Nanopore sequencing of intact, aminoacylated tRNA | LC 25

Biography

Laura White is an RNA biologist at the University of Colorado School of Medicine where she leads nanopore sequencing within the RNA Bioscience Initiative. She completed her PhD in Jay Hesselberth’s laboratory in 2022 where she leveraged nanopore sequencing to detect repaired RNA molecules. Her current research is focused on tRNA biology as well as novel methods development for direct RNA sequencing and RNA modification detection.

Abstract

Transfer RNAs (tRNAs) are the fundamental adaptors of protein synthesis. During translation, tRNAs decode mRNA codons and sequentially deliver their amino acid cargos to the growing polypeptide chain within the ribosome. This process depends on the specific charging, or aminoacylation, of tRNAs with their cognate amino acids.

We developed aa-tRNA-seq, a nanopore-based approach to simultaneously analyze tRNA sequence, modification, and aminoacylation state. aa-tRNA-seq employs a chemical ligation that embeds the charged amino acid into an adapted tRNA for nanopore direct RNA sequencing. The embedded amino acids produce characteristic distortions in ionic current and translocation speed, enabling machine learning models to classify a tRNA’s charging state and identify the attached amino acid for specific tRNA species.

This approach provides a powerful tool for tRNA biology, enabling researchers to interrogate the relationships between tRNA abundance, modification, and aminoacylation within a single, high-throughput experiment.