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: 10612493
• 负责人: Steven Douglas Knutson
• 金额: $ 6.91万
• 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/10612493
• 关键词: Alzheimer' s Disease; Atlases; Benchmarking; Biological; Biological Process; Biotin; Biotinylation; Cell Line; Cell physiology; Cells; Cellular Stress; 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; autism spectrum disorder; 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是一种关键的功能生物分子，跨越生命的所有领域，并在细胞内移动不同的转录本 是细胞用来调节生物事件的一种广泛策略，包括发育，代谢， 细胞迁移和神经功能。考虑到这种广泛的作用， 局部化还与许多疾病有关，包括自闭症、脆性X综合征、阿尔茨海默病， 亨廷顿氏病和几种癌症。尽管细胞内RNA具有重要的生物学意义 运输，我们对这些事件的分子机制，规模和影响的理解显着 受目前可用的亚细胞RNA作图方法的固有缺点的限制和约束 运动高通量追踪转录本分布对于理解RNA如何有助于 细胞功能和疾病的原因，以及实现这些目标的最有效的方法之一， 采用邻近标记，从而将催化剂嵌入不同的亚细胞位置以进行生物素化 附近的分子工程化的生物素连接酶和过氧化物酶已经在这些应用中显示出效用，但是这些应用不限于此。 这些技术还存在对标记的空间和时间控制差的问题，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集团的光催化邻近性 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 并标记感兴趣的生物分子。与酶促方法相比，该方法利用了 无毒蓝光触发，提供对标记的高度时空控制。此外，活化的二氮杂环丙烷 是非常短的寿命（T1/2 ~1 ns），并被水淬灭，导致小的标记半径（~2 nm）产生 生物分子定位和相互作用网络的高分辨率地图。基于这些令人兴奋的结果， 一项提案试图利用这种光催化方法对亚细胞RNA进行高分辨率绘图 本地化和贩运。总之，这种方法将使研究人员能够绘制细胞内转录组 具有更高的空间分辨率，从而更好地了解这些事件如何有助于细胞 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鉴定。最后，开发的方法 这里可以应用于其他生物学问题周围的RNA运输，包括epitranscriptomic 修饰，RNA剪接和代谢/周转，反过来又提供了一种具有广泛影响力的技术， RNA和细胞生物学社区的实用性。

项目成果

Photoproximity Labeling of Sialylated Glycoproteins (GlycoMap) Reveals Sialylation-Dependent Regulation of Ion Transport.

• DOI: 10.1021/jacs.2c11094
• 发表时间: 2022-12
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Claudio F. Meyer;Ciaran P. Seath;Steve D. Knutson;Wenyun Lu;J. Rabinowitz;D. MacMillan