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Molecules 2010, 15(1), 215-225; doi:10.3390/molecules15010215

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.

Received: 6 November 2009; in revised form: 17 December 2009 / Accepted: 31 December 2009 / Published: 7 January 2010

(This article belongs to the Special Issue High-throughput Screening)

Download PDF Full-Text [595 KB, uploaded 7 January 2010 10:54 CET]

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 K_D 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 K_D 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 K_D value of 4.7 × 10² pM.

Keywords: aptamer; cancer diagnosis; sensor element; VEGF₁₂₁; VEGF₁₆₅

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