Characterizing and circumventing sequence restrictions for synthesis of circular RNA in vitro.

Just as eukaryotic circular RNA (circRNA) is a product of intracellular backsplicing, custom circRNA can be synthesized in vitro using a transcription template in which transposed halves of a split group I intron flank the sequence of the RNA to be circularized. Such permuted intron-exon (PIE) constructs have been used to produce circRNA versions of ribozymes, mimics of viral RNA motifs, a streptavidin aptamer, and protein expression vectors for genetic engineering and vaccine development. One limitation of this approach is the obligatory incorporation of small RNA segments (E1 and E2) into nascent circRNA at the site of end-joining. This restriction may preclude synthesis of small circRNA therapeutics and RNA nanoparticles that are sensitive to extraneous sequence, as well as larger circRNA mimics whose sequences must precisely match those of the native species on which they are modelled. In this work, we used serial mutagenesis and in vitro selection to determine how varying E1 and E2 sequences in a thymidylate synthase (td) group I intron PIE transcription template construct affects circRNA synthesis yield. Based on our collective findings, we present guidelines for the design of custom-tailored PIE transcription templates from which synthetic circRNAs of almost any sequence may be efficiently synthesized.

Chemically synthesized circular RNAs with phosphoramidate linkages enable rolling circle translation.

Circular RNA without a stop codon enables rolling circle translation. To produce circular RNAs, we carried out one-pot chemical synthesis of circular RNA from RNA fragments with the use of an EDC/HOBt-based chemical ligation reaction. The synthesized circular RNAs acted as translation templates, despite the presence of unnatural phosphoramidate linkages.

Intracellular build-up RNAi with single-strand circular RNAs as siRNA precursors.

We herein report a new approach for RNA interference, so-called "build-up RNAi" approach, where single-strand circular RNAs with a photocleavable unit or disulfide moiety were used as siRNA precursors. The advantages of using these circular RNA formats for RNAi were presented in aspects of immunogenicity and cellular uptake.