Targeting bacterial riboswitch RNA and viral RNA via NMR-based HTS approaches

This dissertation comprises three projects that are linked by the application of nuclear magnetic resonance (NMR) spectroscopy as the primary biophysical method used. The principal project of this dissertation is the “High-throughput screening of RNA”, which is described in detail in Chapter IV. This project concerns the selective targeting of bacterial and viral RNA constructs with small molecules in order to identify potential active substances that may be used in the treatment of diseases in which the targeted RNA construct plays an important role. Prior to this, screening campaigns have predominantly focused on proteins, whereas RNA molecules have only recently become the subject of research into finding drugs. This is largely due to the inherent difficulty in identifying specific binders for RNA motifs. Furthermore, in addition to binding with high affinity to the RNA, the binder must also alter its function in the desired manner. To date, a considerable number of functional RNAs have been identified which do not encode proteins in the form of messenger RNA (mRNA). Rather, they possess catalytic functions or play a role in genetic control. The following RNAs have been identified as having catalytic activity: ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), micro RNA (miRNA) and small interfering RNA (siRNA). A distinctive category of gene regulatory RNAs are those designated as riboswitch RNAs. The majority of riboswitches are found in prokaryotic cells, although some eukaryotic riboswitches have also been identified. Riboswitches are capable of responding to intercellular signals, such as elevated concentrations of a particular ligand molecule. By binding the ligand with high affinity, riboswitches trigger a structural rearrangement of the nascent RNA, which either influences the transcription of the following genes (transcription 'on' or 'off') or has a similar effect on translation (translation 'on' or 'off'). The system under investigation in this project represents a partial construct of the 2-deoxyguanine-sensing riboswitch (2'dG-switch), which was first identified in the bacterium Mesoplasma florum.  The 39 nucleotides long sub-construct constitutes the terminator hairpin loop (dGterm) and is located in the 3' direction downstream of the expression platform of the riboswitch. The 2'dG-switch functions as a kinetically controlled transcriptional "off-switch." This indicates that the formation of the terminator-hairpin and subsequent transcription inhibition of the downstream genes is dependent on the binding of the ligand and the resulting refolding of the riboswitch RNA. The inhibition of riboswitch function in bacteria, or alternatively the fixation of the riboswitch in its 'off' conformation, could enable the development of a new class of RNA-based antibiotic drugs. The development of new antibiotics is crucial to addressing the growing prevalence of multi-resistant pathogens resulting from the inappropriate use of conventional broad-spectrum antibiotics in medicine and agriculture. In this dissertation, NMR-based screening against a 768 fragments large DSI-poised library (DSI-PL) was employed to demonstrate that riboswitches and, thus, RNAs in general, in addition to proteins, can be targeted by small molecules. The 1H NMR-based screening of dGterm against the DSI-PL was made significantly more time-efficient by utilising mixes comprising 12 fragments per mix. A total of 29 out of the 768 fragments exhibited a change in chemical shift within the NMR spectra, indicating interactions between the fragments and the RNA. In March 2020, a global consortium of NMR spectroscopists, originating from the Schwalbe working group at Goethe University Frankfurt, was established with the objective of elucidating the relevant structures of the RNA genome and proteome of SARS-CoV-2 and screening them against the fragments of the DSI-PL. The majority of the RNA constructs examined are localised in one of the two untranslated regions (UTRs) at either the 5'- or 3'-end of the genomic RNA. The present study concentrated on the screening of the initial four stem loops of the 5'-terminal untranslated region (UTR) of viral RNA. The DRTL-F library, which has been optimised for the binding of RNAs, was provided by Prof. Dr. Hargrove and will be used in subsequent stages of the project. The three NMR experiments conducted for each screening sample, which utilise the detection of the 1H and 19F nuclei, revealed the presence of multiple binding events. Furthermore, the binding affinities and binding sites of the most promising binders were elucidated by NMR spectroscopy. The research yielded evidence of the presence of binding agents with low-micromolar affinity. By analysing the functional groups of the binders identified, it is possible to conclude which groups are most likely to bind to RNA. The objective of this project was to develop and evaluate a cutting-edge system for high-throughput NMR-based screening.