The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5′ end, the ribosomal frameshift segment and the 3′-untranslated region (3′-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.
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 1H‐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.
Previously, we have synthesized a diverse range of 2,5-furandicarboxylic acid (FDCA)-based semiaromatic polyamides via enzymatic polymerization. This novel class of polymers are biobased alternatives to polyphthalamides, which are petrol-based semiaromatic polyamides. From a commercial perspective, they have interesting properties as high-performance materials and engineering thermoplastics. It is even more appealing to explore novel FDCA-based polyamides with added functionality, for the development of sustainable functional materials. Here, a set of FDCA-based heteroatom polyamides have been successfully produced via Novozyme 435 (N435)-catalyzed polymerization of biobased dimethyl 2,5-furandicarboxylate with (potentially)heteroatom diamines, namely, 4,9-dioxa-1,12-dodecanediamine (DODA), diethylenetriamine, and 3,3-ethylenediiminopropylamine. We performed the enzymatic polymerization in solution and bulk. The latter approach is more sustainable and results in higher molecular weight products. Among the tested heteroatom diamines, N435 shows the highest catalytic activity toward DODA. Furthermore, we find that all obtained FDCA-based heteroatom polyamides are amorphous materials with a relatively high thermal stability. These heteroatom polyamides display a glass-transition temperature ranging from 41 to 107 °C.
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.
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