Hana Pivoňková

Aminophenyl‐ and Nitrophenyl‐Labeled Nucleoside Triphosphates: Synthesis, Enzymatic Incorporation, and Electrochemical Detection

DNA biosensors and chips are broadly utilized in the life sciences. Electrochemical detection is a less expensive but comparatively sensitive alternative to common optical methods. Although nucleic acids are electroactive themselves, diverse electroactive tags are used to increase sensitivity and specificity. Besides widely used DNA tags based on metal complexes, quantum dots, or phenothiazine dyes, some simple organic derivatives (such as aromatic amines or nitro compounds) exhibit distinct electrochemical activity, thus making them candidates for nucleic acid labeling. In particular, the nitro group appears promising for sensitive detection because of the high number of electrons (four or six) collected per nitro group reduction. So far, neither amino nor nitro groups have been used as specific electroactive DNA markers. Some modified 2’-deoxyribonucleoside triphosphates (dNTPs) bearing substituents at the nucleobase can be enzymatically incorporated into DNA by polymerases. This approach has been used for the construction of functionalized nucleic acids bearing diverse functional groups. Recently, aqueous-phase cross-coupling reactions of unprotected halogenated nucleoside triphosphates with boronic acids or acetylenes were developed and used in combination with polymerase incorporation for the two-step construction of modified nucleic acids, including ferrocene-labeled oligonucleotides (ONs). Herein, we report the synthesis of nucleoside triphosphates bearing aminophenyl and nitrophenyl groups attached to a nucleobase, their enzymatic incorporation, and the preliminary electrochemical properties of the labeled ONs. We expected that the fully conjugated aromatic system would respond well to the electronic changes arising from incorporation into nucleic acids, and result in changes of the redox potential of the label. The modified dNTPs were prepared by the single-step aqueous-phase cross-coupling reactions of halogenated dNTPs, in analogy to our previously developed procedures. The Suzuki–Miyaura reaction of 7-iodo-7-deaza-2’-deoxyadenosine 5’-triphosphate (7-I-7-deaza-dATP), 5-iodo-2’deoxyuridine 5’-triphosphate (5-I-dUTP), or 5-iodo-2’-deoxycytidine 5’-triphosphate (5-I-dCTP) with either 3-aminophenylor 3-nitrophenylboronic acid (Scheme 1) gave the

Label-Free Sequence-Specific DNA Sensing Using Copper-Enhanced Anodic Stripping of Purine Bases at Boron-Doped Diamond Electrodes

Stripping voltammetric determination of purine bases in the presence of copper ions at mercury, amalgam, or carbon-based electrodes has recently been utilized in analysis of DNA or synthetic oligodeoxynucleotides (ODNs). Here we report on copper-enhanced label-free anodic stripping detection of guanine and adenine bases in acid-hydrolyzed DNA at anodically oxidized boron-doped diamond electrode (AO-BDDE). The AO-BDDE was successfully applied in a three-electrode microcell in which an approximately 50 microL drop of the analyte solution can be efficiently stirred during the accumulation step by streaming of an inert gas. Accelerated mass transport due to the solution motion in the presence of copper resulted in enhancement of the guanine oxidation signal by about 2 orders of magnitude (compared to accumulation of the analyte from still solution not containing copper), allowing an easy detection of approximately 25 fmol of the ODNs. The proposed technique is shown to be suitable for a determination of purine (particularly guanine) content in DNA samples. Applications of the technique in magnetic bead-based DNA assays (such as hybridization with DNA sequences exhibiting asymmetrical distribution of purine/pyrimidine nucleotides between the complementary strands or monitoring of amplification of specific DNA fragments in a duplex polymerase chain reaction) are demonstrated.

A label-free electrochemical test for DNA-binding activities of tumor suppressor protein p53 using immunoprecipitation at magnetic beads.

