Targeting DNA Hairpin Loops with their Partially Complementary Strands

Abstract

Antisense, antigene and siRNA strategies are currently used to control the expression of genes. To this end, our laboratory is currently mimicking the targeting of mRNA by targeting DNA hairpin loops with their partially complementary strands. We use a combination of differential scanning calorimetry (DSC) and temperature-dependent UV spectroscopy to investigate the reaction of a variety of DNA hairpin loops (single end loops, dumbbell, three-way junction with two loops and a hairpin with a bulge of 5 nucleotides) with single strands that are complementary to the bases in the loop and to one strand of their stem. The resulting reaction products form duplexes with dangling ends, nicks or with a displaced strand. We determine standard thermodynamic profiles for the unfolding of the reactants (hairpin loop) and products (duplex) of each reaction. The DSC and UV melting curves show monophasic transitions for the unfolding of all DNA single hairpin loops (reactants) and biphasic transitions for the unfolding of the double hairpin loops (reactants) and duplex products. The resulting unfolding data is then used to create thermodynamic (Hess) cycles that correspond to each targeting reaction. All eight targeting reactions investigated 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 single strand was able to disrupt the hairpin loop structure. Supported by Grant MCB-0616005 from NSF.