The G-quadruplex architecture is a peculiar structure adopted by guanine-rich oligonucleotidic sequences, and, in particular, by several aptamers, including the thrombin-binding aptamer (TBA) that has the highest inhibitory activity against human α-thrombin. A crucial role in determining structure, stability and biological properties of G-quadruplexes is played by ions. In the case of TBA, K+ ions cause an enhancement of the aptamer clotting inhibitory activity. A detailed picture of the interactions of TBA with the protein and with the ions is still lacking, despite the importance of this aptamer in biomedical field for detection and inhibition of α-thrombin. Here, we fill this gap by presenting a high-resolution crystallographic structural characterization of the thrombin–TBA complex formed in the presence of Na+ or K+ and a circular dichroism study of the structural stability of the aptamer both free and complexed with α-thrombin, in the presence of the two ionic species. The results indicate that the different effects exerted by Na+ and K+ on the inhibitory activity of TBA are related to a subtle perturbation of a few key interactions at the protein–aptamer interface. The present data, in combination with those previously obtained on the complex between α-thrombin and a modified aptamer, may allow the design of new TBA variants with a pharmacological performance enhancement.

No abstract

Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human α-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5′-5′ polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin–mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin–aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs.

The reaction between cisplatin and human serum albumin (HSA) was investigated by X-ray crystallography and crystal structures of the cisplatin/HSA adduct were eventually solved for the first time. Structural data unambiguously prove that cisplatin mainly binds to His105 and Met329 side chains; additional binding sites are detected at His288, Met298, and Met548 and at His535, His67 and His247.