Báo cáo khoa học: Binding of berberine to human telomeric quadruplex – spectroscopic, calorimetric and molecular modeling studies pot

Binding of berberine to human telomeric
quadruplex – spectroscopic, calorimetric and
molecular modeling studies
Amit Arora
1
, Chandramouli Balasubramanian
1
, Niti Kumar
1
, Saurabh Agrawal
1
, Rajendra P. Ojha
2
and Souvik Maiti
1
1 Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Delhi, India
2 Biophysics Unit, Department of Physics, DDU Gorakhpur University, India
The telomere is a region of highly repetitive DNA at
the end of a linear chromosome that protects the
terminal ends of chromosomes from being recognized
as damaged DNA and allows faithful chromosome
replication during the cell cycle [1,2]. Mammalian
telomeres consist of several kilobase pairs of double-
stranded G-rich DNA and a 100–200 base single-
stranded overhang on the 3¢-end [3,4]. A host of
telomere-associated proteins, including TRF1, TRF2
and POT1, ensures that the telomeric single-stranded
overhang does not trigger DNA damage response
pathways or lead to abnormal chromosomal rearrange-
ments [5–7]. Exposure of the 3¢-end due to uncapping
results in cellular senescence and apoptosis [8,9]. Telo-

merase, a ribonucleoprotein reverse transcriptase
enzyme (composed of both RNA and proteins), uses
its internal RNA component (complementary to the
telomeric single-stranded overhang) as a template for
synthesis of telomeric DNA A(GGGTTA)
n
, directly at
the ends of chromosomes. Telomerase is present in
most fetal tissues, normal adult male germ cells,
inflammatory cells, proliferative cells of renewal
tissues, and most tumor cells. Importantly, telomerase
is active in a majority of human cancer cells but is
inactive in most normal somatic cells [10]. It has been
shown previously that formation of intramolecular
G-quadruplexes by the telomeric G-rich strand inhibits
the activity of telomerase [10]. Therefore, ligand-
induced stabilization of intramolecular telomeric
G-quadruplexes has become an attractive strategy for
Keywords
berberine; hydration; quadruplex;
quadruplex–ligand interaction;
thermodynamics
Correspondence
S. Maiti, Proteomics and Structural Biology
Unit, Institute of Genomics and Integrative
Biology, CSIR, Mall Road, Delhi 110 007,
India
Fax: +91 11 2766 7471
Tel: +91 11 2766 6156
E-mail:

(Received 11 April 2008, revised 16 May
2008, accepted 9 June 2008)
doi:10.1111/j.1742-4658.2008.06541.x
This study examines the characteristics of binding of berberine to the
human telomeric d[AG
3
(T
2
AG
3
)
3
] quadruplex. By employing UV-visible
spectroscopy, fluorescence spectroscopy and isothermal titration calorime-
try, we found that the binding affinity of berberine to the human telomeric
quadruplex is 10
6
. The complete thermodynamic profile for berberine bind-
ing to the quadruplex, at 25 °C, shows a small negative enthalpy (DH)of
)1.7 kcalÆmol
)1
, an entropy change with TDS of +6.5 kcalÆmol
)1
, and an
overall favorable free energy (DG)of)8.2 kcalÆmol
)1
.Through the temper-
ature dependence of DH, we obtained a heat capacity (DC
p
)of)94

(± 5) calÆmol
)1
ÆK
)1
. The osmotic stress method revealed that there is an
uptake of 13 water molecules in the complex relative to the free reactants.
Furthermore, the molecular modeling studies on different quadruplex–
berberine complexes show that berberine stacking at the external G-quartet
is mainly aided by the p–p interaction and the stabilization of the high
negative charge density of O6 of guanines by the positively charged N7 of
berberine. The theoretical heat capacity (DC
p
) values for quadruplex–
berberine models are )89 and )156 calÆmol
)1
ÆK
)1
.
Abbreviations
H
2
TMPyP
4
, 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine; ITC, isothermal titration calorimetry; MMPBSA, molecular mechanics
Poisson–Bolzmann surface area; SASA, solvent-accessible surface area.
FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS 3971
the development of anticancer ligands. A molecule that
has (a) a p-delocalized system, (b) a partial positive
charge in the center of the molecular scaffold and
(c) positively charged substituents to interact with the

grooves, loops and the negatively charged phosphate
backbone is most likely to interact with, and thus
stabilize, G-quadruplexes. A number of G-quadruplex-
interacting agents with the above-mentioned features,
typically porphyrins [11–16], anthraquinones [17],
perylenes [18] and carbocyanines [19], have been
developed and shown to promote and ⁄ or stabilize
quadruplex structures. In past few years, Neidle and
co-workers have reported a number of trisubstituted
acridine analogs with a variety of side-chain modifi-
cations and stereoisomer variations exhibiting strong
G-quadruplex binding, high selectivity for quadruplex
over duplex DNA, and associated telomerase
inhibitory activity in the nanomolar range [20–22].
The rational design of new therapeutic agents that
bind to quadruplexes in a structure-specific manner is
of considerable interest and urgency. Many small
molecules that bind to quadruplexes have proven to be
effective therapeutic agents, although the exact mode
of binding and nature of thermodynamic forces that
regulate DNA–ligand interactions are often poorly
understood. This limited knowledge hampers many
efforts to rationally modify existing ligands and ⁄ or
design new therapeutic agents that bind to target
quadruplex structures with predictable affinity and
specificity. Characterization of the forces that govern
quadruplex–ligand interactions traditionally relies on
detailed knowledge of the thermodynamic and struc-
tural properties of the ligand, the DNA, and the com-
plex. Berberine, an isoquinoline alkaloid from plants,

