Interpreting Optical Signals from the DNA Fluorescence Probe2-Aminopurine

高荧光核苷酸2AP（2-氨基嘌呤）的Nordlund光谱学是一种相对较新的生化工具，用于监测DNA构象、DNA-蛋白结合和DNA的光物理过程。吸收和荧光光谱，用2AP代替特定的腺嘌呤，可以提供特定位点的详细结构信息，拉曼光谱可以提供更多信息。2AP对氢键、水暴露、分子运动、蛋白质结合、DNA解绕以及DNA熔化和预熔化转变敏感。2AP与其他碱基的链内相互作用产生新的条带，这些条带可以区分A和G碱基以及C和t碱基，从而可以测量DNA中能量转移的效率和序列依赖性；鉴定G和2AP之间的异常近邻相互作用，蛋白质与特定DNA碱基的结合以及其他详细的表征可以完成。现在需要的是组织和解释各种各样的观察结果，形成一个指南，研究人员可以据此计划一套简单的实验，以令人信服地回答结构性问题。这项工作的中心目标是收集一组DNA结构和相互作用的主要问题，这些问题可以很容易地通过2AP光学方法回答，并进一步确定最佳的光谱方法。该方法将应用的系统：寡核苷酸和Eco RI内切酶，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对环境的所有繁殖和适应的核心。人们一度认为DNA只以两种形式存在，要么是高度均匀的沃森-克里克双螺旋结构，要么是无序的随机螺旋结构。但现在很清楚，DNA形成了多种多样的整体结构，局部DNA的不规则性在蛋白质结合并将DNA信息转化为细胞活动时，在信号传导和引导“交通流”方面发挥了核心作用。成熟的方法如x射线晶体学和核磁共振（NMR）可以详细表征这些局部结构，但需要大量的设备、时间和样品。该项目将开发一种新的光学方法用于定量局部和整体DNA结构变化的指南。这种结构不能像x射线或核磁共振那样详细地确定，但在灵敏度、成本、时间和所需设备方面具有明显的优势。该方法是在DNA中插入一种经过修饰的结构成分，当用激光或其他光束激发时，它将发出特征的、结构和环境敏感的荧光灯。＊＊＊

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. ***