Direct RNA Oxford Nanopore sequencing distinguishes between modifications in tRNA at the U34 position

The measurement of tRNA modifications with single transcript resolution has been feasible for only a few modifications due to the lack of available methods. This limitation does not allow to advance basic research studies on the dynamic nature of tRNA modification and its cellular function in time and space, neither to develop modern diagnostic tools for several already known tRNA-dependent human diseases. Nanopore is a well-established sequencing method that has proven to be efficient for the study of RNA. The analysis of tRNA modifications by Nanopore is still under development. We have investigated the efficacy of nanopore technology to discriminate between complex modifications of uridine 34 in tRNA, which affect the base-calling properties of neighbouring bases and are therefore difficult to accurately predict. We have developed new methods to chemically and enzymatically synthesise single modified tRNA molecules with modifications at the anticodon loop. Nanopore technology captures the features produced by uridine with and without a thiol group when present on synthetic tRNA molecules. Thus, Oxford Nanopore Technology (ONT) has great potential for developing strategies to accurately identify the modification status of the tRNA anticodon loop (ACL), which encompasses the most complex modifications on uridine-containing RNA motifs.