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Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy

Abstract

To understand the molecular origins of diseases caused by ultraviolet and visible light, and also to develop photodynamic therapy, it is important to resolve the mechanism of photoinduced DNA damage. Damage to DNA bound to a photosensitizer molecule frequently proceeds by one-electron photo-oxidation of guanine, but the precise dynamics of this process are sensitive to the location and the orientation of the photosensitizer, which are very difficult to define in solution. To overcome this, ultrafast time-resolved infrared (TRIR) spectroscopy was performed on photoexcited ruthenium polypyridyl–DNA crystals, the atomic structure of which was determined by X-ray crystallography. By combining the X-ray and TRIR data we are able to define both the geometry of the reaction site and the rates of individual steps in a reversible photoinduced electron-transfer process. This allows us to propose an individual guanine as the reaction site and, intriguingly, reveals that the dynamics in the crystal state are quite similar to those observed in the solvent medium.

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Figure 1: Crystal structure in the presence of D2O.
Figure 2: Experimental scheme and microscopy controls for the time-resolved infrared spectroscopy experiments on crystal samples.
Figure 3: Transient infrared absorption spectra obtained from D2O-exchanged crystal samples of Λ-[Ru(TAP)2(dppz)]2+ bound to (TCGGCGCCGA)2.
Figure 4: Proposal for the site of reversible electron transfer from guanine to Λ-[Ru(TAP)2(dppz)]2+ bound to (TCGGCGCCGA)2.
Figure 5: Summary of reversible oxidation of the guanine site in Λ-[Ru(TAP)2(dppz)]2+ bound to (TCGGCGCCGA)2.

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References

  1. Smith, N. A. & Sadler, P. J. Photoactivatable metal complexes: from theory to applications in biotechnology and medicine. Phil. Trans. R. Soc. A 371, 20120519 (2013).

    Article  Google Scholar 

  2. Gill, M. R. & Thomas, J. A. Ruthenium(II) polypyridyl complexes and DNA—from structural probes to cellular imaging and therapeutics. Chem. Soc. Rev. 41, 3179–3192 (2012).

    Article  CAS  Google Scholar 

  3. Puckett, C. A. & Barton, J. K. Methods to explore cellular uptake of ruthenium complexes. J. Am. Chem. Soc. 129, 46–47 (2007).

    Article  CAS  Google Scholar 

  4. Marcélis, L., Moucheron, C. & Kirsch-De Mesmaeker, A. Ru–TAP complexes and DNA: from photo-induced electron transfer to gene photo-silencing in living cells. Phil. Trans. R. Soc. A 371, 20120131 (2013).

    Article  Google Scholar 

  5. Ryan, G. J., Quinn, S. & Gunnlaugsson, T. Highly effective DNA photocleavage by novel ‘rigid’ Ru(bpy)3-4-nitro- and -4-amino-1,8-naphthalimide conjugates. Inorg. Chem. 47, 401–403 (2008).

    Article  CAS  Google Scholar 

  6. Elias, B. et al. Photooxidation of guanine by a ruthenium dipyridophenazine complex intercalated in a double-stranded polynucleotide monitored directly by picosecond visible and infrared transient absorption spectroscopy. Chem. Eur. J. 14, 369–375 (2008).

    Article  CAS  Google Scholar 

  7. Towrie, M. et al. ps-TRIR covers all the bases—recent advances in the use of transient IR for the detection of short-lived species in nucleic acids. Analyst 134, 1265–1273 (2009).

    Article  CAS  Google Scholar 

  8. Schreier, W. J. et al. Thymine dimerization in DNA is an ultrafast photoreaction. Science 315, 625–629 (2007).

    Article  CAS  Google Scholar 

  9. Parker, A. W., Lin, C. Y., George, M. W., Towrie, M. & Kuimova, M. K. Infrared characterization of the guanine radical cation: finger printing DNA damage. J. Phys. Chem. B 114, 3660–3667 (2010).

