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. Author manuscript; available in PMC: 2022 Jun 7.
Published in final edited form as: Nat Chem. 2021 Aug 23;13(11):1110–1117. doi: 10.1038/s41557-021-00764-5

Figure 5. Genetically encoding four distinct ncAAs into a protein using a 24 amino acid, 68 codon genetic code.

Figure 5

a, Schematic representation of four engineered mutually orthogonal aaRS/tRNA pairs used for the incorporation of four distinct ncAAs in response to four orthogonal quadruplet codons.

b, Efficient production of full length strepGFP(40PheI, 50AllocK, 136NmH, 150CbzK)His6 was dependent upon the addition of all four ncAAs (Nπ-methyl-L-histidine (NmH) 2, N6-((benzyloxy)carbonyl)-L-lysine (CbzK) 3, N6-((allyloxy)carbonyl)-L-lysine (AllocK) 4, (S)-2-amino-3-(4-iodophenyl)propanoic acid (PheI) 5). Fluorescence from cells containing O1-strepGFP(40CTAG, 50TAGA, 136AGGA, 150AGTA)His6, O-riboQ1, operon aaRS4/tRNA4(quad) (encoding MmPylRS/MspePyltRNAUCUA, MrumPylRS(NMH)/MintPyltRNA(A17,VC10)UCCU, AfTyrRS(PheI)/AftRNATyr-A01CUAG and Mg1PylRS(CbzK)/MalvPyltRNA(8)UACU) in presence or absence of a combination of NmH (2), CbzK (3), AllocK (4), PheI (5). Each bar represents the mean of three biological replicates ± s.d. The individual data points are shown as dots.

c, Positive electrospray TOF-MS of nickel-NTA purified strepGFP(40PheI, 50AllocK, 136NmH, 150CbzK)His6 from cells containing O1-strepGFP(40CTAG, 50TAGA, 136AGGA, 150AGTA)His6, O-riboQ1 and aaRS4_1-2/tRNA4(quad) in presence of the indicated ncAAs. Mass predicted 29470.4 mass found 29468.2. The experiment was performed three times with similar results.