Interferometric Plasmon Ruler for Elucidating Structural Dynamics on the SingleMolecule Level

用于阐明单分子水平结构动力学的干涉等离子体尺

基本信息

批准号：
10707027
负责人：
Bjoern Markus Reinhard
金额：
$ 20.63万
依托单位：
BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-20 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10707027
关键词：
Biological Biological Assay Biophysics Biopolymers Blinking Coupling DNA Data Dependence Detection Development Diameter Dimensions Disadvantaged Disease Dyes Electromagnetics Film Fluorescence Fluorescence Resonance Energy Transfer Gold Individual Length Maps Membrane Membrane Lipids Metals Molecular Molecular Conformation Molecular Probes Molecular Structure Monitor Neurodegenerative Disorders Optical Methods Optics Particle Size Performance Pharmaceutical Preparations Phase Phosphorylation Photobleaching Polymers Process Property Proteins Public Health Research Signal Transduction Single-Stranded DNA Sodium Chloride Structure Surface Techniques Technology Testing Time Work biophysical tools light scattering mechanical properties molecular dynamics nanoGold nanometer nanoparticle particle phase change protein folding single molecule tau Proteins temporal measurement tool

项目摘要

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

概括重要的生物分子的结构本质上是动态的，并且具有光学工具的重要需求可以探测单分子水平的生物聚合物的结构动力学，并具有高时间分辨率并且在最大观察时间内无限制。动态分子统治者，例如荧光共振能量转移（FRET）染料对或等离子体尺（PRS）以及束缚粒子测定当前可用的光学方法来探测单个分子的结构动力学。但是，fret是受到传统有机染料的光物理稳定性的困扰，这些染料是能量供体和受体。光漂白限制了最大的光激发和发射周期的数量，从而定义了连续监测分子结构的基本局限性。传统的PR和系绳粒子测定可以提供高信号强度，而无需眨眼或观察时间限制。警告但是，这些方法是典型尺寸的巨大颗粒，典型的尺寸纳米或更大。该建议开发了一种新的PRS，该PR是基于这样的现象通过生物聚合物调制的金纳米颗粒（NP）的距离依赖性耦合（NP） NP的干涉散射信号。该新PR基于等离子体的干涉测量值耦合并允许使用具有小至5 nm的尺寸的NP，如探针。干涉量PR将使以高时间分辨率的单个生物聚合物的结构动态和无需在时间分辨率和观察持续时间之间妥协。工作该提案中描述的将使用DNA作为生物聚合物实施干涉量PR，并表征其表现。在验证了DNA的干涉量PR概念之后，干涉量PR平台将是扩展以允许表征本质上无序的tau蛋白的结构动力学存在定义成分的脂质膜。干涉量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变化和将测试生物聚合物的动力学。该提案中描述的研究将导致新的动态分子统治者技术克服了常规光学分子尺的长期局限探针的大小和光物理稳定性的术语。该提案的具体目的是： AIM1：实施干涉量PR并测试其适用性以表征结构波动单DNA分子 AIM2：实施并验证一个干涉量PR来探测单个TAU的结构动力学膜附近的蛋白质