Modulation of Aß42 Aggregation Kinetics and Pathway by Low-Molecular-Weight Inhibitors.

The aggregation of amyloid-ß 42 (Aß42) is directly related to the pathogenesis of Alzheimer's disease. Here, we have investigated the early stages of the aggregation process, during which most of the cytotoxic species are formed. Aß42 aggregation kinetics, characterized by the quantification of Aß42 monomer consumption, were tracked by real-time solution NMR spectroscopy (RT-NMR) allowing the impact that low-molecular-weight (LMW) inhibitors and modulators exert on the aggregation process to be analysed. Distinct differences in the Aß42 kinetic profiles were apparent and were further investigated kinetically and structurally by using thioflavin T (ThT) and transmission electron microscopy (TEM), respectively. LMW inhibitors were shown to have a differential impact on early-state aggregation. Insight provided here could direct future therapeutic design based on kinetic profiling of the process of fibril formation.

Site-Specific Labeling of RNAs with Modified and 19 F-Labeled Nucleotides by Chemo-Enzymatic Synthesis.

More than 170 post-transcriptional modifications of RNAs have currently been identified. Detailed biophysical investigations of these modifications have been limited since large RNAs containing these post-transcriptional modifications are difficult to produce. Further, adequate readout of spectroscopic fingerprints are important, necessitating additional labeling procedures beyond the naturally occurring RNA modifications. Here, we report the chemo-enzymatic synthesis of RNA modifications and several structurally similar fluorine-modified analogs further optimizing a recently developed methodology.[1] This chemo-enzymatic method allows synthesis of also large RNAs. We were able to incorporate 16 modified nucleotides and 6 19 F-labeled nucleotides. To showcase the applicability of such modified large RNAs, we incorporated a 19 F-labeled cytidine into the aptamer domain of the 2'dG sensing riboswitch (2'dG-sw) from Mesoplasma florum, enabling characterizing RNA fold, ligand binding and kinetics. Thanks to the large chemical shift dispersion of 19 F, we can detect conformational heterogeneity in the apo state of the riboswitch.

Wavelength-Selective Uncaging of Two Different Photoresponsive Groups on One Effector Molecule for Light-Controlled Activation and Deactivation.

Photocleavable protecting groups (PPGs) play a pivotal role in numerous studies. They enable controlled release of small effector molecules to induce biochemical function. The number of PPGs attached to a variety of effector molecules has grown rapidly in recent years satisfying the high demand for new applications. However, until now molecules carrying PPGs have been designed to activate function only in a single direction, namely the release of the effector molecule. Herein, we present the new approach Two-PPGs-One-Molecule (TPOM) that exploits the orthogonal photolysis of two photoprotecting groups to first release the effector molecule and then to modify it to suppress its induced effect. The moiety resembling the tyrosyl side chain of the translation inhibitor puromycin was synthetically modified to the photosensitive ortho-nitrophenylalanine that cyclizes upon near UV-irradiation to an inactive puromycin cinnoline derivative. Additionally, the modified puromycin analog was protected by the thio-coumarylmethyl group as the second PPG. This TPOM strategy allows an initial wavelength-selective activation followed by a second light-induced deactivation. Both photolysis processes were spectroscopically studied in the UV/vis- and IR-region. In combination with quantum-chemical calculations and time-resolved NMR spectroscopy, the photoproducts of both activation and deactivation steps upon illumination were characterized. We further probed the translation inhibition effect of the new synthesized puromycin analog upon light activation/deactivation in a cell-free GFP translation assay. TPOM as a new method for precise triggering activation/deactivation of effector molecules represents a valuable addition for the control of biological processes with light.

19 F NMR-Based Fragment Screening for 14 Different Biologically Active RNAs and 10 DNA and Protein Counter-Screens.

We report here the nuclear magnetic resonance 19 F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter-screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low-micromolar binding affinity without losing binding specificity between two different terminator structures.

Exploring the Druggability of Conserved RNA Regulatory Elements in the SARS-CoV-2 Genome.

SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1 H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.

