NicOPURE: Nickless RNA Circularization and One-Step Purification with Engineered Group II Introns and Cyclizing UTRs

Circular RNAs (circRNAs) have gained significant attention in recent years due to their diverse biological functions and potential as novel therapeutic modality. Two general mechanisms for generating circRNAs involves the utilization of group 1 and group 2 introns, which are self-splicing ribozymes found in many organisms. Although group 1 intron has been demonstrated to be highly effective in circularization, the reaction requires high temperature plus cofactors such as GTP. Consequently, undesired byproducts such as nicked RNA were generated, which requires complex purification steps before downstream applications. In this study, we have strategically designed structural elements and incorporated sequence features to enhance the efficacy of RNA circularization by group 2 introns. This innovative approach occurred at a reduced temperature and resulted in notably fewer nicks compared to group 1 introns. Furthermore, to streamline the purification process of circular RNA, we incorporated two halves of streptavidin aptamer tags into the enzymatic sites of the group 2 intron. This strategic architecture guarantees the creation of a fully operational streptavidin aptamer tag solely at the circular RNA junction site during in vitro circularization. This unique attribute facilitates a one-step purification process. The resulting "NicOPURE" system emerges as both straightforward and efficient in generating therapeutic circular mRNAs. To understand the structural and functional distinctions existing within Group 1 and Group 2 intron circular mRNAs. 100 μg of circular mRNA (circGLUC) for the same target gene were generated using two distinct approaches: one utilizing a Group 1 intron (Anabaena) and the other employing the NicOPURE system with Group 2 intron (Clostridium tetani). We probed the group 1 and group 2 circular mRNA secondary structure using RNA structure probing method: Selective 2’-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP). SHAPE reactivities were computed for the GLUC open-reading frame (ORF) in both group 1 and group 2 circular mRNA using Shapemapper2.