is a planar molecule with an extended p-delocalized
system having a partial positive charge on N7 [23]
(Fig. 1). It has been shown that berberine and its
analogs bind to telomeric G-quadruplex and inhibit
the telomerase activity [24,25]. Studies show that these
molecules have high selectivity for G-quadruplex over
duplex DNA, and the aromatic moieties of the mole-
cule play a dominant role in quadruplex binding,
implying that this molecule could be an attractive scaf-
fold to develop new ligands targeting G-quadruplex
selectively. To obtain comprehensive knowledge on the
interaction of this scaffold, we performed spectro-
scopic, calorimetric and molecular modeling studies to
obtain thermodynamic and structural details of the
quadruplex–berberine interaction.
Results
Equilibrium binding studies by UV-visible
spectroscopy
To gain insight into the interaction between berberine
and the G-quadruplex formed by the telomere, UV
spectra of berberine in the absence and presence of
quadruplex were obtained. The resulting absorption
spectra are illustrated in Fig. 2. The UV spectra of
berberine show two distinct peaks at 341 and 421 nm.
Addition of increasing amounts of quadruplex results
in hypochromicity (34–40%) and a moderate batho-
chromic shift of 7 nm for the high-energy peak from
341 to 348 nm in the UV-visible spectra of berberine,
and hyperchromicity and a red shift of 19 nm for the
lower-energy peak from 421 to 440 nm, indicating

Fig. 1. Chemical structure of berberine.
Fig. 2. Absorbance spectra of 1 lM berberine in 50 mM MES buffer
(pH 7.4) and 100 m
M KCl in the absence and presence of succes-
sive additions of quadruplex at 25 °C. The inset is the Scatchard
plot of r ⁄ C versus r, where r is the ratio of bound berberine to the
total base pair concentration, and C is the concentration of free
ligand. Data were fitted to the McGhee–von Hippel neighbor exclu-
sion model.
Telomeric quadruplex–berberine interaction A. Arora et al.
3972 FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS
interaction of berberine with quadruplex. The occur-
rence of sharp isobestic points at 359, 383 and 445 nm
clearly indicates the existence of equilibrium in the
binding. Ligand binding with DNA through intercala-
tion usually results in hypochromicity and bathochro-
mism due to strong stacking interactions between an
aromatic chromophore and the base pairs of DNA.
These spectral characteristics suggest a mode of bind-
ing that involves a stacking interaction between berber-
ine and the quartet of quadruplex. The absorbance
change at 341 nm of the berberine absorption spectra
upon successive addition of quadruplex was used to
construct a Scatchard plot. Analysis of this Scatchard
plot yielded a binding affinity of (1.2 ± 0.2) · 10
6
m
)1
and a binding site density of 0.9 at 25 °C.
Equilibrium binding studies by the fluorescence

method
Fluorescence emission spectra for berberine in the
absence and presence of different amounts of quadru-
plex were recorded in order to study the binding event.
Figure 3 shows the effect of successive addition of
quadruplex on the fluorescence emission spectra of
berberine. It is seen that increasing the concentration
of quadruplex results in a gradual increase in the fluo-
rescence intensity of berberine. The ratio of the fluo-
rescence intensity of berberine in the presence and
absence of quadruplex is about 50. The k
max
in the flu-
orescence emission spectra shifts to the blue end by
5 nm. The spectral changes arise from the change in
the environment of berberine, which reveals that
berberine is binding with quadruplex. The change in
fluorescence intensity at 522 nm due to addition of
quadruplex solution was used to construct the binding
isotherm (inset of Fig. 3). Analysis of this isotherm
following 1 : 1 binding stoichiometry using Eqn (7)
(see Experimental procedures) gives a binding affinity
of (1.2 ± 0.1) · 10
6
m
)1
at 25 °C. Thermodynamic
parameters calculated for the quadruplex–berberine
binding are presented in Table 1.
Equilibrium binding studies by the isothermal

titration calorimetry (ITC) method
With recent advances in the sensitivity and reliability
of the calorimeter, ITC has become an important tool
for the direct measurement of thermodynamic para-
meters in various biological interactions [26,27]. ITC
yields thermodynamic parameters such as Gibbs free
energy change (DG), enthalpy change (DH), and
entropy change (DS), along with the number of bind-
ing sites (n) in a single experiment. Also, determination
of binding enthalpy as a function of temperature yields
changes in heat capacity (DC
p
) associated with an
interaction that provides valuable insights into the type
and magnitude of forces involved. Therefore, we have
utilized ITC to characterize the thermodynamics of
binding of berberine to quadruplex. Calorimetric titra-
tions were performed at different temperatures to
directly measure the binding enthalpy. Figure 4A
shows a typical titration curve obtained at 25 °C. The
area under the heat burst curves was determined by
integration to yield the heat of injection associated
with the reaction. These injection heats were corrected
by subtraction of the corresponding dilution heats
derived from the injection of identical amounts of ber-
berine into the buffer alone. The corrected isotherms
obtained at five different temperatures are shown in
Fig. 4B. All related thermodynamic parameters are
presented in Table 2. The binding affinity measured
from ITC is (0.4 ± 0.1) · 10

6
m
)1
at 25 °C. At all
temperatures studied, the binding enthalpies were
found to be negative, with their magnitude increasing
Fig. 3. Fluorescence emission spectra of 0.5 lM berberine in
50 m
M MES buffer (pH 7.4) and 100 mM KCl in the absence and
presence of successive additions of quadruplex at 25 °C. The inset
is the plot of DF versus quadruplex concentration. Data were fitted
to Eqn (7) to extract the binding affinity (Experimental procedures).
Table 1. Thermodynamic parameters obtained for quadruplex–ber-
berine binding at 25 °C. K
b
is the binding constant determined from
spectroscopic titrations in 50 m
M MES buffer (pH 7.4), DG is the
net binding free energy calculated using DG = )RT ln K
b
, and DH is
the binding enthalpy determined directly by ITC and used to calcu-
late the entropy change, using DG = DH)TDS.
K
b
(·10
6
M
)1
) DG (kcalÆmol

