Impact of the Third-Strand Orientation on the Thermodynamic Stability of the Four-Way DNA Junction

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• 作者：Makube ; N. ; Klump ; H. H.
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：2001
• 出版时间：September 1, 2001
• 年：2001
• 卷：393
• 期：1
• 页码：1-13
• 全文大小：205 K

文摘

The physical properties of a triple-helical DNA four-way junction J_T2T4 have been characterized by means of UV spectroscopy, CD spectroscopy, and differential 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 double-helical four-way junctions and finally the binding of third strands to their respective W–C duplexes. CD measurements confirm triplex formation under appropriate pH and ionic strength conditions. The CD spectra also suggest different melting patterns for the triple-helical arms of J_T2T4. The melting temperature as a function of pH or ionic strength characterizes the effect of the third strands on the structural stability. Increased sodium concentration and low pH conditions enhances and stabilizes the overall structure of the junction. The results also indicate that all triplexes in J_T2T4 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 assumed in the presence of salt. Through the deconvolution of DSC data, the calorimetric enthalpies associated with melting of arms of the junctions were 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 in J_T2T4 without changing any of the sequences. Overall, J_T2T4 is slightly more stable than J_T1T3. The differences can be attributed to sequence effects rather than structural effects. All the results illustrate that binding of the third strand in either of the two orientations 5′5′3′ (J_T2T4) or 5′3′3′ (J_T1T3) stabilizes the underlying double-helical four-way junction and its triple-helical arms.