Chemical probe-based Nanopore Sequencing to Selectively Assess the RNA modifications

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 ( Ψ ) 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 Ψ in the in vitro synthesized RNA. Furthermore, we verified the I and Ψ 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. Abstract Figure