The pyrrolysyl-tRNA synthetase/tRNA^Pyl pair is the most versatile and widespread system for the incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. However, low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Here, we generate two tRNA^Pyl variants that significantly boost the performance of the pyrrolysine system. Compared to the original tRNA^Pyl, the engineered tRNAs feature a canonical hinge between D- and T-loop, show higher intracellular concentrations and bear partially distinct post-transcriptional modifications. Using the new tRNAs, we demonstrate efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Moreover, by incorporating last-generation ncAAs for bioorthogonal chemistry, we achieve GPCR labeling with small organic fluorophores on the live cell and visualize stimulus-induced GPCR internalization. Such a robust system for incorporation of single or multiple ncAAs will facilitate the application of a wide pool of chemical tools for structural and functional studies of challenging biological targets in live mammalian cells.

中文翻译：

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设计的tRNA，可通过吡咯赖氨酸系统有效地将非规范氨基酸整合到哺乳动物细胞中

吡咯基-tRNA合成酶/ tRNA ^Pyl对是将非经典氨基酸（ncAAs）掺入哺乳动物细胞蛋白质中功能最广泛，最广泛的系统。然而，ncAA掺入的低产率严重限制了其对相关生物学靶标的适用性。在这里，我们生成了两个tRNA ^Pyl变体，可显着提高吡咯赖氨酸系统的性能。与原始的tRNA ^Pyl相比，经过工程改造的tRNA在D环和T环之间具有典型的铰链，显示较高的细胞内浓度，并具有部分不同的转录后修饰。使用新的tRNA，我们证明了将有效的ncAA掺入G蛋白偶联受体（GPCR）并同时在两个GPCR位点处将ncAA掺入。此外，通过并入用于生物正交化学的上一代ncAA，我们在活细胞上实现了带有小有机荧光团的GPCR标记，并可视化了刺激诱导的GPCR内在化。这种用于结合单个或多个ncAA的强大系统将促进广泛的化学工具在活哺乳动物细胞中对具有挑战性的生物学靶标进行结构和功能研究的应用。