title: Sequence-Specific Incorporation of Enzyme-Nucleotide Chimera by DNA Polymerases.
abstract: DNA polymerases select the right nucleotide for the growing polynucleotide chain based on the shape and geometry of the nascent nucleotide pairs and thereby ensure high DNA replication selectivity. High-fidelity DNA polymerases are believed to possess tight active sites that allow little deviation from the canonical structures. However, DNA polymerases are known to use nucleotides with small modifications as substrates, which is key for numerous core biotechnology applications. We show that even high-fidelity DNA polymerases are capable of efficiently using nucleotide chimera modified with a large protein like horseradish peroxidase as substrates for template-dependent DNA synthesis, despite this "cargo" being more than 100-fold larger than the natural substrates. We exploited this capability for the development of systems that enable naked-eye detection of DNA and RNA at single nucleotide resolution.
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Welter
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Verga
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany. andreas.marx@uni-konstanz.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Marx
mesh_terms: D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004259:DNA-Directed DNA Polymerase; D009711:Nucleotides
keywords: DNA polymerase; DNA replication; colorimetric analysis; horseradish peroxidase; modified nucleotides