Engineering circular RNA for potent and stable translation in eukaryotic cells

• Published in: Nature communications Vol. 9; no. 1; pp. 2629 - 10
• Main Authors: Wesselhoeft, R. Alexander, Kowalski, Piotr S., Anderson, Daniel G.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 06-07-2018; Nature Publishing Group; Nature Portfolio
• Subjects: 13/106; 13/31; 14/35; 14/63; 38; 38/77; 38/88; 42; 42/109; 45/70; 45/90; 631/337; 631/61/391; 631/92/552; 82/16; 96/21; Anabaena - genetics; Base Sequence; Catalysis; Cell Line; Chromatography, High Pressure Liquid; Circular RNA; Circularity; Eukaryotic Cells - metabolism; Exons - genetics; Gene expression; Genetic Engineering - methods; High performance liquid chromatography; Humanities and Social Sciences; Humans; Internal Ribosome Entry Sites; Introns - genetics; Liquid chromatography; mRNA; multidisciplinary; Open Reading Frames - genetics; Protein Biosynthesis; Protein expression; Proteins; Ribonucleic acid; RNA; RNA - genetics; RNA - isolation & purification; RNA Splicing - genetics; RNA, Messenger - genetics; RNA, Messenger - metabolism; Science; Science (multidisciplinary); Sequence Homology, Nucleic Acid; Splicing; Translation
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-018-05096-6

Messenger RNA (mRNA) has broad potential for application in biological systems. However, one fundamental limitation to its use is its relatively short half-life in biological systems. Here we develop exogenous circular RNA (circRNA) to extend the duration of protein expression from full-length RNA messages. First, we engineer a self-splicing intron to efficiently circularize a wide range of RNAs up to 5 kb in length in vitro by rationally designing ubiquitous accessory sequences that aid in splicing. We maximize translation of functional protein from these circRNAs in eukaryotic cells, and we find that engineered circRNA purified by high performance liquid chromatography displays exceptional protein production qualities in terms of both quantity of protein produced and stability of production. This study pioneers the use of exogenous circRNA for robust and stable protein expression in eukaryotic cells and demonstrates that circRNA is a promising alternative to linear mRNA.
Circular RNAs have recently been shown to have protein-coding potential. Here the authors design a self-splicing RNA that, when circularized, provides for stable high-yield protein production.