Programing the formation of DNA and PNA quadruplexes by π-π-stacking interactions

Article abstract of DOI:10.1039/b915955j

Guanine (G) rich G₄T₄G₄ DNA and homologous PNA strands tend to form antiparallel dimeric quadruplexes. In contrast, the same DNA strands carrying planar aromatic 5′-residues preferentially form parallel DNA quadruplex. Con

Full text of DOI:10.1039/b915955j

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Programing the formation of DNA and PNA quadruplexes
by p–p-stacking interactionsw
Sourav Saha, Jianfeng Cai, Daniel Eiler and Andrew D. Hamilton*
Received (in Cambridge, UK) 4th August 2009, Accepted 17th December 2009
First published as an Advance Article on the web 13th January 2010
DOI: 10.1039/b915955j
Guanine (G) rich G
4
T
4
G
4
DNA and homologous PNA strands
microarrays of fluorescent biosensors, as well as energy and
electron transfer materials.
tend to form antiparallel dimeric quadruplexes. In contrast, the
0
same DNA strands carrying planar aromatic 5 -residues
Herein we report that the stability, conformation, and
composition of quadruplexes depend not only on the nucleo-
base sequence, but also on the properties of the head-
preferentially form parallel DNA quadruplex. Conformation
and composition of the DNA quadruplexes can be programed
0
0
by p–p-stacking interaction exerted by the 5 -residues.
groups that are attached to the 5 - and N-termini of the
DNA and PNA strands, respectively (Fig. 2). In order to
investigate the formation of the hybrid quadruplexes by the
The guanine (G) rich telomeric DNA sequence of Oxytricha nova
1
d(G T G ) is known to form stable antiparallel hairpin
G
4
T
4
G
4
DNA and PNA strands in the presence of different
4
4
4
0
dimeric quadruplexes through Hoogsteen hydrogen bond
head-groups, we functionalized the 5 - and N-termini of both
strands with a number of aromatic units, ranging from energy
donating and accepting small p-surfaces, like coumarin-2 (1)
and coumarin-343 (2), to electron donating and accepting
larger p-surfaces, such as porphyrin (3, 4) and perylene
+
formation, in the presence of Na or K ions. Peptide nucleic
+
2
acid (PNA) is a DNA analog that contains oligomeric
N-(2-aminoethyl)glycine units bearing nucleobases at the
glycine nitrogen through acetyl linkers. More recent effort
2
,3
has been directed to the hybridization of complementary as
well as homologous PNA molecules with DNA and RNA for
their importance in sequencing and sensing, as antisense
agents, and in gene technology. The hybrid DNA–PNA
duplexes, triplexes, and quadruplexes display much higher thermal
(5) units.
0
5 -C10-Carboxy-modified d(G
T
G
) oligonucleotide (ODN),
) ODN, and homologous
T G -LysNHBhoc PNA strands were used
4 4 4
4
4
4
0
5 -C6-amino-modified d(G T G
4 4 4
FmocNH-G
0
for 5 -end and N-end functionalization. The coumarin-2
derivative 1, porphyrin derivatives 3 and 4, as well as perylene
diimide 5 were attached to the modified ODNs via solid-state
2
stability over the purely DNA and RNA superstructures.
Enhanced stability of the DNA–PNA hybrid structures is
attributed to the neutral peptidic backbones of the PNA
strands, which in addition to diminishing the electrostatic
repulsion between the phosphate backbones of the DNA
strands, provide an optimal spacing between the nucleobases
that matches the sugar–phosphate backbone of DNA and
RNA. This structural advantage is reflected in the stability
of duplexes formed by complementary DNA, PNA, and RNA
strands (PNA–PNA > PNA–RNA > PNA–DNA >
4
amide coupling reactions, followed by cleavage of the resin
and HPLC purification to obtain three functionalized ODNs.
Basic deprotection of the Fmoc group yielded an amine-
terminated PNA strand, which allowed peptide coupling with
2
RNA–DNA > DNA–DNA).
