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Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF(165) toward pharmaceutical applications

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 t...

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Detalles Bibliográficos
Autores principales: Kimoto, Michiko, Nakamura, Mana, Hirao, Ichiro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009754/
https://www.ncbi.nlm.nih.gov/pubmed/27387284
http://dx.doi.org/10.1093/nar/gkw619
Descripción
Sumario: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-VEGF(165) 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-VEGF(165) RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF(165). Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF(165) 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-VEGF(165) 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 VEGF(165).