Publication details
Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode
| Authors | |
|---|---|
| Year of publication | 2014 |
| Type | Article in Periodical |
| Magazine / Source | Analytical and Bioanalytical chemistry |
| MU Faculty or unit | |
| Citation | |
| Web | 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 |
| Field | Electrochemistry |
| Keywords | Electrochemical analysis; Labeled probes; Osmium complex; Tumor suppressor protein p53; DNA-protein interaction; Immunoprecipitation; Competition assay; Magnetic beads; Mercury electrode |
| Description | 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. |