Published online 1 December 2006 Nucleic Acids Research, 2006, Vol. 34, No. 22 6653–6662 doi:10.1093/nar/gkl915 A new modified thrombin binding aptamer containing a 50–50 inversion of polarity site Luigi Martino, Ada Virno 1 , Antonio Randazzo 1 , Antonella Virgilio1, Veronica Esposito1, Concetta Giancola, Mariarosaria Bucci2, Giuseppe Cirino2 and Luciano Mayol1,* Dipartimento di Chimica, Universita` degli Studi di Napoli ‘Federico II’, via Cintia, I-80126, Napoli, Italy, 1Dipartimento di Chimica delle Sostanze Naturali, Universita` degli Studi di Napoli ‘Federico II’, via D. Montesano 49, I-80131 Napoli, Italy and 2Dipartimento di Farmacologia Sperimentale, Universita` degli Studi di Napoli ‘Federico II’, via D. Montesano 49, I-80131 Napoli, Italy Received July 31, 2006; Revised October 16, 2006; Accepted October 17, 2006 ABSTRACT The solution structure of a new modified thrombin binding aptamer (TBA) containing a 50–50 inversion of polarity site, namely d(30GGT50-50TGGTGTGGT- TGG30), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calcula- tions, have been used to characterize the 3D structure. The modified oligonucleotide is charac- terized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d(30GGT50-50TGGTGTGGTTGG30) is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti- anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4 C higher than the corresponding unmodified TBA. d(30GGT50-50TGGTG- TGGTTGG30) continues to display an anticoagulant activity, even if decreased with respect to the TBA. INTRODUCTION SELEX (Systematic Evolution of Ligands by Exponential Enrichment) (1) is a combinatorial chemistry methodology based on oligonucleotide libraries which are screened for high-afﬁnity binding to a given target. High-afﬁnity ligands can be isolated from the library using iterative rounds afﬁnity-based enrichment, alternating with oligonucleotide ampliﬁcation. Ligands obtained using this methodology have often been referred to as aptamers. In 1992, Block et al. (2) screened a pool of 1013 synthetic oligonucleotides for their interaction with thrombin, which is a serine protease with multiple functions in hemostasis whose roles in coronary heart disease and other thrombotic disorders have promoted efforts toward the identiﬁcation of speciﬁc inhibitors. This study led to the identiﬁcation of a consensus DNA 15mer, namely 50GGTTGGTGTGGTTGG30 (TBA: thrombin binding aptamer), which was found to be a potent inhibitor of thrombin with an EC50 of 20 nM in a ﬁbrinogen clotting assay (3,4). The 3D structure of TBA was solved using NMR techniques (5,6). As Figure 1 shows, TBA is characterized by a chair-like structure consisting of two G-tetrads connected by two TT loops and a single TGT loop. As suggested by others (7), TBA binds at the anion exosite I of thrombin in a conformation little changed from that of the unbound species. In order to improve the properties of TBA, a number of researches (8–10) have been devoted to its structure–activity relationship to post-SELEX modiﬁcations. For example, He et al. (9) synthesized numerous TBA analogues containing modiﬁed guanosine carrying several substituents at 8 position or at the exocyclic amino group. In a different paper (10), the same authors reported the synthesis and the thrombin inhi- biting properties of several TBA-based oligonucleotides con- taining neutral formacetal linkages. However, although TBA was the ﬁrst aptamer to be discovered, no studies dealing with the effects of introduction of sites of polarity inversion have been reported yet, even if such interesting backbone altera- tion has already been utilized in another aptamer, namely Macugen (11) (pegaptanib sodium), that has been recently approved by the US FDA for the treatment of neovascular age-related macular degeneration. In this frame, we have undertaken a study whose aim is to use a biologically driven approach in order to improve the knowledge of the interactions between thrombin and TBA that are critical for the biological activity. Particularly, we have designed and synthesized a TBA-based oligodeoxynu- cleotide containing a 50–50 site of polarity inversion, namely 30GGT50-50TGGTGTGGTTGG30 (1). In this article, we report a detailed structural study, along with thermal stability *To whom correspondence should be addressed. Tel: +39 081 678508; Fax: +39 081 678552; Email: mayoll@unina.it Dedicated to the memory of Prof. Luigi Gomez-Paloma 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.