Structural characterization of the complex between alpha-thrombin and an exosite II specific aptamer

Aptamers are single-stranded DNA or RNA sequences able to recognize a wide variety of natural and synthetic targets including amino acids, peptides, proteins and even cells with high affinity and specificity [1]. They are considered a sort of nucleic acid version of antibodies with improved properties. Aptamers display a large number of structural arrangements, in particular the interesting G-quadruplex architecture that is adopted by many of them. Several cases have been also reported where aptamers are supposed to adopt a mixed duplex/quadruplex structure [1,2]. Interestingly, in every case, the presence of both structural motifs is needed for strong and specific binding of the target. Also potent second-generation thrombin aptamers adopt a duplex/quadruplex bimodular folding [2]. They have been proved to recognize thrombin exosite II [2], whereas best studied thrombin binding aptamers bind exosite I [3,4], and show very high affinity and specificity towards thrombin [2]. A sound model of these oligonucleotides, either free or in complex with thrombin, is not yet available. Here we present a structural study of one of these aptamers, HD22-27mer. Interestingly, the crystal structure of the aptamer in complex with thrombin displays a novel architecture, in which a double helical stem is enchained to a pseudo-G-quadruplex. Our results also underline the role of residues that join the duplex and the quadruplex motifs and control their recruitment in thrombin binding. Finally, our crystallographic model shed light on the contrasting circular dichroism studies reported in the literature [5], suggesting a major role of ions in determining the aptamer properties. [1] B. Gatto, M. Palumbo, C. Sissi Current Medicinal Chemistry, 2009, 16, 1248-1265; [2] D.M. Tasset, M.F. Kubik, W. Steiner Journal of Molecular Biology, 1997, 272(5), 688-698;. [3] I. Russo Krauss, A. Merlino, C. Giancola, A. Randazzo, L. Mazzarella, F. Sica Nucleic Acids Res., 2011, 39(17), 7858–7867 [4] I. Russo Krauss, A. Merlino, A. Randazzo, E. Novellino, L. Mazzarella, F. Sica Nucleic Acids Res., 2012, 40(16), 8119-28 [5] G. Marson, M. Palumbo, C. Sissi Curr Pharm Des., 2012, 18(14), 2027-2035