Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements
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Abstract
SARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome, whose outbreak caused the ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally-conserved coronavirus structural RNA elements have been identified to date. Here, we performed RNA structure probing to obtain single-base resolution secondary structure maps of the full SARS-CoV-2 coronavirus genome both in vitro and in living infected cells. Probing data recapitulate the previously described coronavirus RNA elements (5′ UTR and s2m), and reveal new structures. Of these, ∼10.2% show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. Secondary structure-restrained 3D modeling of these segments further allowed for the identification of putative druggable pockets. In addition, we identify a set of single-stranded segments in vivo, showing high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics. Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.
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SciScore for 10.1101/2020.06.15.151647: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cell culture and SARS-CoV-2 infection: Vero E6 cells were cultured in T-175 flasks in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, cat. 12-604F), supplemented with 8% fetal calf serum (FCS; Bodinco), 2 mM L-glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin (Sigma Aldrich, cat. Vero E6suggested: RRID:CVCL_XD71)Software and Algorithms Sentences Resources Multiplex SHAPE-MaP of SARS-CoV-2 RNA: For multiplex SHAPE-MaP, 70 oligonucleotide pairs, tiling the entire length of the SARS-CoV-2 genome (29,903 nt), were automatically designed using Primer3 (Untergasser et al., … SciScore for 10.1101/2020.06.15.151647: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Cell culture and SARS-CoV-2 infection: Vero E6 cells were cultured in T-175 flasks in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, cat. 12-604F), supplemented with 8% fetal calf serum (FCS; Bodinco), 2 mM L-glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin (Sigma Aldrich, cat. Vero E6suggested: RRID:CVCL_XD71)Software and Algorithms Sentences Resources Multiplex SHAPE-MaP of SARS-CoV-2 RNA: For multiplex SHAPE-MaP, 70 oligonucleotide pairs, tiling the entire length of the SARS-CoV-2 genome (29,903 nt), were automatically designed using Primer3 (Untergasser et al., 2012) and the following parameters: amplicon size between 480 and 520 bp, maximum poly(N) length between 2 and 3, minimum/optimal/maximum oligonucleotide size of 20/25/30, minimum/optimal/maximum Tm of 56/60/62 degrees, minimum/optimal/maximum GC content of 30/50/65 %. Primer3suggested: (Primer3, RRID:SCR_003139)Primers were then searched against the GENCODE v33 human transcriptome, keeping only those with less than 60% predicted base-pairing or more than 60% predicted base-pairing and more than 2 mismatched bases at the 3′ end. GENCODEsuggested: (GENCODE, RRID:SCR_014966)Reads were trimmed of terminal Ns and low-quality bases (Phred < 20) Phredsuggested: (Phred, RRID:SCR_001017)After calibrating the CM using the cmcalibrate module, it was used to search for RNA homologs in a database composed of all the non-redundant coronavirus complete genome sequences from the ViPR database (https://www.viprbrc.org/brc/home.spg?decorator=corona; Pickett et al., 2011), as well as a set of representative coronavirus genomes from NCBI database, using the cmsearch module. ViPRsuggested: (vipR, RRID:SCR_010685)NCBIsuggested: (NCBI, RRID:SCR_006472)Determination of low Shannon – high SHAPE regions’ conservation: To assess the sequence conservation of the identified low Shannon – high SHAPE regions, we computed 4 multiple sequence alignments using MAFFT v7.429 (parameters: --maxiterate 100 --auto; Katoh and Standley, 2013), the reference SARS-CoV-2 sequence and one of the following datasets: 1) SARS-CoV (243 sequences); 2) MERS-CoV (281 sequences); 3) other Beta-CoV (excluding SARS-CoV/SARS-CoV-2/MERS-CoV, 681 sequences); 4) other CoV (excluding Beta-CoV, 1657 sequences). MAFFTsuggested: (MAFFT, RRID:SCR_011811)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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