NMR spectroscopic characterisation of RNA regulatory elements : structural insights into RNA•DNA:DNA triplex formation and conserved elements from the RNA genome of coronaviruses

The thesis “NMR Spectroscopic Characterisation of RNA Regulatory Elements: Structural Insights into RNA•DNA:DNA Triplex Formation and Conserved Elements from the RNA Genome of Coronaviruses” encompasses two main subjects, each addressing distinct regulatory RNA elements. The first section presents the biophysical validation and characterisation of RNA•DNA:DNA triplex formation in vitro. A specific class of non-coding (ncRNAs) RNAs, namely long non-coding RNAs (lncRNAs), plays a crucial role in the formation of triplexes. The binding of the lncRNA to a target dsDNA is facilitated through non-canonical Hoogsteen base pairing and leads to triplex formation, either in parallel or antiparallel orientation of the RNA in relation to the DNA strand to which it binds. Triplexes have the ability to adjust gene regulation and are often associated with disease. The author of this thesis contributed to five research articles describing the biophysical characterisation of formation and stability of RNA•DNA:DNA triplexes. The lncRNA Hypoxia-inducible factor 1-alpha Antisense RNA 1 (HIF1α-AS1) was reported to be downregulated in various vascular diseases. Ephrin receptor A2 (EPHA2) and Adrenomedullin (ADM) were identified as target genes for this lncRNA, and RNA•DNA:DNA triplex formation was proposed as a mechanism of gene regulation by using bioinformatics, molecular biology techniques, and biophysical methods (Research Article 4.1.1). The characterisation of the HIF1α-AS1 binding to the target gene ADM was performed in vitro, in particular by circular dichroism (CD)-spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Different secondary motifs and their spectroscopic signatures were investigated. The use of DNA hairpins results in intramolecular DNA:DNA base pairing and increased stability. Triplex prediction tools, such as Triplexator or Triplex Domain Finder, allow the genome wide prediction of RNA:DNA interactions. The further development of such methods is therefore crucial, which is why the TriplexAligner tool was implemented (Research Article 4.1.2). By using biophysical methods, in particular CD-spectroscopy, the predicted triplexes were validated. The lncRNA FOXF1 Adjacent Noncoding Developmental Regulatory RNA (Fendrr) has been demonstrated to be associated with the development of lung fibrosis through triplex formation. Triplex prediction tools revealed Fendrr target sites in dysregulated genes, implying a direct regulatory relationship (Research Article 4.1.3). CD spectroscopy and thermal melting analysis were applied to confirm potential DNA targets of Fendrr. The aforementioned biophysical methods were refined, and further methods were applied to characterise the triplex formation of two different lncRNAs, the well-known maternally expressed gene 3 (MEG3) RNA and the previously described Fendrr RNA (Research Article 4.1.4). The study employed electrophoretic mobility shift assays (EMSA), CD spectroscopy, thermal melting analysis, single-molecule Förster resonance energy transfer (smFRET), microscale thermophoresis (MST), and NMR spectroscopy. The effect of stabilising the DNA duplex by using a linker loop and converting it into a hairpin construct was shown with MEG3, which forms antiparallel triplex structures with the TGFB2 DNA. The optimised DNA construct design was applied to Emp2 DNA, and triplex formation with Fendrr was investigated. Additionally, the heteroduplex and the DNA hairpin were examined. Combining various biophysical methods allowed for the detailed characterisation of the triplex structures, revealing a heterogeneous mixture of DNA duplexes, RNA:DNA heteroduplexes and RNA:DNA:DNA triplexes. These findings highlight the usefulness of these techniques for distinguishing between structural motifs and highlight the advantages of each method. Based on previous findings, which indicate that triplex formation alters DNA stability, the impact of RNA•DNA:DNA triplex formation on DNA stability was investigated using temperature-dependent imino proton solvent exchange NMR experiments (Research Article 4.1.5). These experiments enabled the determination of individual base pair exchange rates and the derivation of thermodynamic parameters, including the free energy of dissociation (ΔGdiss), enthalpy (ΔHdiss), and entropy (ΔSdiss) for each base pair. The studies used ADM DNA hairpin and the triplex formed with HIF1α-AS1 RNA as a model system for these investigations. Overall, the findings suggest that triplex formation affects DNA stability through Hoogsteen hydrogen bonding, stacking interactions, and solute-solvent effects. The results provide a valuable foundation for refining bioinformatic tools that can predict triplex-formation in the genome. The second project presented insights into conserved elements from the RNA genome of human pathogenic coronaviruses. These belong either to the alpha- or betacoronaviruses. One of the alphacoronaviruses is the human coronavirus 229E (HCoV-229) and one of the betacoronaviruses the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The focus of this thesis is on one structural RNA element of the 5' untranslated region (UTR), the stem loop 5a (SL5a), from HCoV-229E. Research article 4.2.1 presents a 1H, 13C and 15N chemical shift assignment of the SL5a from HCoV-229E using a broad set of NMR experiments. The focus was particularly on the hexaloop, comparing its resonances with the chemical shift assignment of SL5a from SARS-CoV-2. In addition, dynamics were determined that reinforced the general rigidity of the stem in comparison to the more dynamic loop. Using different loop variations, it was shown how these mutations in the SL5a RNA hexaloop affect its overall structure and dynamics. It was determined that alterations at specific positions resulted in more significant structural modifications. The work presented in this article can help to shed light on evolutionary pressures that shape viral RNA sequence, structure, and dynamics.