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是生命各个领域的关键功能生物分子，是不同转录物在细胞内的运动 是一种广泛被细胞用来调节生物事件的策略，包括发育、新陈代谢、 细胞迁移和神经功能。考虑到这一广泛的作用，调节失调的RNA也就不足为奇了 本地化还与一系列疾病有关，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、规模和影响的理解是显著的 受限于当前可用的绘制亚细胞RNA的方法的固有缺陷 有动静。对转录分布的高通量跟踪对于理解RNA如何对 细胞功能和致病原因，以及实现这些目标的最有效方法之一 使用邻近标记，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。工程生物素连接酶和过氧化物酶在这些应用中显示出了有用的作用，但这些 技术还存在标记的空间和时间控制不佳以及在体内表现出毒性的问题。至 克服这些限制，拟议的研究将利用麦克米兰小组的光催化近似性 贴标签的方法。具体地说，这种微映射(µMap)方法利用Ir光催化剂来激活 附近的二氮杂环化合物和标记感兴趣的生物分子。与酶促方法不同，这种方法利用了 无毒的蓝光触发，提供对标签的高度时空控制。此外，活化的二氮杂环丙烷 是非常短暂的(T1/2~1 ns)并且被水淬灭，导致产生小的标记半径(~2 nm 生物分子定位和相互作用网络的高分辨率地图。在这些令人兴奋的结果的基础上，这 一项提案寻求利用这种光催化方法来绘制亚细胞RNA的高分辨率地图 本地化和贩卖。总之，这种方法将使研究人员能够绘制细胞内转录组 更高的空间分辨率，进而更好地理解这些事件如何对细胞 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，并促进确定新的诊断和治疗目标。最后，所开发的方法 这里可以应用于围绕RNA贩运的其他生物学问题，包括表位转录 修饰、RNA剪接和新陈代谢/周转，进而提供了一种具有广泛影响力的技术 对核糖核酸和细胞生物学社区的效用。

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