DYNAMICS OF DNA MOTION AT PHYSIOLOGICAL CONCENTRATIONS

在这项拨款提案中提出的研究重点是开发 并应用理论和光谱工具研究DNA在 生理条件。理论工作的重点是 因此，关于染色质结构和超螺旋的相关问题 DNA在理论工作中，我们将前面的工作扩展到分析大型 相间比例(从105个碱基到3x10[8]碱基)结构 染色体。 试点工作有两个主要抓手：一是开发新的 荧光能量转移技术提高了50-500倍 信噪比高于传统技术，产生(除其他外) 优点)能够测量相对较长的距离(80-130 埃)与蛋白质-DNA复合体相关；第二，发展敏感 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 由细胞自发荧光引起的问题。 这两个实验方面都依赖于 含有稀土元素Tb和Eu的络合物。他们 具有异常长的寿命(Tb寿命1.5-2.2毫秒； Eu(0.6-2.3毫秒)，窄带发射(几纳米)，好到 优异的量子产额(0.1-1)，无自猝灭，且 条件，巨大的斯托克位移(200 NM)。这些特点使得 镧系元素络合发光探针，可用于 细胞上的荧光显微镜。这些特点也使 稀土螯合物在荧光能量转移(FET)中的应用 实验。特别是，它们在信号传输方面产生了改进 比传统FET高出几个数量级的背景 预计可能进行的距离测量大约是这个数字的两倍 以前可以用传统的FET实现。 尽管发展起来的光谱技术不会局限于 涉及DNA的问题，我们建议首先用它们来研究这样的 问题。特别是，我们建议首先应用我们的镧系元素 FET用于DNA-蛋白质的结构(和后来的动态)测量 复合体，包括DNA整合宿主因子复合体和DNA-uvrABC， 蛋白质诱导DNA弯曲的两个模型系统。这样的弯曲现在已为人所知 在原核生物和真核生物的基因调控中具有重要意义。在……里面 此外，IHF是研究重组的一个很好的模型系统，并且 UvrAB是研究DNA修复的一个很好的系统。我们也会申请 FET用于了解血小板增多症的结构和动力学性质 (超级卷曲的)DNA。我们建议首先将镧系元素络合物用作 荧光显微镜中的发光探针用于遗传研究 人类前列腺癌细胞的异常。与更传统的 荧光标记，自体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.