A new light-scattering sensor for screening G-quadruplex stabilizers based on DNA-folding-mediated assembly of gold nanoparticles

Article abstract of DOI:10.1039/c3tb20291g

In this contribution, we constructed a biosensor for screening G-quadruplex stabilizers based on the assembly of gold nanoparticles functionalized with guanine-rich (G-rich) ssDNA using a light-scattering technique. We synthesized a series of metal-terpyridine complexes and investigated their affinity for quadruplex-DNA using the biosensor. Using the ratio of intensity of the light-scattering peak (I - I₀)/I₀, it can intuitively present the sequence of ability of stabilizing G-quadruplex DNA as follows: complex 1 > complex 3 > MTX > complex 2 > complex 5 > complex 6 > complex 7 > complex 4 > methyl green > TO > complex 8 > cisplatin. As our method allows the quantitative analysis of stabilizer affinity, EC₅₀ values obtained are 4.8, 5.3, 6.1, 6.6, 6.7, 18.2, 20.2, 21.5, 32.2, 41.5 and 48.1 μM for complex 1, complex 3, MTX, complex 2, complex 5, complex 6, complex 7, complex 4, methyl green, TO and complex 8, respectively. The results have been verified by G-quadruplex fluorescent intercalator displacement (G4-FID) analysis. The proposed approach is a simple, convenient, intuitive and highly sensitive assay for screening G-quadruplex stabilizers. Our developed biosensor provides a promising tool for screening anticancer drugs. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3tb20291g

Journal of
Materials Chemistry B
View Article Online
View Journal | View Issue
PAPER
A new light-scattering sensor for screening
G-quadruplex stabilizers based on DNA-folding-
Cite this: J. Mater. Chem. B, 2013, 1,
3057
mediated assembly of gold nanoparticles†
a
a
a
a
b
*
Da-Qian Feng,‡ Guoliang Liu,‡ Wenjie Zheng, Tianfeng Chen and Dan Li
In this contribution, we constructed a biosensor for screening G-quadruplex stabilizers based on the
assembly of gold nanoparticles functionalized with guanine-rich (G-rich) ssDNA using a light-scattering
technique. We synthesized a series of metal–terpyridine complexes and investigated their affinity for
quadruplex-DNA using the biosensor. Using the ratio of intensity of the light-scattering peak (I ꢀ I0)/I0,
it can intuitively present the sequence of ability of stabilizing G-quadruplex DNA as follows: complex 1 >
complex 3 > MTX > complex 2 > complex 5 > complex 6 > complex 7 > complex 4 > methyl green >
TO > complex 8 > cisplatin. As our method allows the quantitative analysis of stabilizer affinity, EC50
values obtained are 4.8, 5.3, 6.1, 6.6, 6.7, 18.2, 20.2, 21.5, 32.2, 41.5 and 48.1 mM for complex 1, complex
3, MTX, complex 2, complex 5, complex 6, complex 7, complex 4, methyl green, TO and complex 8,
respectively. The results have been verified by G-quadruplex fluorescent intercalator displacement
(G4-FID) analysis. The proposed approach is a simple, convenient, intuitive and highly sensitive assay for
screening G-quadruplex stabilizers. Our developed biosensor provides a promising tool for screening
anticancer drugs.
Received 3rd March 2013
Accepted 23rd April 2013
DOI: 10.1039/c3tb20291g
www.rsc.org/MaterialsB
pretreatments such as thermal denaturation, complicated
design, demanding expensive machines or requiring many
chemicals including hemin, HPA, H2O2. Thus, it is highly desir-
able to develop a simple, sensitive, and economic method for
screening G-quadruplex stabilizing compounds.
Introduction
Telomerase has been identied as an attractive target for cancer
therapy.1 Telomeric DNA consists of single-stranded tandem
repeated G-rich motifs in the 30-terminal region and can fold into
a G-quadruplex structure in the presence of a stabilizer.2 A novel
anticancer strategy is to stabilize the G-quadruplex conformation
by using a G-quadruplex stabilizer thereby inhibiting activity of
telomerase. A number of organic molecules and some metal
complexes have been reported to efficiently stabilize G-quad-
ruplex structures, but there are few methods for screening
G-quadruplex stabilizers. So far, the interaction between the
G-quadruplex and the stabilizer has been studied by circular
dichroism (CD) spectroscopy,3 UV-vis spectroscopy,4 or uores-
cence spectroscopy.5 However, these methods have some
unavoidable drawbacks: the CD method requires relatively high
DNA and quadruplex stabilizer concentrations; the UV-vis
method suffers from absorbance interference or requires many
reagents such as ABTS, H2O2, hemin or the unstable reactive
product; the uorescence method is limited by cumbersome
In this study, a biosensor for screening G-quadruplex stabi-
lizers based on the assembly of Au-NP-decorated G-rich single-
stranded DNA (G-rich ssDNA) using light-scattering spectros-
copy was fabricated. Nanoparticles can be excellent probes to
monitor biological events and their mechanisms due to their
superior optical properties, especially the enhanced scattering
properties.6 In a typical assay, the G-rich ssDNA sequence,
which is bound on the surface of the Au NPs, folds into
G-quadruplex conformation in the presence of a stabilizer,
inducing the assembly of Au NPs with a concomitant red-to-blue
color change (Scheme 1).7 Moreover, the assembly of Au NPs
generates an enhanced light-scattering signal for the biosensor.
