Large efforts have been devoted to genetic code engineering in the past decade, aiming for unnatural amino acid mutagenesis. Recently, an increasing number of studies were reported to employ quadruplet codons to encode unnatural amino acids. We and others have demonstrated that the quadruplet decoding efficiency could be significantly enhanced by an extensive engineering of tRNAs bearing an extra nucleotide in their anticodon loops. In this work, we report the identification of tRNA mutants derived from directed evolution to efficiently decode a UAGA quadruplet codon in mammalian cells. Intriguingly, the trend of quadruplet codon decoding efficiency among the tested tRNA variants in mammalian cells was largely the same as that in E. coli. We subsequently demonstrate the utility of quadruplet codon decoding by the construction of the first HIV-1 mutant that lacks any in-frame amber nonsense codons and can be precisely activated by the decoding of a genomically embedded UAGA codon with an unnatural amino acid. Such conditionally activatable HIV-1 mutant can likely facilitate both fundamental investigations of HIV-1 as well as vaccine developments. The use of quadruplet codon, instead of an amber nonsense codon, to control HIV-1 replication has the advantage in that the correction of a frameshift caused by a quadruplet codon is much less likely than the reversion of an amber codon back into a sense codon in HIV-1.

中文翻译：

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用哺乳动物细胞中的功能性四联密码子控制基因组编码的HIV-1的复制。

在过去的十年中，已经致力于遗传密码工程，以期进行非天然氨基酸诱变。最近，据报道，越来越多的研究采用四联密码子来编码非天然氨基酸。我们和其他人已经证明，通过广泛工程改造其反密码子环中带有一个额外核苷酸的tRNA，可以大大提高四倍体的解码效率。在这项工作中，我们报告了从定向进化衍生的tRNA突变体的鉴定，以有效地解码哺乳动物细胞中的UAGA四联密码子。有趣的是，哺乳动物细胞中被测试的tRNA变异体中四联体密码子解码效率的趋势与大肠杆菌中的趋势基本相同。。我们随后通过构建第一个HIV-1突变体来证明四联体密码子解码的效用，该突变体缺少任何框架内琥珀色无义密码子，并且可以通过用非天然氨基酸进行基因组嵌入的UAGA密码子的解码而被精确激活。这种有条件可激活的HIV-1突变体可能可以促进对HIV-1的基础研究以及疫苗的开发。使用四倍体密码子而不是琥珀色的无义密码子来控制HIV-1复制的优势在于，校正四倍体密码子引起的移码的可能性远小于将琥珀色密码子还原成有义密码子的可能性。在HIV-1中。