    Article  CAS  Google Scholar 

  10. Bucher, D. B., Pilles, B. M., Carell, T. & Zinth, W. Charge separation and charge delocalization identified in long-living states of photoexcited DNA. Proc. Natl Acad. Sci. USA 111, 4369–4374 (2014).

    Google Scholar 

  11. Khesbak, H., Savchuk, O., Tsushima, S. & Fahmy, K. The role of water H-bond imbalances in B-DNA substate transitions and peptide recognition revealed by time-resolved FTIR spectroscopy. J. Am. Chem. Soc. 133, 5834–5842 (2011).

    Article  CAS  Google Scholar 

  12. Hall, J. P. et al. Structure determination of an intercalating ruthenium dipyridophenazine complex which kinks DNA by semiintercalation of a tetraazaphenanthrene ligand. Proc. Natl Acad. Sci. USA 108, 17610–17614 (2011).

    Google Scholar 

  13. Niyazi, H. et al. Crystal structures of Λ-[Ru(phen)2dppz]2+ with oligonucleotides containing TA/TA and AT/AT steps show two intercalation modes. Nature Chem. 4, 621–628 (2012).

    Article  CAS  Google Scholar 

  14. Song, H., Kaiser, J. T. & Barton, J. K. Crystal structure of Δ-[Ru(bpy)2dppz]2+ bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation. Nature Chem. 4, 615–620 (2013).

    Article  Google Scholar 

  15. Zimmerman, S. B. & Trach, S. O. Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J. Mol. Biol. 222, 599–620 (1991).

    Article  CAS  Google Scholar 

  16. Hall, J. P. et al. Controlled dehydration of a ruthenium complex–DNA crystal induces reversible DNA kinking. J. Am. Chem. Soc. 136, 17505–17512 (2014).

    Article  CAS  Google Scholar 

  17. Greetham, G. M. et al. ULTRA: a unique instrument for time-resolved spectroscopy. Appl. Spectrosc. 64, 1311–1319 (2010).

    Article  CAS  Google Scholar 

  18. Greetham, G. M. et al. Time-resolved multiple probe spectroscopy. Rev. Sci. Instrum. 83, 103107 (2012).

    Article  CAS  Google Scholar 

  19. Banyay, M., Sarkar, M. & Gräslund, A. A library of IR bands of nucleic acids in solution. Biophys. Chem. 104, 477–488 (2003).

    Article  CAS  Google Scholar 

  20. 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, 734–738 (2015).

    Article  CAS  Google Scholar 

  21. Keane, P. M. et al. Reversal of a single base-pair step controls guanine photo-oxidation by an intercalating ruthenium(II) dipyridophenazine complex. Angew. Chem. Int. Ed. 54, 8364–8368 (2015).

    Google Scholar 

  22. Devereux, S. J. et al. Study of picosecond processes of an intercalated dipyridophenazine Cr(III) complex bound to defined sequence DNAs using transient absorption and time-resolved infrared methods. Dalton Trans. 43, 17606–17609 (2014).

    Article  CAS  Google Scholar 

  23. Olmon, E. D. et al. Charge photoinjection in intercalated and covalently bound [Re(CO)3(dppz)(py)]+–DNA constructs monitored by time-resolved visible and infrared spectroscopy. J. Am. Chem. Soc. 133, 13718–13730 (2011).

    Article  CAS  Google Scholar 

  24. Smith, J. A., George, M. W. & Kelly, J. M. Transient spectroscopy of dipyridophenazine metal complexes which undergo photo-induced electron transfer with DNA. Coord. Chem. Rev. 255, 2666–2675 (2011).

    Article  CAS  Google Scholar 

  25. Park, E. S. & Boxer, S. G. Origins of the sensitivity of molecular vibrations to electric fields: carbonyl and nitrosyl stretches in model compounds and proteins. J. Phys. Chem. B 106, 5800–5806 (2002).

    Article  CAS  Google Scholar 

  26. Fried, S. D. & Boxer, S. G. Measuring electric fields and noncovalent interactions using the vibrational Stark effect. Acc. Chem. Res. 48, 998–1006 (2015).

