Project Summary/Abstract RNA modifications, which constitute the epitranscriptome, play vital roles in seemingly all aspects of RNA metabolism and RNA’s role in the Central Dogma. More than 170 naturally occurring chemical modifications to RNA are known, more than 60 of which 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. Despite this significance, no currently existing analytical method is capable of locating multiple RNA modifications simultaneously with precise locus information and stoichiometry. The focus of this application is to de-risk a positional marking approach to RNA modification analysis that is capable of multiplexing, approaching single base resolution. 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 in a given sample.