Thermodynamics of triple helix formation: Spectrophotometric studies on the d(A)₁₀·2d(T)₁₀ and d(C⁺₃T₃C⁺₃)·d(G₃A₄G₃)·d(C₃T₄C₃) triple helices

We have stabilized the d(A)₁₀·2d(T)₁₀ and d(C⁺₃T₄C⁺₃)·d(G₃A₄G₃)·d(C₃T₄C₃) triple helices with either NaCl or MgCl₂ at pH 5.5. UV mixing curves demonstrate a 1:2 stoichtometry of purine to pyrimidine strands under the appropriate conditions of pH and ionic strength. Circular dichroic tltrations suggest a possible sequence-independent spectral signature for triplex formation. Thermal denaturation profiles indicate the initial loss of the third strand followed by dissociation of the underlying duplex with increasing temperature. Depending on the base sequence and ionic conditions, the binding affinity of the third strand for the duplex at 25°C is two to five orders of magnitude lower than that of the two strands forming the duplex. Thermodynamic parameters for triplex formation were determined for both sequences in the presence of 50 mM MgCl₂ and/or 2.0 M NaCl. Hoogsteen base pairs are 0.22 - 0.64 kcal/mole less stable than Watson-Crick base pairs, depending on ionic conditions and base composition. C⁺·G and T·A Hoogsteen base pairs appear to have similar stability in the presence of Mg²⁺ ions at low pH.