Interpreting Optical Signals from the DNA Fluorescence Probe2-Aminopurine

9723278高荧光核苷酸2AP(2-氨基嘌呤)的诺德伦光学光谱是一种相对较新的生物化学工具，用于监测DNA的构象、DNA与蛋白质的结合和光物理过程。吸收和荧光光谱，用2AP代替特定的腺嘌呤，可以提供特定部位的、详细的结构信息，而拉曼光谱可以提供更多。2AP对氢键、水暴露、分子运动、蛋白质结合、DNA解离和DNA熔融及预熔转变敏感。2AP与其他碱基之间的链内相互作用产生了新的区带，将A和G碱基彼此区分开来，并与C和T区分开来。可以测量DNA中能量转移的效率和序列相关性；可以识别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特征。现在需要的是将各种各样的观察组织和解释成一个指南，研究人员可以根据这个指南计划一组简单的实验，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关于DNA结构和相互作用的主要问题，这些问题可以很容易地用2AP光学方法来回答，并进一步确定最佳的光谱方法。该方法将应用于的体系：寡核苷酸和与Eco RI内切酶、CAP和/或T7RNA聚合酶的复合体。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。曾经人们认为只有两种形式存在，要么是高度均匀的Watson-Crick双螺旋，要么是无序的随机卷曲。现在很明显，DNA形成了各种各样的整体结构，局部DNA不规则在结合DNA信息并将其转化为细胞活动时，在信号传递和引导蛋白质的“交通流”方面发挥着核心作用。X射线结晶学和核磁共振等成熟的方法可以详细地表征这些局部结构，但需要花费大量的设备、时间和样品数量。该项目将开发一种使用新的光学方法来量化局部和整体DNA结构变化的指南。这种结构的确定不会像X射线或核磁共振那样详细，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，这种成分将发出特征、结构和环境敏感的荧光。***

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. ***