Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery.
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Meineke B, Heimgärtner J, Caridha R, Block MF, Kimler KJ, Pires MF, Landreh M, Elsässer SJ
Cell Rep Methods 3 (11) 100626 [2023-11-20; online 2023-11-06]
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 (tRNAPyl)/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.
PubMed 37935196
DOI 10.1016/j.crmeth.2023.100626
Crossref 10.1016/j.crmeth.2023.100626
pmc: PMC10694491
pii: S2667-2375(23)00290-4