Improving the Function of RNA by Conformational Restriction

Improving the Function of RNA by Conformational Restriction Philip C. Bevilacqua, Department of Chemistry, Pennsylvania State University, University Park, PA 16802 The malachite green aptamer (MGA) is an RNA that binds the dye molecules tetramethylrosamine (TMR) and malachite green (MG). We have shown through isothermal titration calorimetry, a technique that measures the change in heat upon interaction of two molecules, that MGA specificity for one ligand over the other changes as a function of temperature. The thermodynamic basis for this observation is a larger penalty in entropy (disorder) for binding of MG as compared to TMR. MG, the ligand for which the aptamer was originally selected, consistently has the more favorable enthalpy (heat) as well as a larger heat capacity (the dependence of enthalpy on temperature). Heat capacities for ligand interactions with MGA are double the magnitude for MG, indicating that binding of this ligand requires more conformational rearrangement of the RNA and ligand. This observation explains why the binding stoichiometry for the MGA and MG interaction is only 1: 1 when temperatures are 55 o. C or above. We hypothesize that there is a heterogeneous population of MGA conformations and MG can only bind to a portion of them at lower temperatures. The intramolecular contacts of these diverse RNA conformations have to be weakened before the ligand can bind. In this RNA system, specificity in ligand binding is due to restriction of the RNA conformation from greater entropy loss. TMR MGA DH(T) = DH(T*) + DCp(T-T*) + DDp(T-T*)2 DCp (MG) = -0. 99 kcal mol-1 K-1 DCp (TMR) = -0. 40 kcal mol-1 K-1