UC Santa Cruz

Transfer RNAs (tRNAs) are small RNA molecules responsible for decoding the genetic code into an amino acid sequence. tRNAs bring amino acids to the ribosome, playing an active role in translation. tRNAs are vital to living systems and can impact human health, making them clinically relevant. This motivates the development of high throughput methods to screen and sequence tRNAs. Nanopore sequencing, specifically the commercialized MinION sequencing platform presents an opportunity to directly sequence tRNAs. I have developed a framework for direct RNA sequencing of both biological and synthetic canonical E. coli initiator tRNA that will be expanded to other tRNAs. I discuss the methods developed to sequence tRNAs on the MinION. This includes improving coverage on the terminal ends of the tRNAs, in vitro production of tRNAs, and the detection of modified nucleosides using the MinION platform. The work presented here is intended to be a launching point for further sequencing of tRNAs and modification discrimination on the MinION. This work culminates in presenting distinct differences between the alignment profiles of biological E. coli fmet tRNA and a canonical version due to the presence of modified nucleosides on the biological tRNA. This has implications for both direct sequencing of tRNAs and the broader goal of detecting modified nucleosides.