A New Photocaged Puromycin for an Efficient Labeling of Newly Translated Proteins in Living Neurons.

Monitoring newly synthesized proteins is becoming increasingly important to characterize proteome composition in regulatory networks. Puromycin is a peptidyl transfer inhibitor, widely used in cell biology for tagging newly synthesized proteins. Here, we report synthesis and application of an optimized puromycin carrying a photolabile protecting group as a powerful tool for tagging nascent proteins with high spatiotemporal resolution. The photocaged 7-N,N-(diethylaminocumarin-4-yl)-methoxycarbonyl-puromycin (DEACM-puromycin) was synthesized and compared with the previously developed 6-nitroveratryloxycarbonyl puromycin (NVOC-puromycin). The photochemical behavior as well as the effectiveness in controlling puromycylation in living hippocampal neurons using two-photon excitation is superior to the previously used NVOCpuromycin. We further report on the application of light-controlled puromycylation to visualize new translated proteins in neurons.

1H, 13C and 15N chemical shift assignment of the stem-loops 5b + c from the 5'-UTR of SARS-CoV-2.

The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5'- and 3'-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5'-untranslated region (5'-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5'-UUUCGU-3' hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive 1H, 13C and 15N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy.

1H, 13C and 15N chemical shift assignment of the stem-loop 5a from the 5'-UTR of SARS-CoV-2.

The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain cis-acting RNA elements that affect RNA translation and replication. The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5'-untranslated region (5'-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5'-UUUCGU-3' hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive 1H, 13C and 15N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy.

1H, 13C, 15N and 31P chemical shift assignment for stem-loop 4 from the 5'-UTR of SARS-CoV-2.

The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5'-untranslated region (5'-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1H, 13C, 15N and 31P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy.

1H, 13C and 15N assignment of stem-loop SL1 from the 5'-UTR of SARS-CoV-2.

The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7-33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.

Structural rearrangement of amyloid-ß upon inhibitor binding suppresses formation of Alzheimer's disease related oligomers.

The formation of oligomers of the amyloid-ß peptide plays a key role in the onset of Alzheimer's disease. We describe herein the investigation of disease-relevant small amyloid-ß oligomers by mass spectrometry and ion mobility spectrometry, revealing functionally relevant structural attributes. In particular, we can show that amyloid-ß oligomers develop in two distinct arrangements leading to either neurotoxic oligomers and fibrils or non-toxic amorphous aggregates. Comprehending the key-attributes responsible for those pathways on a molecular level is a pre-requisite to specifically target the peptide's tertiary structure with the aim to promote the emergence of non-toxic aggregates. Here, we show for two fibril inhibiting ligands, an ionic molecular tweezer and a hydrophobic peptide that despite their different interaction mechanisms, the suppression of the fibril pathway can be deduced from the disappearance of the corresponding structure of the first amyloid-ß oligomers.

Phosphate-group recognition by the aptamer domain of the thiamine pyrophosphate sensing riboswitch.

Riboswitches are highly structured RNA elements that control gene expression by binding directly to small metabolite molecules. Remarkably, many of these metabolites contain negatively charged phosphate groups that contribute significantly to the binding affinity. An example is the thiamine pyrophosphate-sensing riboswitch in the 5'-untranslated region of the E. coli thiM mRNA. This riboswitch binds, in order of decreasing affinity, to thiamine pyrophosphate (TPP), thiamine monophosphate (TMP), and thiamine, which contain two, one, and no phosphate groups, respectively. We examined the binding of TPP and TMP to this riboswitch by using (31)P NMR spectroscopy. Chemical-shift changes were observed for the alpha- and beta-phosphate group of TPP and the phosphate group of TMP upon RNA binding; this indicates that they are in close contact with the RNA. Titration experiments with paramagnetic Mn(2+) ions revealed strong line-broadening effects for both (31)P signals of the bound TPP; this indicates a Mg(2+) binding site in close proximity and suggests that the phosphate group(s) of the ligand is/are recognized in a magnesium ion-mediated manner by the RNA.