NMR Chemical Exchange as a Probe for Ligand-Binding Kinetics in a Theophylline-Binding RNA Aptamer

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Abstract
The apparent on and off rate constants for binding of theophylline to its RNA aptamer in the absence of Mg2+ were determined here by 2D 1H−1H ZZ-exchange NMR spectroscopy. Analysis of the buildup rate of the exchange cross peaks for several base-paired imino protons in the RNA yielded an apparent kon of 600 M−1 s−1. This small apparent kon results because the free RNA exist as a dynamic equilibrium of inactive states rapidly interconverting with a low population of active species. The data found here indicate that the RNA aptamer employs a conformational selection mechanism for binding theophylline in the absence of Mg2+. The kinetic data found here also explain a very unusual property of this RNA−theophylline system: slow exchange on the NMR chemical shift time scale for a weakly binding complex. To our knowledge, it is unprecedented to have such a weakly binding complex (Kd ≈ 3.0 mM at 15 °C) show slow exchange on the NMR chemical shift time scale, but the results clearly demonstrate that slow exchange and weak binding are readily rationalized by a small kon. Comparisons with other ligand−receptor interactions are presented.