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1 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 5, David J. Cardin 1, Graeme Winter 2, Thorfinnur Gunnlaugsson 3,4, Igor V. Sazanovich 6, Michael Towrie 6, Christine J. Cardin 1*, John M. Kelly 3* and Susan J. Quinn 7* 1 Department of Chemistry, University of Reading, Whiteknights, Reading RG6 6AD, United Kingdom 2 Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0QX 3 School of Chemistry, Trinity College, University of Dublin, Dublin 2, Ireland 4 Trinity Biomedical Science Institute, (TBSI) Trinity College Dublin, Dublin 2, Ireland 5 Department of Pharmacy, University of Reading, Whiteknights, Reading RG6 6AD, United Kingdom 6 Central Laser Facility, Research Complex at Harwell, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, Oxfordshire, OX11 0QX, UK 7 School of Chemistry, University College Dublin, Dublin 4, Ireland c.j.cardin@rdg.ac.uk, jmkelly@tcd.ie, susan.quinn@ucd.ie NATURE CHEMISTRY 1

2 Table of Contents Item Page Number Methods 3-5 Tables S1-2 and Figure S1 6-8 Figures S2 and S Figure S4 11 Figures S Figure S8 14 Figure S9 15 Figure S10 16 Figure S11 17 Supplementary References 18 Supplementary Video 18 NATURE CHEMISTRY 2

3 Supplementary Methods Crystallization The oligonuclotide d(tcggcgccga) was purchased from ATDBio as a solid purified by double- HPLC. Crystals containing the nucleotide and the complex Λ [Ru(TAP) 2 (dppz)]cl 2 were grown using vapour diffusion from sitting drops at 18 C. The drops contained 1 µl of 2 mm single- stranded oligonuclotide, 1 µl of 4 mm rac [Ru(TAP) 2 (dppz)]cl 2 and 6 µl of the following solution; 10% (vol/vol) 2- Methyl- 2, 4- pentanediol (MPD), 40 mm sodium cacodylate (ph 6.0), 12 mm spermine tetra- HCl, 80 mm sodium chloride and 20 mm barium chloride. The drop was equilibrated against 1 ml of 35% (vol/vol) 2- methyl- 2,4- pentanediol (MPD) in H 2 O and reddish- yellow rhombohedra appeared after approximately one week. The crystals were grown in 24- well CrysChem sitting drop plates. Reference to crystal numbers, in terms of number of plates, is therefore to be taken as a multiple of 24 wells so, for example, two plates would contain 48 wells. D 2 O Crystal Structure A crystal was removed from the crystal growth solution, using a MicroLoop from MiTeGen to minimise liquid around the crystal, and placed into 50 μl of 35% MPD (v/v) in D 2 O. This was allowed to soak for approximately 30 minutes before being flash- cooled in liquid nitrogen. The data were collected on beamline I03 at Diamond Light Source using radiation with a wavelength of Å. The sample, of approximate dimensions 100 x 100 x 80 μm, was kept in the nitrogen cryostream at 100 K and a total of 720 of data were collected in two sweeps, the first at 10% transmission and the second at 100%. Each sweep consisted of frames with a 0.05 s exposure time. The two sweeps were scaled and merged together using xia2 1, with XDS 2 and SCALA 3, with the additional option flags misigma 0.8 and isigma 0.4. These had to be used to extend the data in the highest resolution shell to bring the final I/σI to 2.2 and a final outer shell resolution of 1.21 Å, with a total of unique reflections with an overall fold multiplicity. The R merge is very high, at over 170%, but this was not weighted against the multiplicity of the data. R pim, which is weighted, 4 and CC- half are far more suitable metric and give reasonable values for the outer shell data at 37% and 0.74 respectively. The overall R pim is only 1.3% indicating that the intensities are very precisely known. Initial phases were found, using the anomalous scattering of barium, with the SHELXC/D/E 5 pipeline in CCP4, 6 and the structure was built using Coot. 7 The structure was then refined anisotropically using REFMAC5 8 and hydrogen atoms were placed in riding positions. As the spectroscopic data reported here indicates that hydrogen atoms have been replaced by deuterium, all exchangeable hydrogen atoms and solvent molecules were substituted for D in the final set of coordinates. The model was then refined to give a final R factor of 0.10 and R free of During refinement REFMAC automatically optimised the weight of the X- ray term relative to the experimental restraints. This gave a final weighting of 2.56 to the X- ray data meaning that restraints were only loosely applied. The model is deposited in the Protein Data Bank with ID 4R8J. The structure is not significantly different to our previously reported structure, 3QRN, showing that D 2 O exchange has little effect. Full data collection and refinement statistics are displayed in table S1. NATURE CHEMISTRY 3

4 Crystal Extraction, Washing and Exchange Procedure Crystals were removed from drops by aspiration and pooled into a single tube which then underwent gentle centrifugation (6000rcf for 2 minutes), after which as much of the supernatant solution was removed as possible to leave the crystals in the minimum amount of fluid (1-4 μl). 300 μl of 35% MPD, in H 2 O or D 2 O as appropriate, was then added and the crystals were resuspended in solution by gentle agitation. The sample then underwent gentle centrifugation and again the supernatant solution was removed. This washing process was repeated a minimum of five times, after which the supernatant was removed, leaving the crystals in 1-4 μl of solution. UV/visible spectra, showing a lack of crystal dissolution after washing, can be found in figure S1. Crystal Mounting Procedure: D 2 O Crystals Four trays of crystals were dispersed onto a reduced size CaF 2 plate which consisted of a central area, with an 8 mm diameter for the sample surrounded by a bevel. The sample, between 1-4 μl in volume, was dispensed into the centre of the disk. A second disk was placed on top and was twisted to disperse the sample. The path length of this plate was 20 μm, see Fig. S2. Crystal Mounting Procedure: H 2 O Crystals Two trays were dispersed onto an 8 mm CaF 2 plate. The crystals were suspended in 10 μl of 35% MPD solution and dispensed in the shape of a cross. A second plate was then placed on top and was twisted to disperse the crystals. As no spacer was used, the approximate path length was 3 μm. Crystal Characterisation Controls Characterisation by X- ray Diffraction A solution was needed to maintain the relative humidity around the crystals but which contained no DNA or ruthenium complex. The crystals were equilibrated against 35 % MPD when grown and therefore this was used as a suspension solution. Additionally, the crystals needed to be checked to ensure that they retained the same structure when soaked in 35% MPD and crushed, mimicking what would occur between the CaF 2 plates. Several crystals were removed from their growth solution and were soaked in 35% MPD for at least 30 minutes. The crystals were then mounted on beamline I04 at Diamond Light Source and 3 frames of data were collected with 45 separation. These were then indexed by EDNA and gave the unit cell as approximately 42x42x39 Å, with variance in the first decimal place crystal- to- crystal, and with space group P4, which is consistent with our previously reported structure. Next, crystals which had been soaked in 35% MPD were broken into fragments using a loop. These fragments were mounted in a MicroMesh and exposed to the X- ray beam. In several cases the fragments were approximately 20 μm x 15 μm x 10 μm and as such a 10 μm aperture was used to reduce the exposed area. The diffraction pattern for an approximately 10 μm 3 fragment, illustrated in Fig. S3, was indexed using Mosflm 9 as having a tetragonal lattice with unit cell dimensions of a = 42.3 Å and c = 39.8 Å, which is consistent with the known space group and unit cell for the structure. NATURE CHEMISTRY 4

5 Steady State Infrared Measurements Spectra were recorded using a Perkin- Elmer Spotlight 400 imaging system attached to a Perkin- Elmer 100 FTIR spectrometer, allowing for a 10 μm beam size, at the Chemical Analysis Facility in the University of Reading. Time Resolved Infrared Measurements The ULTRA instrument has been described in detail elsewhere. 10 In brief, the experiments were carried out in the Time Resolved Multiple Probe, TRMPS, mode of the instrument. 11 The mid IR probe was generated by difference frequency conversion of the signal and idler output of a 10 khz, 8W, 40 fs, 800 nm titanium sapphire laser pumped optical parametric amplifier and the pump by second harmonic of a 1 khz, 4W, 120 fs, 800 nm titanium sapphire laser. By operating in TRMPS mode TRIR spectra spanning picosecond to microsecond timescales were produced and exposure of the crystal samples to the pump light minimised. The pump and probe beams were focused to approximately 150 and 100 microns diameter in the sample and set to magic angle. TRMPS data were acquired in sets of 10 probe pulses with the relative delay between pulses of the 1 and 10 khz pulse trains controlled between 0 and 100 μs with sub- ps accuracy using optical and electrical delays. The multiple probing accesses delay times, t, t μs, t μs, etc. up to t μs, for each pump probe delay, t, thus ten probe measurements are made for each pump pulse. The last probe pulse in the set of ten is used as a background and subtracted from the first nine to create the difference spectra presented. Kinetic Treatment of TRIR Data Data was analysed using mono- exponential and bi- exponential fitting functions using Origin 8 software. A feature of all the recorded TRIR spectra is the presence of a broad unstructured transient absorbance spanning the spectral window. The decay of this process was removed from the dynamics of the DNA- bound metal complex by analysing the ratio of transient absorbance and corresponding bleach depletion as a function of time. Thus to obtain the rise/decay trace of the guanine radical cation feature free of that broad spectral offset, we have plotted absorbance expressed as the difference between the absorbance monitored at 1709 cm - 1 and 1684 cm - 1. NATURE CHEMISTRY 5

