DNA-protein crosslinks are significant possible consequences of oxidative damage to DNA. When DNA is subjected to oxidative damage, the covalent adducts formed between DNA and protein can potentially lead to the distortion of the DNA helix and disruptions in fundamental cellular DNA processes. Given that oxidation damage has implications in aging as well as several molecular diseases, it is of increasing importance and interest to have rich structural characterization of these oxidative DNA-protein crosslink products. In this project, we focus on the isolation and structural characterization of physiologically relevant DNA-protein crosslinks. In DNA, the DNA base guanine has the lowest oxidation potential of the DNA bases and is most susceptible to oxidation and formation of DNA-protein crosslinks. In order to prepare DNA-protein crosslinks, a photochemical technique called the Flash-Quench technique is used to selectively oxidize the guanine base(s) in DNA which will in turn form a crosslink with amino acid side chains in protein. In the Flash-Quench experiment, small DNA oligos and histone protein are irradiated in the presence of a photosensitizer, Ru(bpy)₃²⁺ and an oxidative quencher, Co(NH₃)₅Cl²⁺ at 442 nm with a HeCd laser. Following irradiation of Flash-Quench samples with 442 nm light, the products of the reaction are detected and isolated according to a change in retention time via high-performance liquid chromatography (HPLC). The isolated DNA-protein crosslink reaction products will then be further analyzed and identified by quadrupole time-of-flight liquid chromatography/mass spectrometry (LC/Q-TOF), followed by purification by HPLC. The purity of the DNA-protein crosslinks will be confirmed by 1-Dimensional (1D) ¹H Nuclear Magnetic Resonance (NMR) spectroscopy. The structural characterization of the DNA-protein crosslink will be conducted by 1D and 2-Dimensional (2D) ¹H NMR spectroscopy.

Natalie Villalon

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