ARTICLE IN PRESS
International Journal of Biological Macromolecules 00 (2002) 1 /7
www.elsevier.com/locate/ijbiomac
Effect of a modified thymine on the structure and stability of
[d(TGGGT)]4 quadruplex
Luigi Petraccone a, Eva Erra a, Lucia Nasti a, Aldo Galeone b, Antonio Randazzo b,
Luciano Mayol b, Guido Barone a, Concetta Giancola a,
b
a
Dipartimento di Chimica, Via Cintia, Università ‘Federico II’ di Napoli, Monte Sant’ Angelo, 80126 Naples, Italy
Dipartimento di Chimica delle Sostanze Naturali, Via D. Montesano 49, Università ‘Federico II’ di Napoli, 80131 Naples, Italy
Received 15 July 2002; received in revised form 4 September 2002; accepted 5 September 2002
Abstract
Telomeric guanine-rich sequence can adopt quadruplex structures that are important for their biological role in chromosomal
stabilisation. G quartets are characterised by the cyclic hydrogen bonding of four guanine bases in a coplanar arrangement and their
stability is ion-dependent. In this work we compare the stability of [d(TGGGT)]4 and [d(TGGGT)]4 quadruplexes. The last one
contains a modified thymine, where the hydroxyl group substitutes one hydrogen atom of the methyl group of the thymine in the
[d(TGGGT)]4 sequence. We used a combination of spectroscopic, calorimetric and computational techniques to characterise the Gquadruplex formation. NMR and CD spectra of [d(TGGGT)]4 were characteristic of parallel-stranded, tetramolecular quadruplex.
CD and DSC melting experiments reveal that [d(TGGGT)]4 is less stable that unmodified quadruplex. Molecular models suggest
possible explanation for the observed behaviour.
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: DNA quadruple helices; Differential scanning calorimetry; Molecular modelling
1. Introduction
G-rich DNA sequences can adopt unusual fourstranded DNA structures, called DNA G-quadruplex
[1,2]. The fundamental building block of these unusual
structures is the G-tetrad (also called G-quartet) [3]. The
G-tetrad consists of a planar arrangement of four
guanine bases associated through a cyclic array of
hydrogen bonds in which each guanine base both
accepts and donates two hydrogen bonds. The resulting
square /planar array is unique due to the ‘hole’ that is
created in the centre. The G-quadruplex structures have
generated considerable interest recently due to their
potential role in the maintenance of eukaryotic chromosome [4]. Telomeres are DNA protein structures that
exist at the end of chromosomes and are essential for
chromosomal stability. Although most of the telomeric
Corresponding author. Tel.: /39-081-67-4266; fax: /39-081-674090
E-mail address: [email protected] (C. Giancola).
DNA is double-stranded, the extreme 3?-end of the
telomere consists of a single-stranded G-rich DNA
overhang [5,6]. Extensive structural studies with these
sequences by X-ray crystallographic and solution NMR
methods have indicated that the telomeric DNA segments can form a variety of G-quadruplex structures
depending on the exact sequence, the chain length and
the presence of different cations [7 /10]. Telomere
shortening has been implicated in cellular senescence.
Telomerase is an enzyme, which synthesise the G-rich
strand of telomeric DNA. Telomerase activity is highly
correlated with cancer and may allow cancer cells to
escape senescence [11]. One promising approach for
inhibiting telomerase involves targeting the G-quadruplex DNA structures thought to be involved in telomere
and telomerase function [12]. Furthermore, G-quadruplex structures have been found in a number of
aptamers. Particularly, the aptamers against thrombin,
TBA (thrombin binding aptamer), and against HIV-1
integrase (a potent HIV inhibitor) are noteworthy [13 /
15]. Both aptamers containing modified bases have been
also prepared and the resulting G-tetrad-forming oligo-
0141-8130/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 4 1 - 8 1 3 0 ( 0 2 ) 0 0 0 7 3 - 9
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nucleotides showed a significant, functional correlation
between thermal stability of the quadruplex structures
and the capacity to inhibit thrombin and HIV-1
integrase, respectively. In this frame, the study of the
thermodynamic and kinetic stability of the quadruplexes
containing modified bases is of notable significance to
improve the biological properties of active molecules
and to explore the feasibility of new types of tetrads.
