Single molecular fluorescence and force spectroscopy

单分子荧光和力谱

• 批准号: 7551204
• 负责人: Steven Chu
• 金额: $ 18.95万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7551204
• 关键词: Tetrahymena; chemical kinetics; chemical stability; conformation; fluorescence resonance energy transfer; nanotechnology; nucleic acid structure; protein folding; ribozymes; spectrometry; stoichiometry; structural biology

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.

这项工作的长期目标是建立对 生物大分子中的折叠/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模型系统。像蛋白质一样，RNA酶可以进行催化，并在其主动折叠构型中表现出一系列原发性，次级和高等结构元素。但是，与多肽相比，RNA基于4个碱基而不是20个氨基酸的聚合物库。它们还显示了二级和高等结构基序之间的更层次关系。 RNA酶也是 更容易合成，修饰，操纵和建模，所有这些使它们成为生物物理研究的有吸引力的候选者。可以预料，除了提供对核酸生物化学的进一步见解之外，RNA和DNA折叠的某些原理还将推广到蛋白质世界中。该建议将集中于最典型的核糖核酸酶之一，即来自T. thermophila及其衍生物的I组I内含子核酶。我们最近的工作表明，在单分子水平上研究四氢菌核酶中的折叠，展开和催化的可行性。单分子方法，例如单分子 荧光能量转移（FRET）和单分子光学力量光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，这些细节无法通过大量方法容易访问。通过将荧光染料分子放置在核酶上的各个位置，我们建议研究快速崩溃的第一阶段和波动在折叠中的作用，以及搜索新的中间状态，并进一步绘制该酶的折叠景观。我们还计划使用高分辨率光镊，以测量特定力载荷下的核酶剥落。分子的端到端距离作为负载函数的高分辨率测量应允许分配特定 该光谱与特定结构状态的特征。最后，这些基于物理的研究如何表征良好的四膜核酶褶皱和展开将无疑会增加我们对更复杂的核酶和医学上相关的RNA酶（例如核糖体）的理解。