruplex was not changed by the complexes at the high ionic strength of K⁺ buffer.

Full text of DOI:10.1016/j.jinorgbio.2010.10.005

Journal of Inorganic Biochemistry 105 (2011) 149–154
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Studies on synthesis, characterization, and G-quadruplex binding of Ru(II) complexes
containing two dppz ligands
Jing Sun a,b, Yan An c, Li Zhang a, Huo-Yan Chen a, Yan Han a, Yu-Jia Wang a,
a,
Zong-Wan Mao a, , Liang-Nian Ji
⁎
⁎
a
MOE Key laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou, 510275, China
School of Pharmacy, Guangdong Medical College, Dongguan, 523808, China
Institute of Marine Materials Science and Engineering, Shanghai Maritime University, Shanghai 201306, China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 July 2010
Received in revised form 2 October 2010
Accepted 6 October 2010
Available online 14 October 2010
In this work, the interaction between the guanine-rich single-strand oligomer AG3(T2AG3)3 quadruplex and two
Ru(II) complexes, [Ru(L1)(dppz)2](PF6)4 (1) and [Ru(L2)(dppz)2](PF6)4 (2) (L1=5,5′-di(1-(trimethylammonio)
methyl)-2,2′-dipyridyl cation, L2=5,5′-di(1-(triethylammonio)methyl)-2,2′-dipyridyl cation, dppz=dipyrido
[3,2-a:2′,3′-c] phenazine), has been studied by UV–Visible, fluorescence, DNA melting, and circular dichroism in
K
+ buffer. The two complexes after binding to G-quadruplex have shown different DNA stability and fluorescence
enhancement. The results show that both complexes can induce the stabilization of quadruplex DNA. ΔTm values
of complexes 1 and 2 at [Ru]/[DNA] ratio of 1:1 were 9.4 and 7.0, respectively. Binding stoichiometry along with
the quadruplex was investigated through a luminescence-based Job plot. The major inflection points for
complexes 1 and 2 were 0.49 and 0.46, respectively. The data were consistent with the binding mode at a
[quadruplex]/[complex] ratio of 1:1. In addition, the conformation of G-quadruplex was not changed by the
complexes at the high ionic strength of K+ buffer.
Keywords:
Synthesis
Ru(II) complex
dppz
G-quadruplex
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
have been designed to target quadruplex DNA. These complexes
generally have positively charged substituents, which can interact
Guanine-rich sequences of DNA have the propensity to form
tetraplex structures known as G-quadruplexes [1–4]. During the past
decade, these G-quadruplexes have received great attention because
they can inhibit telomere extension by telomerase, which is an enzyme
in over 80% cancer cells [5–7]. This unique activity of telomerase makes
the enzyme an ideal probe for tumor diagnosis and a target for cancer
chemotherapy [8,9]. Molecules, which have (a) a π-delocalized system,
(b) a partial positive charge in the center of the molecular scaffold, and
(c) a positively charged substituent to interact with the grooves, loops,
and the negatively charged phosphate backbone, most likely interact
with and further stabilize G-quadruplexes [10]. Therefore, many
research groups have designed and synthesized some molecules
with such structures, which are believed to be able to interact with
G-quadruplex DNA structures. This interaction plays an important role
to maintain the telomeres [11–15]. Furthermore, inducement/stabili-
zation features of G-quadruplex structures by small molecules directly
prevent elongation of telomeres by disrupting the interaction between
the enzyme and its substrate. A number of transition metal complexes
with the grooves of the quadruplex, and a positively charged center,
which can stay near the center of the guanine quartet. The metal plays a
major structural role in organizing the ligand(s) into an optimal
structure for quadruplex DNA interaction [16–19]. Also, the electropos-
itive metal is in principle positioned at the center of the guanine quartet
and increases electrostatic stabilization by substituting the cationic
charge of the potassium or sodium that normally occupies this site.
Ru(II) complexes have prominent DNA-binding properties, espe-
cially the complex [Ru(bpy)2(dppz)]2+ (dppz=dipyrido[3,2-a:2′,3′-c]
phenazine) known as DNA “light switch”. The complex can intercalate
between the duplex DNA base pairs and stabilize the DNA [20–22].
