Abstract
The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.
Highlights
Decoding of in-frame amber stop codons (UAG), generally referred to as amber suppression, enables the translational incorporation of non-canonical amino acids into target proteins in vitro and in vivo [1,2]
Our results demonstrate that overall iPASS reliably predicts relative non-canonical amino acids (ncAAs) incorporation efficiencies, which we show to be independent of ncAA as well as cell line identity
Efficient amber suppression in mammalian cells has been reported to depend on high suppressor tRNA expression levels [22,23,25,27], whereas two PylS copies are sufficient to expand the genetic code of mice [13]
Summary
Decoding of in-frame amber stop codons (UAG), generally referred to as amber suppression, enables the translational incorporation of non-canonical amino acids (ncAAs) into target proteins in vitro and in vivo [1,2]. The pyrrolysyltRNA synthetase (PylRS, encoded by PylS)/tRNAPylCUA (PylT, encoded by PylT) pair from Methanosarcina species is one of the most commonly used orthogonal translation systems (OTSs) to incorporate ncAAs at amber stop codons in bacteria [3,4,5], yeast [6], mammalian cells [7,8,9] and animals [10,11,12,13]. This expansion of the genetic code allows site-specific introduction of unique moieties into proteins. Depending on the UAG context, high variations in ncAA incorporation rates are frequently observed in bacteria and mammalian cells [1,2,31,32,33,34,35,36,37]
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