)1
) DH (kcalÆmol
)1
) TDS (kcalÆmol
)1
)
1.2 ± 0.1 )8.2 ± 0.8 )1.7 ± 0.2 6.5 ± 0.7
A. Arora et al. Telomeric quadruplex–berberine interaction
FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS 3973
with an increase in temperature. In all cases, the stoi-
chiometry was found to be one mole of ligand binding
per mole of quadruplex.
Apparent discrepancy between spectroscopic and
calorimetric binding constants
Examination of Tables 1 and 2 shows that the K
b
val-
ues determined from the spectroscopic and ITC data
differ by one order at 25 °C, despite the use of identi-
cal salt and buffer conditions. The binding constant
can be determined accurately when titrant is added to
a fixed and constant concentration [Q
0
] of DNA, such
that [Q
0
] is in the range of 1 ⁄ K
A
. In UV and fluores-
cence binding experiment, [Q

0
]=1lm and 0.5 lm
respectively, which is in the range of 1 ⁄ K
A
(1.2 lm).
However, the ITC experiment was performed at
[Q
0
]=10lm, which is much larger than 1 ⁄ K
A
(1.2 lm). ITC could not be used in this case to obtain
an accurate value for K
b
, as the low site concentration
required would give a heat signal below the sensitivity
of the instrument. For a quadruplex concentration of
1 lm in the cell, the heat output was not significantly
greater than the heats of ligand dilution. If the dilution
heats were small and the binding enthalpy was large,
then it would be possible to obtain a binding isotherm
using a quadruplex concentration of 0.5–1 lm. How-
ever, these conditions are not met. Despite these prob-
lems, ITC can still be used to accurately and directly
measure the binding enthalpy and stoichiometry for
this interaction. ITC remains an invaluable technique
for determining binding enthalpies, even in cases where
the binding constant cannot be determined accurately
[28].
Heat capacity measurements
Supplementary Fig. S1 shows the temperature depen-

dence of all thermodynamic parameters. No curvature
in the plots of thermodynamic constants versus tem-
perature is apparent over this temperature range, and
all of the plots are fitted with linear functions. The
heat capacity change (DC
p
) for a binding interaction
Fig. 4. (A) Sample thermogram for the calorimetric titration of
100 l
M berberine into 10 lM quadruplex at 25 °C. (B) Integrated
heats (after subtraction of heat of dilution for berberine) versus ber-
berine to quadruplex molar ratio plot at 10 °C(h), 15 °C(O), 20 °C
(D), 25 °C(,) and 30 °C(e). The first data point was eliminated in
the data fit.
Table 2. Thermodynamic parameters obtained from ITC experiments for quadruplex–berberine binding in 50 mM MES buffer (pH 7.4) buffer
containing 100 m
M KCl. Thermodynamic parameters were obtained for berberine binding to the preformed telomeric quadruplex at 25 °C.
The quadruplex concentration in the cell was 10 l
M and the berberine concentration in the syringe was 100 lM. DG was determined using
the relationship DG = )RT ln K
b
, where R is the universal gas constant, T is temperature, and K
b
is the binding affinity for the quadruplex–
berberine interaction. DH and DS correspond to the enthalpy and entropy change for the binding, respectively, and DG corresponds to the
free energy change of binding. DC
p
is the heat capacity change associated with the quadruplex–berberine interaction and is calculated using
Eqn (1) as described in the text. Values determined from microcalorimetric data.
Temperature (°C) DH (kcalÆmol

)1
) K
b
· 10
)6
(M
)1
) n DG (kcalÆmol
)1
) TDS (kcalÆmol
)1
) DC
p
(calÆmol
)1
ÆK
)1
)
10 )0.2 (±0.2) 4.5 (±0.2) 0.85 )8.5 ± 0.9 8.3 ± 0.8 )94.0 (±5.0)
15 )0.9 (±0.2) 2.0 (±0.1) 0.90 )8.3 ± 0.8 7.4 ± 0.7
20 )1.3 (±0.3) 0.8 (±0.05) 0.95 )7.9 ± 0.8 6.6 ± 0.7
25 )1.7 (±0.3) 0.4 (±0.07) 1.00 )7.7 ± 0.8 6.0 ± 0.6
30 )2.1 (±0.3) 0.2 (±0.05) 0.90 )7.3 ± 0.7 5.2 ± 0.5
Telomeric quadruplex–berberine interaction A. Arora et al.
3974 FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS
can be determined from the temperature dependence
of the observed binding enthalpy using the standard
relationship:
DC
p

¼ dDH
cal
=dT ð1Þ
The slope of the resulting line of DH versus tempera-
ture (T) in supplementary Fig. S1 yields DC
p
of
)94 ± 5 calÆmol
)1
ÆK
)1
for the binding of berberine to
quadruplex. Thus, berberine binding to quadruplex is
associated with a negative heat capacity change that
falls within a range that is frequently observed for
both nucleic acid–ligand and protein–ligand inter-
actions [29,30].
Hydration change due to the binding obtained by
the osmotic stress method
The osmotic stress method has been used extensively
to evaluate the participation of water molecules in a
wide variety of biochemical reactions [30]. Any equilib-
rium that involves changes in the water molecules
associated with a biopolymer is sensitive to changes in
the water activity (a
W
) [31–33]. Water activity can in
turn be manipulated by the addition of low molecular
weight cosolutes, which themselves do not interact
with the biopolymer but are assumed to change the

