Poster abstracts

Poster number 18 submitted by Aastha Gyawali

Simplifying eukaryotic transfer RNA modification mapping via FPLC

Aastha Gyawali (Department of Chemistry, University of Cincinnati), Scott Abernathy (Department of Chemistry, University of Cincinnati), Patrick Limbach (Department of Chemistry, University of Cincinnati)

Abstract:
Organisms generally express about 30-50 tRNA genes, but many eukaryotes can produce hundreds or even thousands of different tRNA genes, even when some differ by just a single nucleotide. Current LC-MS/MS techniques are unable to distinguish between these highly similar tRNAs in a sample. This project aims to simplify complex eukaryotic tRNA mixtures by generating tRNA fractions that contain ~30-50 tRNAs per sample. This fraction size was identified as prior LC-MS/MS modification mapping of total tRNA mixtures was found to be effective for mixtures containing less than 60 unique tRNAs.
Fast protein liquid chromatography (FPLC) was investigated due to its ability to handle larger sample amounts (mass and volume), compatibility with on-line fraction collection, and its general availability and lower cost/expense than conventional HPLC approaches. Various FPLC gradient conditions were tested to identify experimental conditions that would fractionate a complex mixture into a smaller, more manageable number of reproducible fractions (~4-6). Once such conditions were identified, fractions were collected, tRNAs were isolated, and samples were digested to nucleosides for LC-MS/MS analysis. Nucleoside analysis demonstrated that tRNA compositions in each fraction were different, demonstrating proof of concept. Follow-on analysis involved using RNase T1 to perform RNA modification mapping to identify specific tRNAs within each fraction. Additional studies are in the works to expand the complexity of the initial mixture of tRNAs to test the limits of this fractionation method.

References:
Suzuki, T.. Nat. Rev. Mol. Cell Biol. 2021, 22 (6), 375–392.
Edwards, A. M.; Addo, M. A.; Dos Santos, P. C. Genes 2020, 11, 907.
Koubek, J., Lin, K. F., Chen, Y. R., Cheng, R. P., & Huang, J. J. T.. RNA, 2013, 19(10), 1449-1459.

Keywords: tRNA Modifications, FPLC, LC-MSMS