一种双用途聚合酶，可用于假尿嘧啶核苷和 queuosine 的直接测序。
A dual-purpose polymerase engineered for direct sequencing of pseudouridine and queuosine.
机构信息
Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraße 10, 78464Konstanz, Germany.
Institut für Biologie, Humboldt-Universität zu Berlin, Philippstraße 13, Rhoda-Erdmann-Haus, 10099 Berlin, Germany.
出版信息
Nucleic Acids Res. 2023 May 8;51(8):3971-3987. doi: 10.1093/nar/gkad177.
More than 170 posttranscriptional RNA modifications are so far known on both coding and noncoding RNA species. Within this group, pseudouridine (Ψ) and queuosine (Q) represent conserved RNA modifications with fundamental functional roles in regulating translation. Current detection methods of these modifications, which both are reverse transcription (RT)-silent, are mostly based on the chemical treatment of RNA prior to analysis. To overcome the drawbacks associated with indirect detection strategies, we have engineered an RT-active DNA polymerase variant called RT-KTq I614Y that produces error RT signatures specific for Ψ or Q without prior chemical treatment of the RNA samples. Combining this polymerase with next-generation sequencing techniques allows the direct identification of Ψ and Q sites of untreated RNA samples using a single enzymatic tool.
目前已知在编码和非编码 RNA 物种上有超过 170 种转录后 RNA 修饰。在这些修饰中，假尿嘧啶核苷 (Ψ) 和 Queuosine (Q) 是保守的 RNA 修饰，它们在调节翻译方面具有重要的功能作用。目前，这些修饰的检测方法主要基于在分析之前对 RNA 进行化学处理，而这两种修饰都是逆转录（RT）沉默的。为了克服与间接检测策略相关的缺点，我们设计了一种称为 RT-KTq I614Y 的 RT 活性 DNA 聚合酶变体，该变体产生针对 Ψ 或 Q 的特异性错误 RT 特征，而无需对 RNA 样品进行预先的化学处理。将这种聚合酶与下一代测序技术相结合，仅使用单一酶促工具即可直接鉴定未经处理的 RNA 样品中的 Ψ 和 Q 位点。
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