Single molecular fluorescence and force spectroscopy

这项工作的长期目标是更好地理解 在生物大分子中折叠/展开。我们将使用各种最先进的生物物理技术来研究核酸作为一个模型系统。像蛋白质一样，RNA酶可以进行催化，并以其活跃的折叠构型展示一系列的一级、二级和三级结构元素。然而，与多肽不同的是，RNA是基于4个碱基的聚合物库而不是20个氨基酸。它们还表现出二级和三级结构基序之间更具层次性的关系。RNA酶也是 更容易合成、修改、操作和建模，所有这些都使它们成为生物物理研究的候选对象。预计RNA和DNA折叠的一些原理将推广到蛋白质世界，此外还将提供对核酸生物化学的进一步见解。这项建议将集中在最具特性的核糖核酸酶之一，嗜热葡萄球菌及其衍生物的I组内含子核酶。我们最近的工作证明了在单分子水平上研究四膜虫核酶折叠、去折叠和催化的可行性。单分子方法，如单分子 荧光能量转移(FRET)和单分子光力光谱可以揭示折叠中间体、酶的随机性、动力学速率和路径的细节，这些都是通过整体方法不容易获得的。通过将荧光染料分子放置在核酶上的不同位置，我们建议研究快速折叠的第一阶段和波动在折叠中的作用，以及寻找新的中间状态并进一步绘制这种酶的折叠图景。我们还计划使用高分辨率光学镊子来测量核酶在特定的力负荷下如何变性。作为负载函数的分子端到端距离的高分辨率测量应该允许指定特定的 这种光谱的特征表现为特定的结构状态。最后，这些以物理为基础的关于四膜虫核酶如何折叠和展开的研究无疑将增加我们对更复杂的核酶和医学上相关的RNA酶，如核糖体的理解。

The long-term goal of this work is to develop an improved understanding of the mechanics of folding/unfolding in biological macromolecules. We will use a variety of state-of-the-art biophysical techniques to study nucleic acids as a model system. Like proteins, RNA enzymes can carry out catalysis, and exhibit an array of primary, secondary, and tertiary structural elements in their active, folded configurations. However, in contrast to polypeptides, RNA is based on a polymer repertoire of 4 bases instead of 20 amino acids. They also display a more hierarchical relation between secondary and tertiary structural motifs. RNA enzymes are also more easily synthesized, modified, manipulated, and modeled, all of which make them attractive candidates for biophysical studies. It is anticipated that some principles of RNA- and DNA-folding will generalize into the protein world, in addition to providing further insights into nucleic acid biochemistry. This proposal will concentrate on one of the best-characterized ribonucleic acid enzymes, the Group I intron ribozyme from T. thermophila and its derivatives. Our recent work has demonstrated the feasibility of studying folding, unfolding, and catalysis in the Tetrahymena ribozyme at the single molecule level. Single molecule methods such as single-molecule fluorescence energy transfer (FRET) and single-molecule optical force spectroscopy can reveal details of folding intermediates, enzyme stochasticity, kinetic rates and paths, that are not readily accessible through bulk methods. By placing fluorescent dye molecules in a variety of locations on the ribozyme, we propose to study the first stages of rapid collapse and the role of fluctuations in folding, as well as search for new intermediate states and further map the folding landscape of this enzyme. We also plan to use high resolution optical tweezers to measure how the ribozyme denatures under specific force loads. High-resolution measurements of the end-to-end distance of the molecule as a function of load should allow to assign specific features of this spectra to specific structural states. Finally, these physically-based studies of the how the well characterized Tetrahymena ribozyme folds and unfolds will no doubt add to our understanding of more complicated ribozymes and medically relevant RNA enzymes, such as the ribosome.