Emil Paleček

Electrochemistry of Nucleic Acids

Publisher Summary The electroactivity of nucleic acids was discovered several years ago. It was shown that at mercury electrodes, adenine and cytosine were reduced in ssDNA while guanine produced an anodic signal due to the oxidation of guanine reduction product. The DNA signals at mercury electrodes are highly sensitive to changes in DNA structure due to DNA denaturation and renaturation, as well as to minor structural changes resulting from DNA premelting and DNA damage. The changes in DNA structure are reflected not only by the DNA faradaic responses but also by non-faradaic signals due to adsorption/desorption of DNA. Several interesting principles have been used in the development of the DNA sensors—such as amplified electrochemical analysis, investigation of charge transport, and tracing of changes in conformation of DNA. The s ensors for DNA hybridization and DNA damage lead the field of the electrochemistry of nucleic acids. The chapter briefly summarizes some properties of the elimination voltammetry with linear scan (EVLS) method. Oscillographic polarography, at controlled alternating current, was used in the electrochemical analysis of nucleic acids.

Past, present and future of nucleic acids electrochemistry

Electrochemistry of nucleic acids was discovered about 40 years ago. During the first 15 years electrochemistry brought early evidence of DNA premelting and polymorphy of the DNA double helix. At present electrochemical methods working with stationary electrodes are able to detect DNA at attomol and in some cases, even at lower levels. A great progress in the development of electrochemical sensors for DNA hybridization and DNA damage achieved in recent years suggests that these sensors may soon become important tools in medicine and other areas of practical life of the 21st century.

Electrochemical biosensors for DNA hybridization and DNA damage

Recent trends in the development of DNA biosensors for nucleotide sequence-specific DNA hybridization and for the detection of the DNA damage are briefly reviewed. Changes in the redox signals of base residues in DNA immobilized at the surface of carbon or mercury electrodes can be used as a sign of the damage of DNA bases. Some compounds interacting with DNA can produce their own redox signals on binding to DNA. Covalently closed circular (usually supercoiled) DNA attached to the electrode surface can be used for a sensitive detection of a single break of the DNA sugar-phosphate backbone and for detection of agents cleaving the DNA backbone such as hydroxyl radicals, ionizing radiation, nucleases, etc. Using the peptide nucleic acid in the biosensor recognition layer greatly increased the specificity of the DNA hybridization biosensor making it possible to detect point mutations (single-base mismatches) in DNA.

Local supercoil-stabilized DNA structures

The DNA double helix exhibits local sequence-dependent polymorphism at the level of the single base pair and dinucleotide step. Curvature of the DNA molecule occurs in DNA regions with a specific type of nucleotide sequence periodicities. Negative supercoiling induces in vitro local nucleotide sequence-dependent DNA structures such as cruciforms, left-handed DNA, multistranded structures, etc. Techniques based on chemical probes have been proposed that make it possible to study DNA local structures in cells. Recent results suggest that the local DNA structures observed in vitro exist in the cell, but their occurrence and structural details are dependent on the DNA superhelical density in the cell and can be related to some cellular processes.

Magnetic beads as versatile tools for electrochemical DNA and protein biosensing

Magnetic beads (MBs) are versatile tools in the separation of nucleic acids, proteins and other biomacromolecules, their complexes and cells. In this article recent application of MBs in electrochemical biosensing and particularly in the development of DNA hybridization sensors is reviewed. In these sensors MBs serve not only for separation but also as a platform for optimized DNA hybridization. A hybridization event is detected separately at another surface, which is an electrode. The detection is based either on the intrinsic DNA electroactivity or on various kinds of DNA labeling, including chemical modification, enzyme tags, nanoparticles, electroactive beads, etc., greatly amplifying the signals measured. In addition to DNA hybridization, other kinds of biosensing in combination with MBs, such as DNA-protein interactions, are reviewed.

Interactions of antitumor drug daunomycin with DNA in solution and at the surface

Abstract The interaction of the antitumor drug daunomycin with double-stranded (ds) calf thymus DNA was studied in solution and at the electrode surface by means of cyclic voltammetry and particularly by constant-current chronopotentiometric stripping analysis (CPSA) with the carbon paste electrodes (CPE). As a result of intercalation of this drug between the base pairs in dsDNA, the CPSA daunomycin peak δ decreased and a new more positive shoulder δb appeared. This shoulder was attributed to the oxidation of the drug intercalated in DNA. Under the same conditions almost no changes in the DNA peak Gox (due to oxidation of guanine residues) were observed. It was shown that daunomycin adheres strongly to the bare CPE (resisting washing) so that a daunomycin-modified electrode can be easily prepared. Daunomycin immobilized at CPE interacted with DNA on immersion of the modified electrode into the dsDNA solution, showing a decrease of peak δ and a well-separated peak δb instead of the shoulder (which resulted from the interaction of DNA with daunomycin in solution). When the DNA-modified CPE was immersed into a daunomycin solution the peak Gox increased in dependence on daunomycin concentration or on the time of interaction of daunomycin with dsDNA at the electrode surface. Such changes in peak Gox were observed only at submicromolar concentrations of daunomycin. At higher daunomycin concentrations or at longer interaction time intervals a daunomycin peak appeared, which was substantially smaller and more positive than the peak of free daunomycin. The increase of the DNA peak Gox was attributed to interaction of daunomycin from the side of the DNA double helix not contacting the electrode surface. Such binding may induce changes in the DNA structure including bending of the DNA molecule which may result in the increase of peak Gox. Our results thus suggest that the interaction of daunomycin with DNA anchored at the surface may significantly differ from that with DNA in solution. The prospects of using of electroanalytical methods in studies of DNA–drug interactions are discussed.

