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Study of 2-Nitrofluorene Interaction with DNA at a Glassy Carbon Electrode
Skalová, Štěpánka ; Stávková, K. ; Vyskočil, V. ; Barek, J.
2-Nitrofluorene (2-NF) is a nitrated polycyclic aromatic hydrocarbon (NPAH) which occurs as the environmental pollutant. It is a potential carcinogen and mutagen. Interaction of deoxyribonucleic acid (DNA) with 2-NF was monitored using an electrochemical DNA biosensor prepared from a glassy carbon electrode (GCE) and low-molecular-weight DNA by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and square-wave voltammetry (SWV).\nThere were no damaging interactions observed between DNA and 2-NF using EIS. However, CV shows intercalation of this substance into the DNA structure to form the complex 2-NF–DNA. Intercalation was also observed by SWV, confirming intercalation to reduce the number of electroactive sites and thus reducing the peak heights of adenosine and guanosine.
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Searching for Electrochemical Reduction Mechanism of Azidophenyl DNA Labels
Daňhel, Aleš ; Trošanová, Zuzana ; Balintová, Jana ; Hocek, Michal ; Fojta, Miroslav
This contribution brings new information to sporadic scientific results concerned to electrochemical reduction of aromatic azides. Selected compounds used as perspective DNA labels or their structural constituents such as, 4-azidophenyltrifluoroboronic acid, 4-azidophenyl modified deoxycytidine and next probable analogous compounds and/or products of their reduction, were voltammetrically studied and compared to each other in order to reveal a mechanism of their electrochemical reduction at mercury electrodes in aqueous media. Preliminary results obtained by cyclic voltammetry at mercury and carbon based electrodes and by mass spectrometry of the products isolated from batch electrolysis on mercury pool by preparative chromatography are discussed.
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Voltametrická analýza nukleosidtrifosfátů a oligonukleotidů značených antrochinonem na zlatých elektrodách
Vidláková, Pavlína ; Balintová, Jana ; Havran, Luděk ; Hocek, Michal ; Fojta, Miroslav
Elektrochemická aktivita nukleových kyselin byla objevena v 50. letech 20. století a od té doby je používána ke studiu struktury a interakcí přirozených i modifikovaných molekul nukleových kyselin i syntetických oligonukleotidů. Přestože je přirozená DNA sama o sobě elektrochemicky aktivní a je možné ji studovat na různých typech elektrod, je pro řadu analytických aplikací praktické použít DNA značenou elektroaktivními molekulami (například komplexy přechodných kovů, amino- nebo nitroskupinami, antrachinonem apod.). Tyto látky podléhají redoxním reakcím a dávají takto modifikované DNA nové elektrochemické vlastnosti, které je možné využít pro studium struktury a interakcí nukleových kyselin a v oblasti DNA diagnostiky.
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Voltammetric Behavior of 4-Aminophthalimide Label using Hanging Mercury Drop Electrode
Ferenčíková, Z. ; Daňhel, Aleš ; Riedl, Jan ; Hocek, Michal ; Fojta, Miroslav
4-aminophthalimide (API), covalently attached to nucleotide (dCTP or dATP) has recently beendesigned as a fluorescent label for the detection of DNA-protein interaction. However API is electrochemically reducible and therefore it is possible to use alternatively the API-modified nuclotides as DNA redox labels. The electrochemical behavior of labeled nucleotides and labeled DNA was studied using cyclic voltammetry and transfer stripping cyclic voltammetry at hanging mercury drop electrode. Observed CVs showed irreversible reduction signals of 4-aminophthalimide label without production of consequently oxidizable/reducible species.
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Electrochemical Biosensors Based on Silver Solid Amalgam for Analysis in Flow Systems
Josypčuk, Oksana ; Barek, J. ; Josypčuk, Bohdan
In this contribution, the construction and preparation of 3 types of flow amperometric enzymatic biosensors based on silver solid amalgam (AgSA) were presented. Two of them consist of the tubular detector and enzymatic reactor of porous or powdered AgSA. The third biosensor was constructed using polished AgSA electrode. Reactors and polished AgSA electrodes were modified with thiols or chitosan and different enzymes were covalently immobilized by suitable techniques. Prepared biosensors were used for determination of ascorbic acid, glucose, hydrogen peroxide, catechol, pyrogallol, dopamine and sarcosine in model samples and in same cases in the real samples.
