The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis
ABSTRACTCircular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs) following an internal ribosome entry site (IRES) in circRNAs can be translated, and this property has been recently utilized...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2023-12-01
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| Series: | Animal Cells and Systems |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19768354.2023.2265165 |
| _version_ | 1797361682919456768 |
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| author | Yoon-Seob Kim Do-Hyung Kim Daegi An Younghyun Lim Young-Jin Seo Hak Kyun Kim Ho-Young Kang |
| author_facet | Yoon-Seob Kim Do-Hyung Kim Daegi An Younghyun Lim Young-Jin Seo Hak Kyun Kim Ho-Young Kang |
| author_sort | Yoon-Seob Kim |
| collection | DOAJ |
| description | ABSTRACTCircular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs) following an internal ribosome entry site (IRES) in circRNAs can be translated, and this property has been recently utilized to produce proteins as novel therapeutic tools. However, it is difficult to produce large proteins from circRNAs because of the low circularization efficiency of lengthy RNAs. In this study, we report that we successfully synthesized circRNAs with the splint DNA ligation method using RNA ligase 1 and the splint DNAs, which contain complementary sequences to both ends of precursor linear RNAs. This method results in more efficient circularization than the conventional enzymatic method that does not use the splint DNAs, easily generating circRNAs that express relatively large proteins, including IgG heavy and light chains. Longer splint DNA (42 nucleotide) is more effective in circularization. Also, the use of splint DNAs with an adenine analog, 2,6-diaminopurine (DAP), increase the circularization efficiency presumably by strengthening the interaction between the splint DNAs and the precursor RNAs. The splint DNA ligation method requires 5 times more splint DNA than the precursor RNA to efficiently produce circRNAs, but our modified splint DNA ligation method can produce circRNAs using the amount of splint DNA which is equal to that of the precursor RNA. Our modified splint DNA ligation method will help develop novel therapeutic tools using circRNAs, to treat various diseases and to develop human and veterinary vaccines. |
| first_indexed | 2024-03-08T15:58:07Z |
| format | Article |
| id | doaj.art-f6541c3aa50f4d01b4896abfa38ac43b |
| institution | Directory Open Access Journal |
| issn | 1976-8354 2151-2485 |
| language | English |
| last_indexed | 2024-03-08T15:58:07Z |
| publishDate | 2023-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Animal Cells and Systems |
| spelling | doaj.art-f6541c3aa50f4d01b4896abfa38ac43b2024-01-08T17:12:29ZengTaylor & Francis GroupAnimal Cells and Systems1976-83542151-24852023-12-0127120821810.1080/19768354.2023.2265165The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesisYoon-Seob Kim0Do-Hyung Kim1Daegi An2Younghyun Lim3Young-Jin Seo4Hak Kyun Kim5Ho-Young Kang6Drug Discovery Center, NuclixBio, Seoul, Republic of KoreaDrug Discovery Center, NuclixBio, Seoul, Republic of KoreaDepartment of Life Science, Chung-Ang University, Seoul, Republic of KoreaDepartment of Life Science, Chung-Ang University, Seoul, Republic of KoreaDepartment of Life Science, Chung-Ang University, Seoul, Republic of KoreaDepartment of Life Science, Chung-Ang University, Seoul, Republic of KoreaDrug Discovery Center, NuclixBio, Seoul, Republic of KoreaABSTRACTCircular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs) following an internal ribosome entry site (IRES) in circRNAs can be translated, and this property has been recently utilized to produce proteins as novel therapeutic tools. However, it is difficult to produce large proteins from circRNAs because of the low circularization efficiency of lengthy RNAs. In this study, we report that we successfully synthesized circRNAs with the splint DNA ligation method using RNA ligase 1 and the splint DNAs, which contain complementary sequences to both ends of precursor linear RNAs. This method results in more efficient circularization than the conventional enzymatic method that does not use the splint DNAs, easily generating circRNAs that express relatively large proteins, including IgG heavy and light chains. Longer splint DNA (42 nucleotide) is more effective in circularization. Also, the use of splint DNAs with an adenine analog, 2,6-diaminopurine (DAP), increase the circularization efficiency presumably by strengthening the interaction between the splint DNAs and the precursor RNAs. The splint DNA ligation method requires 5 times more splint DNA than the precursor RNA to efficiently produce circRNAs, but our modified splint DNA ligation method can produce circRNAs using the amount of splint DNA which is equal to that of the precursor RNA. Our modified splint DNA ligation method will help develop novel therapeutic tools using circRNAs, to treat various diseases and to develop human and veterinary vaccines.https://www.tandfonline.com/doi/10.1080/19768354.2023.2265165Circular RNAsplint DNA ligation2,6-diaminopurinemRNA vaccine |
| spellingShingle | Yoon-Seob Kim Do-Hyung Kim Daegi An Younghyun Lim Young-Jin Seo Hak Kyun Kim Ho-Young Kang The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis Animal Cells and Systems Circular RNA splint DNA ligation 2,6-diaminopurine mRNA vaccine |
| title | The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis |
| title_full | The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis |
| title_fullStr | The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis |
| title_full_unstemmed | The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis |
| title_short | The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis |
| title_sort | rna ligation method using modified splint dnas significantly improves the efficiency of circular rna synthesis |
| topic | Circular RNA splint DNA ligation 2,6-diaminopurine mRNA vaccine |
| url | https://www.tandfonline.com/doi/10.1080/19768354.2023.2265165 |
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