Electrochemistry of Cobalta Bis(dicarbollide) Ions Substituted at Carbon Atoms with Hydrophilic Alkylhydroxy and Carboxy Groups.

In this study we explore the effect on the electrochemical signals in aqueous buffers of the presence of hydrophilic alkylhydroxy and carboxy groups on the carbon atoms of cobalta bis(dicarbollide) ions. The oxygen-containing exo-skeletal substituents of cobalta bis(dicarbollide) ions belong to the perspective building blocks that are considered for bioconjugation. Carbon substitution provides wider versatility and applicability in terms of the flexibility of possible chemical pathways. However, until recently, the electrochemistry of compounds substituted only on boron atoms could be studied, due to the unavailability of carbon-substituted congeners. In the present study, electrochemistry in aqueous phosphate buffers is considered along with the dependence of electrochemical response on pH and concentration. The compounds used show electrochemical signals around -1.3 and +1.1 V of similar or slightly higher intensities than in the parent cobalta bis(dicarbollide) ion. The signals at positive electrochemical potential correspond to irreversible oxidation of the boron cage (the C2B9 building block) and at negative potential correspond to the reversible redox process of (CoIII/CoII) at the central atom. Although the first signal is typically sharp and its potential can be altered by a number of substituents, the second signal is complex and is composed of three overlapping peaks. This signal shows sigmoidal character at higher concentrations and may be used as a diagnostic tool for aggregation in solution. Surprisingly enough, the observed effects of the site of substitution (boron or carbon) and between individual groups on the electrochemical response were insignificant. Therefore, the substitutions would preserve promising properties of the parent cage for redox labelling, but would not allow for the further tuning of signal position in the electrochemical window.

Carborane- or Metallacarborane-Linked Nucleotides for Redox Labeling. Orthogonal Multipotential Coding of all Four DNA Bases for Electrochemical Analysis and Sequencing.

We report a series of 2'-deoxyribonucleoside triphosphates bearing dicarba-nido-undecaborate ([C2B9H11]1-), [3,3'-iron-bis(1,2-dicarbollide)]- (FESAN, [Fe(C2B9H11)2]2-) or [3,3'-cobalt-bis(1,2-dicarbollide)]- (COSAN, [Co(C2B9H11)2]2-) groups prepared either through the Sonogashira cross-coupling or the CuAAC click reaction. The modified dNXTPs were substrates for KOD XL DNA polymerase in enzymatic synthesis of modified DNA through primer extension (PEX). The nido-carborane- and FESAN-modified nucleotides gave analytically useful oxidation signals in square-wave voltammetry and were used for redox labeling of DNA. The redox-modified DNA probes were prepared by PEX using tailed primers and were hybridized to electrode (gold or glassy carbon) containing capture oligonucleotides. The combination of nido-carborane- and FESAN-linked nucleotides with 7-ferrocenylethynyl-7-deaza-dATP and 7-deaza-dGTP allowed polymerase synthesis of DNA fully modified at all four nucleobases, and each of the redox labels gave four differentiable and ratiometric signals in voltammetry. Thus, the combination of these four redox labels constitutes the first fully orthogonal redox coding of all four canonical nucleobases, which can be used for determination of nucleobase composition of short DNA stretches in one simple PEX experiment with electrochemical readout.

Vicinal Diol-Tethered Nucleobases as Targets for DNA Redox Labeling with Osmate Complexes.

