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.

项目摘要 背景和知识差距：在单分子（SM）空间中揭示生命的细胞内过程 对于监测治疗剂和开发疾病诊断来说，孔尺度是至关重要的。然而， 对这种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。 从根本上说，这是由于模型选择问题而产生的：不可避免的（量子、热、探测器）噪声 在SM尺度上，这意味着数据不能很容易地用于解决“模型”，如分子数量 位于空间的一个小区域内。解决这一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会付出代价。空间或时间分辨率 而样品经常在延长的持续时间内被照射，从而引起样品光损伤。 最近的进展：由于已经达到了我们两个NIGMS R 01的资金中点，我们开发了 数学工具，使我们能够减轻，有时显着，空间（R 01 GM 130745）和时间（R 01）， GM 134426）对模型选择的现有实验解决方案的妥协。我们的工作已经取得了10 出版物，15个合作项目和18个正在进行的项目。这里只有3个项目：1）在最近的出版物， 我们用比通常用来获得的光子少2-3个数量级的光子导出了SM特性， 2）在公认的工作中，我们提供了一种方法， 以确定蛋白质簇化学计量（多达数百个亚基）， 标签属性; 3）在即将提交的工作中，我们以同样的准确性和精度跟踪 一个数量级的标记分子作为自然方法跟踪竞争的获胜者。 未来工作概述：我们已经组织了我们未来的工作作为两个R 01的扩展，项目合并两者 R 01的和方向超越两者。布里说，为了扩展现有的R 01，我们将：1）提供第一个直接的单一- 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。我们将应用这一点， 例如，对于转录因子到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，这可能会大大降低我们的能力， 可靠地跟踪细胞内的分子。在这样做的时候，我们将提供一个计算替代自适应光学 并将我们的工具应用于通常位于细胞深处的microRNA的转移和沉默活性， 原子核当我们合并两个R 01时：我们希望跟踪许多分子的反应扩散事件， SM水平，并将其应用于理解本质无序蛋白质的异质性相互作用。 除了两个R 01：我们将借用数学从SM解决动力学的细菌捕食者， 候选的活抗生素，因为它寻找它的猎物（E。coli）在C.优美的最后，我们建议 广义折射率（RI）映射和结构照明分析目前仅限于缓慢的动态。