AbstractsChemistry

Oligonucleotides are significant tools in chemical biology and show wide applications. This thesis contains two projects concerning the construction of ligandosides and functionalization of oligonucleotides for formylcytosine detection. In the first project, a novel metal-base pair based on the pyrazole ligand was developed. The synthesis of the pyrazole ligandoside comprised the preparation of a protected base building block and a cuprate mediated C-glycosylation as the key step (Figure 1-1). The correct β-configuration of the nucleoside was confirmed by X-ray crystallography. The ligandoside precursor was incorporated into numerous oligonucleotides by automated DNA synthesis (Chapter 4.2). With a homo-pyrazole base pair inserted, duplex stability increased by 9°C after incooperation of one copper ion. The chelating performance depends on deprotonation of the phenol group of the ligandoside. Up to ten copper ions can be coordinated inside the duplex. Compared with the bridging salen base pair, the non-bridging pyrazole base pair shows a kinetical preference for complexation (Figure 1-2, Chapter 4.3). The pyrazole triphosphate is accepted by the Therminator polymerase which extends the primer. The unsatisfactory efficiency, however, hinders its application in PCR (Chapter 4.4.1). A duplex with five pyrazole-copper pairs was applied as a chiral catalyst in a model Diels-Alder reaction, which allowed to reach an ee value of 39% (Chapter 4.4.2). The novel ligandoside sheds light on how DNA may be used as a catalyst in organic reactions and enlightens further design and optimization of ligandosides. The second project is focused on developing a new fdC sequencing method based on an oligonucleotide probe connected to a hydroxylamine linker. The linker was selected using a combinational chemistry strategy. The most suitable linker at an n+4 position of the probe strand was known to react with the fdC in the target strand irreversibly. Because the probe and the target strand hybridized to form a duplex, the probe reacted with the target fdC with high positional specificity (Figure 1-3, Chapter 4.2). The reaction is limited to fdC and can tolerant single nucleotides polymorphisms in the target. Multiple fdC probes can be applied together. Enzymatic digestion and primer extension experiments were performed on the cross-linked oligonucleotide towards LC-MS and PCR detection (Chapter 4.3). After oxime formation, the duplex can be digested into dinucleotides but they cannot be detached. The probe strands hinders the Taq polymerase to pass through the target strand. A method for relative fdC quantification was developed using the described probe (Figure 1-4 Chapter 4.4). After crosslinking, the fdC probe was ligated to an adapter strand, wrapped in nanodroplets and replicated using PCR. The signals were counted and compared to a reference amplicon. 10-fold increase of fdC was observed at one position in an exon in Tdg-/- mES cells compared to Dnmt TKO cells, and 2-fold compared to Tdg+/- cells. The method can be applied to… Advisors/Committee Members: Carell, Thomas (advisor).