Label-free electrochemical monitoring of DNA ligase activity

This study presents a simple, label-free electrochemical technique for the monitoring of DNA ligase activity. DNA ligases are enzymes that catalyze joining of breaks in the backbone of DNA and are of significant scientific interest due to their essential nature in DNA metabolism and their importance to a range of molecular biological methodologies. The electrochemical behavior of DNA at mercury and some amalgam electrodes is strongly influenced by its backbone structure, allowing a perfect discrimination between DNA molecules containing or lacking free ends. This variation in electrochemical behavior has been utilized previously for a sensitive detection of DNA damage involving the sugar-phosphate backbone breakage. Here we show that the same principle can be utilized for monitoring of a reverse process, i.e., the repair of strand breaks by action of the DNA ligases. We demonstrate applications of the electrochemical technique for a distinction between ligatable and unligatable breaks in plasmid DNA using T4 DNA ligase, as well as for studies of the DNA backbone-joining activity in recombinant fragments of E. coli DNA ligase

Sensitive electrochemical assays of DNA structure

Electrochemical methods have been used to study the structure and function of nucleic acids for more than 50 years. These approaches complement other experimental techniques, which we illustrate by using examples from studies of processes involved in the repair of DNA damage. The excellent sensitivity of the electrochemical approaches makes them good candidates for use as biosensors of a wide range of molecules and biological processes

Biophysical and electrochemical studies of protein-nucleic acid interactions

This review is devoted to biophysical and electrochemical methods used for studying protein-nucleic acid (NA) interactions. The importance of NA structure and protein-NA recognition for essential cellular processes, such as replication or transcription, is discussed to provide background for description of a range of biophysical chemistry methods that are applied to study a wide scope of protein-DNA and protein-RNA complexes. These techniques employ different detection principles with specific advantages and limitations and are often combined as mutually complementary approaches to provide a complete description of the interactions. Electrochemical methods have proven to be of great utility in such studies because they provide sensitive measurements and can be combined with other approaches that facilitate the protein-NA interactions. Recent applications of electrochemical methods in studies of protein-NA interactions are discussed in detail

Effects of oxidation agents and metal ions on binding of p53 to supercoiled DNA

Wild type human full length (f.l.) tumor suppressor p53 protein binds preferentially to supercoiled (sc) DNA in vitro both in the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on the agarose gel. Bands revealed by immunoblotting with antibody DO-1 corresponded to the ethidium stained retarded bands. The intensity and the number of bands of p53-scDNA complex were decreased by physiological concentrations of unchelated zinc ions. Nickel and cobalt ions inhibited binding of p53 to scDNA and to p53CON in linear DNA fragments less efficiently than zinc. Compared to the intrinsic zinc strongly bound to Cys 176, Cys 238, Cys 242 and His 179 in the p53 core domain, binding of additional Zn2+ to p53 was much weaker as shown by an easy removal of the latter ions by low concentrations of EDTA. Oxidation of the protein with diamide resulted in a decrease of the number of the retarded bands. Under the same conditions, no binding of oxidized p53 to p53CON in a linear DNA fragment was observed. In agreement with the literature oxidation of f.l. p53 with diamide was irreversible and was not reverted by an excess of DTT. We showed that in the presence of 0.1 mM zinc ions, oxidation of p53 became reversible. Other divalent cations tested (cadmium, cobalt, nickel) exhibited no such effect. We suggested that the irreversibility of p53 oxidation was due, at least in part, to the removal of intrinsic zinc from its position in the DNA binding domain (after oxidation of the three cysteines to which the zinc ion is coordinated in the reduced protein) accompanied by a change in the p53 conformation. Binding of C-terminal anti-p53 antibody also protected bacterially expressed protein against irreversible loss of activity due to diamide oxidation. Binding the human p53 core domain (segment 94-312) to scDNA greatly differed from that observed with the full-length p53. The core domain did not posses the ability to bind strongly to many sites in scDNA regardless of the presence or absence of p53CON suggesting involvement of some other domain (probably C-terminal) in binding of the full-length p53 to scDNA. Supershift experiments using antibodies against p53 N- or C-terminus suggested that in oxidized p53, scDNA binding through the C-terminus gained importance

DNA hybridization on membrane-modified carbon electrodes

The DNA-modified membrane electrode was prepared by casting a mixture of nitrocellulose (NC) with target DNA (tDNA) in organic solvent on glassy carbon electrode (GCE). Unlabeled polymerase chain reaction (PCR)-amplified human genomic sequence (628 bp) or synthetic oligodeoxynucleotides (ODNs) were used as tDNAs, creating a recognition layer. Biotinylated ODNs were used as hybridization probes to recognize specific nucleotide sequences. The hybridization events were detected via an enzyme-linked electrochemical assay involving binding of streptavidin-coupled alkaline phosphatase (SALP) to the biotin labels of the probe bound to tDNA. After the probe hybridization and SALP binding, the electrode was immersed into an electroinactive enzyme substrate (1-naphthyl phosphate). The alkaline phosphatase converted the inactive substrate into electroactive 1-naphthol that penetrated through the NC membrane to the GCE surface and was subsequently detected using an anodic voltammetric signal. The optimized method offered a good discrimination between complementary and nonspecific DNAs and yielded well-defined responses for both single-copy and repetitive tDNA sequences. In contrast to previously published methods using electrodes with mechanically attached membranes, the previously mentioned electrode is easily amenable to parallel DNA analysis. Copyright © Taylor & Francis, Inc