Therefore, here we report the physico-chemical characterisation of [d(TGGGT)]4 quadruplex, where T is
modified thymine: the hydroxyl group substitutes one
hydrogen atom of the methyl group. For comparison
the physico-chemical properties of the same quadruplex,
containing the unmodified thymine base, was also
reported (Fig. 1).
We have used differential scanning calorimetry,
circular dichroism and molecular modelling to characterise the quadruplex dissociation.
2. Experimental
2.1. Preparation of quadruplexes
The synthesis of modified thymine (T) containing
oligonucleotides (T-ODNs) was carried out using the
fully protected 5-hydroxymethyl-2?-deoxyuridine phosphoramidite as synton [[16], and references cited
therein].
The fully protected T was prepared starting from the
hydroxymethylation of 2?-deoxyuridine with paraformaldehyde in a KOH solution to give 5-hydroxymethyl2?-deoxyuridine. In order to protect the 5-hydroxymethyl group, we used the tert-butyldimethylsilyl ether,
a protective group compatible with the acidic treatment
as provided in the 5?-OH deprotection step of the
standard phosphoramidite method. The following reaction was the protection of the 5?-OH function by the
standard 4,4?-dimethoxytrityl group. Finally, the resulting protected intermediate was converted in the desired
phosphoramide using standard procedure. The latter
compound was used for the preparation of 5?TGGGT-3?. The oligonucleotide was synthesised on a
Millipore Cyclon Plus DNA synthesiser, using solid
phase b-cyanoethyl phosphoramidite chemistry. During
the automated syntheses, the T-phosphoramidite exhibited similar coupling efficiencies as the commercially
available phosphoramidites derived from the regular
DNA nucleosides. Final ammonia treatment removed
the protecting groups and detached the T-ODN from
the resin. However, a further treatment with 80% acetic
acid was required to fully remove the 5-hydroxymethyl
protecting group. Purification of the oligonucleotides
was carried out by a HPLC Waters 515 equipped with
an UV detector, by using a VA 50/4.6 Nucleogel SAX
1000-8 column, using linear gradient from 20 mM
Fig. 1. Schematic illustration of parallel-stranded quadruplex (A).
Representation of thymine structure T (B) and of the modified thymine
T (C).
KH2PO4, 20% CH3CN, pH 7/20 mM KH2PO4, 1 M
KCl, 20% CH3CN pH 7 followed by desalting on a Seppak C18 column. 5?-TGGGT-3? was synthesised on a
Millipore Cyclon Plus DNA synthesiser, following the
regular protocols.
The quadruplexes were formed by dissolving the
oligonucleotides in the appropriate buffer and heating
the solution at 90 8C for 5 min. The solution was slowly
cooled to room temperature, then equilibrated for 1 day
at 4 8C. The buffer used was 10 mM KH2PO4, 1.00 M
KCl and 0.1 mM EDTA. Potassium chloride (Sigma),
monopotassium phosphate (Sigma) and ethylenediaminetetraacetic acid (Sigma) were used as obtained from
commercial suppliers. The concentration of oligonucleotide solutions were determined spectrophotometrically,
using the extinction coefficients at 260 nm calculated by
the nearest neighbour model [17].
2.2. Circular dichroism
CD spectra were obtained on a JASCO 715 circular
dichroism spectrophotometer at 20 8C in a 0.1 cm
pathlength cuvette. The wavelength was varied from
200 to 340 nm at 10 nm min1. CD spectra were
recorded with a response of 8 s, at 2.0 nm bandwidth
and normalised by subtraction of the background scan
with buffer. The molar ellipticity was calculated from
the equation [q ] /q /cl where q is the relative intensity,
c the concentration of quadruplex and l is the path
length of the cell in centimeters.
