Use of fluorescein-labeled oligonucleotide for analysis of formation and dissociation kinetics of T:A:T triple-stranded DNA: effect of divalent cations

Abstract

The formation and dissociation kinetics of homonucleotide oligo(dT):oligo(dA):oligo(dT) triplex have been analyzed by fluorescence measurements of fluorescein-labeled oligo(dT) providing considerably higher sensitivity to monitoring reaction kinetics than traditional hypochromicity and circular dichroism. The triplex is concluded to be formed by a bimolecular process corresponding to the addition of the oligo(dT) strand to a preserved oligo(dT):oligo(dA) duplex. The association rate was found to be faster the higher the divalent cation concentration and depends upon the nature of divalent ions in the following order of efficiency: Mn2+ > Mg2+ > Ni2+, Ca2+ > Ba2+. The more efficient metal ions for the triplex formation were found also more efficient for the stabilization. The dissociation kinetics of the third Hoogsteen-bound strand was monitored at below melting temperature by chasing the labeled dT strand from the triplex by excess of non-labeled oligonucleotide. The dissociation rate was found to be almost independent of concentration and nature of cation. The thermodynamic stabilization of triplex by cations is thus a consequence of the increased formation rate.