A comparative study of the interaction of two structurally analogous ruthenium complexes with human telomeric G-quadruplex DNA

Article abstract of DOI:10.1016/j.jinorgbio.2012.12.011

Two new polypyridine ligands and their corresponding ruthenium(II) complexes have been prepared and characterized. The interactions of both complexes with human telomere quadruplex DNA (both the antiparallel basket and the mixed-hybrid G-quadruplex) have been studied by circular dichroism (CD), CD melting, UV-visible (UV-Vis), fluorescent intercalator displacement (FID) assays and molecular docking studies. The results show that both complexes can stabilize G-quadruplexes DNA and two complexes show different binding affinity for different G-quadruplexes DNA. The 1:1 stoichiometry was confirmed in the buffered solutions by the UV-Vis spectrophotometer using Job's plot method and molecular docking studies. We have also investigated the interaction between the complexes and duplex DNA to gain some insight into the selectivity of the complexes for G-quadruplex structures. FID studies have shown that the complexes have a modest selectivity for G-quadruplex versus duplex DNA.

Full text of DOI:10.1016/j.jinorgbio.2012.12.011

Journal of Inorganic Biochemistry 121 (2013) 19–27
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
A comparative study of the interaction of two structurally analogous ruthenium
complexes with human telomeric G-quadruplex DNA
a
a
a
a
a
a
Shuo Shi a, , Hai-Liang Huang , Xing Gao , Jun-Liang Yao , Chun-Yan Lv , Juan Zhao , Wen-Liang Sun ,
⁎
Tian-Ming Yao a, , Liang-Nian Ji
Department of Chemistry, Tongji University, Shanghai 200092, PR China
a,b
⁎
a
b
MOE Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 August 2012
Received in revised form 19 December 2012
Accepted 20 December 2012
Available online 29 December 2012
Two new polypyridine ligands and their corresponding ruthenium(II) complexes have been prepared and
characterized. The interactions of both complexes with human telomere quadruplex DNA (both the antipar-
allel basket and the mixed-hybrid G-quadruplex) have been studied by circular dichroism (CD), CD melting,
UV-visible (UV-Vis), fluorescent intercalator displacement (FID) assays and molecular docking studies. The
results show that both complexes can stabilize G-quadruplexes DNA and two complexes show different bind-
ing affinity for different G-quadruplexes DNA. The 1:1 stoichiometry was confirmed in the buffered solutions
by the UV-Vis spectrophotometer using Job's plot method and molecular docking studies. We have also
investigated the interaction between the complexes and duplex DNA to gain some insight into the selectivity
of the complexes for G-quadruplex structures. FID studies have shown that the complexes have a modest
selectivity for G-quadruplex versus duplex DNA.
Keywords:
Ruthenium complexes
G-quadruplex DNA
Comparative study
Spectral properties
Molecular docking
© 2012 Elsevier Inc. All rights reserved.
1. Introduction
the other hand, formation of G-quadruplex DNA structures in the
human telomere has been shown to inhibit telomerase (an enzyme
G-rich DNA sequences can adopt a special class of DNA structure
called a G-quadruplex, which comprises a stack of G-tetrads, the planar
association of four guanines in a cyclic Hoogsteen hydrogen bond
[1–3]. Recently, bioinformatic studies have shown that in the human ge-
nome there are approximately 350,000 guanine-rich sequences that can
potentially form G-quadruplex DNA structures [4–6]. G-quadruplexes
DNA are highly dynamic and polymorphic DNA structures, and the struc-
tures and the stability of the G-quadruplexes depend on monocations
[7]. The best-studied example is the human telomeric repeat
AG3(T2AG3)3 quadruplex, the NMR structure of AG3(T2AG3)3 in
the presence of Na+ was an antiparallel basket quadruplex [8], the
X-ray structure for the same sequence in the presence of K+ revealed
a parallel propeller quadruplex [9], whereas, it favored a mixed-
hybrid (parallel/antiparallel) structure in the presence of K+ solution
[10,11]. It has been suggested that these secondary DNA structures
could be involved in the regulation of several key biological processes
[12,13]. There is now mounting evidence showing that formation of
quadruplexes in guanine-rich regions of the genome may play impor-
tant roles in regulating gene expression. For example, the promoter
regions of certain oncogenes such as c-myc and c-kit are guanine rich,
and formation of G-quadruplexes in these regions has been proposed
to regulate the corresponding oncogene's transcription [14–18]. On
over expressed in approximately 85% of cancer cells and which plays
an important role in cancer cell immortalization) [19–22]. These poten-
tial roles of G-quadruplex DNA structures have stimulated a search for
specific molecules that stabilize G-quadruplexes in either the promoter
regions of oncogenes or in the telomeric region. Such molecules could
provide a basis for the development of novel anticancer drugs.
Over the past 10 years, a rational approach to design small mole-
cules that can selectively interact with G-quadruplex DNA has emerged.