water activity. Equilibria that are coupled with hydra-
tion changes are influenced by the osmolyte concentra-
tion, as described by Qu & Chaires [33]:
dlnðK
s
=K
0
Þ=d½Osm¼ÀDn
w
=55:6 ð2Þ
where ln(K
s
⁄ K
0
) is the change in the binding free
energy, [Osm] is the osmolality (moles of solute per kg
of solvent) of the solution, and Dn
w
is the difference in
the number of bound water molecules between the
complex and the free reactants. The change in the
binding affinity upon change in osmolyte concentration
is shown in supplementary Fig. S2. As the concentra-
tion of osmolyte increases, the affinity of berberine for
the quadruplex binding site decreases. This observation
is consistent with the acquisition of water by the com-
plex relative to the DNA. Using Eqn (2), the average
number of exchanged water molecules is found to be
13 ± 2.
Molecular modeling studies

Computational methods are widely used to investi-
gate biomolecules and complexes, and have been
shown to be valuable for a deeper understanding of
the structural, dynamic and energetic properties. The
mixed hybrid NMR structure of the human telomeric
quadruplex was used for study (supplementary
Fig. S3). The structure has two external G-quartets
that can act individually as binding sites for berber-
ine [25]. Berberine was docked against these external
G-quartets, and the complexes were simulated in
aqueous solution. The rmsd values of the heavy
atoms of the whole complex (black) and without
loop residues (gray) are shown in supplementary
Fig. S4. The rmsd values of both the MH1 (5¢-end)
and MH2 (3¢-end) complexes remain < 3 A
˚
. The
rmsd values for the G-quartet (without loop
residues) and berberine are conserved in both cases,
and stay at < 1 A
˚
during the last 2 ns (inset in
supplementary Fig. S4). The fluctuations of the loop
residues are obvious, as they are not held tightly by
hydrogen bonds and hence are free to move
during dynamics. This observation has also been
reported in previous studies on G-quadruplex
structures [34,35].
The stacking of berberine over the external G-quartet
is shown in Fig. 5 for the models. Berberine stacking

over the G-quartet plane is aided by the formation of
strong p–p aromatic stacking interactions between the
berberine scaffold and the G-quartet plane. In addition,
the positively charged nitrogen atom in berberine
positions itself on the axis passing through the center of
the G-quartet plane. Hence, a strong electrostatic inter-
action can be expected between the positively charged
nitrogen and the highly electron-rich central area of the
G-quartet plane, due to the guanine carbonyl lone pairs.
The positioning of the nitrogen atom was observed to
be fairly retained in all models during dynamics. This
suggests that the electrostatic interaction between the
negatively charged clouds formed by O6 of guanines
and the positively charged nitrogen atom plays an
important role in this stabilization.
The relative free energy components for the complex
formation were estimated by the molecular mechanics
Poisson–Bolzmann surface area (MMPBSA) approach.
The calculations were performed on the basis of
the single trajectories of the quadruplex–berberine
complexes obtained from the explicit solvent simula-
tions. The estimates are summarized in supplementary
Table S1.
Theoretical heat capacity calculation
The heat capacity change upon complex formation is
an informative measure that can provide insights into
the exchange of water during the process. The relations
connecting the changes in heat capacity to the burial
of polar and nonpolar solvent-accessible surface area
(SASA) during complex formation has been proposed

A. Arora et al. Telomeric quadruplex–berberine interaction
FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS 3975
and applied in a number of previous reports [29,36]. In
our study, we used the relation proposed by Ren et al.
[37], which is given as follows:
DCp ¼ 0:382ðÆ 0:026ÞDA
np
À 0:121ðÆ 0:077ÞDA
p
ð3Þ
Here, DA
np
and DA
p
represent the changes in SASA
for nonpolar and polar groups, respectively. A sum-
mary of the solvent-accessible areas and DC
p
values is
shown in Table 3. The SASA is reduced upon complex
formation, and the majority of the reduction was due
to the burial of nonpolar surface. This is reflected
in the negative values of the calculated heat
capacity change. The calculated values are )89 and
)156 calÆmol
)1
ÆK
)1
for MH1 and MH2 respectively.
Discussion

In order to understand biomolecule–ligand binding in
terms of sequence-specific recognition and affinity, it is
necessary to complement high-resolution structural
data with accurate thermodynamic measurements. By
using a combination of spectroscopic and ITC tech-
niques, we have elucidated a complete thermodynamic
profile (DG, DH, DS, K
b
, DC
p
and Dn
w
) for the binding
of berberine to the telomeric quadruplex. UV-visible
absorption titration experiments show that the binding
of berberine to G-quadruplexes results in a red shift
(10–12 nm) and substantial hypochromicity (34–40%)
in the k
341 nm
of berberine. The red shift in the absorp-
tion maxima and the observed hypochromicity of ber-
berine in the presence of quadruplex may be
interpreted in terms of stacking interactions between
the quartet-forming guanine bases and the aromatic
groups of berberine. Although the observed red shift is
intermediate between what is observed for intercalation
(> 15 nm) and for outside binding (< 8 nm) [38], the
same extent of hypochromicity that is generally seen in
the intercalated binding are observed, revealing that
berberine interacts with quadruplex through stacking

interactions between quartet and berberine, as happens
in case of intercalative binding events. Recently, Wei
Table 3. Summary of the changes in SASA in A
˚
2
and heat capacity changes (DC
p
) in calÆmol
)1
ÆK
)1
calculated for the complex models. DA
tot
is the change in total accessible surface area, DA
np
is the change in nonpolar SASA and DA
p
is the change in polar SASA. DC
p
is calculated
using Eqn (3) as described in the text.
Molecule DA
tot
(A
˚
2
) DA
np
(A
˚

