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Synthesis and studies of modified DNA: (i) development of DNA targeting molecular scissors and (ii) competitive enzymatic incorporation of base-modified nucleotides
Panattoni, Alessandro ; Hocek, Michal (advisor) ; Urban, Milan (referee) ; Fojta, Miroslav (referee)
In the first part of this work, a series of site-specific artificial metallonucleases (AMNs) was developed conjugating clamped-phenanthroline (Clip-Phen) copper complexes to triplex- forming oligonucleotides (TFOs). Several synthetic routes were explored for the synthesis of the TFO-AMNs hybrids, all sharing a copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction as the key step. As a consequence, building blocks for enzymatic or chemical synthesis of oligonucleotides (ONs) containing clickable groups, or already conjugated to the Clip-Phen ligand via CuAAC, were prepared. Two new alkynyl-linked nucleoside-5'-O-triphosphates (dNTPs) were designed and developed in order to obtain an efficient polymerase incorporation of clickable alkynyl-tethers into ONs and, at the same time, enhance the efficiency of CuAAC reactions on modified DNA. The relative 3'-O- phosphormaidites were also prepared in order to insert the same alkynyl-linkers into ONs via solid-phase synthesis. The AMN was linked at the 5'- or 3'-ends or in the middle of the TFO stretch, using diverse likers. The hybridization of all the synthesized TFOs with a target DNA duplex was studied. Finally, an extensive study of cleavage efficiency and specificity of the TFO-AMN conjugates towards the target DNA was performed, exploring the...
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