Splint-free circular RNA synthesis via RNA secondary structure–guided ligation

Circular RNA (circRNA) is a covalently closed RNA molecule in which the 5' and 3' ends are joined. CircRNAs can be generated via RNA circularization, a process that links the termini of linear RNA. The most common in vitro method for RNA circularization is the ribozyme-based permuted introns and exons (PIE) system. However, circRNAs produced by the PIE method retain partial exogenous exon sequences, potentially leading to immunogenicity issues. In this study, we developed an alternative approach that exploits the secondary structure of linear RNA and enzymatic ligation to synthesize circRNA in vitro for targeted gene expression. We first predicted RNA secondary structures and designed linear RNA molecules to form a terminal nick structure. Using T4 RNA ligase 2 (T4 Rnl2), we sealed the nick to connect the RNA ends. This ligase-mediated in vitro circularization achieved high efficiency and enabled functional expression of the encoded target gene. While ligase-based splint-free circularization has been described for small RNA circles, reports demonstrating functional protein expression from such constructs remain limited. This ligase-mediated RNA circularization approach should be an efficient alternative for production of circular RNA. This ligase-mediated, secondary-structure-guided, splint-free strategy using T4 RNA ligase 2 represents an efficient alternative for circRNA production, enabling both high-yield synthesis and validated gene expression in mammalian cells.