A number of promising small organic molecules have been devised to
inhibit telomerase and/or regulate the transcription of certain onco-
genes. These molecules range from acridine derivatives, cationic por-
phyrin derivatives, ethidium derivatives, anthraquinone derivatives,
perylene derivatives, and telomestatin and others planar compounds
[23–28]. These ligands have the common feature of extended planar ar-
omatic electron-deficient chromophore with cationic substituents. In
contrast to the large number of organic molecules reported to bind to
this secondary structure of DNA, metal complexes have only recently
started to be systematically investigated [29–33]. Metal complexes
have clear advantages over their organic counterparts, such as, electro-
positive, modular and facile synthesis, interesting optical and magnetic
properties etc, which offer an ideal platform for sharp drug design and
rationalization of structural interactions [29]. These studies have
shown the great potential metal complexes have in binding to (and
stabilizing) quadruplexes, and in doing so, inhibiting telomerase or
regulating gene expression of certain oncogenes.
⁎
Corresponding authors. Tel.: +86 21 6598 3292; fax: +86 21 6598 2287.
E-mail addresses: shishuo@tongji.edu.cn (S. Shi), tmyao@tongji.edu.cn (T.-M. Yao).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.12.011

20
S. Shi et al. / Journal of Inorganic Biochemistry 121 (2013) 19–27
Ruthenium complexes that bind noncovalently to duplex DNA
of G-quadruplexes DNA recognition and binding by Ru(II) complexes, as
well as laying the foundation for the rational design of new anticancer
therapeutic agents.
have been of great interest for the last 30 years, as this class of mole-
cules has potential as DNA-conformational probes, anticancer drugs,
or initiators for electron-transfer studies on duplex DNA [34–38]. Ru-
thenium complexes containing planar aromatic ligands that bind to
DNA have many convenient features, including the ease with which
the ligand can be attached to the metal in a controlled manner, strong
visible absorbance, due to a localized metal-to-ligand charge transfer
(MLCT) and strong fluorescence emission. Just as the unique proper-
ties of ruthenium complexes have been successfully used to probe
duplex DNA, however, it is important to note that these complexes
are only in the beginning stages of development as G-quadruplex
DNA binders. We reported previously a novel dinuclear complex
Ru2(obip)L4 (obip=2-(2-pyridyl)imidazo[4,5-f][1,10]-phenanthroline,
L=2,2′-bipyridine) which has the remarkable ability to promote the
formation and stabilization of G-quadruplex DNA [31]. Recently, our
laboratory found that [Ru(L)2(dppz)]2+ (L=2,2′-bipyridine or 1,10-
phenanthroline, dppz=dipyrido[3,2-a:2′,3′-c]phenazine) can serve as
a prominent molecular “light switch” for both G-quadruplexes
and i-motif, which prefers binding G-quadruplexes over i-motif
[39,40]. Furthermore, we reported the first example of a reversible
G-quadruplex DNA light switch, the switch can be cycled through
the competition of [Fe(CN)6]4- ions and G-quadruplex DNA [41].
Herein, we report the synthesis, characterization and G-quadruplexes
DNA binding of two new ruthenium(II) complexes (Scheme 1)
[Ru(phen)2(bppp)]2+ (1) and [Ru(phen)2(pppp)]2+ (2) (phen=1,10-
phenanthroline, bppp=12-bromo-pyrido[2′,3′:5,6]pyrazino[2,3-f][1,10]
phenanthroline, pppp=12-phenylpyrido[2′,3′:5,6]pyrazino[2,3-f][1,10]
phenanthroline). We hope that our results will aid in the understanding
2. Materials and methods
2.1. Materials and chemicals
1,10-phenanthroline-5,6-dione, cis-Ru(phen)2Cl2⋅3H2O were syn-
thesized according to the literature methods [42]. The other chemicals
were obtained from commercial sources and used without further puri-
fication unless otherwise noted. DNA oligomers 5′-AGGGTTAGGGTT
AGGGTTAGGG-3′ (22AG) and 16 base-pair complementary duplex DNA
(5′-CCTCGGCCGGCCGACC-3′) were purchased from Sangon (Shanghai,
China) and used without further purification. Concentrations of these
oligomers were determined by measuring the absorbance at 260 nm
after melting. Single-strand extinction coefficients were calculated
from mononucleotide data using a nearest-neighbour approximation
[43]. 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: 100 mM KCl, 10 mM
Tris, pH 7.0; Buffer B: 100 mM NaCl, 10 mM Tris, pH 7.0.
2.2. Synthesis
2.2.1. bppp (12-bromo-pyrido[2′,3′:5,6]pyrazino[2,3-f][1,10]phenanthroline)
A mixture of 1,10-phenanthroline-5,6-dione (640 mg, 3.0 mmol)
and 2,3-Diamino-5-bromopyridine (560 mg, 3.0 mmol) in 20 mL of
Scheme 1. Synthetic routes for the preparation of the complexes [Ru(phen)2(bppp)]2+ (1) and [Ru(phen)2(pppp)]2+ (2).

S. Shi et al. / Journal of Inorganic Biochemistry 121 (2013) 19–27
21
methanol was refluxed for 2 h. Upon cooling, the yellow precipitate
was collected by filtration and further recrystallized from methanol.
