Abstract
Antisense, antigene, and siRNA strategies are currently used to control the expression of genes. To this end, our laboratory is mimicking the targeting of mRNA by reacting DNA stem-loop motifs with their partially complementary strands. Specifically, we used a combination of isothermal titration (ITC), differential scanning calorimetry (DSC), and temperature-dependent UV spectroscopy to investigate: (1) the unfolding of a pseudoknot and a complex containing joined triplex-duplex motifs (shown below); and (2) the reaction of these compact structures with single strands that are complementary to the bases in the loops and to a portion of their stem.
We used DSC to determine the temperature unfolding thermodynamics for the reactants and products of each reaction. The resulting unfolding data is then used to create thermodynamic (Hess) cycles that correspond to each targeting reaction. The resulting enthalpies are compared with the reaction enthalpies, obtained directly from isothermal titration (ITC) experiments. All reactions yielded favorable free energy contributions that were enthalpy-driven. These favorable heat contributions result from the formation of base pair stacks involving the unpaired bases of the loops, indicating that each complementary strand was able to invade and disrupt the secondary structure.
This research is supported by National Science Foundation Grant MCB-1122029 and GAANN grant P200A120231 (C.R.) from the US Department of Education.