title: High-Affinity DNA Aptamer Generation Targeting von Willebrand Factor A1-Domain by Genetic Alphabet Expansion for Systematic Evolution of Ligands by Exponential Enrichment Using Two Types of Libraries Composed of Five Different Bases.
abstract: 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 A1-domain (vWF) with significantly higher affinity (KD= 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 (KD= 61 pM). This new version is expected to consistently produce high-affinity DNA aptamers.
authors.affiliation: Institute of Bioengineering and Nanotechnology , 31 Biopolis Way, The Nanos, #04-01, 138669 Singapore.|TagCyx Biotechnologies , 1-6-126 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.|RIKEN Center for Life Science Technologies , 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Ken-Ichiro
authors.initials: KI
authors.lastname: Matsunaga
authors.affiliation: Institute of Bioengineering and Nanotechnology , 31 Biopolis Way, The Nanos, #04-01, 138669 Singapore.|TagCyx Biotechnologies , 1-6-126 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.|RIKEN Center for Life Science Technologies , 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Kimoto
authors.affiliation: Institute of Bioengineering and Nanotechnology , 31 Biopolis Way, The Nanos, #04-01, 138669 Singapore.|TagCyx Biotechnologies , 1-6-126 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.|RIKEN Center for Life Science Technologies , 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
authors.forename: Ichiro
authors.identifier: 0000-0002-1115-8079
authors.initials: I
authors.lastname: Hirao
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D052156:SELEX Aptamer Technique; D014841:von Willebrand Factor / Q000737:chemistry*
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D014841:von Willebrand Factor