Interpreting Optical Signals from the DNA Fluorescence Probe2-Aminopurine

解读 DNA 荧光探针 2-氨基嘌呤的光信号

• 批准号: 9723278
• 负责人: Thomas Nordlund
• 金额: $ 18万
• 依托单位: University of Alabama at Birmingham
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9723278&HistoricalAwards=false
• 关键词: Interpreting; Optical; Signals; DNA; Fluorescence

9723278 Nordlund Optical spectroscopy of the high-fluorescence nucleotide, 2AP (2-aminopurine), is a relatively new biochemical tool for monitoring DNA conformation, DNA-protein binding and photophysical processes in DNA. Absorption and fluorescence spectroscopy, with 2AP replacing specific adenines, can provide site-specific, detailed structural information, with Raman spectroscopy poised to give even more. 2AP is sensitive to H-bonding, water exposure, molecular motion, protein binding, DNA unwinding and DNA melting and premelting transitions. Intrastrand interaction between 2AP and other bases results in new bands which distinguish A and G bases from each other and from C and T. Efficiency and sequence dependence of energy transfer in DNA can be measured; identification of an anomalous nearest-neighbor interaction between G and 2AP, protein binding to particular DNA bases and other detailed characterizations can be done. What is now needed is to organize and interpret the variety of observations into a guide by which a researcher can plan a simple set of experiments to convincingly answer structural questions. The central objective of this work is to assemble a set of major questions of DNA structure and interactions which can readily be answered by 2AP optical methods and further, to determine the best spectroscopic approach. The systems to which the method will be applied: oligonucleotides and complexes with Eco RI endonuclease, CAP and/or T7 RNA polymerase. Interaction of DNA with proteins and other cellular components lies at the heart of all reproduction and adaption of organisms to the environment. Once believed to exist only in two forms, either the highly uniform Watson-Crick double helix or a disordered random coil, it is now clear that DNA forms a large variety of overall structures, with local DNA irregularities playing a central role in signaling and guiding the "traffic flow" of proteins as they bind and translate DNA information into cellular activity. Well-develo ped methods such as x-rays crystallography and magnetic resonance (NMR) can characterize these local structures in detail, but at great cost of equipment, time and quantity of sample. This project will develop a guide to the use of a new optical method for quantitating local and overall DNA structural changes. The structure will not be determined in as great a detail as with x-ray or NMR, but clear advantages will be gained in sensitivity, cost, time and needed equipment. The approach is to insert a modified structural component into the DNA, which will emit characteristic, structure-and environment-sensitive fluorescent light when excited with a laser or other light beam. ***

9723278 Nordlund高荧光核苷酸2AP（2-氨基嘌呤）的光谱学是一种相对较新的生化工具，用于监测DNA构象，DNA-蛋白质结合和DNA中的生物物理过程。用2AP取代特定腺嘌呤的吸收和荧光光谱可以提供位点特异性的详细结构信息，而拉曼光谱则可以提供更多信息。 2AP对氢键、水暴露、分子运动、蛋白质结合、DNA解旋以及DNA解链和预解链转变敏感。 2AP和其他碱基之间的链内相互作用产生新的条带，这些条带将A和G碱基彼此区分开，并将A和G碱基与C和T区分开。 可以测量DNA中能量转移的效率和序列依赖性;可以进行G和2AP之间的异常最近邻相互作用、蛋白质与特定DNA碱基的结合以及其他详细表征的鉴定。 现在需要的是把各种各样的观察结果组织起来，并加以解释，形成一个指南，使研究人员能够计划一组简单的实验，从而令人信服地回答结构性问题。 这项工作的中心目标是组装一套主要的问题，DNA结构和相互作用，可以很容易地回答2AP光学方法，并进一步确定最佳的光谱方法。 将应用该方法的系统：寡核苷酸和与EcoRI内切核酸酶、CAP和/或T7 RNA聚合酶的复合物。 DNA与蛋白质和其他细胞成分的相互作用是生物体所有繁殖和适应环境的核心。 曾经认为DNA只以两种形式存在，要么是高度均匀的沃森-克里克双螺旋，要么是无序的无规卷曲，现在很清楚，DNA形成了各种各样的整体结构，局部DNA不规则性在蛋白质的信号传导和引导“交通流”中发挥着核心作用，因为它们结合并将DNA信息转化为细胞活性。 发展成熟的方法，如X射线晶体学和磁共振（NMR）可以详细表征这些局部结构，但在设备，时间和样品数量的巨大成本。 该项目将制定一个指南，使用一种新的光学方法来量化局部和整体DNA结构的变化。 结构将不像X射线或NMR那样详细地确定，但在灵敏度、成本、时间和所需设备方面将获得明显的优势。 这种方法是将一种经过修饰的结构成分插入DNA中，当用激光或其他光束激发时，它将发出特征性的、结构和环境敏感的荧光。 ***