    Article  CAS  Google Scholar 

  27. Volk, M. et al. peptide conformational dynamics and vibrational Stark effects following photoinitiated disulfide cleavage. J. Phys. Chem. B 101, 8607–8616 (1997).

    Article  CAS  Google Scholar 

  28. Volk, M. et al. Carbonyl spectator bonds as sensitive sensors for charge transfer reactions on the femtosecond time scale. J. Phys. Chem. A 104, 4984–4988 (2000).

    Article  CAS  Google Scholar 

  29. Movsisyan, L. D. et al. Photophysics of threaded sp-carbon chains: the polyyne is a sink for singlet and triplet excitation. J. Am. Chem. Soc. 136, 17996–18008 (2014).

    Article  CAS  Google Scholar 

  30. Le Gac, S. et al. Photo-reactive RuII–oligonucleotide conjugates: influence of an intercalating ligand on the inter- and intra-strand photo-ligation processes. Dalton Trans. 39, 9672–9683 (2010).

    Article  CAS  Google Scholar 

  31. Walrafen, G. E., Hokmabadi, M. S. & Yang, W. H. Raman investigation of the temperature dependence of the bending ν2 and combination ν2 + νL bands from liquid water. J. Phys. Chem. 92, 2433–2438 (1988).

    Article  CAS  Google Scholar 

  32. Shih, C. et al. Tryptophan-accelerated electron flow through proteins. Science 320, 1760–1762 (2008).

    Article  CAS  Google Scholar 

  33. Kupitz, C. et al. Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser. Nature 513, 261–265 (2014).

    Article  CAS  Google Scholar 

  34. Tenboer, J. et al. Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein. Science 346, 1242–1246 (2014).

    Article  CAS  Google Scholar 

  35. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    Article  CAS  Google Scholar 

  36. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).

    Article  CAS  Google Scholar 

  37. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  38. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).

    Article  CAS  Google Scholar 

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Acknowledgements

The work was supported by the Biotechnology and Biological Sciences Research Council grants BB/K019279/1 and BB/M004635/1 (to C.J.C., J.A.B., M.T. and J.P.H.) and a Royal Irish Academy/Royal Society International Exchange Scheme award (to C.J.C., J.M.K. and T.G.), Science Foundation Ireland (SFI) Principle Investigator awards 10/IN.1/B2999 and 13/IA/1865 (to T.G.) and an Irish Research Council PhD Scholarship (to F.E.P.). These experiments were carried out thanks to the programmed access approved by the CLF (Application No. 14230014). The authors gratefully acknowledge the contributions of H. Beer and K. Buchner to the preparation of the metal complex used in this study. The authors also thank the University of Reading for the provision of the Chemical Analysis Facility. S.J.Q. acknowledges financial support from the University College Dublin College of Science. G.W. thanks K. McAuley for the provision of in-house beam time on I03. We thank T. Sorensen (Diamond Light Source), J. Sanchez-Weatherby (Diamond Light Source) and S. Teixeira (Institut Laue-Langevin and University of Keele) for useful discussions.

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J.P.H., M.T., C.J.C., J.M.K. and S.J.Q. conceived and designed the experiments; J.P.H., S.P.G., P.M.K., G.W., I.V.S. and S.J.Q. each performed some experiments; J.P.H., F.E.P., P.M.K., G.W., M.T. and S.J.Q. analysed the data; F.E.P., J.A.B., T.G, D.J.C. and C.J.C. contributed the materials; M.T. and I.V.S. contributed the analysis tools; S.J.Q. wrote the paper, with contributions from P.M.K. and major contributions from J.P.H., J.M.K. and C.J.C. All the authors reviewed the manuscript.

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Correspondence to Christine J. Cardin, John M. Kelly or Susan J. Quinn.

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Hall, J., Poynton, F., Keane, P. et al. Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy. Nature Chem 7, 961–967 (2015). https://doi.org/10.1038/nchem.2369

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