DYNAMICS OF DNA MOTION AT PHYSIOLOGICAL CONCENTRATIONS

最近的实验表明，在浓溶液中， DNA动力学受分子间相互作用的深刻影响。 这种解释特别有趣，因为DNA执行 是细胞在集中环境中的功能。 的 本文提出的实验旨在探索更大的 深度，DNA动力学的性质，在浓度是 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）--与脉冲电子 将使用电子双共振（ELDOR）技术。 浓溶液中DNA的旋转和平移扩散 将测量溶液和凝胶中的浓度。 我们新引进的 偏振FPR技术和脉冲ELDOR将用于测量 浓度、盐、DNA大小和形式以及 RNA聚合酶等重要酶的结合， 再取向弛豫 极化FPR最近已被证明 要很好地适应监测任务缓慢（长于1亩 sec）再取向弛豫。 脉冲ELDOR技术是 预期产生约10的优异的角分辨率 度，并能够测量旋转相关性 时间从大约200纳秒到一毫秒; 对ELDOR的解释在概念上也相当简单。 为了在ELDOR研究中使用，三个新的电子顺磁 共振（EPR）自旋标记探针--一种氮氧自由基-卟啉化合物， 以及氮氧自由基标记的腺苷和胸苷碱基， 由我们和我们的合作者合成。 后者分子具有 被证明是不干扰的DNA，是忠实的 DNA运动的报告者。 翻译FPR将用于 确定自扩散的相互作用依赖性 浓缩样品中DNA系数;互补FCS 实验将使我们能够测量相互扩散。 的 本文提出的重新定位和翻译研究是值得期待的 揭示了新的信息， 重要的生物学过程如转录、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.