Chemical Synthesis of Circular RNAs with Phosphoramidate Linkages for Rolling‐Circle Translation

Recently, many types of circular RNAs have been reported in human cells. One interesting aspect of circular RNAs is their translation into proteins. We previously discovered that circular RNA without a stop codon can be translated into long repeating peptides via rolling-circle translation in both prokaryotic and eukaryotic systems. Because the rate-limiting step of translation-ribosome binding-occurs only once in rolling-circle translation, the translation efficacy is very efficient compared to translation of linear mRNAs. However, preparation of circular RNAs involves costly and time-consuming enzymatic methods, and there was no practical non-enzymatic method. We recently reported a chemical synthesis strategy using short RNA fragments and one or two phosphoramidate linkages. In this article, we describe the chemical synthesis and purification methods for preparation of circular RNAs for rolling-circle translation. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 3'-amino-modified guanosine controlled-pore glass Basic Protocol 2: Solid-phase synthesis of linear RNA fragments Basic Protocol 3: Chemical synthesis of circular RNAs.