Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

• Published in: Cell reports methods Vol. 3; no. 11; p. 100626
• Main Authors: Meineke, Birthe, Heimgärtner, Johannes, Caridha, Rozina, Block, Matthias F, Kimler, Kyle J, Pires, Maria F, Landreh, Michael, Elsässer, Simon J
• Format: Journal Article
• Language: English
• Published: United States Elsevier 20.11.2023
• Subjects: Amino Acids - genetics; Amino Acyl-tRNA Synthetases - genetics; Codon, Terminator - genetics; CP: Biotechnology; CP: Molecular biology; Genetic Code - genetics; Medicin och hälsovetenskap; RNA, Transfer - genetics; random genomic integration; Genetic code expansion; bioorthogonal labeling; unnatural amino acids; amber suppression; CP: Molecular biology; click chemistry; non-canonical amino acids; mammalian cell culture; stop codon suppression; CP: Biotechnology
• ISSN: 2667-2375; 2667-2375
• DOI: 10.1016/j.crmeth.2023.100626

Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNA )/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.