Trans ligation of RNAs to generate hybrid circular RNAs using highly efficient autocatalytic transcripts.
Trans ligation of RNAs to generate hybrid circular RNAs using highly efficient autocatalytic transcripts.
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DOI：
10.1016/j.ymeth.2021.05.009
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发表时间：
2021-12
期刊：
影响因子：
--
通讯作者：
Jaffrey SR
中科院分区：
文献类型：
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作者：
Litke JL;Jaffrey SR
环状RNA是用于各种生物技术应用的有用实体，例如模板翻译和结合或螯合miRNA和RNA结合蛋白。环状RNA对细胞中的降解具有高度抗性，并且比线性RNA寿命更长。在这里，我们描述了一种用于RNA转录物的细胞内反式连接的方法，该方法可以产生杂合环状RNA。这些杂合环状RNA包含通过连接共价连接以形成环状RNA的两个单独的RNA。通过在每个连接位点掺入自切割核酶，转录物的反式连接在哺乳动物细胞中发生，而不需要额外的材料。我们提供了一个协议，用于设计和测试translation的成绩单，并证明检测杂交环状RNA使用荧光显微镜。
Circular RNAs are useful entities for various biotechnology applications, such as templating translation and binding or sequestering miRNA and RNA binding proteins. Circular RNA as highly resistant to degradation in cells and are more long-lived than linear RNAs. Here, we describe a method for intracellular trans ligation of RNA transcripts that can generate hybrid circular RNAs. These hybrid circular RNAs comprise two separate RNA that are covalently linked by ligation to form a circular RNA. By incorporating self-cleaving ribozymes at each site of ligation, trans ligation of the transcripts occurs in mammalian cells with no additional material. We provide a protocol for designing and testing trans ligation of transcripts and demonstrate detection of hybrid circular RNAs using fluorescence microscopy.