title: Improvement of the VEGF binding ability of DNA aptamers through in silico maturation and multimerization strategy.
abstract: Aptamers are mainly selected by in vitro selection using random nucleic acid libraries. These aptamers have often shown insufficient affinity for biomedical applications. We improved DNA aptamer binding affinity for vascular endothelial growth factor (VEGF) through in silico maturation (ISM) and aptamer multimerization. ISM is one of a number of evolutionary approaches and aptamer multimerization is one of several semi-rational strategies to improve function. We first reselected VEGF-binding aptamers using a partially randomized DNA library and identified two aptamers with higher binding ability than that of a known aptamer. We conducted ISM using the re-selected aptamers to optimize the key loop sequences created by a three-way junction structure. After five ISM rounds, we identified aptamer 2G19 [dissociation constant (Kd), 52 nM] as a local optimum of the defined search space. We characterized the aptamer and found that a specific stem-loop structure was involved in aptamer VEGF recognition. To further improve its affinity for VEGF, we multimerized 2G19 or its stem-loop structure. The designed SL5-trivalent aptamer (Kd, 0.37 nM) with three binding motifs significantly increased binding affinity, representing a 500-fold improvement from systematic evolution of ligands by exponential enrichment-selected aptamers.
authors: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Japan.
authors: Takahiro
authors: T
authors: Fukaya
authors: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Japan.
authors: Koichi
authors: K
authors: Abe
authors: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Japan.
authors: Nasa
authors: N
authors: Savory
authors: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Japan.
authors: Kaori
authors: K
authors: Tsukakoshi
authors: School of Bioscience and Biotechnology, Tokyo University of Technology, Japan.
authors: Wataru
authors: W
authors: Yoshida
authors: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Japan.
authors: Stefano
authors: S
authors: Ferri
authors: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Japan.
authors: Koji
authors: K
authors: Sode
authors: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Japan. Electronic address: ikebu@cc.tuat.ac.jp.
authors: Kazunori
authors: K
authors: Ikebukuro
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D011485:Protein Binding; D055503:Protein Multimerization; D020349:Surface Plasmon Resonance; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D042461:Vascular Endothelial Growth Factor A
keywords: Aptamer; Multimerization; Vascular endothelial growth factor; in silico maturation