Shi, et al. have studied the action of [Ru(bpy)2(dppz)]2+ with 5′-
AGGGTTAGGGTTAGGGTTAGGG-3′ (AG3(T2AG3)3) and 5′-CCCTAACCC-
TAACCCTAACCCT-3′ and found that the complex can serve as a
prominent molecular “light switch” for both G-quadruplexes. It prefers
to bind to G-quadruplexes induced by either Na+ or K+ over an i-motif
[23]. As a star molecule, it would consist of one dppz ligand and two
ancillary ligands. However, the complex combined with two dppz
ligands is rare. Herein, we studied the interaction of two complexes of
[Ru(L1)(dppz)2](PF6)4 (1) and [Ru(L2)(dppz)2](PF6)4 (2) (L1=5,5′-di
(1-(trimethylammonio)methyl)-2,2′-dipyridyl cation; L2=5,5′-di(1-
(triethylammonio)methyl)-2,2′-dipyridyl cation) and G-quadruplexes.
The synthetic route and structure of complex 2 are shown in Scheme 1.
⁎
Corresponding authors. Tel.: +86 20 8411 3788; fax: +86 20 8411 2245.
E-mail addresses: cesmzw@mail.sysu.edu.cn (Z.-W. Mao), cesjln@mail.sysu.edu.cn
(L.-N. Ji).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.10.005

150
J. Sun et al. / Journal of Inorganic Biochemistry 105 (2011) 149–154
N
N
N
N
N
NH2
NH2
L2Br2·4H2O
NH4PF6
N
RuCl3·3H2O
LiCl
N
N
N
N
N
O
O
Cl
N
N
N
N
N
N
N
Ru
N
Ru
N
DMF
CH3OH
N
OHCH2CH2OH
Cl
N
N
N
N
N
(PF6)4
Scheme 1. The synthetic route of complex 2.
2. Experimental section
the solution turned dark red. The solution was cooled down to room
temperature and filtrated. Saturated NH4PF6 was added dropwise to
the solution and red–orange precipitate, which was filtered and
recrystallized with CH3CN/CH3OH (1:1, v/v), was obtained. The yield
of the product was 0.27 g (47%). The atom weight fractions of the
stoichiometric molecule of C128H141.50F48N27O4.50P8Ru2 (3492.08) are
as follows: C of 44.03%, H of 4.08%, and N of 10.83%. The analytical
value of the atom weight fractions were C of 44.01%, H of 4.10%, and N
of 10.80%. The ESI-MS spectra were analyzed at m/z 1484.9 [M-PF6]+
(60), 670.5 [M-2PF6]2+ (100), and 398.5 [M-3PF6]3+ (25). 1H NMR
(DMSO-d6), δ=9.74 (dd, 2 H), 9.62 (dd, 2 H), 9.10 (d, 2 H), 8.51
(m, 6 H), 8.30 (d, 2 H), 8.22 (d, 2 H), 8.12 (m, 6 H), 7.79 (d, 2 H), 7.90
(s, 2 H); 4.34 (s, 4 H), 2.99 (q, 12 H), 1.00 (t, 18 H).
2.1. Materials
DNA oligomers AG3(T2AG3)3 were purchased from Sangon
(Shanghai, China) and used without further purification. Concentra-
tions of these oligomers were measured through the absorbance at
260 nm of melted samples. Single-strand concentrations were
spectroscopically determined and calculated by using the molar
absorption coefficient of quadruplex, ε=2.16×105 M−1 cm−1(per
quadruplex) at 260 nm for G-quadruplex [24]. The formation
procedure of intramolecular G-quadruplexes was carried out as
follows. An oligonucleotide sample dissolved in a buffer solution
was heated to 90 °C for 5 min. The solution was slowly cooled down to
room temperature and then incubated at 4 °C overnight. The buffer
solution was composed of 100 mM KCl, 10 mM KH2PO4/K2HPO4, and
1 mM K2H2EDTA with A pH of 7.0. All reagents and solvents were
purchased commercially and used without further purification unless
specially noted. Doubly distilled water was used to prepare buffer
solutions.
