NMR Structure of the Thrombin-Binding DNA Aptamer Stabilized by Sr 2+ http://www.jbsdonline.com Abstract The structure of thrombin-binding DNA aptamer complexed with a single Sr 2+ ion (Sr 2+ :TBA complex) has been determined using NMR spectroscopy and restrained molecular dynamics simulations. The quadruplex structure for the Sr 2+ :TBA complex is similar in topology, but distinct in structure, from that previously reported for the K + :TBA complex. The inter-tetrad distance of the Sr 2+ :TBA complex is 3.8 Å, or 0.7 Å larger than in the K + :TBA complex. This substantial difference can be attributed to a dif- ferent binding site for Sr 2+ in the Sr 2+ :TBA complex than for K + in the K + :TBA com- plex. The Sr 2+ :TBA complex assumes a 1:1 stoichiometry, and it is very likely that the Sr 2+ ion simultaneously interacts with the eight O6 atoms of the two G-tetrads. The results indicate that quadruplex DNA structures are highly sensitive to the presence of specific metal ions. The binding of specific metal ions may modulate the biological activity of quadruplex DNA structures in vivo. Introduction The thrombin-binding DNA aptamer (TBA) is a 15mer DNA sequence that was identified using the SELEX procedure as a DNA sequence that binds tightly to thrombin (1). Aptamers are relatively short sequences of DNA (or RNA) that fold into three-dimensional structures that have high affinity for a particular molecular target (e.g., thrombin) (2, 3). Thrombin is an important protein that is involved in the enzymatic cascade that results in blood clot formation. Inhibitors of thrombin, such as the TBA, have potential therapeutic value (4). Folded structures of DNA aptamers are essential for their biological activity. Therefore, structural studies are important for understanding how DNA aptamers bind to their molecular targets. NMR spectroscopy (5-11) and X-ray crystallography (12-14) have each been used to determine the structure of the TBA. These studies have shown that the TBA adopts an intramolecular quadruplex DNA structure in which eight of the nine dG nucleotides form two G-tetrads that stack on one another (Figure 1). DNA quadruplex structures are important components in cell biology where they play an important role in the regulation of proto-oncogene transcription (15, 16) and in the maintenance of DNA telomeres (17-21). Several studies have shown that the biological function of DNA quadruplex structures is remarkably sensitive to the presence of particular cations in solution. Thus, the NHE III element from the c-myc proto-oncogene serves as a repressor of gene transcription by adopting a quadruplex DNA structure in the presence of K + (15, 16). Model DNA telomere sequences may form either intramolecular or intermolecular G-quadruplex struc- tures depending on the presence of K + and/or Na + in the solution (22, 23). The propensity for particular cations to stabilize G-quadruplex structures has been attributed to the interior cavity of the G-quadruplex being preferentially occupied Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 22, Issue Number 1, (2004) ©Adenine Press (2004) Xi-an Mao 1,§ Luis A. Marky 2 William H. Gmeiner 1,* 1 Department of Biochemistry Wake Forest University School of Medicine Winston-Salem, NC 27157-1016 2 Department of Pharmaceutical Sciences University of Nebraska Medical Center Omaha, NE 68198 §Present address Cleveland Clinic Cleveland, Ohio (Mailing Address?) 1 * Email: bgmeiner@wfubmc.edu