N¹-methyl adenosine (m¹A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m¹A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m¹A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m¹A can notably modulate RNA structure. It also blocks Watson–Crick–Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m¹A. In this study, we introduce a reduction-based m¹A sequencing (red-m¹A-seq). We report that NaBH₄ reduction of m¹A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m¹A to m⁶A to provide good controls, allowing the detection of m¹A with higher sensitivity and accuracy. We applied red-m¹A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m¹A sites, but also new m¹A sites in mt-tRNA^Asn-GTT and 5.8S rRNA.

中文翻译：

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m1A 的化学操作介导其在人类 tRNA 中的检测

N ¹ -甲基腺苷 (m ¹ A) 是一种广泛存在于 tRNA、rRNA 和 mRNA 中的 RNA 修饰。 tRNA 中的m ¹ A 修饰位点在进化上是保守的，其在 tRNA 上的形成是由甲基转移酶 TRMT61A 和 TRMT6 复合物催化的。 m ¹ A 促进翻译起始和延伸。由于其在生理条件下带正电荷，m ¹ A 可以显着调节 RNA 结构。它还阻断沃森-克里克-富兰克林碱基配对，并在逆转录过程中引起突变和截断。已经开发了几种基于错误掺入的高通量测序方法来对 m ¹ A 进行测序。在本研究中，我们引入了基于还原的 m ¹ A 测序（red-m ¹ A-seq）。我们报告说，NaBH ₄还原 m ¹ A 可以使用市售 RT 酶提高突变和通读率，从而提供更好的阳性信号，而碱催化的 Dimroth 重排可以有效地将 m ¹ A 转化为 m ⁶ A 以提供良好的对照，允许以更高的灵敏度和准确度检测m ¹ A。我们应用red-m ¹ A-seq对人类小RNA进行测序，不仅检测到了之前报道的所有tRNA m ¹ A位点，还检测到了mt-tRNA ^{Asn-GTT和5.8S rRNA中新的m 1} A位点。