Enzymatische PCR-Replikation von vollständig orthogonalen Nukleinsäuren als synthetische Modellgenome
Organische Molekülchemie - Synthese, Charakterisierung
Zusammenfassung der Projektergebnisse
Synthetic biology is a field of research which tries to redesign biological systems artificially to equip these with new and unique functions and properties. One of the most intensively studied molecules are xeno nucleic acids (XNAs) which shall function as biologically orthogonal genetic material. This orthogonality origins from their structural variance from their natural counter parts what prevents the interaction with highly specific natural enzymes (e.g.: polymerases, ligases, etc). Within the framework of this project, a set of four unnatural DNA building blocks (nucleosides) has been synthesized and incorporated into oligonucleotides by automated solid-supported synthesis. The single nucleosides are designed to mimic the hydrogen bonding pattern of the natural AT- and GC-base pairs, but the nucleobase is bound to the 2′-deoxyribose moiety by a C-C glycosidic bond (C-nucleoside). This prevents the artificial building block from enzymatic degradation or exchange. Furthermore, the unnatural adenosine and guanosine mimics are modified to make them highly fluorescent and gives the opportunity for future in vitro and in vivo spectroscopic applications. The synthesis of the artificial 2′-deoxyribo analogues of pseudouridine dψ, the thieno-modified guanosine dthG as well as the N-methylated pseudoisocytidine dψiC (mimics of the natural thymidine, guanosine and cytidine, respectively) were obtained by already published synthetic procedures. The synthesis of the thieno-modified 2′-deoxyribo adenosine building block dthA caused several difficulties. It turned out, that a direct coupling to a 2′-deoxyribose (as performed in case of dthG) was not possible. In an alternative attempt, the RNA analogue of dthA was synthesized and it was tried to convert it into the corresponding DNA building block. Unfortunately, all tested conditions did not yield in the intended molecule. The desired nucleoside was finally obtained by synthesizing an inosine analogue and subsequently converting it into the adenosine dthA. This molecule shows remarkable fluorescence properties which underline its potential for further in vitro and in vivo applications. All four artificial nucleosides in hands, the corresponding phosphoramidite building blocks were synthesized and used for automated solid-supported oligonucleotide synthesis. Their incorporation efficiency was first tested in separate oligonucleotide strands and showed a good reactivity as well as stability. In the coming time, a fully-modified DNA-strand, exclusively containing C-nucleosides, will be synthesized and its structural (melting temperatures, helicity) and spectroscopic (fluorescence) properties will be tested. This study will help to evaluate the potential of artificial C-nucleosides for future synthetic biology applications.
