Fig. 2: Codon competition assays show improved orthogonality with hyperaccurate ribosomes.

a Translation using wild-type ribosomes can be prone to acceptance of near-cognate AA-tRNA, however, the hyperaccurate ribosome (mS12) rejects the near-cognate AA-tRNA, resulting in accurate peptide translation. b A heatmap illustrating the results of the codon competition translation assay using a wt tRNA mixture (left) and t7tRNA mixture (right). The expected cognate and wobble interactions between tRNA and codons are depicted with black borders for Watson-Crick pairing and gold borders for predicted wobble pairing. A green gradient is used to depict the percent of codon readthrough by a given tRNA, and the corresponding value is shown inside the box. Anticodon post-transcriptional modifications on the wt tRNA are depicted with single letter codes: V = 5-oxyacetic acid uridine (cmo5U), B = 2’-O-methyl cytidine (Cm),) = 5-carboxymethylaminomethyl-2’-O-methyl uridine (cmnm5Um). c representative mass spectra of two codon competition experiments: one for the CUA codon and one for the UUA codon. Reactions were performed for thirty minutes at 37 °C with each AA-tRNA supplemented at 5 µM. A table of observed vs expected MS values is shown in Supplementary Table 3. MALDI-MS data showing the tRNA balance is shown in Supplementary Fig.3, and all of the MALDI-MS data used to make the heatmap is shown in Supplementary Fig. 4. Source data are provided as a Source Data file.