Six-valent osmium (osmate) complexes with nitrogenous ligands have previously been used for the modification and redox labeling of biomolecules involving vicinal diol moieties (typically, saccharides or RNA). In this work, aliphatic (3,4-dihydroxybutyl and 3,4-dihydroxybut-1-ynyl) or cyclic (6-oxo-6-(cis-3,4-dihydroxypyrrolidin-1-yl)hex-2-yn-1-yl, PDI) vicinal diols are attached to nucleobases to functionalize DNA for subsequent redox labeling with osmium(VI) complexes. The diol-linked 2'-deoxyribonucleoside triphosphates were used for the polymerase synthesis of diol-linked DNA, which, upon treatment with K2 OsO3 and bidentate nitrogen ligands, gave the desired Os-labeled DNA, which were characterized by means of the gel-shift assay and ESI-MS. Through ex situ square-wave voltammetry at a basal plane pyrolytic graphite electrode, the efficiency of modification/labeling of individual diols was evaluated. The results show that the cyclic cis-diol (PDI) was a better target for osmylation than that of the flexible aliphatic ones (alkyl- or alkynyl-linked). The osmate adduct-specific voltammetric signal obtained for OsVI -treated DNA decorated with PDI showed good proportionality to the number of PDI per DNA molecule. The OsVI reagents (unlike OsO4 ) do not attack nucleobases; thus offering specificity of modification on the introduced glycol targets.

Tuning of Oxidation Potential of Ferrocene for Ratiometric Redox Labeling and Coding of Nucleotides and DNA.

Three sets of 7-deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dNFcX TPs were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox-labelled DNA probes. Square-wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc-labelled A (dAFc ) and FcPa-labelled C (dCFcPa ) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox-labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dAFc and dCFcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.

Phenothiazine-linked nucleosides and nucleotides for redox labelling of DNA.

Nucleosides and 2'-deoxyribonucleoside triphosphates (dNTPs) bearing phenothiazine (PT) attached to a nucleobase (cytosine or 7-deazaadenine) either directly or through an acetylene linker were prepared through Suzuki or Sonogashira cross-coupling and triphosphorylation, and were studied as building blocks for polymerase construction of modified DNA. The directly PT-substituted dNTPs were better substrates for polymerases than the alkyne-linked dNTPs but all of them were used in enzymatic synthesis of DNA using primer extension, nicking enzyme amplification, PCR or 3'-tail labelling by terminal deoxynucleotidyl transferase. The phenothiazine served as an oxidizable redox label (giving two analytically useful signals of oxidation on electrode) for nucleosides and DNA and was also used in orthogonal combination with previously developed benzofurazane or nitrophenyl labels for redox coding of DNA bases. Therefore, the title PT-linked dNTPs are useful additions to the portfolio of nucleotides for enzymatic synthesis of redox-labelled DNA for electrochemical analysis.

G-quadruplex-based structural transitions in 15-mer DNA oligonucleotides varying in lengths of internal oligo(dG) stretches detected by voltammetric techniques.

Electrochemical methods, particularly when applied in connection with mercury-containing electrodes, are excellent tools for studying nucleic acids structure and monitoring structural transitions. We studied the effect of the length of the central (dG) n stretch (varying from 0 to 15 guanine residues) in 15-mer oligodeoxynucleotides (ODN, G0 to G15) on their electrochemical and interfacial behavior at mercury and carbon electrodes. The intensity of guanine oxidation signal at the carbon electrode (peak G(ox)) was observed to increase continuously with number of guanines between 0 and 15, with only a slight positive shift for ODNs with seven or more guanines in the central segment. Very different effects were observed when the peak G(HMDE) was measured at the mercury electrode. Intensity of the latter signal increased with number of guanines up to G5, and decreased sharply with further elongation of the (dG) n stretch. CD spectroscopy and electrophoresis experiments revealed formation of parallel intermolecular quadruplex structures for ODNs containing five or more G residues. Further measurements made by cyclic and alternating-current voltammetry revealed a strong influence of the ODN structure on their behavior at electrically charged surfaces.

Azidophenyl as a click-transformable redox label of DNA suitable for electrochemical detection of DNA-protein interactions.

New redox labelling of DNA by an azido group which can be chemically transformed to nitrophenyltriazole or silenced to phenyltriazole was developed and applied to the electrochemical detection of DNA-protein interactions. 5-(4-Azidophenyl)-2'-deoxycytidine and 7-(4-azidophenyl)-7-deaza-2'-deoxyadenosine nucleosides were prepared by aqueous-phase Suzuki cross-coupling and converted to nucleoside triphosphates (dNTPs) which served as substrates for incorporation into DNA by DNA polymerase. The azidophenyl-modified nucleotides and azidophenyl-modified DNA gave a strong signal in voltammetric studies, at -0.9 V, due to reduction of the azido function. The Cu-catalyzed click reaction of azidophenyl-modified nucleosides or azidophenyl-modified DNA with 4-nitrophenylacetylene gave nitrophenyl-substituted triazoles, exerting a reduction peak at -0.4 V under voltammetry, whereas the click reaction with phenylacetylene gave electrochemically silent phenyltriazoles. The transformation of the azidophenyl label to nitrophenyltriazole was used for electrochemical detection of DNA-protein interactions (p53 protein) since only those azidophenyl groups in the parts of the DNA not shielded by the bound p53 protein were transformed to nitrophenyltriazoles, whereas those covered by the protein were not.

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

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.

Aqueous Heck cross-coupling preparation of acrylate-modified nucleotides and nucleoside triphosphates for polymerase synthesis of acrylate-labeled DNA.

Aqueous-phase Heck coupling methodology was developed for direct attachment of butyl acrylate to 5-iodoracil, 5-iodocytosine, 7-iodo-7-deazaadenine, and 7-iodo-7-deazaguanine 2'-deoxyribonucleoside 5'-O-monophosphates (dNMPs) and 5'-O-triphosphates (dNTPs) and compared with the classical approach of phosphorylation of the corresponding modified nucleosides. The 7-substituted 7-deazapurine nucleotides (dA(BA)MP, dA(BA)TP, dG(BA)MP, and dG(BA)TP) were prepared by the direct Heck coupling of nucleotides in good yields (35-55%), whereas the pyrimidine nucleotides reacted poorly and the corresponding BA-modified dNTPs were prepared by triphosphorylation of the modified nucleosides. The acrylate-modified dN(BA)TPs (N = A, C, and U) were good substrates for DNA polymerases and were used for enzymatic synthesis of acrylate-modified DNA by primer extension, whereas dG(BA)TP was an inhibitor of polymerases. The butyl acrylate group was found to be a useful redox label giving a strong reduction peak at -1.3 to -1.4 V in cyclic voltammetry.

Benzofurazane as a new redox label for electrochemical detection of DNA: towards multipotential redox coding of DNA bases.

Benzofurazane has been attached to nucleosides and dNTPs, either directly or through an acetylene linker, as a new redox label for electrochemical analysis of nucleotide sequences. Primer extension incorporation of the benzofurazane-modified dNTPs by polymerases has been developed for the construction of labeled oligonucleotide probes. In combination with nitrophenyl and aminophenyl labels, we have successfully developed a three-potential coding of DNA bases and have explored the relevant electrochemical potentials. The combination of benzofurazane and nitrophenyl reducible labels has proved to be excellent for ratiometric analysis of nucleotide sequences and is suitable for bioanalytical applications.

Polymerase synthesis of oligonucleotides containing a single chemically modified nucleobase for site-specific redox labelling.

Enzymatic construction of single-nucleobase redox-labelled oligonucleotides was developed either based on polymerase incorporation of a single modified nucleoside triphosphate (dNTP) followed by primer extension (PEX) with natural dNTPs or based on PEX with a biotinylated one-nucleotide overhang template, magnetoseparation and the second PEX with a full-length template.

Anthraquinone as a redox label for DNA: synthesis, enzymatic incorporation, and electrochemistry of anthraquinone-modified nucleosides, nucleotides, and DNA.

