N1-methyladenosine (m¹A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. M¹A is generally located in the T loop of cytosolic tRNA and between acceptor and D stems of mitochondrial tRNAs, it is involved in tertiary interaction that stabilizes tRNA. Human tRNA m¹A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m¹A, a PCR method to assess m¹A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by qPCR method (TL-qPCR) that measures m¹A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template followed by qPCR using the ligation product as the template. M¹A interferes with the ligation in specific ways, allowing for the quantitative assessment of m¹A levels using sub-nanogram amounts of total RNA. We identify the features of specificity and quantitation for m¹A modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.

中文翻译：

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通过模板连接 qPCR 定量 tRNA m1A 修饰

N1-甲基腺苷 (m ¹ A) 是所有真核生物、许多古细菌和一些细菌 tRNA 中广泛存在的修饰。M ¹ A 通常位于胞质 tRNA 的 T 环中以及线粒体 tRNA 的受体和 D 茎之间，它参与稳定 tRNA 的三级相互作用。人类 tRNA m ¹ A 水平受到动态调节，可以微调翻译，也可以作为传染病的生物标志物。尽管已使用许多方法来测量 m ¹ A，但仍缺乏定量评估特定 tRNA 中 m ^{1 A 水平的 PCR 方法。}在这里，我们开发了一种模板化连接后的 qPCR 方法 (TL-qPCR)，用于测量目标 tRNA 中的m ^{1 A 水平。}我们的方法使用 SplintR 连接酶，以 tRNA 作为模板，有效连接两个 tRNA 互补 DNA 寡核苷酸，然后使用连接产物作为模板进行 qPCR。M ¹ A 以特定方式干扰连接，允许使用亚纳克量的总 RNA 定量评估 m ^{1 A 水平。我们确定了 m 1} A 修饰模型 RNA的特异性和定量特征，并将其应用于人类细胞的总 RNA 样本。我们的方法可以轻松研究这种普遍的 tRNA 修饰的动力学和功能。