Illuminating cellular dark matter through the development of novel chemical tools

通过开发新型化学工具照亮细胞暗物质

• 批准号: 10656353
• 负责人: Neal Krishna Devaraj
• 金额: $ 39.5万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656353
• 关键词: Acceleration; Affect; Amyotrophic Lateral Sclerosis; Biological; Biology; C9ORF72; Cell physiology; Cells; Cellular biology; Chemicals; Development; Disease; Genetic; Goals; Image; In Situ; Knowledge; Laboratories; Lipids; Modification; Molecular; Nucleotides; Pathogenicity; Pathway interactions; Permeability; Play; Proteins; RNA; Research; Role; Site; Sphingolipids; Technology; Therapeutic Intervention; cell behavior; cytotoxic; dark matter; human disease; improved; interest; novel; programs; protein complex; recruit; success; tool

PROJECT SUMMARY There is a vast repertoire of species within cells for which we have a poor understanding of their function and biomolecular interactions. These species can be referred to as the “dark matter” of biology, as their mechanism of action is hidden from conventional observation. Our laboratory seeks to illuminate the function of cellular “dark matter” through the development of new chemical technology. The proposed research program will pursue two major research thrusts. First, we plan to develop tools for site-specific RNA modification, and apply these tools for the manipulation, imaging, and isolation of disease relevant RNA protein complexes. We will create tools for use in live cells and develop the ability to covalently recruit proteins to RNA, forming RNA-protein macromolecular conjugates. The technology will be applied to study RNAs implicated in disease. Specifically, we are interested in characterizing the pathways of pathogenicity for the C9orf72 nucleotide repeat expansion RNA, which is thought to play a major role in genetic amyotrophic lateral sclerosis (ALS). In the second thrust, we will carry out the in situ synthesis of lipid species within living cells, with the goal of uncovering the molecular mechanism by which enigmatic lipid species affect cell behavior. We plan to develop approaches enabling the selective and bioorthogonal delivery of sphingolipids to living cells. Building upon technology previously developed in our lab, we will deliver cell permeable lipid precursors which will spontaneously assemble into functional lipids within the cell. Leveraging this approach, we will create photoaffinity probes for the pulldown of sphingolipid-interacting proteins, with the goal of elucidating the protein partners of the non-canonical deoxysphingolipid 1-deoxydihydroceramide, which is cytotoxic and implicated in several diseases. Realization of our research program goals would improve our knowledge of cell biology and lead to the development of new tools for interrogating RNA and lipid species. Our long-term vision is to create and apply technology that enables improved mechanistic understanding of biomolecular interactions, leading to an increased understanding of human disease, and accelerating the development of possible therapeutic interventions.

项目摘要 细胞内有大量的物种，我们对其功能了解甚少， 生物分子相互作用。这些物种可以被称为生物学的“暗物质”， 行为的本质是隐藏在常规观察之外的。我们的实验室试图阐明细胞“黑暗”的功能， 通过发展新的化学技术，“物质”。该研究计划将在两个 主要研究方向。首先，我们计划开发用于位点特异性RNA修饰的工具，并应用这些工具 用于疾病相关RNA蛋白复合物的操作、成像和分离。我们将创建工具， 在活细胞中使用，并发展将蛋白质共价募集到RNA上的能力，形成RNA-蛋白质 大分子共轭物该技术将被应用于研究与疾病有关的RNA。具体地说， 我们感兴趣的是描述C9 orf 72核苷酸重复扩增的致病性途径 RNA被认为在遗传性肌萎缩侧索硬化症（ALS）中起主要作用。在第二次进攻中， 我们将在活细胞内进行脂质物质的原位合成，目的是揭示脂质分子的结构。 神秘的脂质物质影响细胞行为的机制。我们计划制定方法， 将鞘脂选择性和生物正交递送至活细胞。基于以前的技术 在我们的实验室开发，我们将提供细胞渗透脂质前体，将自发组装成 细胞内的功能性脂质。利用这种方法，我们将创建用于下拉的光亲和探针。 鞘脂相互作用蛋白，目的是阐明非典型的蛋白质伴侣， 脱氧鞘脂1-脱氧二氢神经酰胺，其具有细胞毒性并与多种疾病有关。实现 我们的研究计划目标将提高我们的细胞生物学知识，并导致新的发展， 用于询问RNA和脂质种类的工具。我们的长期愿景是创造和应用技术， 提高了对生物分子相互作用的机械理解，从而提高了对 人类疾病，并加快可能的治疗干预措施的发展。

项目成果

Site-Specific and Enzymatic Cross-Linking of sgRNA Enables Wavelength-Selectable Photoactivated Control of CRISPR Gene Editing.

• DOI: 10.1021/jacs.1c12166
• 发表时间: 2022-03-16
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Zhang, Dongyang;Liu, Luping;Jin, Shuaijiang;Tota, Ember;Li, Zijie;Piao, Xijun;Zhang, Xuan;Fu, Xiang-Dong;Devaraj, Neal K.
• 通讯作者: Devaraj, Neal K.

Bioconjugation Strategies for Revealing the Roles of Lipids in Living Cells.

• DOI: 10.1021/acs.accounts.2c00511
• 发表时间: 2022-11-01
• 期刊: ACCOUNTS OF CHEMICAL RESEARCH
• 影响因子: 18.3
• 作者: Devaraj, Neal K.;Knittel, Caroline H.
• 通讯作者: Knittel, Caroline H.

Site-Specific Covalent Labeling of DNA Substrates by an RNA Transglycosylase.

• DOI: 10.1021/jacs.3c00861
• 发表时间: 2023-04-12
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Tota, Ember M.;Devaraj, Neal K.
• 通讯作者: Devaraj, Neal K.

Chemoselective Esterification of Natural and Prebiotic 1,2-Amino Alcohol Amphiphiles in Water.

• DOI: 10.1021/jacs.3c12038
• 发表时间: 2023-12-13
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Bhattacharya, Ahanjit;Tanwar, Lalita;Fracassi, Alessandro;Brea, Roberto J.;Salvador-Castell, Marta;Khanal, Satyam;Sinha, Sunil K.;Devaraj, Neal K.
• 通讯作者: Devaraj, Neal K.