The binding ability of the given compounds to G-quadruplex
can be easily monitored according to the increment of light-
scattering intensity of the biosensor. Therefore, on the basis of
this strategy, a light-scattering method for screening G-quad-
ruplex stabilizers was developed.
aDepartment of Chemistry, College of Life Science and Technology, Jinan University,
Guangzhou 510632, China
bDepartment of Chemistry, Shantou University, Guangdong 515063, China. E-mail:
To date, metal complexes including Ni(II)-salphen, square-
planar platinum(II) phenanthroline complexes, Mn(II)cationic
porphyrins, and metal–terpyridine complexes have recently
been shown to interact effectively with G-quadruplex structure.8
As complexes which can inhibit telomerase activity by inducing/
stabilizing G-quadruplex formation, G-quadruplex DNA is
dli@stu.edu.cn; Fax: +86 754-82902767
† Electronic supplementary information (ESI) available: Experimental details and
supporting gures. See DOI: 10.1039/c3tb20291g
‡ D. Q. Feng and G. L. Liu contributed equally to this work.
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 3057–3063 | 3057

View Article Online
Journal of Materials Chemistry B
Paper
Experimental section
Chemicals
The oligonucleotides (HPLC purication) used in this paper
were purchased from TaKaRa Biotechnology (Dalian, P. R.
China) Co., Ltd and used without further purication. Tetra-
chloroauric acid (HAuCl4$3H2O), trisodium citrate, tris-(2-car-
boxyethyl)-phosphine (TCEP) was obtained from Alfa Aesar
(UK). All starting materials and solvents were commercially
available and used as received without further purication. The
solutions were prepared with Milli-Q water (18.2 MU cm) from a
Millipore system.
The Tris–HCl buffer solution was used to control the acidity
of the solution, which was made up of 10 mM Tris solution, and
proper volume of hydrochloric acid. The pH values of solutions
were adjusted by a pH meter.
Scheme 1 A schematic illustration of the light-scattering (LS) sensor with the
maximum peak located at 388 nm for screening G-quadruplex stabilizers based
on the irreversible assembly of Au NPs decorated with G-rich ssDNA at room
temperature.
Preparation of gold nanoparticles
Gold nanoparticles (16 nm diameter) were synthesized accord-
ing to our reported procedure.9a An aqueous solution of HAuCl4
(100 mL, 0.01%) was brought to a vigorous boil with stirring in a
conical ask, and then 3.5 mL of trisodium citrate (1.0%)
solution was added rapidly. The color of the solution changed to
deep red in seconds and the reduction of HAuCl4 was practically
completed aer boiling for 7 min. The solution was cooled
naturally to room temperature and then diluted to 100 mL. The
average diameter of the prepared gold nanoparticles was about
16 nm as characterized by transmission electron microscope
(TEM). Besides, the absorbance values at 520 nm in the UV-vis
spectrum suggested it was the single size gold colloid.
Scheme 2 Reaction scheme for the syntheses of 40-R-2,20:60,200-terpyridine
ligands L1–L4.
Preparation of Au NPs functionalized with G-rich ssDNA
Gold nanoparticles functionalized with G-rich unit ssDNA were
prepared according to the literature method with a little
modication.9 The Au NPs (4 mL) were rst modied with 50-
thiol-capped G-rich ssDNA (100 mL, 10 mM, HPLC grade) which
was activated by a TCEP (40 mM, freshly prepared) solution
before use. The molar ratio of nanoparticles, G-rich unit ssDNA,
and space ssDNA is 1 : 60 : 60. The T10 tether in ssDNA is used
in the design of the sensor to push a recognition sequence away
from the particle surface to enhance recognition and hybrid-
ization.10 The mixture solution was magnetically stirred at room
temperature for 24 hours to facilitate the hybridization. Then,
the solution was stored in a drawer at room temperature for at
Fig. 1 The turning-on light-scattering (LS) spectra of DNA folding-mediated
irreversible assembly of gold nanoparticles in the presence of metal–terpyridine
complex 1 in Tris–HCl buffer (10 mM Tris, pH 7.4). The inserted figure displays the
curve between the increments of light-scattering (DILS ¼ I ꢀ I0) with the maximum
peak located at 388 nm and various concentrations of complex 1 at room
temperature. Conditions: (1) the buffer solution (blank); (2) Au NPs functionalized least 24 hours. Aer that, the nanoparticle solutions were
G-rich unit ssDNA; (3)–(8): as for (2) + complex 1: 0.5, 1.0, 2.0, 4.0, 5.0 and 8.0 mM.
