Project Summary/Abstract Epitranscriptomics is the study of RNA modifications, which include more than 170 naturally occurring chemical alternations to the nucleotides. More than 60 are found in human RNA of all types: mRNA, tRNA, rRNA, lncRNA, and the others. These modifications are dynamic; their global quantities change in development and during disease progression. They are installed by writer enzymes, read by reader proteins and removed by eraser enzymes, and they have an intrinsic capacity to alter RNA structure and dynamics. They influence translation initiation and termination, translation fidelity, alternative splicing, trafficking between cellular compartments, and regulate RNA degradation. RNA reader, writer and eraser proteins are promising drug targets of high current interest to pharma. In this project, we will test and validate a new approach to detecting, identifying, and mapping RNA modifications in a multiplex and with high sensitivity—suitable for clinical samples (e.g. needle biopsies, FFPE samples) in which only sub-nanogram quantities of RNA may be available. This technology will be significant because it will provide the first commercial method for profiling and correlating changes of multiple RNA modification types across the entire transcriptome using low sample input.