Fundamental Molecular Mechanisms of DNA Damage

DNA 损伤的基本分子机制

• 批准号: RGPIN-2015-06247
• 负责人: Loppnow, Glen
• 金额: $ 2.04万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=614035
• 关键词: Fundamental; Molecular; Mechanisms; DNA; Damage

DNA and RNA are linear oligomers of adenosine (A), cytidine (C), and guanidine (G) nucleotides, with either uridine (U) or thymidine (T) as the fourth nucleotide in RNA and DNA, respectively. In DNA, hydrogen bonding between nucleobases on two oligomeric strands forms the iconic double-stranded B-form helix, with C pairing with G and A pairing with T. The consequent overlap and interactions between the nucleobase electronic states results in hypochromism, exciton splitting and ultrafast (sub-ps) electronic excited-state relaxation, and leads to difficulties in predicting DNA's chemical and photophysical behavior.      DNA exhibits many damage products from such insults as UV and other radiation, reactive oxygen species and other chemicals. Our research program goal is to measure and understand molecular-level damage in nucleic acids. Our approach combines measurement of damage from fluorescent probes and multiplex platforms, with elucidation of the femtosecond (fs) initial excited-state structural dynamics and damage reaction mechanisms from resonance Raman spectroscopy.      Most techniques for DNA damage detection suffer from either too specific or too general detection of damage, are complex or time-consuming, can only detect damage in a few sequences simultaneously, and/or introduce additional lesions. We have developed simple fluorescent mix-and-read assays of single- and double-stranded DNA damage and applied them in a microplate platform to characterize damage in over 20 oligomers simultaneously. Our assays are selective for a single damage lesion within 10 kb and have sensitivity in the fmol range. Expanding this approach to hundreds and thousands of sequences simultaneously, as proposed here, can yield a critical understanding of how damage depends on DNA structure and composition.       Current techniques for characterizing initial excited-state structural dynamics, such as time-resolved spectroscopy, suffer from non-linear artifacts and insufficient time or spectral resolution. We use resonance Raman intensities to probe the fs initial excited-state structural dynamics of nucleic acid components. Our previous work has shown that UV-induced damage in nucleobases is highly dependent on structure, particularly the mass of nucleobase substituents. In this proposal, we continue to explore the causal relationship between nucleobase structure, initial excited-state structural dynamics and photochemistry. These experiments provide useful tests of computational models, many of which remain controversial and unproven.      Specific objectives for this proposal include (1) characterizing the fs initial excited-state structural dynamics of nucleic acid components, (2) expanding our assays of damage to cellular DNA, and (3) developing parallel platforms to simultaneously probe sequence- and structure-dependent damage in multiple DNA sequences to a wide range of insults.

DNA和RNA是腺苷(A)、胞苷(C)和胍(G)核苷酸的线性低聚物，尿苷(U)或胸苷(T)分别是RNA和DNA中的第四个核苷酸。在DNA中，碱基之间的氢键形成了标志性的双链B型螺旋，C与G配对，A与T配对。碱基电子态之间的重叠和相互作用导致了减色、激子分裂和超快(亚PS)电子激发态弛豫，导致了预测DNA的化学和光物理行为的困难。 他们的DNA展示了许多来自紫外线和其他辐射、活性氧物种和其他化学物质等侮辱的损害产物。我们的研究计划目标是测量和了解核酸分子水平的损伤。我们的方法结合了对荧光探针和多路平台的损伤测量，以及从共振拉曼光谱解释飞秒(Fs)初始激发态结构动力学和损伤反应机制。 然而，大多数DNA损伤检测技术要么过于具体，要么过于笼统地检测损伤，而且复杂或耗时，只能同时检测几个序列中的损伤，和/或引入额外的损伤。我们已经开发了简单的单链和双链DNA损伤的荧光混合读取分析方法，并在微板平台上应用它们来同时表征20多种低聚物的损伤。我们的检测方法对10kb范围内的单个损伤损伤具有选择性，并且在fmol范围内具有灵敏度。将这种方法同时扩展到成百上千个序列，可以产生对损伤如何依赖于DNA结构和组成的关键理解。 目前用于表征初始激发态结构动力学的技术，如时间分辨光谱学，存在非线性伪影和时间或光谱分辨率不足的问题。我们使用共振拉曼强度来探测核酸组分的飞秒初始激发态结构动力学。我们以前的工作表明，紫外线对碱基的损伤高度依赖于结构，特别是碱基取代基的质量。在这个提议中，我们继续探索核碱基结构、初始激发态结构动力学和光化学之间的因果关系。这些实验提供了对计算模型的有用测试，其中许多仍然存在争议和未经证实。 这项建议的具体目标包括(1)表征核酸成分的FS初始激发态结构动力学，(2)扩大我们对细胞DNA的损伤分析，以及(3)开发并行平台，以同时探测多个DNA序列中的序列和结构依赖的损伤，以进行广泛的侮辱。