The ellipsoidal electron density implies the Ba 2+ ion disorder.

1. Wavelength (Å) Wavelength (Å) Wavelength (Å) Wavelength (Å) 0.8126 0.8416 0.8416 0.8416 Unit Cell (Å) Unit Cell (Å) Unit Cell (Å) Unit Cell (Å) a=24.81 b=24.81 c=92.08 a=22.53 b=58.47 c=24.15 a=24.79 b=24.79 c=91.82 a=22.01 b=59.54 c=47.67 Spacegroup Spacegroup Spacegroup Spacegroup C2221 P6222 C222 P6222 Resolution Range (Å) Resolution Range (Å) Resolution Range (Å) Resolution Range (Å) 20-1.40 5.80-1.00 30.57-2.85 30.71-2.18 Completeness (%) Completeness (%) Completeness (%) Completeness (%) 96.8 98.5 95.5 98.4 Rmerge(%) Rmerge(%) Rmeas (%) Rmerge(%) 9.5 8.20 15.72 7.30 R-factor (%) R-factor (%) R-factor (%) R-factor (%) 24.80 14.93 22.40 18.62 R-free (%) R-free (%) R-free (%) R-free (%) 20.93 30.90 18.38 24.60 Refinement statistics Refinement statistics Refinement statistics The ellipsoidal electron density implies the Ba2+ ion disorder. 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 withRefmac, 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: 8335.6eV;1.4874Å • Inflection Point: 8329.8eV;1.4884Å • Remote: 9029.9eV;1.3730Å 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, 5%MPD, 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, 10%MPD 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.