Functional Engineering of Synthetic RNA Through Circularization

Abstract

Many types of RNAs exist in the cells, such as messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), noncoding RNA (ncRNA), and microRNA (miRNA). Most of these endogenous RNAs have linear structures, while some circular types have been recently founded. Before the findings of endogenous circular RNAs, we have recognized the unique molecular properties of circular RNAs and advanced studies on the design, synthesis, and biological application of circular RNAs. Specifically, we have created circular formats of RNA for RNA interference (RNAi) and protein production (translation). In the former application, a dumbbell-type circular RNA and single-stranded circular RNA with some functional molecular linkers were newly developed. These circular RNAs were well designed so that the advantage of this structural format would be demonstrated in biological systems, thereby achieving higher and prolonged gene-silencing effects, reduced immunogenicity, and enhanced cellular uptake compared with canonical siRNA. In the latter application, circular RNA coding proteins were designed and prepared, expecting that the avoidance of the repetitive rate-limiting step (initiation) in translation reaction and continuous protein production would be achieved on the circular format of mRNA. In accordance with our speculation, greatly enhanced protein production could be achieved by circular mRNA in both prokaryotic and eukaryotic translation systems. In addition, total chemical synthesis of circular RNA that could be translated with high efficiency was achieved, thus widening the application of circular RNA in biotechnological and pharmaceutical contexts. These beneficial properties that arise from circular format would have high versatility and a pivotal RNA molecular design for enhancing the molecular function and solving inherent problems associated with the synthetic RNA applied for biological systems.