Yield: 880 mg, 82%. Anal. Calcd for C17H8BrN5: C, 56.38; H, 2.23; N,
19.34. Found: C, 56.37; H, 2.24; N, 19.36. FAB-MS: m/z=362.0
titration processes were repeated until there was almost no change,
indicating binding saturation had been achieved. For each sample, at
least four spectrum scans were accumulated over the wavelength
range of 200–350 nm at the temperature 25 °C in a 1.0 cm path
length cell at a scanning rate of 50 nm/min. The instrument was
flushed continuously with evaporated liquid nitrogen throughout
the experiment. The scan of the buffer alone was subtracted from the
average scan for each sample. In the melting studies, the temperature
of the sample was maintained by a Julabo HD-25 temperature control-
ler. The melting curves of the G-quadruplex were measured with the
intensity at 295 nm. Before the CD spectroscopy, all the samples
were thermally treated as described above. The heating rate was
[M+H]+
.
2.2.2. pppp (12-phenylpyrido[2′,3′:5,6]pyrazino[2,3-f][1,10]phenanthroline)
A solution of bppp (362 mg, 1.0 mmol), phenylboronic acid (134 mg,
1.1 mmol) and potassium carbonate (15 mL, 2 M) in 20 mL of toluene
and 10 mL of ethanol was fully degassed and refluxed at 90 °C under
argon 3 h. Then tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4)
(116 mg, 0.1 mmol) was added and another 12 h of heating, the reac-
tion mixture was cooled to room temperature. The solvents were evapo-
rated, water was added and extracted with chloroform. The organic
fractions were collected and evaporated to dryness, giving 251 mg of a
yellow solid (70% yield). Anal. Calcd for C23H13N5: C, 76.87; H, 3.65; N,
1.0 °C min−1
.
2.3.2. Absorption spectra titrations
Absorption spectra titrations were carried out at room tempera-
ture to determine the binding affinity between DNA and complex.
Initially, 3000 μL solutions of the blank buffer and the ruthenium
complex sample (10 μM) were placed in the reference and sample
cuvettes (1.0 cm path length), respectively, and then first spectrum
was recorded in the range of 200–600 nm. During the titration,
aliquot (1–10 μL) of buffered DNA solution was added to each cuvette
to eliminate the absorbance of DNA itself, and the solutions were
mixed by repeated inversion. After the solutions were mixed for
~5 min, the absorption spectra were recorded. The titration processes
were repeated until there was no change in the spectra for four titra-
tions 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.
19.49. Found: C, 76.84; H, 3.64; N, 19.47. FAB-MS: m/z=360.4 [M+H]+
.
2.2.3. [Ru(phen)2(bppp)](PF6)2 (1)
Cis-[Ru(phen)2Cl2].3H2O (170 mg, 0.30 mmol) and bppp (109 mg,
0.30 mmol) were added to 20 ml ethylene glycol–water (9:1, v/v).
The mixture was refluxed for 6 h under an argon atmosphere. The
cooled reaction mixture was diluted with water (50 ml) and filtered
to remove solid impurities. And then, to the filtrate was added am-
monium hexafluorophosphate. The precipitated complex was dried,
dissolved in a small amount of acetonitrile, and purified by chromatog-
raphy over alumina, using MeCN–toluene (3:1, v/v) as eluent and
further recrystallized from acetone/diethyl ether (1:5, v/v). Yield:
280 mg, 84%. 1H NMR [(CD3) 2SO]: δ 9.62 (1H, d), 9.60 (1H, d), 9.54
(1H, d), 9.34 (1H, d), 8.81 (4H, t), 8.42 (4H, s), 8.29 (2H, d), 8.25
(2H, d), 8.07 (2H, d), 7.95 (2H, t), 7.85–7.77 (4H, m). Calc. for C41H24-
BrF12N9P2Ru: C, 44.22; H, 2.17; N, 11.32. Found: C, 44.20; H, 2.19; N,
11.38. ESI-MS: m/z 824.0 ([M-2PF6 +H]+).
2.3.3. Job's plots of UV-visible (UV-Vis) absorption
According to the literature procedure, equimolecular solutions of
compounds and G-quadruplex DNA are mixed in various proportions.
The mole fraction of each molecular was varied from 0.1 to 1.0, in 0.1
increments, while the total concentration was kept at 10 μM. The dif-
ference (ΔA) between each experimental value and that calculated as
the sum of the contributions of the two separate components is plot-
ted against the molar fractions. The resulting curves display a break
point at the molar fraction corresponding to the stoichiometry of
the complex. The absorption of each sample was recorded at room
temperature and baseline values were routinely subtracted from the
spectra.
2.2.4. [Ru(phen)2(pppp)](PF6)2 (2)
With 0.30 mmol, 0.108 g pppp in place of bppp, this complex was
obtained by a procedure similar to that described for 1. Yield: 290 mg,
87%. 1H NMR [(CD3) 2SO]: 9,94(1H, d), 9.67(1H, d), 9.61(1H, d), 9.19
(1H, d), 8.82(4H, t), 8.42 (4H, s), 8.30 (2H, t), 8.25 (2H, d), 8.20
(2H, d), 8.08 (2H, d), 7.96 (2H, t), 7.83 (2H, m), 7.79 (2H, m), 7.68
(2H, t), 7.62 (1H, d). Calc. for C47H29F12N9P2Ru: C, 50.82; H, 2.63;
N, 11.35. Found: C, 50.80; H, 2.63; N, 11.39. ESI-MS: m/z 821.2
[M-2PF6+H]+
.
