The epitranscriptomic mark N⁶‐methyladenosine (m⁶A) is the most abundant RNA modification in eukaryotic mRNA, but various limitations in currently available m⁶A detection methods have precluded routine identification of m⁶A marks at the single‐site level in mRNA transcripts. Herein, we report a single‐base elongation‐ and ligation‐based qPCR amplification method (termed “SELECT”) that exploits the ability of m⁶A to hinder 1) the single‐base elongation activity of DNA polymerases and 2) the nick ligation efficiency of ligases; SELECT employs qPCR for quantitation. Following optimization and validation, SELECT was applied on three highly relevant proof‐of‐concept cases: determining 1) if a putative m⁶A site is m⁶A‐modified in mRNAs and lncRNAs from biological samples, 2) the m⁶A fraction at biological sites, and 3) if a particular m⁶A modification enzyme functions on a specific target site. In summary, the rapid and flexible SELECT method facilitates the identification and verification of m⁶A marks with unprecedented ease.

中文翻译：

---

用于位点特异性 N6-甲基腺苷修饰的无放射性标记检测的基于延伸和连接的 qPCR 扩增方法

表观转录组标记N ⁶甲基腺苷(m ⁶ A) 是真核 mRNA 中最丰富的 RNA 修饰，但目前可用的 m ⁶ A 检测方法的各种限制已经排除了在 mRNA 的单位点水平上常规识别 m ⁶ A 标记成绩单。在此，我们报告了一种基于单碱基延伸和连接的 qPCR 扩增方法（称为“SELECT”），该方法利用了 m ⁶A 阻碍 1) DNA 聚合酶的单碱基延伸活性和 2) 连接酶的切口连接效率；SELECT 采用 qPCR 进行定量。在优化和验证之后，SELECT 应用于三个高度相关的概念验证案例：确定 1) 假定的 m ⁶ A 位点是否在来自生物样本的 mRNA 和 lncRNA 中被 m ⁶ A 修饰，2) m ⁶ A 分数在生物位点，以及 3) 如果特定的 m ⁶ A 修饰酶在特定目标位点上起作用。总之，快速灵活的 SELECT 方法以前所未有的简便性促进了 m ⁶ A 标记的识别和验证。