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CD melting curves were recorded at 263 nm. Samples
were heated at a rate of 1 8C min 1, in the range 20/
100 8C of temperature.
Temperature was kept constant with a thermoelectrically controlled cell holder (JASCO PTC-348).
2.3. Differential scanning calorimetry
DSC measurements were performed on a second
generation Setaram Micro-DSC at scan rate of 0.1, 0.5
and 1 8C min 1. The calorimetric unit was interfaced
to an IBM PC computer for automatic data collection
and analysis using the software previously described
[18]. The apparent molar heat capacity versus temperature profiles were obtained by subtracting buffer versus
buffer curves from the sample versus buffer curves. The
data were normalised with regard to the concentration,
sample volume and scan rate. The performance of the
instrument was calibrated periodically with an electrical
pulse. The excess heat capacity function DC p was
obtained after baseline subtraction, assuming that the
baseline is given by the linear temperature dependence
of the native state heat capacity [19]. The reversibility of
the thermal processes was verified by checking the
reproducibility of the calorimetric trace in a second
heating of the samples immediately after cooling from
the first scan. The transition enthalpies, DH 8(Tm), were
obtained by integrating the area under the heat capacity
versus temperature curves. Tm is the temperature
corresponding to the maximum of each DSC peak.
The reported errors for DH 8(Tm) and Tm are the
standard deviations of the mean from the multiple
determinations.
3
MD runs were started by assigning random velocities,
that followed a Gaussian distribution at 10 K. The
systems were heated from 10 to 300 K by coupling to
heat bath over a period of 6 ps. Then for both the
quadruplexes a constant temperature dynamics were
performed over a period of 74 ps without any restraints.
A 0.5 fs integration time step was used. After 20 ps of
equilibration at 300 K snapshots were collected every
0.1 ps for 60 ps.
3. Results and discussion
3.1. CD spectra
The CD spectra for the two quadruplexes at pH 7.0
and C /1.11 /10 5 M are shown in Fig. 2 Panel A.
The spectra of the [d(TGGGT)]4 (dashed line) and
[d(TGGGT)]4 (solid line) are similar but not identical
and show slight differences attributable to non equivalent conformation in solution. The spectra are characteristic of parallel-stranded quadruplex structures with a
positive band at 263 nm and a negative band at 245 nm
[23].
Fig. 2 Panel B shows the CD melting profiles
measured at 263 nm. [d(TGGGT)]4 (dashed line) and
2.4. Molecular modelling
Models of the two quadruplexes were built modifying
the AG3T structure reported by P.K. Patel, A.S.R. Koti,
V. Hosur [20]. The A-tetrad was substituted with a Ttetrad or with a T-tetrad.
The two quadruplexes were neutralised by adding
potassium counterions. Each K counterion was placed
on the phosphate bisector 3 Å from the P atom and then
each quadruplexes was individually placed in a 35 /
35 /35 Å box of Monte Carlo TIP3P water [21], with
periodic boundary conditions. The water molecules that
were nearest of 2.3 Å from any solute atoms were
removed. For both quadruplexes the same energy
minimisations and dynamics procedure were used.
The Amber force field was utilised [22]. The nonbonded interactions were cut off using a switching
function at 16.0 Å. Starting structures were minimised
using 1200 steps of the steepest descent method followed
by the conjugate method until convergence to a r.m.s.
gradient of 0.1 kcal mol1 Å 1.
Fig. 2. CD spectra of [d(TGGGT)]4 (dashed line) and d[(TGGGT)]4
(solid line) (panel A). CD melting curves for [d(TGGGT)]4 (dashed
line) and [d(TGGGT)]4 (solid line) at 263 nm (panel B).
