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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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Novel conformationally locked nucleosides and nucleotides based on bicyclo[3.2.1]octane scaffold as a pseudosugar moiety
Šála, Michal ; Dejmek, Milan ; Procházková, Eliška ; Hřebabecký, Hubert ; Rybáček, Jiří ; Dračínský, Martin ; Novák, Pavel ; Rosenbergová, Šárka ; Fukal, J. ; Sychrovský, Vladimír ; Rosenberg, Ivan ; Nencka, Radim
A route to a series of novel carbocyclic nucleosides locked in North conformation with bicyclo[3.2.1]octane scaffold was developed. Prepared nucleosides served as a starting material for the synthesis of modified oligomers [d(GCATATCAC), r(GCAUAUCAC), and A9]. Biological effects of the prepared nucleosides as well as the hybridization properties of the appropriate duplexes were evaluated.
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Acyclic nucleoside bis-phosphonates as potent inhibitors of 6-oxopurine phosphoribosyltransferases
Špaček, Petr ; Keough, D. T. ; Vrbková, Silvie ; Slavětínská, Lenka ; Janeba, Zlatko ; Naesens, L. ; Edstein, M. D. ; Chavchich, M. ; Wang, T. H. ; de Jersey, J. ; Guddat, L. W. ; Hocková, Dana
Hypoxanthine-guanin-(xanthine) phosphoribosyltransferase (HG(X)PRT) is critical for the survival of malarial parasites Plasmodium falciparum and Plasmodium vivax. These parasites rely on HG(X)PRT to make 6-oxopurine nucleoside monophosphates. Specific acyclic nucleoside phosphonates (ANPs) inhibit HG(X)PRT and thus have an anti-plasmodial activity. Crystal structures of human HGPRT in complex with several ANP-based inhibitors suggested that attachment of the second phosphonate group which could occupy the pyrophosphate binding site may lead to increased affinity of these compounds.
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