A Simple and Efficient One-Step Synthesis System for Flexible Production of Circular RNA in <i>E. coli</i>
Circular RNA (circRNA) exhibits a higher stability and intracellular half-life than linear RNA and has better potential in the fields of RNA vaccines and RNAi drugs. The current strategies for circRNA preparation have low efficiency, high costs, and high complexity, which significantly limits their...
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2024-11-01
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| author | Xiayang Zhao Yiqing Liu Huanhui Huang Yue Sun Fangli Wu Weibo Jin |
| author_facet | Xiayang Zhao Yiqing Liu Huanhui Huang Yue Sun Fangli Wu Weibo Jin |
| author_sort | Xiayang Zhao |
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| description | Circular RNA (circRNA) exhibits a higher stability and intracellular half-life than linear RNA and has better potential in the fields of RNA vaccines and RNAi drugs. The current strategies for circRNA preparation have low efficiency, high costs, and high complexity, which significantly limits their applications. In this paper, we propose a one-step synthesis of circRNA based on <i>E. coli.</i> The four RNA sequence lengths of 1700, 1400, 500, and 64 nt were connected to group II intron elements from the surface protein region of <i>Clostridium tetani</i> and then inserted downstream of the T7 promoter in the pET28a plasmid to assist in cyclization. Then, circRNA was produced in HT115, where the yields of pET28-1700, pET28-1400, pET28-500, and pET28-64 were improved to 820, 783, 691, and 460 ng/1 mL, respectively. Consequently, this system could achieve the mass production of circRNA using only a simple <i>E. coli</i> culture and inducible expression. Meanwhile, the overexpressed circRNA and small circular interference RNA (sciRNA) maintained their biological functions in the protein translation and RNAi. Therefore, this simple and efficient one-step synthesis system can be applied to the functional study and preparation of circRNA in the future. |
| format | Article |
| id | doaj-art-e911e12f6ef8476c8e294512ca10816d |
| institution | OA Journals |
| issn | 2218-273X |
| language | English |
| publishDate | 2024-11-01 |
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| series | Biomolecules |
| spelling | doaj-art-e911e12f6ef8476c8e294512ca10816d2025-08-20T01:53:40ZengMDPI AGBiomolecules2218-273X2024-11-011411141610.3390/biom14111416A Simple and Efficient One-Step Synthesis System for Flexible Production of Circular RNA in <i>E. coli</i>Xiayang Zhao0Yiqing Liu1Huanhui Huang2Yue Sun3Fangli Wu4Weibo Jin5Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, ChinaKey Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, ChinaKey Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, ChinaZhejiang Sci-Tech University Shaoxing Academy of Biomedicine, Zhejiang Sci-Tech University, Shaoxing 312366, ChinaKey Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, ChinaKey Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, ChinaCircular RNA (circRNA) exhibits a higher stability and intracellular half-life than linear RNA and has better potential in the fields of RNA vaccines and RNAi drugs. The current strategies for circRNA preparation have low efficiency, high costs, and high complexity, which significantly limits their applications. In this paper, we propose a one-step synthesis of circRNA based on <i>E. coli.</i> The four RNA sequence lengths of 1700, 1400, 500, and 64 nt were connected to group II intron elements from the surface protein region of <i>Clostridium tetani</i> and then inserted downstream of the T7 promoter in the pET28a plasmid to assist in cyclization. Then, circRNA was produced in HT115, where the yields of pET28-1700, pET28-1400, pET28-500, and pET28-64 were improved to 820, 783, 691, and 460 ng/1 mL, respectively. Consequently, this system could achieve the mass production of circRNA using only a simple <i>E. coli</i> culture and inducible expression. Meanwhile, the overexpressed circRNA and small circular interference RNA (sciRNA) maintained their biological functions in the protein translation and RNAi. Therefore, this simple and efficient one-step synthesis system can be applied to the functional study and preparation of circRNA in the future.https://www.mdpi.com/2218-273X/14/11/1416group II introncircularization in vivoone-step synthesissciRNAcondition optimization |
| spellingShingle | Xiayang Zhao Yiqing Liu Huanhui Huang Yue Sun Fangli Wu Weibo Jin A Simple and Efficient One-Step Synthesis System for Flexible Production of Circular RNA in <i>E. coli</i> Biomolecules group II intron circularization in vivo one-step synthesis sciRNA condition optimization |
| title | A Simple and Efficient One-Step Synthesis System for Flexible Production of Circular RNA in <i>E. coli</i> |
| title_full | A Simple and Efficient One-Step Synthesis System for Flexible Production of Circular RNA in <i>E. coli</i> |
| title_fullStr | A Simple and Efficient One-Step Synthesis System for Flexible Production of Circular RNA in <i>E. coli</i> |
| title_full_unstemmed | A Simple and Efficient One-Step Synthesis System for Flexible Production of Circular RNA in <i>E. coli</i> |
| title_short | A Simple and Efficient One-Step Synthesis System for Flexible Production of Circular RNA in <i>E. coli</i> |
| title_sort | simple and efficient one step synthesis system for flexible production of circular rna in i e coli i |
| topic | group II intron circularization in vivo one-step synthesis sciRNA condition optimization |
| url | https://www.mdpi.com/2218-273X/14/11/1416 |
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