centrifuged and redispersed in the buffer solution. The particles
were washed three more times and then redispersed in the
detection buffer solution. Finally, the solution was kept in dark
becoming an important target for the development of anti- at room temperature for another 24 hours before used. The nal
cancer drugs.8b Numerous metal complexes have been reported concentrations of the probe solutions were estimated from their
as potent telomerase inhibitors which are potential antitumor measured absorption at 520 nm and published values for
drugs. Here, we synthesized a series of metal–terpyridine extinction coefficients of the unmodied particles.11 The UV-vis
complexes (Scheme 2 and Fig. S1, ESI†) and studied their ability spectra were obtained by monitoring the extinction at 520 nm
to stabilize G-quadruplex using our proposed method.
for the dispersed gold nanoparticles (16 nm).
3058 | J. Mater. Chem. B, 2013, 1, 3057–3063
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Journal of Materials Chemistry B
based on the data collected on the rst minute aer adding the
metal complexes or drugs.
Instrument
1H NMR spectra were recorded on a Bruker AVANCE400 spec-
trometer and referenced to TMS. Infrared spectra were collected
from KBr disk on a Nicolet Avatar 360 FTIR spectrometer in the
range of 4000–400 cmꢀ1. Elemental analyses of C, H, and N were
determined with a Perkin-Elmer 2400C elemental analyzer. ESI-
MS analyses were carried out using an ABI4000 Q TRAP Liquid
Chromatography-Mass Spectrometer. The light-scattering
spectra were measured with a model LS-55 spectrouorometer
(Perkin-Elmer, USA). Fluorescence spectra were measured with
an FLS-920 picosecond uorescence lifetime spectrometer
(Edinburgh Instruments, UK). The TEM images of the colloidal
gold nanoparticles were acquired on a JEM-1400 transmission
electron microscope (JEOL, Japan). CD spectra were measured
using a MOS-450 Circular Dichroism Spectrometer (Bio-Logic,
France) equipped with a Peltier temperature controller. UV-vis
spectra were recorded on an Agilent 8453 UV-vis spectropho-
tometer (Agilent Technologies Co. Ltd., USA). Each spectrum is
an average of three scans.
Fig. 2 UV-vis spectra of gold nanoparticles functionalized with human telomeric
G-rich ssDNA (TAGGG(TTAGGG)3) in the absence and presence of increasing
concentrations of metal complex 1 in Tris–HCl buffer (10 mM Tris, pH 7.4).
Conditions: (1) the buffer solution (blank); (2) the solution of Au NPs function-
alized with G-rich ssDNA; (3)–(7) as for (2) + complex 1: 5, 10, 15, 20 and 25 mM.
Results and discussion
Strategy for screening G-quadruplex stabilizers via a light-
scattering sensor
In a typical assay, Au NPs functionalized with G-rich unit ssDNA
or space ssDNA were used (see Table S1 in the ESI†). Compared
to space ssDNA, G-rich unit ssDNA had an additional three-
guanine at the termini. By the formation of Au–S covalent bond,
Au NPs are decorated with G-rich unit ssDNA with a random
distribution in this case. In the absence of a stabilizer, the light-
scattering signal is low as an “OFF” state. In this state, gold
nanoparticles are well-dispersed. On the contrary, aer intro-
ducing a stabilizer, it appeared the assembly of Au NPs in a
cross-linking manner, which is attributed to the formation of
G-quadruplex structure between Au NPs, thereby activating
light-scattering signal of the biosensor and representing an
“ON” state (Scheme 1).
Fig. 3 Typical TEM images of samples of gold nanoparticles in the absence (a)
and presence (b) of complex 1. The inserted photos display the visual color
change. Conditions: (a) the biosensor: Au NPs functionalized G-rich unit ssDNA;
(b) as for (a) + 1.0 mM metal complex 1.
Synthesis of ligands and metal complexes
The ligands L1–L4 and metal–terpyridine complexes 1–8 were
synthesized according to previously reported work.12 The
synthesis details are listed in the ESI (see Scheme 2 and
Fig. S1†). Stock solutions (4 mM) of metal complexes for titra-
tion studies were prepared in DMSO and diluteꢁd with Milli-Q
water. All the stock solutions were kept at ꢀ20 C in the dark
between experiments.
