Extended Data Fig. 8: Z-AisoK tripeptides allow efficient incorporation of Z and dual encoding of two ncAAs using a single tripeptide.

a. Left: SDS-PAGE analysis of sfGFP-N150TAG expression in the presence of AcK or the corresponding Z-AisoK peptide AcK-AisoK (3) with AcKRS3/PylT⁵³. Full-length sfGFP expression is significantly higher when supplementing 3 instead of AcK. Right: LC-MS analysis of sfGFP purified from cultures grown in the presence of 3. Observed mass indicates AcK incorporation. Consistent results were obtained over three distinct replicates. b. Left: SDS-PAGE analysis of sfGFP-N150TAG expression in the presence of CouK or the corresponding Z-AisoK tripeptide CouK-AisoK (6). Full-length sfGFP expression is higher in K12-Z2 cells in presence of 6 compared to wt-K12 or compared to supplementation with CouK. Middle: LC-MS analysis of sfGFP purified from K12-Z2 cells grown in the presence of tripeptide 6. Observed mass indicates incorporation of CouK with a second peak indicating incorporation of lysine likely due to photo-decaging of CouK. Right: SDS-PAGE analysis of sfGFP-N150TAG expression in the presence of ONBK or the corresponding Z-AisoK tripeptide (5). Full-length sfGFP expression is higher in K12-Z2 cells with tripeptide 5 in comparison to the same in wt-K12 cells or with ONBK. Consistent results were obtained over three distinct replicates. c. Left: Chemical structures of ncAAs for dual stop codon suppression to allow for orthogonal incorporation of AcK and pLisoK. Right: SDS-PAGE analysis of sfGFP-N150TAG expression in the presence of 2 mM AcK or 2 mM G-pLisoK and their corresponding PylRS/PylT pairs to confirm substrate orthogonality between the pairs. Full-length sfGFP expression in presence of AcK is only observed with AcKRS3/PylT and in presence of G-pLisoK only with MaPylRS(H227I/Y228P)/PylT, hence confirming orthogonality. d. Left: Scheme of Ub-SUMO fusion construct used to confirm dual and orthogonal incorporation of pLisoK and AcK. SDS-PAGE analysis shows purified full-length construct and products of TEV protease cleavage. Right: LC-MS analysis of full-length construct and cleaved products confirming pLisoK and AcK incorporation at the intended positions.