Conformation and thermodynamics of DNA "Necks": Models for three-arm branch formation in a duplex

The properties of three-arm DNA junctions differ from those of four-arm junctions in several respects. Most apparently, bases flanking the branch are reactive to single strand specific agents in three-arm junctions but not four-arm junctions. To determine the basis for this, we have designed and synthesized a series of complexes in which a short duplex, a neck, progressively extends from a parent 16-mer DNA duplex. These structures allow us to investigate how a three-arm branch forms, and how its properties change as the neck extends. Comparison of the properties of a nicked duplex with those of the neck structures using native gel electrophoresis with reporter arms attached reveals progressively greater geometrical perturbation of the complexes as the number of base-pairs in the neck increases. Footprinting by single-strand specific reagents indicates that the reactivity to single-strand reagents near the branch occurs when only a single pair is possible. The branch in each neck interacts tightly with ethidium, as does a nick in the same duplex. The thermodynamics of neck formation have been evaluated by calorimetry and from the concentration dependence of absorbance temperature profiles. Each neck complex is destabilized with respect to duplex DNA or a nicked duplex, and has a lower enthalpy of formation despite the increased number of base-pairs present. A model is proposed to account for these properties in which the bases in the pair adjacent to the duplex interact directly with the duplex, via transient insertion.