On 25th April 1941, at a meeting of the Czech Chemical Society in Prague, Jaroslav Heyrovský delivered his lecture "Use of oscillograph in polarography". This lecture was published in Chemické Listy in December 1941. His lecture marked the beginning of the oscillographic polarography with controlled alternating current (OP). In the first half of the 1950's, a simple instrument Polaroskop P 524 became available, stimulating wide application of OP as a method of fast and simple chemical analysis of various compounds, including biomolecules. In the 1960's and in the following decades a number of papers on chronopotentiometry were published but J. Heyrovsky's OP was almost forgotten. At present we can witness another step in the development of chronopotentiometry in the application of CPS in the analysis of biomacromolecules and particularly in the structure-sensitive analysis of proteins based on catalytic hydrogen evolution reaction (CHER). This analysis requires mercury-containing electrodes, such as HMDE (hanging mercury drop electrode) or solid amalgam electrodes, because the CHER has not been observed with any other electrode. We believe that Professor J. Heyrovský would be satisfied by the fruitful development of chronopotentiometry and its application in bioelectrochemistry, stimulated by his OP.
Electrochemistry of nucleic acid is at present a booming field producing about 800 papers published per year. First papers in this field were published in 1958–1961 in Brno (Czech Republic) showing that purine and pyrimidine base residues in single-stranded DNA and RNA were reduced at Hg electrodes and the guanine residue produced an anodic signal when cyclic modes were used. The reduction sites of the base residues in native double-stranded (ds) DNA are hidden in the interior of the dsDNA molecule, which made their reduction difficult. At that time oscillographic polarography (ac chronopotentiometry) showed excellent sensitivity to changes in DNA structure and allowed to investigate DNA denaturation and hybridization. Later on also other electrochemical methods and electrodes were applied. In the following three decades basic principles were found which are at present used in the development of DNA hybridization sensors.
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- chromozomy chemie MeSH
- dějiny 20. století MeSH
- dějiny 21. století MeSH
- DNA * analýza chemie MeSH
- elektrochemické techniky dějiny metody MeSH
- elektrochemie * dějiny metody MeSH
- plazmidy analýza chemie MeSH
- polarografie dějiny metody MeSH
- potenciometrie dějiny metody MeSH
- RNA transferová analýza chemie MeSH
- RNA virová analýza chemie MeSH
- Check Tag
- dějiny 20. století MeSH
- dějiny 21. století MeSH
- Publikační typ
- historické články MeSH
- práce podpořená grantem MeSH
Elektrochemická analýza nukleových kyselin, bílkovin i polysacharidů představuje zajímavou, i když zatím méně používanou alternativu ve srovnání se stávajícími metodami založenými zejména na optické detekci. Nabízí totiž relativně levnou, rychlou a přístrojově nenáročnou možnost paralelní detekce na miniaturizovaných čipech, ideální pro personalizovanou medicínu 21. století. Elektrochemie nukleových kyselin umožňuje např. detekci konkrétních sekvencí DNA (pro určení genů, stanovení přítomnosti bakterií a virů, atd.), analýzu poškození DNA a interakcí s jinými molekulami, DNA metylaci, nebo detekci mikroRNA jako nádorových biomarkerů. V elektrochemii bílkovin je v současnosti kladen důraz zejména na konstrukci tzv. imunosenzorů schopných detekce konkrétních proteinů (antigenů) pomocí jejich vazby na protilátku, s potenciálem pro diagnostiku. Z biofyzikálního hlediska je zajímavý vlastní elektrokatalytický signál bílkovin citlivý k jejich konformačním změnám, který by mohl nalézt uplatnění při rozlišení mutantních forem proteinů (např. u p53) nebo při jejich agregaci (α-synuklein v případě Parkinsonovy nemoci), popřípadě při studiu interakcí s nízkomolekulárními látkami či DNA. Díky zvýšenému zájmu o glykoproteiny se začínají objevovat elektrochemické práce zabývající se detekcí oligo-sacharidů a polysacharidů (tzv. glykanů, pokud jsou součástí bílkovin), a to pomocí elektroaktivních značek specifických pro sacharidy anebo konstrukcí lektinových biosenzorů využívajících lektinů, které se silně váží právě na glykany. Elektrochemická analýza se tak jeví jako zajímavý nástroj v současném výzkumu na poli genomiky, proteomiky i glykomiky, včetně diagnostiky nádorových onemocnění.
