title: Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode.
abstract: 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 dT20 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.
authors: Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65, Brno, Czech Republic.
authors: Kateřina
authors: K
authors: Němcová
authors: Peter
authors: P
authors: Sebest
authors: Luděk
authors: L
authors: Havran
authors: Petr
authors: P
authors: Orság
authors: Miroslav
authors: M
authors: Fojta
authors: Hana
authors: H
authors: Pivoňková
mesh_terms: D002384:Catalysis; D004247:DNA / Q000737:chemistry*; D055664:Electrochemical Techniques / Q000295:instrumentation / Q000379:methods*; D004566:Electrodes; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D008628:Mercury / Q000737:chemistry*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D009992:Osmium / Q000737:chemistry; D011485:Protein Binding; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*
chemical_list: D015345:Oligonucleotide Probes; D016159:Tumor Suppressor Protein p53; D009992:Osmium; D006859:Hydrogen; D004247:DNA; D008628:Mercury