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L. Petraccone et al. / International Journal of Biological Macromolecules 00 (2002) 1 /7
[d(TGGGT)]4 (solid line) exhibit sharp transitions,
with midpoint temperatures, Tm, of about 60 and
58 8C, respectively. At 20 8C ellipticity at 263 nm is
more
intense
for
[d(TGGGT)]4
respect
to
[d(TGGGT)]4 and become equal at 80 8C when the
dissociation process is complete for both the quadruplexes.
3.2. Thermodynamics of quadruplex formation
The unfolding of the two quadruplexes was investigated using microcalorimetry. The corresponding DSC
melting profiles are shown in Fig. 3. These curves
indicate that both quadruplexes dissociate in biphasic
transitions, as indicated by the presence of an additional
peak as shoulder at lower temperature. The dissociation
process is reversible for both quadruplexes since the
melting profiles are recovered upon cooling. Nevertheless, the melting temperatures and the calorimetric
profiles are influenced by scan rate indicating a kinetic
control on the dissociation process. In particular, on
decreasing the scan rate, for both quadruplexes, the
apparent melting temperature of the main peak does not
significantly change but the Tm of the secondary peak
decreases of about 108. Consequently, the equilibrium
can be reached only at very slow scan rates. Calorimetric
measurements performed at 0.1 8C min 1 and slower
do not show good profiles and it is not possible to
obtain correct thermodynamic data. On the other hand,
the model-dependent DHVH values cannot be derived
from the shape of CD and UV melting curves at slow
scan rate, being the quadruplex dissociation a biphasic
process. Hence, in order to compare the melting
behaviour of the quadruplexes, we focus on the enthalpy
and Tm data obtained from DSC experiments in the
same solution conditions. DH and apparent Tm values
obtained from analysis of the shape of the DSC curve
are shown in Table 1. The measurements were performed at pH 7.0 in 10 mM KH2PO4, 1.00 M KCl and
0.1 mM EDTA buffer and 1 8C min 1 scan rate. Both
DH and apparent Tm values are lower for the quadruplexes containing modified thymine. DH value for
[d(TGGGT)]4 quadruplex is 231 kJ mol1, slightly less
than that found by Breslauer and co-workers and
corresponds to a value of 77 kJ mol 1 per tetrad [23].
For [d(TG3T)]4 this value is lower indicating the lost of
interactions in the quadruplex structure. It is evident
that association of four strands is enthalpically favourable and highly unfavourable from entropic point of
view. The entropic contributions to Gibbs energies
cannot be calculated since the equilibrium is not reached
in these conditions.
NMR measurements for both quadruplexes were
performed at a concentration of 1.0 mM (0.5 ml, 90%
H2O/10% D2O), having 10 mM potassium phosphate, 1
M KCl, 0.1 mM EDTA (pH 7.0). 1H-NMR spectra
show that a single, well-defined species, are plainly
observable in solution for both structures, and that both
quadruplex possess a 4-fold symmetry with all strands
parallel to each other (data not shown).
Hence, the presence of biphasic calorimetric profiles is
not imputable to different topological arrangements. It
has been already noted that one of the two component
peaks shifts at lower temperature on decreasing scan
rate while the other one remains fixed.
It is possible to rationalise these observations supposing a mechanism with two sequential dissociation steps
for quadruplex melting in according with the following
scheme:
S4 0 2S2
slow
U
fast equilibrium
4S
(1)
where S indicates the single strand, S2 the dimer and S4
the tetramer. The first step in which the tetramer
dissociate in two dimers is slow and represents the rate
limiting step for the quadruplex dissociation. The
Table 1
Temperature and enthalpy values for the dissociation process of the
two quadruplexes
Fig. 3. Calorimetric heat capacity vs. temperature profiles for the
[d(TGGGT)]4 (curve A) and [d(TGGGT)]4 (curve B). Excess capacity
values have been shifted along the y -axis for ease of presentation. The
measurements were performed at pH 7.0 in 10 mM KH2PO4, 1.00 M
KCl and 0.1 mM EDTA buffer and 1 8C min 1 scan rate.
[d(TGGGT)]4
[d(TGGGT)]4
a
b
Shoulder peak.
