Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode
Language English Country Germany Media print-electronic
Document type Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- DNA chemistry MeSH
- Electrochemical Techniques instrumentation methods MeSH
- Electrodes MeSH
- Catalysis MeSH
- Humans MeSH
- Tumor Suppressor Protein p53 chemistry MeSH
- Oligonucleotide Probes chemistry MeSH
- Osmium chemistry MeSH
- Mercury chemistry MeSH
- Protein Binding MeSH
- Hydrogen chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA MeSH
- Tumor Suppressor Protein p53 MeSH
- Oligonucleotide Probes MeSH
- Osmium MeSH
- Mercury MeSH
- Hydrogen MeSH
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.
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