DYNAMICS OF DNA MOTION AT PHYSIOLOGICAL CONCENTRATIONS

本拨款申请中提出的研究重点是开发 并应用理论和光谱工具来研究DNA， 生理条件。理论工作的重点是 因此，在与染色质结构和超螺旋相关的问题上， DNA.在理论工作中，我们扩展了以前的工作，分析了大的 规模（从105个碱基到3x 10 [8]个碱基）的界面结构 染色体 实验工作有两个主要的推动力：第一，开发新的 荧光能量转移技术， 信噪比优于常规技术，产生（除其它外 优点）测量相对较长距离（80-130 血管紧张素）相关的蛋白质-DNA复合物;第二，发展敏感的 用于标记细胞和DNA细胞器的发光探针， 荧光显微镜这些探头旨在克服对比度 由于细胞自发荧光的问题。 这两个实验方面都依赖于 含有镧系元素铽和铕的螯合物。他们 具有异常长的寿命（铽寿命1.5-2.2毫秒; 铕0.6-2.3毫秒），窄带发射（几纳米），良好的 优异的量子产率（0.1-1），无自猝灭，且在合适的 条件下，巨大的斯托克位移（200 nm）。这些特点使 镧系螯合物几乎理想的发光探针， 荧光显微镜下观察细胞。 这些特点也使得 镧系元素螯合物在荧光能量转移（FET）中的优异供体 实验特别地，它们产生信号的改善， 背景技术的几个数量级超过传统的FET， 预计可以在大约两倍于 这是以前用传统FET可以达到的。 尽管所开发的光谱技术将不限于 涉及DNA的问题，我们建议首先使用它们来研究 问题.特别是，我们建议首先应用我们的镧系元素基 FET到DNA-蛋白质的结构（和后来的动态）测量 复合物，包括DNA -整合宿主因子复合物和DNA-uvrABC， 蛋白质诱导DNA弯曲的两个模型系统。这种弯曲现在已知 在原核和真核基因调控中起重要作用。在 此外，IHF是研究重组的极好模型系统， uvrAB是研究DNA修复的优秀系统。我们还将应用 场效应晶体管对了解plectonemic的结构和动力学性质 （超螺旋）DNA。我们建议首先使用镧系元素螯合物作为 荧光显微镜中的发光探针， 人类前列腺癌细胞的异常。与更传统 荧光标记，自发荧光阻止了这种成像。

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.