Development of novel research tools and a database for mapping human mitochondrial tRNA modifications by mass spectrometry

开发新的研究工具和数据库，用于通过质谱绘制人类线粒体 tRNA 修饰图

• 批准号: 9185063
• 负责人: PATRICK A LIMBACH
• 金额: $ 15万
• 依托单位: RIBONOVA, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9185063
• 关键词: Aging; Biological; Biological Assay; Biological Markers; Cell Line; Cells; Central Nervous System Diseases; Clinical; Collaborations; Databases; Detection; Development; Drug Targeting; Drug abuse; Fibroblasts; Functional disorder; Goals; Harvest; Health; Heart Arrest; Human; Inherited; Isomerism; Link; Maps; Mass Spectrum Analysis; Medicine; Methods; Mining; Mitochondria; Mitochondrial Diseases; Modification; Molecular; Molecular Biology; Monitor; Nervous System Physiology; Neuraxis; Nucleosides; Nucleotide Mapping; Nucleotides; Patients; Pediatric Hospitals; Pharmaceutical Preparations; Phase; Philadelphia; Physiological; Production; Protein Biosynthesis; Protocols documentation; Pseudouridine; RNA Databases; Reaction; Reagent; Research; Resources; Role; Sample Size; Sampling; Site; Small Business Technology Transfer Research; Stroke; Structure; Tissues; Transfer RNA; Universities; Uridine; Work; base; candidate marker; clinical biomarkers; design; human disease; human tissue; improved; insight; meetings; mitochondrial DNA mutation; mitochondrial dysfunction; new therapeutic target; novel; precision medicine; product development; small molecule; success; tool

Summary In this fast-track Phase I/II STTR project, a novel mass spectrometry method and a matched reagent kit will be developed for mapping modified nucleotides in human mitochondrial transfer RNA (mt-tRNA), including the most abundant modification, pseudouridine, which hitherto has proved to be a technical challenge because it is an isomer of uridine. These new research tools will be harnessed to build the first database of modification maps for all 22 human mt-tRNAs from ten normal subjects and twenty patients with mitochondrial disease who have disorders of the central nervous system (CNS). Phase I of this project will involve the development of a mass spectrometry method for the site-specific quantification of pseudouridine and other nucleotide modifications in mt-tRNAs (Aim 1) and a method for isolating mitochondria from practical quantities of human tissues and cell lines (Aim 2). Phase II will involve the development of a method for isolating human mt-tRNAs from mitochondria (Aim 3), the modification mapping of normal mt-tRNAs from healthy subjects (Aim 4), and the mapping of modifications in abnormal mt- tRNAs from mitochondrial disease patients with CNS disorders (Aim 5). The resulting database of more than 600 mt-tRNA modification maps will provide valuable insights into the molecular links between mitochondrial dysfunction and CNS disorders, including those caused by physiological insults such as drug abuse. Success in this project will enable the Phase III completion of product development and mining of the modification database for clinical biomarkers and drug targets on mt-tRNAs for the discovery of small-molecule precision medicines that recognize the abnormal modification structures. This new class of drugs will be designed to selectively terminate protein synthesis in, and cull, abnormal mitochondria, whereupon the healthy mitochondria will automatically repopulate the cell and restore normal CNS function. This work will be conducted as a collaborative project between RiboNova, the University of Cincinnati, and The Children's Hospital of Philadelphia, who have the relevant resources and expertise in molecular biology, mass spectrometry and mitochondrial medicine, respectively, to accomplish the project goals.

概括 在这个快速轨道I/II STTR项目中，一种新型的质谱法和匹配的试剂套件将是 开发用于在人线粒体转移RNA（MT-tRNA）中绘制修饰的核苷酸的开发，包括 大多数丰富的修改，伪源，迄今已被证明是技术挑战，因为它是 尿苷的异构体。这些新的研究工具将被利用以构建第一个修改数据库 来自10名正常受试者和20例线粒体疾病患者的所有22个人类MT-TRNA的地图 患有中枢神经系统（CNS）的疾病。 该项目的第一阶段将涉及针对特定地点的质谱法的开发 MT-TRNA中的假喹啉和其他核苷酸修饰的定量（AIM 1）和一种方法 从实际数量的人体组织和细胞系中分离线粒体（AIM 2）。第二阶段将涉及 从线粒体中隔离人类mt-trnas的方法的开发（AIM 3），修改 来自健康受试者的正常MT-TRNA（AIM 4）的映射，以及异常MT-的修饰映射 线粒体疾病患者中枢神经系统疾病的TRNA（AIM 5）。最终的数据库超过 600 MT-tRNA修饰地图将为线粒体之间的分子链接提供有价值的见解 功能障碍和中枢神经系统疾病，包括由药物滥用等生理侮辱引起的。 该项目的成功将使第三阶段的产品开发和采矿的完成 用于MT-TRNA的临床生物标志物和药物靶标的修改数据库，以发现小分子 识别异常修饰结构的精确药物。这种新的药物将是 设计为选择性终止蛋白质的合成，并在线粒体异常中脱颖而出，因此健康 线粒体将自动重新填充细胞并恢复正常的中枢神经系统功能。 这项工作将作为Ribonova，Cincinnati大学和 费城儿童医院，他们拥有分子生物学的相关资源和专业知识，弥撒 光谱法和线粒体医学分别实现了项目目标。