The NMR spectra of [Ru(phen)2(bppp)]2+ (1) and [Ru(phen)2
(pppp)]2+ (2) as well as their definition have been given in the supple-
mentary information (Fig. s1).
2.3.4. Fluorescent intercalator displacement (FID) assay
The 22AG strand 5′-AGGGTTAGGGTTAGGGTTAGGG-3′ and 16
base-pair complementary strand 5′-CCTCGGCCGGCCGACC-3′ were
used for the human telomeric G-quadruplex and duplex DNA, re-
spectively. FID experiments were performed as follows: onto a mix-
ture of prefolded DNA (1 μM) and thiazole orange (TO) (2 μM), in
100 mM K+ buffer or 100 mM Na+ buffer, an increasing amount of
the corresponding molecule under study was added (0.25–20 μM)
by a 3 min equilibration period before the fluorescence spectrum is
recorded. Emission spectra were measured on a Shimadzu RF-5000
spectrofluorophotometer. The excitation wavelength was 492 nm,
and the emission spectrum was collected from 500 to 750 nm. Exci-
tation and emission slits were set at 10 and 10 nm, respectively. The
fluorescence of samples was measured at 25 °C. The titration pro-
cesses were repeated until there was no change in the spectra
for at least four titrations indicating binding saturation had been
achieved.
2.3. Physical measurements
Elemental analyses (C, H and N) were carried out with a Perkin–
Elmer 240C elemental analyzer. 1H NMR spectra were recorded on a
Bruker DRX-400 NMR spectrometer with (CD3)2SO as solvent and
SiMe4 as an internal standard. Electrospray ionisation mass spectra
(ESI-MS) were acquired on a Thermo Finnigan LCQ DECA XP ion
trap mass spectrometer, equipped with an ESI source.
2.3.1. CD measurements and CD melting profiles
Circular dichroism (CD) spectra were measured on a Jasco J-810
spectropolarimeter. The oligonucleotide samples were dissolved
in two different buffered solutions in this study: (a) in K+ buffer
(pH 7.0); (b) in Na+ buffer, (pH 7.0). The corresponding samples
of the DNA (22AG) at a concentration of 5 μM were dissolved in dif-
ferent solutions and placed in a quartz cuvette. During the titration,
aliquot (1–10 μL) of [Ru(phen)2(L)]2+ solution was added to the cu-
vette, and the solutions were mixed by repeated inversion. After the
solutions were mixed for ~5 min, the CD spectra were recorded. The
2.3.5. Molecular docking studies
Both the antiparallel basket quadruplex (Protein Data Bank (PDB)
ID: 143D) and the mixed parallel/antiparallel structure (PDB ID:
2HY9) were used as an initial model to study the interaction be-
tween [Ru(phen)2(L)]2+ and 22-mer telomeric G-quadruplex DNA.

22
S. Shi et al. / Journal of Inorganic Biochemistry 121 (2013) 19–27
[Ru(phen)2(L)]2+ was optimized using density functional theory
(DFT) with a LanL2MB basis set [44]. The optimized structure of the
ruthenium complex was used to do the docking. When preparing
for docking DNA and complex ([Ru(phen)2(L)]2+), necessary modi-
fications were carried out: (1) add all hydrogens or just non-polar
hydrogens; (2) assign partial atomic charges to the complex and
the macromolecule (Gasteiger or Kollman United Atom charges);
(3) merge non-polar hydrogens and Set up rotatable bonds in the
complex; (4) output PDBQT (Protein Data Bank, Partial Charge (Q),
& Atom Type (T)) files from traditional PDB files are also created
for the side chain coordinates. Docking was carried out with the
AutoDock 4.2 Lamarckian Genetic Algorithm (LGA) [45–47]. For
antiparallel basket G-quadruplex structure, to create a pseudo-
intercalation complex binding site between the diagonal loop and
the G-quartet segment of the structure (at the 5′ AG step) in the
human intramolecular G-quadruplex NMR structure (PDB code:
143D), the following steps have been employed: (1) two phosphate
backbones are broken at the 5′ AG step; (2) the two halves of the
structure are separated so that the separation of the A:A base pair
and the G-quartet is increased from 3.4 to 6.8 Å; (3) the sugar-
phosphate backbones are reconnected [48,49]. As for the mixed-
hybrid G-quadruplex structure, two adenines from each end of the
mixed-hybrid type structure (PDB code: 2HY9) were removed and
similar operations to increase the separation between loop base
pairs and the G-quartet were performed [50]. In the autodocking,
DNA was enclosed in the grid defined by Auto Grid having 0.375 Å
spacing and parameters (supplied with the program package) were
used for dispersion/repulsion, hydrogen bonding, electrostatics, and
desolvation, respectively. Auto Grid performed a precalculated atomic
affinity grid maps for each atom type in the complex plus an electrostat-
ics map and a separate desolvation map present in the substrate
molecule. Then, during the AutoDock calculation, the energetics of a
particular complex configuration is evaluated using the values from
the grids. The output from AutoDock was rendered with Accelrys
Discovery Studio 3.0 Client [51].
