Thermodynamic and kinetic studies of DNA triplex formation of an oligohomopyrimidine and a matched duplex by filter binding assay.
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.
Shindo
H
H
Tokyo College of Pharmacy, Japan.
Torigoe
H
H
Sarai
A
A
D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D003101:Collodion; D004247:DNA / Q000737:chemistry*; D005374:Filtration / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011742:Pyrimidine Nucleotides / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics
D011742:Pyrimidine Nucleotides; D003101:Collodion; D004247:DNA