In Vitro Fluorogenic Real‐Time Assay of the Repair of Oxidative DNA Damage |
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| Authors: | Sarah K Edwards Dr Toshikazu Ono Dr Shenliang Wang Dr Wei Jiang Dr Raphael M Franzini Dr Jong Wha Jung Ke Min Chan Prof Eric T Kool |
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| Affiliation: | 1. Department of Chemistry, Stanford University, Stanford, CA 94305 (USA);2. Present Address: Department of Chemistry and Biochemistry, Graduate School of Engineering, Center for Molecular Systems (CMS), Kyushu University, 744 Motooka, Nishi‐ku, Fukuoka, 819‐0395 (Japan);3. Present Address: College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, 702‐701 (Republic of Korea) |
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| Abstract: | The repair of oxidative damage to DNA is essential to avoid mutations that lead to cancer. Oxidized DNA bases, such as 8‐oxoguanine, are a main source of these mutations, and the enzyme 8‐oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8‐oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel‐based measurements of radiolabeled DNAs. Here we report the design and properties of fluorogenic probes that directly report on the activity of OGG1 (and its bacterial homologue Fpg) in real time as the oxidized base is excised. The probes are short, modified DNA oligomers containing fluorescent DNA bases and are designed to utilize 8‐oxoguanine itself as a fluorescence quencher. Screening of combinations of fluorophores and 8‐oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probes containing these fluorophores: the optimum probe, OGR1, yields a 60‐fold light‐up signal in vitro with OGG1 and Fpg. It can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes might prove useful in quantifying enzyme activity and performing competitive inhibition assays. |
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| Keywords: | DNA damage fluorescent probes Fpg OGG1 oxoguanine |
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