London Calling 2023: Nanopore sequencing of tRNA modifications

Transfer (t) RNAs are extensively decorated with a wide variety of post-translational modifications. The steady-state level of each tRNA and its modification status are dynamically regulated in different cells and tissues in a spatiotemporal manner. To explore the physiological importance of tRNAs in various biological contexts, it is necessary to establish an innovative technology to profile and analyze cellular tRNAs with their modification status. Although next-generation sequencing (NGS)-based methods are highly sensitive and widely available for profiling tRNAs and detecting several tRNA modifications from limited specimens, they are applied only to specific tRNA modifications, because most of the information on RNA modifications is eliminated during cDNA conversion. Nanopore-based sequencing is a unique method capable of directly analyzing RNA molecules without cDNA conversion. We have been developing a handy and practical method for classifying cellular tRNAs with their modification status using nanopore sequencing.