The focus of the research presented in this grant proposal is to develop and apply both theoretical and spectroscopic tools for studying DNA under physiological conditions. The emphasis in the theoretical work is therefore on problems relevant to chromatin structure and to supercoiled DNA. In the theoretical work, we extend earlier work to analyze the large scale (from 105 bases to 3x10[8] bases) structure of the interphase chromosome. The experimental work has two main thrusts: first, to develop new fluorescence energy transfer techniques with 50-500 fold improvement in signal to noise over conventional techniques, yielding (among other benefits) the ability to measure the relatively long distances (80-130 Angstroms) relevant in protein-DNA complexes; second, to develop sensitive luminescent probes for labeling of cellular and DNA organelles for fluorescence microscopy. These probes are intended to overcome contrast problems due to cellular autofluorescence. Both experimental aspects rely on the unusual luminescent properties of chelates containing the lanthanide elements Terbium and Europium. They have unusually long lifetimes (Terbium lifetime 1.5-2.2 milliseconds; Europium 0.6-2.3 msec), narrow band emissions (a few nanometers), good to excellent quantum yields (0.1-1), no self-quenching, and under the right conditions, huge Stoke shifts (200nm). These characteristics make lanthanide chelates nearly ideal luminescent probes for use in fluorescence microscopy on cells. These characteristics also make the lanthanide chelates excellent donors in fluorescence energy transfer (FET) experiments. In particular, they yield an improvement in signal to background of several orders of magnitude over conventional FET and are expected to make possible measurements over distances roughly twice that previously attainable with conventional FET. Although the spectroscopic techniques developed will not be limited to questions involving DNA, we propose to first use them to study such questions. In particular, we propose to first apply our lanthanide-based FET to structural (and later dynamic) measurements of DNA-protein complexes, including DNA -Integration Host Factor complex, and DNA-uvrABC, two model systems for protein-induced DNA bends. Such bends are now known to be important in prokaryotic and eukaryotic gene regulation. In addition, IHF is an excellent model system for studying recombination, and uvrAB is an excellent system for studying DNA repair. We will also apply FET to understanding the structural and dynamic properties of plectonemic (supercoiled) DNA. We propose to first use the lanthanide chelates as luminescent probes in fluorescence microscopy to study genetic abnormalities in human prostate cancer cells. With more conventional fluorescent labels, autofluorescence has prevented such imaging.
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