Solution structure of a thrombin binding aptamer complex with a non-planar platinum(ii) compound

Abstract

Thrombin Binding Aptamer (TBA) is a monomolecular well-defined two G-tetrad antiparallel G-quadruplex DNA that inhibits the activity of human α-thrombin. In this report, we synthesized a quasi-cross-shaped platinum(ii) compound (L'₂LPt) with one cyclometalated and two carbene ligands. We found L'₂LPt has selective affinity to bind the TBA G-quadruplex. A fibrinogen clotting assay revealed that L'₂LPt can abrogate the inhibitory activity of TBA against thrombin. We solved the 1 : 1 L'₂LPt-TBA complex structure by NMR, which revealed a unique self-adaptive property of L'₂LPt upon binding to TBA. In the complex, a carbene ligand of L'₂LPt rotates to pair with the cyclometalated ligand to form a plane stacking over half of the TBA G-tetrad and covered by lateral TT loops. It is notable that the heavy atom Pt stays out of the G-tetrad. Meanwhile, the other carbene ligand remains relatively perpendicular and forms a hydrogen bond with a guanine to anchor the L'₂LPt position. This structure exhibits a quasi-cross-shaped Pt(ii) compound bound to the G-quadruplex with an unusual "wall-mounted" binding mode. Our structures provide insights into the specific recognition of antiparallel G-quadruplex DNA by a self-adaptive Pt(ii) compound and useful information for the design of selective G-quadruplex targeting non-planar molecules.

Fig. 1. (A) Schematic diagram of the chemical structure of L′₂LPt. (B) The folding topology of TBA G4. Deep red box = (anti)-guanine, light red box = (syn)-adenine, blue ball = thymine.

Fig. 2. (A) ΔT_m value from FRET and CD melting of different G4s and duplex DNA. FRET data were tested in 0.40 μM DNA interacting with 0.40 μM L′₂LPt in 10 mM tris–HCl buffer (pH 7.4) containing 60 mM potassium cacodylate. CD melting data were tested in 3 μM DNA interacting with 3 μM L′₂LPt in 10 mM tris–HCl and 100 mM KCl buffer (pH 7.4). (B) CD spectra of L′₂LPt titrated into TBA G4 DNA solution at different L′₂LPt/TBA ratios. Conditions: pH 7.4, 10 mM tris–HCl, 100 mM KCl solution. (C) Top: ESI-MS titrations of 10 μm TBA with L′₂LPt in 150 mM ammonium acetate (CH₃CO₂NH₄) buffer (pH 7.4) showing a zoom-in of the 4⁻ charged state. Bottom: Quantification and fitting of the ESI-MS data based on the number of bound ligands (0 L′₂LPt and 1 L′₂LPt).

Fig. 3. ITC data and fitting curves of different titrations of L′₂LPt, TBA and thrombin at a cell concentration of 6 μM interacting with a 100 μM syringe in PBS buffer (pH 7.4) at 25 °C.

Fig. 4. (A) Imino proton regions of the 1D ¹H NMR titration spectra of TBA interacting with L′₂LPt. The imino proton assignments are labeled. (B) The H1′–H6/H8 region of the 2D NOESY spectrum of the TBA G4 bond to 1.0 equivalents of L′₂LPt. The complete sequential assignment pathway is shown. Residues with syn glycosidic conformations are marked in black, and those with anti glycosidic conformations are marked in blue. (C) The chemical shift difference of TBA G4 protons between the free TBA and the 1 : 1 L′₂LPt–TBA complex. Conditions: 25 mM K-phosphate, 70 mM KCl solution, pH 7.0, 25 °C.

Fig. 5. (A) The ensemble of the superimposed 10 NMR restraint-refined structures of the 1 : 1 L′₂LPt–TBA complex in the stereo view (PDB ID 7V3T). (B) A representative structure of the 1 : 1 complex shown in stereo view. (C–E) Different views of the L′₂LPt-induced binding pockets. (F) Potential hydrogen bonding between the L′₂LPt carbene ligand L′-2 and G11 of TBA G4. The distance between the N atom on the pyridine of the carbene ligand L′-2 and the H atom on the NH₂ of the G11 base is 2.0 Å.

Fig. 6. (A) Overlapping structures of free TBA G4 (PDB ID 148D) and 1 : 1 L′₂LPt–TBA G4 complex (PDB ID 7V3T) showing the structural changes of TBA G4. (B) Overlapping structures of free and bound L′₂LPt showing structural changes upon TBA G4 binding.

Fig. 7. Fibrinogen clotting time of thrombin alone (1.0 ml of PBS containing 2.0 mg ml⁻¹ of fibrinogen added to 100 μl/10 NIH per ml of human thrombin) or in the presence of 0, 200, 500 and 1000 nM concentrations of TBA and different equivalents of L′₂LPt. TMPyP₄ and PDS were used as positive control. The bars of the control and vehicle represent the fibrinogen clotting time values of the system in the absence of any L′₂LPt/TBA and diluted with the buffer alone, respectively.