In this paper we extend the application area of the label-free structure-sensitive electrochemical DNA sensing with mercury-based electrodes which is for the first time used, in combination with immunoprecipitation at magnetic beads (MB), for the probing of DNA interactions with tumor suppressor protein p53. The technique relies on capture of the p53-DNA complexes at MB via anti-p53 antibodies, followed by salt-induced dissociation of linear DNA from the complex and its voltammetric detection. Competitive binding of p53 to various plasmid DNA substrates, including lin or scDNAs with or without a specific target site, can easily be followed by ex situ electrochemical analysis of DNA recovered from the immunoprecipitated complexes. Compared to gel electrophoresis which is usually applied to analyze different plasmid DNA forms and their complexes with proteins, the electrochemical detection is faster and allows simpler quantitation of DNA containing free ends at submicrogram levels. We demonstrate applicability of the proposed technique to monitor different DNA-binding activities of wild type and mutant p53 proteins.

The potential of the cruciform structure formation as an important factor influencing p53 sequence-specific binding to natural DNA targets.

p53 is one of the most important tumor suppressors which responds to DNA damage by binding to DNA and regulating the transcription of genes involved in cell cycle arrest, apoptosis, or senescence. As it was shown previously, p53 binding to DNA is strongly influenced by DNA topology. DNA supercoiling is fundamentally important for a wide range of biological processes including DNA transcription, replication, recombination, control of gene expression and genome organization. In this study, we investigated the cruciform structures formation of various inverted repeats in p53-responsive sequences from p21, RGC, mdm2 and GADD45 promoters under negative superhelical stress, and analyzed the effects of these DNA topology changes on p53-DNA binding. We demonstrated using three different methods (gel retardation analyses, ELISA and magnetic immunoprecipitation assay) that the p53 protein binds preferentially to negatively supercoiled plasmid DNAs with p53-responsive sequence presented as a cruciform structure. Not only the appearance of the cruciform structures within naked supercoiled DNA, but also the potential of the binding sites for adopting the non-B structures can contribute to a more favorable p53-DNA complex.

Direct voltammetric analysis of DNA modified with enzymatically incorporated 7-deazapurines.

Nucleic acids studies use 7-deazaguanine (G*) and 7-deazaadenine (A*) as analogues of natural purine bases incapable of forming Hoogsteen base pairs, which prevents them from being involved in DNA triplexes and tetraplexes. Reduced propensity of the G*- and/or A*-modified DNA to form alternative DNA structures is utilized, for example, in PCR amplification of guanine-rich sequences. Both G* and A* exhibit significantly lower potentials of their oxidation, compared to the respective natural nucleobases. At carbon electrodes, A* yields an oxidation peak which is by about 200-250 mV less positive than the peak due to adenine, but coincides with oxidation peak produced by natural guanine residues. On the other hand, oxidation signal of G* occurs at a potential by about 300 mV less positive than the peak due to guanine, being well separated from electrochemical signals of any natural DNA component. We show that enzymatic incorporation of G* and A* can easily be monitored by simple ex situ voltammetric analysis of the modified DNA at carbon electrodes. Particularly G* is shown as an attractive electroactive marker for DNA, efficiently incorporable by PCR. While densely G*-modified DNA fragments exhibit strong quenching of fluorescence of SYBR dyes, commonly used as fluorescent indicators in both gel staining and real time PCR applications, the electrochemical detection provides G*-specific signal suitable for the quantitation of the amplified DNA as well as for the determination of the DNA modification extent. Determination of DNA amplicons based on the measurement of peak G*(ox) is not affected by signals produced by residual oligonucleotide primers or primary templates containing natural purines.

Selective binding of tumor suppressor p53 protein to topologically constrained DNA: Modulation by intercalative drugs

Selective binding of the wild type tumor suppressor protein p53 to negatively and positively supercoiled (sc) DNA was studied using intercalative drugs chloroquine (CQ), ethidium bromide, acridine derivatives and doxorubicin as a modulators of the level of DNA supercoiling. The p53 was found to lose gradually its preferential binding to negatively scDNA with increasing concentrations of intercalators until the DNA negative superhelix turns were relaxed. Formation of positive superhelices (due to further increasing intercalator concentrations) rendered the circular duplex DNA to be preferentially bound by the p53 again. CQ at concentrations modulating the closed circular DNA topology did not prevent the p53 from recognizing a specific target sequence within topologically unconstrained linear DNA. Experiments with DNA topoisomer distributions differing in their superhelix densities revealed the p53 to bind selectively DNA molecules possessing higher number of negative or positive superturns. Possible modes of the p53 binding to the negatively or positively supercoiled DNA and tentative biological consequences are discussed.

