Acrylonitrile reactivity based detection of inosine and Pseudouridine using nanopore sequencing

Current methods to identify RNA modifications with short-read sequencing are laborious and direct RNA sequencing gets proclaimed as the viable alternative. Herein, we harness the selective reactivity of the acrylonitrile towards the Inosine (I) and pseudouridine (?) modifications and developed a chemical probe-based direct RNA sequencing method. We first demonstrated that the chemical probe-induced differential signature profile using nanopore sequencing could facilitate the selective assessment of I and ? in the in vitro synthesized RNA. Furthermore, we verified the I and ? modification with single- nucleotide resolution using RNA derived from mouse brain without the need for a null dataset using knockouts. Our chemical probe-based nanopore sequencing strategy can be extended to profile multiple RNA modifications on a single RNA and may facilitate the diagnosis of disease-associated epitranscriptome markers by generating a comparative dataset in clinical scenarios.