Circular RNA (circRNA) is a candidate for next-generation messenger RNA therapeutics owing to its remarkable stability. Here we describe trans-splicing-based methods for the synthesis of circRNAs over 8,000 nucleotides. The methods are independent of bacterial sequences, outperform the permuted intron–exon method and allow for the incorporation of RNA modifications. The resulting unmodified circRNAs, which incorporate sequences from human 28S ribosomal RNA, display low immunogenicity and are translated more efficiently than permuted intron–exon-derived circRNAs. Additionally, by using viral internal ribosomal entry sites for rolling circle translation, we show that ribosomes can efficiently read through highly structured internal ribosomal entry sites, enhancing the efficiency of rolling circle translation by over 7,000-fold with respect to previous constructs. The efficient and reliable production of circRNA may facilitate its therapeutic use.

环状 RNA （circRNA） 因其卓越的稳定性而成为下一代信使 RNA 疗法的候选药物。在这里，我们描述了基于反式剪接的方法，用于合成超过 8,000 个核苷酸的 circRNA。这些方法独立于细菌序列，优于排列的内含子-外显子方法，并允许掺入 RNA 修饰。所得的未修饰 circRNA 包含来自人 28S 核糖体 RNA 的序列，显示出低免疫原性，并且比排列的内含子-外显子衍生的 circRNA 更有效地翻译。此外，通过使用病毒内部核糖体进入位点进行滚环翻译，我们表明核糖体可以有效地读取高度结构化的内部核糖体进入位点，与以前的构建体相比，将滚环翻译的效率提高了 7,000 倍以上。circRNA 的高效可靠生产可能有助于其治疗用途。