Impact of the third-strand orientation on the thermodynamic stability of the four-way DNA junction

The physical properties of a triple-helical DNA four-way junction J(T2T4) h ave been characterized by means of UV spectroscopy, CD spectroscopy, and di fferential scanning calorimetry (DSC). J(T2T4) is another four-way junction that was designed in addition to J(T1T3) (N. Makube and H. H. Klump (2000) Arch. Biochem. Biophys. 377, 31-42) to study the effects of third strands on the stability of the four-way junction with triple-helical arms. The pH titration curves illustrate the sequential folding of single strands to dou ble-helical four-way junctions and finally the binding of third strands to their respective W-C duplexes. CD measurements confirm triplex formation un der appropriate pH and ionic strength conditions. The CD spectra also sugge st different melting patterns for the triple-helical arms of J(T2T4). The m elting temperature as a function of pH or ionic strength characterizes the effect of the third strands on the structural stability. Increased sodium c oncentration and low pH conditions enhances and stabilizes the overall stru cture of the junction. The results also indicate that all triplexes in J(T2 T4) are formed in the absence of salt and at low pH; however, the junction may, under these conditions, assume a conformation different from the one a ssumed in the presence of salt. Through the deconvolution of DSC data, the calorimetric enthalpies associated with melting of arms of the junctions we re determined. The loops are designed to have the same enthalpic effect on the different arms. The stabilizing effect of the loops is more pronounced when those loops are shifted from arms 1 and 3 in J(T1T3) to arms 2 and 4 i n J(T2T4) without changing any of the sequences. Overall, J(T2T4) is slight ly more stable than J(T1T3). The differences can be attributed to sequence effects rather than structural effects. All the results illustrate that bin ding of the third strand in either of the two orientations 5 '5 '3 ' (J(T2T 4)) or 5 '3 '3 ' (J(T1T3)) stabilizes the underlying double-helical four-wa y junction and its triple-helical arms. (C) 2001 Academic Press.