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Utilisation of enzymatic labelling with 4-aminophtalimide and 4-hydroxybenzylideneimidazolinone fluorescent derivates for monitoring of DNA-protein interaction
Orság, Petr ; Pivoňková, Hana ; Riedl, Jan ; Hocek, Michal ; Fojta, Miroslav
The 5’-substituted deoxycytosine triphosphates with conjugated solvatochromic derivates of 4-aminophtalimide (API) and derivates of the green fluorescent protein, 4-hydroxybenzylideneimidazolinone (HBI) were synthetized and successfully tested for enzymatic incorporation using primer extension assay. Site specifically labelled oligonucleotide probes were prepared and tested for interaction with p53 and SSB proteins, displaying distinct DNA-binding properties. The incorporation of multiple fluorescent labels did not interfere with natural protein binding and protein interaction leaded in both cases the to the gradual ratiometric increase of the fluorescence intensity moreover accompanied with the changes of the fluorescence emission spectra profile. Neither effect was observed after incubation with BSA, non-DNA binding protein, confirming the specificity of the interaction. Modified nucleoside triphosphates with conjugated fluorescence labels derivates of API and HBI can be used as substrates for preparation of the specific oligonucleotide labelled probes and provide the novel tool for studying and monitoring the DNA-protein interaction.
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Electrochemical analysis of DNA using switchable redox moieties
Fojta, Miroslav ; Daňhel, Aleš ; Horáková Brázdilová, Petra ; Plucnara, Medard ; Pivoňková, Hana ; Havran, Luděk ; Vidláková, Pavlína ; Raindlová, Veronika ; Balintová, Jana ; Macíčková-Cahová, Hana ; Hocek, Michal
Labelling of DNA with electrochemically active moieties proved to be a convenient way to the development of electrochemical techniques for the sequence-specific DNA sensing. Through combinations of various labels differing in redox potentials, independent redox coding of different DNA sequences or individual nucleobases can be attained. Applications possibilities of electrochemistry in analysis of modified DNAs are further extended by facile monitoring of chemical conversion of reactive groups on DNA during post-labelling with ultimate redox labels. In addition, controlled in situ electrochemical conversions of specific intrinsic and extrinsic DNA components can be utilized to switch their electrochemical signals and improve signal resolution.
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Polymerase synthesis of new photocaged DNA
Vaníková, Zuzana ; Hocek, Michal
5-[(2-nitrobenzyl)oxymethyl]-2’-deoxyuridine 5’-O-triphosphate (dUNBTP) and 5-hydroxymethyl-2’- deoxyuridine 5’-O-triphosphate (dUHMTP) were incorporated to diverse DNA sequences by polymerase synthesis (PEX or PCR). UHM modified DNA was synthesized also through the photolysis of photocaged, UNB-linked DNA. The presence of bulky NB-modification in the recognition sequence of DNA resulted in blocking of cleavage by all restriction endonucleases (REs), however sequences with small HM-modification (formed after irradiation by UV) were perfectly cleaved by all enzymes. Using of photoremovable protecting group in the DNA sequence presents bioorthogonal chemical masking and it has a potential in gene manipulation and cloning.
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Preparation of modified oligonucleotides by nicking enzyme amplification reaction
Ménová, Petra ; Hocek, Michal
A method for the enzymatic synthesis of short (10-22 nt) base-modified oligonucleotides was developed, based on the nicking enzyme amplification reaction. The methodology, including product isolation and purification, was scaled up to nanomolar amounts. The modified oligonucleotides were successfully used as primers in primer extension and PCR, affording primer-modified oligonucleotides and DNA. Two simple and efficient methods for fluorescent labelling of the PCR products were also developed.
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