Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR
Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR
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DOI：
10.1038/s41587-022-01534-5
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发表时间：
2022-10-27
影响因子：
46.9
通讯作者：
Abudayyeh, Omar O.
中科院分区：
文献类型：
--
作者：
Jiang, Kaiyi;Koob, Jeremy;Abudayyeh, Omar O.
在生物系统中感知和响应特定RNA存在的可编程方法在研究、诊断和治疗中具有广泛的应用。在这里，我们设计了一种可编程的RNA传感技术，可重新编程的阿达尔传感器（RADARS），它利用腺苷脱氨酶作用于RNA（阿达尔）的RNA编辑，通过内源性RNA转录物的存在来门控货物蛋白的翻译。在货物上游的向导中引入终止密码子使得翻译取决于内源转录物与向导的结合，导致终止密码子的阿达尔编辑并允许翻译通读。通过系统的传感器工程，我们在传感器激活方面实现了277倍的改进，并利用不同的货物蛋白（包括荧光蛋白、重组酶和半胱天冬酶）设计了RADARS，从而能够对表达水平低至每百万份13份转录物的内源性转录物进行检测灵敏度。我们发现RADARS作为表达的DNA或合成的mRNA以及与外源性或内源性阿达尔一起起作用。我们将RADARS应用于多种环境中，包括跟踪转录状态、RNA传感诱导的细胞死亡、细胞类型鉴定和控制合成mRNA翻译。RNA传感器利用阿达尔来控制靶蛋白的翻译。
Programmable approaches to sense and respond to the presence of specific RNAs in biological systems have broad applications in research, diagnostics, and therapeutics. Here we engineer a programmable RNA-sensing technology, reprogrammable ADAR sensors (RADARS), which harnesses RNA editing by adenosine deaminases acting on RNA (ADAR) to gate translation of a cargo protein by the presence of endogenous RNA transcripts. Introduction of a stop codon in a guide upstream of the cargo makes translation contingent on binding of an endogenous transcript to the guide, leading to ADAR editing of the stop codon and allowing translational readthrough. Through systematic sensor engineering, we achieve 277 fold improvement in sensor activation and engineer RADARS with diverse cargo proteins, including luciferases, fluorescent proteins, recombinases, and caspases, enabling detection sensitivity on endogenous transcripts expressed at levels as low as 13 transcripts per million. We show that RADARS are functional as either expressed DNA or synthetic mRNA and with either exogenous or endogenous ADAR. We apply RADARS in multiple contexts, including tracking transcriptional states, RNA-sensing-induced cell death, cell-type identification, and control of synthetic mRNA translation.An RNA sensor harnesses ADAR to control translation of a target protein.