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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 4. Four orthogonal aaRS/tRNA pairs decoding four orthogonal quadruplet codons are expressed from aaRS operons and computationally generated tRNA operons and are mutually orthogonal in their aminoacylation specificity, recognize distinct ncAAs, and decode distinct orthogonal codons.

Figure 4

a-d, Fluorescence from cells containing O1-strepGFP(40XXXX)His6, with XXXX being the codon at position 40 in sfGFP: TAGA, CTAG, AGGA or AGTA. E. coli also contained O-riboQ1 and the aaRS and tRNA operons (aaRS4_1-2/tRNA4(quad)); these operons expressed MmPylRS/MspetRNAPyl-evolUCUA, MrumPylRS(NmH)/MinttRNAPyl-A17VC10UCCU, AfTyrRS(PheI)/AftRNATyr-A01CUAG and Mg1PylRS(CbzK)/MalvtRNAPyl-8UACU. The indicated 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 were added to cells or omitted (-). Each codon was only efficiently decoded in the presence of cognate ncAA of the aaRS/tRNA pair assigned to the respective quadruplet codon: (a) O1-strepGFP(40TAGA)His6 decoded by MmPylRS/MspetRNAPyl-evolUCUA, (b) O1-strepGFP(40AGGA)His6 decoded by MrumPylRS(NMH)/MinttRNAPyl-A17VC10UCCU, (c) O1-strepGFP(40AGTA)His6 decoded by Mg1PylRS(CbzK)/ MalvtRNAPyl-8UACU, and (d) O1-strepGFP(40CTAG)His6 decoded by AfTyrRS(PheI)/AftRNATyr-A01CUAG. Each bar represents the mean of three biological replicates ± s.d. The individual data points are shown as dots. e-h, Positive electrospray TOF-MS of nickel-NTA-purified strepGFPHis6, expressed from O1-strepGFP(40XXXX)His6, with XXXX being either TAGA (e), AGGA (f), AGTA (g) or CTAG (h), in the presence of NmH 2, CbzK 3, AllocK 4, PheI 5. Cells also contained O-riboQ1 and operon aaRS4_2-1/tRNA4(quad). strepGFP(40AllocK)His6: mass predicted 29113.2, mass found 29114.8. strepGFP(40NmH)His6: mass predicted 29052.1, mass found 29052.5. strepGFP(40CbzK)His6: mass predicted 29163.3, mass found 29164.2. strepGFP(40PheI)His6: mass predicted 29174.03, mass found 29174.2. The experiment was performed three times with similar results.