Trans ligation of RNAs to generate hybrid circular RNAs using highly efficient autocatalytic transcripts

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.