Fig. 4. Double and triple incorporation of distinct noncanonical amino acids into TCG, TCA, and TAG codons in Syn61Δ3 cells.

(A) Reassignment of TCG (blue box), TCA (gold box) and TAG (green box) codons to distinct ncAAs in Syn61Δ3. Reassigning all three codons to distinct ncAAs in a single cell requires three engineered triply orthogonal aaRS/tRNA pairs. Each pair must recognize a distinct ncAA and decode a distinct codon. The tRNAs from these triply orthogonal pairs are labelled O-tRNA^1-3.

(C) ESI-MS analyses of purified Ub-(11CbzK, 65p-I-Phe) (black trace) and Ub-(11CbzK, 14CbzK, 57p-I-Phe, 65p-I-Phe) (gray trace), expressed in the presence of CbzK and p-I-Phe, as described in (E) and purified by nickel-nitrilotriacetic acid chromatography. These data confirm the quantitative incorporation of CbzK and p-I-Phe in response to TCG and TAG codons, respectively. Ub-(11CbzK, 65p-I-Phe), theoretical mass: 9707.81 Da; measured mass: 9707.40 Da. Ub-(11CbzK, 14CbzK, 57p-I-Phe, 65p-I-Phe), theoretical mass: 10,055.00 Da; measured mass: 10,054.60 Da.

(D) The incorporation of three distinct noncanonical amino acids into TCG, TCA, and TAG codons in a single gene. Syn61Δ3(ev4) – containing the 1R26PylRS(CbzK)/AlvtRNA^ΔNPyl(8) _CGA pair, the MmPylRS/MmtRNA^Pyl _UGA pair and the AfTyrRS(p-I-Phe)/AftRNA^Tyr(A01) _CUA pair – were provided with CbzK, BocK and p-I-Phe. Cells also contained Ub_{9TAG,11TCG,14TCA} (TCG/TCA/TAG). Expression of this gene was performed in the absence (-) or presence (+) of the ncAAs. Full-length Ub-(9p-I-Phe, 11CbzK, 14BocK)-His₆ was detected in cell lysate from an equal number of cells with an anti-His₆ antibody.

(E) ESI-MS of purified Ub-(9p-I-Phe, 11CbzK, 14BocK), theoretical mass: 9820.97 Da; measured mass: 9820.80 Da. Western blot experiments [(B) and (D)] were performed in five biological replicates with similar results. The ESI-MS data [(C) and (E)] were collected once.