Michiko Kimoto, Mana Nakamura, Ichiro Hirao, Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications, Nucleic Acids Research, Volume 44, Issue 15, 6 September 2016, Pages 7487–7494, https://doi.org/10.1093/nar/gkw619
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.
License type:
http://creativecommons.org/licenses/by-nc/4.0/
Funding Info:
Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology [KAKENHI 26248043 to I.H.];grants for projects focused on developing key technologies for discovering and manufacturing drugs for next-generation treatment and diagnosis from the Ministry of Economy, Trade and Industry [to I.H.]; Japan Science and Technology Agency (JST) Precursory Research for Embryonic Science and Technology (PRESTO) [to M.K.]. Funding for open access charge: Institute of Bioengineering and Nanotechnology (IBN). Conflict of interest statement. M.K. and I.H. own stock in TagCyx Biotechnologies.