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Updated: Jun 16, 2026
A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer
Published on: September 13, 2022
Screening and improvement of an anti-VEGF DNA aptamer
Yoshihiko Nonaka1, Koji Sode, Kazunori Ikebukuro
1Department of Biotechnology & Life science, Tokyo University of Agriculture and Technology, 2-24-21 Naka Cho, Koganei, Tokyo 1848588, Japan.
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 VEGF(121) and VEGF(165) 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 VEGF(121) and VEGF(165) 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 VEGF(165) with a K(D) value of 4.7 x 10(2) pM.
