Binding of tetracycline to its aptamer determined by 2D-correlated Mn hyperfine spectroscopy.

The tetracycline-binding RNA aptamer (TC-aptamer) binds its cognate ligand the antibiotic tetracycline (TC) via a Mg or Mn ion with high affinity at high divalent metal ion concentrations (K=800pM, ⩾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 Mn concentration of 1 mM and 1D-hyperfine experiments reveal two pronounced P couplings from the RNA besides the C signal of C-labeled TC. From these 1D-hyperfine data alone, however, no conclusions can be drawn on the binding of TC. Either TC may bind via Mn to the aptamer or TC may form a free Mn-TC complex and some Mn also binds to the aptamer. In this work, we show using 2D-correlated hyperfine spectroscopy at Q-band frequencies (34 GHz), that the C and P 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 and P) on two different molecules (RNA and TC) to the same electron spin (Mn). Out of the two observed P-hyperfine couplings, only one shows a clear correlation to C. Although THYCOS and 2D EDNMR yield identical results, 2D EDNMR is far more sensitive. THYCOS spectra needed a time factor of ×20 in comparison to 2D EDNMR to achieve a comparable signal-to-noise.