Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells

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.

Engineered Human Induced Pluripotent Cells Enable Genetic Code Expansion in Brain Organoids

“…ncAA incorporation into endogenous stop codons is thought to be disfavored by strong termination signals associated with terminal stop codons and quality control mechanisms that mitigate aberrantly elongated protein products. [32][33][34] While this result hinted that the proteome of CTL07-II-AS hiPSCs was not collaterally affected by the amber suppression machinery, we also confirmed that expression of the pluripotency marker OCT4 was not affected by 24 h incubation with 0.2 mM CpK (Figure S2). Nevertheless, we note that applications of genetic code expansion in hiPSCs or derived cell lines need to consider inference from off-target effects of amber suppression activity, and it will be important to optimize concentration and incubation time of ncAA to minimize such effects.…”

Direct fluorescent labeling of NF186 and NaV1.6 in living primary neurons using bioorthogonal click chemistry

“…Among other factors, the UAA incorporation efficiency depends on the sequence surrounding the TAG amber codon (Bartoschek et al, 2021). We used a recently developed tool (Bartoschek et al, 2021) to predict the UAA incorporation efficiency rate in the three remaining amber mutants. The iPASS (identification of p ermissive a mber s ites for s uppression) score was higher for NF186 K680TAG -HA (1.83) than for NF186 K519TAG -HA (1.61) and NF186 K604TAG -HA (1.63).…”

Alteration in H-bond strength affects the stability of codon-anticodon interaction at in-frame UAG stop codon during in vitro translation

“…Our study highlights the importance of anticodon base modification in the context of hydrogen bonding. It is well-known that the yield of modified protein depends on the UAG site selection, called mRNA context effect 41 . One report suggested that RF1 interactions during translation depend on mRNA sequence context, and contributed approximately half of the observed difference in reassignment efficiency 42 .…”