A Four-Way Junction with Triple-Helical Arms: Design, Characterization, and Stability

Abstract

The formation of the four-way junction containing four triple-helical arms has been demonstrated using chemical methods (polyacrylamide gel electrophoresis and chemical footprinting using OsO4 as a probe) and physical methods (UV absorbance melting and DSC). The junction JT1T3 was assembled from two 20-mer purine strands and two 44-mer pyrimidine strands. To determine the contribution of the different arms to the stability of the complete structure of JT1T3, the junction was compared to two simplified substructures, JT1 and JT3, respectively. Common to these complexes is the underlying double-helical four-way junction Js. Addition of Na+ had a profound effect on stabilizing and subsequently folding the junctions into the stacked X-structures. The following results support the structure present: (i) The native polyacrylamide electrophoresis exhibits only a single band(s) corresponding to one species present when all four single strands are mixed in equal amounts. (ii) OsO4 modifications were investigated at pH 5.0 and in the presence of 10 mM Mg2+ and 100 mM Na+. There is no cleavage of thymine residues at the branch point and throughout the structure. (iii) The thermal unfolding of JT1 and JT3 illustrates that the triple-helical arms are more stable than the double-helical arms which are contained in these junctions and that JT1T3 with four triple-helical arms is slightly more stable than JT1 and JT3. (iv) The calorimetric transition enthalpies determined for the arms of JT1T3 are comparable to those associated with the unfolding of its corresponding arms in JT1 and JT3. The results also illustrate that the formation of the junctions is not restricted by the pH, [Na+], sequence composition of the arms, and/or the loop position.