Binding of divalent metal ions plays a key role in the structure and function of ribozymes and other RNAs. In turn, the energetics and kinetics of the specific binding process are dominated by the balance between the cost of dehydrating the aqueous ion and the energy gained from inner-sphere interactions with the macromolecule. In this work, we introduce the use of the pulsed EPR technique of 2H Electron Spin-Echo Envelope Modulation (ESEEM) to determine the hydration level of Mn2+ ions bound to nucleotides and nucleic acids. Mn2+ is an excellent structural and functional mimic for Mg2+, the most common divalent ion of physiological interest. Comparison of data in D2O and H2O, with aqueous Mn2+ as a reference standard, allows a robust and precise determination of the number of bound water molecules, and therefore the number of RNA-derived ligands. Examples of applications to the mononucleotide models MnGMP and MnATP, as well as to the paradigmatic RNA system tRNAPhe, are shown.
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