A four-way junction with triple-helical arms: Design, characterization, and stability

The formation of the four-way junction containing four triple-helical arms has been demonstrated using chemical methods (polyacrylamide gel electropho resis and chemical footprinting using OsO4 as a probe) and physical methods (UV absorbance melting and DSC). The junction J(T1T3) was assembled from t wo 20-mer purine strands and two 44-mer pyrimidine strands. To determine th e contribution of the different arms to the stability of the complete struc ture of J(T1T3), the junction was compared to two simplified substructures, J(T1) and J(T3), 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-s tructures. The following results support the structure present: (i) The nat ive polyacrylamide electrophoresis exhibits only a single band(s) correspon ding 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 pr esence of 10 mM Mg2+ and 100 mM Na+. There is no cleavage of thymine residu es at the branch point and throughout the structure. (iii) The thermal unfo lding of J(T1) and J(T3) illustrates that the triple-helical arms are more stable than the double-helical arms which are contained in these junctions and that J(T1T3) With four triple-helical arms is slightly more stable than J(T1) and J(T3). (iv) The calorimetric transition enthalpies determined fo r the arms of J(T1T3) are comparable to those associated with the unfolding of its corresponding arms in J(T1) and J(T3) The results also illustrate t hat the formation of the junctions is not restricted by the pH, [Na+], sequ ence composition of the arms, and/or the loop position. (C) 2000 Academic P ress.