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• W244100535 abstract "Structure determination is one of the most fundamental fields of research, as it provides insights in biological systems and their mechanisms. Therein, NMR spectroscopy is an essential tool for investigations under physiological conditions and the development of new techniques, which enable the detection of more detailed information, is an ongoing topic of research. By using paramagnetic tagging of biomolecules, highly valuable NMR parameters, e.g. residual dipolar couplings (RDCs) and pseudocontact shifts (PCSs), can be determined, which provide angle and distance information about the target molecule. Therefore, several lanthanide-binding tags have been developed and successfully attached to proteins and oligosaccharides. Due to the paramagnetic properties of the lanthanide ions, an alignment of the target molecule is induced, which facilitates the determination of RDCs. Additionally, the NMR signals are shifted due to a dipole-dipole interaction between the nuclei and the lanthanide ion (PCS). Both of these effects can not be detected in conventional NMR spectroscopy, due to the isotropic distribution of orientations of a molecule in solution. A successful transfer of the protein tagging method to oligonucleotides has of yet not been reported, although DNA and RNA are two of the most important groups of biomolecules. Based on the recently developed Cys-Ph-TAHA tag, a reliable protocol for the determination of paramagnetic effects in a DNA molecule is presented in this work. To achieve this, a modified nucleobase was synthesized, which provides a suitable binding site for the Cys-Ph-TAHA tag. A tagging and purification protocol was established, by which two paramagnetic samples (thulium and terbium) and a diamagnetic reference sample (lutetium) were prepared. High resolution NMR spectroscopy revealed paramagnetically-induced PCSs and RDCs in the tagged oligonucleotide. The data evaluation proved a high quality of the determined PCSs for both samples. As 13C/15N labeling of synthesized DNA strands is unreasonable, due to the expensive starting materials and the low overall yield of the DNA synthesizer, all NMR spectra were performed using samples with natural abundance. Consequently, the signal-to-noise ratio of the NMR signals was relatively low and together with the paramagnetic relaxation enhancement, the determined RDCs have a significant error. Nevertheless, a suitable ensemble model was created, by which the combined PCS and RDC data was satisfactorily described. In conclusion, the technique of paramagnetic tagging was successfully transferred to an oligonucleotide. To do this, a reliable strategy for the incorporation of a binding site and sufficient tagging and purification protocols were established. This approach was verified by the determination and evaluation of PCSs and RDCs in a DNA strand." @default.
• W244100535 created "2016-06-24" @default.
• W244100535 creator A5088310326 @default.
• W244100535 date "2022-02-20" @default.
• W244100535 modified "2023-09-30" @default.
• W244100535 title "Paramagnetic Tagging of Oligonucleotides for Structure Determination using NMR-Spectroscopy" @default.
• W244100535 doi "https://doi.org/10.53846/goediss-5002" @default.
• W244100535 hasPublicationYear "2022" @default.
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