Oxidative damage is a causative factor in cancer and many diseases, and 8-oxo-guanine (8-oxo-G) is a common oxidation product in DNA that causes missense mutations. Here, we examined whether an osmium metallointercalator, Os(phen)₂dppz³⁺ can selectively oxidize 8-oxo-G to form DNA-protein cross-linking. With a 3+/2+ couple of 1.15V, Os(phen)₂dppz³⁺should be able to oxidize 8-oxo-G (~0.70V) without oxidizing guanine (~1.30V). 8-oxo-G was produced by photoexcitation and subsequent oxidative quenching of Ru(bpy)₃²⁺ (bpy = bipyridine) in the presence of DNA. This treated DNA was then mixed with histone protein, Os(phen)₂dppz²⁺ and methyl viologen (MV²⁺) in a pH7 phosphate buffer and irradiated for 0-32 minutes to induce DNA-protein cross-linking. The sample was then analyzed by the chloroform extraction assay, in which the organic solvent extracts the proteinaceous material. The amount of cross-link can be determined from the 260 nm absorbance of the free DNA remaining, and was much larger for the pretreated DNA reacted with the osmium complex, relative to untreated DNA. In an analogous experiment using pUC19 DNA, the treated plasmid showed more accumulation of lower mobility material as irradiation times increased. The relative reactivities of Os(phen)₂dppz³⁺ and its ruthenium analogue were compared using transient absorption spectroscopy. While a signal indicative of Ru(phen)₂dppz³⁺ is observed in DNA without guanine, a signal with the spectral characteristics of guanine radical is seen in DNA containing guanine. In contrast, Os(phen)₂dppz³⁺ displays the same spectrum in both types of DNA, consistent with a molecule that can oxidize 8-oxo-G but not guanine.

Katie Ramirez

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