Mutations that introduce premature termination codons (PTCs) within protein-coding genes are associated with incurable and severe genetic diseases. Many PTC-associated disorders are life-threatening and have no approved medical treatment options. Suppressor transfer RNAs (sup-tRNAs) with the capacity to translate PTCs represent a promising therapeutic strategy to treat these conditions; however, developing novel sup-tRNAs with high efficiency and specificity often requires extensive engineering and screening. Moreover, these efforts are not always successful at producing more efficient sup-tRNAs. Here we show that a pyrrolysine (Pyl) tRNA (tRNAPyl), which naturally translates the UAG stop codon, offers a favorable scaffold for developing sup-tRNAs that restore protein synthesis from PTC-containing genes. We created a series of rationally designed Pyl tRNAScaffold Suppressor-tRNAs (PASS-tRNAs) that are substrates of bacterial and human alanyl-tRNA synthetase. Using a PTC-containing fluorescent reporter gene, PASS-tRNAs restore protein synthesis to wild-type levels in bacterial cells. In human cells, PASS-tRNAs display robust and consistent PTC suppression in multiple reporter genes, including pathogenic mutations in the tumor suppressor gene BRCA1 associated with breast and ovarian cancer. Moreover, PTC suppression occurred with high codon specificity and no observed cellular dysregulation. Collectively, these results unveil a new class of sup-tRNAs with encouraging potential for tRNA-based therapeutic applications.

中文翻译：

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通过工程化嵌合吡咯赖氨酸 tRNA 有效抑制丙氨酸的过早终止密码子


在蛋白质编码基因中引入过早终止密码子 （PTC） 的突变与无法治愈的严重遗传疾病有关。许多 PTC 相关疾病会危及生命，并且没有批准的药物治疗方案。具有翻译 PTC 能力的抑制转移 RNA （sup-tRNA） 代表了治疗这些疾病的一种有前途的治疗策略;然而，开发具有高效率和特异性的新型 sup-tRNA 通常需要广泛的工程和筛选。此外，这些努力并不总是成功地产生更有效的 sup-tRNA。在这里，我们展示了天然翻译 UAG 终止密码子的吡咯赖氨酸 （Pyl） tRNA （tRNAPyl） 为开发恢复含 PTC 基因的蛋白质合成的 sup-tRNA 提供了有利的支架。我们创建了一系列合理设计的 Pyl tRNAScaffold Suppressor-tRNAs （PASS-tRNA），它们是细菌和人丙氨酰-tRNA 合成酶的底物。使用含 PTC 的荧光报告基因，PASS-tRNA 将细菌细胞中的蛋白质合成恢复到野生型水平。在人类细胞中，PASS-tRNA 在多个报告基因中表现出强大且一致的 PTC 抑制作用，包括与乳腺癌和卵巢癌相关的抑癌基因 BRCA1 的致病性突变。此外，PTC 抑制具有高密码子特异性，未观察到细胞失调。总的来说，这些结果揭示了一类新的 sup-tRNA，在基于 tRNA 的治疗应用中具有令人鼓舞的潜力。