DYNAMICS OF DNA MOTION AT PHYSIOLOGICAL CONCENTRATIONS

最近的实验表明，在浓缩溶液中， DNA动力学深受分子间相互作用的影响。 这种解释特别耐人寻味，因为DNA执行 是细胞在集中环境中的功能。这个 这里提出的实验旨在探索，在更大的 深度，DNA动力学的本质在浓度为 代表生理状态。两种荧光 技术--荧光光漂白恢复(FPR)和 荧光相关光谱(FCS)--和一个脉冲电子 将使用电子双共振(Eldor)技术。 浓缩液中DNA的转动扩散和平移扩散 将测量溶液和凝胶中的浓度。我们新引进的 将使用偏振FPR技术和脉冲Eldor来测量 浓度、盐、DNA大小和形态以及 RNA聚合酶等重要酶的结合 重新定向放松。偏振fpr最近被展示出来。 能很好地适应慢速(1亩以上)的监控任务 SEC)重新定向放松。脉冲Eldor技术是 预计将产生出色的角度分辨率，约为10 度，并能够测量旋转相关性 时间从大约200纳秒到1毫秒； 对Eldor的解释在概念上也相当简单。 用于Eldor研究的三个新的电子顺磁 共振(EPR)自旋标记探针-氮氧化物-补骨脂素化合物， 和氮氧化物标记的腺苷和胸苷碱基-已经被 由我们和我们的合作者合成。后者的分子具有 被证明对DNA没有干扰，并且是一个忠诚的人 DNA运动的记者。将使用翻译的FPR来 确定自扩散的相互作用相关性 浓缩样品的DNA系数；互补FCS 实验将使我们能够测量相互扩散。这个 这里提出的重新定位和翻译研究是值得期待的 以揭示有关其背后的动力的新信息 重要的生物过程，如转录、DNA 复制和DNA缩合。

Recent experiments have suggested that, in concentrated solutions, DNA dynamics is profoundly affected by intermolecular interactions. This interpretation is especially intriguing because DNA performs is cellular functions in a concentrated environment. The experiments proposed herein are designed to explore, in greater depth, the nature of DNA dynamics at concentrations that are representative of the physiological state. Two fluorescence techniques--Fluorescence Photobleaching Recovery (FPR) and Fluorescence Correlation Spectroscopy (FCS)-- and a pulsed Electron Electron Double Resonance (ELDOR) technique will be used. Rotational and translational diffusion of DNA in concentrated solutions and in gels will be measured. Our newly introduced polarized FPR technique and pulsed ELDOR will be used to measure the effects that concentration, salt, DNA size and form and the binding of important enzymes such as RNA polymerase have on reorientational relaxation. Polarized FPR has recently been shown to be well suited to the task of monitoring slow (longer than 1 mu sec) reorientational relaxation. The pulsed ELDOR technique is expected to yield excellent angular resolution approximately 10 degrees, and to be capable of measuring rotational correlation times from about 200 nanoseconds to one millisecond; the interpretation of ELDOR is also conceptually quite straightforward. For use in the ELDOR studies, three new Electron Paramagnetic Resonance (EPR) spin label probes--a nitroxide-psoralen compound, and nitroxide-labeled adenosine and thymidine bases--have been synthesized by us and our collaborators. The latter molecule has been shown to be nonperturbing to the DNA and to be a faithful reporter of DNA motion. Translational FPR will be used to determine the interaction dependence of the self-diffusion coefficient of DNA in concentrated samples; complementary FCS experiments will allow us to measure mutual diffusion. The reorientation and translation studies proposed here are expected to reveal novel information about the dynamics that underlies important biological processes such as transcription, DNA replication and DNA condensation.