Preparation and Application of Enzyme-Nucleotide Conjugates.

In this unit the preparation and application of enzyme-nucleotide conjugates is depicted. First, a modified nucleoside triphosphate is synthesized bearing a long and flexible linker equipped with a thiol group. The nucleotide is then reacted with maleimide-activated horseradish peroxidase to yield an enzyme-nucleotide conjugate, which due to the long linker, can be used as a substrate by DNA polymerases in primer extension reactions. Finally, an assay based on these findings is described that provides a fast and easy nucleic acid detection and genotyping platform. © 2018 by John Wiley & Sons, Inc.

Sequence-Specific Incorporation of Enzyme-Nucleotide Chimera by DNA Polymerases.

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.

Sequence selective naked-eye detection of DNA harnessing extension of oligonucleotide-modified nucleotides.

DNA polymerases can efficiently and sequence selectively incorporate oligonucleotide (ODN)-modified nucleotides and the incorporated oligonucleotide strand can be employed as primer in rolling circle amplification (RCA). The effective amplification of the DNA primer by Φ29 DNA polymerase allows the sequence-selective hybridisation of the amplified strand with a G-quadruplex DNA sequence that has horse radish peroxidase-like activity. Based on these findings we develop a system that allows DNA detection with single-base resolution by naked eye.

Synthesis of γ-Phosphate-Labeled and Doubly Labeled Adenosine Triphosphate Analogs.

This unit describes the synthesis of γ-phosphate-labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide-containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N-hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD.

Carbohydrate-based Cu(I) stabilizing ligands and their use in the synthesis of carbohydrate-ferrocene conjugates.

A series of carbohydrate-ferrocene conjugates have been synthesized by copper(I)-catalyzed cycloaddition of carbohydrate-azides and ethynylferrocene (CuAAC). Newly carbohydrate-based tris-triazoles have been used as Cu(I) stabilizing ligands and showed at least comparable, in some cases even better results compared to the use of tris-(benzyltriazolylmethyl)amine (TBTA).