FIGURE 4.

Strategies for ribozyme methods. (A) Group I intron self-splicing system requires only the addition of GTP and Mg²⁺ as cofactors and shows great potential for protein synthesis. This method realized RNA ligation through a normal group I intron self-splicing reaction, including two transesterifications at defined splice sites. The final circRNA will contain exogenous exon sequences. (B) Group II intron self-splicing system involves the joining of the 5′ splice site at the end of an exon to the 3′ splice site at the beginning of the same exon. All exon sequences are dispensable for group II intron catalyzed inverse splicing. This method can enable more accurate linear RNA precursor ligation. (C) Hairpin ribozyme method can produce circRNA through the rolling circle reaction and the self-splicing reaction. The linear RNA precursor with HPR will fold into two alternative cleavage-active conformations to remove the 3′-end and the 5′-end. As a result, the intermediate will contain a 5′-OH and a 2′, 3′-cyclic phosphate to produce target circRNA.