Mechanisms of Nucleic Acid Oxidation and Cross-linking

在化学系有机动力学项目的支持下，犹他大学化学系的辛西娅·J·伯罗斯教授将绘制一张详细的分子图，说明在蛋白质、多胺和儿茶酚胺等活性物种存在的情况下，DNA是如何被氧化的。为了理解基因组DNA损伤的基本过程，了解这些加合物如何形成的完整机制图是很重要的。这项建议中概述的研究基于最近的结果，表明8-氧代-7，8-二氢鸟苷对单电子氧化剂的进一步氧化很敏感，导致一种喹酮中间体被亲核捕获以生成海因产物。最近的进展表明，其他被氧化的碱也受到相同的易氧化/亲核捕获机制的影响。实验内容包括核磁共振和LC/MS表征核苷和DNA低聚物上的氨基酸和多胺加合物，并研究它们的形成机理和化学稳定性。通过DNA氧化和蛋白质氧化了解DNA-蛋白质交联物的化学结构。将在DNA中制备酚和儿茶酚与鸟苷的加合物，并分析它们介导DNA进一步氧化损伤的能力。新研究的主要部分将集中在8-氧鸟苷和尿酸氧化反应中的活性喹酮中间体的表征。核苷和人工合成的寡核苷酸将通过停流分光光度和光谱电化学进行研究，以破译苯二酚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提出了机械上的相似之处，导致了一种变革性的假设，即RNA损伤产物是核苷酸辅酶进化的祖先。犹他大学的布伦斯教授的这项研究将包括对本科生和研究生的培训，他们将精通机械有机化学，获得操纵核酸和蛋白质的生物技术技能，并有助于我们对DNA损伤的分子理解，DNA损伤是导致衰老、癌症和神经疾病的基础过程。这个项目的一个主要新努力是开发一个DNA加合物文库，该文库将由本科生准备。有机合成是直截了当的，适合以前几乎没有研究经验的学生。学生将通过从各种商业亲核试剂(例如，伯胺，如氨基酸)中选择，然后用高效液相色谱法从每个加合物中提纯5毫克，并将它们装入96孔板中，来制备新的核苷加合物。然后，学生们将通过UPLC/ESI-MS分析这些平板，并将它们提交给犹他大学S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大学？S通道项目招募的，该项目在大学一年级时快速追踪成绩优异的女高中生进入研究实验室。布伦斯教授是化学-生物学接口培训项目的主任，将从该项目招聘少数族裔本科生研究人员。

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.