Fig. 4: Defining the ncAA and hydroxy acid specificity of aaRSs derived from mutually orthogonal pairs.
From: Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles
a, Seven orthogonal aaRS variants and their cognate tRNAs, which span three mutually orthogonal aaRS classes, were tested for their ability to incorporate 16 monomers (5 ncAAs and 11 alpha hydroxy acids with aliphatic and aromatic side chains) into sfGFP. The fluorescent protein was expressed from sfGFP-3-XXX (where XXX is TCG for Class +N pyrrolysyl-derived pairs, and TAG for ΔN Class A pyrrolysyl-derived pairs and tyrosyl-derived pairs) in Syn61∆3(ev5) cells. Fluorescence levels within a row are normalized to the maximum activity of the active-site variant assayed in that row; raw data are provided in Supplementary Fig. 6b–h. Hashed squares indicate that the data results from amino acid incorporation in cells supplemented with the corresponding hydroxy acid. 1R26PylRS(CbzK) is 1R26PylRS-Y126G-M129L18, AfTyrRS(pIF) is AfTyrRS−Y36I-L69M-H74L-Q116E-D165T-I166G17 and AfTyrRS(pAzF) is AfTyrRS-Y36T-H74L-Q116E-D165T-I166G-N190K17. b–f, Further characterizing five pairs of mutually orthogonal active sites derived from the different synthetase types (Class + N pyrrolysyl, ΔN Class A pyrrolysyl and tyrosyl) shown in a. In the presence of both aaRS/tRNA pairs and their cognate substrates (b, MmPylRS WT and AfTyrRS(pIF) with 7 and 3; c, MmPylRS(ArOH) and 1R26PylRS WT with 16 and 1; d, MmPylRS WT and AfTyrR(pAzF) with 8 and 5; e, MmPylRS(PheOH) and 1R26PylRS(CbzK) with 14 and 4; f, MmPylRS WT and 1R26PylRS(CbzK) with 7 and 12), selective incorporation of each monomer by each pair was confirmed by ESI–MS of the sfGFP expressed from sfGFP-3-TCG or sfGFP-3-TAG reporters. The underlined monomer was selectively incorporated. WT, wild type.