3. Results and discussion
CD spectroscopy was employed to determine the solution forma-
tion of G-quadruplex in the absence or presence of synthetic com-
plexes in either K+ or Na+ buffers. As shown in Fig. 1(a), the CD
spectrum of the human telomeric sequence 22AG in the presence of
100 mM K+ exhibited a large positive band at 290 nm, a small posi-
tive band at 270 nm, and a negative band at 235 nm, which suggested
that 22AG might exist as a mixed-hybrid quadruplex DNA containing
parallel and antiparallel structure. On the other hand, the CD spec-
trum of the same sequence in the presence of 100 mM Na+ ions
had a 295 nm positive band and a 265 nm negative band, which
was characteristic of an antiparallel G-quartet structure consistent
with the results of previous NMR studies [52–55]. Upon addition
of either 1 or 2 to 22AG aqueous solution in K+ buffer (Fig. 1(a))
resulted in significant changes to the CD spectrum, including the
observation of an isoelliptical point at 259 nm, which suggested the
presence of both free and bound quadruplex DNA in solution. With
increasing 1 or 2 binding, the negative band at about 260 nm
(characteristic of antiparallel G-quartet structure) started to appear
and increased sharply. The emergence of homogeneous antiparallel
basket type characteristics with the distinctive minima pattern of
265 nm indicated that both 1 and 2 converted the preformed mixture
of G-quadruplexes into the antiparallel basket type G-quadruplex
under K+ conditions. These significant changes in the CD spectrum of
22AG after the addition of 1 or 2 were consistent with both complexes
binding to the DNA and thus causing substantial change(s) in the con-
formation of the DNA. However, upon addition of 1 or 2 to 22AG in
buffer containing Na+, no obvious spectral changes were observed
(Fig 1(c, d)), which implied that the conformation of G-quadruplex
was stabilized by Na+, and either 1 or 2 could not change the conforma-
tion of G-quadruplex at high ionic strength.
The stability of the adducts of the complexes with the G-quadruplex
was studied by CD spectroscopy at variable temperature using 22AG in-
cubated with either complex 1 or 2 in the presence of K+ or Na+
.
(b)
(a)
10
10
5
0
5
0
-5
-5
-10
-10
-15
240 260 280 300 320 340
Wavelength/nm
240 260 280 300 320 340
Wavelength/nm
(d)10
(c) 10
5
0
5
0
-5
-5
-10
-10
240 260 280 300 320 340
Wavelength/nm
240 260 280 300 320 340
Wavelength/nm
Fig. 1. CD titration of 22AG by adding complex 1 or 2 in K+ buffer (100 mM KCl, 10 mM Tris, pH 7.0) or Na+ buffer (100 mM NaCl, 10 mM Tris, pH 7.0) at 25 °C. CD spectra of
(a) 22AG+1 in K+ buffer; (b) 22AG+2 in K+ buffer; (c) 22AG+1 in Na+ buffer and (d) 22AG+2 in Na+ buffer.

S. Shi et al. / Journal of Inorganic Biochemistry 121 (2013) 19–27
23
Consistent with previous studies [56,57], 295 nm was chosen to study
the influence of the complex on the stability of G-quadruplex DNA.
The normalized CD intensity of 22AG with 5 uM complex in different
buffers at 295 nm vs the temperature was shown in Fig. 2. The transi-
tion temperature of the G-quadruplex increased from 55.3 to 61.3 °C
in Na+ buffer and increased from 65.2 to 71.7 °C in K+ buffer induced
by complex 2, in which an increase in the melting temperature of the
G-quadruplex indicated a stabilizing effect. Under the same conditions,
the transition temperature of the G-quadruplex increased from 55.3 to
60.2 °C in Na+ buffer and increased from 65.2 to 69.8 °C in K+ buffer in-
duced by complex 1. These data are indicative of the strong propensity
of both complexes to form stable interactions with G-quadruplexes
resulting in the formation of bound complexes whose thermal stabili-
ties are markedly enhanced from native G-quadruplexes. As for com-
plex 2, it had bigger influence on the thermal stability G-quadruplex
DNA either in Na+ buffer or in K+ buffer (Fig. 2) compared with com-
plex 1, which implies that a higher affinity of 2 for the human telomeric
sequence and resulting in bigger stability of the telomeric G-quadruplex
than that of complex 1. Since the ancillary ligand (phen) of the com-
plexes 1 and 2 is the same, their differential DNA-binding affinities
must be attributed to the different main ligands. On going from the
main ligand bppp to pppp, the hydrophobicity of the ligand pppp is
greater than the ligand bppp and the square π-aromatic surface of
pppp is larger than bppp. Both factors are advantageous to the
G-quadruplexes DNA-binding. Therefore, synthetically considering
these factors, the difference of the G-quadruplexes DNA-binding af-
finity of complexes 1 and 2 can be well understood.
addition of 22AG to a solution of 2 in K+ buffer led to a 22 nm red
shift and 28.9% hypochromism of the band at 392 nm; however addi-
tion of 22 AG to the Na+ buffered solution led to a 20 nm red shift
and 23.7% hypochromism. The similar case could be seen for complex
1, too. Hypochromism and red shift indicated strong interactions be-
tween the DNA bases and the complexes. Here the representative
electronic spectral traces of both complexes titrated with 22AG in
K+ buffered solutions were given in Fig. 3. The others and related
quantitative data were shown in Table 1.
