Improving the Function of RNA by Conformational Restriction

1
Improving the Function of RNA by Conformational
Restriction Philip C. Bevilacqua, Department of
Chemistry, Pennsylvania State University,
University Park, PA 16802
TMR ? MGA
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 11 when temperatures are
55oC 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.
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