Characteristics of light-scattering spectra
As shown in Fig. 1, the light-scattering intensity of the
biosensor solution (G-rich unit ssDNA-S–Au NPs) is low in the
absence of complex 1 in the whole range of scanning wave-
lengths. Light-scattering, which occurs when a light beam with
the wavelength close to the absorption region of species, is
Light-scattering screening assay
A certain volume of G-rich ssDNA-S–Au NPs solution was added usually applied in the detection of bioassembly processes and
into a test tube. Then, a series of dilutions of metal complexes so on.13 It is reasonable because the diameter of Au NPs
or drugs were pipetted into the test tubes by using micro- (approximately 16 nm) is smaller than 1/20th of the incident
syringes before light-scattering measurement. The light-scat- wavelengths in the range of 250–700 nm, therefore the signal
tering spectrum was then obtained by simultaneously scanning would only come from Rayleigh scattering.14 However, in the
the excitation and emission monochromators (Dl ¼ 0.0 nm) presence of complex 1, the aggregations of Au-NP-based
from 250 to 700 nm with the excitation and emission slits 5 nm. biosensor were rapidly formed, which produces the enhanced
Based on the spectra, LS intensities were measured with the light-scattering signals.15 The light-scattering intensity of the
maximum peak located at 388 nm. Spectrum curves were made biosensor gradually increases with the addition of increasing
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 3057–3063 | 3059

View Article Online
Journal of Materials Chemistry B
Paper
suggest that complex 1 has a similar effect to K+ or Na+ ions
(1000 equal) for promoting the stabilization of G-quadruplex
(see Fig. S2 in the ESI†). Besides, we did the experimental
assays for the studied gold nanoparticles in the presence of
Cu(NO3)2. The results suggest that Cu2+ ions (even 100 equiv-
alents) do not interfere with the detection of metal complex
[Cu(L)(NO3)2] by the Au-NP-based biosensor. Notably, light-
scattering technique is a quite sensitive method for the
determination of assembly.9a,15a,16 Thus, the current method
would be a sensitive approach for the detection of G-quad-
ruplex stabilizer.
Characteristics of CD spectra
In order to obtain proof of the assembly of Au NPs decorated
with G-rich unit ssDNA, CD spectra were recorded. In the
absence of metal complexes or metal cations, CD spectrum of
the human telomeric DNA (TAGGG(TTAGGG)3) exhibited a
positive peak at 256 nm and a negative one at 235 nm, which
suggests that human telomeric DNA exists as a single-stranded
structure under these conditions. However, upon the addition
of complex 1, a dramatic change in the CD spectrum was
observed. Specically, the intensity of the positive CD band at
256 nm decreased gradually from positive to negative with
continually blue shi. Meanwhile, a remarkable increase of
the CD band at 295 nm and an emergence of a shoulder peak
at 265 nm were observed, which reveal that complex 1 can
promote G-rich single-stranded oligonucleotide to form and
stabilize an antiparallel–parallel hybrid G-quadruplex struc-
ture (see Fig. S3 in the ESI†).17 Considering the existence of
steric hindrance in the G-quadruplex formation, it is inclined
to form the intermolecular G-quadruplex structure instead of
the intramolecular one.18 In addition, the characteristic peak
Fig. 4 Bar chart of the light-scattering response of the G-quadruplex stabilizers,
where I0 and I are the intensities in the absence and presence of the G-quadruplex
stabilizers. (1) The control group: the biosensor, Au NPs functionalized with G-rich
unit ssDNA; (2) the control group: Au NPs functionalized with space DNA in the
presence of complex 1 (5 mM); (3) as for (1) + Cisplatin; (4) as for (1) + MTX; (5) as
for (1) + Berberine; (6) as for (1) + Acetaminophen; (7) as for (1) + Amoxicillin;
(8) as for (1) + TO; (9) as for (1) + Methyl green; (10) as for (1) + Hemin; (11) as for
(1) + L1 ligand; (12) as for (1) + complex 1; (13) as for (1) + complex 2; (14) as
for (1) + L2 ligand; (15) as for (1) + complex 3; (16) as for (1) + complex 4; (17)
as for (1) + L3 ligand; (18) as for (1) + complex 5; (19) as for (1) + complex 6; (20) as
for (1) + L4 ligand; (21) as for (1) + complex 7; (22) as for (1) + complex 8. The final
concentration of the stabilizers is 5 mM.
concentration of complex 1, which discloses a concentration-
dependent relationship. In other words, complex 1 activates
the light-scattering signal of the biosensor. In order to further
verify the reasoning, K+ or Na+ ions, G-quadruplex stabilizer,
was added in the solution of the biosensor. The results showed
that an enhanced light-scattering signal also appeared, which
Table 1 The comparisons of the proposed method with G-quadruplex fluorescent intercalator displacement (G4-FID) assay
Screening
results
LS assay
(EC50, mM)
Candidate drugs
G4-FID assay (DC50,a mM)
Cisplatin
MTX
Methyl green
TO
Berberine
Acetaminophen
Amoxicillin
Hemin
Positive
Positive
Positive
Positive
Negative
Negative
Negative
Negative
Positive
Positive
Positive
Negative
Positive
Positive
Negative
Positive
Positive
Negative
Positive
Positive
>70
6.1
32.2
41.5
—
—
—
—
14.35
4.8
6.6
>70
5.3
21.5
>100
6.7
18.2
>90
20.2
48.1
—
6.3
30.5
—
—
—
—
—
15.1
5
6.5
—
5.6
22.5
—
6.8
19.5
—
L1
Complex 1
Complex 2
L2
Complex 3
Complex 4
L3
Complex 5
Complex 6
L4
Complex 7
Complex 8
22.1
50.2
a
DC50 value is expressed as the concentration required to displace 50% of the TO from G-rich ssDNA.
