Abstract 2412 "Design of a Model System for Studying Long Range Oxidation in DNA"

Oxidative damage plays a causative role disease, and one type is DNA-protein crosslinking. Among the four DNA bases, guanine is the most susceptible to one-electron oxidation due to its low redox potential of 1.29V. In double stranded DNA, the ionization potential for the 5'G decreases as follows: GT>GC>GA>GG>GGG; thus, GG and GGG sites are particularly prone to one-electron oxidation. Previous work has shown charge transport (CT), visualized by oxidative damage to guanine, occurring at distances up to 200Å, but attempts to study CT at even longer ranges have been unsuccessful due to technical limitation 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 a pUC19 vector. The strand 5'CATATG CACTAGACTGACTGACTG TAATATAATT 2-AP GTGCGA GGATCC3' contained a 2-Aminopurine (2-AP), which is capable of guanine photooxidation. The 2-AP was inserted near a group of GG-containing restriction sites. In the opposite direction, an insulating AT sequence was incorporated to force the charge transport in one direction. The guanine ionization potentials were deliberately stepped down as the distance from the 2-AP increased, to facilitate CT by hindering back electron transfer. In the presence of protein, guanine radicals undergo efficient trapping reactions with the sidechains of basic amino acids to form DNA-protein crosslinks. To detect this damage, we will utilize an assay based on restriction enzyme cleavage in which the guanine-protein crosslinks impede cleavage. We give thanks to the following foundations/ organizations for their support in providing funding used towards our research: NSF Building Capacities Grant EastonFoundation Fletcher Jones Foundation John StaufferFoundation Loring-Denault Endowed Chair in Chemistry