Energetic contributions for the formation of TAT/TAT, TAT/CGC⁺, and CGC^-/CGC⁺ base triplet stacks

We used a combination of spectroscopic and calorimetric techniques to determine complete thermodynamic profiles accompanying the folding of a set of triple helices and control duplexes. Specifically, we studied the sequences: d(A₇C₅T₇C₅T₇), d(A₆C₅T₆C₅T₆), d(A₆C₅T₆), d(AGAGAGAC₅TCTCTCTC₅-TCTCTCT), d(AGAGAC₅TCTCTC₅TCTCT), d(AGAGAC₅TCTCTC₂), d(AAGGAC₅TCCTTC₅TTCCT), d(AGG- AAC₅TTCCTC₅TCCTT), and d(GAAAGC₅CTTTCC₅CTTTC). Circular dichroism spectroscopy indicated that all triplexes and duplexes are in the "B" conformation. DSC melting experiments revealed that the formation of triplexes is accompanied by a favorable free energy change, which arises from the compensation of a large and favorable enthalpic contribution with an unfavorable entropic contribution. Comparison of the thermodynamic profiles of these triplexes yielded enthalpic contributions of -24 kcal/mol, -23 kcal/mol, and -22 kcal/mol for the formation of TAT/TAT, TAT/CGC⁺, and CGC⁺/CGC⁺ base triplet stacks, respectively. UV melts as a function of sodium concentration show sodium ions stabilize the triplexes that contain only TAT triplets but destabilize the triplexes that contain CGC⁺ triplets. UV melts as a function of pH indicate that the protonation of the third strand and loop cytosines stabilizes the triplexes that contain CGC⁺ and TAT triplets, respectively. Our overall results suggest that the triplex to duplex transition of triplexes that contain CGC⁺ triplets is accompanied by a release of protons and an uptake of sodium, while their duplex to random coil transition is accompanied by a release of sodium ions. A consequence of this opposite sodium dependence is that their coupled transitions are nearly independent of sodium concentration but are dependent on the experimental pH.