Characterization of group I introns in generating circular RNAs as vaccines
Group 1 introns can be utilized for efficient circularization of RNA molecules.  However different group 1 introns are known to have different properties, including having different efficiencies of circularization and generating different levels of immune response inside cells (reactogenicity).  Here, we investigated generating circular RNAs using permuted intron-exon (PIE) systems of group 1 introns from different organisms. We observed that group 1 intron sequences have variable circularization efficiency that is size dependent and circularize with high accuracy. We also determined the sequence properties of the exon1 and exon2 domains of different group1 introns and showed that we can engineer elements such as S1 tag into this domain for better purification. Structural probing and mutational analysis in group 1 introns and their PIE RNAs showed that structural features in P2 and P9 domains are correlated with circularization efficiency and that swapping sequences can improve circularization efficiency. Lastly, we showed that circular RNAs made by Sd group 1 introns elicit lower amount of reactogenicity and either An or Sd can act as effective RNA vaccines. Our work deepens the understanding of the properties of group 1 introns and their use in circular RNA production and medicine. Overall design: To investigate whether different group 1 introns can enable RNA circularization upon PIE re-arrangement, we tested 8 group 1 intron sequences across a range of different buffers, differing in salt and temperature to identify the condition for efficient circularization (Supp Table 1). These 8 group 1 introns are from T4 phage (T4), Anabaena (An), Scytalidium dimidiatum (Sd), Clostridium botulinum (Cb), Scytonema hofmanni (Sh), Geosmithia virida (Gv), Penicillium oblatum (Po), and Barrmaelia oxyacanthae (Bo) (15-18). PIE constructs were made by splitting these group 1 introns at the P6 region and moving 3'-intron and exon 2 (3'I-E2) sequences to the 5' end and exon 1 and 5'-intron (E1-5'I) sequences to the 3' end (Figure 1A, B). For initial testing purpose, a 0.5kb Gaussia luciferase (Gluc) gene is used as an insert between 3'I-E2 and E1-5'I. To test circularization, all T4, An, Sd, Cb, Sh, Gv, Po, Bo PIE Gluc RNAs were in-vitro transcribed using T7 RNA polymerase and were incubated in different buffer conditions at different temperatures for circularization (A to G, Methods). To determine of exon sequence requirement for circularization,  T4 PIE N60 Gluc, An PIE N60 Gluc, Sh PIE N60 Gluc, and Po PIE N60 Gluc IVT RNA were folded in condition B for 15 minutes or 1 hour. These samples were resolved by agarose gel and the lower band in each sample containing circular RNA was gel extracted for sequencing To analyze the circular RNA formation accuracy, synthesis of first-stand cDNA from circular Gluc RNA was performed using SuperScript III reverse transcriptase (Thermo Fisher) with primer junction R according to manufacturer's instructions. Subsequently, this cDNA was used as template for PCR amplification of 6 junction fragments with respective T4, An, Sd, Cb, Sh, and Po junction F and R primers. Each junction PCR fragment was processed using NEBNext Ultra II DNA Library Prep Kit to construct libraries compatible for Illumina sequencing. To investigate sequences of full circular RNA, same cDNA template was used for full circular PCR amplification with respective T4, An, Sd, Cb, Sh and Po full circle F and R primers. Then these full circle fragments were sequenced using Nanopore ligation sequencing kit.