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

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 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 and a method for isolating mitochondria from practical quantities of human tissues and cell lines Aim Phase II will involve the development of a method for isolating human mt tRNAs from mitochondria Aim the modification mapping of normal mt tRNAs from healthy subjects Aim and the mapping of modifications in abnormal mt tRNAs from mitochondrial disease patients with CNS disorders Aim The resulting database of more than 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 Childrenandapos 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 Project Narrative A novel mass spectrometry method and a matched reagent kit will be developed for mapping modified nucleotides in human mitochondrial transfer RNA These research tools will be harnessed to build the first comprehensive database of modification maps of mitochondrial transfer RNAs from healthy people and patients with mitochondrial disease who have disorders of the central nervous system This database will facilitate the discovery of drugs to treat CNS disorders including those caused by drug abuse