PCR Incorporation of Polyoxometalate Modified Deoxynucleotide Triphosphates and Their Application in Molecular Electrochemical Sensing of Yersinia pestis

Abstract

Redox-labeled nucleotides are of increasing interest for the fabrication of next generation molecular tools and should meet requirements of being thermally stable, sensitive, and compatible with polymerase-mediated incorporation while also being electrochemically discriminable. The synthesis and characterization of Keggin and Dawson polyoxometalate-deoxynucleotide (POM-dNTP) bioconjugates linked through 7-deaza-modified purines is described. The modified POM-dNTPs were used for polymerase-based amplification of a DNA sequence specific for Yersinia pestis and the amplified DNA detected using an electrochemical DNA sensor. This highlights the potential of polyoxometalates as thermally stable, sensitive and polymerase-compatible redox labels for exploitation in bioanalytical applications.

Enhancing the biological activity of polyoxometalate–peptide nano-fibrils by spacer design

“…4 Nevertheless, the potential of hybrid POMs for cell targeting is yet undisclosed, and only a few bio-conjugates are known. [5][6][7] Among hybrid POMs, the Anderson-Evans [XMo 6 O 24 ] 3À polyoxomolybdates are disc-shaped polyanions of approximately D 3d symmetry, with average dimensions of 8.6 Â 8.6 Â 2.7 A. 8 Being oxidatively and hydrolytically stable, they are suitable for biomedical applications.…”

Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides

“…From the portfolio of our modified dNTPs bearing polyoxometalates, 54 benzofurazane, nitrobenzene, 55 , 56 and other electroactive substituents, we selected ferrocenylethynyl derivatives of dNTPs, which are available with all four nucleobases ( Figure 1 b). 49 − 51 , 57 , 58 …”

“…This may be attributable to a better substrate specificity of the Klenow (exo-) DNA polymerase for dA Fc TP as compared to dG Fc TP and dU Fc TP, as well as the possible steric hindrance between consecutive ferrocene-labeled bases at homopolymeric regions of the amplicon, which could inhibit the elongation at higher percentages of dN Fc TP, as was previously observed for other bulky electroactive labels. 54 …”

Duplex Electrochemical DNA Sensor to Detect Bacillus anthracis CAP and PAG DNA Targets Based on the Incorporation of Tailed Primers and Ferrocene-Labeled dATP

“…Several redox labeled dNTPs have been reported including ferrocene, 16 anthraquinone, 17 benzofurazane, 18 and polyoxometalates. 19 Ferrocene is particularly of interest because it is a reversible and stable label with redox peaks within the potential window of a gold electrode, and furthermore, Fc-labeled dNTPs are efficiently incorporated by several DNA polymerases such as Klenow (exo-), DyNAzyme, 16 and Vent (exo-). 20 We have chosen 7-ferrocenyl-ethynyl-7-deaza-dATP ( dA EFc TP ) because it was previously shown that 7-alkynyl-7-deazaadeninen are particularly good substrates for polymerases, even better than natural dATP.…”