Informace o publikaci

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

Autoři	NĚMCOVÁ Kateřina ŠEBEST Peter HAVRAN Luděk ORSÁG Petr FOJTA Miroslav PIVOŇKOVÁ Hana
Rok publikování	2014
Druh	Článek v odborném periodiku
Časopis / Zdroj	Analytical and Bioanalytical chemistry
Fakulta / Pracoviště MU	Středoevropský technologický institut
Citace
www	http://download.springer.com/static/pdf/605/art%253A10.1007%252Fs00216-014-7996-0.pdf?auth66=1424693573_e49fe0b8548f527d28393c19da86150b&ext=.pdf
Doi	http://dx.doi.org/10.1007/s00216-014-7996-0
Obor	Elektrochemie
Klíčová slova	Electrochemical analysis; Labeled probes; Osmium complex; Tumor suppressor protein p53; DNA-protein interaction; Immunoprecipitation; Competition assay; Magnetic beads; Mercury electrode
Popis	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. Versatility of the electrochemical MBIP technique and its general applicability in studies of any DNA-binding protein is discussed.