8-oxoguanine Production and Oxidation in Duplex DNA
Authors:
Kelsey Miller, Zitadel PerezMentor:
Eric Stemp, Department Chair, Physical Sciences, Mount St. Mary's College8-oxoguanine is a common oxidation product in DNA and can lead to mutation. The metallointercalator Ru(phen)2dppz2+ is a useful luminescent probe for DNA that has also found use as a guanine-selective oxidizing agent via the flash-quench technique. Here, we use its osmium analogue as a selective way to oxidize 8-oxoguanine in double-stranded DNA, as visualized by oxidative DNA-protein crosslinking. With the 3+/2+ couple at 1.15 V for Os(phen)2dppz2+, this metallointercalator should be able to oxidize 8-oxo-G (~0.7 V) without oxidizing guanine (~1.3V). MALDI mass spectrometry data confirms that oxidizing guanine using Ru(NH3)63+ and Ru(phen)2dppz2+ produces 8-oxoguanine. In plasmid DNA where 8-oxo-G has been incorporated, flash-quench treatment with Os(phen)2dppz2+ and Co(NH3)5Cl2+ leads to crosslinking with histone. Furthermore, in gel shift experiments with a duplex of the oligonucleotide 5’-ATATGATAT8GATATGATAT-3’ (8 = 8-oxo-G) and its complement, flash-quench treatment with Ru(phen)2dppz2+ in the presence of histone leads to a band of intermediate mobility (presumably 1:1 crosslink) and to a band of well-shifted material. In contrast, analogous treatment with Os(phen)2dppz2+ produces only the band of intermediate mobility, consistent with the presence of a single site that is oxidizable by the osmium complex.