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Switching on the fluorescence of 2-aminopurine by site-selective microhydration

Nature Chemistry volume 6, pages 989–993 (2014)Cite this article

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Abstract

2-Aminopurine (2AP) is a fluorescent isomer of adenine and has a fluorescence lifetime of ~11 ns in water. It is widely used in biochemical settings as a site-specific fluorescent probe of DNA and RNA structure and base-flipping and -folding. These assays assume that 2AP is intrinsically strongly fluorescent. Here, we show this not to be the case, observing that gas-phase, jet-cooled 2-aminopurine and 9-methyl-2-aminopurine have very short fluorescence lifetimes (156 ps and 210 ps, respectively); they are, to all intents and purposes, non-fluorescent. We find that the lifetime of 2-aminopurine increases dramatically when it is part of a hydrate cluster, 2AP·(H2O)n, where n = 1–3. Not only does it depend on the presence of water molecules, it also depends on the specific hydrogen-bonding site to which they attach and on the number of H2O molecules at that site. We selectively microhydrate 2-aminopurine at its sugar-edge, cis-amino or trans-amino sites and see that its fluorescence lifetime increases by 4, 50 and 95 times (to 14.5 ns), respectively.

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Figure 1: Pump/ionization delay measurements of 9H-2AP, 9M-2AP and their mono-, di- and tri-hydrated water clusters.
Figure 2: Comparison of the calculated and experimental adiabatic excitation energies.
Figure 3: Correlation of the experimental 1π π* excited-state lifetime τ of 9H-2AP (9H), 9M-2AP (9M) and their mono-, di- and trihydrate clusters (1A to 3B) with the difference of the 1n π* and 1π π* state minimum energies.

References

  1. Ward, D. C., Reich, E. & Stryer, L. Fluorescence studies of nucleotides and polynucleotides. J. Biol. Chem. 244, 1228–1239 (1969).

    CAS  PubMed  Google Scholar 

  2. Guest, C. R., Hochstrasser, R. A., Sowers, L. C. & Millar, D. P. Dynamics of mismatched base pairs in DNA. Biochemistry 30, 3271–3279 (1991).

    Article  CAS  Google Scholar 

  3. Allan, B. W. & Reich, N. O. Targeted base stacking disruption by the EcoRI DNA methyltransferase. Biochemistry 35, 14757–14762 (1996).

    Article  CAS  Google Scholar 

  4. Stivers, J. T. 2-Aminopurine fluorescence studies of base stacking interactions at abasic sites in DNA: metal-ion and base sequence effects. Nucleic Acids Res. 26, 3837–3844 (1998).

    Article  CAS  Google Scholar 

  5. Kelley, S. O. & Barton, J. K. Electron transfer between bases on double helical DNA. Science 283, 375–381 (1999).

    Article  CAS  Google Scholar 

  6. Wan, C., Fiebig, T., Schiemann, O., Barton, J. K. & Zewail, A. H. Femtosecond direct observation of charge transfer between bases in DNA. Proc. Natl Acad. Sci. USA 97, 14052–14055 (2000).

    Article  CAS  Google Scholar 

  7. Jiao, Y., Stringfellow, S. & Yu, H. Distinguishing ‘looped-out’ and ‘stacked-in’ DNA bulge conformation using fluorescent 2-aminopurine replacing a purine base. J. Biomol. Struct. Dyn. 19, 929–934 (2002).

    Article  CAS  Google Scholar 

  8. Pal, S. K., Peon, J. & Zewail, A. H. Ultrafast decay and hydration dynamics of DNA bases and mimics. Chem. Phys. Lett. 363, 57–63 (2002).

    Article  CAS  Google Scholar 

  9. Pal, S. K., Zhao, L., Xia, T. & Zewail, A. H. Site- and sequence-selective ultrafast hydration of DNA. Proc. Natl Acad. Sci. USA 100, 13746–13751 (2003).

    Article  CAS  Google Scholar 

  10. Lee, B. J., Barch, M., Castner, E. W., Völker, J. & Breslauer, K. J. Structure and dynamics in DNA looped domains: CAG triplet repeat sequence dynamics probed by 2-aminopurine fluorescence. Biochemistry 46, 10756–10766 (2007).

