Fundamental Molecular Mechanisms of DNA Damage

DNA和RNA是腺苷（A）、胞苷（C）和胍（G）核苷酸的线性寡聚体，其中尿苷（U）或胸苷（T）分别作为RNA和DNA中的第四个核苷酸。 在DNA中，两条寡聚链上的核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。 核碱基电子态之间的重叠和相互作用导致减色、激子分裂和超快（亚ps）电子激发态弛豫，并导致难以预测DNA的化学和物理行为。 DNA表现出许多损伤产物，如紫外线和其他辐射，活性氧和其他化学物质。 我们的研究计划目标是测量和了解核酸分子水平的损伤。我们的方法结合了荧光探针和多重平台的损伤测量，以及共振拉曼光谱的飞秒（fs）初始激发态结构动力学和损伤反应机制的阐明。 用于DNA损伤检测的大多数技术遭受太特异或太一般的损伤检测，是复杂或耗时的，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荧光混合和读取检测单链和双链DNA损伤，并将其应用于微孔板平台，以同时表征20多个寡聚体的损伤。我们的检测对10 kb内的单个损伤病变具有选择性，并具有fmol范围内的灵敏度。 如本文所提出的，将这种方法同时扩展到成百上千个序列，可以对损伤如何取决于DNA结构和组成产生关键性的理解。 当前用于表征初始激发态结构动力学的技术，例如时间分辨光谱学，遭受非线性伪影和不足的时间或光谱分辨率。 我们使用共振拉曼强度来探测核酸组分的fs初始激发态结构动力学。我们以前的工作表明，紫外线诱导的核碱基损伤是高度依赖于结构，特别是核碱基取代基的质量。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。 这些实验为计算模型提供了有用的测试，其中许多仍然存在争议和未经证实。 该提案的具体目标包括：（1）表征核酸组分的fs初始激发态结构动力学，（2）扩展我们对细胞DNA损伤的测定，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性损伤，以适应广泛的损伤。

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.**