Poster abstracts
Poster number 155 submitted by Hina Zain
Liquid Chromatography-Mass Spectrometry Analysis of Total tRNA using RNase 4
Hina Zain (Department of Chemistry, University of Cincinnati, OH), Bibek Hamal (Department of Chemistry, University of Cincinnati, OH), Asif Rayhan (Department of Chemistry, University of Cincinnati, OH), Patrick A. Limbach (Department of Chemistry, University of Cincinnati, OH)
Abstract:
Tandem liquid chromatography-mass spectrometry (LC-MS/MS) is one of the best tools for the analysis of the RNA sequence without amplification and converting it into cDNA. Identifying the RNA sequence and locating the specific ribonucleoside modifications requires the digestion of RNA into oligonucleotides that can then be analyzed by LC-MS/MS. Various ribonucleases with different cleavage specificity can be used to generate these oligonucleotides. Recently, human RNase 4, which cleaves at the sequence motif Up(A/G), became available commercially. Past work has focused on the use of this enzyme for the digestion of messenger RNA (mRNA). However, transfer RNAs (tRNAs) contain far more modifications than are found in mRNAs. In the present study, different incubation periods and enzyme concentrations for human RNase 4 were optimized with a 78-nt tRNA mimic. Once the conditions were optimized, the reaction was applied to yeast total tRNA. We found that RNase 4 cleavage motif can include the modified uridines, pseudouridine and dihydrouridine. Additionally, a Cp(A/G) cleavage motif was also found. Optimization of reaction conditions for human RNase 4 digestion of complex tRNA mixtures will enable improved tRNA modification mapping by LC-MS/MS
Keywords: Human RNase 4, tRNA, LC-MSMS