SUPPLEMENTARY INFORMATION

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1 DOI: /NCHEM.1397 Crystal structures of Λ-[Ru(phen) 2 dppz] 2+ with oligonucleotides containing TA/TA and AT/AT steps show two intercalation modes Hakan Niyazi a, 1 James P. Hall a, Kyra O Sullivan c, Graeme Winter b, Thomas Sorensen b, John M. Kelly c and Christine J. Cardin a. a Department of Chemistry, University of Reading, Whiteknights, Reading, RG6 6AD, United Kingdom b Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom c Department of Chemistry, Trinity College, Dublin 2, Ireland. 1 Current address : Institut Laue-Langevin, 6 Rue Jules Horowitz, Grenoble, France; European Synthrotron Radiation Facility, 6 Rue Jules Horowitz, Grenoble, France NATURE CHEMISTRY 1

2 Materials and Methods Text T1 Preparation of Crystals Containing CCGGTACCGG Racemic [Ru(phen) 2 (dppz)].2cl was prepared according to a literature method 1. The oligonucleotide d(ccggtaccgg) was custom synthesised and doubly purified by hplc by ATDBio (Southampton) Ltd. The crystallization was performed, using the sitting drop method, by adding 1µl of 4mM rac-[ru(phen) 2 (dppz)] 2+ to a drop containing 1 µl of 1 mm d(ccggtaccgg) 2, 1µl of 12mM spermine, 1µL of 10% 2-methyl-2,4-pentanediol, 1µL of 40mM sodium cacodylate ph 7.0, 1µL of 80mM NaCl and 1µL of 20mM BaCl 2. This was equilibrated against 1ml of 35% 2-methyl-2,4-pentanediol. Orange crystals, approximately 30x30x30 µm in size and only containing Λ-[Ru(phen) 2 (dppz)] 2+, appeared after 4 days at room temperature. The data were collected on beamline I04 at Diamond Light Source from a flash-cooled crystal at 100 K. As on I04 the wavelength determines the size of the X-ray beam, the wavelength chosen was Å, which gave a beam size of 9µm x 2µm, and gave anomalous signal with a mid-slope of anomalous normal probability of A single 90 wedge was collected giving 180 frames with a 0.5 oscillation. The data were processed using xia2 3-5 with XDS 6-8 giving 2908 unique reflections to 2.10 Å resolution. The phasing was determined using single wavelength anomalous dispersion using the anomalous scattering of barium using SHELXC/D/E 9 with the hkl2map 10 interface. Three heavy atoms were located per asymmetric unit and were assigned to be one barium cation and one ruthenium cation at 100% occupancy and one ruthenium cation at 50% occupancy due to it being on a crystallographic two-fold axis. The model was built using Coot 11 and refined using Refmac 12 version 5.6 from the CCP4 13 suite to give a final R cryst of 0.17 and R free of 0.23 with 5% of reflections reserved for the R free test. Figures were drawn using PyMol 14. The model was deposited in the Protein Data Bank with ID code 3U38. NATURE CHEMISTRY 2

3 Text T2 Preparation of Crystals Containing CCGGATCCGG The oligonucletide d(ccggatccgg) was synthesised and doubly purified by HPLC by ATDBio (Southampton) Ltd. The crystallization was performed, using the sitting drop method, by adding 1ul of 3 mm Λ-[Ru(phen) 2 (dppz)].cl 2, 1ul of 3 mm Δ-[Ru(TAP)2(dppz- (Me) 2 )].Cl 2, 1ul of 1 mm d(ccggatccgg) 2 and 6 ul of 10% (v/v) 2-methyl-2,4-pentanediol, 40 mm sodium cacodylate ph 7, 12 mm spermine tetra-hcl, 80 mm potassium chloride and 20 mm barium chloride. Crystals of approximate dimensions 0.2 x 0.2 x 0.1mm grew after several weeks. The crystals grew in space group P with cell dimensions a = b = Å, c = Å, α = β= γ = 90. A single crystal of approximate dimensions 0.2 x 0.2 x 0.1 mm was mounted on a MicroMesh (MiTeGen). Data were collected on I02 at Diamond Light Source Ltd. Data were collected from a flash-cooled crystal at 100 K using radiation with a wavelength of Å with a beam size of 75 x 25 µm. The data gave anomalous signal with a mid-slope of anomalous normal probability of A single 120 wedge was collected giving 120 images with 1 oscillation. Data were processed using Xia2 3, with XDS 6 and Scala 15, giving 4747 unique reflections to 1.70 Å resolution. The phasing was determined by singlewavelength anomalous dispersion, using the anomalous scattering of barium, using the SHELXC/D/E 9 pipeline in the CCP4 13 suite. Two heavy atoms were located per asymmetric unit and were assigned to be one ruthenium cation at 100% occupancy and one barium cation at 100% occupancy. An initial model was build using Coot 11 and refined using REFMAC to give a final R cryst of and Rfree of with 5% of reflections reserved for the R free test. The model was deposited in the Protein Data Bank with ID code 4E7Y. References 1. Hiort, C., Lincoln, P. & Norden, B. DNA binding of Δ- and Λ-Ru(phen) 2 dppz] 2+. J. Am. Chem. Soc. 115, (1993). 2. Evans, P. R. Proceedings of the CCP4 study weekend (1997). NATURE CHEMISTRY 3