6 Supplementary Tables S1-2 and Figure S1 The majority of the volume of heavy water resides in a channel which runs through the crystal lattice, and is also found in the DNA grooves where the majority of ordered water in the sample resides, Table 2 and Fig. S1. The D 2 O- soaked crystal structure has been deposited in the Protein Data Bank with ID 4R8J. This table lists the information regarding the data collection and refinement statistics for the D 2 O exchanged crystals and the water contributions in the crystal structure (v/v) unit cell. Table S1: Data collection and refinement statistics for 4R8J Data Collection Space group P Cell dimensions, Å 42.37, 42.37, Resolution, Å ( )* R merge (1.756) R pim (0.378) I/σI 29.6 (2.2) Completeness, % 99.9 (100) Multiplicity 42.8 (23.0) *Outer shell statistics are given in parentheses Refinement No. Reflections R work /R free 0.10/0.12 No. Atoms DNA 315 Ligands 75 D 2 O 74 Average B- Factors DNA Ligands D 2 O rmsd Bond lengths, Å Bond angles, PDB ID 4R8J NATURE CHEMISTRY 6

7 The volume of the unit cell contents were calculated using 3V 12 with a probe radius of 0 to obtain the volume of the van de Waals surface. The PDB file containing the asymmetric unit of the crystal was edited to remove different components, resulting in three models, from which the volumes were obtained. The volumes were then multiplied by 8 as the unit cell consists of eight asymmetric units. The three models contained: 1. The DNA and ligands only 2. DNA, ligands and H 2 O/D 2 O molecules which form a direct hydrogen bond to the biomolecule ( bound waters ) 3. DNA, ligands and all ordered water molecules in the crystal structure From this, subtractions were made to find the volume of each of the following components: 1. All ordered water 2. DNA- bound water 3. Unbound, ordered water 4. All solvent (including disordered void space) 5. Non- water components (DNA, ligand, Ba 2+ ) 65.17% of the crystal is made up from ordered water and 35% MPD in D 2 O. This includes all bound water molecules and void space within the lattice. Therefore, D 2 O would make up 42.38% of the crystal volume with 22.79% of the crystal consisting of MPD. The intercalated DNA makes up the remaining 34.83% of the crystal volume. Table S2: Analysis of the unit cell contents Number of Water Molecules: In the crystallographic model 74 In the model and with polar contacts to the DNA 58 Percentage of observable waters bound to the DNA (%) 78 Volumes of the model: DNA and ligands only, Å DNA, ligands and bound water, Å DNA, ligands and all water in model, Å Unit Cell Contents Volume, Å 3 Percentage volume of unit cell, % All ordered water, Å Only DNA- bound water, Å Unbound water (ordered and void space), Å All water (ordered and void space), Å Non- water components (DNA and ligands), Å Percentage of total water which is bound to DNA, % NATURE CHEMISTRY 7

8 Figure S1: The surface from which the volume of the intercalated DNA, in the asymmetric unit, was calculated. The model of the asymmetric unit was edited to remove all water molecules. T 1 is shown in orange with A 10 in purple. The rest of the bases are coloured on a gradient. Calculations were performed using 3V. 12 NATURE CHEMISTRY 8

