Characterization of group I introns in generating circular RNAs as vaccines [SPLASH]

Group I introns can be utilized for efficient circularization of RNA molecules; however different introns show variations in their efficiencies of circularization and immune response inside cells. Here, we generated circular RNAs using permuted intron-exon (PIE) systems of group I introns from eight different organisms. We observed that group I introns have variable circularization efficiencies, circularize with high accuracies, and form circular RNAs with distinct reactogenicities. Group I introns also exhibit different sequence requirements for their exon1 and exon2 domains, enabling us to engineer elements such as a S1 tag for better purification. Structural probing and mutational analysis in group I introns and their PIE RNAs showed that structural features in P2 and P9 domains are correlated with circularization efficiency and that swapping sequences can restore pairing to improve circularization. Additionally, RNA modifications do not increase protein production but decrease the reactogenicity of circular RNAs. We demonstrated that circular RNAs made by Scytalidium dimidiatum group I intron elicit strong immunogenicity against SPIKE protein and serve as effective RNA vaccines. Our work deepens the understanding of the properties of group I introns and expands the panel of introns that can be used to generate circular RNAs for vaccines and therapeutics. Overall design: To determine how the bases are paired to each other in the Po and Gv PIE precursor forms, we performed proximity ligation sequencing using SPLASH on Po and Gv PIE systems.