pH and cation effects on the properties of parallel pyrimidine motif DNA triplexes

The effects of cytosine protonation and various cations on the properties o f parallel pyrimidine motif DNA triplexes were intensively investigated and characterized by several different techniques, such as circular dichroism (CD) conformation, ultraviolet (UV) melting, differential scanning calorime try (DSC) thermal denaturation, and surface plasmon resonance (SPR) real-ti me dynamics. The comparative CD spectra of the triplex and the correspondin g homoduplexes showed that the negative peak at similar to 218 nm would be the eigenpeak of the Hoogsteen paired strand, and moreover, the formation p athway of a triplex was significantly pH-dependent and fell into three grou ps: under acidic conditions, the triplex is formed by a one-step docking, u nder near physiological conditions, the Watson-Crick duplex is first struct ured and then accepts the Hoogsteen third strand into its major groove, and under basic conditions, the triplex is not formed. The pH-dependent thermo dynamics of the global triplex, the Watson-Crick antiparallel duplex, and t he Crick-Hoogsteen parallel duplex were comparatively discussed for the fir st time. These data revealed that the thermodynamic stabilities of the Wats on-Crick-Hoogsteen triplex and the Crick-Hoogsteen duplex would be strongly dependent on cytosine protonation, but a low-pH environment somewhat desta bilized the Watson-Crick duplex. The binding energy of triplex formation wo uld be different from the unfolding energy of triplex melting under acidic conditions due to the disparity in the pathway between the formation and un folding of a triplex. Real-time dynamic measurements showed that the associ ation and dissociation rate constants of a duplex-to-triplex formation are (1.98 +/- 0.24) x 10(3) M-1 s(-1) and (4.09 +/- 0.96) x 10(-4) s(-1) at 20 degreesC and pH 6.0, respectively. The formation energy of the duplex-to-tr iplex transition derived from SPR measurements was in agreement with the un folding energy of the free Hoogsteen paired duplex derived from UV measurem ents. The calorimetric enthalpies of the triplex-to-duplex-to-single transi tion were 39.3 and 75.3 kcal/mol under near physiological conditions (pH 7. 0), respectively, which were underestimated relative to the van't Hoff enth alpies. In addition, the effects of various cations, ionic strength, mixed- valent cations, and the position of the C(+)xG.C triplets on the thermodyna mics of the triplexes were addressed under near physiological conditions. T he interaction of metal ions with the triplexes clearly depended on the typ e and ionic strength of the cations, and the efficiency with which the cati ons stabilized the global triplex was in the order Mg2+ > Mn2+ > Ca2+ > Ba2 + much greater than Na+. These observations would be useful for the design of triplex-forming oligonucleotides for antigene drugs and therapeutic purp oses.