Global Profiling of the Oxidative Stress Induced Effects on RNA Modifications by Liquid Chromatography‐Tandem Mass Spectrometry

Abstract

The goal of this work was to profile the oxidative damage exerted by ultraviolet‐A radiation (UVA, 370 nm) or H2O2 on RNA modifications by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Oxidative damage to RNA impacts biological systems due to its association with metabolic, mitochondrial, neurological diseases and cancer.1,2 In the presence of photosensitizer (e.g., riboflavin), UVA radiation induces the production of reactive oxygen species (ROS, O2•, HOO•, HO•) in vivo.3 Likewise, H2O2 generates ROS under physiological conditions, and formation of radicals is accelerated via Fenton reactions in the presence of catalysts (e.g., Fe2+, Cu2+).4Oxidation pathways of canonical nucleosides are well documented.5 However, the effect of ROS on modified ribonucleosides is not well known, and information about RNA damage is largely extrapolated from studies on DNA. More than 160 modified nucleosides have been reported in various types of RNA of different organisms.6 E. coli with 46 modifications in its tRNA serve as a good model system to investigate the stress‐induced effects. Apart from the known oxidative products arising from canonical nucleosides, we have documented a list of modified nucleosides that are adversely affected by UVA or H2O2. To understand the oxidative environment generated by UVA exposure, we exposed E. coli total tRNA with or without a photosensitizer for defined period of time. In a similar manner, the effect of H2O2 on RNA modifications was investigated by incubating E. coli total tRNA and H2O2, with or without the presence of transition metal ion catalysts. The oxidative damage observed on various modifications was measured by subjecting it to LC‐MS/MS following complete hydrolysis of tRNA. We will present data that describes the susceptibility of various functional groups of these modifications and underlying patterns. Further, these studies also document a range of RNA photoproducts and chemical alterations arising due to the oxidative conditions.Support or Funding InformationFinancial support of this work was provided by the NSF (CHE1507357) and DTRA (HDTRA1‐15‐1‐0033), both to Patrick A. Limbach, and the University of Cincinnati.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.