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 Mg²⁺ or Mn²⁺ ion with high affinity at high divalent metal ion concentrations (K_D=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 Mn²⁺ concentration of 1 mM and 1D-hyperfine experiments reveal two pronounced ³¹P couplings from the RNA besides the ¹³C signal of ¹³C-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 ¹³C and ³¹P 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 (¹³C and ³¹P) on two different molecules (RNA and TC) to the same electron spin (Mn²⁺). Out of the two observed ³¹P-hyperfine couplings, only one shows a clear correlation to ¹³C. 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.