A Simple and Efficient One-Step Synthesis System for Flexible Production of Circular RNA in E. coli

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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Bibliographic Details
Published in:Biomolecules (Basel, Switzerland) Switzerland), 2024-11, Vol.14 (11), p.1416
Main Authors: Zhao, Xiayang, Liu, Yiqing, Huang, Huanhui, Sun, Yue, Wu, Fangli, Jin, Weibo
Format: Article
Language:English
Subjects:
B cells
Circular RNA
circularization in vivo
condition optimization
E coli
Efficiency
Escherichia coli
Escherichia coli - genetics
Escherichia coli - metabolism
group II intron
Methods
Nucleotide sequence
one-step synthesis
Plasmids - genetics
Plasmids - metabolism
Protein biosynthesis
Protein expression
Proteins
Ribonucleic acid
RNA
RNA, Circular - genetics
RNA, Circular - metabolism
RNA-mediated interference
sciRNA
Transcription
Vaccines
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Summary: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 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 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 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.
ISSN:2218-273X
2218-273X
DOI:10.3390/biom14111416