High-Resolution Mapping of Subcellular RNA Dynamics Using Photocatalytic Proximity Labeling

使用光催化邻近标记对亚细胞 RNA 动力学进行高分辨率绘图

• 批准号: 10382258
• 负责人: Steven Douglas Knutson
• 金额: $ 6.72万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10382258
• 关键词: Alzheimer' s Disease; Atlases; Benchmarking; Biological; Biological Process; Biotin; Cell Line; Cell physiology; Cells; Cellular Stress Response; Cellular biology; Chemicals; Communities; Development; Diagnostic; Diazomethane; Diffuse; Disease; Electrons; Engineering; Event; Exhibits; Fluorescence Microscopy; Fragile X Syndrome; Goals; Huntington Disease; Hydrogen Peroxide; In Vitro; Individual; Intracellular Transport; Iridium; Label; Life; Ligase; Light; Link; Location; Maps; Metabolism; Methodology; Methods; Microscopy; Modification; Molecular; Monitor; Morphologic artifacts; Movement; Nervous System Physiology; Pattern; Peroxidases; Phenols; Process; Proteins; Proteomics; RNA; RNA Splicing; RNA Transport; Radial; Research; Research Personnel; Resolution; Role; Series; Stimulus; Stress; Techniques; Technology; Therapeutic; Time; Toxic effect; Transcript; Water; base; cancer type; carbene; catalyst; cell behavior; cell motility; epitranscriptomics; experimental study; genetic information; in vivo; interest; irradiation; nanometer; new therapeutic target; novel diagnostics; oxidation; response; spatiotemporal; trafficking; transcriptome; transcriptome sequencing

Project Abstract RNA is a key functional biomolecule across all domains of life, and intracellular movement of different transcripts is a widespread strategy employed by cells to regulate biological events, including development, metabolism, cell migration, and neurological function. Given this broad role, it is unsurprising that dysregulated RNA localization is also linked to a host of diseases, including autism, fragile X syndrome, Alzheimer’s disease, Huntington’s disease, and several types of cancer. Despite the critical biological importance of intracellular RNA transport, our understanding of the molecular mechanisms, scale, and impact of these events is significantly limited and confined by the inherent shortcomings in currently available methods for mapping subcellular RNA movement. High-throughput tracking of transcript distribution is vital to understanding how RNA contributes to cellular function and causes disease, and one of the most effective approaches for achieving these goals employs proximity labeling, whereby a catalyst is embedded into different subcellular locations to biotinylate nearby molecules. Engineered biotin ligases and peroxidases have shown utility in these applications, but these techniques also suffer from poor spatial and temporal control over labeling and exhibit in vivo toxicity. To overcome these limitations, the proposed research will leverage the MacMillan group’s photocatalytic proximity labeling approach. In particular, this “micromapping” (µMap) method utilizes an iridium photocatalyst to activate nearby diazirines and label biomolecules of interest. In contrast to enzymatic approaches, this method utilizes a non-toxic blue light trigger, providing high spatiotemporal control over labeling. In addition, activated diazirines are very short-lived (T1/2 ~1 ns) and quenched by water, resulting in a small labeling radius (~2 nm) to generate high-resolution maps of biomolecule localization and interaction networks. Building off these exciting results, this proposal seeks to leverage this photocatalytic approach toward high-resolution mapping of subcellular RNA localization and trafficking. Together, this method will enable researchers to map the intracellular transcriptome with higher spatial resolution, in turn providing better understanding of how these events contribute to cellular function. In addition, these experiments will help elucidate disease-causing mechanisms related to RNA transport, and facilitate identification of new diagnostic and therapeutic targets. Lastly, the methods developed here can be applied to other biological questions surrounding RNA trafficking, including epitranscriptomic modifications, RNA splicing, and metabolism/turnover, in turn providing an impactful technology that is of broad utility to the RNA and cell biology community.

项目摘要 RNA 是生命各个领域的关键功能生物分子，以及不同转录物的细胞内运动 是细胞用来调节生物事件的广泛策略，包括发育、代谢、 细胞迁移和神经功能。鉴于这种广泛的作用，RNA 失调也就不足为奇了。 定位还与许多疾病有关，包括自闭症、脆性 X 综合征、阿尔茨海默病、 亨廷顿舞蹈症和几种癌症。尽管细胞内 RNA 具有至关重要的生物学重要性 运输，我们对这些事件的分子机制、规模和影响的理解显着 受限于目前可用的亚细胞 RNA 作图方法的固有缺陷 移动。转录本分布的高通量跟踪对于理解 RNA 如何贡献至关重要 细胞功能和引起疾病，以及实现这些目标的最有效方法之一 采用邻近标记，将催化剂嵌入不同的亚细胞位置以进行生物素化 附近的分子。工程生物素连接酶和过氧化物酶已在这些应用中显示出效用，但这些 技术还存在对标记的空间和时间控制较差的问题，并表现出体内毒性。到 为了克服这些限制，拟议的研究将利用麦克米伦小组的光催化邻近性 标签方法。特别是，这种“微映射”（μMap）方法利用铱光催化剂来激活 附近的二氮丙啶并标记感兴趣的生物分子。与酶法相比，该方法利用 无毒蓝光触发，提供对标记的高时空控制。此外，活化的二氮丙啶 寿命非常短（T1/2 ~1 ns）并被水淬灭，导致标记半径很小（~2 nm） 生物分子定位和相互作用网络的高分辨率图。在这些令人兴奋的结果的基础上， 该提案旨在利用这种光催化方法来绘制亚细胞 RNA 的高分辨率图谱 本地化和贩运。总之，该方法将使研究人员能够绘制细胞内转录组图谱 具有更高的空间分辨率，从而更好地了解这些事件如何影响细胞 功能。此外，这些实验将有助于阐明与RNA相关的致病机制 运输，并促进新的诊断和治疗靶点的识别。最后，开发的方法 这里可以应用于围绕 RNA 运输的其他生物学问题，包括表观转录组学 修饰、RNA 剪接和代谢/周转，进而提供了一种具有广泛影响力的技术 对 RNA 和细胞生物学界的实用性。