    Article  CAS  Google Scholar 

  11. Wilcox, J. L. & Bevilacqua, P. C. A simple fluorescence method for pKa determination in RNA and DNA reveals highly shifted pKas. J. Am. Chem. Soc. 135, 7390–7393 (2013).

    Article  CAS  Google Scholar 

  12. Fagan, P. A., Fàbrega, C., Eritja, R., Goodman, M. F. & Wemmer, D. E. NMR study of the conformation of the 2-aminopurine:cytosine mismatch in DNA. Biochemistry 35, 4026–4033 (1996).

    Article  CAS  Google Scholar 

  13. Sowers, L. C., Fazakerley, G. V., Eritja, R., Kaplan, B. E. & Goodman, M. F. Base pairing and mutagenesis: observation of a protonated base pair between 2-aminopurine and cytosine in an oligonucleotide by proton NMR. Proc. Natl Acad. Sci. USA 83, 5434–5438 (1986).

    Article  CAS  Google Scholar 

  14. Rachofsky, E. L., Osman, R. & Ross, J. B. Probing structure and dynamics of DNA with 2-aminopurine: effects of local environment on fluorescence. Biochemistry 40, 946–949 (2001).

    Article  CAS  Google Scholar 

  15. Neely, R. K. et al. Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.Hhal–DNA complexes. Nucleic Acids Res. 33, 6953–6960 (2005).

    Article  CAS  Google Scholar 

  16. Smagowicz, J. & Wierzchowski, K. L. Lowest excited states of 2-aminopurine. J. Lumin. 8, 210–218 (1974).

    Article  CAS  Google Scholar 

  17. Allan, B. W., Reich, N. O. & Beechem, J. M. Measurement of the absolute temporal coupling between DNA binding and base flipping. Biochemistry 38, 5308–5314 (1999).

    Article  CAS  Google Scholar 

  18. Jean, J. M. & Hall, K. B. 2-Aminopurine fluorescence quenching and lifetimes: role of base stacking. Proc. Natl Acad. Sci. USA 98, 37–41 (2001).

    Article  CAS  Google Scholar 

  19. Rachofsky, E. L., Seibert, E., Stivers, J. T., Osman, R. & Ross, J. B. Conformation and dynamics of abasic sites in DNA investigated by time-resolved fluorescence of 2-aminopurine. Biochemistry 40, 957–967 (2001).

    Article  CAS  Google Scholar 

  20. Souliére, M. F., Haller, A., Rieder, R. & Micura, R. A Powerful approach for the selection of 2-aminopurine substitution sites to investigate RNA folding. J. Am. Chem. Soc. 133, 16161–16167 (2011).

    Article  Google Scholar 

  21. Rist, M. J. & Marino, J. P. Fluorescent nucleotide base analogs as probes of nucleic acid structure, dynamics and interaction. Curr. Org. Chem. 6, 775–793 (2002).

    Article  CAS  Google Scholar 

  22. Broo, A. A theoretical investigation of the physical reason for the very different luminescence properties of the two isomers adenine and 2-aminopurine. J. Phys. Chem. A 102, 526–531 (1998).

    Article  CAS  Google Scholar 

  23. Jean, J. M. & Hall, K. B. Theoretical study of the excited state properties and transitions of 2-aminopurine in the gas phase and in solution. J. Phys. Chem. A 104, 1930–1937 (2000).

    Article  CAS  Google Scholar 

  24. Rachofsky, E. L., Ross, J. B. A., Krauss, M. & Osman, R. CASSCF investigation of electronic excited states of 2-aminopurine. J. Phys. Chem. A 105, 190–197 (2001).

    Article  CAS  Google Scholar 

  25. Serrano-Andrés, L., Merchán, M. & Borin, A. Adenine and 2-aminopurine: paradigms of modern theoretical photochemistry. Proc. Natl Acad. Sci. USA 103, 8691–8696 (2006).

    Article  Google Scholar 

  26. Perun, S., Sobolewski, A. L. & Domcke, W. Ab initio studies of the photophysics of 2-aminopurine. Mol. Phys. 104, 1113–1121 (2006).

    Article  CAS  Google Scholar 

  27. Ludwig, V., Serrou do Amaral, M., da Costa, Z. M., Borin, A. C., Canuto, S. & Serrano-Andrés, L. 2-Aminopurine non-radiative decay and emission in aqueous solution: a theoretical study. Chem. Phys. Lett. 463, 201–205 (2008).

