Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
Nature. 2010 Mar 18;464(7287):441-4. doi: 10.1038/nature08817. Epub 2010 Feb 14.
The in vivo, genetically programmed incorporation of designer amino acids allows the properties of proteins to be tailored with molecular precision. The Methanococcus jannaschii tyrosyl-transfer-RNA synthetase-tRNA(CUA) (MjTyrRS-tRNA(CUA)) and the Methanosarcina barkeri pyrrolysyl-tRNA synthetase-tRNA(CUA) (MbPylRS-tRNA(CUA)) orthogonal pairs have been evolved to incorporate a range of unnatural amino acids in response to the amber codon in Escherichia coli. However, the potential of synthetic genetic code expansion is generally limited to the low efficiency incorporation of a single type of unnatural amino acid at a time, because every triplet codon in the universal genetic code is used in encoding the synthesis of the proteome. To encode efficiently many distinct unnatural amino acids into proteins we require blank codons and mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs that recognize unnatural amino acids and decode the new codons. Here we synthetically evolve an orthogonal ribosome (ribo-Q1) that efficiently decodes a series of quadruplet codons and the amber codon, providing several blank codons on an orthogonal messenger RNA, which it specifically translates. By creating mutually orthogonal aminoacyl-tRNA synthetase-tRNA pairs and combining them with ribo-Q1 we direct the incorporation of distinct unnatural amino acids in response to two of the new blank codons on the orthogonal mRNA. Using this code, we genetically direct the formation of a specific, redox-insensitive, nanoscale protein cross-link by the bio-orthogonal cycloaddition of encoded azide- and alkyne-containing amino acids. Because the synthetase-tRNA pairs used have been evolved to incorporate numerous unnatural amino acids, it will be possible to encode more than 200 unnatural amino acid combinations using this approach. As ribo-Q1 independently decodes a series of quadruplet codons, this work provides foundational technologies for the encoded synthesis and synthetic evolution of unnatural polymers in cells.
体内，经基因编程的设计氨基酸的掺入使蛋白质的性质能够以分子精度进行定制。已经进化出了 Methanococcus jannaschii 酪氨酸转移 RNA 合成酶-tRNA(CUA)(MjTyrRS-tRNA(CUA))和 Methanosarcina barkeri 吡咯赖氨酸 tRNA 合成酶-tRNA(CUA)(MbPylRS-tRNA(CUA))正交对，以响应大肠杆菌中的琥珀色密码子掺入一系列非天然氨基酸。然而，合成遗传密码扩展的潜力通常限于每次低效掺入一种类型的非天然氨基酸，因为通用遗传密码中的每个三联体密码子都用于编码蛋白质组的合成。为了有效地将许多不同的非天然氨基酸编码到蛋白质中，我们需要空白密码子和相互正交的氨酰-tRNA 合成酶-tRNA 对，它们可以识别非天然氨基酸并解码新密码子。在这里，我们通过合成进化出一种正交核糖体（ribo-Q1），它可以有效地解码一系列四联体密码子和琥珀色密码子，在正交信使 RNA 上提供几个空白密码子，它专门翻译这些密码子。通过创建相互正交的氨酰-tRNA 合成酶-tRNA 对并将其与 ribo-Q1 结合使用，我们可以指导根据正交 mRNA 上的两个新空白密码子掺入不同的非天然氨基酸。使用该密码子，我们通过生物正交的叠氮化物和炔烃的环加成，遗传指导形成特定的、氧化还原不敏感的纳米级蛋白质交联。由于所使用的氨酰-tRNA 合成酶对已进化为掺入许多非天然氨基酸，因此使用这种方法有可能编码超过 200 种非天然氨基酸组合。由于 ribo-Q1 可以独立地解码一系列四联体密码子，因此这项工作为细胞中非天然聚合物的编码合成和合成进化提供了基础技术。
