title: Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates.
abstract: To expand the chemical functionality of DNAzymes and aptamers, several new modified deoxyuridine triphosphates have been synthesized. An important precursor that enables this aim is 5-aminomethyl dUTP, whereby the pendent amine serves as a handle for further synthetic functionalization. Five functional groups were conjugated to 5-aminomethyl dUTP. Incorporation assays were performed on several templates that demand 2-5 sequential incorporation events using several commercially available DNA polymerases. It was found that Vent (exo-) DNA polymerase efficiently incorporates all five modified dUTPs. In addition, all nucleoside triphosphates were capable of supporting a double-stranded exponential PCR amplification. Modified PCR amplicons were PCR amplified into unmodified DNA and sequenced to verify that genetic information was conserved through incorporation, amplification, and reamplification. Overall these modified dUTPs represent new candidate substrates for use in selections using modified nucleotide libraries.
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T1Z1, Canada. lekluck@chem.ubc.ca.
authors.forename: Erkai
authors.initials: E
authors.lastname: Liu
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T1Z1, Canada. curtishonminglam@hotmail.com.
authors.forename: Curtis H
authors.initials: CH
authors.lastname: Lam
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T1Z1, Canada. dperrin@chem.ubc.ca.
authors.forename: David M
authors.initials: DM
authors.lastname: Perrin
mesh_terms: D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase
keywords: Vent (exo-) DNA polymerase; incorporation; modified nucleoside triphosphates