Modified 2'-deoxynucleosides and deoxynucleoside triphosphates (dNTPs) bearing anthraquinone (AQ) attached through an acetylene or propargylcarbamoyl linker at the 5-position of pyrimidine (C) or at the 7-position of 7-deazaadenine were prepared by Sonogashira cross-coupling of halogenated dNTPs with 2-ethynylanthraquinone or 2-(2-propynylcarbamoyl)anthraquinone. Polymerase incorporations of the AQ-labeled dNTPs into DNA by primer extension with KOD XL polymerase have been successfully developed. The electrochemical properties of the AQ-labeled nucleosides, nucleotides, and DNA were studied by cyclic and square-wave voltammetry, which show a distinct reversible couple of peaks around -0.4 V that make the AQ a suitable redox label for DNA.

Alkylsulfanylphenyl derivatives of cytosine and 7-deazaadenine nucleosides, nucleotides and nucleoside triphosphates: synthesis, polymerase incorporation to DNA and electrochemical study.

Aqueous Suzuki-Miyaura cross-coupling reactions of halogenated nucleosides, nucleotides and nucleoside triphosphates derived from 5-iodocytosine and 7-iodo-7-deazaadenine with methyl-, benzyl- and tritylsufanylphenylboronic acids gave the corresponding alkylsulfanylphenyl derivatives of nucleosides and nucleotides. The modified nucleoside triphosphates were incorporated into DNA by primer extension by using Vent(exo-) polymerase. The electrochemical behaviour of the alkylsulfanylphenyl nucleosides indicated formation of compact layers on the electrode. Modified nucleotides and DNA with incorporated benzyl- or tritylsulfanylphenyl moieties produced signals in [Co(NH(3))(6)](3+) ammonium buffer, attributed to the Brdicka catalytic response, depending on the negative potential applied. Repeated constant current chronopotentiometric scans in this medium showed increased Brdicka catalytic response, which suggests the deprotection of the alkylsulfanyl derivatives to free thiols under the conditions.

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

A complex OsO(4), 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.

Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl transferase. Application in electrochemical DNA hybridization and protein-DNA binding assays.

A simple approach to DNA tail-labelling using terminal deoxynucleotidyl transferase and modified deoxynucleoside triphosphates is presented. Amino- and nitrophenyl-modified dNTPs were found to be good substrates for this enzyme giving 3'-end stretches of different lengths depending on the nucleotide and concentration. 3-Nitrophenyl-7-deazaG was selected as the most useful label because its dNTP was efficiently incorporated by the transferase to form long tail-labels at any oligonucleotide. Accumulation of many nitrophenyl tags per oligonucleotide resulted in a considerable enhancement of voltammetric signals due to the nitro group reduction, thus improving the sensitivity of electrochemical detection of the tail-labelled probes. We demonstrate a perfect discrimination between complementary and non-complementary target DNAs sequences by tail-labelled hybridization probes as well as the ability of tumour suppressor p53 protein to recognize a specific binding site within tail-labelled DNA substrates, making the methodology useful in electrochemical DNA hybridization and DNA-protein interaction assays.

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.

Determination of the level of DNA modification with cisplatin by catalytic hydrogen evolution at mercury-based electrodes.

Electrochemical methods proved useful as simple and inexpensive tools for the analysis of natural as well as chemically modified nucleic acids. In particular, covalently attached metal-containing groups usually render the DNA well-pronounced electrochemical activity related to redox processes of the metal moieties, which can in some cases be coupled to catalytic hydrogen evolution at mercury or some types of amalgam electrodes. In this paper we used voltammetry at the mercury-based electrodes for the monitoring of DNA modification with cis-diamminedichloroplatinum (cisplatin), a representative of metallodrugs used in the treatment of various types of cancer or being developed for such purpose. In cyclic voltammetry at the mercury electrode, the cisplatin-modified DNA yielded catalytic currents the intensity of which reflected DNA modification extent. In square-wave voltammetry, during anodic polarization after prereduction of the cisplatinated DNA, a well-developed, symmetrical signal (peak P) was obtained. Intensity of the peak P linearly responded to the extent of DNA modification at levels relevant for biochemical studies (rb = 0.01-0.10, where rb is the number of platinum atoms bound per DNA nucleotide). We demonstrate a correlation between the peak P intensity and a loss of sequence-specific DNA binding by tumor suppressor protein p53, as well as blockage of DNA digestion by a restriction endonuclease Msp I (both caused by the DNA cisplatination). Application of the electrochemical technique in studies of DNA reactivity with various anticancer platinum compounds, as well as for an easy determination of the extent of DNA platination in studies of its biochemical effects, is discussed.

