Interferometric Plasmon Ruler for Elucidating Structural Dynamics on the SingleMolecule Level

摘要 重要生物分子的结构本质上是动态的，对光学工具有重要需求 它可以在单分子水平上以高时间分辨率探测生物聚合物的结构动力学 并且不受最长观察时间的限制。动态分子尺子，如荧光 共振能量转移(FRET)染料对或等离子尺子(PR)以及拴系粒子分析 目前可用的光学方法来探测单个分子的结构动力学。然而，令人担忧的是， 作为能量供体的传统有机染料的有限光物理稳定性和 接受者。光漂白限制了光激发和发射循环的最大次数，从而定义了 连续监测分子结构的基本限制。传统PR和系留 颗粒分析可以提供高信号强度，而不会闪烁，也不会限制观察时间。告诫： 然而，在这些方法中，有一种是大尺寸的粒子，其典型尺寸约为几十 纳米或更大。这项提议开发了一种新的公关类别，它基于这样的现象 金纳米粒子(NP)通过生物聚合物与金膜的距离依赖耦合调制 NP的干涉散射信号。这一新的PR是基于对等离子体的干涉检测 耦合，并允许使用尺寸小至5纳米的纳米粒子作为探针。干涉测量PR将 使以高时间分辨率监测单个生物聚合物的结构动力学成为可能 不需要在时间分辨率和观测持续时间之间妥协。这项工作 将使用DNA作为生物聚合物来实现干涉测量PR，并表征其 性能。在用DNA验证干涉PR概念后，干涉PR平台将是 扩展以允许表征固有无序的tau蛋白的结构动力学 存在特定成分的脂膜。干涉型PR监测的能力 单个tau分子的结构动力学，并检测膜诱导的结构和 将测试生物聚合物的动力学。这项提案中描述的研究将产生一种新的动力 分子尺技术，克服了传统光学分子尺子长期以来的局限性 探测器的尺寸和光物理稳定性方面的条件。这项建议的具体目的是： AIM1：实现干涉测量PR并测试其对表征结构波动的适用性 单个DNA分子 AIM2：实现并验证用于探测单个Tau的结构动力学的干涉PR 膜附近的蛋白质

Summary The structure of important biomolecules is intrinsically dynamic, and there is an important need for optical tools that can probe the structural dynamics of biopolymers at the single molecule level with high temporal resolution and without limitation in maximum observation time. Dynamic molecular rulers, such as Fluorescence Resonance Energy Transfer (FRET) dye pairs or plasmon rulers (PRs), as well as tethered particle assays are currently available optical methods to probe the structural dynamics of individual molecules. FRET is, however, plagued by the limited photophysical stability of conventional organic dyes that serve as energy donor and acceptor. Photobleaching limits the maximum number of photoexcitation and emission cycles and, thus, defines fundamental limitations for a continuous monitoring of molecular structure. Conventional PRs and tethered particle assays can provide high signal intensities without blinking or limitation in observation time. The caveat of these approaches is, however, the large size of the particles with typical dimensions on the order of tens of nanometers or larger. This proposal develops a new class of PRs that is based on the phenomenon that the distance-dependent coupling of a gold nanoparticle (NP) tethered to a gold film through a biopolymer modulates the interferometric scattering signal of the NP. This new PR is based on an interferometric detection of plasmon coupling and allows the use of NPs with dimensions as small as 5 nm as probes. The interferometric PRs will make it possible to monitor the structural dynamics of individual biopolymers with high temporal resolution and with no need to compromise between temporal resolution and the duration of the observation. The work described in this proposal will implement interferometric PRs using DNA as biopolymer and characterize their performance. After validating the interferometric PR concept with DNA, the interferometric PR platform will be expanded to allow the characterization of the structural dynamics of the intrinsically disordered tau protein in the presence of a lipid membrane of defined composition. The ability of the interferometric PR to monitor the structural dynamics of a single tau molecule and to detect membrane-induced changes in the structure and dynamics of the biopolymer will be tested. The research described in this proposal will result in a new dynamic molecular ruler technology that overcomes longstanding limitations of conventional optical molecular rulers in terms of the size and photophysical stability of the probes. The specific Aims of this proposal are to: Aim1: Implement the Interferometric PR and Test Its Applicability to Characterize Structural Fluctuations in Single DNA Molecules Aim2: Implement and Validate an Interferometric PR for Probing the Structural Dynamics of a Single Tau Protein in the Vicinity of a Membrane