The diversity of splicing modifiers acting on A-1 bulged 5'-splice sites reveals rules for rational drug design.

Pharmacological modulation of RNA splicing by small molecules is an emerging facet of drug discovery. In this context, the SMN2 splicing modifier SMN-C5 was used as a prototype to understand the mode of action of small molecule splicing modifiers and propose the concept of 5'-splice site bulge repair. In this study, we combined in vitro binding assays and structure determination by NMR spectroscopy to identify the binding modes of four other small molecule splicing modifiers that switch the splicing of either the SMN2 or the HTT gene. Here, we determined the solution structures of risdiplam, branaplam, SMN-CX and SMN-CY bound to the intermolecular RNA helix epitope containing an unpaired adenine within the G-2A-1G+1U+2 motif of the 5'-splice site. Despite notable differences in their scaffolds, risdiplam, SMN-CX, SMN-CY and branaplam contact the RNA epitope similarly to SMN-C5, suggesting that the 5'-splice site bulge repair mechanism can be generalised. These findings not only deepen our understanding of the chemical diversity of splicing modifiers that target A-1 bulged 5'-splice sites, but also identify common pharmacophores required for modulating 5'-splice site selection with small molecules.

A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2.

The pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further fragment expansion and drug development.

Novel 13 C-detected NMR Experiments for the Precise Detection of RNA Structure.

Up to now, NMR spectroscopic investigations of RNA have utilized imino proton resonances as reporters for base pairing and RNA structure. The nucleobase amino groups are often neglected, since most of their resonances are broadened beyond detection due to rotational motion around the C-NH2 bond. Here, we present 13 C-detected NMR experiments for the characterization of all RNA amino groups irrespective of their motional behavior. We have developed a C(N)H-HDQC experiment that enables the observation of a complete set of sharp amino resonances through the detection of proton-NH2 double quantum coherences. Further, we present an "amino"-NOESY experiment to detect NOEs to amino protons, which are undetectable by any other conventional NOESY experiment. Together, these experiments allow the exploration of additional chemical shift information and inter-residual proton distances important for high-resolution RNA secondary and tertiary structure determination.

NMR resonance assignments for the GTP-binding RNA aptamer 9-12 in complex with GTP.

Ligand binding RNAs such as artificially created RNA-aptamers are structurally highly diverse. Therefore, they represent important model systems for investigating RNA-folding, RNA-dynamics and the molecular recognition of chemically very different ligands, ranging from small molecules to whole cells. High-resolution structures of RNA-aptamers in complex with their cognate ligands often reveal unexpected tertiary structure elements. Recent studies on different classes of aptamers binding the nucleotide triphosphate GTP as a ligand showed that these systems not only differ widely in binding affinity but also in their ligand binding modes and structural complexity. We initiated the NMR-based structure determination of the high-affinity binding GTP-aptamer 9-12 in order to gain further insights into the diversity of ligand binding modes and structural variability of those aptamers. Here, we report 1H, 13C and 15N resonance assignments for the GTP 9-12-aptamer bound to GTP as the prerequisite for the structure determination by solution NMR.

RNA structure refinement using NMR solvent accessibility data.

NMR spectroscopy is a powerful technique to study ribonucleic acids (RNAs) which are key players in a plethora of cellular processes. Although the NMR toolbox for structural studies of RNAs expanded during the last decades, they often remain challenging. Here, we show that solvent paramagnetic relaxation enhancements (sPRE) induced by the soluble, paramagnetic compound Gd(DTPA-BMA) provide a quantitative measure for RNA solvent accessibility and encode distance-to-surface information that correlates well with RNA structure and improves accuracy and convergence of RNA structure determination. Moreover, we show that sPRE data can be easily obtained for RNAs with any isotope labeling scheme and is advantageous regarding sample preparation, stability and recovery. sPRE data show a large dynamic range and reflect the global fold of the RNA suggesting that they are well suited to identify interaction surfaces, to score structural models and as restraints in RNA structure determination.

A Stably Protonated Adenine Nucleotide with a Highly Shifted pKa Value Stabilizes the Tertiary Structure of a GTP-Binding RNA Aptamer.

RNA tertiary structure motifs are stabilized by a wide variety of hydrogen-bonding interactions. Protonated A and C nucleotides are normally not considered to be suitable building blocks for such motifs since their pKa values are far from physiological pH. Here, we report the NMR solution structure of an in vitro selected GTP-binding RNA aptamer bound to GTP with an intricate tertiary structure. It contains a novel kind of base quartet stabilized by a protonated A residue. Owing to its unique structural environment in the base quartet, the pKa value for the protonation of this A residue in the complex is shifted by more than 5 pH units compared to the pKa for A nucleotides in single-stranded RNA. This is the largest pKa shift for an A residue in structured nucleic acids reported so far, and similar in size to the largest pKa shifts observed for amino acid side chains in proteins. Both RNA pre-folding and ligand binding contribute to the pKa shift.

NMR resonance assignments for the class II GTP binding RNA aptamer in complex with GTP.

The structures of RNA-aptamer-ligand complexes solved in the last two decades were instrumental in realizing the amazing potential of RNA for forming complex tertiary structures and for molecular recognition of small molecules. For GTP as ligand the sequences and secondary structures for multiple families of aptamers were reported which differ widely in their structural complexity, ligand affinity and ligand functional groups involved in RNA-binding. However, for only one of these families the structure of the GTP-RNA complex was solved. In order to gain further insights into the variability of ligand recognition modes we are currently determining the structure of another GTP-aptamer--the so-called class II aptamer--bound to GTP using NMR-spectroscopy in solution. As a prerequisite for a full structure determination, we report here (1)H, (13)C, (15)N and partial (31)P-NMR resonance assignments for the class II GTP-aptamer bound to GTP.