Fig. 1: Covalent tethering of ligand–RNA complexes.
From: Engineering covalent small molecule–RNA complexes in living cells
a, Key is the ligand modification with a short handle and an electrophile (E) retaining initial ligand–RNA contacts. b, Secondary structure of Tt preQ1-I riboswitch (minimal aptamer motif, black; reactive guanosine, yellow; ligand, cyan). c, Stick representation of preQ1 binding pocket (Protein Data Bank (PDB) 3Q50). The ligand (cyan) is in close proximity to the N7 nucleophile of G5 (yellow). d, Structure-based design for ligand derivatization suggests 3-bromopropyl as reactive handle (Brc3DPQ1). e, Incubation of Brc3DPQ1 and 33 nt Tt C15U preQ1 RNA aptamer analyzed by AE-HPLC indicates a major alkylation product. f, Time course of the reaction (2.5 μM RNA, 125 μM Brc3DPQ1, 100 mM KCl, 2.0 mM MgCl2, 50 mM MES pH 6.0, 37 °C). Individual data points are shown as open circles. Mean values (filled circles) ± s.e.m. are shown. Measurements were performed in three independent experiments. g, pH dependence of the reaction rate (conditions as in f, except 60 μM Brc3DPQ1; pH values as indicated; for a relative conversion–time plot at different pH values see Extended Data Fig. 1a; for HPLC traces see Extended Data Fig. 1b. h, FT-ICR mass spectrometric characterization of the covalent c3DPQ1–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 (top). MS signals of c4, c5 and complementary y29, y28 fragments from CAD of (M–9H)9− and (M–8H)8− ions reveal the site of alkylation (G5); calculated isotopic profiles (red open circles). i, Loss of c3DPQ1 alkylated guanine (right) in spectra from CAD of (M–12H)12− ions of RNA is direct evidence for G nucleobase alkylation. Alkylated guanine is lost as a deprotonated species such that the product carries only 11 charges and appears at a higher m/z (~947.5). SM, small molecule.
