We designed and synthesized a set of six 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) bearing functional groups mimicking amino acid side chains in enzyme active sites (OH, SH, COOH, and imidazole) attached to position 5 of pyrimidines or position 7 of 7-deazapurines through different linkers. These modified dNTPs were studied as substrates in enzymatic synthesis of modified and hypermodified DNA using several DNA polymerases. In primer extension (PEX), all modified dNTPs provided DNA containing one, two, three, or, (all) four modified nucleotides each bearing a different modification, although the thiol-modified dNTPs were worse substrates compared to the others. In PCR, we observed exponential amplification for any combination of one, two, or three nonsulfur dNTPs but the thiol-modified dNTP did not work well in any combinations. Sequencing of the hypermodified DNA confirmed the good fidelity of the incorporation of all the modified nucleotides. This set of modified dNTPs extends the portfolio of building blocks for prospective use in selections of functional nucleic acids.

• Keywords
• DNA, enzymatic syntheses, nucleotides, polymerases,
• MeSH
• DNA-Directed DNA Polymerase * metabolism   chemistry   MeSH
• DNA * chemistry   chemical synthesis   MeSH
• Imidazoles * chemistry   MeSH
• Catalytic Domain MeSH
• Carboxylic Acids * chemistry   MeSH
• Polymerase Chain Reaction MeSH
• Sulfhydryl Compounds * chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 7-deazapurine MeSH Browser
• DNA-Directed DNA Polymerase * MeSH
• DNA * MeSH
• imidazole MeSH Browser
• Imidazoles * MeSH
• Carboxylic Acids * MeSH
• Purines MeSH
• Sulfhydryl 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