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

摘要

Oligonucleotides 3'-d(GT)(5)-(CH(2)CH(2)O)(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)(4)G-(CH(2)CH(2)O)(3)-dG(GT)(4)G-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. Dyn. 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 (tau) of Et:qGT (q = 0.15 +/- 0.01 and tau = 24 +/- 1 ns) and Et:hp-GT (q = 0.10 +/- 0.01 and tau = 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 omega = 3.9 +/- 0.1 and the binding constant Kappa = (6.4 +/- 0.1).10(4) M(-1). The maximum number of Et molecules intercalating into GT-quadruplex is as high as twice the number of innerspaces 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 Kappa = (1.5 +/- 0.1).10(5) M(-1). Thermodynamic parameters of formation of Et:qGT and EtBr:hp-GT complexes were calculated from UV thermal denaturation profiles.