Mild and ultrafast GLORI enables absolute quantification of m6A methylome from low-input samples

Methods that enable absolute quantification of N6-methyladenosine (m6A) RNA modification have emerged as powerful tools in the field of epitranscriptomics. We previously reported GLORI, a chemical-assisted approach firstly achieved quantitatively transcriptome-wide m6A measurement at single-base resolution. Despite its advantages, GLORI suffers from lengthy reaction time and severe RNA degradation. Here, we present two updated GLORI approaches: GLORI 2.0 is an ultra-fast and mild version that preserves RNA integrity and enhances sensitivity for both transcriptome-wide and locus-specific m6A detection; GLORI 3.0 further utilizes a novel reverse transcription-silent carrier RNA to achieve high-quality m6A quantification from ~ 1,000 cells. Using limited RNA input extracted from single mouse dorsal hippocampus, we measure m6A methylome in the synaptic and cytoplasmic fractions and reveal a high modification level in synapse-related gene sets. We envision that the updated GLORI methods will greatly expand the applicability of absolute quantification of m6A in biology.

可实现N6-甲基腺嘌呤（m6A，N6-methyladenosine）RNA修饰绝对定量的方法，现已成为表观转录组学（epitranscriptomics）领域的强力研究工具。我们此前曾报道GLORI技术——一种化学辅助型方法，首次实现了单碱基分辨率下全转录组范围的m6A定量检测。尽管该技术具备诸多优势，但仍存在反应耗时过长、RNA降解严重的缺陷。本研究中我们推出两款升级后的GLORI技术方案：GLORI 2.0为超快速温和版本，可维持RNA完整性，并同时提升全转录组及位点特异性m6A检测的灵敏度；GLORI 3.0则进一步采用了新型逆转录静默载体RNA，可从约1000个细胞中实现高质量的m6A定量检测。我们利用从单只小鼠背侧海马提取的少量RNA输入样本，对突触组分与胞质组分中的m6A甲基化组进行了检测，并揭示了突触相关基因集的高修饰水平。我们预计，升级后的GLORI技术将极大拓展m6A绝对定量方法在生物学研究中的应用场景。