Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode

0435469 - BFÚ 2015 RIV DE eng J - Journal Article
Němcová, Kateřina - Šebest, Peter - Havran, Luděk - Orság, Petr - Fojta, Miroslav - Pivoňková, Hana
Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode.
Analytical and Bioanalytical Chemistry. Roč. 406, č. 24 (2014), s. 5843-5852. ISSN 1618-2642. E-ISSN 1618-2650
R&D Projects: GA ČR(CZ) GAP301/11/2076; GA AV ČR(CZ) IAA400040901
Institutional support: RVO:68081707
Keywords : Electrochemical analysis * Labeled probes * Osmium complex
Subject RIV: BO - Biophysics
Impact factor: 3.436, year: 2014

In this paper, we present an electrochemical DNA-protein interaction assay based on a combination of protein-specific immunoprecipitation at magnetic beads (MBIP) with application of oligonucleotide (ON) probes labeled with an electroactive oxoosmium complex (Os,bipy). We show that double-stranded ONs bearing a dT(20) tail labeled with Os,bipy are specifically recognized by the tumor suppressor p53 protein according to the presence or absence of a specific binding site (p53CON) in the double-stranded segment. We demonstrate the applicability of the Os,bipy-labeled probes in titration as well as competition MBIP assays to evaluate p53 relative affinity to various sequence-specific or structurally distinct unlabeled DNA substrates upon modulation of the p53-DNA binding by monoclonal antibodies used for the immunoprecipitation. To detect the p53-bound osmium-labeled probes, we took advantage of a catalytic peak yielded by Os,bipy-modified DNA at the mercury-based electrodes, allowing facile determination of subnanogram quantities of the labeled oligonucleotides.
Permanent Link: http://hdl.handle.net/11104/0239337