Extraction of information on the buildup and consumption of reactive intermediates from quadruplex DNA assembly time courses.

A method was developed to detect the time course of the overall presence of intermediate species during K+-induced DNA quadruplex assembly from single-stranded d(TG4) oligonucleotides in experiments in which only the combined circular dichroisms (CD) of all species present could be measured directly. The presence of intermediate species is determined unambiguously but quantitative estimates can be made only to the extent that the CD characteristics of all intermediates are known. The method consists of (i) obtaining CD spectra of known concentrations of initial and final species to determine their molar ellipticity coefficients, (ii) carrying out CD measurements of the kinetics of quadruplex assembly reactions at two different wavelengths, chosen to give optimal differentiation between the initial and final species, and (iii) using the results of (ii) to detect discrepancies between the rates of consumption of single strands and the generation of quadruplex to infer the presence of intermediate species. The analysis was facilitated by the validation and use of biphasic exponential expressions obtained from the SAS nonlinear curve fitting procedure NLIN in place of the raw CD data. The general method is described, then applied to data from [d(TG4)4.(K+)3] quadruplex assembly experiments.
(Copyright 2004 Elsevier B.V.)