In order to compare quantitatively the binding strength of
[Ru(phen)2(L)]2+ to each G-quadruplexes DNA, the intrinsic bind-
ing constants K with each DNA at 25 °C were obtained using the
following Eqs. (2a) and (2b) [61–63],
ꢂ
ꢃ
ꢀ
ꢁ
1=2
ðεa−εf Þ=ðεb−εf Þ ¼ b− b2−2K2Ct½DNAꢀ=s
b ¼ 1 þ KCt þ K½DNAꢀ=2s
=2KCt
ð2aÞ
ð2bÞ
where [DNA] is the concentration of DNA in base pair, εa, εf and εb
are, the apparent extinction coefficient (Aabs/[M]), the extinction
coefficient for free metal (M) complex and the extinction coeffi-
cient for the metal (M) complex in the fully bound form, respec-
tively. K is the equilibrium binding constant in M−1, Ct is the total
metal complex concentration, and s is the binding size. As shown
in Table 1, both complexes bind the mixed-hybrid quadruplex
more avidly than that of antiparallel basket quadruplex DNA and
complex 2 bind to the G-quadruplexes DNA more tightly than com-
plex 1 does. The results obtained by absorption spectra titrations
are overall consistent with those obtained by CD melting profiles.
To find out the stoichiometry interactions between complex and
G-quadruplex DNA, we used the method of continuous variation
known as Job plot. The results are displayed in Fig. 4. It is evident
that the binding is determined to be 1:1. This binding stoichiometry
suggests both complexes are interacting with each molecule of
G-quadruplex DNA.
For both complexes 1 and 2, no emission was observed either in
Tris buffer or in the presence of G-quadruplexes DNA. To further
clarify the nature of the interaction between the complex and DNA,
G-quadruplex FID was carried out. FID is a simple and fast method
to evaluate the affinity of a compound for G-quadruplex DNA [64–67].
This assay is based on the loss of fluorescence of thiazole orange
(TO) upon competitive displacement from DNA by a putative ligand.
Upon interaction with quadruplex DNA, TO exhibits high affinity
(K=3×106 M−1) and displays a significant increase in its fluores-
cence, whereas when free in solution, the fluorescence is quenched.
Therefore, the affinity of a molecule for quadruplex DNA is estimated
by the DC50 value, which corresponds to the required concentration
of complex to induce a 50% fluorescence decrease. The fluorescence
area (FA, 500–750 nm), converted in percentage displacement
(PD, with PD=100−[(FA/FA0)×100], FA0 being FA before addition
of complex), is then plotted versus the concentration of added
complex.
The application of electronic absorption spectroscopy in DNA-
binding studies is one of the most useful techniques. The high-
⁎
energy band around 262 nm is attributed to the π→π transitions
corresponding to the phenanthroline moiety of the ligands. The
low-energy band at 438 nm for complex 1 is attributed to the overlap
⁎
⁎
of Ru(dπ)→phen (π ) and Ru(dπ)→ bppp (π ). Apart from these,
absorption bands centered at 376 and 392 nm for complexes 1 and
⁎
2 can be assigned to the intraligand (IL) π →π transition of bppp
and pppp, respectively, by comparison with the spectrum of other
polypyridyl Ru(II) complexes [58–60].
Absorption spectra titrations were performed to determine the
binding affinity of complex to 22AG too. DNA sample was added in
aliquots sequentially to complex solutions, with absorbance spectra
recorded after each addition. The changes in the spectral profiles
during titration were shown in Fig. 3. When 22AG is added into com-
plex 2 solutions, notable red shift and hypochromism are observed.
Furthermore, bigger hypochromism is observed for complexes upon
addition of the mixed-hybrid quadruplex DNA than the antiparallel
basket quadruplex DNA under the same conditions. For example,
1.0
0.8
0.6
0.4
0.2
0.0
We were first interested in comparing the quadruplex DNA binding
abilities of the free ligands and the corresponding metal complexes. The
emission spectra of TO bound to DNA in the absence and the presence of
complex or ligand are shown in Fig. 5. We can see that the addition of
either complex 1 or complex 2 to DNA pretreated TO causes appreciable
reduction in the emission intensity (Fig. 5(a), (b)). The DC50 values
are summarized in Table 2. Several interesting trends emerge from
these studies: both complexes displace TO at low μM concentrations
suggesting strong interactions between these complexes and telomeric
quadruplex DNA, which are comparable to those found for other com-
pounds reported to be good quadruplex DNA binders [67–69]. In
contrast, no obvious displacement was observed induced by bppp. Sim-
ilarly, pppp is not able to fully displace TO, even at concentrations as
40
50
60
70
80
90
Temperature /oC
Fig. 2. Normalized CD melting curves for G-quadruplex DNA ( ), G-quadruplex
DNA+1 ( ), G-quadruplex DNA+2 ( ) in Na+ buffer (pH 7.0); G-quadruplex DNA
( ), G-quadruplex DNA+1 ( ), G-quadruplex DNA+2 ( ) in K+ buffer (pH 7.0).
[DNA]=5 μM, [complex]=5 μM. The stability of G-quadruplexes DNA was assessed
by CD at 295 nm.

