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, 46 (1), 1-10

Designer tRNAs for Efficient Incorporation of Non-Canonical Amino Acids by the Pyrrolysine System in Mammalian Cells

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Designer tRNAs for Efficient Incorporation of Non-Canonical Amino Acids by the Pyrrolysine System in Mammalian Cells

Robert Serfling et al. Nucleic Acids Res.

Abstract

The pyrrolysyl-tRNA synthetase/tRNAPyl pair is the most versatile and widespread system for the incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. However, low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Here, we generate two tRNAPyl variants that significantly boost the performance of the pyrrolysine system. Compared to the original tRNAPyl, the engineered tRNAs feature a canonical hinge between D- and T-loop, show higher intracellular concentrations and bear partially distinct post-transcriptional modifications. Using the new tRNAs, we demonstrate efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Moreover, by incorporating last-generation ncAAs for bioorthogonal chemistry, we achieve GPCR labeling with small organic fluorophores on the live cell and visualize stimulus-induced GPCR internalization. Such a robust system for incorporation of single or multiple ncAAs will facilitate the application of a wide pool of chemical tools for structural and functional studies of challenging biological targets in live mammalian cells.

Figures

Figure 1.
Figure 1.
Cloverleaf structures of tRNAs. (A) M. mazei tRNAPyl. Compared to canonical tRNAs, tRNAPyl misses nucleotides represented by small circles and features a longer anticodon stem (six base pairs instead of five). To conserve the canonical numbering (28,29), position 29:41 is counted twice as a/b. Dashed lines indicate tertiary interactions inferred from the crystal structure of D. hafniense tRNAPyl (11). (B) Canonical human tRNA with tertiary interactions indicated as dashed lines (–28). Positions that are 100% conserved as a purine (R) or a pyrimidine (Y), and single nucleotides (A,U,G,C) that are >90% conserved are indicated. Nucleotides corresponding to the consensus internal promoter sequences (A-box, TRGCNNAGY for positions 8–16 and G18G19; B-box, GGTTCGANTCC for positions 52–62) are highlighted by bold circles. (C) Stabilized B. taurus mt-tRNASerCUA amber suppressor (29). (D) tRNAM15. Gray shades indicate mutations from wild type tRNAPyl. (E) tRNAC15. Gray shading indicates nucleotides imported from tRNAPyl.
Figure 2.
Figure 2.
Selection of enhanced tRNAPyl variants for ncAA incorporation. (A) ncAAs used in this study. (B–D) Amber codon suppression in EGFPY183TAG by tRNAPyl* and engineered tRNA variants. Bars represent the fluorescence of the probe relative to the fluorescence intensity of wild type EGFP expression. All values were normalized to mCherry fluorescence. Data represent the average ± SD of biological triplicates. All tRNA sequences are reported in Supplementary Table S1. (B) First round of screening. Mutant and chimera tRNAs (1 copy) were combined with MbPylRSF (natural gene with archaeal codon usage) for the incorporation of Lys(Boc). (C) Second round of screening. tRNA variants (1 copy) were combined with either MbPylRSF or MbPylRSAF (genes optimized for human codon usage) for incorporation of either Lys(Boc) or Lys(Z). (D) Incorporation efficiency of different ncAAs by tRNAPyl*, tRNAM15, tRNAC15, using constructs bearing either one (1×) or four (4×) copies of the tRNA expression cassette.
Figure 3.
Figure 3.
Northern blots. Total RNA from HEK293 cells expressing the indicated tRNA were isolated under acidic conditions to preserve the AA-tRNA ester linkage (39), resolved on 12.5% acidic urea-PA gels and electroblotted. (A) tRNAPyl* and tRNAM15 were detected using the same 5′-biotinylated oligonucleotide probe (in gray), whereas tRNAC15 was detected with a different oligonucleotide targeted to the same tRNA region. (B and C) # indicates RNA probes extracted from non-transfected cells; iv indicates in vitro transcripts (loading controls, 25 ng); (B) cells co-expressed MbPylRSF, (C) cells co-expressed MbPylRSAF. The results are representative of at least 3 independent experiments.
Figure 4.
Figure 4.
Single and multiple ncAA incorporation into a GPCR. ncAAs were incorporated into the CRF1R bearing FLAG at the C-terminus. (A) Amber suppression by the indicated tRNAs combined to either MbPylRSF or MbPylRSAF. # indicates non-transfected cells. (B) Amber (TAG), opal (TGA) or ochre (TAA) suppression by tRNAM15CUA, tRNAM15UCA, tRNAM15UUA, respectively, measured by fluorescence assay as described above. Data represent the average ± SD of biological triplicates. (C) Opal and amber suppression by the MbPylRSAF/tRNAM15UCA pair combined with the EcAziRS/tRNATyrCUA pair. In A and C, * indicates half amount and one fourth amount loaded, respectively.
Figure 5.
Figure 5.
GPCR labeling via SPIEDAC on live cells. Representative images in the green, red and blue channel. (A) Cells expressing CRF1R95TCO*K-EGFP were treated with Cy3-tetrazine (1.5 μM) for 5 min. Only cells expressing the receptor (green) showed Cy3 labeling (red). (B) Cy3-CRF1R95TCO*K-EGFP was activated with Urocortin1 (200 nM) and imaged after 15 min. Intracellular vesicles correspond to the internalized receptor. Scale bar: 10 μm.

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