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.

Among the three major classes of biomacromolecules (DNA, RNA, and proteins) RNA's pronounced dynamics are the most explicitly linked to its wide variety of functions, which include catalysis and the regulation of transcription, translation, and splicing. These functions are mediated by a range of RNA biomachinery, including such varied examples as macromolecular noncoding RNAs, microRNAs, small interfering RNAs, riboswitch RNAs, and RNA thermometers. In each case, the functional dynamics of an interconversion is characterized by an associated rate constant. In this Account, we provide an introduction to NMR spectroscopic characterization of the landscape of RNA dynamics. We introduce strategies for measuring NMR parameters at various time scales as well as the underlying models for describing the corresponding rate constants. RNA exhibits significant dynamic motion, which can be modulated by (i) intermolecular interactions, including specific and nonspecific binding of ions (such as Mg(2+) and tertiary amines), (ii) metabolites in riboswitches or RNA aptamers, and (iii) macromolecular interactions within ribonucleic protein particles, including the ribosome and the spliceosome. Our understanding of the nature of these dynamic changes in RNA targets is now being incorporated into RNA-specific approaches in the design of RNA inhibitors. Interactions of RNA with proteins, other RNAs, or small molecules often occur through binding mechanisms that follow an induced fit mechanism or a conformational selection mechanism, in which one of several populated RNA conformations is selected through ligand binding. The extent of functional dynamics, including the kinetic formation of a specific RNA tertiary fold, is dependent on the messenger RNA (mRNA) chain length. Thus, during de novo synthesis of mRNA, both in prokaryotes and eukaryotes, nascent mRNA of various lengths will adopt different secondary and tertiary structures. The speed of transcription has a critical influence on the functional dynamics of the RNA being synthesized. In addition to modulating the local dynamics of a conformational RNA ensemble, a given RNA sequence may adopt more than one global, three-dimensional structure. RNA modification is one way to select among these alternative structures, which are often characterized by nearly equal stability, but with high energy barriers for conformational interconversion. The refolding of different secondary and tertiary structures has been found to be a major regulatory mechanism for transcription and translation. These conformational transitions can be characterized with NMR spectroscopy, for any given RNA sequence, in response to external stimuli.

In a combined NMR/MD study, the temperature-dependent changes in the conformation of two members of the RNA YNMG-tetraloop motif (cUUCGg and uCACGg) have been investigated at temperatures of 298, 317 and 325 K. The two members have considerable different thermal stability and biological functions. In order to address these differences, the combined NMR/MD study was performed. The large temperature range represents a challenge for both, NMR relaxation analysis (consistent choice of effective bond length and CSA parameter) and all-atom MD simulation with explicit solvent (necessity to rescale the temperature). A convincing agreement of experiment and theory is found. Employing a principle component analysis of the MD trajectories, the conformational distribution of both hairpins at various temperatures is investigated. The ground state conformation and dynamics of the two tetraloops are indeed found to be very similar. Furthermore, both systems are initially destabilized by a loss of the stacking interactions between the first and the third nucleobase in the loop region. While the global fold is still preserved, this initiation of unfolding is already observed at 317 K for the uCACGg hairpin but at a significantly higher temperature for the cUUCGg hairpin.

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.

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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.

The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein with the RNA packaging signal Psi ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV-1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. We previously selected peptides binding to HIV-1 Psi-RNA or stem loops (SL) thereof by phage display. Herein, we describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize Psi-RNA binding. The optimized peptide, psi-pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the Psi packaging signal by NMR and tryptophan fluorescence quenching. Functional analysis revealed that psi-pepB caused a strong reduction of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compound to develop new drugs targeting HIV-1.

Tying up the package: The HIV‐1 RNA packaging signal ψ is a complex RNA structure located within the 5′‐end of unspliced HIV‐1 RNAs (see picture). Upon binding of the viral polyprotein Gag to the ψ RNA, a process mediated by the nucleocapsid protein NCp7, two RNA genomes are packaged into the assembling virions. By using phage display technology peptides were selected that bind specifically to different RNA structures derived from the ψ RNA.