Fig. 4.

Schematic illustration of in vitro transcription (IVT) technology and circularization of linear RNA precursor (circRNA synthesis). The process of IVT include plasmid construction, plasmid linearization and IVT (production of linear precursors). a Chemical ligation methods by the conjugation of 5’-end phosphate with 3’-end hydroxyl catalyzed by BrCN or EDC treatment. Enzymatic ligation method, catalyzed by RNA ligases, use complementary template to bridge specific ligations in two flanks. And then the DNase strips the template. b Group I intron-based permuted intron‒exon (PIE) system. Permutation of a native group I intron and insertion of a custom sequence into the exonic region (E1 and E2). And then this PIE system spontaneously ligates in the presence of free guanosine, making it circular and freeing the two half-intron fragments. c Group II intron-based PIE system. The custom sequence is flanked by the 5’- and 3’-self-cleaving native group II intron. And then, this system automatically ligates and excise lariat intermediate