2
) DA
p
(A
˚
2
) Calculated DC
p
(calÆmol
)1
ÆK
)1
)
MH1 4306 1533 2773 )89
Quadruplex 4248 1370 2879
Berberine 550 459 91
DA
tot
= )493 DA
np
= )296 DA
p
= )197
MH2 4079 1419 2659 )156
Quadruplex 4172 1424 2748
Berberine 551 461 90
DA
tot
= )645 DA
np

= )466 DA
p
= )179
A
B
G 20
G 2
G 8
G 16
G 4
G 10
G 14
G
Fig. 5. Stacking of berberine on the G-quartet face A-MH1, B-MH2.
The G-quartet is shown as a stick model. Berberine is shown as a
ball and stick model.
Telomeric quadruplex–berberine interaction A. Arora et al.
3976 FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS
et al. [15] have studied the interaction of cationic
porphyrin [5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,
23H-porphine] (H
2
TMPyP
4
) with three distinct
G-quadruplex DNAs, parallel-stranded (TG
4
T)
4
,

dimer-hairpin-folded (G
4
T
4
G
4
)
2
, and monomer-folded
AG
3
(T
2
AG
3
)
3
, by UV resonance Raman spectroscopy,
UV-visible absorption spectroscopy, fluorescence spec-
troscopy, and surface-enhanced Raman spectroscopy.
In their UV-visible absorption titration experiments,
the same extent of red shift (11–13 nm) but larger
hypochromicities (56–62%) were observed, indicating
intercalative (containing the end stacking) binding of
H
2
TMPyP
4
to these G-quadruplexes. Comparing these
observations with ours, it can be concluded that

berberine binds to quadruplex at external quartets
through stacking interactions between the quartet-
forming guanine bases and the aromatic groups of
berberine. The lower red shift and moderate hypo-
chromicity can be accounted for by the partial inter-
calation by end stacking with the quartet. The 1 : 1
binding stoichiometry, as estimated from the present
results, limits the interaction to a single binding mode.
It was seen that addition of increasing concentrations
of quadruplex results in a gradual increase in the
fluorescence intensity of berberine, indicating the trans-
fer of berberine from an aqueous environment to a
hydrophobic environment. This observation rules out
the possibility of outside stacking of berberine, where
quenching of the chromophore fluorescence by solvent
molecules could have been continued. In a recent
study, Franceschin et al. [25], through molecular
modeling, have shown that piperidino-berberine stacks
on the terminal G-tetrad of the quadruplex.
The sigmoidal binding isotherms obtained from ITC
experiments are indicative of the existence of either a
single binding site per quadruplex or a number of
equivalent, but not necessarily independent, binding
sites. At all temperatures, the stoichiometry for the
quadruplex–berberine binding was found to be 1 : 1,
confirming the results of our spectroscopic binding
studies. The binding enthalpies were found to be neg-
ative with increasing magnitudes upon increase in
temperature. Berberine binds to quadruplex at 25 °C
with a small, negative enthalpy (DH)of)1.7

kcalÆmol
)1
and an entropy change with TDS of +6.5
kcalÆmol
)1
with an overall favorable free energy (D G )
of )8.2 kcalÆmol
)1
. The favorable binding of berberine
comes from a combination of enthalpy and entropy
terms that vary with temperature. The negative values
of DH and positive values of DS are consistent with
the characteristics of a combination of van der Waals,
hydrophobic and electrostatic interactions in the
binding process. The temperature dependence of the
binding affinity was used to calculate the van’t Hoff
enthalpy, which did not match the calorimetric
enthalpy. The obtained van’t Hoff enthalpy was
)12 kcalÆmol
)1
, giving DH
vH
⁄ DH
cal
ratios in excess of
1. A large difference between the van’t Hoff and calo-
rimetric enthalpies could be due to substantial temper-
ature-dependent behavior of associated reactions. This
might originate from changes in hydrophobic hydra-
tion [39], in which release of water molecules from

hydrophobic surfaces upon binding results in loss of
enthalpy (due to stronger hydrogen bonds of struc-
tured water) and gain in entropy, a phenomenon
known to be temperature-dependent [40–42]. Measure-
ment of heat capacity changes (DC
p
) associated with
ligand–macromolecule binding can help to differentiate
the nature of hydration changes, i.e. hydrophobic ver-
sus polar hydration [43]. Unlike other thermodynamic
parameters, which have contributions from various
sources, DC
p
is believed to arise purely from molecular
hydration associated with binding [44]. This parameter
can thus be utilized to estimate the extent of burial or
exposure of polar and nonpolar groups to bulk water
upon molecular binding. The obtained DC
p
for
the quadruplex–berberine interaction was )94 ± 5
calÆmol
)1
ÆK
)1
. In the case of intercalative as well as
minor groove binding ligands, it was shown that DC
p
varies from )100 to )400 calÆmol
)1

ÆK
)1
[29,44]. How-
ever, the large negative heat capacity change is highly
correlated with hydration heat capacity changes that
arise from burial of the hydrophobic area. As we have
observed in our UV binding experiment, berberine
binds to quadruplex by stacking on the terminal G-tet-
rad of the quadruplex, so burial of the hydrophobic
group is not extensive enough to show a sufficiently
negative heat capacity change upon interaction. The
free energy (DG
hyd
) for the hydrophobic transfer of a
ligand from aqueous solution to its macromolecular
binding pocket is a function of the DC
p
for the binding
reaction, and is given by the following relationship
[44]:
DG
hyd
¼ 80 Â DC
p
ð4Þ
According to this relationship, negative heat capacity
changes result in a large negative (favorable) DG
hyd
value, which, in turn, shows a significant driving force
for complex formation. As an illustrative example, the

