Angewandte
Chemie
DOI: 10.1002/anie.201307594
G-Quadruplex DNA
Reversible Stabilization of Transition-Metal-Binding DNA
G-Quadruplexes**
David M. Engelhard, Roberta Pievo, and Guido H. Clever*
For the biological function of oligonucleotides, not only the
primary sequence but also the precise secondary and tertiary
structure is essential, especially in the context of protein
interactions. DNA is known to form a number of secondary
structures beyond the double helix such as triplexes, the four-
stranded i-motif, and G-quadruplexes.[1] The latter result from
the self-assembly of guanine-rich oligonucleotides by Hoog-
steen base pairing, thus forming stacked guanine tetrads
stabilized by central cations. G-quadruplexes have gained
increasing attention,[2,3] as studies indicate that their in vivo
formation both prevents human telomere elongation, and
therefore shortens cancer cell lifetimes, and also plays a role
in the gene expression of oncogenes.[4]
Although the G-quartets themselves show little structural
diversity (syn/anti conformation of guanosine), complexity is
introduced by the relative strand orientation (parallel/anti-
parallel) and variations in the topology and sequence of
single-stranded loops.[5] Biomimetic G-quadruplexes with an
increased and switchable stability as well as diagnostically
exploitable properties such as fluorescence[6] and magnetism
may be useful for the labeling and capturing of specific
binders from a biological matrix.[7] It is thus of great interest
to design discrete G-quadruplex probes which allow for
control over the de- and rehybridization thermodynamics and
kinetics, the choice of loop sequence and topology, and the
introduction of nonbiogenic functionalities by automated
DNA synthesis.[8] In addition, the selective formation of such
functionalized, bioartificial G-quadruplex constructs may
prove valuable in the emerging field of DNA nanotechnol-
ogy.[9,10]
metal–base pairing has demonstrated that the synthetic
exchange of the natural nucleobases for ligands allows for
the incorporation of transition metals inside duplex
DNA.[12,13] Both the higher stability of the metal–ligand
bonding (as compared to the H-bonding in Watson–Crick
base pairs) and the unique magnetic and electronic properties
of the incorporated metals have led to a number of functional
nanodevices. Examples include a system capable of a rever-
sible hairpin–duplex transformation,[14] Ag/Hg-selective mul-
tiplex sensors,[15] ferro- and antiferromagnetically coupled
linear arrays,[16] programmable mixed-metal stacks,[17] and
metal-controlled single-molecule conductors.[18]
All of these functional systems, however, are based on
double-stranded DNA. With respect to other DNA secondary
structures, only a small number of metal-binding architectures
such as triplexes[19,13a] and three-way junctions[20] have been
reported.[21] In particular, only very few examples of G-
quadruplexes carrying covalently bound metal complexes
have been reported including the binding of mercury to
thymine bases in the loops of a bimolecular G-quadruplex,[22]
the cleavage of human telomeric sequences by a covalently
attached chelate–cerium(IV) complex,[23] and a bimolecular
G-quadruplex with bipyridine units in the loops.[24]
Here, we report the first tetramolecular G-quadruplex
terminated by a “metal–base tetrad”, in which each of the
guanosines in one guanine-tetrad motif is formally replaced
by a monodentate pyridine ligand. Together the four pyridine
units coordinate to a transition metal ion such as CuII or NiII in
a square-planar fashion, thus significantly stabilizing the
whole assembly towards thermal denaturation. The reversi-
bility of the metal-triggered quadruplex stabilization as
monitored by melting temperature studies, circular dichroism
(CD), and gel electrophoresis is presented. Additionally,
electron paramagnetic resonance (EPR) spectroscopic evi-
dence for the nature of the metal coordination is given.[16a,b]
The oligonucleotides reported in this work contain
a guanine sequence (n = 3–5), which is necessary for the
quadruplex formation, and a pyridyl donor functionality
attached to the 5’-end of the strand by means of a propane-
1,3-diyl linker. This linker length was chosen based on
a molecular modeling study (see Figure 1c and the Support-
ing Information). The synthesis commences with the attach-
ment of the linker to 4-chloropyridine hydrochloride (1).
Subsequent phosphorylation leads to phosphoramidite 2,
which is then used in the automated solid-state oligonucleo-
tide synthesis yielding the single strands L1d(Gn) (Figure 1a,
see the Supporting Information for experimental details).
As with unmodified G-quadruplexes, the formation of
[L1d(Gn)]4 requires a high electrolyte concentration and low
temperatures (Figure 1b).[25] The annealing process is slow
Up until now studies concerned with the stabilization and
labeling of G-quadruplexes focused mainly on the binding of
planar organic compounds and flat metal complexes through
noncovalent interactions such as end-on p-stacking and
intercalation.[2c,11] In recent years, however, the concept of
[*] D. M. Engelhard, Prof. Dr. G. H. Clever
Institute of Inorganic Chemistry
Georg-August University Gçttingen
Tammannstrasse 4, 37077 Gçttingen (Germany)
E-mail: gclever@gwdg.de
Dr. R. Pievo
Max Planck Institute for Biophysical Chemistry
Am Fassberg 11, 37077 Gçttingen (Germany)
[**] This work was funded by the DFG IRTG 1422. D.M.E. thanks the
Fonds der Chemischen Industrie for a PhD scholarship. We thank
Prof. Dr. U. Diederichsen and Prof. Dr. M. Bennati for access to their
spectrometers.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2013, 52, 12843 –12847
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
12843