Work Group Prof. Dr. F. Temps

Photoexcited DNA Building Blocks

 B-DNA double helix

Photochemical damage of the DNA by ultraviolet (UV) light is one of the two main causes for mutagenesis and carcinogenic cell growth in all eukariotic organisms exposed to the sun. The high UV-photostability of the DNA is therefore of utmost importance for the stability of the genomic information of life on Earth. 

While nature has selected the most photostable pyrimidine and purine nucleobases for its genetic code - molecules which exhibit ultrafast radiationless electronic deactivation mechanisms to ensure very rapid dissipation of the harmful energy of an absorbed UV photon - the photophysical and photochemical properties of the DNA are extraordinarily complex and still far from understood. This is true, in particular, regarding the unusual supramolecular intra- and interstrand coupling mechanisms among the nucleobases arising from base stacking interactions, coupling of the (transition) dipole moments, and the hydrogen bonding in the Watson-Crick base pairs. Beyond these effects, the situation is even further complicated by the dependence of the photo-induced dynamics on the nucleobase sequence and on the tertiary and quarternary macromolecular structure of the DNA.

In effect, the excited electronic state lifetimes in DNA can be orders of magnitude longer than in the free bases! 

Our research is driven by the following crucial questions:

  • What is the nature of the different photo-excited states in the DNA?
  • How is the energy supplied by a UV photon dissipated in the DNA?  
  

To solve these challenging problems, we are investigating the ultrafast photophysical and photochemical dynamics in the four main DNA bases, in rare natural DNA and RNA bases, in hydrogen-bonded base pairs, and in short single- and double-stranded DNA oligonucleotides from dimers to 20-mers. Our toolbox consists of femtosecond fluorescence and transient electronic and vibrational absorption spectroscopies in solution, femtosecond time-resolved mass spectrometry and photoelectron imaging, and REMPI and UV/UV double resonance spectroscopy of model compounds for hydrogen-bonded and stacked nucleobases. 

   

Isolated DNA & RNA bases

The four main natural DNA bases adenine (Ade), cytosine (Cyt), guanine (Guo) and thymine (Thy), the RNA base uracil (Ura) stand out for extraordinarily high UV-photostabilities owing to ultrafast, highly efficient radiationless electronic deactivation mechanisms, dissipating the dangerous energy of an absorbed UV photon before profound damage of the molecules by a photo-induced chemical reaction can occur. It has been speculated that this high photostability has been a deciding factor in the selection of the major DNA and RNA bases for the genomic code during the evolution of life on Earth at the earliest times before the ozone layer provided efficient UV protection. The underlying basic photophysical mechanisms are now understood to involve conical intersections (CoIns) between the excited and ground electronic states mediating the ultrafast relaxation processes. Much less is known about the fine differences of the dynamics among the many tautomers and isomers of the canonical nucleobases, e.g. 9H-adenine, 7H-adenine, or 2-aminopurine as a highly fluorescent structural isomer of adenine (= 6-aminopurine).   

 DNA bases 
 
Structures of the canonical natural DNA and RNA bases and selected rare RNA bases.

 

To elucidate the fine structural details affecting the photophysical dynamics, we investigate the main bases and their nucleosides and nucleotides in solution in water and other solvents in comparison to selected tautomeric and isomeric structures and the many rare natural DNA and RNA bases. The currrent record holder for the fastest electronic deactivation is inosine. Particular interest is currently paid to the difference between 9H- and 7H-adenine.

Important papers:

  • K. Röttger, R. Siewertsen, F. Temps, "Ultrafast Electronic Deactivation Dynamics of the Rare Nucleobase Hypoxanthine", Chem. Phys. Lett. 536, 140 - 146 (2012). DOI: 10.1016/j.cplett.2012.03.106
  • T. Pancur, N. K. Schwalb, F. Renth, F. Temps, "Femtosecond Fluorescence Up-Conversion Spectroscopy of Adenine and Adenosine: Experimental Evidence for the πσ* State?" Chem. Phys. 313, 199 - 212 (2005).

  

H-bonded base pairs & aggregates

Proton transfer along the intermolecular hydrogen bonds between the conjugate nucleobases in the Watson-Crick (WC) base pairs has long been assumed as a possible precursor state for mutagenesis and carcinogenesis. However, recent theoretical studies postulated that ultrafast inter-strand electron-driven proton transfer (EDPT) instead contributes to the prevention of mutagenic photolesions in UV-photoexcited DNA.   

G-C Deactivation Mechanism 
Proposed deactivation mechanism of UV-photoexcited G-C Watson-Crick base pairs.