Chaires [37] and Wilson [29,45] groups applied Eqn (3)
to show that the major driving force for the binding of
various heterocyclic ligands (e.g. Hoechst 33258 and
penta-amidine) to the minor groove of duplex DNA
stems from the hydrophobic transfer of the ligands
from solution to the DNA-binding site.
A. Arora et al. Telomeric quadruplex–berberine interaction
FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS 3977
Careful inspection of Table 1 clearly shows that the
binding of berberine to the human telomeric quadru-
plex having a small negative value of DH and a large
positive value of TDS is predominantly entropically
driven. Recently, Chaires has analyzed the binding
data for 26 DNA–ligand interactions and discussed
distinctive thermodynamic signatures for groove bind-
ing and intercalation [46]. Groove-binding interactions
are largely entropically driven, whereas intercalation
reactions are driven by large favorable enthalpy con-
tributions and are opposed by entropy. In the present
study, the observed low negative enthalpy and high
positive entropy change could constitute further evi-
dence in support of stacking with the terminal quartet
rather than intercalation. Rigidifying the DNA ought
to exert an entropic cost, and this is the most likely
explanation for the unfavorable entropy associated
with intercalation [47]. In contrast, terminal stacking
in the quadruplex structures should not rigidify the
quadruplex structure, thus reducing the entropic cost
as compared to intercalation. The origin of the favor-
able entropic term in the present study is not also

apparent. It has been argued that groove binding
shows the favorable entropy because of the release of
water molecules upon complex formation, by dis-
placement of the ‘spine of hydration’ within the
minor groove [48]. However, the osmotic stress study
shows that the quadruplex–berberine complex
acquires 13 molecules of water on average per mole
of complex at 25 °C. Similar kinds of observation
have been reported recently by Kiser et al. [49], where
uptake of water molecules as well as a positive
entropy change were observed when Hoechst, a
groove binder, bound to oligomeric DNA. This dis-
crepancy may be due to the inefficiency of the osmo-
tic stress method in measuring the overall hydration
change, as mentioned by Chaires [46] as well as by
Kiser et al. [49].
The simulated structures show that the planar ber-
berine molecule is stacked onto the G-quartet, posi-
tioning the N7 positive charge above the center of the
G-tetrad in the region of high negative charge density
generated by the carbonyl groups to stabilize the
complex via favorable p-stacked interactions between
aromatic residues without significant disruption of
the guanine tetrads. The binding enthalpy (DH) for the
quadruplex–berberine complexes originates from the
combination of polar solvation energy and favorable
solute electrostatic, van der Waals and nonpolar sol-
vation energy. The conformational entropy for the
complexes arises from the loss of translational and
rotational degrees of freedom. The overall free energy

change (DG) for the quadruplex–berberine binding is
found to be more favorable for the MH2 berberine–
quadruplex complex (supplementary Table S1). Fur-
thermore, the calculated DC
p
is highly negative and
arises due to the loss of nonpolar SASA, whereas the
accompanying uptake of water is associated with gain
of polar SASA. This is because berberine is nonpolar
in nature, and hence the more nonpolar accessible sur-
face gets buried upon interacting with the quadruplex.
On the other hand, the positive change in the polar
surface area (DA
p
) confirms that there is exposure of
the polar surface on complex formation, further imply-
ing the uptake of water molecules within the hydration
layer.
Conclusion
The single-stranded G-rich telomeric 3¢-overhang at
the ends of chromosomes can form unique secondary
DNA structures, such as G-quadruplexes, which are
known to inhibit telomerase activity and have thus
become attractive targets for new anticancer ligands.
However, a structure-based approach needs to be
developed to design a new generation of binding
agents that can selectively target such unique second-
ary DNA structures. Ultimately, a comprehensive
understanding of the thermodynamic and structural
parameters of quadruplex–ligand complexes would aid

in the design of new quadruplex-selective molecules
and help to rationalize their in vivo performance. In
this study, we have obtained comprehensive data
on the thermodynamic and structural parameters
involved in the quadruplex–ligand interaction, using a
well-characterized human telomeric quadruplex and
an alkaloid, berberine. It has been observed that bind-
ing of berberine to the human telomeric quadruplex
is associated with a small, negative enthalpy (DH)
of )1.7 kcalÆmol
)1
, a favorable free energy (DG)
of )8.2 kcalÆmol
)1
and a favorable entropy with TDS
value of +6.5 kcalÆmol
)1
ÆK
)1
at 25 °C. A negative
heat capacity change was observed when it was calcu-
lated using two independent methods experimentally,
from the temperature dependence of DH values, and
theoretically based on surface area calculations. The
theoretical value is more negative than the experimen-
tal value. There was an uptake of 13 water molecules
on average per complex, which provides an unfavor-
able contribution to the free energy of the binding.
Structural studies of the complex obtained from mole-
cular dynamic studies reveal that berberine stacks over

the G-tetrad, allowing overlap of the p-system of
berberine primarily with two bases of each G-tetrad.
The partial positive charge on the berberine N7
appears to act as a ‘pseudo’ potassium ion, and is
Telomeric quadruplex–berberine interaction A. Arora et al.
3978 FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS
positioned above the center of the G-tetrad in the
region of high negative charge density generated by
the carbonyl groups. Extension of this study to other
known and well-established ligands, such as porphyrin
and telomestatin, will be reported in due course.
Experimental procedures
Berberine chloride was obtained from Sigma and was used
without any further purification. The 22-mer oligonucleo-
tide from the telomere end, d(AGGGTTAGGGTTAGG
GTTAGGG), was obtained from Sigma Genosys USA.
Concentrations of oligonucleotide solutions were deter-
mined from the absorbance at 260 nm, using the molar
extinction coefficient for the G-rich strand, calculated by
extrapolation of tabulated values of the dimers and mono-
mer bases [50] at 25 °C, using procedures reported earlier
[51]. All other reagents were of analytical grade. Milli Q
water was used throughout all the experiments. All
experiments were performed in 50 mm MES buffer (pH 7.4)
containing 100 mm KCl at 25 °C, unless otherwise
specified.
UV-visible and fluorescence spectroscopy
Quadruplex–berberine binding constants were determined
by UV fluorescence, and Cary 400 (Varian) and Fluoro-
max 4 (Spex) instruments were used for UV and fluores-

