Energetic contributions for the formation of TAT/TAT, TAT/CGC(+), and CGC(+)/CGC(+) base triplet stacks.

ABSTRACT

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(A(7)C(5)T(7)C(5)T(7)), d(A(6)C(5)T(6)C(5)T(6)), d(A(6)C(5)T(6)), d(AGAGAGAC(5)TCTCTCTC(5)TCTCTCT), d(AGAGAC(5)TCTCTC(5)TCTCT), d(AGAGAC(5)TCTCTC(2)), d(AAGGAC(5)TCCTTC(5)TTCCT), d(AGGAAC(5)TTCCTC(5)TCCTT), and d(GAAAGC(5)CTTTCC(5)CTTTC). 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.