Sensing mispaired thymines in DNA heteroduplexes using an electroactive osmium marker: towards electrochemical SNP probing

AbstractA complex OsO4, 2,2′-bipyridine (Os,bipy), has been used for electroactive labeling of biopolymers as well as for probing of nucleic acids and protein structure and interactions. In DNA, Os,bipy forms electrochemically active adducts with pyrimidine nucleobases, exhibiting highly selective modification of thymine residues in single-stranded DNA. Here, we show that modification of rare thymine residues (one thymine among several tens of unreactive purine bases) can easily be detected by means of a simple ex situ voltammetric analysis using carbon electrodes. Based on this remarkable sensitivity of detection, Os,bipy has been used as an electroactive probe for unpaired and/or mismatched thymine residues within DNA heteroduplexes. Site-specific chemical modification of the DNA with the Os,bipy has allowed a clear distinction between perfectly base-paired DNA homoduplexes and mismatched heteroduplexes, as well as discrimination among heteroduplexes containing one or two mispaired thymines, a single thymine insertion, or combination of a mispair and an insertion. FigureSensing of single base mismatches using osmium tetroxide, 2,2′-bipyridine (Os,bipy). Os,bipy binds selectively to mispaired thymines, giving electroactive adducts easily detectable at a pyrolytic graphite electrode

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

AbstractIn 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. Figureᅟ

DNA modification with cisplatin affects sequence-specific DNA binding of p53 and p73 proteins in a target site-dependent manner.

Proteins p53 and p73 act as transcription factors in cell cycle control, regulation of cell development and/or in apoptotic pathways. Both proteins bind to response elements (p53 DNA‐binding sites), typically consisting of two copies of a motif RRRCWWGYYY. It has been demonstrated previously that DNA modification with the antitumor drug cisplatin inhibits p53 binding to a synthetic p53 DNA‐binding site. Here we demonstrate that the effects of global DNA modification with cisplatin on binding of the p53 or p73 proteins to various p53 DNA‐binding sites differed significantly, depending on the nucleotide sequence of the given target site. The relative sensitivities of protein–DNA binding to cisplatin DNA treatment correlated with the occurrence of sequence motifs forming stable bifunctional adducts with the drug (namely, GG and AG doublets) within the target sites. Binding of both proteins to mutated p53 DNA‐binding sites from which these motifs had been eliminated was only negligibly affected by cisplatin treatment, suggesting that formation of the cisplatin adducts within the target sites was primarily responsible for inhibition of the p53 or p73 sequence‐specific DNA binding. Distinct effects of cisplatin DNA modification on the recognition of different response elements by the p53 family proteins may have impacts on regulation pathways in cisplatin‐treated cells.

Enzyme-linked electrochemical detection of DNA fragments amplified by PCR in the presence of a biotinylated deoxynucleoside triphosphate using disposable pencil graphite electrodes

In this report, we present a simple electrochemical detection protocol for the detection of specific PCR-amplified DNA fragments, based on incorporation of biotin tags into DNA amplicons during PCR run in the presence of a biotinylated nucleoside triphosphate. For detection, an enzyme-linked electrochemical system involving streptavidin–alkaline phosphatase conjugate attached to the biotinylated DNA, adsorbed at the surface of a disposable pencil graphite electrode, is used. The enzyme converts an inactive indicator, 1-naphthyl phosphate, into electrochemically oxidizable indicator 1-naphthol that is subsequently detected. Excellent selectivity of this fast, facile, and inexpensive analysis not requiring any sophisticated electrode modification and its applicability for off-line monitoring of DNA amplification is demonstrated. Applications of the technique include detection of the presence of specific nucleotide sequences in biological samples, such as sequences related to pathogenic microorganism or transgenes.Graphical abstract