Use of fluorescein-labeled oligonucleotide for analysis of formation and dissociation kinetics of T:A:T triple-stranded DNA: effect of divalent cations.
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.
Ellouze
C
C
Groupe d'Etude de Mutagénèse et Cancérogénèse, UMR 216, Institut Curie & CNRS, Orsay, France.
Piot
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F
Takahashi
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M
D002413:Cations, Divalent; D004247:DNA / Q000737:chemistry*; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry*; D005454:Fluorescence Polarization; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry*
D002413:Cations, Divalent; D005452:Fluoresceins; D009841:Oligonucleotides; C051802:triplex DNA; D004247:DNA; D019793:Fluorescein