Extended Data Fig. 4: Directed evolution of Pyl tRNAs and resulting quadruply orthogonal pairs.
From: Quintuply orthogonal pyrrolysyl-tRNA synthetase/tRNAPyl pairs
a. Heatmap showing the activity of hits obtained from positive selection of the library with S∆B-ClosPylRS followed by successive negative screening with the PylRS enzymes from classes N, A, C and S. Each heatmap value represents the average of three biological replicates. All numerical values and bar charts including error bars showing s.d. are provided (Supplementary Table 2). b. Heatmap showing the activity of the tRNAPyl hit S-I2tRNAPyl-S52 obtained from positive selection of the library with S+-DebPylRS followed by successive negative screening with the PylRS enzymes from the other classes. This tRNAPyl exhibits selective activity with S+-DebPylRS. Each heatmap value represents the average of three biological replicates. All numerical values and bar charts including error bars showing s.d. are provided (Supplementary Table 2). c. Activity heatmaps from each family of quadruply orthogonal PylRS/tRNAPyl pairs obtained following the tRNAPyl directed evolution strategy; the quadruplets with the highest o.c. are shown. Dark grey box: quadruply orthogonal families discovered in Fig. 5. Light grey box: quadruply orthogonal family discovered in Fig. 5, but which has a higher o.c. when incorporating a newly evolved tRNAPyl. Orthogonality coefficient, o.c., is shown in grey. Each heatmap value represents the average of three biological replicates. All numerical values and bar charts including error bars showing s.d. are provided (Supplementary Table 2).