 

Particular interest arises in the guanosine-cytidine (G--C) WC base pair, where an ultrafast transition from the photoexcited πGπG* state to a G-to-C charge transfer (CT) state followed by the transfer of a proton along the central G--C hydrogen bond has been proposed. In collaboration with the Orr-Ewing group at Bristol, we recently succeded to observe the resulting G-H]--[C+H] biradical by transient electronic absorption spectroscopy and by transient vibrational absorption spectroscopy. In our ongoing work, we investigate the possible role of EDPT processes in selected DNA sequences.

Important papers:

  • K. Röttger, H. J. B. Marroux, M. P. Grubb, P. M. Coulter, H. Böhnke, A. S. Henderson, M. C. Galan, F. Temps, A. J. Orr-Ewing, G. M. Roberts, "Ultraviolet Absorption Induces Hydrogen-Atom Transfer in GC Watson-Crick DNA Base Pairs in Solution", Angew. Chem. Int. Ed. 54, 14719 - 14722 (2015).  DOI: 10.1002/anie.201506940
  • K. Röttger, F. D. Sönnichsen, F. Temps, "Ultrafast Electronic Deactivation Dynamics of the Inosine Dimer - A Model Case for H-Bonded Purine Base Pairs", Photochem. Photobiol. Sci. 12, 1466 - 1473 (2013).  DOI:  10.1039/C3PP50093D
  • K. Röttger, N. K. Schwalb, F. Temps, "Electronic Deactivation of Guanosine in Extended Hydrogen-Bonded Self-Assemblies", J. Phys. Chem. A 117, 2469 - 2478 (2013), DOI: 10.1021/jp3095193
  • N. K. Schwalb, F. Temps, "Ultrafast Electronic Relaxation in Guanosine is Promoted by Hydrogen Bonding with Cytidine", J. Am. Chem. Soc. 129, 9272 - 9273 (2007).  DOI: 10.1021/ja073448+

    

ss- and ds-DNA oligonucleotides

    (Ade)2
The adenine dincucleotide in water
adopts  the  B-DNA  configuration.

As shown by femtosecond time-resolved fluorescence and absorption measurements, the excited-state lifetimes of single- and double-stranded DNA oligonucleotides and natural DNA can be three to four orders of magnitude longer than the lifetimes of the free nucleobases in solution. These huge differences have to be attributed to strong electronic coupling mechanisms between the nearly coplanar stacked bases in a DNA strand. It is highly controversally debated, however, whether the long lifetimes are due to dipole-coupled (Frenkel) excitons, which can be delocalized over a number of bases, or whether the local ππ* photoexcited states transform to long-lived excimer- resp. exciplex-like states with (partial) charge transfer character involving a neighboring base.

Since the excited state structures of long oligonucleotides with > 4 - 5 bases are far too complex to elucidate, we have turned to the investigation of small di-, tri- and tetranucleotides as nearly ideal model systems. For the adenine dinucleotide as an example, we have recently been able to time-resolve the transition from the initially excited exciton state to the long-lived (380 ps) excimer state which takes place in the first 100 - 500 fs after excitation. A sizable energetic stabilization of the excimer is reflected by strong red- and blue-shifts by ~1 eV, respectively, in the observed fluorescence and absorption spectra. Distinctive spectral features of the exciton and excimer states were identified.

In a bottom-up approach, we are studying the related dynamics in selected other di- and oligonucleotides to elucidate the excited-staet dynamics in larger DNA molecules. 

Important papers:

  • M. C. Stuhldreier, K. Röttger, F. Temps, "Distinctive Spectral Features of Exciton and Excimer States in the Ultrafast Electronic Deactivation of the Adenine Dinucleotide", in: Ultrafast Phenomena XIX, K. Yamanouchi, S. Cundiff, R. de Vivie-Riedle, M. Kuwata-Gonokami, L. DiMauro (Eds.), Springer Proceedings in Physics, Band 162, 452 - 454 (2015).  DOI:  10.1007/978-3-319-13242-6_110
  • M. C. Stuhldreier, F. Temps, "Ultrafast Photo-Initated Molecular Quantum Dynamics in the DNA Dincleotide d(ApG) Revealed by Broadband Transient Absorption Spectroscopy", Faraday Discuss. 163, 173 - 188 (2013).  DOI: 10.1039/C3FD00003F
  • N. K. Schwalb, F. Temps, "Base Sequence and Higher-Order Structure Induce the Complex Excited-State Dynamics in DNA", Science 322, 243 - 245 (2008).  DOI: 10.1126/science.1161651