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 involve 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 with group 1 introns. Furthermore, to streamline the purification process of circRNA, 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 circRNA junction site during in vitro circularization. This unique attribute facilitates a one-step purification process. The resulting "nickless one-step purification of engineered circular messenger RNA" system emerges as both straightforward and efficient in generating therapeutic circular messenger RNAs using simple, scalable in vitro systems.