2.4. X-ray crystallography
Single crystal X-ray diffraction (XRD) experiments were carried
out with a Bruker Smart Apex CCD area detector at 173(2) K for
complex 2. The dimensions of the crystal of complex 2 used for X-ray
diffraction analysis were 0.48×0.30×0.21 mm. XRD pattern was
recorded with Mo Kα radiation (λ=0.71073 Å). The data were
collected in the θ range of 1.79–25.00°. Total 57149 independent
reflections were obtained, and 27,464 reflections with |Fo|≥2σ (Fo)
were used for the further calculations. Absorption corrections were
applied by the SADABS program [31]. The structure was analyzed by
direct methods. All non-hydrogen atoms were refined anisotropically
by least-squares on F2 using the SHELXTL program [32]. These were
refined isotropically first and then anisotropically. The hydrogen
atoms of the ligands were placed in calculated positions with fixed
isotropic thermal parameters and the structure factors of calculation
were included in the final stage of full-matrix least-squares
refinement. The final discrepancy factors were R1 =∑||Fo|−|Fc||/
∑|Fo|=0.1086 and wR2 =[∑w(F2o −Fc2)2/∑w(F2o)2]1/2 =0.2374
with weight w=1/[σ2(Fo2)+(0.0410 P)2 +83.4000 P] where P=
(F2o +2F2c)/3. The crystal data is summarized in Table 1.
2.2. Physical measurement
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-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 (Finngan MAT, USA) using CH3CN as a
mobile phase. UV–Visible (UV–Vis) and emission spectra were
recorded on Perkin-Elmer Lambda-850 spectrophotometer and Ls55
spectrofluorophotometer. The circular dichroism (CD) spectra were
recorded on a Jasco J-810 spectropolarimeter.
2.3. Preparation of ligands and complexes
5,5′-Dimethyl-2,2′-dipyridyl was purchased from Aldrich Chemi-
cal Co. 5,5′-Dibromomethyl-2,2′-dipyridyl, L1Br2 and L2Br2·4H2O
were synthesized and characterized according to our previous
method [25,26]. The compounds of 1,10-phenanthroline-5,6-dione
[27], dppz [28], cis-[Ru(dppz)2Cl2]·2H2O [29], and complex 1 [30]
were prepared and characterized according to methods in the
literature.
2.5. Absorption and emission spectra
The absorption and emission spectra as well as titration curves
were recorded through a constant concentration of complexes, to
which the DNA stock solution were added step by step at room
temperature. The concentrations of the [Ru(L)(dppz)2]4+ solution
were 10.0 μM and 2.0 μM, and the volume of the complex was
3000 μL. Complex-DNA solutions were incubated for 5 min before
absorption spectra were recorded. The titration processes were
repeated several times until no change was observed in the spectra.
It indicated that binding saturation was achieved. The changes in the
2.3.1. [Ru(L2)(dppz)2](PF6)4 ·CH3OH·1.25H2O·1.5CH3CN (2)
A solution of cis-[Ru(dppz)2Cl2]·2H2O (0.26 g, 0.34 mmol) and
L2Br2·4H2O (0.21 g, 0.34 mmol) in ethylene glycol (30.0 cm3) was
heated at 130 °C under the protection of argon for 6 h. In the process,

J. Sun et al. / Journal of Inorganic Biochemistry 105 (2011) 149–154
151
Table 1
spectrum of the buffer was subtracted from the average spectrum for
each sample.
Crystallographic data of complex 2.
Complex
(2·CH3OH·1.25H2O·1.5CH3CN)×2
3. Results and discussion
Empirical formula
Formula weight
C128H141.50F48N27O4.50P8Ru2
3492.08
Fig. 1 depicts the structure of complex 2. Selected bond distances
and bond angles of complex 2 are listed in Table 2. The complex
molecule contains a six-coordinated ruthenium atom chelated by one
dicationic L ligand and two dppz ligands. The coordination geometry
of the ruthenium atom is a distorted octahedron with a mean bite
angle of 79.67° over three bidentate ligands. The mean Ru–N bond
length of 2.061 Å is typical for six-coordinated Ru(II) complexes,
which is similar to complex 1 [30].
Temperature (K)
Wavelength (Å)
Crystal system, Space group
173(2)
0.71073
Triclinic, P-1
20.094(3)
21.299(3)
a (Å)
b (Å)
c (Å)
α/°
β (°)
γ/°
22.377(3)
108.560(2)
114.511(2)
97.221(2)
7883.4(2)
2
Volume (Å3)
Z
Titration was performed to determine the binding affinity of the
complexes to G-quadruplex. The DNA sample was added sequentially
to complex solutions. The absorbance spectra were recorded after
each addition. The changes in the spectral profiles during titration are
shown in Fig. 2. The absorbance in the ligand absorption region, as
well as the MLCT (metal-to-ligand charge transfer) band, decreased
with increasing concentration of DNA. The hypochromisms (H%),
defined as H%=100%∙(Afree −Abound)/Afree, of MLCT bands of com-
plexes 1 and 2, were calculated as about 17.94% and 13.33%,
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) [35–38], where [DNA] is the
concentration of DNA in nucleotides, εa is the molar absorption
coefficient (Aabs/[M]) of the MLCT absorption band at a given DNA
concentration, εf and εb are the molar absorption coefficients of the
free Ru(II) complex and the molar absorption coefficient of the Ru(II)
complex in the fully bound form, respectively. Kb is the equilibrium
binding constant in M−1, Ct is the total Ru(II) complex concentration,
and s is the binding site size. Eq. (1) has been applied to titration data
for noncooperative metallointercalator binding to DNA.
