Fig. 2: Genetically encoding hydroxy acids in response to TAG and TCG codons in Syn61∆3(ev5).
From: Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles
a, Structures of the ‘aliphatic’ hydroxy acids 6–13. b, Intracellular detection of 6 and its amino acid counterpart (BocK). Left: standards (100 µM) were used to define the retention times of BocK and 6. The minor peak at 2 min detected with the 6 channel corresponds to the 13C isotope of BocK, which has the same mass as 6. After the incubation of DH10B cells with the relevant ‘added’ compound (2 mM 6 (red) or 2 mM BocK (grey)), extracts were subjected to LC–MS assays, with selected ion monitoring, to detect BocK (middle) or 6 (right). In cells with BocK added (grey), only BocK was detected. In cells with 6 added (red), both 6 and BocK were detected. c, The production of sfGFP-His6 incorporating hydroxy acid 6, sfGFP(3BocK-OH)-His6, from a sfGFP-3-TCG gene in Syn61∆3(ev5) cells harbouring the MmPylRS/MmtRNAPylCGA orthogonal pair was confirmed by ESI–MS—only a mass that corresponds to hydroxy acid incorporation followed by ester bond cleavage at position 3 was detected. The minor peak at −100 Da results from loss of tert-butoxycarbonyl from 6. d, Orthogonal PylRS active-site variants direct the incorporation of hydroxy acids 6–13 into sfGFP. All the MmPylRS variants were paired with MmtRNAPylCGA to decode TCG, and all the 1R26PylRS variants were paired with AlvtRNAΔNPyl(8)CUA to decode TAG (the data show sfGFP fluorescence in the absence (–) or presence of hydroxy acids). Error bars represent standard deviation from the mean of three biological replicates. PylRS active-site mutations18,19,35,36 are provided in Supplementary Table 2. e, Production of sfGFP-His6 incorporating hydroxy acids 6–13, as confirmed by ESI–MS. Expressions were performed in Syn61∆3(ev5) cells supplemented with a hydroxy acid and its cognate PylRS/tRNA pair. 6 (hydrolysed): expected mass (exp) 27,826.26 Da, actual mass (act) 27,826.40 Da. 7 (hydrolysed): exp 27,810.23 Da, act 27,809.80 Da. 8 (hydrolysed): exp 27,808.20 Da, act 27,809.60 Da. 9 (hydrolysed): exp 27,822.26 Da, act 27,822.50 Da. 10 (hydrolysed): exp 27,836.29 Da, act 27,836.00 Da. 11 (hydrolysed): exp 27,876.32 Da, act 27,876.60 Da. 12 (hydrolysed): exp 27,860.28 Da, act 27,860.20 Da. 13 (hydrolysed): exp 27,768.18 Da, act 27,767.80 Da. a.u., arbitrary units.
