Engineering circular RNA for potent and stable translation in eukaryotic cells

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.

Past, present, and future of circ RNA s

“…As a side note, the coding ability and stability of circRNAs could be used in biotechnological applications that require production of peptides (Wesselhoeft et al, 2018(Wesselhoeft et al, , 2019. The asymmetric distribution of circRNAs in neurons suggests the possibility of a directed transport of these RNAs across the cell.…”

“…Although physiological functions for most of these possible proteins have not yet been identified, it is likely that they will share some of the abilities of their full-length protein counterparts encoded by the linear form of the transcript. As a side note, the coding ability and stability of circRNAs could be used in biotechnological applications that require production of peptides (Wesselhoeft et al, 2018(Wesselhoeft et al, , 2019.…”

“…Moreover, poly(I:C) (polyinosinic:polycytidylic acid) stimulation, which mimics viral infection, promotes the export of NF90/NF110 to the cytosol. However, the magnitude of RIG-1 activation due to exogenous circRNA was challenged by later reports (Wesselhoeft et al, 2018(Wesselhoeft et al, , 2019. NF90/NF110 binds not only to nascent circRNAs in the nucleus but also to at least two circRNAs in the cytoplasm (circPOLR2A and circDHX34).…”

“…This protein is at the core of a mechanism that allows the distinction between endogenous and foreign circRNAs . However, the magnitude of RIG-1 activation due to exogenous circRNA was challenged by later reports (Wesselhoeft et al, 2018(Wesselhoeft et al, , 2019.…”

Current status and potential role of circular RNAs in neurological disorders

“…Some circRNAs have been hypothesized to function as ‘sponges’ to sequester microRNAs (miRNAs), thus preventing interactions between miRNAs and messenger RNAs (mRNAs) (Hansen et al ; Memczak et al ; Zheng et al ; Han et al ). In addition, circRNAs contribute to regulation of intracellular RBPs, RNA transport, peptide translation, and they can act as a source of pseudogenes (Hansen et al ; Jeck and Sharpless ; Dong et al ; Pamudurti et al ; Wesselhoeft et al ).…”