A procedure is described for the complete resolution of concentration profiles of oligonucleotide triplexes as a function of pH and temperature. The pH and temperature ranges at which triplexes are present and the relative concentrations of all the species involved in acid-base and conformational equilibria are successfully estimated from Multivariate Curve Resolution analysis of UV absorbance spectra recorded along acid-base titrations and melting experiments of single stranded, hairpin and their mixtures. The dependence of formation constants upon pH was successfully estimated. The hairpin h26 (5?-GAAGGAGGAGA-TTTT-TCTCCTCCTTC-3?), and the single stranded oligonucleotides s11CT (5?-CTTCCTCCTCT-3?), s11AG (5?-AGAGGAGGAAG-3?) and s11TG (5?-TGTGGTGGTTG-3?) were synthesized and their protonation and conformational equilibria were studied in detail. The procedure was shown to be especially useful for the study of triplexes with a low hypochromism upon formation.

Key words: Triplex forming oligonucleotides, Multivariate Curve Resolution, Factor analysis, Protonation studies, Melting studies.