4
Armitage et al. established that G T G DNA and PNA
4
4
4
form a stable 2 : 2 hybrid quadruplex (Fig. 1c), in which each
of the two negatively charged DNA strands is flanked by two
0
neutral PNA strands and the 5 -ends of the two DNAs are
aligned with the N-termini of both PNA strands. As part of
5
our interest in controlling the characteristics of quadruplexes,
we set out to investigate whether or not the formation of homo
and hybrid quadruplexes can be biased in either direction
through chemical modification of the DNA and PNA strands.
If the structural integrity of the functionalized DNA and PNA
quadruplexes can be controlled, they can be employed to form
Department of Chemistry, Yale University, 225 Prospect Street,
New Haven, CT 06520-8107, USA. E-mail: andrew.hamilton@yale.edu;
Fax: +1 203-432-3221; Tel: +1 203-432-5570
w Electronic supplementary information (ESI) available: CD melting
and control studies, as well as syntheses and characterizations of
compounds 1 and 5 as well as those of functionalized DNA and PNA
strands with compounds 1 through 5. See DOI: 10.1039/b915955j
Fig.
1
4 4 4
Graphical representation of G T G DNA and PNA
0
quadruplexes, modified at the 5 - and N-terminals, respectively.
(
a) An antiparallel hairpin dimeric DNA quadruplex, (b) an antiparallel
hairpin dimeric PNA quadruplex, (c) a parallel DNA –PNA hybrid
quadruplex, (d) a tetramolecular parallel DNA quadruplex, and (e) a
dimeric parallel DNA quadruplex.
2
2
4
2
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Fig. 3 CD spectra demonstrating the formation of DNA, PNA, and
hybrid quadruplexes at 20 mM single-strand concentration of each
species.
Fig. 2 Coumarin-2 derivative (1), coumarin-343 (2), porphyrin
0
derivatives (3 and 4), and perylene diimide (5) used as 5 - and
recorded for 5-DNA, which carries a hydrophobic planar
0
N-head-groups for DNA and PNA, respectively.
head-group, perylene diimide 5, at the 5 -end.
To destabilize the strong p–p-stacking interactions between
the hydrophobic porphyrin units by electrostatic repulsion, we
coumarin-343 (2). Cleavage of the resin and HPLC purification
4
provided two functionalized PNA strands. All compounds
installed tetra(4-carboxyphenyl) porphyrin 4 head-groups via
0
were characterized by high resolution MALDI-TOF mass
spectrometry (MS) and UV-Vis spectroscopy.
amide bond formation on 5 -C6-amino-modified d(G
4
T
G
4 4
)
ODN. The CD spectrum of the annealed sample showed a
significant departure from that of 3-DNA. Although CD
spectra of 3-DNA, 4-DNA and 5-DNA indicate the formation
of respective parallel qudruplexes that show characteristic
peaks at 265 nm and troughs at 245 nm, the 4-DNA
quadruplex shows only 50% intensity of the 265 nm peak
compared to that of the 3-DNA and 5-DNA quadruplexes
(Fig. 3) at the same SS concentrations (20 and 40 mM).
Electrostatic repulsion between the negatively charged
porphyrin residues in 4-DNA presumably destabilizes the
formation of quadruplexes leading to the formation of a
parallel hairpin dimer (Fig. 1e), allowing only two negatively
charged porphyrins to come in close proximity. In contrast,
the 3-DNA and 5-DNA strands predominantly form tetra-
molecular parallel quadruplexes (Fig. 1d), by virtue of
p–p-stacking interaction between neutral aromatic groups.
The quadruplex formed by coumarin-343 (2) appended
Formation of the DNA, PNA, and hybrid quadruplexes is a
dynamic process, owing to hydrogen bonding, metal ion
coordination, as well as p–p-stacking interactions between
4
the nucleobases. All quadruplexes were formed by incubating
single-stranded (SS) DNA and/or PNA at 20 mM concentrations
in pH = 7.0 buffer, containing 10 mM sodium phosphate,
1
00 mM NaCl, and 0.1 mM EDTA at 90 1C for 15 min, then
cooling them slowly to 25 1C and storing them at 4 1C for 16 h.
