(405e) Tracking Air Pollution Induced RNA Oxidation Using an Improved Biochemical Methodology

Exposure to Reactive Oxygen Species (ROS), universally present in the environment, poses a threat to the structural stability and function of several key biomolecules within the cell. Among these biomolecules are RNAs, which have been shown to become chemically oxidized under conditions of ROS stress. 8-oxo-7,8-dihydroguanine (8-oxoG), an oxidized derivative of the canonical guanine (G) nucleobase, is the most prevalent RNA modification associated with cellular exposure to ROS stress. In targeted studies, the 8-oxoG modification has been shown to impact key cellular processes such as translation and RNA degradation. As a result, it is of particular interest to investigate where this modification differentially accumulates within the cell under conditions of ROS stress. Common methodologies for investigating the location of RNA modifications, including 8-oxoG, largely involve the use of antibody-based RNA ImmunoPrecipitation and Sequencing (RIP-Seq) pipelines. These detection methods have notable limitations, including high experimental costs, low nucleotide resolution, and significant rates of false positives. In this work, we have developed an alternative, cost-effective method to probe for 8-oxoG RNA modifications by covalently functionalizing 8-oxoG modification sites, thereby enabling antibody-free enrichment of 8-oxoG-containing RNAs. We demonstrate the high specificity of this method for detecting 8-oxoG modifications over unmodified nucleobases and benchmark this specificity to an antibody-based detection approach. We further apply this modification detection platform to sequence the location of 8-oxoG modification sites in mRNA isolated from a model of environmental ROS exposure—air pollution exposure to the human lung epithelium. In this model system, human bronchial epithelial (BEAS-2B) cells are exposed to liquid suspensions of standardized Particulate Matter (PM) mixtures to mimic the deposition of PM within the human lung. Our results suggest that a select pool of key mRNAs become differentially oxidized in response to this air pollution exposure when compared to a growth media-only control. These findings have furthered our understanding of the specific molecular mechanisms by which air pollution impacts human health.