Pseudouridine detection with single-base resolution in a multiplexed epitranscriptomics assay

ABSTRACT Chemical modifications to the canonical nucleosides are found throughout human RNA and collectively constitute the epitranscriptome. They are critical regulators of RNA function in many cellular processes, including RNA translation, splicing, processing, trafficking, stability, and degradation. RNA modifications play a central role in cellular and organismal development, homeostasis, and disease and have been linked to cancer, metabolic and neurological diseases, and viral infection. RNA modifications, and the pathways in which they are involved, are now being exploited in the development of novel therapeutics, especially cancer chemotherapies, and present new opportunities to develop clinical biomarkers. However, much of the opportunity to understand and use the epitranscriptome to advance human health is held back by limitations of the available tools for detecting and locating these modifications. There are no currently available high-throughput methods that can simultaneously profile multiple modifications and are readily accessible to researchers doing RNA sequencing. In this project, we will develop and commercialize an assay for the simultaneous detection and quantification of the three most prevalent modifications in RNA: N6-methyladenosine (m6A), inosine (I), and pseudouridine (Ψ). Our strategy leverages a combination of engineered RNA modification-specific binders and enzymatic conversion of Ψ to enable pull-down of modified RNA fragments onto mod-specific beads, barcoding to record the presence of the modification, next-gen sequencing of the resulting library, and analysis with custom bioinformatics tools. The expected outcome of this project is the full commercial launch of the AlidaBio EpiPlex v2 assay, which adds Ψ detection to our platform, enabling any researchers doing RNA-seq to include the detection, location, and relative quantification of m6A, I, and Ψ in their analysis with ≥ 10 ng of polyA-selected RNA or ≥ 200 ng total RNA input.