Mechanisms of Nucleic Acid Oxidation and Cross-linking

核酸氧化和交联的机制

• 批准号: 0809483
• 负责人: Cynthia Burrows
• 金额: $ 49万
• 依托单位: University of Utah
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0809483&HistoricalAwards=false
• 关键词: Mechanisms; Nucleic; Acid; Oxidation; Cross

With the support of the Organic Dynamics Program in the Chemistry Division, Professor Cynthia J. Burrows of the Chemistry Department at the University of Utah will formulate a detailed molecular picture of how DNA is oxidized in the presence of reactive species such as protein, polyamine and catecholamine nucleophiles. A complete mechanistic picture of how such adducts form is important in order to understand the fundamental processes in damage to genomic DNA. Studies outlined in this proposal build upon recent results showing that 8-oxo-7, 8-dihydroguanosine is sensitive to further oxidation by one-electron oxidants leading to a quinonoid intermediate that is nucleophilically trapped to generate hydantoin products. Recent progress has shown that other oxidized bases are subject to the same facile oxidation/nucleophilic trapping mechanism. Experiments proposed include NMR and LC/MS characterization of amino acid and polyamine adducts to nucleosides and to DNA oligomers, and investigation of their mechanisms of formation and chemical stability. Methods will be developed to understand the chemical structures responsible for DNA-protein cross-linking via both DNA oxidation and protein oxidation. Adducts of phenols and catechols to guanosine in DNA will be prepared and analyzed for their ability to mediate further oxidative damage to DNA. A major part of the new research will focus on characterization of reactive quinonoid intermediates in 8-oxoguanosine and uric acid oxidation. Nucleosides and synthetic oligonucleotides will be studied by stopped-flow spectrophotometry and spectroelectrochemistry to decipher individual steps in the formation and decay of the quinones. Mechanistic parallels are proposed between purine oxidation and the pterin and flavin redox cofactors, leading to the transformative hypothesis that RNA damage products were ancestral to the evolution of nucleotide coenzymes.This research by Professor Burrows of the University of Utah will include the training of undergraduate and graduate students who will be well versed in mechanistic organic chemistry, gain biotechnical skills in the manipulation of nucleic acids and proteins, and contribute to our molecular understanding of DNA damage, which underlies processes leading to aging, cancer, and neurological disorders. A major new effort in this project is the development of a library of DNA adducts that will be prepared by undergraduate research students. The organic synthesis is straightforward and appropriate for students with little or no prior research experience. Students will prepare new nucleoside adducts by selecting from a wide variety of commercially available nucleophiles (e.g. primary amines such as amino acids), then purifing 5 mg of each adduct by HPLC, and loading them onto 96-well plates. Students will then analyze the plates by UPLC/ESI-MS and submit them for a battery of screening tests for biological activity at the University of Utah?s new core facility for biomolecular screening. Undergraduate researchers are recruited from the University?s ACCESS program, which fast-tracks high achieving women high school students into research labs in their freshman year. Professor Burrows is the Director of the Chemistry-Biology Interface Training Program from which minority undergraduate researchers will be recruited.

在化学系有机动力学项目的支持下，犹他大学化学系的 Cynthia J. Burrows 教授将详细描绘 DNA 在蛋白质、多胺等活性物质存在下如何被氧化的分子图。儿茶酚胺亲核试剂。为了了解基因组 DNA 损伤的基本过程，了解此类加合物如何形成的完整机制非常重要。该提案中概述的研究基于最近的结果，表明 8-oxo-7, 8-二氢鸟苷对单电子氧化剂的进一步氧化敏感，导致醌类中间体被亲核捕获，生成乙内酰脲产物。最近的进展表明，其他氧化碱基也受到相同的容易氧化/亲核捕获机制的影响。提议的实验包括核苷和 DNA 寡聚物的氨基酸和多胺加合物的 NMR 和 LC/MS 表征，以及它们的形成机制和化学稳定性的研究。我们将开发方法来了解通过 DNA 氧化和蛋白质氧化导致 DNA-蛋白质交联的化学结构。将制备酚和儿茶酚与 DNA 中鸟苷的加合物，并分析其介导 DNA 进一步氧化损伤的能力。新研究的主要部分将集中于 8-氧代鸟苷和尿酸氧化中反应性醌类中间体的表征。将通过停流分光光度法和分光电化学来研究核苷和合成寡核苷酸，以破译醌形成和衰变的各个步骤。嘌呤氧化与蝶呤和黄素氧化还原辅因子之间存在机制相似性，从而提出了一个变革性的假设：RNA 损伤产物是核苷酸辅酶进化的祖先。犹他大学 Burrows 教授的这项研究将包括对本科生和黄素氧化还原辅因子的培训。研究生将精通机械有机化学，获得操作核酸和蛋白质的生物技术技能，并为我们对 DNA 损伤的分子理解做出贡献，这是导致衰老、癌症和神经系统疾病的过程的基础。该项目的一项重大新工作是开发 DNA 加合物库，该库将由本科生准备。有机合成非常简单，适合那些很少或没有研究经验的学生。学生将通过从多种市售亲核试剂（例如氨基酸等伯胺）中进行选择来制备新的核苷加合物，然后通过 HPLC 纯化 5 mg 每种加合物，并将其加载到 96 孔板上。然后，学生将通过 UPLC/ESI-MS 分析这些板，并将其提交到犹他大学新的生物分子筛选核心设施进行一系列生物活性筛选测试。本科生研究人员是从该大学的 ACCESS 计划中招募的，该计划可让成绩优异的女高中生在大一时进入研究实验室。伯罗斯教授是化学-生物学界面培训项目的主任，该项目将招募少数族裔本科生研究人员。