J 2014

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

NĚMCOVÁ, Kateřina, Peter ŠEBEST, Luděk HAVRAN, Petr ORSÁG, Miroslav FOJTA et. al.

Basic information

Original name

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

Authors

NĚMCOVÁ, Kateřina (203 Czech Republic), Peter ŠEBEST (203 Czech Republic), Luděk HAVRAN (203 Czech Republic), Petr ORSÁG (203 Czech Republic, belonging to the institution), Miroslav FOJTA (203 Czech Republic, guarantor, belonging to the institution) and Hana PIVOŇKOVÁ (203 Czech Republic)

Edition

Analytical and Bioanalytical chemistry, HEIDELBERG, SPRINGER HEIDELBERG, 2014, 1618-2642

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10405 Electrochemistry

Country of publisher

Germany

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Impact factor

Impact factor: 3.436

RIV identification code

RIV/00216224:14740/14:00079183

Organization unit

Central European Institute of Technology

UT WoS

000341834500011

Keywords in English

Electrochemical analysis; Labeled probes; Osmium complex; Tumor suppressor protein p53; DNA-protein interaction; Immunoprecipitation; Competition assay; Magnetic beads; Mercury electrode

Tags

International impact, Reviewed
Změněno: 23/2/2015 13:18, Martina Prášilová

Abstract

V originále

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.