direct detection of thrombin binding to 8-bromodeoxyguanosine-modified aptamer effects of modification on affinity and kinetics直接检测凝血酶结合8-bromodeoxyguanosine-modified适配子修改亲和力和动力学的影响.pdfVIP

SAGE-Hindawi Access to Research Journal of Nucleic Acids Volume 2011, Article ID 316079, 5 pages doi:10.4061/2011/316079 Research Article Direct Detection of Thrombin Binding to 8-Bromodeoxyguanosine-Modified Aptamer: Effects of Modification on Affinity and Kinetics Shou Goji and Jun Matsui Department of Nanobiochemistry, FIRST, Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan Correspondence should be addressed to Jun Matsui, matsui@konan-u.ac.jp Received 14 May 2011; Revised 15 July 2011; Accepted 21 July 2011 Academic Editor: Daisuke Miyoshi Copyright © 2011 S. Goji and J. Matsui. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The affinity of an 8-bromodeoxyguanosine- (8-BrdG-) substituted thrombin-binding aptamer (TBA-Br), which has the 1st and 10th guanosine residues replaced with 8-BrdG, was estimated using reflectometric interference spectroscopy (RIfS). When comparing TBA-Br with unmodified TBA (TBA-H), it was demonstrated that the modification effectively improved the affinity of TBA; dissociation constants (KD ) of TBA-H and TBA-Br were 45.4 nM and 1.99 nM, respectively. These values, which were obtained by direct observation of thrombin binding using RIfS, have the same order of magnitude as those obtained in our previous study utilizing conformational changes in TBA to detect thrombin binding, thus confirming the validity of the obtained KD values. RIfS measurements also revealed that the 8-BrdG modification resulted in a lower dissociation rate constant (kd ), which suggests that the enhancement of affinity can be attributed to the stabilization of the