Thermodynamic and kinetic studies of DNA triplex formation of an oligohomopyrimidine and a matched duplex by filter binding assay

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Abstract
The filter binding method was found to be a powerful method for studying the formation of triplexes composed of a single-stranded homopyrimidine and a duplex with a matched purine-pyrimidine tract. With this technique, we were able to determine thermodynamic and kinetic parameters for triplex formation between a homopyrimidine 19-mer (5'-TCCTCTTCTTTTCTTTCTT-3') and a duplex with sequence 5'-GCAGGAGAAGAAAAGAAAGAACG-3' for the purine strand. The experiments were performed over a wide pH range (3.8-7.4) and a temperature range of 0-35 degrees C. pH and temperature dependencies of the thermodynamic parameters were best explained in terms of a three-state model for triplex formation at low temperatures relative to the melting point. The main results were as follows: (1) pH dependence of the dissociation constants of the triplex is a result of the rapid acid-base equilibrium of pyrimidine single strands; (2) the association rate for triplex formation decreases with increasing pH in accordance with the dissociation constants; (3) the dissociation constant is virtually temperature-independent at low pH, while it becomes strongly temperature-dependent with increasing pH (these results can be explained in terms of a negative, non-zero delta Cp for triplex formation at low pH); (4) the association rate decreases with increasing temperature, and the resulting negative activation energy indicates that the triplex formation process involves a quasi-stable intermediate; (5) the triplex formation is a second-order reaction at low pH, whereas it can be interpreted as a third-order reaction at neutral pH, suggesting that different triplex formation pathways are observed depending on the pH.