Accurate analysis of Mg2+ binding to RNA: From classical methods to a novel iterative calculation procedure

Mg²⁺ acts as a catalytic cofactor in many ribozymes and specifically bound divalent metal ions have been implicated in the stabilization of structural motifs that are essential for RNA folding. The accurate calculation of intrinsic affinity constants of M²⁺ to specific binding sites in nucleic acids is therefore of high importance. Methods classically applied to determine the affinity constants of metal ions to RNAs are summarized in the first part of this review, e.g. hydrolytic cleavage experiments, equilibrium dialysis, and spectroscopic techniques like EPR and NMR. However, the fact that several binding sites of similar affinities are often present in a single RNA molecule is mostly neglected. The most immediate consequence of several binding sites is that less than the total amount of M²⁺ is available to bind to a particular binding site at a given total concentration. We have recently introduced a new iterative procedure that tackles this problem and have developed a rapid calculation tool (ISTAR) that is available from the authors. Here, we explain this procedure in detail under different assumptions and illustrate how the intrinsic affinity constants for Mg²⁺ to a short RNA hairpin, a minimal domain 6 from the group II intron Sc.ai5γ, change. We use ISTAR to calculate intrinsic affinities and to validate a particular binding stoichiometry by judging the quality of the fit to the experimental data for a given model. This is important since weak coordination sites exhibiting similar binding affinities, and being thus in direct competition to each other, are a characteristic feature of nucleic acids. With ISTAR these binding affinities can be calculated more accurately within minutes and we can gain a better understanding of these crucial metal ion–nucleic acid interactions.