The constant assault on cellular constituents by endogenous and exogenous agents leads to a large range of DNA alterations. These lesions are often related to conformational changes and relative movements of domains on the nanometre scale that may have a high biological impact in terms of lethality and mutagenicity. The analysis of lesion-induced structural changes will lead to a better elucidation of the structure-toxicity relationship. We have developed a new strategy to probe such conformational changes by recording long distances (up to 6 nm) between two spin labels grafted on damaged oligonucleotides by pulsed EPR. This approach in which chemists and spectroscopists work in close collaboration has recently demonstrated its efficiency.
Up to now, determining distance changes related to DNA structure modifications, have mainly been determined by X-ray crystallography, high-resolution NMR spectroscopy and by Fluorescence Resonance Energy Transfer (FRET). However, although such techniques can provide exhaustive information for the characterization of DNA structures, they still exhibit some drawbacks. Pulsed EPR based on the DEER sequence is a powerful, recently developed method which can quantitatively measure long distance distributions (DD) up to 8 nm between two paramagnetic spin labels in a disordered state with a good sensitivity.
Since oligonucleotides are diamagnetic, the first challenge for the chemists involved in this project was to develop new methods to efficiently graft two spin labels (nitroxides) onto an oligonucleotide and introduce a biologically relevant chemical modification on the same molecule. This was done using specific solid state DNA synthesis with modified synthons and new DNA base functionalisation approaches. Using these molecules, we were able to compare the DD (center, width, asymmetry...) between undamaged and damaged systems by pulsed EPR.
The results obtained clearly demonstrate that while some lesions (8-oxoguanine, nick, gap, A1-bulge) do not noticeably modify the conformation of oligonucléotides, others induce important modifications, in particular in the case of abasic site analogues ("THF", "propyl", "ethyl"). This effect is demonstrated by the differences between the DD of undamaged and damaged oligonucleotides (Fig 1, insert). While the undamaged system exhibits a narrow symmetrical DD, the other systems produce much broader, asymmetrical patterns with a shorter average distance. This means that the flexibility of damaged systems is greatly increased with a possible coexistence of several conformers (asymmetry).
To further analyse our results, we turned to molecular dynamics (MD). For example, in the case of the "THF" system, different conformations in which the THF ring and the opposite base are intra or extrahelical (Fig 2) are possible. For each case, we were able to compare the theoretical DD obtained by MD and the experimental DD obtained by pulsed EPR. We found that only the conformation in which the lesion and the opposite base are extrahelical produce comparable distance measurements (Fig 2c). This analysis can also be performed on the other systems and can be correlated to the mutagenic properties of the different lesions.
Thus we have demonstrated that this interdisciplinary approach combining spin labeling, chemical synthesis of modified oligonucleotides, pulsed EPR analyses and molecular dynamics simulations provide a powerful tool for the analysis of lesion-induced conformational changes in DNA.
Selected publications: G. Sicoli et al., Ang. Chem. Int. Ed. 47 (2008) 735-37; G. Sicoli et al. Nucl. Acids. Res. 37 (2009) 3165-76.
Last update : 05/30 2013 (780)