摘要
环状RNA(circRNA)是具有共价闭合的环状结构的RNA,其中有些circRNA具有翻译能力。然而,调节circRNA翻译效率的方法及其应用仍需我们进一步探索。本文利用T7 RNA聚合酶和优化的PIE方法,转录形成含有CVB3 IRES翻译起始元件和荧光素酶蛋白编码序列的RNA,并在体外环化形成环状RNA。我们将环状RNA转染到细胞中,并成功翻译成具有活性的荧光素酶。在荧光素酶编码序列两侧插入miRNA结合位点显著降低了circRNA的翻译效率。随后萤火虫荧光素酶编码序列中的miRNA结合位点的同义突变导致体外合成的circRNA翻译效率增加。我们还证明了特定miRNA结合位点的突变也可以增强合成circRNA的翻译效率。进一步的体内实验通过生物发光成像表明,miRNA结合位点的同义突变促进了体外合成circRNA在体内的翻译。本研究表明,调节circRNA内的miRNA结合位点影响了circRNA的翻译效率,这有望作为未来临床成像应用的多功能工具。
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.
作者
张可维
单革
陈亮
Kewei Zhang;Ge Shan;Liang Chen(Department of Laboratory Medicine,the First Affiliated Hospital of USTC,the CAS Key Laboratory of Innate Immunity and Chronic Disease,School of Basic Medical Sciences,Division of Life Sciences and Medicine,University of Science and Technology of China,Hefei 230027,China)
出处
《中国科学技术大学学报》
CAS
CSCD
北大核心
2023年第9期39-50,66,67,共14页
JUSTC
基金
This work was supported by the National Natural Science Foundation of China(32270590,31930019).
