Fig. 3.

Design of linear RNA precursor (pre-circRNA) for efficient circularization, translation initiation and avoidance of immunogenicity. a The untranslated region (UTR) containing the RNA-binding proteins (RBPs) motif can be added upstream and downstream of the IRES-ORF cassettes of linear RNA precursor to promote translation efficiency, and homologous arms and unstructured spacer sequences can be added to promote circularization and to further improve translation efficiency. b During in vitro transcription (IVT), nucleotide modification can be achieved by the addition of N6-methyl-ATP (m6ATP) to the reaction buffer, which allows the host to recognize the circRNA as its own, significantly reducing or even abrogating the immunogenicity of circRNAs