Mechanistic insights into circularization via Anabaena group I intron-based scarless circular RNA.

Circular RNA (circRNA) offers significant advantages in stability, storage, manufacturing, and pharmacokinetics, making it an attractive option for therapeutic applications over linear RNA. However, the commonly used permuted intron-exon (PIE) method for constructing circRNA introduces an exogenous "scar" sequence during splicing initiation, potentially compromising circRNA potency and inducing immunogenicity. Through exploration of the molecular mechanism of the Anabaena group I intron splicing, we conclude the sequence characterization of splice sites and the recognition rules of IG sequence. Leveraging these principles, we successfully prepared "scarless" circRNA without sacrificing its circularization efficiency using standard in vitro transcription procedures. Immunogenicity analysis of scarless circRNAs revealed that the scar will not induce an immune response, consistent with previous findings, after complete removal of linear byproducts, that circRNAs are naturally low immunogenicity. Finally, we find that the incorporation of modified nucleotides in circRNAs disrupts not only splicing function but also internal ribosome entry site function, with a low percentage of modified nucleotides destroying translation capacity.