Terminology of bioanalytical methods (IUPAC Recommendations 2018)

Recommendations are given concerning the terminology of methods of bioanalytical chemistry. With respect to dynamic development particularly in the analysis and investigation of biomacromolecules, terms related to bioanalytical samples, enzymatic methods, immunoanalytical methods, methods used in genomics and nucleic acid analysis, proteomics, metabolomics, glycomics, lipidomics, and biomolecules interaction studies are introduced

Manganese Superoxide Dismutase: Guardian of the Powerhouse

The mitochondrion is vital for many metabolic pathways in the cell, contributing all or important constituent enzymes for diverse functions such as β-oxidation of fatty acids, the urea cycle, the citric acid cycle, and ATP synthesis. The mitochondrion is also a major site of reactive oxygen species (ROS) production in the cell. Aberrant production of mitochondrial ROS can have dramatic effects on cellular function, in part, due to oxidative modification of key metabolic proteins localized in the mitochondrion. The cell is equipped with myriad antioxidant enzyme systems to combat deleterious ROS production in mitochondria, with the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) acting as the chief ROS scavenging enzyme in the cell. Factors that affect the expression and/or the activity of MnSOD, resulting in diminished antioxidant capacity of the cell, can have extraordinary consequences on the overall health of the cell by altering mitochondrial metabolic function, leading to the development and progression of numerous diseases. A better understanding of the mechanisms by which MnSOD protects cells from the harmful effects of overproduction of ROS, in particular, the effects of ROS on mitochondrial metabolic enzymes, may contribute to the development of novel treatments for various diseases in which ROS are an important component

Effects of halide anions on adsorption and 2D condensation of 5-fluorocytosine at hanging mercury drop electrode

Self-assembled layers, and 2D condensed molecular films as their specific cases, are known to be strongly sensitive to external conditions such as temperature, concentration of self-assembling substance(s) in solution, as well as composition of the supporting electrolyte in terms of the kind and concentration of present ions. In this work we have studied effects of a series of four sodium halides on the 2D condensation of 5-fluorocytosine (5-FC) at the hanging mercury drop electrode. This fluorinated nucleobase was selected for the study with respect to its unique 2D condensation properties (previously described in NaCl), showing remarkable capacitance pits in two potential regions (one around the potential of zero charge and the other within highly negative potential region). NaF electrolyte was not able to support 2D condensation of 5-FC, while in NaI and NaBr the 2D condensation was facilitated in comparison with the NaCl electrolyte. Furthermore, we have studied the formation of the 2D condensed film at different temperatures and ac perturbation frequencies to obtaining a complex view into the 5-FC 2D condensation processes. Our results show great importance of the composition of supporting electrolyte, and/or other experimental conditions on the properties of 2D condensed layers.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Electrochemical sensors based on stationary electrodes and immobilized DNA or its fragments and the assessment of their analytical potentials

Amperometric biosensors were developed on the basis of stationary mercury-film and glassy-carbon electrodes and DNA or its fragments, oligodeoxynucleotides (ODNs), immobilized in a nitrocellulose matrix. Taking into account the high affinity of Cu(II) and Fe(III) ions to denatured DNA ((19.1 ± 0.1) ×105 and (1.4 ± 0.3) × 105 L/mol, respectively), a procedure was proposed for the voltammetric determination of these ions in natural materials and blood serum at a level of n × 10-11 M. This procedure involves analyte pre-concentration on a DNA-containing biosensor. An ODN-containing biosensor (DNA probe) was used in the study of DNA hybridization for the highly specific determination of its nucleotide sequence. © 2005 Pleiades Publishing, Inc

Electrochemical sensors based on stationary electrodes and immobilized DNA or its fragments and the assessment of their analytical potentials

Amperometric biosensors were developed on the basis of stationary mercury-film and glassy-carbon electrodes and DNA or its fragments, oligodeoxynucleotides (ODNs), immobilized in a nitrocellulose matrix. Taking into account the high affinity of Cu(II) and Fe(III) ions to denatured DNA ((19.1 ± 0.1) ×105 and (1.4 ± 0.3) × 105 L/mol, respectively), a procedure was proposed for the voltammetric determination of these ions in natural materials and blood serum at a level of n × 10-11 M. This procedure involves analyte pre-concentration on a DNA-containing biosensor. An ODN-containing biosensor (DNA probe) was used in the study of DNA hybridization for the highly specific determination of its nucleotide sequence. © 2005 Pleiades Publishing, Inc