Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme
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-targeti...
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| Format: | Article |
| Language: | English |
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Elsevier
2023-09-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S216225312300207X |
| _version_ | 1797743375058731008 |
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| author | Kyung Hyun Lee Seongcheol Kim Jaehwi Song Seung Ryul Han Ji Hyun Kim Seong-Wook Lee |
| author_facet | Kyung Hyun Lee Seongcheol Kim Jaehwi Song Seung Ryul Han Ji Hyun Kim Seong-Wook Lee |
| author_sort | Kyung Hyun Lee |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-12T14:54:35Z |
| format | Article |
| id | doaj.art-5ce9744c0190496cb778acfb7abcd3eb |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-03-12T14:54:35Z |
| publishDate | 2023-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-5ce9744c0190496cb778acfb7abcd3eb2023-08-15T04:04:16ZengElsevierMolecular Therapy: Nucleic Acids2162-25312023-09-0133587598Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozymeKyung Hyun Lee0Seongcheol Kim1Jaehwi Song2Seung Ryul Han3Ji Hyun Kim4Seong-Wook Lee5R&D Center, Rznomics Inc, Seongnam 13486, Republic of Korea; Corresponding author: Kyung Hyun Lee, R&D Center, Rznomics Inc, Seongnam 13486, Republic of Korea.R&D Center, Rznomics Inc, Seongnam 13486, Republic of KoreaR&D Center, Rznomics Inc, Seongnam 13486, Republic of KoreaR&D Center, Rznomics Inc, Seongnam 13486, Republic of KoreaR&D Center, Rznomics Inc, Seongnam 13486, Republic of KoreaR&D Center, Rznomics Inc, Seongnam 13486, Republic of Korea; Department of Bioconvergence Engineering, Research Institute of Advanced Omics, Dankook University, Yongin 16890, Republic of Korea; Corresponding author: Seong-Wook Lee, R&D Center, Rznomics Inc, Seongnam 13486 and Department of Bioconvergence Engineering, Dankook University, Yongin 16890, Republic of Korea.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.http://www.sciencedirect.com/science/article/pii/S216225312300207XMT: non-coding RNAscircular RNAmRNAself-circularizationTetrahymenagroup I intron |
| spellingShingle | Kyung Hyun Lee Seongcheol Kim Jaehwi Song Seung Ryul Han Ji Hyun Kim Seong-Wook Lee Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme Molecular Therapy: Nucleic Acids MT: non-coding RNAs circular RNA mRNA self-circularization Tetrahymena group I intron |
| title | Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme |
| title_full | Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme |
| title_fullStr | Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme |
| title_full_unstemmed | Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme |
| title_short | Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme |
| title_sort | efficient circular rna engineering by end to end self targeting and splicing reaction using tetrahymena group i intron ribozyme |
| topic | MT: non-coding RNAs circular RNA mRNA self-circularization Tetrahymena group I intron |
| url | http://www.sciencedirect.com/science/article/pii/S216225312300207X |
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