We present a novel dual redox labeling approach enabling a facile relative evaluation of protein-DNA interactions based on immunoprecipitation at magnetic beads (MBIP) with subsequent electrochemical detection. DNA probes labeled with two different electroactive markers, benzofurazane and nitrobenzene, which yield reduction peaks at distinct potentials, were synthesized using primer extension (PEX) reaction. We show that using the labeled DNA probes, specific and non-specific binding of the p53 protein can be distinguished in a simple competition binding experiment, as a strong preference of the p53 protein was observed towards DNA probes bearing a specific p53 binding site (p53CON), which is in agreement with known binding properties of the p53 protein. The p53 binding to the individual DNA probes can be modulated by specific monoclonal antibodies used for the immunoprecipitation. This approach can potentially be applied, after selection of appropriate DNA probes and monoclonal antibodies, for investigations of DNA-binding properties of other proteins and thus represents a versatile tool for studies of any DNA-binding proteins.
- Klíčová slova
- DNA modification, DNA-protein interactions, Immunoprecipitation, Redox coding, Voltammetry,
- MeSH
- DNA sondy chemie MeSH
- DNA chemie MeSH
- elektrochemické techniky * MeSH
- imunoprecipitace MeSH
- lidé MeSH
- molekulární struktura MeSH
- monoklonální protilátky chemie MeSH
- nádorový supresorový protein p53 chemie MeSH
- oxidace-redukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA sondy MeSH
- DNA MeSH
- monoklonální protilátky MeSH
- nádorový supresorový protein p53 MeSH
- TP53 protein, human MeSH Prohlížeč
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.
- MeSH
- DNA chemie MeSH
- elektrochemické techniky přístrojové vybavení metody MeSH
- elektrody MeSH
- katalýza MeSH
- lidé MeSH
- nádorový supresorový protein p53 chemie MeSH
- oligonukleotidové sondy chemie MeSH
- osmium chemie MeSH
- rtuť chemie MeSH
- vazba proteinů MeSH
- vodík chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
- nádorový supresorový protein p53 MeSH
- oligonukleotidové sondy MeSH
- osmium MeSH
- rtuť MeSH
- vodík MeSH
Bioorthogonal covalent cross-linking of DNA-binding proteins (p53) to DNA was achieved through novel DNA probes bearing a reactive vinylsulfonamide (VS) group. The VS-modified dCTP served as building block for polymerase synthesis of modified DNA, which was readily conjugated with cysteine-containing peptides and proteins by Michael addition.
- Klíčová slova
- DNA, DNA polymerase, Michael additions, bioorthogonal chemistry, proteins,
- MeSH
- akrylamid chemická syntéza chemie MeSH
- DNA-dependentní DNA-polymerasy chemie MeSH
- DNA chemická syntéza chemie MeSH
- ethyleny chemie MeSH
- kyseliny sulfonové chemie MeSH
- molekulární modely MeSH
- proteiny chemie metabolismus MeSH
- reagencia zkříženě vázaná chemie MeSH
- sulfonamidy chemická syntéza chemie MeSH
- vinylové sloučeniny chemická syntéza chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- akrylamid MeSH
- DNA-dependentní DNA-polymerasy MeSH
- DNA MeSH
- ethylenesulfonic acid MeSH Prohlížeč
- ethyleny MeSH
- kyseliny sulfonové MeSH
- proteiny MeSH
- reagencia zkříženě vázaná MeSH
- sulfonamidy MeSH
- vinylové sloučeniny MeSH
GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (FBI) and 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone (MBI) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (dC(MBI)TP and dC(FBI)TP) were used for a facile enzymatic synthesis of oligonucleotide or DNA probes by polymerase-catalyzed primer extension. The MBI/FBI-labeled DNA probes exerted low fluorescence that was increased 2-3.2 times upon binding of a protein. The concept was demonstrated on sequence-specific binding of p53 to dsDNA and on nonspecific binding of single strand binding protein to an oligonucleotide. The FBI label was also used for a time-resolved experiment monitoring a single-nucleotide incorporation followed by primer extension by Vent(exo-) polymerase.
- MeSH
- benzylové sloučeniny chemie MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- DNA-dependentní DNA-polymerasy chemie metabolismus MeSH
- DNA chemie metabolismus MeSH
- fluorescenční barviva chemie MeSH
- imidazoliny MeSH
- imidazoly chemie MeSH
- molekulární sekvence - údaje MeSH
- nukleotidy chemie metabolismus MeSH
- párování bází MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone MeSH Prohlížeč
- 3,5-difluoro-4-hydroxybenzylidene imidazolinone MeSH Prohlížeč
- benzylové sloučeniny MeSH
- DNA vazebné proteiny MeSH
- DNA-dependentní DNA-polymerasy MeSH
- DNA MeSH
- fluorescenční barviva MeSH
- imidazoliny MeSH
- imidazoly MeSH
- nukleotidy MeSH
- Tli polymerase MeSH Prohlížeč
BACKGROUND: Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects. METHODS: A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 - 10(9) copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively. RESULTS: Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242-292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes. CONCLUSION: Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials.
- Publikační typ
- časopisecké články MeSH
