Fig. 5: tRNA display selection of orthogonal synthetases that charge ncMs.
From: Adding α,α-disubstituted and β-linked monomers to the genetic code of an organism
a, ncMs for which selective PylRS mutants were discovered. b, Fluoro-tREX. A representative gel is shown for each PylRS variant. Experiments performed in independent triplicates with similar results. c, Selected PylRS variants acylate \({{\rm{tRNA}}}_{{\rm{C}}{\rm{U}}{\rm{A}}}^{{\rm{P}}{\rm{y}}{\rm{l}}}\) with 13. LC–MS traces (scanning ion mode on 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) adduct of 13) of AQC-derivative eluted from tRNA pulldowns. Cells harbouring the corresponding PylRS variant and \({{\rm{tRNA}}}_{{\rm{C}}{\rm{U}}{\rm{A}}}^{{\rm{P}}{\rm{y}}{\rm{l}}}\) (or only \({{\rm{tRNA}}}_{{\rm{C}}{\rm{U}}{\rm{A}}}^{{\rm{P}}{\rm{y}}{\rm{l}}}\) (−)) were grown with 13. Pulldowns used a biotinylated probe against \({{\rm{tRNA}}}_{{\rm{C}}{\rm{U}}{\rm{A}}}^{{\rm{P}}{\rm{y}}{\rm{l}}}\). Representative traces are shown. d,f,h,j,l,n,p, As in b, but with indicated PylRS variants and 14 (d), 15 (f), 16 (h), 17 (j), 18 (l), 19 (n) or 20 (p). e,g,i,k,m,o,q, As in c, but with indicated PylRS variants and 14 (e), 15 (g), 16 (i), 17 (k), 18 (m), 19 (o) or 20 (q). e,g,i, Performed in duplicate, all replicates yielded similar results. c,k,m,o,q, Performed in triplicate, all replicates yielded similar results. r, Fluorescence from cells containing GFP(150TAG)His6, indicated PylRS variant and \({{\rm{tRNA}}}_{{\rm{C}}{\rm{U}}{\rm{A}}}^{{\rm{P}}{\rm{y}}{\rm{l}}}\), and grown in the presence or absence of ncM (13–20, 4 mM). Fluorescence is shown as a fraction of the fluorescence generated by the wild-type PylRS–\({{\rm{tRNA}}}_{{\rm{C}}{\rm{U}}{\rm{A}}}^{{\rm{P}}{\rm{y}}{\rm{l}}}\) pair with 4 mM BocK (1) and GFP(150TAG)His6. Bar graphs represent mean of three independent measurements, individual data points are shown as dots and error bars indicate s.d. s, ESI–MS of GFP(150(S)β3mBrF)–His6 purified from cells harbouring PylRS(13_1evol1), \({{\rm{tRNA}}}_{{\rm{C}}{\rm{U}}{\rm{A}}}^{{\rm{P}}{\rm{y}}{\rm{l}}}\) and GFP(150TAG)His6 grown with 13. Found mass: 27,939.0 Da, predicted mass: 27,938.2 Da. Spectra acquired once. t, ESI–MS of GFP(150(S)α-Me-pIF)–His6 purified from cells harbouring PylRS(19_1), \({{\rm{tRNA}}}_{{\rm{C}}{\rm{U}}{\rm{A}}}^{{\rm{P}}{\rm{y}}{\rm{l}}}\) and GFP(150TAG)His6 grown with 19. Found mass: 28,000.5 Da, predicted mass: 28,000.2 Da. Spectra were acquired once. u, Close up on residue 150 of GFP(150(S)β3mBrF)–His6, from a crystal structure determined at 1.5 Å. The 2Fo − Fc map is shown at contour level of σ = 2 (Protein Data Bank (PDB) ID 8OVY), electron density (blue).
