Electron Nuclear Double Resonance (ENDOR) approach for studying radiation damage in DNA

The ultimate ambition of this project is to perform an EMR study of radiation-induced radicals on DNA samples, comparably successful as for sucrose. This would provide the most direct way to determine the precise role of sugar radicals in radiation damage to DNA. Mainly because of the lack of DNA single crystals (DNA fibers are only partially oriented), literature studies have so far been restricted to EPR studies of polycrystalline or frozen solution DNA, with high risk of erroneous conclusions based only on isotropic parameters, as the UGent group demonstrated recently for trehalose . This could be avoided if information on the anisotropy of g and hyperfine tensors is collected for confrontation with DFT calculations. Therefore we are exploring the possibilities of powder ENDOR (and/or pulsed EPR), first on DNA-related materials and later in the project on DNA. It is known, but still remarkable that anisotropic information can be retrieved from powder materials exploiting the orientation selectivity of ENDOR and pulsed EPR. Before tackling DNA, we have started our research on sucrose.

Sucrose is used as a test case where both single crystal and powder EMR spectra can be directly compared, taking advantage of the detailed and reliable knowledge, and experience gained already in the past few years. Being universally present, sucrose is most promising for dosimetry and nowadays the best understood sugar in this context. In recent years our team has studied four, RT stable radicals in great detail via their g and 1H hyperfine tensors obtained by ENDOR techniques. Very recently the most reliable reproduction to-date of the experimental EPR powder spectrum was reported using the single crystal spin Hamiltonian parameters of the aforementioned radicals.

The multi-composite character of both sugar and DNA EPR spectra, exhibiting HF splitting dominant over g-anisotropy, present important challenges for the envisaged powder ENDOR study. Existing powder ENDOR simulation software is extensively tested on sucrose spectra.

Even pushing the computer and software possibilities to the limit, it is apparent that the available set of experimental spectra has to be optimized. This implies exploring the use of higher microwave frequencies to increase the g-anisotropy and the orientation selection and pulsed EPR techniques (e.g. electron spin-echo envelope modulation, ESEEM) to resolve smaller hyperfine couplings. Linking ENDOR (and ESEEM) signals to the individual EPR components presents another challenge. Setups for in situ annealing treatments during EPR/ENDOR experiments is in development, in order to realize an experimental simplification of the spectra.