Oligonucleotide-modified nucleotides

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Nucleic acid-based diagnostics span a wide field reaching from the detection of pathogen-derived nucleic acids (such as viruses) to the analysis of single nucleotide variations in the entire genome such as point mutations and single nucleotide polymorphisms (SNPs). For most analytical approach are based on the polymerase chain reaction (PCR) and require sophisticated equipment. The aim of this project is to further develop methods that allows the detection of a nucleic acid target by the naked eye with single nucleotide precision without requiring PCR-based technology. Such a system would be highly useful for point-of-care testing or the detection of pathogens in the field. DNA polymerase based reactions hold great potential in this regard, since they are catalyzing nucleotide incorporation in a template-dependent fashion with high sequence selectivity. In the first funding period we demonstrated that nucleotides that are modified with large functional entities for signal generation such as DNA constructs with enzymatic activity (i.e. DNAzymes) or proteins (i.e. horse radish peroxidase) are processed by DNA polymerases and sequence selectively incorporated into a growing DNA strand. Thereby the functional entities were connected to solid supports and resulting in sequence-selective signal generation that is detectable by naked eye. These studies were supported by structural investigations on the mechanism of how such large modifications are accepted by DNA polymerases. In future studies we aim at increasing the sensitivity of the systems by exploiting loop-mediated isothermal amplification (LAMP) and antibody-based detection systems. First preliminary results indicate that both approaches are highly promising. Thus, several DNA- and antibody-modified nucleoside triphosphates will be synthesized and thoroughly investigated. Furthermore, we aim at thoroughly investigating the incorporation of the modified nucleotides by DNA polymerases by functional and structural means. This will result in the development of a broader understanding of the mechanisms by which these enzymes process nucleotides that are modified with entities being up to several times larger than the diameter of the DNA polymerases itself. On the other hand, new insights into the complex mechanisms by which DNA polymerases function will be obtained due to planned structural investigations of relevant DNA polymerases that have not been crystallized before with bound modified substrates.