Engineered transfer RNAs for suppression of premature termination codons
Engineered transfer RNAs for suppression of premature termination codons
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DOI：
10.1038/s41467-019-08329-4
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发表时间：
2019-02-18
影响因子：
16.6
通讯作者：
Ahern, Christopher A.
中科院分区：
文献类型：
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作者：
Lueck, John D.;Yoon, Jae Seok;Ahern, Christopher A.
过早终止密码子 (PTC) 导致 10-15% 的遗传性疾病。翻译过程中的 PTC 抑制为治疗多种遗传性疾病提供了一种有前途的方法，但促进 PTC 通读的小分子在临床试验中表现不一。在这里，我们提出了一种高通量、基于细胞的检测方法来鉴定反密码子工程转移 RNA (ACE-tRNA)，它可以有效抑制框内 PTC 并忠实地编码其同源氨基酸。总的来说，我们确定了针对最常见的人类致病无义密码子具有高度抑制活性的 ACE-tRNA。对表达 ACE-tRNA 的细胞进行全基因组转录组核糖体分析，其水平可修复 PTC，表明与翻译终止密码子的相互作用有限。这些 ACE-tRNA 在哺乳动物细胞、非洲爪蟾卵母细胞和小鼠体内表现出高抑制效力，在多个基因中产生 PTC 修复，包括囊性纤维化跨膜电导调节器 (CFTR) 内引起疾病的突变。
Premature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. Here we present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identify ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within cystic fibrosis transmembrane conductance regulator (CFTR).