Fig. 3: A coFLAP of Pepper.
From: Engineering covalent small molecule–RNA complexes in living cells
a, Sequence and secondary structure of the Pepper aptamer (nucleosides in gray and blue form the binding pocket; ligand (HBC) in cyan; reactive guanosine in yellow; same color code is used in b). b, Stick representation of the fluorophore binding pocket (PDB 7EOM). The HBC ligand (cyan) is in close proximity to the N7 nucleophile of G41 (yellow). c, Tested HBC ligands, with and without reactive handles (Brc3HBC, MsOc3HBC and MsOc3HBC-vinyl). d, Incubation of Brc3HBC and 49-nt Pepper RNA results in clean reaction to a major alkylation product as analyzed by AE-HPLC. e, Time course of the reaction (2.5 μM RNA, 50 μM MsOc3HBC or Brc3HBC, 150 mM KCl, 2.0 mM MgCl2, 50 mM MES buffer pH 6.0 (15% DMSO in case of Brc3HBC), 37 °C). Mean values (filled circles) ± s.e.m. are shown (n = 3). f, pH dependence of the MsOc3HBC reaction rate (conditions as in e, pH values as indicated; for relative conversion–time plot at different pH values see Extended Data Fig. 3a). g, FT-ICR mass spectrometric characterization of the covalent c3HBC–RNA complex. CAD of (M–nH)n– ions of RNA produces c and y fragment ions from RNA backbone cleavage. Fragment-ion map illustrating sequence coverage from CAD of the alkylated Pepper RNA (top). MS signals of unmodified and alkylated c40, c41 and complementary y9, y8 fragments from CAD (M–11H)11− and (M–12H)12− ions reveal site of alkylation (G41); calculated isotopic profiles for unmodified and singly alkylated RNA are indicated by red open circles. h, Fluorescence absorption and emission (em) spectra of coPepper with c3HBC (cyan) and c3HBC-vinyl (dark gray). Ex, excitation. i, Pepper aptamer reactivity analysis using diverse conditions; bars show mean ± s.e.m. (n = 3).