3060 | J. Mater. Chem. B, 2013, 1, 3057–3063
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Journal of Materials Chemistry B
of G-quadruplex does not appear when replacing complex 1 complex 5 > complex 6 > complex 7 > complex 4 > complex 8 >
with ligand L1. Besides, mimicking the peroxidase-like DNA- L1 > cisplatin > L2 > L4 > L3. The higher the affinity ability for
zyme assay revealed that it appeared G-quadruplex structure G-quadruplex, the higher the light-scattering intensity was.
aer the addition of complex 1 (see Fig. S4 in the ESI†), which Thus, it was easy to obtain the sequence of the ability of
is consistent with the results of CD spectra.19 Therefore, it is stabilizing G-quadruplex structure as follows: complex 1 >
concluded that the assembly of the Au-NP-based biosensor is complex 3 > complex 2 > complex 5 > complex 6 > complex 7 >
attributed to the formation of an intermolecular G-quadruplex complex 4 > complex 8 > L1 > cisplatin > L2 > L4 > L3. The
structure between the NPs in the presence of the stabilizer experimental results displayed that complexes are much more
complex 1.
potent G-quadruplex binders than the corresponding free
terpyridine ligands. It is due to the electron-decient planar
aromatic system and high polarization through the coordina-
tion interaction between metal ions and ligand, which favors
p-stacking on the external G-quartet and an electrostatic
interaction with negatively charged DNA. Moreover, Cu-
complexes (complexes 1, 3, 5 and 7) were found to have higher
ability of stabilizing G-quadruplex than Zn-complexes
(complexes 2, 4, 6 and 8) (Fig. S7, ESI†). This is due to the
different geometries of the complexes: Cu-complexes have
geometries of pseudo-square pyramidal structure which
features one at face, promoting p-stacking on the external
G-quartet while Zn-complexes adopt trigonal bipyramidal
geometries, leading to the steric hindrance of both faces.20
Fig. S8† suggests that it induced signicantly enhanced light-
scattering intensity of the sensor in presence of drugs (MTX,
methyl green, TO, or cisplatin), which reveals that four drugs
are strong G-quadruplex binders. Because of having an
extended planar aromatic electron-decient chromophore,
MTX promotes the formation and stabilization of G-quad-
ruplex conformation, leading to the enhancement of light-
scattering signal of the biosensor. However, as amoxicillin and
acetaminophen only contain one aromatic ring, they could not
strongly bind to G-quadruplex.
The inuences of these compounds on the light-scattering
signal of the biosensor were demonstrated by comparing the
ratios of LS intensity (I ꢀ I0)/I0, where I0 and I are the intensities
in absence and presence of G-quadruplex stabilizers. Using the
ratio of light-scattering peak intensity (I ꢀ I0)/I0, the sequence of
affinity ability of G-quadruplex stabilizers can easily be obtained
as follows: complex 1 > complex 3 > MTX > complex 2 > complex
5 > complex 6 > complex 7 > complex 4 > methyl green > TO >
complex 8 > cisplatin (Fig. 4). The bar gure suggests that four
drugs (amoxicillin, acetaminophen, berberine, and hemin) gave
negative results, which were consistent with the previous
report.5b Moreover, our developed method allows the quantita-
tive analysis of stabilizer affinity. According to the curves of
optical density versus drug concentration, EC50 value, which
represent the drug concentration required for 50% enhance-
Characteristics of UV-vis spectra
In order to investigate the interaction between the G-rich unit
ssDNA-S–Au-NPs and complex 1, UV-vis spectra were subse-
quently carried out in 10 mM Tris–HCl buffer (pH 7.4). As
shown in Fig. 2, a persistent bathochromic shi appears for
the maximum absorbance peak of the sensor aer intro-
ducing the increasing concentrations of complex 1. It is due to
the fact that complex 1 stabilizes the formation of G-quad-
ruplex conformation, leading to the aggregation of the
DNA-linked Au NPs, and then inducing the red shi of the
absorbance peak and the reduction of the absorbance inten-
sity. As a comparison, the effect of complex 2, K+, and Na+ ions
on the formation of G-quadruplex was also investigated (see
Fig. S5, ESI†). The results are consistent with those of the
light-scattering spectra.
