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1.
Biomol NMR Assign ; 15(2): 467-474, 2021 10.
Article in English | MEDLINE | ID: covidwho-1442185

ABSTRACT

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.


Subject(s)
5' Untranslated Regions , Nuclear Magnetic Resonance, Biomolecular , SARS-CoV-2/genetics , Nucleic Acid Conformation , RNA, Spliced Leader
2.
Biomol NMR Assign ; 15(2): 335-340, 2021 10.
Article in English | MEDLINE | ID: covidwho-1442184

ABSTRACT

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.


Subject(s)
5' Untranslated Regions , Nuclear Magnetic Resonance, Biomolecular , SARS-CoV-2/genetics , Inverted Repeat Sequences/genetics
4.
Angew Chem Int Ed Engl ; 60(35): 19191-19200, 2021 08 23.
Article in English | MEDLINE | ID: covidwho-1279344

ABSTRACT

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.


Subject(s)
Genome , RNA, Viral/metabolism , SARS-CoV-2/genetics , Small Molecule Libraries/metabolism , Drug Evaluation, Preclinical , Ligands , Molecular Structure , Nucleic Acid Conformation , Proton Magnetic Resonance Spectroscopy , RNA, Viral/chemistry , Small Molecule Libraries/chemistry
5.
Nucleic Acids Res ; 48(22): 12415-12435, 2020 12 16.
Article in English | MEDLINE | ID: covidwho-917705

ABSTRACT

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.


Subject(s)
COVID-19/prevention & control , Magnetic Resonance Spectroscopy/methods , Nucleic Acid Conformation , RNA, Viral/chemistry , SARS-CoV-2/genetics , 3' Untranslated Regions/genetics , Base Sequence , COVID-19/epidemiology , COVID-19/virology , Frameshifting, Ribosomal/genetics , Genome, Viral/genetics , Humans , Models, Molecular , Pandemics , SARS-CoV-2/physiology
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