Dcalcd (g cm−3
F(000)
)
1.471
3549
Crystal size (mm)
θ range for data collection (°)
Limiting indices
Reflections collected
Independent reflections
Goodness-of-fit on F2
R1/wR2 [IN2σ(I)]a
0.48×0.30×0.21
1.79 to 25.00
−23≤h≤23, −25≤k≤25,−25≤l≤25
57,149
27,464 (Rint =0.0516)
1.045
0.1086/0.2374
0.1893/0. 2803
1.340/−0.953
R1/wR2 (all data)
Largest diff. peak (eA−3
)
Ru(II) complex concentration caused by dilution at the end of each
titration were negligible.
2.6. Thermal denaturation of DNA
Thermal denaturation of DNA was carried out with a PerkinElmer
Lambda 850 spectrophotometer equipped with a Peltier temperature-
control programmer ( 0.1 °C). The temperature of the solution was
increased from 40 to 90 °C at a rate of 1 °C min−1, and the absorbance
at 295 nm was continuously monitored for solutions of DNA
(10.0 μM) in the absence and presence of the Ru(II) complex
(10.0 μM). The data were presented as (A−Af)/(A0 −Af) vs. temper-
ature, where A, Af and A0 were the observed absorbances at
temperature T, at 90 °C, and at 40 °C, respectively, at 295 nm.
ꢂ
ꢀ
ꢁ
ꢀ
ꢁ
ꢀ
ꢁ
εa−εf
=
εb−εf
=
b− b2−2Kb2Ct½DNAꢀ=s 1=2 = 2KbCt
ð1aÞ
ð1bÞ
b = 1 + KbCt + Kb½DNAꢀ= 2s
The intrinsic binding constants Kb of complexes 1 and 2 were
(9.14 0.47)×107 M−1 and (4.50 0.19)×107 M−1, respectively,
from the decay of the absorbance (Table 3). The binding constant Kb
of complex 1 is larger than that of complex 2. It indicated that complex
1 bound to the DNA more tightly than complex 2 did. The two
complexes have the same intercalative ligand. This trend is mostly
because of the difference between the ancillary ligands, which can be
explained by the less sterical hindrance of methyl in complex 1 than
2.7. Continuous variation analysis
Binding stoichiometries were obtained for the two complexes
using the method of continuous variation [33,34]. The concentrations
of both metal complexes and DNA varied, while the sum of the
concentrations of the two reactants was kept constant at 10 μM. In the
solutions, the mole fraction of the Ru(II) complex varied from 1 to 0 in
0.1 increments. Each mixture was equilibrated at 5 °C for 12 h in the
dark. The fluorescence intensities of these mixtures were measured at
25 °C using an excitation wavelength of 446 and 449 nm for
complexes 1 and 2, respectively. The Fmax (fluorescence) was
recorded in the range of 500–750 nm. Binding stoichiometries were
obtained from the intercepts of the linear plot obtained by linear
least-squares fits to the left- and right-hand portions of the Job plots.
2.8. Circular dichroism measurements
CD titration procedure was described as follows: 4.0 μL Ru(II)
(200 μM) complex was added sequentially to solutions containing
G-quadruplex (5.0 μM). 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
Fig. 1. X-ray crystal structure of complex 2. All the hydrogen atoms are omitted for
clarity.

152
J. Sun et al. / Journal of Inorganic Biochemistry 105 (2011) 149–154
Table 2
Table 3
Selected bond lengths (Å) and angles (°) for metal environments of complex 2.
Absorption spectra (λmax/nm) and DNA-binding constants Kb (×107 M−1
) of
complexes 1 and 2.