Main peak.
T1 (8C)a
T2 (8C)b
DH 8 (kJ mol 1)
62.290.2
58.590.1
64.090.1
62.090.1
23198
130910
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Fig. 4. Quadruple helical structures of the [d(TGGGT)]4 (on the left) and [d(TGGGT)]4 generated by molecular mechanics. For clarity, at the top,
the T-tetrad and the corresponding unmodified T-tetrad are also reported. The four strands are reported in different colours.
second step represents a fast equilibrium of dissociation
of the dimers in two single strands. This dissociation
scheme is already reported in literature for the quadruplex [d(T2G4T2)]4 [24]. The scan rate dependent peak
should correspond to the first step of scheme (1) while
the scan rate independent peak should corresponds to
the dimer-single strands fast equilibrium.
3.3. Molecular modelling
To gain more insight into the structural difference
between the [d(TGGGT)]4 and the unmodified oligonucleotide, it was performed a molecular mechanics
calculation in presence of explicit water and counterions. The minimised structures are shown in Fig. 4. For
Fig. 5. Plot of potential energy for the MD simulation of the [d(TGGGT)]4. A similar plot was obtained for the unmodified quadruplex.
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Fig. 6. Hydrogen bond distances N3(T) O4(T) for each thymine in the plane of the T-tetrad (panel A) and T-tetrad (panel B).
both quadruplexes the T-tetrad and the G-tetrad are
formed. Analysing the T-tetrads, it can be noted that the
modified thymines are able to form the extra H-bonds
with the hydroxyl groups. Hence the modified T-tetrad
has four additional H-bonds respect to the unmodified
T-tetrad, at least in the energy minimised structure. On
the other hand, in order to form these H-bonds the
carbonyl groups of thymine residues are forced outside
the plane of the T-tetrad. Hence the T-tetrad appears
slightly more open towards the solvent respect to the
unmodified T-tetrad. It is possible that the extra Hbonds formation could facilitate the opening of the T-
tetrad in high temperature condition and this could
cause a destabilisation rather than a stabilisation of the
modified quadruplex. To check this hypothesis the two
minimised structures were subjected to a constant
temperature MD simulation at 300 K, in the presence
of explicit water and counterions. Inspection of the plot
of potential energy against time (Fig. 5) reveals that the
system became energetically stable after an equilibration
phase involving the first 20 ps of simulation. The
integrity of the two quadruplexes is maintained throughout all the simulation, even though no constrains are
introduced to reinforce the H-bonds. However, the
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analysis of the H-bond distances during the MD
trajectory showed that the extra H-bonds found in the
minimised [d(TGGGT)]4 structure are completely loss
at 300 K and there is a free rotation of the C /OH bonds
of the hydroxy-methyl groups of the modified thymine.
In order to put in evidence the possible opening of the
terminal T-tetrad it has been monitored the planar
circular H-bonds pattern formed by the T bases.
The plot of Fig. 6 shows each H-bond distance
N3(T) O4(T) inside the T-tetrad and the T-tetrad
during the simulation. The anomalous values above 4.5
Å (red line in Panel A) are associated with a displacement of one modified thymine outside the tetrad plane.
Hence the T-tetrad appear to be broken at 300 K. It is
important to underline that the perturbation of the Ttetrad directly affects the stacking energy of the
neighbour G-tetrad. On the other hand the behaviour
of H-bonds distances for the unmodified thymines don’t
show drastic opening of the T-tetrad. These findings
could justify the difference in enthalpy observed for the
formation of the [d(TGGGT)]4 respect to the
[d(TGGGT)]4.
4. Conclusions
Results from NMR, CD and DSC experiments clearly
demonstrated that [d(TGGGT)]4 acquires a quadruple
helical structure.
