Fig. 4: Aminoacyl-thiol side-chain scope.
From: Thioester-mediated RNA aminoacylation and peptidyl-RNA synthesis in water
a, Aminoacylation (%, mean ± s.d.; n = 3) for the reaction of: black, 16U (20 mM) in water at pH 6.5 and room temperature after 24 h; blue, ss-ON5 (0.5 µM) in MES buffer (400 mM, pH 6.5) and KCl (1 M) with l-\({{\bf{1}}}_{{\bf{A}}{\bf{a}}{\bf{a}}}^{{\bf{e}}}\) (200 mM) at room temperature after 16 h; and green, ds-ON5/ON7 (0.5 µM) in MES buffer (400 mM, pH 6.5) and KCl (1 M) with l-\({{\bf{1}}}_{{\bf{A}}{\bf{a}}{\bf{a}}}^{{\bf{e}}}\)(200 mM) at room temperature after 16 h. †l-\({{\bf{1}}}_{{\bf{P}}{\bf{h}}{\bf{e}}}^{{\bf{e}}}\) (100 mM). *l-\({{\bf{1}}}_{{\bf{A}}{\bf{r}}{\bf{g}}}^{{\bf{e}}}\) (60 mM). See Fig. 5c for l-\({{\bf{1}}}_{{\bf{A}}{\bf{s}}{\bf{p}}}^{{\bf{e}}}\) and Supplementary Figs. 81–83 for l-\({{\bf{1}}}_{{\bf{G}}{\bf{l}}{\bf{u}}}^{{\bf{e}}}\), l-\({{\bf{1}}}_{{\bf{G}}{\bf{l}}{\bf{n}}}^{{\bf{e}}}\) and l-\({{\bf{1}}}_{{\bf{M}}{\bf{e}}{\bf{t}}}^{{\bf{e}}}\). b, The reaction of arginine thioester l-\({{\bf{1}}}_{{\bf{A}}{\bf{r}}{\bf{g}}}^{{\bf{e}}}\) was enhanced by side-chain catalysis. 1H NMR spectra show: (1) adenosine 16A (2 mM); (2) the reaction of 16A (2 mM) with l-\({{\bf{1}}}_{{\bf{A}}{\bf{r}}{\bf{g}}}^{{\bf{e}}}\) (120 mM) at pH 6.5 and room temperature after 6 h, which formed arginyl-adenosine \({{\bf{17}}}_{{\bf{A}}{\bf{r}}{\bf{g}}}^{{\bf{A}}}\) (76%) in excellent yield. See Fig. 1b (2) for l-\({{\bf{1}}}_{{\bf{A}}{\bf{r}}{\bf{g}}}^{{\bf{e}}}\) reaction time course. The Arg side chain (pKaH 13.8) is drawn neutral but will be fully protonated at pH 6.0–7.0. c, Minizyme-enhanced aminoacylation of oligonucleotide 5′-FAM-UGAGAGAGCCU (ON10). Aminoacylation (%, mean ± s.d.; n = 3) of ON10 as a blunt end duplex (+5′-AGGCUCUCUCA (ON11) 0.5 µM), underhang (+5′-GGCUCUCUCA (ON12) 0.5 µM) and ‘minizyme’ complex (+5′-GUGGCUCUCUCA (ON13) 0.5 µM) with l-\({{\bf{1}}}_{{\bf{P}}{\bf{h}}{\bf{e}}}^{{\bf{e}}}\) or l-\({{\bf{1}}}_{{\bf{A}}{\bf{l}}{\bf{a}}}^{{\bf{e}}}\) (100 mM) in MES buffer (200 mM, pH 6.5) and KCl (500 mM) after 16 h at room temperature. See Supplementary Figs. 122–124 for further experimental details.
