Ms. Yang et al., DIRECT MEASUREMENT OF THERMODYNAMIC AND KINETIC-PARAMETERS OF DNA TRIPLE-HELIX FORMATION BY FLUORESCENCE SPECTROSCOPY, Biochemistry, 33(51), 1994, pp. 15329-15337
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.