cence titration experiments respectively. A fixed berberine
concentration was titrated by increasing the quadruplex
concentration in 50 mm MES buffer (pH 7.4) containing
100 mm KCl. Data were transformed into a Scatchard plot
of r ⁄ C versus r, where r is the ratio of bound berberine to
the total quadruplex concentration, and C
f
is the concen-
tration of free ligand. In the Scatchard equation,
r ⁄ C
f
= K
b
(n)r), where r is the number of moles of
berberine bound to 1 mol of quadruplex (C
b
⁄ C
qua
), n is the
number of equivalent binding sites, and K
b
is the affinity of
ligand for those sites [52]. Data were fitted to the McGhee–
von Hippel neighbor exclusion model.
To determine the affinity of binding between berberine
and quadruplex, fluorescence experiments were carried out
at 25 °C using a fixed concentration (500 nm) of berberine
and varying the quadruplex concentration (0–5 lm). For
analysis of data, the observed fluorescence intensity was
considered as the sum of the weighted contributions from

free berberine and berberine bound to quadruplex:
F ¼ð1 À a
b
ÞF
0
þ a
b
F
b
ð5Þ
where F is the observed fluorescence intensity at each
titrant concentration, F
0
and F
b
are the respective fluores-
cence intensities of the initial and final states of titration,
and a
b
is the mole fraction of berberine in bound form.
Assuming 1 : 1 stoichiometry for the interaction as
observed in the Scatchard plot, it can be shown that:
½L
0
a
2
b
Àð½L
0
þ½Qþ1=K

b
Þa
b
þ½Q¼0 ð6Þ
where K
b
is the binding constant, [L]
0
is the total berberine
concentration, and [Q] is the added quadruplex concentra-
tion.
From Eqns (4,5), it can be shown that:
DF ¼ðDF
max
=2½L
0
Þð½L
0
þ½Qþ1=K
A
ÞÀ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð½L
0
þ½Qþ1=K
A
Þ
2
À 4½L
0

½Q
q

ð7Þ
where DF = F)F
0
and DF
max
= F
max
)F
0
, and F and F
0
are the initial and subsequent fluorescence intensities of the
berberine at 522 nm, upon quadruplex addition.
ITC
ITC measurements were carried out on a VP-ITC titration
calorimeter (MicroCal, Northampton, MA, USA). Before
loading, the solutions were thoroughly degassed. The refer-
ence cell was filled with the respective degassed buffer. The
preformed quadruplex concentration (10 l m) was kept in
the sample cell, and berberine (100 lm) in the same buffer
was placed in a syringe of volume 300 lL. The berberine
solution was added sequentially in 10 lL aliquots (for a
total of 25 injections, 20 s duration each) at 4 min intervals.
Sequential titrations were performed to ensure full occu-
pancy of the binding sites by loading and titrating with the
same ligand without removing the samples from the cell
until the titration signal was essentially constant. The heats

of dilution were determined in parallel experiments by
injecting a berberine solution of the same concentration in
the same buffer. The respective heat of dilution is sub-
tracted from the corresponding binding isotherm prior to
curve fitting. origin 5.0 software was used to fit the
thermodynamic parameters to the heat profiles.
Molecular modeling
Literature studies based on NMR data reveal that the 22-
mer human telomeric sequence assumes multiple intercon-
vertible conformations, comprising the parallel, antiparallel
and hybrid-type G-quadruplexes, in the presence of potas-
sium ions [53]. However, no solution structure obtained in
the presence of potassium ions is available for the 22-mer
human telomeric sequence. Furthermore, the telomeric
quadruplex adopts a mixed hybrid conformation in the pres-
ence of berberine (1 : 1 molar ratio of berberine: telomeric
quadruplex), as shown in the CD spectra (supplementary
Fig. S5). We chose the hybrid-type NMR structure (Protein
Data Bank ID 2hy9) [54] of the human telomeric quadru-
plex, as shown in supplementary Fig. S3. The 2hy9 is a 26-
mer mixed-hybrid structure of the human telomeric sequence
in the presence of potassium ions. The four adenine residues,
two from each terminal end, were removed for comparison
A. Arora et al. Telomeric quadruplex–berberine interaction
FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS 3979
purposes. The initial 3D coordinate for berberine was
extracted from the crystal structure of the transcriptional
receptor QacR from the Protein Data Bank (1jum) [55]. The
extracted structure was then optimized, and the partial
charges were derived with the HF ⁄ 6-31G* basis set in

gaussian 03 [56]; this was followed by restrained electro-
static potential calculation in the antechamber module
of amber8. The remaining parameters for berberine were
taken from the GAFF forcefield in amber8 [57]. The NMR
structures possess two external G-quartets, shown in supple-
mentary Fig. S3, that can independently act as binding sites
for berberine stacking. The optimized structure of berberine
was docked against the binding sites by defining the external
G-quartet as the active site using the suflexdock module
in sybyl 7.3 [58]. This resulted in two quadruplex–
berberine complex models, referred to as MH1 and MH2.
surflexdock provides multiple docked conformations, and
the one with the lowest docking energy was considered as
the starting structure for further simulation experiments.
Molecular dynamics
Two potassium ions were manually placed in the central
channel between the G-quartet planes in the complex mod-
els. The complex models were simulated using the amber8
[59] suite of programs with the Cornell et al. all-atom force
field ff99 [60]. The complexes were neutralized with potas-
sium ions, with the two inner ions retained inside. The sys-
tems were then immersed in a periodic box of TIP3P water
model, which extended approximately 8 A
˚
(in each direc-
tion) from the solute in a truncated octahedron unit cell
[61]. Simulations were performed with periodic boundary
conditions, and the particle-mesh Ewald method was used
to treat long-range electrostatics [62]. Bond lengths
involving bonds to hydrogen atoms were constrained using