4 3. Winter, G. Xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, (2010). 4. Sauter, N. K., Grosse-Kunstleve, R. W. & Adams, P. D. Robust indexing for automatic data collection. J. Appl. Cryst. 37, (2004). 5. Zhang, Z., Sauterm N. K., Van den Bedem, H., Snell, G. & Deacon, A. M. Automated diffraction image analysis and spot searching for high throughput crystal screening. J. Appl. Cryst. 39, (2006). 6. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, (1993). 7. Kabsch, W. Automatic indexing of rotation diffraction patterns. J. Appl. Cryst. 21, (1988). 8. Kabsch, W. Evolution of single-crystal X-ray diffraction data from a position sensitive detector. J. Appl. Cryst. 21, (1988). 9. Sheldrick, G. M. A short history of SHELX. Acta Cryst. D. 64, (2008). 10. Pape, T. & Schneider, T. R. HKL2MAP: a graphical user interface for phasing with SHELX programs. J. Appl. Cryst. 37, (2004). 11. Emsley, P., Lohkamp, B., Scott, W. & Corwan, K. Features and development of Coot. Acta. Cryst. D. 66, (2010). 12. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta. Cryst. D. 53, (1997). 13. Collaborative computational project, number 4. The CCP4 suite: programs for protein crystallography. Acta Cryst. D. 50, (1994). 14. The PyMOL Molecular Graphics System, Version 1.2r3Pre, Schrödinger, LLC. 15. Evans, P. Scaling and assessment of data quality. Acta. Cryst. D. 62, (2006). NATURE CHEMISTRY 4

5 Table S1 Data collection and refinement statistics for PDB entries 3U38 and 4E7Y Data Collection CCGGTACCGG CCGGATCCGG Space group P P Cell dimensions, Å a=b=52.55, c=32.39 a=b=47.72, c=34.66 Resolution, Å *( ) ( ) R merge (0.552) (0.804) I/σI 14.7 (3.0) 13.2 (3.2) Completeness, % 99.7 (100.0) (100.0) Multiplicity 6.3 (6.6) 8.9 (9.2) *Outer shell statistics shown in parentheses Refinement No. Reflections R work /R free 0.17/ /0.18 No. Atoms DNA Ligands Water B factors DNA Ligands Water rmsd Bond lengths, Å Bond angles, PDB ID 3U38 4E7Y NATURE CHEMISTRY 5

6 Table S2 Local base-pair step parameters, calculated by 3DNA. This program is available at URL CCGGTACCGG Step Shift Slide Rise Tilt Roll Twist Context1 Context2 C+GC/ CCG CGG- CG/CG CCGG CCGG GG/CC CGGT ACCG GT/AC GGTA TACC TA/TA GTAC GTAC AC/GT CC/GG TACC GGTA ACCG CGGT CG/CG CCGG CCGG GG+C/ CGG- -CCG CCGGATCCGG Step Shift Slide Rise Tilt Roll Twist Context1 Context2 C+GC/ CCG CGG- CG/CG GG/CC GA/TC CCGG CCGG CGGA TCCG GGAT ATCC AT/AT GATC GATC TC/GA ATCC GGAT CC/GG TCCG CGGA CG/CG CCGG CCGG GG+C/ CGG- -CCG NATURE CHEMISTRY 6

The orientation of the long axis of each basepair is defined by the position of the C1 carbon atoms of the ribose sugar rings.

7 Figure S1 a) The centroid of the four atoms T5(C4) and A6(C8) used to define an estimated distance between the ruthenium atom and the helix axis. The distance is 5.27 Å. b) the perpendicular intercalation geometry. The orientation of the long axis of each basepair is defined by the position of the C1 carbon atoms of the ribose sugar rings. These axes cross at an angle of approximately 40 in this case. The long axis of the dppz ligand is perpendicular to the mean of these two basepair axes, which in this case is parallel also to the P-P vector. a b NATURE CHEMISTRY 7

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy James P. Hall 1,2, Fergus E. Poynton 3,4, Páraic M. Keane 1,3, Sarah P. Gurung 1,2, John A. Brazier