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- RNA (2)
- SARS-CoV-2 (2)
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- 1H, 13C and 15N assignment of stem-loop SL1 from the 5'-UTR of SARS-CoV-2 (2021)
- 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.
- NMR spectroscopy of large functional RNAs: from sample preparation to low‐gamma detection (2020)
- NMR spectroscopy is a potent method for the structural and biophysical characterization of RNAs. The application of NMR spectroscopy is restricted in RNA size and most often requires isotope‐labeled or even selectively labeled RNAs. Additionally, new NMR pulse sequences, such as the heteronuclear‐detected NMR experiments, are introduced. We herein provide detailed protocols for the preparation of isotope‐labeled RNA for NMR spectroscopy via in vitro transcription. This protocol covers all steps, from the preparation of DNA template to the transcription of milligram RNA quantities. Moreover, we present a protocol for a chemo‐enzymatic approach to introduce a single modified nucleotide at any position of any RNA. Regarding NMR methodology, we share protocols for the implementation of a suite of heteronuclear‐detected NMR experiments including 13C‐detected experiments for ribose assignment and amino groups, the CN‐spin filter heteronuclear single quantum coherence (HSQC) for imino groups and the 15N‐detected band‐selective excitation short transient transverse‐relaxation‐optimized spectroscopy (BEST‐TROSY) experiment. Basic Protocol 1: Preparation of isotope‐labeled RNA samples with in vitro transcription using T7 RNAP, DEAE chromatography, and RP‐HPLC purification Alternate Protocol 1: Purification of isotope‐labeled RNA from in vitro transcription with preparative PAGE Alternate Protocol 2: Purification of isotope‐labeled RNA samples from in vitro transcription via centrifugal concentration Support Protocol 1: Preparation of DNA template from plasmid Support Protocol 2: Preparation of PCR DNA as template Support Protocol 3: Preparation of T7 RNA Polymerase (T7 RNAP) Support Protocol 4: Preparation of yeast inorganic pyrophosphatase (YIPP) Basic Protocol 2: Preparation of site‐specific labeled RNAs using a chemo‐enzymatic synthesis Support Protocol 5: Synthesis of modified nucleoside 3′,5′‐bisphosphates Support Protocol 6: Preparation of T4 RNA Ligase 2 Support Protocol 7: Setup of NMR spectrometer for heteronuclear‐detected NMR experiments Support Protocol 8: IPAP and DIPAP for homonuclear decoupling Basic Protocol 3: 13C‐detected 3D (H)CC‐TOCSY, (H)CPC, and (H)CPC‐CCH‐TOCSY experiments for ribose assignment Basic Protocol 4: 13C‐detected 2D CN‐spin filter HSQC experiment Basic Protocol 5: 13C‐detected C(N)H‐HDQC experiment for the detection of amino groups Support Protocol 9: 13C‐detected CN‐HSQC experiment for amino groups Basic Protocol 6: 13C‐detected “amino”‐NOESY experiment Basic Protocol 7: 15N‐detected BEST‐TROSY experiment
- Exploring the druggability of conserved RNA regulatory elements in the SARS-CoV-2 genome (2021)
- 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.
