title: Affinity improvement of a VEGF aptamer by in silico maturation for a sensitive VEGF-detection system.
abstract: Systematic evolution of ligands by exponential enrichment (SELEX) is an efficient method to identify aptamers; however, it sometimes fails to identify aptamers that bind to their target with high affinity. Thus, post-SELEX optimization of aptamers is required to improve aptamer binding affinity. We developed in silico maturation based on a genetic algorithm (1) as an efficient mutagenesis method to improve aptamer binding affinity. In silico maturation was performed to improve a VEGF-binding DNA aptamer (VEap121). The VEap121 aptamer is considered to fold into a G-quadruplex structure and this structure may be important for VEGF recognition. Using in silico maturation, VEap121 was mutated with the exception of the guanine tracts that are considered to form the G-quartet. As a result, four aptamers were obtained that showed higher affinity compared with VEap121. The dissociation constant (K(d)) of the most improved aptamer (3R02) was 300 pM. The affinity of 3R02 was 16-fold higher than that of VEap121. Moreover, a bivalent aptamer was constructed by connecting two identical 3R02s through a 10-mer thymine linker for further improvement of affinity. The bivalent aptamer (3R02 Bivalent) bound to VEGF with a K(d) value of 30 pM. Finally, by constructing a VEGF-detection system using a VEGF antibody as the capture molecule and monovalent 3R02 as the detection molecule, a more sensitive assay was developed compared with the system using VEap121. These results indicate that in silico maturation could be an efficient method to improve aptamer affinity for construction of sensitive detection systems.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Nonaka
authors.forename: Wataru
authors.initials: W
authors.lastname: Yoshida
authors.forename: Koichi
authors.initials: K
authors.lastname: Abe
authors.forename: Stefano
authors.initials: S
authors.lastname: Ferri
authors.forename: Holger
authors.initials: H
authors.lastname: Schulze
authors.forename: Till T
authors.initials: TT
authors.lastname: Bachmann
authors.forename: Kazunori
authors.initials: K
authors.lastname: Ikebukuro
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D002942:Circular Dichroism; D006801:Humans; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis*
chemical_list: D052157:Aptamers, Nucleotide; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A