Binding of tetracycline to its aptamer determined by 2D-correlated Mn2+ hyperfine spectroscopy

The tetracycline-binding RNA aptamer (TC-aptamer) binds its cognate ligand the antibiotic tetracycline (TC) via a Me2+ or Mn2+ ion with high affinity at high divalent metal ion concentrations (K-D = 800 pM, >= 10 mM). These concentrations lie above the physiological divalent metal ion concentration of ca. 1 mM and it is known from literature, that the binding affinity decreases upon decreasing the divalent metal ion concentration. This work uses a Mn2+ concentration of 1 mM and 1D-hyperfine experiments reveal two pronounced P-31 couplings from the RNA besides the C-13 signal of C-13-labeled TC. From these 1D-hyperfine data alone, however, no conclusions can be drawn on the binding of TC. Either TC may bind via Mn2+ to the aptamer or TC may form a free Mn-TC complex and some Mn2+ also binds to the aptamer. In this work, we show using 2D-correlated hyperfine spectroscopy at Q-band frequencies (34 GHz), that the C-13 and P-31 signals can be correlated; thus arising from a single species. We use THYCOS (triple hyperfine correlation spectroscopy) and 2D ELDOR-detected NMR (2D electron electron double resonance detected NMR) for this purpose showing that they are suitable techniques to correlate two different nuclear spin species (C-13 and P-31) on two different molecules (RNA and TC) to the same electron spin (Mn2+) Out of the two observed P-31-hyperfine couplings, only one shows a clear correlation to C-13. Although THYCOS and 2D EDNMR yield identical results, 2D EDNMR is far more sensitive. THYCOS spectra needed a time factor of x20 in comparison to 2D EDNMR to achieve a comparable signal-to-noise. (C) 2019 Elsevier Inc. All rights reserved.