通过四联体密码子解码实现哺乳动物细胞中的遗传密码扩展。
Genetic Code Expansion in Mammalian Cells Through Quadruplet Codon Decoding.
机构信息
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA.
Department of Chemical & Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE, USA.
出版信息
Methods Mol Biol. 2023;2676:181-190. doi: 10.1007/978-1-0716-3251-2_13.
Genetic code expansion enables the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins both in vitro and in vivo. In addition to a widely applied nonsense suppression strategy, the use of quadruplet codons could further expand the genetic code. A general approach to genetically incorporate ncAAs in response to quadruplet codons is achieved by utilizing an engineered aminoacyl-tRNA synthetase (aaRS) together with a tRNA variant containing an expanded anticodon loop. Here we provide a protocol to decode quadruplet UAGA codon with a ncAA in mammalian cells. We also describe microscopy imaging and flow cytometry analysis of ncAA mutagenesis in response to quadruplet codons.
遗传密码扩展使非天然氨基酸（ncAAs）能够在体外和体内进行定点掺入蛋白质。除了广泛应用的无意义抑制策略外，四联体密码子的使用可以进一步扩展遗传密码。通过利用工程化的氨酰-tRNA 合成酶（aaRS）和含有扩展反密码子环的 tRNA 变体，实现了对四联体密码子的 ncAA 的遗传掺入。在这里，我们提供了一种在哺乳动物细胞中用 ncAA 解码四联体 UAGA 密码子的方案。我们还描述了针对四联体密码子的 ncAA 诱变的显微镜成像和流式细胞术分析。
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