We designed and synthesized a set of four 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) derived from 5-substituted pyrimidines and 7-substituted 7-deazapurines bearing anionic substituents (carboxylate, sulfonate, phosphonate, and phosphate). The anion-linked dNTPs were used for enzymatic synthesis of modified and hypermodified DNA using KOD XL DNA polymerase containing one, two, three, or four modified nucleotides. The polymerase was able to synthesize even long sequences of >100 modified nucleotides in a row by primer extension (PEX). We also successfully combined two anionic and two hydrophobic dNTPs bearing phenyl and indole moieties. In PCR, the combinations of one or two modified dNTPs gave exponential amplification, while most of the combinations of three or four modified dNTPs gave only linear amplification in asymmetric PCR. The hypermodified ONs were successfully re-PCRed and sequenced by Sanger sequencing. Biophysical studies including hybridization, denaturation, CD spectroscopy and molecular modelling and dynamics suggest that the presence of anionic modifications in one strand decreases the stability of duplexes while still preserving the B-DNA conformation, whilst the DNA hypermodified in both strands adopts a different secondary structure.

• MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA * chemistry   MeSH
• Nucleotides * chemistry   MeSH
• Pyrimidines MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA-Directed DNA Polymerase MeSH
• DNA * MeSH
• Nucleotides * MeSH
• Pyrimidines MeSH

The previously reported approach of orthogonal multipotential redox coding of all four DNA bases allowed only analysis of the relative nucleotide composition of short DNA stretches. Here, we present two methods for normalization of the electrochemical readout to facilitate the determination of the total nucleotide composition. The first method is based on the presence or absence of an internal standard of 7-deaza-2'-deoxyguanosine in a DNA primer. The exact composition of the DNA was elucidated upon two parallel analyses and the subtraction of the electrochemical signal intensities. The second approach took advantage of a 5'-viologen modified primer, with this fifth orthogonal redox label acting as a reference for signal normalization, thus allowing accurate electrochemical sequence analysis in a single read. Both approaches were tested using various sequences, and the voltammetric signals obtained were normalized using either the internal standard or the reference label and demonstrated to be in perfect agreement with the actual nucleotide composition, highlighting the potential for targeted DNA sequence analysis.

• MeSH
• DNA Primers MeSH
• DNA * chemistry   MeSH
• Nucleotides * chemistry   MeSH
• Oxidation-Reduction MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Primers MeSH
• DNA * MeSH
• Nucleotides * MeSH

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.

• Keywords
• DNA, electrochemistry, ferrocenes, nucleobases, redox labelling,
• MeSH
• Staining and Labeling methods   MeSH
• Cytidine Triphosphate chemistry   MeSH
• DNA Probes chemical synthesis   chemistry   MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Electrochemical Techniques MeSH
• Metallocenes chemistry   MeSH
• Nucleotides chemistry   MeSH
• Oxidation-Reduction MeSH
• Substrate Specificity MeSH
• Ferrous Compounds chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytidine Triphosphate MeSH
• DNA Probes MeSH
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH
• ferrocene MeSH Browser
• Metallocenes MeSH
• Nucleotides MeSH
• Ferrous Compounds MeSH

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.

• Publication type
• Journal Article MeSH

A set of 6 base-modified 2'-deoxyadenosine derivatives was incorporated to diverse DNA sequences by primer extension using Vent (exo-) polymerase and the influence of the modification on cleavage by diverse restriction endonucleases was studied. While 8-substituted (Br or methyl) adenine derivatives were well tolerated by the restriction enzymes and the corresponding sequences were cleaved, the presence of 7-substituted 7-deazaadenine in the recognition sequence resulted in blocking of cleavage by some enzymes depending on the nature and size of the 7-substituent. All sequences with modifications outside of the recognition sequence were perfectly cleaved by all the restriction enzymes. The results are useful both for protection of some sequences from cleavage and for manipulation of functionalized DNA by restriction cleavage.

• MeSH
• Deoxyadenine Nucleotides chemistry   MeSH
• Deoxyadenosines chemistry   MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA chemistry   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Deoxyribonucleases, Type II Site-Specific metabolism   MeSH
• DNA Cleavage * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2'-deoxyadenosine MeSH Browser
• Deoxyadenine Nucleotides MeSH
• Deoxyadenosines MeSH
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH
• Deoxyribonucleases, Type II Site-Specific MeSH
• Tli polymerase MeSH Browser

Electrochemical enzyme-linked techniques for sequence-specific DNA sensingare presented. These techniques are based on attachment of streptavidin-alkalinephosphatase conjugate to biotin tags tethered to DNA immobilized at the surface ofdisposable screen-printed carbon electrodes (SPCE), followed by production andelectrochemical determination of an electroactive indicator, 1-naphthol. Via hybridizationof SPCE surface-confined target DNAs with end-biotinylated probes, highly specificdiscrimination between complementary and non-complementary nucleotide sequences wasachieved. The enzyme-linked DNA hybridization assay has been successfully applied inanalysis of PCR-amplified real genomic DNA sequences, as well as in monitoring of planttissue-specific gene expression. In addition, we present an alternative approach involvingsequence-specific incorporation of biotin-labeled nucleotides into DNA by primerextension. Introduction of multiple biotin tags per probe primer resulted in considerableenhancement of the signal intensity and improvement of the specificity of detection.

• Keywords
• DNA hybridization, PCR, electrochemical detection, enzyme-linked assay, gene expression, primer extension,
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH