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是生命的所有领域的关键功能性生物分子，也是不同转录本的细胞内运动 是细胞制定的一种广泛的策略，用于调节生物学事件，包括发展，代谢， 细胞迁移和神经功能。鉴于这种广泛的作用，不足为奇的RNA失调 本地化还与许多疾病有关，包括自闭症，脆弱X综合征，阿尔茨海默氏病， 亨廷顿氏病和几种类型的癌症。尽管细胞内RNA至关重要 运输，我们对这些事件的分子机制的理解，规模和影响很大 在当前可用的映射亚细胞RNA的方法中的固有缺点限制和限制 移动。转录本分布的高通量跟踪对于理解RNA如何贡献 细胞功能并引起疾病，也是实现这些目标的最有效的方法之一 员工接近标签，从而将催化剂嵌入到不同的亚细胞位置中以生物素基因 附近的分子。工程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 技术还遭受了对标记和体内毒性暴露的空间和临时控制不良的影响。到 克服这些局限性，拟议的研究将利用Macmillan集团的光催化接近度 标签方法。特别是，这种“微型绘制”（µMAP）方法利用虹膜光催化剂激活 附近的重氮嗪和标签生物分子。与酶促方法相反，此方法利用 无毒的蓝光触发器，为标记提供了高时空控制。另外，活化的重氮 非常短（T1/2〜1 ns），并用水淬火，导致小标记半径（〜2 nm）产生 生物分子定位和相互作用网络的高分辨率图。建立这些令人兴奋的结果，这个 提案旨在利用这种光催化方法用于亚细胞RNA的高分辨率映射 本地化和贩运。这种方法将使研究人员能够映射细胞内转录组 通过较高的空间分辨率，反过来又可以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相关的引起疾病的机制 运输，并促进鉴定新的诊断和治疗靶标。最后，方法开发了 这里可以应用于围绕RNA贩运的其他生物学问题，包括表面转录组 修改，RNA剪接和新陈代谢/营业额，反过来提供了一种有影响力的技术 RNA和细胞生物学界的实用程序。

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