    Article  CAS  Google Scholar 

  28. Lim, E. C. Proximity effect in molecular photophysics: dynamic consequences of pseudo-Jahn–Teller interaction. J. Phys. Chem. 90, 6770–6777 (1986).

    Article  CAS  Google Scholar 

  29. Seefeld, K. A. et al. Tautomers and electronic states of jet-cooled 2-aminopurine investigated by double resonance spectroscopy and theory. Phys. Chem. Chem. Phys. 7, 3021–3026 (2005).

    Article  CAS  Google Scholar 

  30. Sinha, R. K., Lobsiger, S., Trachsel, M. & Leutwyler, S. Vibronic spectra of jet-cooled 2-aminopurine·H2O clusters studied by UV resonant two-photon ionization spectroscopy and quantum chemical calculations. J. Phys. Chem. A 115, 6208–6217 (2011).

    Article  CAS  Google Scholar 

  31. Trachsel, M., Lobsiger, S., Schär, T. & Leutwyler, S. Low-lying excited-states and nonradiative processes of 9-methyl-2-aminopurine. J. Chem. Phys. 140, 044331 (2014).

    Article  Google Scholar 

  32. Sinha, R. K., Lobsiger, S. & Leutwyler, S. Isomer- and species-selective infrared spectroscopy of jet-cooled 7H- and 9H-2-aminopurine and 2-aminopurine·H2O clusters. J. Phys. Chem. A 116, 1129–1136 (2012).

    Article  CAS  Google Scholar 

  33. Lobsiger, S., Sinha, R. K. & Leutwyler, S. Building up water-wire clusters: isomer-selective ultraviolet and infrared spectra of jet-cooled 2-aminopurine (H2O)n, n = 2 and 3. J. Phys. Chem. B 117, 12410–12421 (2013).

    Article  CAS  Google Scholar 

  34. TURBOMOLE V6.3 2011 (Universität Karlsruhe, Forschungszentrum Karlsruhe, TURBOMOLE); available from http://www.turbomole.com

  35. Improta, R. & Barone, V. Absorption and fluorescence spectra of uracil in the gas phase and in aqueous solution: a TD-DFT quantum mechanical study. J. Am. Chem. Soc. 126, 14320–14321 (2004).

    Article  CAS  Google Scholar 

  36. Etinski, M. & Marian, C. M. Ab initio investigation of the methylation and hydration effects on the electronic spectra of uracil and thymine. Phys. Chem. Chem. Phys. 12, 4915–4923 (2010).

    Article  CAS  Google Scholar 

  37. Lobsiger, S., Sinha, R. K., Trachsel, M. & Leutwyler, S. Low-lying excited-states and nonradiative processes of the adenine analogues 7H- and 9H-2-aminopurine. J. Chem. Phys. 134, 114307 (2011).

    Article  Google Scholar 

Download references

Acknowledgements

The authors acknowledge financial support from the Schweiz Nationalfonds (SNSF; project numbers 20020-121993 and 200021-132540).

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, Bern, CH-3012, Switzerland

    Simon Lobsiger, Susan Blaser, Rajeev K. Sinha, Hans-Martin Frey & Samuel Leutwyler

  2. Department of Atomic and Molecular Physics, Manipal University, Manipal-576104, Karnataka, India

    Rajeev K. Sinha

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  1. Simon Lobsiger
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  2. Susan Blaser
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  4. Hans-Martin Frey
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Contributions

S.Lo. and R.K.S. performed the nanosecond spectroscopic and pump–probe experiments as well as the DFT calculations, and analysed the spectroscopic and nanosecond kinetic data. S.B. co-designed the picosecond pump–probe experiment and performed the picosecond pump–probe measurements. H.M.F. conceived and designed the picosecond pump–probe experiments and programmed the kinetic fitting analysis. S.Lo. and S.Le. wrote major parts of the manuscript.

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Correspondence to Samuel Leutwyler.

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Lobsiger, S., Blaser, S., Sinha, R. et al. Switching on the fluorescence of 2-aminopurine by site-selective microhydration. Nature Chem 6, 989–993 (2014). https://doi.org/10.1038/nchem.2086

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