Moreover, the ability of complex 1 to bind G-quadruplex was
evaluated by the melting temperature (Tm) assay which is based
on the monitoring of the stability imparted by a ligand to a
G-quadruplex structure. In the presence of complex 1, the Au
NPs decorated with G-rich ssDNA form aggregates and exhibit
the characteristic melting transitions with a high melting
temperature, which suggests the transition of DNA conforma-
tion (see Fig. S6, ESI†).
Characteristics of TEM
The size and morphology of the Au-NP-based biosensor were
characterized with transmission electron microscopy (TEM).
TEM images (Fig. 3a) showed that gold nanoparticles are
generally spherical and well proportioned with an average
diameter around 16 nm. The dispersed Au NPs formed large
DNA-linked aggregation particles when the addition of the
metal complex 1 with a concomitant of red-to-blue colour
change (Fig. 3b), which is consistent with the results of light-
scattering spectra and UV-vis spectra.
Application-screening as G-quadruplex stabilizers
To test the utility of the proposed method, metal complexes ment of the light-scattering intensity, can be calculated. The
and drugs were used as G-quadruplex stabilizers. The light- obtained EC50 values are 4.8, 5.3, 6.1, 6.6, 6.7, 18.2, 20.2, 21.5,
scattering spectra of screening G-quadruplex stabilizers are 32.2, 41.5 and 48.1 mM for complex 1, complex 3, MTX, complex
displayed in Fig. S7 and S8.† The rst curve is the blank group 2, complex 5, complex 6, complex 7, complex 4, methyl green,
which is the buffer solution while the second curve is the TO and complex 8, respectively (Table 1). The results were
control group which is the solution of Au NPs functionalized veried by G-quadruplex uorescent intercalator displacement
with G-rich unit ssDNA without adding any drugs (or (G4-FID) in vitro analysis that can evaluate the interaction
complexes). As shown in Fig. S7,† according to the light-scat- between the metal complex and the G-quadruplex,21 which
tering intensity at the maximum peak, the sequence of demonstrates the availability of light-scattering screening
compounds is as follows: complex 1 > complex 3 > complex 2 > method (Table 1).
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 3057–3063 | 3061

View Article Online
Journal of Materials Chemistry B
Paper
Scholars of China (no. 20825102) and the National Natural
Science Foundation of China (no. 91222202, 21171114).
Mechanism
The principle of screening G-quadruplex stabilizers by using a
light-scattering biosensor is shown in Scheme 1. In the absence
of a stabilizer, Au-NP-based biosensors are well-dispersed,
giving the low light-scattering signal. Upon the addition of a
stabilizer, the “TTAGGG” at the terminal of ssDNA on the
surface of gold nanoparticles can fold into a G-quadruplex
between nanoparticles, which promote the DNA-mediated
cross-linking assembly of gold nanoparticles (Scheme 1).7a The
results from the CD spectra, peroxidase-like DNAzyme assay,
and the melting transitions assay display the formation of
G-quadruplex structure. The quadruplex structures contain
guanine quartets (G-quartets). As a G-quartet consists of four
guanines, it is only likely to produce the intermolecular
G-quadruplex for the “TTAGGG” fragment decorated on gold
nanoparticles. The aggregation turns on the light-scattering
signal of the biosensor. In our assays, strong G-quadruplex
stabilizers can promote G-quadruplex mediated aggregation of
Au NPs, which produces a signicantly enhanced light-scat-
tering signal. In contrast, non- or weak G-quadruplex ligands
cannot form the G-quadruplex structure, which generates a low
light-scattering signal. As different stabilizers have different
abilities to stabilize G-quadruplex conformation, they create the
distinct enhancement of light-scattering. The ability of the
stabilizing G-quadruplex can be intuitively monitored according
to the increment of the light-scattering intensity of the Au NPs-
based biosensor. Therefore, the proposed approach may be
used as a primary screening technique for G-quadruplex
stabilizers.
Notes and references
1 (a) J. W. Shay and W. E. Wright, Nat. Rev. Drug Discovery,
2006, 5, 577–584; (b) L. R. Kelland, Eur. J. Cancer, 2005, 41,
971–979; (c) J. L. Mergny, J. F. Riou, P. Mailliet,
M. P. Teulade-Fichou and E. Gilson, Nucleic Acids Res.,
2002, 30, 839–865.
2 (a) Y. Wang and D. J. Patel, Structure, 1993, 1, 263–282; (b)
G. N. Parkinson, M. P. H. Lee and S. Neidle, Nature, 2002,
417, 876–880; (c) A. Ambrus, D. Chen, J. Dai, T. Bialis,
R. A. Jones and D. Yang, Nucleic Acids Res., 2006, 34, 2723–
`
2735; (d) I. M. Dixon, F. Lopez, A. M. Tejera, J. P. Esteve,
M. A. Blasco, G. Pratviel and B. Meunier, J. Am. Chem. Soc.,
2007, 129, 1502–1503.
