Refine
Has Fulltext
- yes (3)
Is part of the Bibliography
- yes (3)
Year of publication
- 2023 (3) (remove)
Document Type
- Doctoral Thesis (3)
Language
- English (3) (remove)
Keywords
- Chemische Synthese (3) (remove)
As central components of life, DNA and RNA encode the genetic information. However, RNA performs several functions that exceed the competences stated in the ‘central dogma of life‘. RNAs undergo extensive post-transcriptional processing like chemical modifications. Among all classes of RNA, tRNAs are the most extensively modified. Their modifications are chemically diverse and vary from simple methylations (e.g. m3C, m6A) to more complex residues, like isopentenyl group (e.g. i6A, hypermodifications: e.g. ms2i6A) or even amino acids (e.g. t6A). Depending on their location within the overall structure, modifications can have an impact on tRNA stability and structure, as well as affinity for the ribosome and translation efficiency and fidelity. Given the importance of tRNA modifications new tools are needed for their detection and to study their recognition by proteins and enzymatic transformations.
The chemical synthesis of these naturally occurring tRNA modifications as phosphoramidite building blocks is a prerequisite to incorporate the desired modification via solid-phase synthesis into oligonucleotides. With the help of the m3C, (ms2)i6A, and t6A oligonucleotides, the importance and impact of tRNA modifications was investigated in this thesis. To this end, the role of METTL8 as the methyltransferase responsible for the installation of the methyl group at C32 for mt-tRNAThr and mt-tRNASer(UCN) was resolved. Thereby, the respective adenosine modification on position 37 is essential for the effectiveness of the enzyme. Besides, by means of NMR analysis, CD spectroscopy, thermal denaturation experiments, and native page separation, the impact of m3C32 on the structure of the tRNA ASLs was shown. The modification appeared to fine-tune the tRNA structure to optimize mitochondrial translation. To investigate the regulation of the dynamic modification pathway of m3C, demethylation assays were performed with the modified tRNA-ASLs and the (α-KG)- and Fe(II)-dependent dioxygenase ALKBH1 and ALKHB3. A demethylation activity of ALKBH3 on the mt-tRNAs was observed, even though it has so far only been described as a cytoplasmic enzyme. Whether this is physiologically relevant and ALKBH3 present a mitochondrial localization needs further validation. In addition, ALKBH1 was confirmed to not be able to demethylate m3C on mt-tRNAs, but indications for a deprenylation and exonuclease activity were found. Furthermore, the aforementioned naturally occurring modifications were utilized to find analytical tools that can determine the modification levels by DNAzymes, which cleave RNA in the presence of a specific modification. Selective DNA enzymes for i6A, as well as the three cytidine isomers m3C, m4C, and m5C have been identified and characterized.
Besides the naturally occurring tRNA modifications, the investigation on artificially modified nucleosides is also part of this thesis. Nucleosides with specific properties for desired applications can be created by modifying the scaffold of native nucleosides.
During the pandemic, the potential of antiviral nucleoside analogues was highlighted for the treatment of the SARS-CoV-2 infection. For examinations of the potential drug-candidate Molnupiravir, the N4-hydroxycytidine phosphoramidite building block was synthesized and incorporated into several RNA oligonucleotides. A two-step model for the NHC-induced mutagenesis of SARS-CoV-2 was proposed based on RNA elongation, thermal denaturation, and cryo-EM experiments using the modified RNA strands with the recombinant SARS-CoV-2 RNA-dependent RNA polymerase. Two tautomeric forms of NHC enable base pairing with guanosine in the amino and with adenosine in the imino form, leading to error catastrophe after the incorporation into viral RNA. These findings were further corroborated by thermal melting curve analysis and NMR spectroscopy of the NHC-containing Dickerson Drew sequence. In conclusion, the anti-amino form in the NHC-G base pair was assigned by NMR analysis using a 15N-labeld NHC building block incorporated into the Dickerson Drew sequence.
This thesis also addressed the synthesis of a 7-deazaguanosine crosslinker with a masked aldehyde as a diol linker for investigations of DNA-protein interactions. The diol functional group can be unmasked to release the reactive aldehyde, which can specifically form a covalent bond with amino acids Lys or Arg within the protein complex condensin. The incorporation of the synthesized phosphoramidite and triphosphate building blocks were shown and the functionality of the PCR product containing the crosslinker was demonstrated by oxidation and the formation of a covalent bond with a fluorescein label.
The development of assays that detect changes in this methylation pattern of m6A could provide new insights into important biological processes. In the last project of this thesis, the influence of RNA methylation states on the structural properties of RNA was analyzed and a fluorescent nucleoside analog (8-vinyladenosine) as molecular tools for such assays was developed. Initial experiments with the fluorescent nucleoside analog N6-methyl-8-vinyladenosine (m6v8A) were performed and revealed a strong fluorescence enhancement of the free m6v8A nucleoside by the installation of the vinyl moiety at position 8.
Overall, this thesis contributes to various research topics regarding the application of naturally occurring and artificial nucleoside analogues. Starting with the chemical synthesis of RNA and DNA modifications, this thesis has unveiled several open questions regarding the dynamic (de-)methylation pathway of m3C and the mechanism of action of molnupiravir through in-depth analysis and provided the basis for further investigations of the protein complex condensin, and a new fluorescent nucleoside analog m6v8A.
The 1st chapter provides a detailed review of the development of synthetic approaches to triarylboranes from their first report nearly 135 years ago to the present. In the 2nd chapter, a novel and convenient methodology is reported for the one-pot synthesis of sterically-congested triarylboranes, using bench-stable aryltrifluoroborates as the boron source. The new procedure gives access to symmetrically- and unsymmetrically-substituted triarylboranes. The borylated triarylboranes are suggested as building blocks for the design of functional materials. In the 3rd chapter, four luminescent tetracationic bis-triarylborane DNA and RNA sensors that show high binding affinities, in several cases even in the nM range, are investigated. The molecular structures of two of the neutral precursors reveal some structural flexibility for these compounds in the solid state. The compounds were found to be highly emissive even in water and DNA and RNA binding affinities were found to be dependent on linker length and flexibility. Strong SERS responses for three of the four compounds demonstrate the importance of triple bonds for strong Raman activity in molecules of this compound class. In chapter 4, the compound class of water-soluble tetracationic bis-triarylborane chromophores is extended by EDOT-linked compounds and those are compared to their thiophene-containing analogs. Absorption and emission are significantly red-shifted in these compounds, compared to their thiophene-containing analogs and, due to a large Stokes shift, one of the reported compounds exhibits the most bathochromically shifted emission, observable well into the near infrared region, of all tetracationic water-soluble bis-triarylborane chromophores reported to date. Long-lived excited states, completely quenched by oxygen, were observed for the water-stable compounds of this study via transient absorption spectroscopy and a quantum yield for singlet oxygen formation of 0.6 was determined for one of them.
The human African trypanosomiasis is a neglected tropical disease, which is caused by the protozoan Trypanosoma brucei and transmitted by the bite of the tsetse fly. An untreated infection leads to death. However, only a few drugs with significant drawbacks are currently available for treatment. In this thesis, quinolone amides with an antitrypanosomal activity were synthesized and their biological and physicochemical properties were measured. New structure-activity relationships and a promising lead structure were discovered.