Modulating miRNA binding sites within circRNA for enhanced translation efficiency

Circular RNAs (circRNAs) are covalently closed circular RNAs, and some of them preserve translation potency. However, modulation of circRNA translation efficiency and its applications need to be explored. In this study, RNAs containing the translation initiation element CVB3 IRES and the coding sequence of luciferase protein were transcribed and circularized in vitro by T7 RNA polymerase and an optimized permutated intron‒exon (PIE) splicing strategy. The circularized RNAs were then transfected and translated into active luciferase in the cultured cells. Insertion of miRNA binding sites at the flanking region of the luciferase coding sequence significantly reduced the translation efficiency of the circRNAs. Mutations of the miRNA binding sites in the firefly luciferase coding sequence led to increased translation efficiency of synthetic circRNAs in cells. We also proved that mutations of the binding sites of specific miRNAs also enhanced the translation efficiency of synthetic circRNAs. Further in vivo experiments via bioluminescence imaging showed that synonymous mutation of the miRNA binding sites promoted synthetic circRNA translation in nude mice. This study demonstrates that the modulation of miRNA binding sites affects the translation efficiency of synthetic circRNAs in vitro and in vivo, which could be used as versatile tools for future applications in clinical imaging.