Both Ba2 and K ions stabilise T4 residues directly through their O2 and O4 positions. K also mediate

1
INFLUENCE OF METAL ION COORDINATION ON THE
d(GCATGCT) QUADRUPLEX STRUCTURE
Y.Gan, J. H. Thorpe, S. C. M. Teixeira, B. C.
Gale, M. I. A. Moraes, N. Hopcroft and C. J.
Cardin School of Chemistry, University of
Reading, UK.
The structure and dynamics of nucleic acid
molecules are influenced by a variety of
contributions. Among them, the interactions
(either directly or indirectly) present between
metal ions and bases, phosphates and sugar
moieties are particularly significant. The
affinity of a metal ion for a specific site on a
nucleic acid is a general function of its charge,
hydration-free energy, coordination geometry, and
coordinate bond-forming capacity. Here we report
the X-ray crystallographic structure solution of
four DNA quadruplex structures formed by the DNA
heptanucleotide sequence d(GCATGCT), under the
influence of Ba2, K, V3, Ni2, and Co2 to
resolutions of 1.4 Å, 1.0Å, 2.18Å and 2.85Å
respectively.
X Y
Z B Z
F.O.M. Solution Ni2 0.173 0.094
0.074 35.90 4.94 0.53
d(GCATGCT) Ni2 Crystals grew from sitting
drops containing 40mM Na Cacodylate (pH 5.7), 5mM
NiCl2, 5mM KCl, 2 MPD, and 1mM DNA equilibrated
against a 1ml reservoir of 35 MPD, to dimensions
of approximately 0.6 x 0.1 x 0.1 mm. The
structure was solved through MAD phasing at the
Ni edge. The MAD data was collected with the
anomalous diffraction conditions for the nickel K
edge at the tuneable beam line BW7A. The phasing
was calculated with the program Solve-Resolve and
the native data set was collected on beam line
X11 to 2.18 Å, utilising a MAR-CCD detector, and
then processed and scaled with Mosflm and Scala.
The refinement was carried out with Refmac, water
divination achieved with Xtalview from sigma-A
and difference maps. The structure has been
deposited with the NDB (ID no. UD0043) .
d(GCATGCT) V3 Crystals grew from sitting drops
containing 40 mM Na Cacodylate (pH 5.7), 25mM
VCl2, 10mM NaCl, 10 MPD, and 1mM DNA
equilibrated against a 1ml reservoir of 35 MPD,
to dimensions of approximately 0.5 x 0.5 x 0.3 mm
and synchrotron data was collected on beam line
BW7B. The data was processed and scaled with XDS.
Anisotropic structural refinement was carried out
with Shelx-97, utilising the model UDG028.(3)

• From the fluorescence scan of the nickel (above),
  three wavelengths for MAD data collection were
  chosen
• Peak 8335.6eV1.4874Å
• Inflection Point 8329.8eV1.4884Å
• Remote 9029.9eV1.3730Å

The first map calculated by MAD phasing already
showed clear solvent regions and a stacking
direction. A zoom on the electron density showed
where to place the first 5 guanine (G1)
Refinement statistics
The vanadium cation tethers the DNA residues A3,
T4 and their symmetry related residues indirectly
through its primary and secondary hydration
spheres.
Normal probability plot of anomalous difference,
showing clear anomalous signal for the peak data.
Unlike the G-quadruplexes (1,2), which usually
have a coplanar geometry, the G/C pairs in this
quadruplex structure cross at an angle of 38º
approximately.
Both Ba2 and K ions stabilise T4 residues
directly through their O2 and O4 positions. K
also mediates the repulsion between the
intra-strand residues C2 and C6 through their O1P
positions. The Ba2 ion also interacts indirectly
with the DNA residues through its primary solvent
sphere.
d(GCATGCT) Co2 Crystals grew from sitting
drops containing 40mM Na Cacodylate (pH 6.5), 3mM
CoCl2, 2mM KCl, 5MPD, and 1mM DNA equilibrated
against a 1ml reservoir of 25 MPD, to dimensions
of approximately 0.6 x 0.1 x 0.1 mm. Synchrotron
data was collection on beam line BW7B, utilising
a MAR-345 image plate to a resolution of 2.85Å.
The data was then processed with Mosflm and
scaled with Scala. Structural refinement was
carried out with Refmac utilising the model
obtained from the Ni2 derivative (NDB ID
UD0043), with model fitting and water divination
achieved with Xtalview from sigma-A and
difference maps.The structure has been deposited
with the NDB (ID no. UD0042).
d(GCATGCT) Ba2 Crystals grew from sitting
drops containing 40 mM, Na Cacodylate (pH 5.9),
15mM BaCl2, 10mM KCl, 10MPD and 1mM DNA
equilibrated against a 1ml reservoir of 35 MPD,
to dimensions of approximately 0.6 x 0.5 x 0.2
mm. Synchrotron data was collected on beam line
BW7B, utilising a MAR-345 image plate to a
resolution of 1.4Å. The data was processed with
Mosflm and scaled with Scala. Anisotropic
refinement was carried out with Shelx-97
utilising the model UDG028, with model fitting
and water divination achieved with Xtalview from
sigma-A and difference maps calculated from
Shelxpro. The structure has been deposited with
the NDB (ID no. UD0040)
Ni2 binding cavity, illustrating the hydration
and symmetry related residues. The Ni2 bridges
guanine residues, G1 and G5 directly through
their N7 positions, with the primary hydration
shell coordinating, the octahedral geometry
expected for the cation.
Co2 bridging guanine residues G1 and G5 directly
through through their N7 positions.

• References
• Keniry, M. A. (2000), Biopolymers., 56, 123-146.
• 2. Kuryavyi, V., Majumdar, A., Shallop, A.,
  Chernichenko, N., Skripkin, E., Jones, R and
  Patel, D. J. (2001). J. Mol. Biol., 310, 181-194.
  
• Hunter, W. H., Leonard, G. A., Zhnag, S.,
  Perterson, M. R., Harrop, S. J., Helliwell, J. R.
  (1995) Structure, 3, 335-340.
• Thorpe, J. H., Teixeira, C. M., Gale, B. C.,
  Cardin, C.J., Nucleic Acids Res, 31, 1-6

Acknowledgments I would like to thank the School
of Chemistry at the University of Reading for
their generous funding of my work and to Syngenta
for kindly funding me to attend this meeting.
Also thanks to the staff at DESY synchrotron
facility, my supervisor Dr. Christine Cardin, Dr.
J. H. Thorpe and Dr. S. C. M. Teixeira and
everyone else in our group. For funding of the
research I would like to thank the Association
for International Cancer Research and Xenova Ltd.
Results and Conclusion This G/C quadruplex motif
was first reported by Hunter et al (3) in space
group C222, with a Mg2 in the lattice. Then
Thorpe et al (4) in our group reported a similar
structure under the influence of cobalt hexammine
in space group C2221. Here, similar structures
are observed under the influence of Ba2, K
(C2221) and V3 (C222), however, water molecules
were located in the heart of the quadruplex
structure to stretch the quadruplex open up in
both cases . Furthermore, in the presence of more
toxic metal ions Ni2, and Co2 the crystals
packed into space group P6222, and the structure
is distorted. Only one G/C quadruplex similar to
those found in the barium and vanadium cases is
retained due to the direct interactions between
these ions and the N7 of guanine residues. From
these results we can conclude that firstly,
cations can bind to the base edges either at
nitrogen or oxygen positions and they can also
bind to phosphate oxygen atoms. Secondly, the
hydrated ions should be viewed as hydrogen bond
donors in addition to their counter-ion
condensation effect.