M.G. DeGroote Institute for Infectious Disease Research Department of Biochemistry and Biomedical Sciences DeGroote School of Medicine, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4 K1, Canada.
Department of Medicine DeGroote School of Medicine, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4 K1, Canada.
Chembiochem. 2020 Jul 16;21(14):2029-2036. doi: 10.1002/cbic.202000024. Epub 2020 Mar 30.
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.
先前已经报道过两种与人类血管内皮生长因子 165（VEGF-165）的肝素结合域（HBD）结合的 DNA 适体。尽管 VEGF-165 是一种同源二聚体蛋白，并且这两种适体具有不同的序列和二级结构，但它们似乎占据相同的结合位点，并且不能形成 2:1 的适体/蛋白复合物，因此它们不适合用于创建具有更高亲和力的二聚体 DNA 适体。这促使我们进行了新的体外选择实验，以寻找具有不同性质的新的 VEGF-165 结合 DNA 适体。我们采用了多流选择策略，其中 VEGF-165 的浓度有很大的变化。我们进行了 11 轮选择，并且在每个流的每一轮都进行了下一代测序。通过全面的序列分析，我们确定了四类 DNA 适体，其中两种以前已经报道过，但两种是新的 DNA 适体。其中一种新的适体表现出一种以前从未观察到的独特性质：它能够与 VEGF-165 形成 2:1 的适体/蛋白复合物。这项工作扩展了与 VEGF-165 结合的 DNA 适体的种类，并为工程化 VEGF-165 的更高亲和力同源二聚体适体创造了可能性。
