Screening and improvement of an anti-VEGF DNA aptamer.
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.
Nonaka
Yoshihiko
Y
Department of Biotechnology & Life science, Tokyo University of Agriculture and Technology, 2-24-21 Naka Cho, Koganei, Tokyo 1848588, Japan.
Sode
Koji
K
Ikebukuro
Kazunori
K
D052157:Aptamers, Nucleotide / Q000032:analysis* / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D052156:SELEX Aptamer Technique / Q000379:methods*; D020349:Surface Plasmon Resonance; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D052157:Aptamers, Nucleotide; D050505:Mutant Proteins; D042461:Vascular Endothelial Growth Factor A