Optimized chemical labeling method for isolation of 8-oxoG-modified RNA, ChLoRox-Seq, identifies mRNAs enriched in oxidation and transcriptome-wide distribution biases of oxidation events post environmental stress

Bulk increases in nucleobase oxidation, most commonly manifesting as the guanine (G) nucleobase modification 8-oxo-7,8-dihydroguanine (8-oxoG), have been linked to several disease pathologies. Elucidating the effects of RNA oxidation on cellular homoeostasis is limited by a lack of effective tools for detecting specific regions modified with 8-oxoG. Building on a previously published method for studying 8-oxoG in DNA, we developed ChLoRox-Seq, which works by covalently functionalizing 8-oxoG sites in RNA with biotin. Importantly, this method enables antibody-free enrichment of 8-oxoG-containing RNA fragments for Next Generation Sequencing-based detection of modified regions transcriptome-wide. We demonstrate the high specificity of ChLoRox-Seq for functionalizing 8-oxoG over unmodified nucleobases in RNA and benchmark this specificity to a commonly used antibody-based approach. Key advantages of ChLoRox-Seq include: (1) heightened resolution of RNA oxidation regions (e.g. exon-level) and (2) lower experimental costs. By applying ChLoRox-Seq to mRNA extracted from human lung epithelial cells (BEAS-2B) after exposure to environmentally relevant stress, we observe that 8-oxoG modifications tend to cluster in regions that are G-rich and within mRNA transcripts possessing longer 5’ UTR and CDS regions. These findings provide new insight into the complex mechanisms that bias the accumulation of RNA oxidation across the transcriptome. Notably, our analysis suggests the possibility that most mRNA oxidation events are probabilistically driven and that mRNAs that possess more favourable intrinsic properties are prone to incur oxidation events at elevated rates. ChLoRox-Seq can be readily applied in future studies to identify regions of elevated RNA oxidation in any cellular model of interest.

核碱基氧化的整体水平升高，最常见表现为鸟嘌呤（G）核碱基的修饰产物8-氧代-7,8-二氢鸟嘌呤（8-oxoG），已与多种疾病的病理过程密切相关。目前，阐明RNA氧化对细胞稳态（cellular homoeostasis）的影响，因缺乏可特异性检测8-oxoG修饰位点的有效工具而受到极大限制。本研究基于此前发表的DNA中8-oxoG检测方法，开发了ChLoRox-Seq技术：该技术通过生物素（biotin）对RNA中的8-oxoG位点进行共价修饰，以此实现后续检测。尤为关键的是，该方法无需使用抗体即可富集携带8-oxoG的RNA片段，进而可基于下一代测序（Next Generation Sequencing）实现全转录组范围内修饰位点的检测。本研究验证了ChLoRox-Seq在RNA中对8-oxoG的共价修饰相较于未修饰核碱基具有极高特异性，并将该特异性与常用的基于抗体的检测方法进行了基准对比。ChLoRox-Seq的核心优势包括：(1) 可实现RNA氧化区域的更高分辨率检测（例如外显子水平）；(2) 实验成本更低。本研究将ChLoRox-Seq应用于经环境相关应激处理后人肺上皮细胞（BEAS-2B）提取的信使RNA（mRNA），发现8-oxoG修饰倾向于富集在G碱基富集区域，以及拥有更长5'非翻译区（5' UTR）和编码区（CDS）的mRNA转录本中。上述研究结果为解析全转录组范围内RNA氧化积累的偏倚性复杂机制提供了全新视角。值得注意的是，本研究分析表明，大多数mRNA氧化事件可能由概率驱动，而具备更有利内在特性的mRNA更易以更高频率发生氧化修饰。未来研究中，ChLoRox-Seq可便捷应用于任意目标细胞模型中RNA氧化升高区域的识别。