Jana Balintová

Benzofurazane as a New Redox Label for Electrochemical Detection of DNA: Towards Multipotential Redox Coding of DNA Bases

Benzofurazane has been attached to nucleosides and dNTPs, either directly or through an acetylene linker, as a new redox label for electrochemical analysis of nucleotide sequences. Primer extension incorporation of the benzofurazane-modified dNTPs by polymerases has been developed for the construction of labeled oligonucleotide probes. In combination with nitrophenyl and aminophenyl labels, we have successfully developed a three-potential coding of DNA bases and have explored the relevant electrochemical potentials. The combination of benzofurazane and nitrophenyl reducible labels has proved to be excellent for ratiometric analysis of nucleotide sequences and is suitable for bioanalytical applications.

Dual redox labeling of DNA as a tool for electrochemical detection OF P53 protein-DNA interactions

We present a novel dual redox labeling approach enabling a facile relative evaluation of protein-DNA interactions based on immunoprecipitation at magnetic beads (MBIP) with subsequent electrochemical detection. DNA probes labeled with two different electroactive markers, benzofurazane and nitrobenzene, which yield reduction peaks at distinct potentials, were synthesized using primer extension (PEX) reaction. We show that using the labeled DNA probes, specific and non-specific binding of the p53 protein can be distinguished in a simple competition binding experiment, as a strong preference of the p53 protein was observed towards DNA probes bearing a specific p53 binding site (p53CON), which is in agreement with known binding properties of the p53 protein. The p53 binding to the individual DNA probes can be modulated by specific monoclonal antibodies used for the immunoprecipitation. This approach can potentially be applied, after selection of appropriate DNA probes and monoclonal antibodies, for investigations of DNA-binding properties of other proteins and thus represents a versatile tool for studies of any DNA-binding proteins.