Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2 0 -C-piperazino-UNA monomer Troels B. Jensen a , Jonas R. Henriksen b , Bjarne E. Rasmussen c , Lars M. Rasmussen c , Thomas L. Andresen b , Jesper Wengel a , Anna Pasternak a,⇑ a Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark b Department of Micro and Nanotechnology, Ørsteds Plads Building 345 East, DK-2800 Kgs. Lyngby, Denmark c Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, University of Southern Denmark, Sdr. Boulevard 29, 5000 Odense C, Denmark article info Article history: Received 10 May 2011 Revised 27 June 2011 Accepted 30 June 2011 Available online 7 July 2011 Keywords: Thrombin binding aptamer Unlocked nucleic acid Isothermal titration calorimetry Thermodynamics Thrombin time assay abstract Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promis- ing anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2 0 -C-piperazino-UNA residue and UNA residues incorporated in several positions on thermo- dynamics, kinetics and biological properties of the aptamer. 2 0 -C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermo- dynamic stability of TBA by 0.28–0.44 kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest sta- bilization of G-quadruplex structure (DDG  37 = 1.03 kcal/mol). On the contrary, the largest destabiliza- tion mounting to 1.79 kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Thrombin is a serine protease involved in the blood coagulation cascade. 1 Inhibition of thrombin is applied in coronary surgery, prevention and treatment of cardiovascular diseases, and cancer therapy. 2–5 Development of novel thrombin inhibitors is of interest due to a number of side effects of anticoagulants used at present. Thrombin binding aptamer (TBA) is a short DNA sequence (5 0 GGTTGGTGTGGTTGG3 0 ) which was found as a result of in vitro selection targeted towards thrombin. 6 This consensus 15-mer forms an intramolecular, antiparallel G-quadruplex structure with a chair-like conformation (Fig. 1). 7,8 The core constitutes of two G-quartets linked at one end by a TGT loop and by two TT loops at the other end. TBA is characterized by strong anticoagulant properties in vitro, short in vivo lifetime and rapid onset of action facilitating reversal of its activity and excluding dose-adjusting complications observed for other, commonly used anticoagulants. 9 Recently, TBA progressed through preclinical to clinical develop- ment (ARC183, Archemix). 10 According to NMR and X-ray studies, TBA forms a complex with two thrombin molecules inactivating only one of them. 7,11,12 The inhibitory properties of TBA are attributed to specific interaction of the aptamer with the thrombin anion exosite I of one of the thrombin molecules. The electropositive heparin exosite of the sec- ond thrombin molecule is involved in neutralization of the nega- 0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2011.06.087 Abbreviations: TBA, thrombin binding aptamer; UNA, unlocked nucleic acid; ITC, isothermal titration calorimetry; SPR, surface plasmon resonance; CD, circular dichroism; TDS, thermal difference spectra. ⇑ Corresponding author. Tel.: +45 65502510; fax: +45 66158780. E-mail address: apa@ifk.sdu.dk (A. Pasternak). Figure 1. Quadruplex structure of thrombin binding aptamer (TBA). Bioorganic & Medicinal Chemistry 19 (2011) 4739–4745 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc