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 背景和知识鸿沟：在单分子(SM)时空解开生命的细胞内过程-- 门脉鳞片对监测治疗药物和发展疾病诊断至关重要。然而，画画 对这种尺度上的生物分子事件的洞察对现有的fl荧光成像提出了深刻的挑战。 从根本上说，这是由模型选择问题引起的：不可避免的(量子、热、探测器)噪声 在SM尺度上意味着数据不能很容易地用来解析诸如分子数量之类的“模型” 位于一小块空间内的。解决这一问题的试验性解决方案赢得了2014年 不过，这样的解决方案必然是要付出代价的。无论是空间分辨率还是时间分辨率 当样品经常被长时间照射而导致样品光损伤时，样品会受到损害。 最新进展：由于已经达到我们两个NIGMS R01的资金中点，我们开发了 数学工具使我们能够减少空间(R01GM130745)和时间(R01)，有时甚至是戏剧性的 GM134426)在模型选择上折衷了现有的实验解决方案。我们的工作已经取得了10项成果 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)在最近的出版物中， 我们用比通常要少2-3个数量级的光子来获得SM性质 fl荧光相关光谱的体积性质；2)在公认的工作中，我们提供了一种方法 要确定蛋白质簇化学计量比(最多数百个亚基)，不需要控制 fl荧光标签属性；3)在即将提交的工作中，我们以同样的准确率和精确度跟踪关于 更多标记分子成为自然方法跟踪竞赛的获胜者。 未来工作概述：我们将未来的工作组织为R01和S两个项目的延伸，合并了两个项目 R01的S和超越两者的方向。Briefly，为了扩展现有的R01‘S，我们将：1)提供fi首个直接单- 单分子fl荧光共振能量转移数据的光子分析 学习生物分子的状态数，甚至取消离散状态的假设。我们将应用这一点， 例如，对于转录因子到DNA的未解决的旋转和翻译动力学；2)寻找 像差和照明伪影的计算解决方案，这些伪影会显著降低我们的 可靠地跟踪细胞内的分子。在此过程中，我们将为自适应光学提供一种计算替代方案 并将我们的工具应用于通常位于细胞深处的microRNA的Traffi转录和沉默活动 原子核。随着我们合并R01和S：我们希望跟踪许多分子的反应-扩散事件，在 SM水平，并将其应用于理解本质上无序的蛋白质的异质相互作用。 超越两个R01：我们将借用SM的数学知识来解决细菌捕食者的动力学问题 候选的活性抗生素，在线虫的肠道内寻找猎物(大肠杆菌)。最后，我们建议 概括折射率(RI)映射和结构化照明分析，目前仅限于慢速动力学。