Interferometric Plasmon Ruler for Elucidating Structural Dynamics on the SingleMolecule Level

总结 重要生物分子的结构本质上是动态的，对光学工具有重要的需求 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 并且没有最大观察时间的限制。动态分子标尺，如荧光 共振能量转移（FRET）染料对或等离子体标尺（PR），以及系留颗粒测定法， 目前可用的光学方法来探测单个分子的结构动力学。然而，FRET， 受到用作能量供体的常规有机染料的有限的物理化学稳定性的困扰， 接受者光漂白限制了光激发和发射循环的最大数量，因此， 分子结构连续监测的基本限制。传统PR和系留PR 颗粒测定可提供高信号强度而不闪烁或不受观察时间的限制。知会备忘 然而，这些方法中的一个缺点是颗粒的大尺寸， 纳米或更大。这一建议开发了一种新的PR类，其基于以下现象： 通过生物聚合物将金纳米颗粒（NP）拴系到金膜上的距离依赖性偶联调节 NP的干涉散射信号。这种新的PR是基于等离子体激元的干涉检测 耦合，并允许使用尺寸小至5 nm的NP作为探针。干涉PR将 使得能够以高时间分辨率监测单个生物聚合物的结构动力学， 不需要在时间分辨率和观察持续时间之间折衷。工作 将使用DNA作为生物聚合物来实现干涉PR，并表征其特征。 性能在用DNA验证干涉式PR概念之后，干涉式PR平台将 扩展到允许表征的结构动力学的内在无序的tau蛋白在 存在确定组成的脂质膜。干涉式PR监测 单个tau分子的结构动力学，并检测膜诱导的结构变化， 将测试生物聚合物的动力学。本提案中描述的研究将产生一种新的动态 分子标尺技术，克服了传统光学分子标尺的长期局限性， 探针的尺寸和物理稳定性。该提案的具体目标是： 目标1：实施干涉PR并测试其在表征结构波动方面的适用性， 单个DNA分子 目标2：实施和验证干涉PR，用于探测单个Tau的结构动力学 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