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, T_M, of the lower transition depends on strand concentration, while the T_M 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.