Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 139, Issue 1, Pages 324-334Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.6b10767
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- Ministry of Education, Culture, Sports, Science and Technology [KAKENHI 26248043]
- Ministry of Economy, Trade, and Industry
- Japan Science and Technology Agency (JST) Precursory Research for Embryonic Science and Technology (PRESTO)
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The novel evolutionary engineering method ExSELEX (genetic alphabet expansion for systematic evolution of ligands by exponential enrichment) provides high-affinity DNA aptamers that specifically bind to target molecules, by introducing an artificial hydrophobic base analogue as a fifth component into DNA aptamers. Here, we present a newer version of ExSELEX, using a library with completely randomized sequences consisting of five components: four natural bases and one unnatural hydrophobic base, 7-(2-thienyl)imidazo [4,5-b]pyridine (Ds). In contrast to the limited number of Ds-containing sequence combinations in our previous library, the increased complexity of the new randomized library could improve the success rates of high-affinity aptamer generation. To this end, we developed a sequencing method for each clone in the enriched library after several rounds of selection. Using the improved library, we generated a Ds-containing DNA aptamer targeting von Willebrand factor Al-domain (vWF) with significantly higher affinity (K-D = 75 pM), relative to those generated by the initial version of ExSELEX, as well as that of the known DNA aptamer consisting of only the natural bases. In addition, the Ds-containing DNA aptamer was stabilized by introducing a mini-hairpin DNA resistant to nucleases, without any loss of affinity (K-D = 61 pM). This new version is expected to consistently produce high-affinity DNA aptamers.
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