Efficient Circularization of Protein-Encoding RNAs via a Novel Cis-Splicing System

Circular RNAs (circRNAs) have emerged as a promising alternative to linear mRNA, owing to their unique properties and potential therapeutic applications, driving the development of novel approaches for their production. This study introduces a cis-splicing system that efficiently produces circRNAs by incorporating a ribozyme core at one end of the precursor, thereby eliminating the need for additional spacer elements between the ribozyme and the gene of interest (GOI). In this cis-splicing system, sequences resembling homologous arms at both ends of the precursor are crucial for forming the P9.0 duplex, which in turn facilitates effective self-splicing and circularization. We demonstrate that the precise recognition of the second transesterification site depends more on the structural characteristics of P9.0 adjacent to the ?G position than on the nucleotide composition of the P9.0-?G itself. Further optimization of structural elements, like P10 and P1-ex, significantly improves circularization efficiency. The circRNAs generated through the cissplicing system exhibit prolonged protein expression and minimal activation of the innate immune response. This study provides a comprehensive exploration of circRNA generation via a novel strategy and offers valuable insights into the structural engineering of RNA, paving the way for future advancements in circRNA-based applications. Overall design: To assess the sequence integrity at the splicing sites following the two-step transesterification reactions of various precursors, we conducted RNA-Seq analysis on the circularized products. The differences among the precursors primarily stem from the designs of the recognition elements (Recognizers) at both ends, including ?spacers-?G, Loop-?G, Arm-Loop-?G, Arm-?G, and ?C. In the in vitro transcription reactions (IVT), the products resulting from the cotranscriptional circularization of all precursors were individually subjected to RNA-Seq analysis.