Current methods to identify RNA modifications with short-read sequencing are laborious and direct RNA sequencing gets proclaimed as the viable alternative. Herein, we harness the selective reactivity of the acrylonitrile towards the Inosine (I) and pseudouridine (Ψ) modifications and developed a chemical probe-based direct RNA sequencing method. We first demonstrated that the chemical probe-induced differential signature profile using nanopore sequencing could facilitate the selective assessment of I and Ψ in the in vitro synthesized RNA. Furthermore, we verified the I and Ψ modification with single-nucleotide resolution using RNA derived from mouse brain without the need for a null dataset using knockouts. Our chemical probe-based nanopore sequencing strategy can be extended to profile multiple RNA modifications on a single RNA and may facilitate the diagnosis of disease-associated epitranscriptome markers by generating a comparative dataset in clinical scenarios.

中文翻译：

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基于化学探针的纳米孔测序，选择性评估RNA修饰

用短读测序鉴定RNA修饰的当前方法费力，并且直接RNA测序被认为是可行的选择。在本文中，我们利用丙烯腈对肌苷（I）和假尿苷（Ψ）修饰的选择性反应性，开发了一种基于化学探针的直接RNA测序方法。我们首先证明了使用纳米孔测序的化学探针诱导的差异特征谱可以促进体外合成RNA中I和Ψ的选择性评估。此外，我们使用源自小鼠大脑的RNA以单核苷酸分辨率验证了I和Ψ修饰，而无需使用基因敲除的空数据集。