In the presence of Na ions, the circular dichroism (CD)
+
4
spectrum of unmodified d(G
4
T
G
4 4
)
ODN displays
a
maximum at 295 nm and a minimum at 265 nm, indicating
the formation of an antiparallel dimeric hairpin DNA
quadruplex (ESIw). CD spectroscopy further confirmed that
the dimeric antiparallel conformation was preserved (Fig. 3)
for the modified d(G T G ) ODNs functionalized at the
4
4
4
0
4
5
-end with smaller aromatic units, such as fluorescein and
coumarin-2 (1), that cannot exert strong p–p-stacking inter-
4 4 4
G T G -Lys PNA strands under identical conditions did not
actions. The d(G
0
5
trough at 245 nm, signals that are characteristic of a parallel
4
T
4
G
4
) ODNs modified with porphyrin 3 at
show (Fig. 3) any CD signal since the PNA backbone lacks a
4,6
stereogenic center other than at the lysine residue. Armitage
the 5 -end, however, showed a CD peak at 265 nm and a
4
et al. demonstrated that akin to d(G
4
T
4
4
G ) ODN, the
DNA quadruplex. Two different quadruplex conformations
can be explained by the difference in the strength of
p–p-stacking interactions between the aromatic head-groups
and their influence on the quadruplex formation. In the case of
H-G
quadruplex. We employed the 2-PNA strand as a probe to
4
T
4
G
4
-Lys PNA strand forms a dimeric antiparallel
examine the structural integrity (Fig. 3) of the modified DNA
0
quadruplexes carrying different 5 -residues. For this study, an
1
-DNA, the coumarin-2 unit is not large enough to provide
equimolar amount (10 mM of each SS in the final solution
mixture) of each of the preformed quadruplexes of 1-DNA
and 5-DNA was treated separately with 2-PNA (10 mM SS in
the final solution) under the same annealing conditions. In the
presence of the 2-PNA probe, the CD spectrum of the dimeric
antiparallel quadruplex of 1-DNA changed dramatically,
showing complete disappearance of the 295 nm peak and
265 nm trough and emergence of a peak at 265 nm and a
trough at 245 nm. These spectroscopic changes indicate that
the dimeric antiparallel 1-DNA quadruplex (Fig. 1a) is
perturbed by two homologous PNA strands forming a
any significant p–p-stacking interaction, allowing the formation
of a dimeric antiparallel quadruplex (Fig. 1a). In 3-DNA, the
hydrophobic porphyrin head-group presents a large planar
p-surface that templates the formation of a highly stable
parallel quadruplex involving four or two DNA strands
(
Fig. 1d and e), via p–p-stacking interactions in the aqueous
5
environment. Porphyrin units are known to template G-quartet
formation by preorganizing four guanine bases through
p-stacking interactions. An almost identical CD spectrum—
characteristic of a parallel DNA quadruplex (Fig. 1d and e)—was
1
686 | Chem. Commun., 2010, 46, 1685–1687
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Fig. 5 CD spectra demonstrating the formation (RT), thermal dis-
sociation (95 1C), and reformation (25 1C) of 3-DNA and 4-DNA
quadruplexes at (a) 40 mM and (b) 20 mM SS concentrations of each
species.
Fig.
quadruplexes of 5 -TG
single-strand concentration.
