We analyzed the thermodynamics of purine motif triplex formations involving single mismatches of four different base triplets (A x A:T, T x A:T, A x T:A and T x T:A) by isothermal titration calorimetry. The T x A:T and A x A:T base triplets are the most stable and the A x T:A base triplet is the least stable among the four base triplets. The magnitude of the Gibbs free energy change varies within 1.0 kcal mol(-1) for the single mismatches of the base triplet. On the other hand, the magnitude of the enthalpy change exhibits very large sequence dependence with up to 14 kcal mol(-1) variation per the single mismatches among the four base triplets, which is 14 times larger than the sequence-dependent variation of the magnitude of the Gibbs free energy change. Thus, the single mismatches in the purine motif triplex do not have strong effects on the magnitude of the Gibbs free energy change, but the magnitude of the enthalpy change exhibits much larger sequence-dependent variations. The present results support the previously proposed nucleation-elongation mechanism of the triplex formation and offer some hints to design novel triplex-forming oligonucleotides with enhanced sequence specificity.
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