shake [63]. A time step of 2 fs was used except for the
equilibration phase, which was 1 fs. The direct-space cutoff
used was 10 A
˚
. Simulations were performed at a constant
temperature of 300 K. The Langevin coupling with a
collision frequency of 1.0 was used for temperature regula-
tion [64]. A constant pressure of 1 atm with isotropic mole-
cule-based scaling with a relaxation time of 1 ps was used.
The equilibration step involves multiple optimization and
relaxation of the solvent and potassium ions in the bulk
solvent with the solute and the two inner potassium ions
fixed with restraints that include 500, 100, 250, 50, 100, 25,
50 and 10 kcalÆmol
)1
A
˚
respectively. Then, the whole sys-
tem was heated from 0 to 300 K at constant volume, and
this was followed by equilibration for 25 ps at a constant
temperature of 300 K and a pressure of 1 atm. The produc-
tion phase was started at this stage and continued for 5 ns.
All simulations were performed in an SGI Altix 450 cluster.
The conformations in the trajectories were collected at
intervals of 2 ps. Trajectory analyses were done using the
ptraj program in amber 8.
SASA calculation
The SASA calculation was done using grasp 1.3 [65]. The
lowest-energy structure evolved during simulation was used
for the SASA calculation of the complexes. Surfaces for

carbon, carbon-bound hydrogen and phosphorus are
defined as nonpolar, and the remaining hydrophilic atoms
are defined as polar. The grasp radii set was used in the
calculation.
Thermodynamics calculation
The free energy estimates were performed by the MMPBSA
approach, where the total free energy of binding is
expressed as the sum of the contributions from the gas
phase and solvation energies plus an additional term for
the solute entropic contribution. This can be expressed in
the following equation:
G ¼ E
gas
þ E
solv
þ TS
solute
ð8Þ
where E
gas
is the total gas-phase energy, E
solv
is the total
solvation energy (polar + nonpolar), and TS
solute
corre-
sponds to solute entropic effects. The analysis was done for
the last 2 ns trajectory of the complexes. The snapshots for
quadruplex and berberine were extracted from the complex
trajectories at intervals of 10 ps. This yielded 100 snapshots

in total. All counterions (except the two spanning the cen-
tral channel of the G-quartet planes) and water molecules
were stripped out from the trajectory prior to the thermo-
dynamic analysis. The gas-phase energies of the solutes
were calculated using the Cornell et al. force field [60] with
no cutoff. Solvation free energies were computed as the
sum of polar and nonpolar contributions using a contin-
uum solvent representation.
The polar contribution was calculated with the pbsa
program in amber8. The dielectric constants used for the
solute and the surrounding solvent were 1 and 80, respec-
tively. The Cornell et al. radii set was used to define
atom-centered spheres for the solute atoms, and a probe
radius of 1.4 A
˚
was used for the solvent to define the
dielectric boundary around the molecular surface. A lat-
tice spacing of two grid points per A
˚
was used, and 1000
finite difference iterations were performed, excluding salt
effect. The nonpolar solvent contribution was estimated
from an SASA-dependent term, DE
snp
= c. SASA + b,
where c was set to 0.0075 kcalÆA
˚
)2
and b to 0. The cal-
culation for solute entropic contribution was performed

with the nmode module in amber8. The snapshots were
minimized in the gas phase using the conjugate gradient
method for 5000 steps, using a distance-dependent dielec-
tric of 4r (r is the interatomic distance) and with a con-
vergence criterion of 0.1 kcalÆ(molÆA
˚
)
)1
for the energy
gradient. The frequencies of the vibrational modes were
computed for these minimized structures at 300 K, using
normal mode analysis methodology. The thermodynamic
Telomeric quadruplex–berberine interaction A. Arora et al.
3980 FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS
parameters calculated are presented in supplementary
Table S1.
Acknowledgements
The authors acknowledge CSIR for funding this
research. A. Arora acknowledges a research fellowship
from UGC, India. C. Balasubramanian, N. Kumar
and S. Agrawal acknowledge a research fellowship
from CSIR, India.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Temperature dependence of DG (h ), DH (D)
and TDS (s) for binding of berberine to the human
telomeric quadruplex.
Fig. S2. The natural logarithm of the ratio of the bind-
ing constant at a given osmolyte concentration (K
s
)
relative to the binding constant in 50 mm MES buffer
(pH 7.4).
Fig. S3. Mixed hybrid-type NMR structures of the
human telomeric quadruplex (Protein Data Bank ID:
2hy9).
Fig. S4. rmsd plots for the models (A) MH1 and (B)
MH2.

Fig. S5. CD spectra of 5 lm telomeric quadruplex in
the absence (h) and presence (s)of5lm berberine in
50 mm MES buffer (pH 7.4) containing 100 mm KCl.
Table S1. Free energy estimates for quadruplex–
berberine complex formation.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
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than missing material) should be directed to the corre-
sponding author for the article.
A. Arora et al. Telomeric quadruplex–berberine interaction
FEBS Journal 275 (2008) 3971–3983 ª 2008 The Authors Journal compilation ª 2008 FEBS 3983