Screening and Improvement of an Anti-VEGF DNA Aptamer 英文参考文献.docVIP

Molecules 2010, 15, 215-225; doi:10.3390/moleculeOPEN ACCESS molecules ISSN 1420-3049 /journal/molecules Article Screening and Improvement of an Anti-VEGF DNA Aptamer Yoshihiko Nonaka, Koji Sode and Kazunori Ikebukuro * Department of Biotechnology Life science, Tokyo University of Agriculture and Technology, 2-24-21 Naka Cho, Koganei, Tokyo, 1848588, Japan * Author to whom correspondence should be addressed; E-Mail: ikebu@cc.tuat.ac.jp; Tel./Fax: +81-42-388-7030. Received: 6 November 2009; in revised form: 17 December 2009 / Accepted: 31 December 2009 / Published: 7 January 2010 Abstract: To obtain an aptamer with a high affinity for vascular endothelial growth factor (VEGF), we focused on the receptor-binding domain (RBD) of VEGF as a target epitope. Three rounds of screening gave Vap7, which bound to the VEGF isoforms VEGF121 and VEGF165 with KD values of 1.0 nM and 20 nM, respectively. Moreover, Vap7 showed specificity within the VEGF family. Secondary structure predictions and circular dicrhoism suggested that Vap7 folds into a G-quadruplex structure. We obtained a mutant aptamer that contains only this region of the aptamer sequence. This truncated mutant (V7t1) bound to both VEGF121 and VEGF165 with KD values of 1.1 nM and 1.4 nM, respectively. Its sequence was 5-TGTGGGGGTGGACGGGCCGGGTAGA-3, and it appeared to form a G-quadruplex structure. We also produced an aptamer heterodimer consisting of our previously derived aptamer (del5-1), which binds to the heparin-binding domain of VEGF, linked to V7t1. The resulting heterodimer bound strongly to VEGF165 with a KD value of 4.7 × 102 pM. Keywords: aptamer; cancer diagnosis; sensor element; VEGF121; VEGF165 1. Introduction Aptamers are nucleic acids that bind to target molecules [1,2]. Recently, aptamers have drawn a great deal of attention as sensing elements in biosensors for small molecules and proteins [3–5]. DNA aptamers can be r