Oxidative damage to DNA plays a role in the progression of many diseases, including cancer. This damage is observed primarily at guanine because it is the most easily oxidized base. Base excision repair enzymes, like Endonuclease III and MutY, readily detect these DNA base lesions and contain [FeS] clusters that are active in electron transfer. In our experiment, we chose to use cytochrome c as a cheaper model for redox-active repair proteins. Here, we investigated whether oligonucleotides could be designed such that a long-lived guanine radical could be created by photooxidation, allowing cytochrome c to reduce the guanine radical. Toward that end, we synthesized a DNA strand with a fluorescent base analogue, 2-aminopurine(AP), inserted near the 5’ end. The propensity for oxidation for the 5’ G of 5’-GX-3’ steps is known to decrease in the following order: GT &gt; GC &gt; GA &gt; GG &gt; GGG. Thus, the oligonucleotide sequence was designed to contain guanines that decrease in ionization potential as the distance from AP increases, to prevent back electron transfer and thus increase the lifetime of the guanine radical, in order to facilitate reaction with protein. The oligonucleotide duplex containing AP was then excited with the 325 nm output from a HeCd laser. Using uv-visible spectroscopy, the 550 nm absorption band characteristic of reduced cytochrome c was seen to decrease upon irradiation. Additional experiments provide further evidence for a long-lived guanine radical that can react with protein. When the oligonucleotide was conjugated with Alexafluor546, a fluorescent dye, to the 3’ end, it was observed that upon irradiation, the intensity of the free DNA band decreased and lower mobility material became visible, indicating the formation of oxidative DNA-protein crosslinks. Future experiments will target the redox-active DNA repair proteins.

Reactions of DNA with protein following photooxidation of guanine by 2-aminopurine

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.