Enzymatic aminoacylation of tRNA with unnatural amino acids

Fig. 16. Normalized charging efficiency of all of the analogs at 3.3 mM concentration (t = 5 min) using the labeled tRNA assay. The analog percent charging was normalized to the ratio of AA-AMP to AMP of its cognate, natural amino acid, except the Tyr analogs that, because of the low efficiency observed with Tyr itself, were normalized to the most efficient Tyr analog, b11.

Supporting Figure 17

Fig. 17. Quantitation of Met-tRNA^Met formation upon addition of 50 mM concentration of the specified analogs. For analogs N1–N4, N6–N11 the Met-tRNA^Met was quantified using the standard (H₁₅) phosphonium benzaldehyde. Exogenously added d₁₅-Met-AMP served as the internal standard. Because of the proximity of their derivatized analog-AMP masses to the d₁₅-Met-AMP peak, analogs b9, N5, P6, and P7, were tested with the inverse assay setup (see Supporting Experimental Procedures for details). Error bars denote the range of duplicate experiments.

Supporting Figure 18

Fig. 18. IC₅₀ determinations for analogs N6 and P7. Each analog was varied over a wide variety of concentrations while the concentration of Met was kept constant at 50 mM. The curves shown were determined by fitting the data points to the equation Y = B + ((A – B)/(1 + 10ˆ(X – logIC₅₀))), where A, B, and logIC₅₀ are constants determined by the fitting, and X is the log of the analog concentration.

Supporting Figure 19

Fig. 19. Codon table showing charged analogs. The number of analogs for each amino acid is shown in parentheses.

Supporting Experimental Procedures

Synthesis of 4-Formylphenoxypropyl Triphenylphosphonium-d₁₅ Bromide. The synthesis was carried our according to ref. 1, except triphenylphosphine-d₁₅ (Aldrich) was used in place of standard triphenylphosphine.

Labeled tRNA AARS Assay. Assays were performed for 5 min according to procedures described in ref. 2. Each analog was tested at 3.3 mM with its corresponding synthetase only. AARS were at the following concentrations: AlaRS (780 nM), AsnRS (350 nM), AspRS (980 nM), GluRS (1220 nM), HisRS (2050 nM), IleRS (467 nM), LeuRS (380 nM), LysRS (1800 nM), MetRS (890 nM), PheRS (133 nM), ProRS (117 nM), ThrRS (400 nM), TrpRS (4000 nM), TyrRS (480 nM), and ValRS (416 nM).

Quantitative MALDI Assays. Quantitative assays were performed according to the general screening assay procedure in Materials and Methods with several important modifications. The assays were done on a 50-m l scale and contained 50 mM methionine, 145 mM total tRNA, 1.1 mM MetRS, and 50 mM appropriate analog. The internal standard was added as described below.

Internal standard preparation. To prepare the internal standards, assays identical to those described above were performed, except that no analog was added. These assays were treated in the same way as the regular assays, except they were reacted with 4-formylphenoxypropyl triphenylphosphonium-d₁₅ bromide in the derivatization step. Immediately after the reductive amination step, 25 ml of these internal standard assays was added to the analog-containing assays, and subsequent steps were performed as described in the general screening assay.

Standard curve. Derivatized Met-AMP and d₁₅-Met-AMP were mixed in known ratios and analyzed by MS. After baseline subtraction, the determined peak height ratios were plotted vs. the known ratios giving a line with a slope of 0.9168, intercept 0.0327, and an R² value of 0.99. These values were used to correct all subsequent MS data.

Quantification. After baseline subtraction, the relative peak heights of standard Met-AMP and d₁₅-Met-AMP in the MALDI-MS spectra (256 scans) were determined. The peak heights then were further corrected based on the standard curve described above.

Inverse assays. Because all of the analogs tested are substrates, a peak was observed in the MS corresponding to the derivatized analog-AMP. In certain cases (for b9, N5, P6, and P7), this peak would obscure the mass of the d₁₅-Met-AMP internal standard. To overcome this problem, the assay was done in reverse: the d₁₅-phosphonium benzaldehyde was used to label the assay mixture, and standard, nondeuterated phosphonium-Met-AMP was added as the internal standard.

IC₅₀ determinations. IC₅₀ experiments were carried out exactly as described above, except the analogs N6 and P7 were varied over seven different concentrations ranging from 0 to 100 mM. N6 used the standard procedure, whereas P7 used the inverse assay procedure. The curves shown were determined by fitting the data points to the equation Y = B + ((A – B)/(1 + 10ˆ(X – logIC₅₀))), where A, B, and logIC₅₀ are constants determined by the fitting and X is the log of the analog concentration.