Graduate School of Science, Nagoya University, Aichi, Japan.
Curr Protoc. 2021 Mar;1(3):e43. doi: 10.1002/cpz1.43.
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.
最近，在人类细胞中已经报道了许多种环状 RNA。环状 RNA 的一个有趣的方面是它们可以翻译为蛋白质。我们之前发现，没有终止密码子的环状 RNA 可以通过原核和真核系统中的滚环翻译翻译成长重复肽。由于在滚环翻译中，翻译的限速步骤-核糖体结合-仅发生一次，因此与线性 mRNA 的翻译相比，翻译效率非常高。然而，环状 RNA 的制备涉及昂贵且耗时的酶法，并且没有实用的非酶法。我们最近报道了一种使用短 RNA 片段和一个或两个亚磷酰胺键的化学合成策略。在本文中，我们描述了用于滚环翻译的环状 RNA 的化学合成和纯化方法。© 2021 Wiley Periodicals LLC. 基本方案 1：3'-氨基修饰鸟嘌呤控孔玻璃的合成 基本方案 2：线性 RNA 片段的固相合成 基本方案 3：环状 RNA 的化学合成。
