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)碱基发生8-氧代-7,8-二氢鸟嘌呤(8-oxoG)修饰，已被证实与多种疾病的病理进程相关。目前，解析RNA氧化对细胞稳态的调控作用仍存在瓶颈，核心限制因素是缺乏能够特异性检测8-oxoG修饰区域的有效工具。本研究基于此前已发表的DNA中8-oxoG检测方法，开发了ChLoRox-Seq技术：该技术通过将生物素(biotin)与RNA中的8-oxoG位点进行共价偶联，实现对修饰位点的标记。尤为关键的是，该方法无需依赖抗体即可富集携带8-oxoG的RNA片段，从而可基于下一代测序(Next Generation Sequencing)在全转录组范围内实现修饰区域的精准检测。我们验证了ChLoRox-Seq对RNA中8-oxoG的共价修饰靶向特异性，相较于未修饰的核碱基，其仅高效结合8-oxoG，并将该特异性与当前常用的抗体依赖检测方法进行了基准对比。ChLoRox-Seq的核心优势包括：(1) 可实现更高分辨率的RNA氧化区域定位（例如外显子级分辨率）；(2) 实验成本显著降低。我们将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mRNA，结果发现8-oxoG修饰倾向于富集在G碱基富集区域，以及拥有较长5'非翻译区(5' UTR)和编码区(CDS)的mRNA转录本中。上述发现为阐释跨转录组的RNA氧化积累偏好性的复杂分子机制提供了全新视角。值得注意的是，我们的分析表明，多数mRNA氧化事件可能是概率驱动的，而具备更有利内在特性的mRNA更易以较高速率发生氧化修饰。ChLoRox-Seq技术可在未来研究中便捷推广应用，以在任意目标细胞模型中鉴定RNA氧化水平升高的区域。