title: Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications.
abstract: A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G-C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF165 unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF165 RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF165 Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF165 and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF165 DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF165.
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, 138669, Singapore RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan TagCyx Biotechnologies, 1-6-126 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan PRESTO, JST, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Kimoto
authors.affiliation: RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan Department of Medical Life Science, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Mana
authors.initials: M
authors.lastname: Nakamura
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, 138669, Singapore RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan TagCyx Biotechnologies, 1-6-126 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan ichiro@ibn.a-star.edu.sg.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hirao
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D003362:Cost-Benefit Analysis; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D040262:Receptors, Vascular Endothelial Growth Factor / Q000378:metabolism; D052156:SELEX Aptamer Technique; D013379:Substrate Specificity; D013816:Thermodynamics; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C495058:pegaptanib; D004247:DNA; D040262:Receptors, Vascular Endothelial Growth Factor; D002118:Calcium