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. ***
