title: New synthetic route to ethynyl-dUTP: A means to avoid formation of acetyl and chloro vinyl base-modified triphosphates that could poison SELEX experiments.
abstract: 5-Ethynyl-2'-deoxyuridine is a common base-modified nucleoside analogue that has served in various applications including selection experiments for potent aptamers and in biosensing. The synthesis of the corresponding triphosphates involves a mild acidic deprotection step. Herein, we show that this deprotection leads to the formation of other nucleoside analogs which are easily converted to triphosphates. The modified nucleoside triphosphates are excellent substrates for numerous DNA polymerases under both primer extension and PCR conditions and could thus poison selection experiments by blocking sites that need to be further modified. The formation of these nucleoside analogs can be circumvented by application of a new synthetic route that is described herein.
authors.affiliation: Laboratory for Bioorganic Chemistry of Nucleic Acids, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France; CNRS UMR3523 Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Röthlisberger
authors.affiliation: Laboratory for Bioorganic Chemistry of Nucleic Acids, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France; CNRS UMR3523 Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Levi-Acobas
authors.affiliation: Laboratory for Bioorganic Chemistry of Nucleic Acids, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France; CNRS UMR3523 Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Electronic address: marcel.hollenstein@pasteur.fr.
authors.forename: Marcel
authors.initials: M
authors.lastname: Hollenstein
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011122:Polyphosphates / Q000737:chemistry*; D052156:SELEX Aptamer Technique
chemical_list: D003856:Deoxyuracil Nucleotides; D011122:Polyphosphates; C027078:deoxyuridine triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase; C005692:triphosphoric acid
keywords: In vitro selection; Nucleobase modification; Nucleoside triphosphates; PCR; Primer extension