Přístupy k enzymové syntéze hypermodifikovaných DNA polymerů

Ondruš, Marek

The aim of this thesis was to synthesize new series of modified 2'-deoxynucleoside triphosphates (dNTPs) bearing hydrophobic modifications, use them in enzymatic synthesis of fully-modified DNA and study its bio-physical properties. In the first part of the thesis, a set of four 2'-deoxynucleosides bearing a linear or branched alkane, indole or phenyl group were synthesized by Sonogashira cross-coupling reactions using alkynes and iodinated nucleosides. Ethynyl linkers of corresponding nucleosides were reduced by catalytic hydrogenation to obtain another four nucleosides bearing the modifications through alkyl linker. All eight nucleosides were then transformed into 2'-deoxynucleoside triphosphates (dNTPs) by Yoshikawa phosphorylation and individually tested as substrates for polymerase synthesis by primer extension (PEX). Their combinations were systematically tested to generate DNA containing one or even four modified nucleotides. It was possible to replace all four canonical nucleotides with hydrophobically-modified counterparts and thus synthesize DNA with high density of modifications. Nevertheless, only nucleotides bearing modifications through rigid ethynyl linker were suitable for synthesis of longer hypermodified DNA. Nucleotides with more flexible alkyl linker destabilized duplex during...

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