Ru(1)–N(1)
Ru(1)–N(5)
Ru(1)–N(9)
2.057(7)
2.068(9)
2.059(8)
78.7(3)
173.6(3)
91.6(3)
96.7(3)
95.8(3)
94.6(3)
86.7(3)
80.2(3)
Ru(1)–N(2)
Ru(1)–N(6)
Ru(1)–N(10)
N(1)–Ru(1)–N(5)
N(1)–Ru(1)–N(9)
N(2)–Ru(1)–N(5)
N(2)–Ru(1)–N(9)
N(5)–Ru(1)–N(6)
N(5)–Ru(1)–N(10)
N(6)–Ru(1)–N(10)
2.040(8)
2.084(7)
2.060(8)
95.4(3)
98.2(3)
89.7(3)
174.9(3)
80.1(3)
171.8(3)
93.2(3)
Complex
1
λ
max(free)
λ
max(bound) Δλ/nm H/(%) Kb /107 M−1
s
446
361
281
449
361
282
457
359
289
453
363
290
11
−2
8
4
2
17.94 9.14 0.47
29.04
33.40
13.33 4.50 0.19
23.12
26.00
3.19 0.18
N(1)–Ru(1)–N(2)
N(1)–Ru(1)–N(6)
N(1)–Ru(1)–N(10)
N(2)–Ru(1)–N(6)
N(2)–Ru(1)–N(10)
N(5)–Ru(1)–N(9)
N(6)–Ru(1)–N(9)
N(9)–Ru(1)–N(10)
2
2.90 0.22
8
Since the hydrophobic environment inside the DNA helix reduces the
accessibility of solvent water molecules to the complex thus the
complex mobility is restricted at the binding site, leading to the
decrease of vibrational modes of relaxation [39]. This phenomenon
can be explained by the hydrophilic property of the complexes.
Complex 2 showed less solubility than complex 1 in water at the same
condition. The property of complex 1 most likely caused the
fluorescence be quenched much more easily than complex 2.
The thermal behavior of G-quadruplex DNA in the presence of
complexes can provide insight into their conformational changes with
the temperature and the interaction between the complexes and DNA.
As shown in Fig. 4, the Tm of G-quadruplex in buffer solutions was
68.9 °C. After addition of complexes 1 and 2, the Tm of the DNA
increased to 78.3 °C and 75.9 °C, respectively, at a concentration ratio
[Ru]/[DNA]=1:1. The ΔTm values of complexes 1 and 2 were 9.4 °C
and 7.0 °C, respectively. The results indicated that both complexes can
that of ethyl in complex 2. In general, the larger sterical hindrance in
ancillary ligand will reduce the interaction of the complexes with
DNA.
Luminescence measurements were used to further clarify the
nature of the interaction between these complexes and G-quadruplex
DNA. The results of the fluorescence titration for these complexes
with DNA are shown in Fig. 3. Both complexes showed fluorescence in
buffer solutions with a maximum wavelength of about 630 nm. The
fluorescence intensity of these complexes would reach a maximum
with increasing DNA concentration. The intensities at the maximum
were 4.8 and 7.6 times than that in the absence of DNA for complexes
1 and 2, respectively. It is worth noting that the increasing extent of
the fluorescence intensity of complex 2 is bigger than complex 1.
1.0
5
0.8
0.8
60
4
0.6
3
1
0.4
0.6
50
2
0.2
1
40
30
20
10
0
1
0.0
0.0
0.4
0.8
1.2
1.6
2.0
0.4
0
1
2
3
4
5
[DNA]/[Ru]
[DNA] (uM)
0.2
0.0
300
400
500
Wavelength (nm)
550
600
650
700
750
Wavelength (nm)
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
8
7
6
5
4
3
2
1
250
200
150
100
50
2
2
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0
1
2
3
4
5
[DNA]/[Ru]
[DNA] (uM)
300
400
500
0
Wavelength (nm)
550
600
650
700
750
Wavelength (nm)
Fig. 2. Absorption spectra of complexes 1 and 2 in buffer with increasing amounts of
G-quadruplex. [Ru]=10.0 μM, [DNA]=0–4.5 μM from top to bottom. Arrows refer to
the change in absorbance upon increasing the DNA concentration. Inset: plot of
(εa −εf)/(εb −εf) vs. [DNA] and the nonlinear fit for the titration of DNA to Ru(II)
complexes.
Fig. 3. Emission spectra of complexes 1 and 2 in buffer with increasing amounts of
G-quadruplex, [Ru]=2.0 μM, [DNA]=0–5.0 μM. Arrow refers to the emission intensity
changes upon increasing DNA concentrations.