3 C. C. Chang, J. Y. Wu, C. W. Chien, W. S. Wu, H. Liu,
C. C. Kang, L. J. Yu and T. C. Chang, Anal. Chem., 2003, 75,
6177–6183.
4 (a) J. L. Mergny, A. T. Phan and L. Lacroix, FEBS Lett., 1998,
435, 74–78; (b) D. M. Kong, J. Wu, Y. E. Ma and H. X. Shen,
Analyst, 2008, 133, 1158–1160.
`
5 (a) A. De Cian, L. Guittat, M. Kaiser, B. Sacca, S. Amrane,
A. Bourdoncle, P. Alberti, M. P. Teulade-Fichou, L. Lacroix
and J. L. Mergny, Methods, 2007, 42, 183–195; (b) L. H. Fu,
B. X. Li and Y. F. Zhang, Anal. Biochem., 2012, 421, 198–202.
6 (a) G. L. Liu, D. Q. Feng, X. Y. Mu, W. J. Zheng, T. F. Chen,
L. Qi and D. Li, J. Mater. Chem. B, 2013, 1, 2128–2131; (b)
G. L. Liu, D. Q. Feng, T. F. Chen, D. Li and W. J. Zheng,
J. Mater. Chem., 2012, 22, 20885–20888; (c) L. Zhang, Y. Li,
D. W. Li, C. Jing, X. Y. Chen, M. Lv, Q. Huang, Y. T. Long
and I. Willner, Angew. Chem., Int. Ed., 2011, 50, 6789–6792.
7 (a) Z. Li and C. A. Mirkin, J. Am. Chem. Soc., 2005, 127, 11568–
11569; (b) I. Lubitz and A. Kotlyar, Bioconjugate Chem., 2011,
22, 2043–2047; (c) X. Hou, W. Guo, F. Xia, F. Q. Nie, H. Dong,
Y. Tian, L. P. Wen, L. Wang, L. X. Cao, Y. Yang, J. M. Xue,
Y. L. Song, Y. G. Wang, D. S. Liu and L. Jiang, J. Am. Chem.
Soc., 2009, 131, 7800–7805; (d) C. Z. Huang, Q. G. Liao,
L. H. Gan, F. L. Guo and Y. F. Li, Anal. Chim. Acta, 2007,
604, 165–169.
8 (a) S. N. Georgiades, N. H. Abd Karim, K. Suntharalingam
and R. Vilar, Angew. Chem., Int. Ed., 2010, 49, 4020–4034;
(b) D. L. Ma, C. M. Che and S. C. Yan, J. Am. Chem. Soc.,
2009, 131, 1835–1846; (c) T. C. Jenkins, Curr. Med. Chem.,
2000, 7, 99–115; (d) L. R. Kelland, Eur. J. Cancer, 2005, 41,
971–979; (e) B. Fu, J. Huang, L. Ren, X. Weng, Y. Zhou,
Y. Du, X. Wu, X. Zhou and G. Yang, Chem. Commun., 2007,
3264–3266; (f) Y. J. Lu, T. M. Ou, J. H. Tan, D. Peng,
N. Sun, X. D. Wang, W. Y. Shao, W. b. Wu, Z. S. Huang,
X. Z. Bu, D. L. Ma, K. Y. Wong and L. Q. Gu, J. Med. Chem.,
2008, 51, 6381–6392; (g) H. J. Yu, X. H. Wang, M. L. Fu,
J. S. Ren and X. G. Qu, Nucleic Acids Res., 2008, 36, 5695–5703.
9 (a) D. Q. Feng, G. L. Liu, W. J. Zheng, J. Liu, T. F. Chen and
D. Li, Chem. Commun., 2011, 47, 8557–8559; (b)
J. J. Sorhoff, R. Elghanian, R. C. Mucic, C. A. Mirkin and
R. L. Letsinger, J. Am. Chem. Soc., 1998, 120, 1959–1964.
Conclusions
In summary, a new method for screening G-quadruplex stabi-
lizers based on Au-NP-based biosensors using a light-scattering
technique was proposed. We synthesized a series of metal–ter-
pyridine complexes and investigated their affinity for G-quad-
ruplex DNA by our developed method. As comparisons, clinical
drugs were used as G-quadruplex binder candidates in screening
assays. The differentiation of G-quadruplex stabilizers including
drugs and metal–terpyridine complexes by using the light-scat-
tering sensor was intuitively observed, and was veried by G4-FID
analysis. Compared with the reported uorescence method and
UV-vis method, our developed method has the following advan-
tages: rst, it is a simple and fast process without requiring many
reagents or cumbersome pretreatment. Second, it is a convenient
and rapid screening method due to having intuitive screening
results. Third, it only requires low DNA and complex concentra-
tion. Last but not least, the reactive product is stable. Moreover,
as for the anticancer activity of G-quadruplex stabilizers, the
nanosensor provides a useful tool for the primary screening of
anticancer drugs.
