A cis-acting ligase ribozyme generates circular RNA in vitro for ectopic protein functioning
Chan-I Su, Zih-Shiuan Chuang, Chi-Ting Shie, Hsin-I Wang, Yu-Ting Kao, Chia-Yi Yu
Abstract
Delivering synthetic protein-coding RNA bypassing the DNA stage for ectopic protein functioning is a novel therapeutic strategy. Joining the linear RNA head-to-tail covalently could be a state-of-the-art strategy for functioning longer. Here we enroll a cis-acting ligase ribozyme (RzL) to generate circular RNA (circRNA) in vitro for ectopic protein expression. The RNA circularization is confirmed by masking the 5’ phosphate group, resisting exonuclease RNase R digestion, failing for further tailing, and sequencing the RT-PCR products of the joined region. Interestingly, one internal ribosome entry site (IRES) renders circRNA translation competent, but two IRES in cis, not trans, hamper the translation. The circRNA with highly potent in translation is conferred for antiviral functioning. Accompanying specific guided RNA, a circRNA expressing ribonuclease Cas13 shows excellent potential against the corresponding RNA virus, further extending circRNA functioning in its growing list of applications. Unlike conventional methods of generating circular RNA through RNA splicing, the authors here utilize RNA itself as a ligase ribozyme to directly link a protein-encoding RNA head-to-tail. This approach enhances the circular RNA’s antiviral potential compared to mRNAs.