Base-modified DNA labeled by [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes: construction by polymerase incorporation of modified nucleoside triphosphates, electrochemical and luminescent properties, and applications.

Modified 2'-deoxynucleoside triphosphates (dNTPs) bearing [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes attached via an acetylene linker to the 5-position of pyrimidines (C and U) or to the 7-position of 7-deazapurines (7-deaza-A and 7-deaza-G) have been prepared in one step by aqueous cross-couplings of halogenated dNTPs with the corresponding terminal acetylenes. Polymerase incorporation by primer extension using Vent (exo-) or Pwo polymerases gave DNA labeled in specific positions with Ru(2+) or Os(2+) complexes. Square-wave voltammetry could be efficiently used to detect these labeled nucleic acids by reversible oxidations of Ru(2+/3+) or Os(2+/3+). The redox potentials of the Ru(2+) complexes (1.1-1.25 V) are very close to that of G oxidation (1.1 V), while the potentials of Os(2+) complexes (0.75 V) are sufficiently different to enable their independent detection. On the other hand, Ru(2+)-labeled DNA can be independently analyzed by luminescence. In combination with previously reported dNTPs bearing ferrocene, aminophenyl, and nitrophenyl tags, the Os-labeled dATP has been successfully used for "multicolor" redox labeling of DNA and for DNA minisequencing.

Novel base-functionalized DNA. Efficient methodology for construction and bioanalytical applications.

A novel efficient two-step methodology for the construction of base-functionalized DNA is based on direct aqueous cross-coupling reactions of unprotected nucleoside triphosphates followed by polymerase incorporation. Preliminary applications of the modified DNA in electrochemical detection and bioanalysis are outlined.

Effect of spin-orbit coupling on reduction potentials of octahedral ruthenium(II/III) and osmium(II/III) complexes.

Reduction potentials of several M(2+/3+) (M = Ru, Os) octahedral complexes, namely, [M(H2O)6](2+/3+), [MCl6](4-/3-), [M(NH3)6](2+/3+), [M(en)3](2+/3+) [M(bipy)3](2+/3+), and [M(CN)6](4-/3-), were calculated using the CASSCF/CASPT2/CASSI and MRCI methods including spin-orbit coupling (SOC) by means of first-order quasi-degenerate perturbation theory. It was shown that the effect of SOC accounts for a systematic shift of approximately -70 mV in the reduction potentials of the studied ruthenium (II/III) complexes and an approximately -300 mV shift for the osmium(II/III) complexes. SOC splits the sixfold-degenerate (2)T(2g) ground electronic state (in ideal octahedral symmetry) of the M(3+) ions into the E((5/2)g) Kramers doublet and G((3/2)g) quartet, which were calculated to split by 1354-1573 cm(-1) in the Ru(3+) complexes and 4155-5061 cm(-1) in the Os(3+) complexes. It was demonstrated that this splitting represents the main contribution to the stabilization of the M(3+) ground state with respect to the closed-shell (1)A(1g) ground state in M(2+) systems. Moreover, it was shown that the accuracy of the calculated reduction potentials depends on the calculated solvation energies of both the oxidized and reduced forms. For smaller ligands, it involves explicit inclusion of the second solvation sphere into the calculations, whereas implicit solvation models yield results of sufficient accuracy for complexes with larger ligands. In such cases (e.g., [M(bipy)3](2+/3+) and its derivatives), very good agreement between the calculated (SOC-corrected) values of the reduction potentials and the available experimental values was obtained. These results led us to the conclusion that especially for Os(2+/3+) complexes, inclusion of SOC is necessary to avoid systematic errors of approximately 300 mV in the calculated reduction potentials.