Ultrafast Spectroscopic Methods to Probe Photodamage and Unfolding in Biopolymers

描述(申请人提供)：需要新的光谱方法来确定生物系统在飞秒(10-15 S)到纳秒(10-9 S)时间尺度上发生的结构变化。紫外光对核酸的光损伤发生在100‘S飞秒内，而肽、蛋白质和寡核苷酸的众多功能依赖于发生在皮秒到纳秒甚至更长时间的结构波动。在每一种情况下，几乎没有实验技术可以解析进化系统的动态分子结构。我们的工作旨在通过开发能够收集振动光谱的超快拉曼光谱来纠正这一问题，该光谱可以直接与生物分子的光化学和热激活动力学的分子结构相关。这项提议的具体目标#1将开发新的飞秒拉曼仪器，该仪器可以收集时间分辨率优于100飞秒的高分辨率振动光谱。这一方法将被应用于对DNA中嘧啶类化合物在紫外光激发下的超快二聚化的新的理解，以及对核酸聚合物中激发的量子力学性质的新的基本理解。带着明确的目标#2，我们将开发新的方法来冲动地启动生物聚合物的热解折叠。这将使目前应用仅限于光化学的时间分辨光谱学的众多工具能够在皮秒到纳秒的时间尺度上解开热驱动的二级结构变化的动力学。拉曼光谱在这些领域具有独特的地位，因为它能够在不受水环境干扰的情况下，在宽光谱窗口内收集生物聚合物的振动光谱。生物聚合物的拉曼光谱表现出特定的峰，这些峰是聚合物的二级结构和侧链或核酸的特定环境的特征。因此，通过收集生化过程中的时间分辨拉曼光谱，我们可以确定形成的时间尺度和动力学中间体的结构。这项实验工作将补充许多关于光激发DNA的超快结构变化和更长时间尺度结构涨落的理论预测。这项拟议的研究意义重大，因为这些技术可以解决光生物学的广度问题，并且需要对对生物学和人类健康重要的快速结构变化进行实验探测。 公共卫生相关性(由申请人提供)：拟议的研究通过帮助建立对紫外线损伤DNA的机制及其对公共健康的影响的新理解，直接支持国家卫生研究院的使命。紫外线是地球上最普遍的环境诱变剂之一，对人类健康具有重大的有害影响。这些研究还将提高生物医学研究人员研究对蛋白质和DNA功能至关重要的快速结构运动的能力，从而加速新疗法的开发。

DESCRIPTION (provided by applicant): New spectroscopic methods are needed to determine the structural changes that occur in biological systems on the femtosecond (10-15 s) to nanosecond (10-9 s) time-scale. Photodamage to nucleic acids by ultraviolet (UV) light occurs in 100's of femtoseconds and numerous functions of peptides, proteins and oligonucleotides depend on structural fluctuations occurring over picoseconds to nanoseconds and longer. In each of these cases, there are few experimental techniques that can resolve the dynamic molecular structure of the evolving system. Our work aims to correct this by developing ultrafast Raman spectroscopy capable of collecting vibrational spectra, which can be directly related to molecular structure, of photochemically and thermally activated dynamics in biomolecules. Specific Aim #1 of this proposal will develop new femtosecond Raman instrumentation that can collect high-resolution vibrational spectra with time resolution better than 100 fs. This methodology will be applied to gain new understanding of the ultrafast dimerization of pyrimidines in DNA following excitation by UV light, as well as new fundamental understanding of the quantum mechanical nature of the excitation in nucleic acid polymers. With Specific Aim #2, we will develop new methodologies to impulsively initiate thermal unfolding of biopolymers. This will allow the numerous tools of time-resolved spectroscopy, which currently have applications limited to photochemistry, to unravel the dynamics of thermally driven secondary structural changes on the picosecond to nanosecond time-scale. Raman spectroscopy is uniquely positioned to contribute to these areas because of its ability to collect vibrational spectra of biopolymers over a wide spectral window without interference from the aqueous environment. Raman spectra of biopolymers exhibit particular peaks that are characteristic of the secondary structure of the polymer and the particular environment of the side chains or nucleic acids. Hence by collecting time-resolved Raman spectra as biochemical processes proceed, we can determine both the time-scales of formation and the structures of kinetic intermediates. This experimental work will complement the many theoretical predictions that have been made about ultrafast structural changes in photoexcited DNA and longer time-scale structural fluctuations. The proposed research is significant because of the breadth of photobiology that can be addressed by these techniques and the need for experimental probes of rapid structural changes important to biology and human health. PUBLIC HEALTH RELEVANCE (provided by applicant): The proposed research directly supports the mission of the NIH by helping to establish new understanding of the mechanisms of ultraviolet light damage to DNA, and its implication for public health. Ultraviolet light is one of the most prevalent environmental mutagens on earth, with significant deleterious effects on human health. These studies will also increase the capability of biomedical researchers to investigate the rapid structural motions important to the functions of proteins and DNA, thereby accelerating the development of new therapeutics.