Figure 5. Identifying triply orthogonal and active Class B ^ΔNPyltRNAs.

a, Schematic of the evolution of class B Int ^ΔNPyltRNA. Clover leaf structures of Int ^ΔNPyltRNA_CUA libraries are shown with randomized nucleotides highlighted in light yellow (variable loop library) or dark yellow (acceptor stem library). Int ^ΔNPyltRNA_CUA hybrids result from combining the best hits from each library. b, GFP fluorescence from GFP(150TAG)_His6 in cells containing BocK 1 and Int ^ΔNPyltRNA_CUA hybrids with each PylRS. Each heatmap value represents the average of three biological replicates. Bar charts including error bars showing s.d. are provided in Supplementary Fig. 17 and all numerical values are provided in Supplementary Table 3. c, Schematic of the progress made in generating triply orthogonal PylRS/^PyltRNA pairs when substituting wild-type Int ^ΔNPyltRNA (top) with selected Int ^ΔNPyltRNA hybrids (bottom). Black arrows indicate high activity, grey arrows indicate low activity and dashed grey arrows indicate minimal activity corresponding to functional orthogonality. The interactions of Int ^ΔNPyltRNA that were manipulated in these experiments are highlighted. d, Heatmap representation of all sets of triply orthogonal PylRS/^PyltRNA pairs generated in this study. Each heatmap value represents the average of three biological replicates. Bar charts including error bars showing s.d. are provided in Supplementary Figs. 17 and 18. Each triplet is composed of MmPylRS and Spe ^PyltRNA, a specific class A ΔNPylRS and an evolved Alv ^ΔNPyltRNA variant, and a specific class B ΔNPylRS and an evolved Int ^ΔNPyltRNA hybrid.