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.

总结