title: Cation Coordination Alters the Conformation of a Thrombin-Binding G-Quadruplex DNA Aptamer That Affects Inhibition of Thrombin.
abstract: Thrombin-binding aptamers are promising anticoagulants. HD1 is a monomolecular antiparallel G-quadruplex with two G-quartets linked by three loops. Aptamer-thrombin interactions are mediated with two TT-loops that bind thrombin exosite I. Several cations were shown to be coordinated inside the G-quadruplex, including K+, Na+, NH4+, Ba2+, and Sr2+; on the contrary, Mn2+was coordinated in the grooves, outside the G-quadruplex. K+or Na+coordination provides aptamer functional activity. The effect of other cations on aptamer functional activity has not yet been described, because of a lack of relevant tests. Interactions between aptamer HD1 and a series of cations were studied. A previously developed enzymatic method was applied to evaluate aptamer inhibitory activity. The structure-function correlation was studied using the characterization of G-quadruplex conformation by circular dichroism spectroscopy. K+coordination provided the well-known high inhibitory activity of the aptamer, whereas Na+coordination supported low activity. Although NH4+coordination yielded a typical antiparallel G-quadruplex, no inhibitory activity was shown; a similar effect was observed for Ba2+and Sr2+coordination. Mn2+coordination destabilized the G-quadruplex that drastically diminished aptamer inhibitory activity. Therefore, G-quadruplex existence per se is insufficient for aptamer inhibitory activity. To elicit the nature of these effects, we thoroughly analyzed nuclear magnetic resonance (NMR) and X-ray data on the structure of the HD1 G-quadruplex with various cations. The most reasonable explanation is that cation coordination changes the conformation of TT-loops, affecting thrombin binding and inhibition. HD1 counterparts, aptamers 31-TBA and NU172, behaved similarly with some distinctions. In 31-TBA, an additional duplex module stabilized antiparallel G-quadruplex conformation at high concentrations of divalent cations; whereas in NU172, a different sequence of loops in the G-quadruplex module provided an equilibrium of antiparallel and parallel G-quadruplexes that shifted with cation binding. In conclusion, structures of G-quadruplex aptamers are flexible enough and are fine-tuned with different cation coordination.
authors.affiliation: 1 Chemistry Department, Lomonosov Moscow State University , Moscow, Russia .
authors.forename: Elena
authors.initials: E
authors.lastname: Zavyalova
authors.affiliation: 1 Chemistry Department, Lomonosov Moscow State University , Moscow, Russia .
authors.forename: Grigory
authors.initials: G
authors.lastname: Tagiltsev
authors.affiliation: 2 Belozersky Institute of Physical-Chemical Biology, Lomonosov Moscow State University , Moscow, Russia .
authors.forename: Roman
authors.initials: R
authors.lastname: Reshetnikov
authors.affiliation: 2 Belozersky Institute of Physical-Chemical Biology, Lomonosov Moscow State University , Moscow, Russia .
authors.forename: Alexander
authors.initials: A
authors.lastname: Arutyunyan
authors.affiliation: 1 Chemistry Department, Lomonosov Moscow State University , Moscow, Russia .
authors.forename: Alexey
authors.initials: A
authors.lastname: Kopylov
mesh_terms: D000641:Ammonia / Q000737:chemistry; D000925:Anticoagulants / Q000737:chemistry*; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001464:Barium / Q000737:chemistry; D001665:Binding Sites; D002413:Cations, Divalent; D002414:Cations, Monovalent; D056831:Coordination Complexes / Q000737:chemistry*; D054856:G-Quadruplexes; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D012964:Sodium / Q000737:chemistry; D013324:Strontium / Q000737:chemistry; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D000925:Anticoagulants; D052157:Aptamers, Nucleotide; D002413:Cations, Divalent; D002414:Cations, Monovalent; D056831:Coordination Complexes; C081676:thrombin aptamer; D001464:Barium; D008345:Manganese; D000641:Ammonia; D012964:Sodium; D013917:Thrombin; D011188:Potassium; D013324:Strontium
keywords: aptamer; cation coordination; circular dichroism; thrombin