National Repository of Grey Literature 5 records found  Search took 0.01 seconds. 
New functionalized nucleic acids for application in chemical biology
Kielkowski, Pavel ; Hocek, Michal (advisor) ; Stiborová, Marie (referee) ; Moravcová, Jitka (referee)
4 Abstract This work is focused on the synthesis of the modified 2'-deoxyribonucleoside triphosphates, their incorporation into DNA and use in chemical biology applications. The synthetic routes to the double-headed nucleosides and nucleotide triphosphates in which the two nucleobases were connected via ethynyl or propargyl linker has been developed. (Cytosin-5-yl)ethynyl, 3-(cytosin-1-yl)prop-1-yn-1-yl and 3-(5-fluorocytosin-1-yl)prop-1-yn- 1-yl derivatives of pyrimidine and 7-deazaadenine 2'-deoxyribonucleosides and nucleoside triphosphates were prepared by aqueous palladium-catalyzed cross-coupling reactions. The double-headed modified nucleoside triphosphates were good substrates for DNA polymerases suitable for primer extension and PCR construction of DNA bearing linked cytosine or 5- fluorocytosine in the major groove mimicking the flliped-out nucleotide. The assay for the testing of the inhibition of DNA methyltransferases was developed. Next, the transient protection of DNA against cleavage by restriction endonucleases (REs) using (trialkylsilyl)ethynyl modified DNA was developed. A series of 7-(trialkylsilyl)ethynyl-7- deaza-2'-deoxyadenosine triphosphates was prepared and they were shown to be incorporated into DNA by primer extension and/or PCR using KOD XL polymerase. The deprotection conditions...
Competitive incorporations of modified versus natural nucleotides by DNA polymerases
Kielkowski, Pavel ; Hocek, Michal
A series of 7-substituted 7-deazaadenosine and 5-substituted cytosine triphosphates were tested in competitive incorporations with different DNA polymerases with their natural analogs and analyzed using restriction endonucleases and compared with steady state kinetics of the modified and natural dNTPs. Data sets correlation is up to 99 % and gave us to find modified dATPs with high incorporation ratios.
Polymerase synthesis of base-modified DNA: New methods and new applications
Balintová, Jana ; Daďová, Jitka ; Kielkowski, Pavel ; Ménová, Petra ; Vaníková, Zuzana ; Riedl, Jan ; Raindlová, Veronika ; Fojta, Miroslav ; Hocek, Michal
Diverse base-modified oligonucleotides and double-stranded DNA molecules were prepared by polymerase incorporation of modified nucleoside triphosphates. The methods include primer extension, PCR, nicking enzyme amplification reaction and mixed incorporations. The modified nucleic acids were used in redox and fluorescence labelling and coding, as well as regulation of binding and cross-linking with proteins.
Polymerase synthesis of modified DNA containing cytosine in major groove
Kielkowski, Pavel ; Hocek, Michal
(Cytosine-5-yl)ethynyl derivatives of pyrimidine and 7-deazaadenine 2-deoxyribonucleosides and nucleoside triphosphates (dNTPs) were prepared by aqueous Sonogashira coupling. All modified dNTPs were excellent substrates for DNA polymerases to give DNA bearing acetylene- linked cytosine(s) in the major groove.
Polymerase construction of base-modified DNA for chemical biology
Hocek, Michal ; Macíčková-Cahová, Hana ; Kielkowski, Pavel ; Raindlová, Veronika ; Kalachová, Lubica ; Riedl, Jan ; Balintová, Jana ; Ménová, Petra
An efficient two-step methodology for construction of base-modified DNA was developed based on aqueous-phase cross-coupling reactions of halogenated nucleoside triphosphates (dNTPs) followed by polymerase incorporation. A number of diverse chemical modifications have been successfully incorporated in this way for the use in chemical biology (modulation of cleavage by restriction enzymes, interactions with diverse DNA-binding proteins and bioconjugations).

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