Modified dATP (2'-deoxyadenosine-5'-triphosphate) and dUTP (2'-deoxyuridine-5'-triphosphate) bearing ferrocene (Fc) labels linked via a conjugate acetylene spacer were prepared by single-step aqueous-phase cross-coupling reactions of 7-iodo-7-deaza-dATP or 5-iodo-dUTP with ethynylferrocene. The Fc-labeled dNTPs were good substrates for DNA polymerases and were efficiently incorporated to DNA by primer extension (PEX). Electrochemical analysis of the 2'-deoxyribonucleoside triphosphates (dNTPs) and PEX products revealed significant differences in redox potentials of the Fc label bound either to U or to 7-deazaA and between isolated dNTPs and conjugates incorporated to DNA. Prospective bioanalytical applications are outlined.
Hereditary neurodegenerative diseases are connected with the expansion of trinucleotide repetitive sequences in genomic DNA. Molecular diagnosis of these diseases is based on the determination of the triplet repeat length. Currently used methods involve PCR amplification followed by electrophoretic determination of the amplicon size. We propose a novel electrochemical technique based on hybridization of target DNA (tDNA) immobilized at magnetic beads with a reporter probe (RP) complementary to the triplet repeats (12 units per RP). The biotin-labeled RP is detected via an enzyme-linked electrochemical assay involving binding of streptavidin-alkaline phosphatase conjugate and transformation of electroinactive 1-naphthyl phosphate to electroactive 1-naphthol. Pyrimidine residues within sequences flanking the homopurine (GAA)n repeat in tDNA are premodified with osmium tetroxide, 2,2'-bipyridine (Os,bipy), introducing electroactive labels in tDNA. The length of the triplet expansion is calculated from the ratio of the intensities of electrochemical signals of hybridized RP/tDNA-Os,bipy. The normalized signal increases linearly with the repeat length between 0 and about 200 triplet units, allowing for discrimination between normal, premutated, and mutated alleles. Application of this method for the detection of the asymptomatic heterozygous carrier of expanded alleles is demonstrated.
Labeling of oligonucleotide reporter probes (RP) with electroactive markers has frequently been utilized in electrochemical detection of DNA hybridization. Osmium tetroxide complexes with tertiary amines (Os,L) bind covalently to pyrimidine (predominantly thymine) bases in DNA, forming stable, electrochemically active adducts. We propose a technique of electrochemical "multicolor" DNA coding based on RP labeling with Os,L markers involving different nitrogenous ligands (such as 2,2' -bipyridine, 1,10-phenanthroline derivatives or N,N,N',N'-tetramethylethylenediamine). At carbon electrodes the Os,L-labeled RPs produce specific signals, with the potentials of these differing depending on the ligand type. When using Os,L markers providing sufficiently large differences in their peak potentials, parallel analysis of multiple target DNA sequences can easily be performed via DNA hybridization at magnetic beads followed by voltammetric detection at carbon electrodes. Os,L labeling of oligonucleotide probes comprising a segment complementary to target DNA and an oligo(T) tail (to be modified with the osmium complex) does not require any organic chemistry facilities and can be achieved in any molecular biological laboratory. We also for the first time show that this technology can be used for labeling of oligonucleotide probes hybridizing with target DNAs that contain both purine and pyrimidine bases.
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.
Primer extension is used to incorporate labeled nucleoside triphosphates into oligonucleotides (ONs). Aminophenyl and nitrophenyl modifications serve as electrochemical labels that are detectable by either oxidation or reduction, respectively (see representation of the ON double strand and redox curves). The redox potentials of the labels differ depending on the nucleobase and respond to incorporation into ONs.
Target DNAs, including a 71-mer oligonucleotide, a PCR product and a plasmid DNA, all containing oligo(A) stretches, were hybridized at magnetic Dynabeads oligo(dT) 25 (DBT). The hybridization events were detected using a technique based on chemical modification of the target DNA with a complex of osmium tetroxide with 2,2'-bipyridine (Os, bipy) and voltammetric detection at carbon electrodes. DNA was modified with Os, bipy prior to capture at DBT, at the beads, or after release from the beads. In the latter case, DNA-Os, bipy was detected in the reaction mixture using adsorptive transfer stripping voltammetry involving extraction of unreacted Os, bipy from the electrode by organic solvents. Pre-labeling of the target plasmid DNA and the PCR product with Os, bipy significantly increased the yield of DNA captured at the beads. Tens of femtomoles of both short (the 71-mer oligonucleotide) and long (the 3-kilobase plasmid) target DNAs in a 20-microliter hybridization sample can be easily detected by means of these techniques. Various carbon electrode materials, including pyrolytic graphite (PGE), highly oriented pyrolytic graphite (HOPGE), carbon paste (CPE), glassy carbon and pencil graphite, were tested regarding their suitability for the detection of osmium-labeled DNA. Among them, PGE and HOPGE appeared usable in the measurements of both purified DNA-Os, bipy and its mixtures with unreacted Os, bipy while CPE was suitable for the detection purified osmium-labeled DNA.
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