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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...
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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.
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