J. Sun et al. / Journal of Inorganic Biochemistry 105 (2011) 149–154
153
1.0
0.8
0.6
0.4
0.2
0.0
before CD data were used alone to determine the structure of
quadruplex molecular definitively when CD studies are useful in
establishing the presence of quadruplex structures [42,43]. Fig. 6
shows the CD spectra of G-quadruplex at the absence or presence of
two Ru(II) complexes. Upon addition of complexes 1 and 2 to the
G-quadruplex aqueous solution, the maximum of absorbance at
290 nm was gradually suppressed and shifted to 285 nm. It implied
that those two complexes have the tendency to form the anti-parallel
conformation. The characteristic positive peak near 295 nm also
supported the implication [44,45]. In addition, the conformation of
G-quadruplex was not changed at a high ionic strength.
DNA
1
2
4. Conclusions
40
50
60
70
80
90
T (0C)
In this paper, we studied the interaction between two Ru(II)
complexes and G-quadruplex. The results showed that complex 1
bound to the DNA more tightly than complex 2 did. Both complexes
can induce the stabilization of quadruplex DNA. The ΔTm were 9.4 and
7.0 at a [Ru]/[DNA] ratio of 1:1 for complexes 1 and 2, respectively. It
implies that two complexes could be used as potential anticancer
drugs. However, the conformation of G-quadruplex was not changed
by the complexes at a high ionic strength in K+ buffer. The details of
the binding modes of these complexes with G-quadruplex and the
structure of G-quadruplex are not clear yet and further studies are
needed.
Fig. 4. Normalized UV melting curves of G-quadruplex in the absence (■) or presence of
complexes 1 ( ) and 2 ( ) at a [complex]/[DNA] ratio of 1:1. The absorbance was
measured at 295 nm and the temperature was varied from 40 to 90 °C at a heating rate
of 1 °C min−1
.
stabilize G-quadruplex DNA and complex 1 affected the stability of
G-quadruplex more than complex 2. The difference may originate
from the different DNA-binding affinity. It should be noted that the
ΔTm values of the two complexes in this work are higher than that of
[Ru(bpy)2(dppz)]2+ [23] at the same condition. The mechanism is not
clear yet, however, it is important for determining the number of dppz
ligands, which enhance the stability of G-quadruplex DNA. It was
reported that the telomerase inhibition activity of drugs was strongly
related to the stabilization of the quadruplex structure. Therefore,
both complexes might be potential anticancer drugs.
Fig. 5 shows the Job plots of the two complexes. Two major
inflection points for both complexes, at x=0.49 for complex 1 and at
x=0.46 for complex 2, were observed. These data are consistent with
the binding mode at a [quadruplex]/[complex] ratio of 1:1 [24,40].
In order to determine the selectivity of the complexes for any
particular G-quadruplex conformation, circular dichroism (CD)
experiments were carried out. K+ ions induce and stabilize the
parallel and anti-parallel conformations of the human telomeric
G-quadruplex [41]. However, it is controversial to explain the optical
properties, such as hypochromicity and the shape and sign of CD
bands [42]. Bates, et al. believed that a much better understanding of
the various contributions to quadruplex CD spectra would be needed
25
20
15
10
5
DNA
DNA+5 μM 1
DNA+10 μM 1
DNA+13 μM 1
DNA+15 μM 1
0
250
275
300
325
Wavelength (nm)
800
600
20
15
10
5
DNA
DNA+5 μM 2
DNA+10 μM 2
DNA+13 μM 2
DNA+15 μM 2
400
1
2
200
0
0
0.0
0.2
0.4
0.6
0.8
1.0
-5
X
250
275
300
325
Wavelength (nm)
Fig. 5. Job plot using luminescence data for complexes 1 (■) and 2 ( ) with a final
G-quadruplex at 10.0 μM using a buffer solution of 100 mM KCl, 100 mM KH2PO4/
Na2HPO4, and 1 mM K2H2EDTA with pH of 7.0. X=mole fraction of complex added to
DNA.
Fig. 6. CD spectra of G-quadruplex in the absence and presence of complexes 1 and 2,
[DNA]=5.0 μM, [Ru]=0–15.0 μM.

154
J. Sun et al. / Journal of Inorganic Biochemistry 105 (2011) 149–154
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We are grateful for financial support from the National Natural
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China (973 Program No. 2007CB815306), and the Doctoral Program of
Guangdong Medical College (B2009003).
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CCDC 782113 contains the supplementary crystallographic data
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