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

Abstract

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 E. coli. 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 Clostridium tetani 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 E. coli 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.

Keywords

• group II intron
• circularization in vivo
• one-step synthesis
• sciRNA
• condition optimization