4 CD spectra demonstrating the formation of parallel
0
0
8 3
T -3 ODN and 3-DNA at 20 and 40 mM
tetramolecular hybrid parallel quadruplex (Fig. 1c), which is a
4
thermodynamically more stable assembly. The preformed
the CD signals of 4-DNA quadruplex disappeared completely
at 95 1C as a result of thermal decomplexation, the intensity of
3-DNA quadruplex signals faded only partially, suggesting
a higher thermal stability of the latter. Cooling of each
quadruplex solution to 25 1C reproduced the corresponding
initial CD spectra recorded at room temperature, confirming
the reversibility of the thermal decomplexation–reformation
processes. We assert that a neutral porphyrin residue at the
parallel quadruplex of 5-DNA, however, did not show any
change in the CD spectrum after treatment with an equimolar
amount of the 2-PNA probe, apart from a reduction in the
intensities of the 265 and 245 nm signals by 50%, presumably
because of a two-fold dilution of the original solution. Had a
(
5-DNA)
CD spectrum would have been similar to that recorded for the
1-DNA) (2-PNA) quadruplex as a result of having the same
2 2
(2-PNA) hybrid quadruplex formed, the resulting
0
5 -end of 3-DNA leads to the formation of a stable tetra-
(
2
2
molecular parallel quadruplex, which cannot be completely
+
dissociated thermally in an aqueous buffer, containing Na
helical pitch. These results suggest that the p–p-stacking
interactions between the larger aromatic head-groups, which
5
template the formation of parallel homo-quadruplexes, are
ions. In contrast, to minimize the electrostatic repulsion
between the charged porphyrin residues, 4-DNA forms
a dimeric parallel quadruplex which can be thermally disso-
ciated completely.
strong enough to resist a disruption by the PNA probe.
Therefore, such p–p-stacking interactions or lack thereof can
be exploited to bias the formation of either an all-DNA homo-
quadruplex or a hybrid with a homologous PNA.
In summary, we have demonstrated that the perturbation of
a DNA quadruplex by a homologous PNA strand can be
programed by equipping the DNA strands with large planar
To confirm the difference in compositions of the 3-DNA
and 4-DNA parallel quadruplexes, we studied the CD
0
aromatic head-groups. The nature of the 5 -modifications of
0
0
response of the quadruplex formed from 5 -TG
T
8 3
-3 ODN
4 4 4
d(G T G ) ODNs determines whether this telomeric sequence
7
at 20 and 40 mM SS concentrations in identical buffer condi-
tions and compared them with those of 3-DNA at the same
concentrations (Fig. 4). The intensities of the 265 and 245 nm
CD signals of both quadruplexes were virtually the same
at respective SS concentrations although the tetramolecular
parallel quadruplex of TG T presents eight contiguous planes
would form antiparallel or parallel quadruplexes. We could
also control the stability, geometry, and molecularity of the
parallel quadruplexes formed by the modified d(G T G )
4
4
4
ODNs by changing the nature of aromatic residues at their
0
5 -ends.
We thank the National Science Foundation for financial
support of this research.
8
3
of G-quartets, whereas the 3-DNA quadruplex possesses two
sets of four contiguous G-quartet planes separated by a TTTT
segment. Therefore, it is unlikely that the TTTT segment,
separating two GGGG segments in 3-DNA, interferes with
exciton-coupling among the G-quartet planes. This also
confirms that while 3-DNA forms a tetramolecular quadru-
plex with eight G-quartet planes, 4-DNA forms a dimeric
quadruplex with only four G-quartet planes, resulting in 50%
reduction of the CD intensity for the latter at various
concentrations (Fig. 5).
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3
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3
4
V. L. Marin and B. A. Armitage, Biochemistry, 2006, 45, 1745.
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0
To understand the effects of 5 -residues on 3-DNA and
4
-DNA quadruplexes, we investigated their thermal stabilities
at two different concentrations (20 mM and 40 mM of SS) by
CD spectroscopy. As expected, the intensities of the 265 nm
peak and 245 trough at 25 1C for both 3-DNA and 4-DNA
quadruplexes were twice as high at 40 mM compared to those
at 20 mM of initial SS concentrations (Fig. 5). This suggests
that 3-DNA and 4-DNA SS are completely consumed to form
respective parallel quadruplexes at both concentrations. While
5
6
7 A. Benz and J. S. Hartig, Chem. Commun., 2008, 4010.
This journal is ꢀc The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 1685–1687 | 1687