Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells. Issue 1 (21st November 2017)

• Record Type: Journal Article
• Authors: Serfling, Robert; Lorenz, Christian; Etzel, Maja; Schicht, Gerda; Böttke, Thore; Mörl, Mario; Coin, Irene
• Is Part Of: Nucleic acids research. Volume 46:Issue 1(2018)
• Page Start: 1
• Page End: 10
• Publication Date: 2017-11-21
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkx1156
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: 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.