Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and anticoagulant activity

10602–10611 Nucleic Acids Research, 2015, Vol. 43, No. 22 doi: 10.1093/nar/gkv1224 Published online 17 November 2015 Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and anticoagulant activity Antonella Virgilio1 , Luigi Petraccone2 , Valentina Vellecco1 , Mariarosaria Bucci1 , Michela Varra1 , Carlo Irace1 , Rita Santamaria1 , Antonietta Pepe3 , Luciano Mayol1 , Veronica Esposito1,* and Aldo Galeone1,* 1 Dipartimento di Farmacia, Università degli Studi di Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy, Dipartimento di Scienze Chimiche, Università degli Studi di Napoli Federico II,via Cintia, I-80126 Napoli, Italy and 3 Dipartimento di Scienze, Università degli Studi della Basilicata, Viale dell’Ateneo Lucano 10, I-85100 Potenza, Italy 2 Received July 13, 2015; Revised October 28, 2015; Accepted October 29, 2015 Here we report investigations, based on circular dichroism, nuclear magnetic resonance spectroscopy, molecular modelling, differential scanning calorimetry and prothrombin time assay, on analogues of the thrombin binding aptamer (TBA) in which individual thymidines were replaced by 5fluoro-2 -deoxyuridine residues. The whole of the data clearly indicate that all derivatives are able to fold in a G-quadruplex structure very similar to the ‘chair-like’ conformation typical of the TBA. However, only ODNs TBA-F4 and TBA-F13 have shown a remarkable improvement both in the melting temperature (Tm ≈ +10) and in the anticoagulant activity in comparison with the original TBA. These findings are unusual, particularly considering previously reported studies in which modifications of T4 and T13 residues in TBA sequence have clearly proven to be always detrimental for the structural stability and biological activity of the aptamer. Our results strongly suggest the possibility to enhance TBA properties through tiny straightforward modifications. INTRODUCTION DNA and RNA aptamers are nucleic acid ligands characterized by an outstanding ability to bind with high affinity and specificity to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers can be discovered and engineered through repeated rounds of an in vitro selection strategy called SELEX (Systematic Evolution of Ligands by Exponential enrichment) (1,2). Among the DNA aptamers the thrombin binding aptamer (TBA, 5 GGTTGGTGTGGTTGG-3 ) has been one of the first to be discovered (3,4). However, despite this fact, TBA is still the subject of several researches, both in therapeutics, being endowed with anticoagulant properties, and in analytics, thank to its ability of binding potassium ions and, more importantly, also heavy metal ions of toxicological interest. According to nuclear magnetic resonance (NMR) and Xray structural investigations TBA adopts a monomolecular, antiparallel G-quadruplex structure with a ‘chair-like’ conformation (5,6). The central part of the G-quadruplex consists of two syn-anti-syn-anti stacked G-quartets, which are connected to each other by three edge-wise loops: a central TGT loop and two lateral TT loops (Figure 1). Quite soon after its discovery, TBA has been subjected to several chemical modifications, most of which with the targets to: (i) improve its thermal stability in physiological conditions, which is connected to the anticoagulant activity; (ii) render it resistant to the exonucleases, ubiquitous in biological environments and (iii) enhance its affinity to thrombin. As far as the TBA/thrombin interaction is concerned, a compelling number of investigations have highlighted that the loops are the parts of the aptamer mostly involved in the interaction with the protein (6–8). In particular, these studies indicated that the minor loops TT interact with the thrombin anion exosite I, while the larger TGT loop is in close proximity to the heparin binding site of a further thrombin molecule. Therefore, TBA seems to interact with two thrombin molecules, inactivating only one of them. The availability of rather detailed information on which residues were involved in the interaction of TBA with its target allowed researchers to propose a number of interesting site specific modifications aimed at obtaining data concerning the quantitative structure-activity and/or structurestability relationship. In fact, by limiting our discussion to * To whom correspondence should be addressed. Tel: +39 081678542; Fax: +39 081678552; Email: Correspondence may also be addressed to Veronica Esposito. Tel: +39 081678746; Fax: +39 081678552; Email:  C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact ABSTRACT Nucleic Acids Research, 2015, Vol. 43, No. 22 10603 lar dichroism (CD) experiments, NMR techniques (1 H and 19 F), molecular modelling, differential scanning calorimetry (DSC) and prothrombin time (PT) assay measurements have clearly shown significant improvements for both the physical-chemical and biological properties in TBA derivatives, where residues T4 and T13 have been replaced with F. These results are particularly noteworthy taking into account that they have been obtained with TBA analogues in which a single fluorine atom replaces a methyl group in the original structure. MATERIALS AND METHODS Oligonucleotides synthesis and purification Figure 1. Schematic representation of the TBA G-quadruplex and chemical structure of 5-fluoro-2 -deoxyuridine (F). Guanosines in syn and anti glycosidic conformations are in dark and light grey, respectively. CD and UV spectroscopy CD samples of modified oligonucleotides and their natural counterpart were prepared at an oligodeoxynucleotide (ODN) concentration of 100 ␮M using a potassium phosphate buffer (10 mM KH2 PO4 /K2 HPO4 , 70 mM KCl, pH 7.0) and submitted to the annealing procedure (heating at 90◦ C and slowly cooling at room temperature). CD spectra of all quadruplexes and CD melting curves were registered on a Jasco J-715 CD spectrophotometer. For the CD spectra, the wavelength was varied from 220 to 320 nm at 100 nm min−1 scan rate, and the spectra recorded with a response of 16 s, at 2.0 nm bandwidth and normalized by subtraction of the background scan with buffer. The temperature was kept constant at 20◦ C with a thermoelectrically-controlled cell holder (Jasco PTC-348). CD melting curves were registered as a function of temperature from 20 to 80◦ C for all quadruplexes at their maximum Cotton effect wavelengths. The CD data were recorded in a 0.1 cm pathlength cuvette with a scan rate of 0.5◦ C/min. The C (...truncated)