24
S. Shi et al. / Journal of Inorganic Biochemistry 121 (2013) 19–27
(a)
(b)1.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
0.8
0.6
0.4
0.2
0.0
0
1 2 3 4 5 6 7 8 9 10
0.0
2.5
5.0
7.5 10.0
[DNA] uM
[DNA] uM
250 300 350 400 450 500 550 600
Wavelength/nm
250 300 350 400 450 500 550 600
Wavelength/nm
Fig. 3. Absorption spectra of complex 1 (a) and complex 2 (b) in K+ buffered solutions in the presence of 22AG ([complex]=10 μM, [DNA]=0–10 μM). Arrow shows the absor-
bance change upon increasing DNA concentration. Inset: plot of (εa−εf)/(εb−εf) vs. [DNA] for the titration of Ru(II) complexes.
high at 10 μM and only 5–10% displacement at 2.5 μM. The results indi-
cate that the free ligands do not display significant intermolecular π-π
stacking interaction while the corresponding complexes do. Another
important observation from the data shown in Table 2 is that DC50
values in K+ buffer (for the mixed-hybrid quadruplex) are smaller
than those of Na+ buffer (antiparallel basket quadruplex). However,
no marked buffer effect was observed in absorption spectra studies.
This difference might be caused by the different spectroscopy method.
Compared with absorption spectra studies, FID assay is more sensitive
to buffers and DNA structures. So buffer effect was observed from FID
data.
The selectivity of both complexes for quadruplex DNA versus
duplex DNA was then carried out. Though both complexes showed
prominent G-quadruplex binding affinity, a modest selectivity for
quadruplex over duplex was observed (Table 2). It is also worth
pointing out that, although the differences are small, complex 2 was
found to have a stronger preference for binding to the mixed-hybrid
G-quadruplex over duplex DNA (binding up to 3 times better to the
mixed-hybrid quadruplex DNA than to duplex DNA). This indicated
that modifying the complex could create some interesting differences
in the DNA-binding properties, therefore, such structural information
of the complexes was still important for a more comprehensive
understanding of the biological implications of these structures and
for designing new drugs with enhanced activity and minimized
undesired toxicity.
G-quadruplex molecule binds to one [Ru(phen)2(L)]2+ molecule. It
has been previously shown that G-quadruplex binders can stack on
the surface of both terminal G-quartet planes. Complexes 1 and 2
contain a square π-aromatic surface, both bppp and pppp preferred
to stack in the center of a terminal G-quartet end. For antiparallel
basket G-quadruplex structure, both diagonal loop and parallel
loop binding positions were considered. When [Ru(phen)2(L)]2+
binds in the diagonal loop position, π-π stacking become less stable
than in the parallel loop binding position as showed energy calcula-
tion results. So both complexes prefer to stack on the center of be-
tween the parallel loop and terminal G-quartet (Fig. 6(a), (c)). As
for the mixed-hybrid G-quadruplex DNA, the predicted most favor-
able binding site between [Ru(phen)2(L)]2+ and G-quadruplex
DNA was stacking on the external G-quartets at the 5′ end of oligo-
nucleotide (Fig. 6(b), (d)). A higher planar area, an extended π sys-
tem, hydrophobicity, and aromaticity of complex 2 lead to better
stacking within the base-pairs of DNA than complex 1 does. This
proposed model is supported by molecular modeling study of
the binding interactions between complex and G-quadruplex DNA.
The favorable calculated binding free energies of complex 1 with
G-quadruplex DNA are −11.2 and −9.3 kcal/mol for the mixed-
hybrid G-quadruplex and the antiparallel basket G-quadruplex, re-
spectively. The favorable calculated binding free energies of complex
2 with G-quadruplex DNA are −12.2 and −10.1 kcal/mol for the
mixed-hybrid G-quadruplex and the antiparallel basket G-quadruplex,
respectively. The lower binding free energy of complex 2 also suggests
more favorable binding interactions with G-quadruplexes DNA than
complex 1. It was also noteworthy that both complexes possessed
To gain insight into interaction between complex and G-quadruplex,
molecular docking studies were carried out, which could corroborate
the experimental results [49,70,71]. The antiparallel basket NMR
G-quadruplex structure (PDB ID: 143D) and a 26-mer mixed-hybrid
type G-quadruplex structure (PDB ID: 2HY9) were used as the tem-
plates for the docking studies. To compare both conformations, we re-
moved two adenines from each end of the mixed-hybrid structure. As
shown in Fig. 6, the docking study confirms that each intramolecular
0.00
-0.02
-0.04
-0.06
Table 1
Absorption spectra (λmax/nm) and G-quadruplexes DNA-binding constants Kb (×105 M−1
of 1 and 2 in different buffers.
)
Complex
1(K+
λ
max/free
λ
max/bound
Δλ/nm
H (%)a
K/105 M−1
s
)
262
376
438
262
376
438
262
392
262
392
263
376
438
262
376
438
265
413
263
412
1
0
0
0
0
0
3
22
1
22.6
35.9
27.8
22.0
27.1
18.4
40.5
28.9
34.9
23.7
3.8 0.3
1.2
1(Na+
)
3.3 0.2
0.9
0.0
0.2
0.4
0.6
0.8
1.0
Mole fraction of complex
2(K+
)
8.3 0.5
6.1 0.2
1.4
1.3
Fig. 4. Job's plots of complex 1 binding to 22AG in Na+
(
) and K+ ( ) buffer; Job's
2(Na+
)
plots of complex 2 binding to 22AG in Na+ ) and K+
(
( ) buffer. Total concentration
20
(complex+DNA)=10 μM. ΔA is the calculated absorption difference at wavelengths
376 and 392 nm for complexes 1 and 2, respectively.
a
H: hypochromism.

