In vitro incorporation of nonnatural amino acids into protein using tRNA^Cys-derived opal, ochre, and amber suppressor tRNAs

Incorporation of nonnatural amino acids by tRNA^Cys-derived amber, opal, and ochre tRNAs. (A) Translation of AQP4-P(98) mRNA containing an amber, opal, or ochre codon at residue 44 was carried out in WG extract in the presence of cognate aa-tRNAs (white bars) or NBD-modified aa-tRNAs (gray bars). Incorporation of [¹⁴C]Lys or [¹⁴C]Cys into translated protein was measured by liquid scintillation counting of hot acid precipitable ¹⁴C counts. (B) Translation of the same mRNA was carried out as in A, but in the absence (white bars) or presence (gray bars) of 1 μM RNA aptamer. Incorporation of NBD- or MBB-¹⁴C-labeled amino acids was determined as in A. Results show mean ± SEM (n ≥ 3).