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

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Site-specific incorporation of non-canonical amino acids (ncAAs) enables protein labeling and modification.
• Dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs are used for stop codon suppression to incorporate ncAAs.
• Mammalian cell line applications require efficient and homogeneous ncAA incorporation.

Purpose of the Study:

• To demonstrate efficient and homogeneous ncAA incorporation in mammalian cells using piggyBac-mediated genomic integration.
• To explore the use of ncAAs for fluorescent labeling applications in stable cell lines.
• To achieve site-specific, dual-fluorophore labeling of cell surface receptors using mutually orthogonal click chemistries.

Main Methods:

• Utilized piggyBac transposon system for genomic integration of tRNA/aaRS pairs.
• Employed modular plasmid design with multi-copy tRNA arrays for enhanced expression.
• Investigated suppression of ochre and opal stop codons.
• Incorporated two distinct ncAAs with orthogonal click chemistries.

Main Results:

• Achieved homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines.
• Demonstrated successful fluorescent labeling of proteins using incorporated ncAAs.
• Showcased site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.

Conclusions:

• PiggyBac-mediated genomic integration provides an efficient platform for ncAA incorporation in mammalian cells.
• ncAAs serve as valuable chemical handles for protein labeling and modification, including dual-labeling applications.
• This technology facilitates advanced studies of protein function and localization in live mammalian systems.