Direct quantitation of Mg2+-RNA interactions by use of a fluorescent dye

The ionic composition of a solution strongly influences the folding of an RNA into its native structure; of particular importance, the stabilities of RNA tertiary structures are sharply dependent on the concentration of Mg2+. Most measurements of the extent of Mg2+ interaction with an RNA have relied on equilibrium dialysis or indirect measurements. Here we describe an approach, based on titrations in the presence of a fluorescent indicator dye, that accurately measures the excess Mg2+ ion neutralizing the charge of an RNA (the interaction or Donnan coefficient, Gamma2+) and the total free energy of Mg2+-RNA interactions (DeltaG(RNA-2+)). Automated data collection with computer-controlled titrators enables the collection of much larger data sets in a short time, compared to equilibrium dialysis. Gamma2+ and DeltaG(RNA-2+) are thermodynamically rigorous quantities that are directly comparable with the results of theoretical calculations and simulations. In the event that RNA folding is coupled to the addition of MgCl2, the method directly monitors the uptake of Mg2+ associated with the folding transition.