DIRECT MEASUREMENT OF THERMODYNAMIC AND KINETIC-PARAMETERS OF DNA TRIPLE-HELIX FORMATION BY FLUORESCENCE SPECTROSCOPY

Direct measurement of thermodynamic and kinetic parameters of oligonuc leotide-directed DNA triple helix formation has been achieved by fluor escence spectroscopic methods. Fluorescence resonance energy transfer (FRET) was used to study the binding of an acceptor-labeled single-str anded oligonucleotide to a donor-labeled DNA duplex. Equilibrium bindi ng constants and association rate constants for tripler formation betw een 5'-tetramethylrhodamine-labeled 11-mer, 13-mer, and 15-mer homopyr imidine oligonucleotides and a 5'-fluorescein-labeled, 25-bp DNA duple x containing a 15-bp homopurine site were determined by FRET measureme nts, and the values were in close agreement with those determined by e stablished methods. The thermal dissociation profile of the tripler-to -duplex transition was also directly observed by FRET and was consiste nt with the tripler melting curves obtained by UV absorbance measureme nts. In addition, a homogeneous fluorescence anisotropy assay is descr ibed which enables determination of the binding constants between 5'-t etramethylrhodamine-labeled Il-mer and 13-mer homopyrimidine oligonucl eotides and unlabeled 25-, 30-, and 50-bp double-stranded DNA containi ng a homopurine target site. These results demonstrate the utility of nonradioactive fluorescence measurements as an efficient method for st udying triple helix formation under homogeneous solution conditions an d highlight the uniqueness of the FRET method for obtaining equilibriu m, kinetic, and thermal dissociation data ina straightforward manner.