A central challenge in expanding the genetic code of cells to incorporate noncanonical amino acids into proteins is the scalable discovery of aminoacyl-tRNA synthetase (aaRS)–tRNA pairs that are orthogonal in their aminoacylation specificity. Here we computationally identify candidate orthogonal tRNAs from millions of sequences and develop a rapid, scalable approach—named tRNA Extension (tREX)—to determine the in vivo aminoacylation status of tRNAs. Using tREX, we test 243 candidate tRNAs in Escherichia coli and identify 71 orthogonal tRNAs, covering 16 isoacceptor classes, and 23 functional orthogonal tRNA–cognate aaRS pairs. We discover five orthogonal pairs, including three highly active amber suppressors, and evolve new amino acid substrate specificities for two pairs. Finally, we use tREX to characterize a matrix of 64 orthogonal synthetase–orthogonal tRNA specificities. This work expands the number of orthogonal pairs available for genetic code expansion and provides a pipeline for the discovery of additional orthogonal pairs and a foundation for encoding the cellular synthesis of noncanonical biopolymers.
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The tREX screening data used in this study are available in Supplementary Figs. 2–10, with tREX screening data for tRNA orthogonality in E. coli shown in Supplementary Figs. 2–7 and tREX screening data for aaRS activity on cognate tRNAs shown in Supplementary Figs. 8–10. Supplementary Table 4 has a complete list of the tRNAs generated by the filter described. Supplementary Table 5 lists tRNAs that were selected for experimental investigation, including tRNA accession numbers from tRNA-DB-CE and cognate synthetase accession numbers from NCBI Protein together with the sequences of the corresponding tREX probes. Source data for Figs. 3 and 7 are presented with the paper. All other datasets and material generated or analyzed in this study are available from the corresponding author upon reasonable request.
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This work was supported by the UK Medical Research Council (MRC; MC_U105181009 and MC_UP_A024_1008) and ERC-Advanced Grant SGCR, all to J.W.C. We thank W. Schmied, W. Robertson and R. Hegde for helpful discussions.
The authors declare no competing interests.
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Supplementary Figs. 1–19, Table 1–7 legends and References
Complete list of identity elements used in this study, as described in the literature, thought to be recognized by the E. coli aaRSs.
Alignment of the D loop of each possible sequence in the tRNA-DB-CE database.
Complete list of tRNAs passing our filtering scheme, sorted by isoacceptor class.
tRNA scoring, tRNA detection, tRNA orthogonality, and synthetase and tREX probe sequences for experimentally tested tRNAs.
Raw data for quantification of sfGFP150TAG expression resulting from amber suppression by a given tRNA and aaRS.
Theoretical mass of the sfGFP variant used in this study after maturation of the chromophore.
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Cervettini, D., Tang, S., Fried, S.D. et al. Rapid discovery and evolution of orthogonal aminoacyl-tRNA synthetase–tRNA pairs. Nat Biotechnol 38, 989–999 (2020). https://doi.org/10.1038/s41587-020-0479-2
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