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, NH, Ba, and Sr; on the contrary, Mn 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 NH coordination yielded a typical antiparallel G-quadruplex, no inhibitory activity was shown; a similar effect was observed for Ba and Sr coordination. Mn 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.

Novel isoguanine derivative of unlocked nucleic acid—Investigations of thermodynamics and biological potential of modified thrombin binding aptamer

“…In this case, however, it seems the thrombin acts as a molecular chaperone by stabilizing G-quadruplex structure and consequently an anticoagulant activity of TBA is retained [ 36 , 37 ]. Importantly, the formation of thermodynamically stable G-quadruplex structure of TBA with disrupted loop geometry could be detrimental for TBA anticoagulant properties [ 38 ]. Taken together, the literature data indicate that thermodynamic stability is not the only factor that determines inhibitory activity of TBA.…”

Mechanically rigid supramolecular assemblies formed from an Fmoc-guanine conjugated peptide nucleic acid

“…The G-quartets further stack to produce a G-quadruplex structure, generally stabilized by metal cations, e.g., K + , Na + , Ba 2+ , or Sr 2+ , inside the core, though exceptions have been reported 53,54 . However, exceptions to this traditional structural pattern have been observed in many natural/synthetic G-quadruplexes 39,[55][56][57][58] . For example, Krauss et al reported the crystal structure of a thrombin-bound DNA aptamer, in which the orientation of guanine bases broke the Hoogsteen hydrogen-bonded cyclic pattern, but a G-quartet structure was still produced 55 .…”

G-quadruplex-based aptamers against protein targets in therapy and diagnostics

“…NU172. Discovered through SELEX and subsequently truncated to 26 nucleotides, NU172 (ARCA Biopharma) is a DNA aptamer targeting thrombin exosite I [127], able to fold into a G4 structure [128]. In preclinical studies, NU172 has shown the ability to prolong thrombin clotting time.…”