Petra Horáková

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 Brdička catalytic response, depending on the negative potential applied. Repeated constant current chronopotentiometric scans in this medium showed increased Brdička catalytic response, which suggests the deprotection of the alkylsulfanyl derivatives to free thiols under the conditions.

Direct voltammetric analysis of DNA modified with enzymatically incorporated 7-deazapurines.

Nucleic acids studies use 7-deazaguanine (G*) and 7-deazaadenine (A*) as analogues of natural purine bases incapable of forming Hoogsteen base pairs, which prevents them from being involved in DNA triplexes and tetraplexes. Reduced propensity of the G*- and/or A*-modified DNA to form alternative DNA structures is utilized, for example, in PCR amplification of guanine-rich sequences. Both G* and A* exhibit significantly lower potentials of their oxidation, compared to the respective natural nucleobases. At carbon electrodes, A* yields an oxidation peak which is by about 200-250 mV less positive than the peak due to adenine, but coincides with oxidation peak produced by natural guanine residues. On the other hand, oxidation signal of G* occurs at a potential by about 300 mV less positive than the peak due to guanine, being well separated from electrochemical signals of any natural DNA component. We show that enzymatic incorporation of G* and A* can easily be monitored by simple ex situ voltammetric analysis of the modified DNA at carbon electrodes. Particularly G* is shown as an attractive electroactive marker for DNA, efficiently incorporable by PCR. While densely G*-modified DNA fragments exhibit strong quenching of fluorescence of SYBR dyes, commonly used as fluorescent indicators in both gel staining and real time PCR applications, the electrochemical detection provides G*-specific signal suitable for the quantitation of the amplified DNA as well as for the determination of the DNA modification extent. Determination of DNA amplicons based on the measurement of peak G*(ox) is not affected by signals produced by residual oligonucleotide primers or primary templates containing natural purines.

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.

Enzyme-linked electrochemical detection of DNA fragments amplified by PCR in the presence of a biotinylated deoxynucleoside triphosphate using disposable pencil graphite electrodes

In this report, we present a simple electrochemical detection protocol for the detection of specific PCR-amplified DNA fragments, based on incorporation of biotin tags into DNA amplicons during PCR run in the presence of a biotinylated nucleoside triphosphate. For detection, an enzyme-linked electrochemical system involving streptavidin–alkaline phosphatase conjugate attached to the biotinylated DNA, adsorbed at the surface of a disposable pencil graphite electrode, is used. The enzyme converts an inactive indicator, 1-naphthyl phosphate, into electrochemically oxidizable indicator 1-naphthol that is subsequently detected. Excellent selectivity of this fast, facile, and inexpensive analysis not requiring any sophisticated electrode modification and its applicability for off-line monitoring of DNA amplification is demonstrated. Applications of the technique include detection of the presence of specific nucleotide sequences in biological samples, such as sequences related to pathogenic microorganism or transgenes.Graphical abstract

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.