9 Supplementary Figure S2 and Figure S3 The crystals are found to be stable to washing procedures with no trace of the metal complex detected in the washings by UV- visible spectroscopy. The unit cell for the structure shows that the structure of the crystal is unaffected by soaking or mechanical dispersion, Fig. S2 and Fig. S3. This figure shows comparative UV/visible spectra, which demonstrate a lack of crystal dissolution after washing. Figure S2: UV/visible absorbance spectra of several supernatant solutions obtained during crystal washing. The absorbance spectrum of 100% 2- methyl- 2,4- pentanediol (MPD) shows a clear absorbance at 210 nm. This matches to the spectrum in red for 35% MPD in H 2 O where a similar absorbance can be observed. A spectrum of the crystal growth solution, which has been diluted by a factor of three using H 2 O, is displayed in blue. A large absorbance peak at 274 nm is attributed to a combination of absorbances from both the Λ [Ru(TAP) 2 (dppz)] 2+ complex and DNA. Absorbance bands at 414 nm and 465 nm are attributed to the Λ [Ru(TAP) 2 (dppz)] 2+ complex. Note that only the MPD absorbance band is present in the final crystal wash solution (pink) indicating that the crystals do not dissolve in the solution. Spectra were recorded using a PerkinElmer 1040 spectrophotometer in a 1 mm path length quartz cell. NATURE CHEMISTRY 9

10 Supplementary Figure S3 This figure provides crystallographic data showing that the structure of the crystal is not affected by soaking or mechanical dispersion Figure S3: (Top) A fragment from a crystal of d(tcggcgccga) with Λ- [Ru(TAP)2(dppz)]2+ which has been soaked in 35% (v/v) MPD. The beam size is indicated by the crosshair which has a 10 μm diameter. {Bottom) A quarter of the resulting diffraction image, collected on beamline I04 at Diamond Light Source, collected from the fragment. This has been indexed by Mosflm as having point group P 4 with unit cell dimensions of a = 42.3 Å and c = 39.8 Å. This is consistent with known space group and unit cell for the structure showing that the structure of the crystal is not affected by soaking or mechanical dispersion. The image of the crystal fragment has been sharpened, and had its brightness and contrast increased. NATURE CHEMISTRY

11 Supplementary Figure S4 This figure shows the ground- state infrared spectra recorded for the crystal samples. Figure S4: Single- crystal FTIR spectra of Λ- [Ru(TAP) 2 (dppz)] 2+ bound to (TCGGCGCCGA) 2 in (a) H 2 O and (b) D 2 O. NATURE CHEMISTRY 11

12 Supplementary Figures S5-7 This figure shows the solution TRIR spectra for the complex Figure S5 TRIR spectrum of 400 µm Λ- [Ru(TAP) 2 (dppz)] 2+ at 2 ns delay after 400 nm (120 fs, 1 µj, 10 khz) excitation. In phosphate buffered (50 mm, ph 7) D 2 O Figure S6 TRIR spectra of Λ- [Ru(TAP) 2 (dppz)] 2+ bound to (TCGGCGCCGA) 2 in (a) crystal state and (b) solution at 2 ns delay after 400 nm (120 fs, 1 µj, 1 khz) excitation. 13 Solution conditions = 400 µm Λ- [Ru(TAP) 2 (dppz)] 2+, 500 µm (TCGGCGCCGA) 2 in phosphate buffered (50 mm, ph 7) D 2 O. a a The concentrations for the solution work are 400 µm metal complex; 1 mm ss- ODN (i.e. 500 µm duplex ODN). These conditions were chosen so as to minimise the concentration of un- bound metal complex, as the presence of free metal complex would complicate the kinetic analysis. NATURE CHEMISTRY 12

13 Supplementary Figure S7 Figure S7 FTIR spectra of Λ- [Ru(TAP) 2 (dppz)] 2+ bound to (TCGGCGCCGA) 2 in D 2 O crystal state and in 400 µm Λ- [Ru(TAP) 2 (dppz)] 2+, 500 µm (TCGGCGCCGA) 2 in phosphate buffered (50 mm, ph 7) D 2 O. NATURE CHEMISTRY 13

14 Supplementary Figures S8 This figure profiles the dynamics of the broad background absorption observed upon excitation at 400 nm. Figure S8 Kinetics showing the time- dependent absorption of the broad transient signal for uncorrected spectra after laser excitation (400 nm, 120 fs) of Λ- [Ru(TAP) 2 (dppz)] 2+ bound to (TCGGCGCCGA) 2 in the D 2 O crystal. b b A feature of all the recorded TRIR spectra is the presence of a broad unstructured transient absorbance that spans the spectral window. This is attributed to low energy intraband transitions which have been previously observed for TRIR spectra of transition metal complexes. 14 The kinetics of this process are determined by monitoring the decay of this signal at wavenumbers where neither the DNA nor the metal complex absorb (1750 cm - 1 ). NATURE CHEMISTRY 14

15 Supplementary Figures S9 This figure shows the TRIR spectra showing grow in of the oxidised guanine photoproduct after laser excitation and the corresponding kinetic analysis. Figure S9 TRIR spectra showing grow in of the oxidised guanine photoproduct after laser excitation (400 nm, 120 fs) of 400 µm Λ- [Ru(TAP) 2 (dppz)] 2+ bound to (TCGGCGCCGA) 2 in the D 2 O crystal. Kinetic analysis obtained by plotting the difference between the transient band at 1709 cm - 1 and bleach at 1684 cm - 1. c c A feature of all the recorded TRIR spectra is the presence of a broad unstructured transient absorbance that spans the spectral window. This is attributed to low energy intraband transitions which have been previously observed for TRIR spectra of transition metal complexes. This absorbance is found to decay over 6.5 ns. The kinetics of this phenomenon are subtracted from the system kinetics by performing a band ratio analysis of transient and corresponding bleach bands. 14 NATURE CHEMISTRY 15

16 Supplementary Figure S10 Figure S10 Relationship of the guanine G 9 and the dppz planes. Program Mercury was used to define the geometric relationships between these planes. The figure shows the projection onto the plane of the guanine G 9 ring system. The perpendicular distances to this plane from the two separate phenazine nitrogen atoms are as shown, 3.38 Å and 3.24 Å. Therefore the mean of these distances is a good estimate of the interplanar distance, 3.32 Å. NATURE CHEMISTRY 16

17 Supplementary Figure S11 This figure shows the TRIR spectra showing grow in of the oxidised guanine photoproduct after laser excitation and the corresponding kinetic analysis for H 2 0 crystals. Figure S9 Transient infrared absorption spectra for H 2 O Crystals. TRIR spectra of Λ- [Ru(TAP) 2 (dppz)] 2+ bound to (TCGGCGCCGA) 2 after 400 nm (120 fs, 1µJ, 1 khz) at delays (a- d) and kinetic analysis of the loss of the absorbance at 1712 cm - 1. NATURE CHEMISTRY 17

18 References 1. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, (2010). 2. Kabsch, W. XDS. Acta. Crystallogr. D66, (2010). 3. Evans, P. Scaling and assessment of data quality. Acta. Crystallogr. D62, (2006). 4. Karplus, P. A., & Diederichs, K. Linking cystallographic model and data quality. Science. 336, (2012). 5. Sheldrick, G. M. A short history of SHELX. Acta. Crystallogr. A64, (2008). 6. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta. Crystallogr. D50, (1994). 7. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta. Crystallogr, D66, (2010). 8. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of Macromolecular Structures by the Maximum- Likelihood Method. Acta. Crystallogr. D53, (1997). 9. Leslie, A. G. W. & Powell, H. R. Processing Diffraction Data with Mosflm in Evolving Methods for Macromolecular Crystallography. Edited by Read, R.J. & Sussman, L.J (2007). Dordrecht: Springer. 10. Greetham, G. M. et.al. ULTRA: a unique instrument for time- resolved spectroscopy. Appl. Spectrosc. 64, (2010) 11. Greetham, G. M., Sole, D., Clark, I. P., Parker, A. W., Pollard, M. R. & Towrie, M. Time- resolved multiple probe spectroscopy. Rev. Sci. Instrum. 83, (2012) 12. Voss, N. R. & Gerstein, M. 3V: cavity, channel and cleft volume calculator and extractor. Nucleic Acids Res. 38, W555- W562 (2010). 13. Keane, P. M. et al. Enantiomeric conformation controls rate and yield of photo- induced electron transfer in DNA sensitized by Ru(II) dipyridophenazine complexes, J. Phys. Chem. Lett. 6, (2015). 14. Sazanovich, I. V., Alamiry, M. A. H., Meijer A. J. H. M., Towrie, M., E. S. Davies, E. S., R. D. Bennett, R. D. & Weinstein, J. A. Photoinduced charge separation in a PtII acetylide donor- acceptor triad based on 2- (1- pyrazole)- pyridine modified with naphthalene mono- imide electron acceptor, Pure Appl. Chem., 85, , (2013) Video 1 The crystal structure of the DNA duplex with bound ruthenium complexes and the site of photo- oxidation. NATURE CHEMISTRY 18