Poster abstracts

Poster number 57 submitted by Manasses Jora

Global Profiling of the Effects of Oxidative Stress on RNA Modifications by Liquid Chromatography-Mass Spectrometry

Manasses Jora (Department of Chemistry, University of Cincinnati), Andrew Burns (Department of Chemistry, University of Cincinnati), Patrick A. Limback (Department of Chemistry, University of Cincinnati), Balasubrahmanyam Addepalli (Department of Chemistry, University of Cincinnati)

Abstract:
The goal of this work was to profile by liquid chromatography-tandem mass spectrometry (LC-MS/MS) the oxidative damage exerted by H2O2 on RNA modifications1 using E. coli tRNA as a model system. Oxidative damage to RNA impacts biological systems due to its association with metabolic, mitochondrial, neurological diseases and cancer.2,3 Oxidation pathways of canonical nucleosides are well documented.4 However, the effect of reactive oxygen species (ROS) on modified ribonucleosides is not well known.5 Hydrogen peroxide generates ROS (e.g., HOO•, HO•) under physiological conditions. Formation of hydroxyl radicals is accelerated via Fenton reactions in the presence of catalysts (e.g., Fe2+, Cu2+). Apart from the known oxidative products arising from canonical nucleosides, we have documented a list of modified nucleosides that are adversely affected by oxidative stress. These results indicated some modifications (e.g., 2-methylthio-N6-isopentenyladenosine, epoxyqueuosine, 2-thiocytidine) are more susceptible to oxidation than others. It was also observed that oxidation damage is more predominant with a copper catalyst. In addition, a new oxidation product with m/z 260.0877 was noticed to increase in abundance upon increasing ROS. Even though this m/z value is identical to that observed for 5-hydroxycytidine, it differs in retention time and MS/MS fragmentation data. The exact molecular structure of this unknown oxidation product is currently under investigation.

References:
1. modomics.genesilico.pl. A Database of RNA Modification Pathways.
2. Sarin, P.; et al.. RNA Biology 2015, 11, 1555.
3. Gu, C.; et al.. FEBS lett. 2014, 588, 4287.
4. Alshykhly, O, R.; et al.. J. Org. Chem. 2016, 80, 6996.
5. Nawrot, B.; et al.. Cell. Mol. Life Sci. 2011, 68, 4023.

Keywords: Oxidative Stress, RNA Damage, Nucleoside Modifications