Oxidative damage plays a causative role in many diseases, and DNA-protein cross-linking is one consequence of such damage. Guanine is the most easily oxidized base and the ionization potential for the 5’G decreases as follows: GT&gt;GC&gt;GA&gt;GG&gt;GGG; thus GG and GGG sites are particularly prone to one-electron oxidation. Previous work has shown charge transport (CT), as visualized by oxidative damage to guanine, can occur at distances up to 200 Å, but attempts to study CT at even longer range have been thwarted by technical limitations of artificial DNA synthesis and the weak ability of water to trap the guanine radical. Here, we designed a system for the detection of long-range oxidation in DNA. Two oligonucleotides were artificially synthesized and inserted into the pNEB206A and pUC19 plasmids. The sense strand of the oligonucleotides contained 2-Aminopurine (2-AP), which is capable of guanine photooxidation. In the first system, 2-AP was inserted near a group of GG and GGG-containing restriction sites. In the opposite direction, an insulating AT sequence was incorporated to force charge transport in one direction. In the second system, as in the first, the 2-AP was proximal to guanine containing recognition sites. Moreover, the oligo incorporated two new, unique, guanine-rich restriction sites to facilitate CT in the other direction in relation to 2-AP as well. For both sequences, the guanine ionization potentials were deliberately stepped down as the distance from aminopurine increased, in order to facilitate CT by hindering back electron transfer between guanine cation radical and 2-AP anion radical. In the presence of protein, guanine radicals undergo efficient trapping reactions with the sidechains of basic amino acids such as lysine to form DNA-protein crosslinks. Exploiting this fact, we will use an assay based on restriction enzyme cleavage, whereby guanine-protein crosslinks will inhibit cleavage, to detect this damage. These experiments are underway.

Designing a new system for studying long range oxidation in DNA

251st National Meeting (2016)

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