CD spectrum of the [d(TGGGT)]4 complex shows
characteristic bands of quadruplex and is comparable
with that of [d(TGGGT)]4 sequence which exists as
tetramolecular system. Further, 1H-NMR spectra show
that the modified quadruplex possesses a 4-fold symmetry with all strands parallel to each other. Having
stabilised the formation of the complex, we were able to
follow the dissociation process by DSC. A two sequential dissociation mechanism was invoked to interpret the
biphasic behaviour of the quadruplex melting curves.
The results provide indication of a minor stability of
[d(TGGGT)]4 quadruplex in comparison to
[d(TGGGT)]4 quadruplex. Hence the introduction of
the modification results in a destabilisation rather than
stabilisation of the quadruplex. The hypothesis is that
the modification facilitates the opening of the T-tetrad,
this hypothesis is reinforced by molecular modelling
studies. Indeed, MD simulation reveals that no extra Hbonds are formed by the modified thymines and the Ttetrad is partially broken at 300 K.
7
In conclusion, we proved that a modified thymine
destabilises the quadruplex and confirmed that the
physico-chemical methodologies are powerful tools to
gain information on the effect of little modifications on
quadruple helical structures.
Acknowledgements
This work was supported by a PRIN-MURST grant
from the Italian Ministry of University and Scientific
and Technological Research (Rome).
References
[1] Lee JS, Evans DH, Morgan AR. Nucleic Acids Res 1980;8:4305.
[2] Arnott S, Chandrasekaran R, Martilla CM. Biochem J
1974;141:537.
[3] Phillips K, Dauter Z, Murchie AIH, Lilley DMJ, Luisi B. J Mol
Biol 1997;273:171.
[4] Wellinger RJ, Sen D. Eur J Cancer 1997;33:735.
[5] Blackburn EH. Nature 1991;350:569.
[6] Rhodes D, Giraldo R. Curr Opin Struct Biol 1995;5:311.
[7] Shafer RH. Prog Nucleic Acids Res Mol Biol 1998;50:55.
[8] Kankia I, Marky LA. J Am Chem Soc 2001;123:10799.
[9] Hud NV, Smith FW, Anet AL. Feigon J Biochem 1996;35:15383.
[10] Töhl J, Eimer W. Biophys Chem 1997;67:177.
[11] Ishikawa F. Biochem Biophys Res Commun 1997;230:1.
[12] Borman S. Chem Eng News 1999;5:36.
[13] He GX, Krawczyk SH, Swaminathan S, Shea RS, Dougherty JP,
Terhosrst T, Law VS, Griffin LC, Coutrè S. Bischofberger New J
Med Chem 1998;41:2234.
[14] Jing N, De Clercq E, Rando RF, Pallansch L, Lackman-Smith C,
Lee S, Hogan ME. J Biol Chem 2000;275:3421.
[15] Wyatt JR, Vickers TA, Roberson JL, Buckheit RW, Klimkait T,
DeBaets E, Davis PW, Rayner B, Imbach JL, Ecker DJ. Proc
Natl Acad Sci USA 1994;91:1356.
[16] Conte MR, Galeone A, Avizonis D, Hsu VL, Mayol L, Kearns
DR. Bioorg Med Chem Lett 1992;2(1):79 (references cited
therein).
[17] Cantor CR, Warshaw MM, Shapiro H. Biopolymers 1970;9:1059.
[18] Barone G, Del Vecchio P, Fessas D, Giancola C, Graziano G. J
Thermal Anal 1993;39:2779.
[19] Freire E, Biltonen RL. Biopolymers 1978;17:463.
[20] Patel PK, Koti ASR, Hosur V. Nucleic Acids Res 1999;27:3836.
[21] Jorgensen WL. J Chem Phys 1983;79:926.
[22] Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM,
Ferguson DM, Spellmeyer DC, Fox T, Caldwell JW, Kollman
PA. J Am Chem Soc 1995;117:5179.
[23] Renzhe J, Gaffney BL, Wang C, Jones AR, Breslauer KJ.
Biochemistry 1992;89:8832.
[24] Wyatt JR, Davis PW, Freir SM. Biochemistry 1996;35:8002.