Enzymatic Synthesis of Functionalized DNA

Abstract

In conclusion, the synthesis of several triphosphates with different modifications at the C5 position of the nucleobase was developed. In enzyme catalysed reactions I was able to find conditions whereby functionalised nucleotides can act as surrogate for natural nucleotides. The triphosphates synthesised in the first part of this work were functionalised with polyethylene glycol monomethyl ether residues or with polyamido dendrons bearing different end groups. Highly modified DNA fragments were generated by primer extension and PCR employing the described modified nucleotides. These modified DNA fragments were used to investigate the influence of the modifications on DNA properties. Thus, I was able to establish a method to generate DNA fragments that are grafted with linear polyethylene glycol monomethyl ethers or branched polyamido dendrons leading to entities with high molecular weight, modification density and structural accuracy.In the second part of this work, I was able to establish the synthesis of nucleotides harbouring a DNA strand (up to 38 nucleotides) at the nucleobase. These DNA building blocks were used successfully to introduce an additional DNA strand with a free 3’-OH in a nascent primer strand by primer extension. By this method highly branched DNA is feasible by enzyme-mediated reactions. I was able to find conditions to use the incorporated DNA strands as primer in primer extension or rolling circle amplification. This method was enhanced and transferred successful on DNA microarrays. With these findings the eligibility of these nucleotides as diagnostic tool was evidenced.