Adsorptive stripping voltammetry of biomacromolecules with transfer of the adsorbed layer

Abstract A new analytical method, based on the adsorptive preconcentration of biomacromolecules on an electrode, the transfer of the adsorbed layer into a new medium (containing background electrolyte) and subsequent voltammetric analysis, is proposed. This method is called adsorptive transfer stripping (inverse) voltammetry, AdTSV (AdTIV). The adsorption of nucleic acids, some proteins, polysaccharides, and lipids on a mercury electrode can be carried out on open circuit from drops of solution; owing to this fact, it is possible to reduce the volume of the sample usually used in voltammetric analysis by a factor of fifty or more. In addition, AdTSV makes it possible (a) to carry out the voltammetric analysis of biomacromolecules adsorbed from media not suitable for voltammetric analysis of the conventional type, (b) to exploit the differences in adsorbability of substances to separate them on the electrode, (c) to study the interaction of biomacromolecules immobilised on the surface of the electrode with substances contained in the solution without the results of the voltammetric measurement being affected by the interactions in the bulk of the solution, and (d) to study the effect of electrode potential on the properties and interactions of the adsorbed macromolecules.

Adsorptive transfer stripping voltammetry: Determination of nanogram quantities of DNA immobilized at the electrode surface

In adsorptive transfer stripping voltammetry (AdTSV), DNA is first adsorbed at the electrode, the electrode is washed and transferred (with the adsorbed layer) in the medium not containing DNA, and voltammetric analysis is performed in this medium. Adsorption can be performed from a drop of DNA solution, which makes it possible to reduce the volume of the analyzed sample by two orders of magnitude as compared to that of conventional voltammetry. With the hanging mercury drop electrode the limit of detection of single-stranded DNA is below 0.1 micrograms/ml; thus if the adsorption is performed from a 10-microliter drop of DNA solution subnanogram quantities of single-stranded DNA are sufficient for the analysis. In AdTSV the behavior of single- and double-stranded DNAs markedly differ from each other in a manner similar to that in the conventional voltammetric or polarographic analysis; AdTSV can thus be used in DNA structure analysis. In AdTSV the DNA transport and its adsorption at the electrode are separated from the electrode process; due to this fact it is possible (a) to perform the voltammetric analysis of DNA from media not suitable for voltammetric analysis of the conventional type, (b) to study the interaction of immobilized DNA with other substances in solution without the results of the voltammetric analysis being influenced by DNA interactions in the bulk of solution, and (c) to exploit the differences of adsorbability of DNA and other substances in order to separate them on the electrode.

Electrochemistry of Nucleic Acids and Development of DNA Sensors

In recent years our knowledge regarding the nucleotide sequences of a number of genomes has increased tremendously. With the completion of an increasing number of genomic sequences attention is currently focused on how the sequence data might be interpreted in terms of the structure, function, and control of biological systems. It now appears that methods of electrochemical analysis may find application in this exciting research area.

Interaction of nucleic acids with electrically charged surfaces: II. Conformational changes in double-helical polynucleotides

The influence of adsorption of double-stranded (ds) DNA, ds RNA and homopolymeric pairs at a mercury electrode on conformation of these polynucleotides was studied. Changes in the polarographic reducibility of polynucleotides, which were followed by means of normal pulse polarography and linear sweep peak voltammetry at the dropping mercury electrode were exploited to indicate conformational changes. It was found that, as a consequence of adsorption of ds polynuclotides on the negatively charged electrode conformational changes similar to denaturation take place in a narrow potential region around -1.2 V (the region U). After sufficiently long time of the contact with the electrode (under our conditions about 10 s) these changes reach limiting values, which can approach total denaturation. Upon adsorption of ds polynucleotides on the electrode charged to more positive potentials than the region U either (1) no conformational changes occur or (2) only a small part of the polynucleotide (probably labile regions of the ds molecule) is very quickly denatured - the remainder of the molecule preserves its ds structure. Conformational changes of adsorbed ds polynucleotides are influenced by factors which change the stability of ds polynucleotides in solution. It is supposed that denaturation of ds polynucleotides in the region U might result from the strains connected with the repulsion of certain segments of the molecule anchored on the electrode from the negatively charged surface.