Journal of Biomolecular Structure & Dynamics 2003, 21, 267 - 278
J. Jaumot, A. Aviρσ, R. Eritja, R. Tauler, R. Gargallo
Abstract
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.
Keywords
triplex forming oligonucleotides, Multivariate Curve Resolution, factor analysis, protonation studies, melting studies
Acid-base titration of the mixture of h26 and s11CT. (a) Experimental absorbance data, D. (b) Concentration profiles C calculated by MCR. 1: mixture of protonated h26 and protonated s11CT. 2: mixture of several species containing the triplex as majority species. 3: mixture of deprotonated h26 and deprotonated s11CT. (c) Concentration profiles Ctriplex calculated by MCR according to the simultaneous analysis. (d) Pure spectra ST, (a): triplex, (b) protonated s11CT, (c) deprotonated s11CT, (d) protonated h26, (e) deprotonated h26.