Electrochemical analysis of nucleic acids, proteins and polysaccharides represents an interesting, although not widely spread alternative to current methods based predominantly on optical detection because it offers a relatively inexpensive, fast and instrumentally simple detection of parallel samples on miniaturized chips, ideal for personalized medicine of the 21st century. Nucleic acid electrochemistry enables, for example, detection of specific DNA sequences (for determination of genes or presence of bacteria and viruses, etc.), DNA damage analysis and interaction with other molecules, DNA methylation or detection of microRNAs as potential cancer biomarkers. In the electrochemistry of proteins, great emphasis is put on construction of immunosensors for capturing specific proteins (antigens) using antibodies, suitable for diagnostics. From a biophysical point of view, intrinsic electrocatalytic signal of proteins sensitive to conformational changes could be useful in discrimination of mutant proteins (e. g. p53), native and aggregated forms (α-synuclein in Parkinson‘s disease) or for studies of protein interactions with low molecular‑weight ligands and DNA. Due to an increased interest of scientists in glycoproteins, new electrochemical papers emerged aiming at detection of oligosaccharides and polysaccharides (i.e. glycans, when part of the protein). These assays employ for instance electroactive labels specific for saccharides or lectin biosensors using lectins which strongly bind glycans. Electrochemical analysis thus appears as an interesting tool in current genomics, proteomics and glycomics, as well as for cancer diagnostics. Key words: electrochemistry – electrodes – nucleic acid hybridization – DNA sensors – electrocatalysis – biological markers This work was supported by the Czech Science Foundation projects No. P301/11/2055 (to EP) and 14-24931P (to MB), by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101) and by MH CZ – DRO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 15. 1. 2014 Accepted: 27. 2. 2014
- Klíčová slova
- elektrokatalýza, DNA senzory,
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- biomedicínský výzkum MeSH
- elektrochemické techniky * dějiny metody trendy MeSH
- elektrochemie MeSH
- hybridizace nukleových kyselin MeSH
- lidé MeSH
- nádorové biomarkery analýza MeSH
- nukleové kyseliny * analýza MeSH
- polysacharidy * analýza MeSH
- proteiny * analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.
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- lidé MeSH
- mutace * MeSH
- mutantní proteiny chemie genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 chemie genetika metabolismus MeSH
- plazmidy genetika MeSH
- promotorové oblasti (genetika) genetika MeSH
- protein X asociovaný s bcl-2 genetika MeSH
- protein-serin-threoninkinasy genetika MeSH
- regulace genové exprese genetika MeSH
- substrátová specifita MeSH
- superhelikální DNA chemie metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
First papers on electroactivity of DNA and RNA were published more then 50 years ago. For about 8 years oscillographic polarography at controlled a.c. (OP, proposed by J. Heyrovský already in 1941) was the method of choice for DNA analysis. Since approximately 1954 Robert Kalvoda developed OP for wide application in various fields. It is shown that already before 1960 it was possible to detect damage to DNA in X-ray-irradiated rats by means of OP. DNA samples from irradiated animals produced significantly larger OP anodic guanine signal indicating changes in the DNA structure. At present, radiation-induced strand breaks and damage to bases in DNA can be electrochemically detected at high sensitivity.
History of electrochemistry of proteins and nucleic acids is briefly reviewed. The ability of proteins to catalyze hydrogen evolution at Hg electrodes was discovered almost 80 years ago in J. Heyrovský's laboratory. This phenomenon was not sufficiently appreciated for several decades. Recently it has been shown that using constant current chronopotentiometric stripping (CPS) with hanging mercury drop, solid amalgam or Hg-film electrodes the CPS peak H is obtained with nanomolar concentrations of peptides and proteins. This peak is derived from the presodium wave but it has some new properties useful in protein research. It is sensitive to changes in protein structures and to protein redox states, representing a new tool for protein analysis applicable in biomedicine. Electroactivity of nucleic acids was discovered about 50 years ago. Electrochemistry of DNA and RNA is now a booming field because of its potential use in sensors for DNA hybridization and DNA damage. Quite recently it has been shown that electrochemistry can be applied also in polysaccharide analysis. A review with 99 references.
