Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme

Kyung Hyun Lee; Seongcheol Kim; Jaehwi Song; Seung Ryul Han; Ji Hyun Kim; Seong-Wook Lee

doi:10.1016/j.omtn.2023.07.034

Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme

Mol Ther Nucleic Acids. 2023 Aug 1:33:587-598. doi: 10.1016/j.omtn.2023.07.034. eCollection 2023 Sep 12.

Authors

Kyung Hyun Lee¹, Seongcheol Kim¹, Jaehwi Song¹, Seung Ryul Han¹, Ji Hyun Kim¹, Seong-Wook Lee^{1

2}

Affiliations

¹ R&D Center, Rznomics Inc, Seongnam 13486, Republic of Korea.
² Department of Bioconvergence Engineering, Research Institute of Advanced Omics, Dankook University, Yongin 16890, Republic of Korea.

Abstract

Circular RNA (circRNA) has various advantages over linear mRNA that is gaining success as a new vaccine and therapeutic agent. Thus, circRNA and its engineering methods have attracted attention recently. In this study, we developed a new in vitro circRNA engineering method by end-to-end self-targeting and splicing (STS) reaction using Tetrahymena group I intron ribozyme. We found that only the P1 helix structure of the group I intron was enough to generate circRNA by STS reaction. The efficacy of circRNA generation by STS reaction was comparable to the method using a permuted intron-exon (PIE) reaction. However, an end-to-end STS reaction does not introduce any extraneous fragments, such as an intronic scar that can be generated by PIE reaction and might trigger unwanted innate immune responses in cells, into circRNA sequences. Moreover, generated circRNA was efficiently purified by ion pair-reversed phase high-pressure liquid chromatography and used for cell-based analysis. Of note, efficient protein expression and stability with least innate immune induction by the circRNA with coxsackievirus B3 IRES were observed in cells. In conclusion, our new in vitro circRNA strategy can effectively generate highly useful circRNAs in vitro as an alternative circRNA engineering method.

Keywords: MT: non-coding RNAs; RNA replacement; RNA therapeutics; RNA vaccine; Tetrahymena; circular RNA; group I intron; mRNA; self-circularization; trans-splicing ribozyme.