In Vitro Selection of New DNA Aptamers for Human Vascular Endothelial Growth Factor 165

Abstract

Two DNA aptamers that bind the heparin‐binding domain (HBD) of the human vascular endothelial growth factor 165 (VEGF‐165) have been previously reported. Although VEGF‐165 is a homodimeric protein and the two aptamers have different sequences and secondary structures, the aptamers appear to occupy the same binding site and cannot form a 2 : 1 aptamer/protein complex, thus making them unsuitable for creating a higher‐affinity dimeric DNA aptamer. This has motivated us to conduct a new in vitro selection experiment to search for new VEGF‐165‐binding DNA aptamers with different properties. We undertook a multistream selection strategy in which the concentration of VEGF‐165 was varied significantly. We carried out 11 rounds of selection, and next‐generation sequencing was conducted for every round in each stream. From comprehensive sequence analysis, we identified four classes of DNA aptamers, of which two were reported before, but two are new DNA aptamers. One of the new aptamers exhibits a unique property that has never been observed before: it is capable of forming the 2 : 1 aptamer/protein complex with VEGF‐165. This work has expanded the repertoire of VEGF‐165‐binding DNA aptamers and creates a possibility to engineer a higher affinity homodimeric aptamer for VEGF‐165.

Anti‐VEGF DNA‐based aptamers in cancer therapeutics and diagnostics

“…After 11 rounds of selection by next‐generation sequencing for every round in each stream and sequence analysis, they found some DNA aptamers already known, but also novel sequences. Among these, the best candidate was H4r3 (or simply H4; Table 3), which showed a K d value of 4 nM, as determined by EMSA experiments 191 …”

“…This trimeric aptamer showed a 140-fold enhanced binding affinity to the protein target compared to monomeric 2G19 (K d = 0.37 nM; Table 5). 186 T A B L E 5 Overview of the described anti-VEGF multimeric constructs More recently, starting from the H4 sequence selected by Manochehry et al, 191 the same research group proposed a homodimeric construct in which two H4 sequences were connected via very long linkers containing 100 thymidine residues. When evaluated in its binding ability to VEGF 165 by EMSA assay, this dimer exhibited a K d value of ca.…”

“…threefold improvement in binding affinity compared to its monomeric counterpart ( Table 5). 191 In another work, starting from the aptamer 3R02, a homodimeric derivative was designed by connecting two 3R02 sequences through a 10-mer thymidine linker, which showed a ca. 10-fold higher affinity for the protein than the monomeric aptamer (K d = 0.03 nM; Table 5).…”

“…More recently, starting from the H4 sequence selected by Manochehry et al, 191 the same research group proposed a homodimeric construct in which two H4 sequences were connected via very long linkers containing 100 thymidine residues. When evaluated in its binding ability to VEGF 165 by EMSA assay, this dimer exhibited a K d value of ca.…”

An Ensemble Docking Approach for Analyzing and Designing Aptamer Heterodimers Targeting VEGF165

“…Several DNA aptamers, including VEa5, 2G19, and Vap7, have been further developed as anti-angiogenic agents against VEGF [20][21][22][23]. In addition, efforts have been made to enhance the binding affinity of aptamers to VEGF by designing G-quadruplex-forming aptamers [23][24][25] and aptamer dimers [20,23,[26][27][28][29]. Naturally, it is important to find an aptamer with high binding affinity to VEGF.…”