Chen Yan, Wan Yanmin, Wang Nanxi, Yuan Zhe, Niu Wei, Li Qingsheng, Guo Jiantao
ACS Synth Biol. 2018 Jun 15;7(6):1612-1617. doi: 10.1021/acssynbio.8b00096. Epub 2018 Jun 5.
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.
在过去十年中，人们为遗传密码工程付出了巨大努力，目标是实现非天然氨基酸诱变。最近，越来越多的研究报道采用四联体密码子来编码非天然氨基酸。我们和其他人已经证明，通过对反密码子环带有额外核苷酸的tRNA进行广泛改造，四联体解码效率可以显著提高。在这项工作中，我们报告了通过定向进化获得的tRNA突变体的鉴定结果，这些突变体能够在哺乳动物细胞中高效解码UAGA四联体密码子。有趣的是，在哺乳动物细胞中测试的tRNA变体之间，四联体密码子解码效率的趋势与在大肠杆菌中基本相同。随后，我们通过构建首个没有任何框内琥珀色无义密码子、并且可以通过用非天然氨基酸解码基因组嵌入的UAGA密码子而被精确激活的HIV-1突变体，证明了四联体密码子解码的实用性。这种条件可激活的HIV-1突变体可能有助于对HIV-1的基础研究以及疫苗开发。使用四联体密码子而非琥珀色无义密码子来控制HIV-1复制的优势在于，由四联体密码子引起的移码校正比HIV-1中琥珀色密码子回复为有义密码子的可能性要小得多。