Acknowledgements
This work was nancially supported by the National Basic
Research Program of China (973 Program, no. 2013CB834803),
the National Science Foundation for Distinguished Young
3062 | J. Mater. Chem. B, 2013, 1, 3057–3063
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Journal of Materials Chemistry B
10 R. C. Jin, G. S. Wu, Z. Li, C. A. Mirkin and G. C. Schatz, J. Am.
Chem. Soc., 2003, 125, 1643–1654.
11 J. Yguerabide and E. E. Yguerabide, Anal. Biochem., 1998,
262, 137–156.
12 J. H. Wang and G. S. Hanan, Synlett, 2005, 1251–1254.
13 L. Ling, C. Z. Huang, Y. F. Li, Y. F. Long and Q. G. Liao, Appl.
Spectrosc. Rev., 2007, 42, 177–201.
1999, 103, 8474–8481; (b) C. Z. Huang, Q. G. Liao,
L. H. Gan, F. L. Guo and Y. F. Li, Anal. Chim. Acta, 2007,
604, 165–169; (c) J. Ling, C. Z. Huang and Y. F. Li, Anal.
Chim. Acta, 2006, 567, 143–151; (d) J. Ling, C. Z. Huang,
Y. F. Li, L. Zhang, L. Q. Chen and S. J. Zhen, TrAC, Trends
Anal. Chem., 2009, 28, 447–453; (e) G. T. Song, C. Chen,
X. G. Qu, D. Miyoshi, J. S. Ren and N. Sugimoto, Adv.
Mater., 2008, 20, 706–710.
14 G. A. Miller, J. Phys. Chem., 1978, 82, 616–618.
15 (a) B. A. Du, Z. P. Li and C. H. Liu, Angew. Chem., Int. Ed., 17 (a) D. M. Gray, J. D. Wen, C. W. Gray, R. Repges, C. Repges,
2006, 45, 8022–8023; (b) C. Z. Huang, K. A. Li and
S. Y. Tong, Anal. Chem., 1996, 68, 2259–2263; (c)
C. Z. Huang and Y. F. Li, Anal. Chim. Acta, 2003, 500, 105–
G. Raabe and J. Fleischhauer, Chirality, 2008, 20, 431–440;
(b) A. N. Lane, J. B. Chaires, R. D. Gray and J. O. Trent,
Nucleic Acids Res., 2008, 36, 5482–5515.
117; (d) W. Lu, B. S. F. Band, Y. Yu, Q. G. Li, J. C. Shang, 18 (a) J. H. Oh and J. S. Lee, Chem. Commun., 2010, 46, 6382–
C. Wang, Y. Fang, R. Tian, L. P. Zhou, L. L. Sun, Y. Tang,
S. H. Jing, W. Huang and J. P. Zhang, Microchim. Acta,
6384; (b) K. Sato, K. Hosokawa and M. Maeda, J. Am. Chem.
Soc., 2003, 125, 8102–8103.
2007, 158, 29–58; (e) R. F. Pasternack, C. Bustamante, 19 (a) L. Stefan, F. Denat and D. Monchaud, J. Am. Chem. Soc.,
P. J. Collings, A. Giannetto and E. J. Gibbs, J. Am. Chem.
Soc., 1993, 115, 5393–5399; (f) R. F. Pasternack and
P. J. Collings, Science, 1995, 269, 935–939; (g) L. Shang,
H. J. Chen, L. Deng and S. J. Dong, Biosens. Bioelectron.,
2011, 133, 20405–20415; (b) D. M. Kong, W. Yang, J. Wu,
C. X. Li and H. X. Shen, Analyst, 2010, 135, 321–326; (c)
Y. Xiao, V. Pavlov, T. Niazov, A. Dishon, M. Kotler and
I. Willner, J. Am. Chem. Soc., 2004, 126, 7430–7431.
2008, 23, 1180–1184; (h) Q. C. Zou, Q. J. Yan, G. W. Song, 20 H. Bertrand, D. Monchaud, A. D. Cian, R. Guillot,
S. L. Zhang and L. M. Wu, Biosens. Bioelectron., 2007, 22,
1461–1465.
J. L. Mergny and M. P. Teulade-Fichou, Org. Biomol. Chem.,
2007, 5, 2555–2559.
16 (a) P. J. Collings, E. J. Gibbs, T. E. Starr, O. Vafek, C. Yee, 21 D. Monchaud, C. Allain and M. P. Teulade-Fichou, Bioorg.
L. A. Pomerance and R. F. Pasternack, J. Phys. Chem. B, Med. Chem. Lett., 2006, 16, 4842–4845.
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 3057–3063 | 3063