Nucleic acid oxidation encompasses myriad chemical processes, notably ionizing radiation and chronic inflammation, which are at the heart of a variety of diseases and aging. DNA oxidation is recognized as a contributing factor in an increasing number of diseases, including cancer, cardiovascular, and most recently triplet repeat diseases (e.g. Huntington's disease). RNA oxidation is also associated with neurodegenerative diseases. Nucleic acid damage is a double-edged sword because cancer is treated by ?-radiolysis and drugs that oxidatively damage DNA. Furthermore, nucleic acid oxidation is used to study nucleic acid structure, noncovalent binding, and RNA folding, and is an important tool in biotechnology. The research program described in this proposal relies largely on organic chemistry to improve our understanding of how nucleic acids are oxidatively damaged, a fundamentally important biomedical research topic. Our goal is to undertake fundamental research to develop a detailed understanding of how nucleic acids are oxidatively damaged, and to apply the knowledge gained in these investigations to the design of research tools and possible therapeutic agents. Our general approach utilizes organic synthesis to independently generate reactive intermediates that are involved in nucleic acid damage. This method simplifies studies on nucleic acid damage by controlling which reactive intermediates are produced and where they are generated. We propose to use this approach to be the first to independently generate reactive intermediates in DNA that are produced by the direct effect of ionizing radiation and that are involved in electron transfer (Aims 1 & 2). We will explore the rol that protein radicals in nucleosomes play in DNA damage (Aim 3). We also propose to advance our understanding of a novel mechanism for DNA double-strand cleavage that we discovered during the previous funding period (Aim 4). This could lead to the development of molecules that produce double- strand breaks via a single oxidation event. Finally, Aim 5 builds upon our experiments to develop a family of nucleotide analogues that are radiosensitizing agents and form interstrand cross-links in DNA selectively under hypoxic conditions. In summary, the project combines organic chemistry and biochemistry to increase our understanding of fundamentally important chemical processes that occur in living organisms, and potentially improve human health.