S. Shi et al. / Journal of Inorganic Biochemistry 121 (2013) 19–27
25
(a)
(b)
1.0
0.8
0.6
0.4
0.2
0.0
1.0
500
0.8
500
0.6
400
300
200
100
0
0.4
400
0.2
0.0
300
0
2 4 6 8 10 12 14 16 18
0
2
4
6
8 10 12
Complex uM
Complex uM
200
100
0
500
550
600
650
700
500
550
600
650
700
Wavelength/nm
Wavelength/nm
(c)
(d)
0 uM
1 uM
2 uM
4 uM
0 uM
1 uM
2 uM
4 uM
10 uM
500
400
300
200
100
0
500
400
300
200
100
0
10 uM
500
550
600
650
700
500
550
600
650
700
Wavelength/nm
Wavelength/nm
Fig. 5. Fluorescence displacement of TO bound G-quadruplex DNA by complex 1 (a), 2 (b), ligand bppp (c) and pppp (d) in K+ buffer. Arrow shows the intensity changes upon
increasing the concentration of complex 1 or complex 2. Inset: FID results onto 22AG for complex 1 or complex 2.
much more favorable binding interactions (lower binding free energy)
with the mixed-hybrid G-quadruplex than antiparallel basket
G-quadruplex. Such reliable end-stacking of compounds onto the
G-quartet are in agreement with previous aromatic quadruplex
binders [72]. Thus, molecular modeling studies explained the dif-
ferent binding affinities and confirmed the excellent complemen-
tarity in binding modes.
modification of main ligand could create some interesting differences
in the DNA-binding properties. Designing new complexes with en-
hanced activity and minimized undesired toxicity is currently being
investigated in our laboratory.
Abbreviations
phen
bppp
1,10-phenanthroline
12-bromo-pyrido[2′,3′:5,6]pyrazino[2,3-f][1,10]
phenanthroline
4. Conclusions
pppp
dppz
12-phenylpyrido[2′,3′:5,6]pyrazino[2,3-f][1,10]
phenanthroline
dipyrido[3,2-a:2′,3′-c]phenazine
In summary, two new polypyridyl ligands (bppp and pppp), and
their mixed-ligand ruthenium(II) complexes [Ru(phen)2(bppp)]2+
(1) and [Ru(phen)2(pppp)]2+ (2), have been synthesized and charac-
terized. A comparison of the G-quadruplex DNA binding abilities of
the free ligands and the corresponding metal complexes, has shown
the important role played by the octahedral ruthenium(II) center in
yielding good G-quadruplex DNA binders. The 1:1 stoichiometry sug-
gests that both complexes are interacting with each molecule of
G-quadruplex DNA. Furthermore, both complexes bind the mixed-
hybrid G-quadruplex more avidly than that of antiparallel basket
G-quadruplex DNA and complex 2 bind to the G-quadruplexes DNA
more tightly than complex 1 does. However, the selectivity of the
complexes for G-quadruplex versus duplex DNA is not as high as
initially expected. Studies carried out so far have revealed that
Pd(PPh3)4 tetrakis(triphenylphosphine) palladium(0)
TO
Thiazole orange
5′-AGGGTTAGGGTTAGGGTTAGGG-3′
G-quadruplex
Duplex strand
Metal-to-ligand charge transfer
Intraligand
22AG
G-4
ds
MLCT
IL
ESI-MS Electrospray ionisation mass spectra
FAB-MS Fast atom bombardment mass spectra
UV–Vis UV-visible
CD
Circular dichroism
FID
PDB
DFT
Fluorescent intercalator displacement
Protein Data Bank
Density Functional Theory
Table 2
PDBQT Protein Data Bank, Partial Charge (Q), & Atom Type (T)
G−4DC50 and dsDC50 values (μM) determined using FID assay for complexes 1 and 2a.
Complex
TO displacement
Selectivity
G−4DC50 (μM)
dsDC50 (μM)
dsDC50 G−4DC50
/
Acknowledgments
1(K+
1(Na+
)
0.96 0.15
1.55 0.24
0.42 0.10
1.46 0.19
1.45 0.21
1.96 0.22
1.34 0.17
1.64 0.13
1.5
1.3
3.2
1.1
)
)
2(K+
)
This work was supported by the National Natural Science Foundation
of China (20901060, 31170776, 81171646) and the Fundamental Re-
search Funds for the Central Universities.
2(Na+
a
Values are average of three independent measurements.

26
S. Shi et al. / Journal of Inorganic Biochemistry 121 (2013) 19–27
Fig. 6. Schematic diagram of the interaction of complex 1 with the antiparallel G-quadruplex structure (a) and the mixed-hybrid G-quadruplex structure (b); schematic diagram of
the interaction of complex 2 with the antiparallel G-quadruplex structure (c) and the mixed-hybrid G-quadruplex structure (d). The G-quadruplexes and complexes are shown in
ribbon and CPK mode, respectively.
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