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.

摘要 在这个快速通道的第一阶段/第二阶段STTR项目中，一种新的质谱学方法和匹配的试剂盒将 为定位人类线粒体转移RNA(mt-tRNA)中的修饰核苷酸而开发的，包括 最丰富的修饰，伪尿苷，到目前为止被证明是一个技术挑战，因为它是 尿苷的一种异构体。这些新的研究工具将被利用来建立第一个修改数据库 来自10名正常人和20名线粒体疾病患者的全部22个人类mt-tRNA的图谱 有中枢神经系统(CNS)障碍。 该项目的第一阶段将涉及为特定地点开发一种质谱学方法。 Mt-tRNA中假尿苷和其他核苷酸修饰的定量(目标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(目标2)。第二阶段将涉及 人线粒体mt-tRNA分离方法的建立(目标3)--改良 健康受试者正常mt-tRNA图谱(目标4)，异常mt-tRNA的修饰图谱 线粒体疾病伴中枢神经系统疾病患者的tRNA(目标5)。由此产生的数据库超过 600 mt-tRNA修饰图谱将对线粒体之间的分子联系提供有价值的见解 功能障碍和中枢神经系统障碍，包括由药物滥用等生理侮辱引起的疾病。 该项目的成功将使产品开发和采矿的第三阶段完成 用于发现小分子的mt-tRNAs上临床生物标志物和药物靶点的修饰数据库 识别异常修饰结构的精准药物。这类新的药物将是 旨在选择性地终止并剔除异常线粒体中的蛋白质合成，因此健康的 线粒体会自动重新填充细胞，恢复正常的中枢神经系统功能。 这项工作将作为RiboNova、辛辛那提大学和 费城儿童医院，他们在分子生物学方面拥有相关的资源和专业知识，麻省 光谱学和线粒体医学分别完成了项目目标。