Abstract
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.
Highlights
Messenger RNA has broad potential for application in biological systems
Circularization may allow for the stabilization of Messenger RNA (mRNA) that generally suffer from short half lives[10, 11] and may improve the overall efficacy of exogenous mRNA in a variety of applications[12]
A ribozymatic method utilizing a permuted group I catalytic intron has been reported to be more applicable to long RNA circularization and requires only the addition of GTP and Mg2+ as cofactors[17]. This permuted intron-exon (PIE) splicing strategy consists of fused partial exons flanked by half-intron sequences[20]
Summary
Messenger RNA (mRNA) has broad potential for application in biological systems. one fundamental limitation to its use is its relatively short half-life in biological systems. In addition to having protein-coding potential, endogenous circRNAs lack the free ends necessary for exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and granting them extended lifespans as compared to their linear mRNA counterparts[2, 9]. For this reason, circularization may allow for the stabilization of mRNAs that generally suffer from short half lives[10, 11] and may improve the overall efficacy of exogenous mRNA in a variety of applications[12]. We present an engineering approach to generating exogenous circRNAs for potent and durable protein expression in eukaryotic cells
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