Quantifying the Number and Affinity of Mn²⁺-Binding Sites with EPR Spectroscopy

During the last decades, various functional oligonucleotides have been discovered including DNAzymes, ribozymes, and riboswitches. Their function is based on their ability to form and change their three-dimensional structure. Binding of divalent ions to specific binding pockets was found to be important for the global structure and function. Here, we present a protocol that allows counting the number of Mn²⁺-binding sites and to determine their dissociation constants by means of continuous wave X-band Electron Paramagnetic Resonance (EPR) spectroscopy. In this method, Mn²⁺ is titrated into the oligonucleotide-containing sample and the intensity of the EPR spectrum is recorded. By comparison with a Mn²⁺-only reference sample, the binding isotherm can be constructed and fitted to binding models yielding the number and affinities of the binding sites. This method has been successfully applied to several functional oligonucleotides.