Development and comprehensive evaluation of synthetic scarless circular RNAs

Circular RNAs (circRNAs) are a unique class of single-stranded RNA molecules with a closed-loop structure that confer enhanced stability, extended protein expression, and resistance to exonucleases, making them promising candidates for RNA therapeutics. In recent years, several methods have been developed to generate circRNAs, including the traditional, scar-containing Anabaena Permuted Intron-Exon (Ana-PIE) method and newer “scarless” circularization approaches. This study introduces a novel scarless circularization method, Split-Coxsackievirus B3-Anabaena Permuted Intron-Exon (Split-CVB3 Ana-PIE, SCAP). Scarless circular RNAs generated using the SCAP system were systematically compared to their scarred counterparts produced by the Ana-PIE system in terms of circularization efficiency, protein expression, stability, and immune response. The SCAP system achieved circularization efficiencies comparable to those of the Ana-PIE system while significantly enhancing protein expression. Scarless circular RNAs exhibited similar stability to scarred circular RNAs and did not trigger significant immune responses. These findings highlight the potential of scarless circular RNAs in gene therapy and vaccine development, demonstrating that removing extraneous sequences improves translation efficiency without compromising stability or immunogenicity. This study provides a foundation for the rational design of circular RNAs, with future efforts focusing on diverse target genes, optimized delivery platforms, and in vivo validation. Overall design: RNA-seq profiling of HEK293T cells transfected with linear RNA and circRNA at 24 hours