Single molecular fluorescence and force spectroscopy

这项工作的长期目标是提高对 生物大分子中的折叠/解折叠。我们将使用各种最先进的生物物理技术来研究核酸作为模型系统。像蛋白质一样，RNA酶可以进行催化，并以其活性折叠构型展示一系列一级、二级和三级结构元件。然而，与多肽相反，RNA基于4个碱基而不是20个氨基酸的聚合物库。它们还显示出二级和三级结构基序之间的层次关系。RNA酶也是 更容易合成，修改，操纵和建模，所有这些都使它们成为生物物理研究的有吸引力的候选人。预计RNA和DNA折叠的一些原理将推广到蛋白质世界，除了提供对核酸生物化学的进一步见解。这项建议将集中在一个最好的特点，核糖核酸酶，第一组内含子核酶从T。嗜热菌及其衍生物。我们最近的工作证明了在单分子水平上研究四膜虫核酶的折叠、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）和单分子光学力谱可以揭示折叠中间体、酶随机性、动力学速率和路径的细节，这些是通过本体方法不容易获得的。通过将荧光染料分子放置在核酶的不同位置，我们建议研究快速崩溃的第一阶段和折叠中波动的作用，以及寻找新的中间状态，并进一步绘制这种酶的折叠景观。我们还计划使用高分辨率的光镊来测量核酶在特定的力载荷下如何变性。作为负载函数的分子的端到端距离的高分辨率测量应允许分配特定的分子量。 这些光谱的特征与特定的结构状态有关。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.