Splint and Ribozyme-Free Enzymatic Synthesis and Purification of Long Circular RNA for in vitro Translation in Human Cells

Aisha Svihla1, Christopher B. Rohde2, Matthew Angel2

1Northeastern University, Boston, MA, 2Factor Bioscience Inc., Cambridge, MA

Mol Ther, Vol 29, No 4S1, 2021

Messenger RNA (mRNA) has many applications in biological research and therapeutic development. However, its relatively short half-life limits the use of mRNA in applications where long-term protein translation is desired. Circular RNAs (circRNAs) are a novel class of RNA molecules with enhanced stability that exist in many organisms, including C. elegans, Drosophila melanogaster, mice, and humans. Engineered circRNAs have been used for a variety of applications including microRNA sponges and protein expression. The covalently closed structure of circRNAs makes them resistant to degradation by exonucleases, leading to extended protein translation in cells. Methods for synthesizing circRNAs include the use of a splint molecule to bring the 5’ and 3’ ends of linear RNA in close proximity for ligation and the use of ribozymatic methods in conjunction with self-splicing introns to covalently link the 5’ and 3’ ends of an in vitro-transcribed RNA molecule. However, these methods suffer from low efficiency or result in cytotoxic byproducts that must be removed using high-performance liquid chromatography (HPLC). We show that circRNA can be efficiently synthesized without cytotoxic byproducts using T4 RNA Ligase 1 and rationally designed short RNA sequences that form secondary structures designed to enhance ligation efficiency. Specifically, we show that RNA sequences that form a hairpin loop in close proximity to the 5’ and 3’ ends better support intramolecular ligation than RNA sequences without such a hairpin loop. We demonstrate that this method of ligation is applicable to a variety of RNA sequences, including long RNAs. CircRNA containing an internal ribosome entry site (IRES) 5’ to a sequence encoding green fluorescent protein is readily translated in primary human fibroblasts with minimal cytotoxicity. In summary, we present a simple, cost effective method of producing protein-encoding circRNA sequences that removes the need for complex purification methods, which may find use in biomedical applications that can benefit from extended RNA stability and translation.