Improving the Function of RNA by Conformational Restriction Philip C. Bevilacqua, Department of Chemistry, Pennsylvania State University, University Park, PA 16802 MGA The malachite green aptamer (MGA) binds the non-cognate dye molecule tetramethylrosamine (TMR) tighter than the cognate ligand that it was selected for, malachite green (MG). We have shown through isothermal titration calorimetry that the thermodynamic basis for this reverse specificity is the larger entropic penalty of binding for the cognate ligand, as MG binding actually has a more negative DH compared to TMR binding. Heat capacities for ligand interactions with MGA are double the magnitude for MG, suggesting that binding of this ligand requires more conformational rearrangement of the RNA and ligand. Our hypothesis is supported by comparison of the structures (PDB 1Q8N, 1F1T) which reveal three syn bases required for MG binding while TMR binding only involves two syn bases. The next phase of our project is to substitute the conformationally restricted nucleotide 8-bromoguanosine at one or more of these positions in order to manipulate the entropy of binding. 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 MG binding at 350C Kd = 250 +/- 10 nM DH = -52.9 +/- 0.6 kcal/mol DS = -141+/- 4 e.u. TMR binding at 350C Kd = 93 +/- 7 nM DH = -21.0 +/- 0.3 kcal/mol DS = -36 +/- 1 e.u. VS