Ethidium Probing of the Parallel Double- and Four-stranded Structures Formed by the Telomeric DNA Sequences dG(GT)₄G and d(GT)₅

Abstract

Oligonucleotides 3′-d(GT)₅-(CH₂CH₂O)₃-d(GT)₅-3′ (parGT), containing GT repeats present in the telomeric DNA from Saccharomyces cerevisiae, had been demonstrated to form bimolecular structure, GT-quadruplex (qGT) [O. F. Borisova et al. FEBS Letters 306, 140–142 (1992)]. Four d(GT)₅ strands of the GT-quadruplex are parallel and form five G-quartets while thymines are bulged out. The four GT repeats when flanked by guanines, 3′-dG(TG)₄G-(CH₂CH₂O)₃- dG(GT)₄G-3′ (hp-GT), had been shown to form a novel parallel-stranded (ps) double helix with G·G and T·T base pairs (hp-GT ps-DNA) [A. K. Shchyolkina et al. J. Biomol. Struct. Dynam. 18, 493–503 (2001)]. In the present study the intercalator ethidium bromide (Et) was used for probing the two structures. The mode of Et binding and its effect on thermostability of qGT and hp-GT were compared. The quantum yield (q) and the fluorescence lifetime (τ) of Et:qGT (q = 0.15 ±0.01 and τ = 24 ±1 ns) and Et:hp-GT (q = 0.10 ± 0.01 and τ = 16.5 ± 1 ns) indicative of intercalation mode of Et binding were determined. Et binding to qGT was found to be cooperative with corresponding coefficient ω = 3.9 ± 0.1 and the binding constant K= (6.4 ± 0.1)·10M⁻¹. The maximum number of Et molecules intercalating into GT-quadruplex is as high as twice the number of inerspaces between G-quartets (eight in our case). The data conform to the model of Et association with GT-quadruplex suggested earlier [O. F. Borisova et al. Mol. Biol. (Russ) 35, 732–739 (2001)]. The anticooperative type of Et binding was observed in case of hp- GT ps-DNA, with the maximum number of bound Et molecules, N = 4 ÷ 5, and the association constant K = (1.5 ± 0.1)·10⁵ M⁻¹. Thermodynamic parameters of formation of Et:qGT and EtBr:hp-GT complexes were calculated from UV thermal denaturation profiles.