G-Quadruplexes from Human Telomeric DNA: How Many Conformations in PEG Containing Solutions? Luigi Petraccone,* ,† Anna Malafronte, † Jussara Amato, ‡ and Concetta Giancola † † Dipartimento di Chimica “P. Corradini”, Via Cintia, Universita ̀ “Federico II” di Napoli, 80126, Naples, Italy ‡ Dipartimento di Chimica delle Sostanze Naturali, Via D. Montesano 49, Universita ̀ “Federico II” di Napoli, 80131, Naples, Italy * S Supporting Information ABSTRACT: G-quadruplex structures are an attractive target for the development of anticancer drugs, as their formation in human telomere induces a DNA damage response followed by apoptosis in cancer cells. However, the development of new anticancer drugs by means of structural- based drug design is hampered by a lack of accurate information on the exact G-quadruplex conformation adopted by the human telomeric DNA under physiological conditions. Several groups reported that, in a molecular crowded, cell-like environment, simulated by polyethylene glycol (PEG), the human telomeric DNA adopts the parallel G-quadruplex conformation. These studies have suggested that 40% (w/v) PEG concentration induces complete structural conversion from the other known human telomeric G-quadruplex conformations to the parallel G-quadruplex, thus simplifying the high structural polymorphism existing in the absence of PEG. In this study, we demonstrate that the structural conversion to the parallel G-quadruplex is not a complete reaction at physiological temperature. We report a complete kinetic and thermodynamic characterization of the conformational transitions involving the (TTAGGG) 4 TT and (TTAGGG) 8 TT human telomeric DNA sequences in K + solution containing PEG. Our data show that the hybrid-type and parallel conformations coexist at equilibrium in the presence of PEG at physiological temperature and the degree of the quadruplex interconversion depends on the PEG molecular weight. Further, we find that telomeric DNA folds in the parallel quadruplex in the seconds time scale, a much larger time scale than the one reported for the hybrid quadruplex folding (∼ms). The whole of our data allow us to predict the relative amount of each G-quadruplex conformation as a function of temperature and time. The effect of other crowding agents like Ficoll 400 and glycerol on the quadruplex interconversion has been also explored. ■ INTRODUCTION Human telomeric DNA contains thousands of tandem repeats of the sequence TTAGGG that terminates at the 3′-end as single-stranded G-rich overhangs of 100-200 nt. 1,2 The human telomeric overhangs can fold into G-quadruplex structures stabilized by consecutive G-tetrads connected by loops. 3 Several cell-based studies have shown that folding of the telomeric G-rich single strand into a quadruplex structure alters the structure of the telomere, inducing a DNA damage response followed by apoptosis in cancer cells. 4,5 For this reason, G-quadruplex structures are currently an attractive target for development of anticancer drugs. 4,6 However, the development of new anticancer drugs by means of structural- based drug design is hampered by the lack of accurate information on the exact G-quadruplex conformation adopted by the human telomeric DNA under physiological conditions. Extensive structural investigations have been carried out by NMR on human telomeric sequences containing four G-tracts in physiological K + solution. 7-11 The majority of the explored sequences form the hybrid-1 or hybrid-2 structures (or a mix of both of them). These two topologies, also named (3 + 1), have three G-tracts oriented in one direction and the fourth in the opposite direction, but they differ in the order of the loop arrangements. 8 The GGG(TTAGGG) 3 sequence, a particular truncation of the human telomeric DNA, has been shown to form an antiparallel basket type form (called Form-3) containing just two G-quartets. 11 The crystal structure of the human telomeric quadruplex in K + differs from the NMR structures and has all four G-tracts parallel and connected by three propeller loops. 12 Although the parallel fold seems not to be the predominant conformation in dilute solution, 13 several groups reported that in molecular crowding conditions, simulated by polyethylene glycol (PEG), the human telomeric DNA adopts the parallel G-quadruplex conformation. 13-17 Since molecular crowding is the typical condition in cells, 18 these results have raised much attention and have been used to suggest that the parallel quadruplex is biologically relevant. However, recently, doubts have been raised on the use of PEG to simulate crowding conditions, 19 as it could interact with macromolecules rather than being an inherent partner. 20 Other authors have suggested that the PEG acts on DNA more as a dehydrating agent rather than as a molecular crowding agent. 21 For these reasons, careful considerations are required when using agents such as PEG to mimic molecular crowding. Besides this, the detailed kinetics and thermodynamics of the Received: September 22, 2011 Revised: January 19, 2012 Published: January 23, 2012 Article pubs.acs.org/JPCB © 2012 American Chemical Society 2294 dx.doi.org/10.1021/jp209170v | J. Phys. Chem. B 2012, 116, 2294-2305