Calorimetric unfolding of the bimolecular and i-motif complexes of the human telomere complementary strand, d(C3TA2)4

A combination of spectroscopic and calorimetric techniques is used to determine the unfolding thermodynamics of the complexes formed by the complementary sequence of the human telomere, d(C3TA2)4, in the pH range of 4.2 to 6. Calorimetric melting curves show biphasic transitions; both transitions are shifted to higher temperatures as the pH is decreased, indicative of cytosine protonation, which favors the formation of C•C+ base pairs. Furthermore, the transition temperature, TM, of the lower transition depends on strand concentration, while the TM of the higher transition is independent of strand concentration, indicating the following sequential melting: bimolecular complex(s)→intramolecular complex→random coil. The thermodynamic profiles for the formation of each complex, bimolecular and i-motif reveals small favorable free energy terms resulting from favorable enthalpy-unfavorable entropy compensations, uptake of protons, marginal uptake of counterions (i-motif) and marginal release of water molecules (i-motif). Furthermore, an enthalpy of 3.2 kcal/mol (bimolecular complex) and 5.0 kcal/mol (i-motif) is estimated for a single C•C+/C•C+ base-pair stack.