Toward high spatiotemporal resolution models of single molecules for in vivo applications

用于体内应用的单分子高时空分辨率模型

• 批准号: 10552322
• 负责人: Steve Presse
• 金额: $ 27.4万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552322
• 关键词: Antibiotics; Biological; Caenorhabditis elegans; Cell Nucleus; Chemistry; Collaborations; DNA; Data; Deterioration; Diffusion; Disease; Energy Transfer; Escherichia coli; Event; Fluorescence; Funding; Future; Image; Knowledge; Label; Learning; Life; Lifting; Light; Lighting; Maps; Mathematics; Methods; MicroRNAs; Modeling; Monitor; Morphologic artifacts; National Institute of General Medical Sciences; Nature; Nobel Prize; Noise; Photons; Process; Property; Proteins; Publications; Reaction; Refractive Indices; Resolution; Sampling; Spectrum Analysis; Stress; Structure; Techniques; Therapeutic Agents; Work; adaptive optics; cost; data acquisition; data exchange; detector; disease diagnostic; fluorescence imaging; in vivo; insight; novel; quantum; single molecule; spatiotemporal; stoichiometry; temporal measurement; tool; trafficking; transcription factor

Project Summary Background and Knowledge Gap: Unraveling life's intracellular processes at single molecule (SM) spatiotem- poral scales is critical toward monitoring therapeutic agents and developing disease diagnostics. Yet drawing insight on biomolecular events at such scales presents profound challenges to existing fluorescence imaging. Fundamentally, this arises due to the model selection problem: unavoidable (quantum, thermal, detector) noise at the SM scale means that the data cannot easily be used to resolve “models" such as the number of molecules located within a small region of space. An experimental solution toward resolving this problem earned the 2014 Chemistry Nobel prize though such solutions necessarily come at a cost. Either spatial or temporal resolution is compromised while samples are often irradiated over extended durations inducing sample photodamage. Recent Progress: Thanks to having reached the funding midpoint of both our NIGMS R01s, we developed mathematical tools allowing us to mitigate, sometimes dramatically, spatial (R01GM130745) and temporal (R01 GM134426) compromises of existing experimental solutions to model selection. Our work has resulted in 10 publications, 15 collaborations, and 18 ongoing projects. Here are just 3 projects: 1) in recent publications, we derived SM properties using 2-3 orders of magnitude fewer photons than would normally be used to obtain bulk properties from fluorescence correlation spectroscopy (FCS); 2) in accepted work, we provide a means to determine protein cluster stoichiometry (up to hundreds of subunits) eliminating the requirement to control fluorescent label properties; 3) in work about to be submitted, we track with equal accuracy and precision about an order of magnitude more labeled molecules as winners of the Nature Methods tracking competition. Overview of Future Work: We've organized our future work as extensions of both R01's, projects merging both R01's and directions beyond both. Briefly, to extend existing R01's, we will: 1) provide the first direct single- photon analysis of single molecule fluorescence resonant energy transfer (smFRET) data that simultaneously learns the number of states of biomolecules even lifting the assumption of discrete states. We will apply this, for example, to the unresolved rotational and translational dynamics of a transcription factor to DNA; 2) seek computational solutions to aberration and illumination artifacts that can dramatically deteriorate our ability to reliably track molecules intracellularly. In doing so, we will provide a computational alternative to adaptive optics and apply our tools to the trafficking and silencing activity of microRNAs often located deep within the cellular nucleus. As we merge both R01's: we hope to track reaction-diffusion events of many molecules, resolved at the SM level, and apply them toward understanding heterogeneous interactions of intrinsically disordered proteins. Beyond both R01s: we will borrow Mathematics from SM to resolve the dynamics of a bacterial predator, a candidate living antibiotic, as it hunts for its prey (E. coli) within the gut of c. elegans. Finally, we propose to generalize refractive index (RI) mapping and structured illumination analyses currently limited to slow dynamics.

项目概要 背景和知识差距：在单分子（SM）空间温度下揭示生命的细胞内过程 口腔鳞片对于监测治疗药物和开发疾病诊断至关重要。还画画 对如此尺度的生物分子事件的洞察对现有的荧光成像提出了深刻的挑战。 从根本上来说，这是由于模型选择问题造成的：不可避免的（量子、热、探测器）噪声 SM 规模意味着数据不能轻易用于解析“模型”，例如分子数量 位于空间的一个小区域内。解决这个问题的实验性解决方案赢得了 2014 年 诺贝尔化学奖虽然这样的解决方案必然是有代价的。空间或时间分辨率是 样品经常受到长时间照射，导致样品光损伤。 最新进展：由于已达到 NIGMS R01 的资金中点，我们开发了 数学工具使我们能够减轻（有时是戏剧性的）空间（R01GM130745）和时间（R01 GM134426）对模型选择现有实验解决方案的妥协。我们的工作已取得 10 出版物、15 个合作项目和 18 个正在进行的项目。这里只是 3 个项目：1) 在最近的出版物中， 我们使用比通常用于获得 SM 属性少 2-3 个数量级的光子来导出 SM 属性 荧光相关光谱 (FCS) 的整体特性； 2）在接受的工作中，我们提供了一种手段 确定蛋白质簇化学计量（多达数百个亚基），无需控制 荧光标记特性； 3）在即将提交的工作中，我们以同样的准确度和精确度跟踪 作为自然方法追踪竞赛获胜者的标记分子数量级增加了一个数量级。 未来工作概述：我们将未来的工作组织为两个 R01 的扩展，项目合并了两个 R01 R01 和超越两者的方向。简而言之，为了扩展现有的 R01，我们将： 1）提供第一个直接单 单分子荧光共振能量转移 (smFRET) 数据的光子分析，同时 学习生物分子的状态数，甚至解除离散状态的假设。我们将应用这个， 例如，转录因子到 DNA 的未解决的旋转和平移动力学； 2）寻求 像差和照明伪影的计算解决方案可能会极大地降低我们的能力 可靠地追踪细胞内的分子。在此过程中，我们将为自适应光学提供计算替代方案 并将我们的工具应用于通常位于细胞深处的 microRNA 的运输和沉默活动 核。当我们合并两个 R01 时：我们希望跟踪许多分子的反应扩散事件，并在 SM 水平，并将其应用于理解本质上无序蛋白质的异质相互作用。 除了 R01 之外：我们将借用 SM 的数学来解决细菌捕食者的动力学问题， 候选活抗生素，因为它在 c 的肠道内寻找猎物（大肠杆菌）。线虫。最后，我们建议 推广目前仅限于慢速动态的折射率 (RI) 映射和结构照明分析。