Multiplexed Analysis of the Epitranscriptome - 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. Currently available analytical methods either do not report on sequence context or provide sequence information but at the expense of multiplexing capability. Despite these limitations, it is now known that modifications are dynamically tied to phenotypic changes in cancer progression, drug resistance, aging, and viral infection. Alida Biosciences is developing a new commercial platform 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 low quantities of RNA may be available. Following completion of our Phase I milestones focused on creating the new multiplexed assay and proof-of-concept testing, this proposal aims to (1) complete assay development, optimizing sensitivity, specificity, and a robust and user-friendly workflow, (2) develop assay automation, and (3) perform assay validation in preparation for commercial launch. This technology will be significant because it will provide the first commercial platform capable of profiling and correlating changes of multiple RNA modification types across the entire transcriptome in a given sample.