Engineering circular RNA with Tetrahymena group I intron ribozyme

Linear mRNA vaccines are constrained by exonuclease susceptibility and instability, leading to compromised antigen expression. Circular RNA (circRNA) lacking canonical 5′ and 3′ untranslated regions demonstrates intrinsic exonuclease resistance. Current circularization strategies face three principal limitations: chemical methods produce non-native 2′,5′-phosphodiester bonds; ribozyme-mediated approaches are restricted to RNA fragments shorter than 500 nucleotides; the Anabaena Group I intron system retains immunogenic exon sequences. In contrast, the self-splicing Group I intron ribozyme from Tetrahymena enables precisely controlled circularization through autonomous structural rearrangement, yielding exon-free constructs. Through optimized purification protocols, historical scalability challenges are systematically addressed. This Perspective establishes the mechanistic rationale and therapeutic superiority of this engineered RNA circularization platform.