Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome
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SciScore for 10.1101/2020.07.18.209270: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Frameshifting levels were calculated by taking the ratio: The resulting mean fold-change frameshifting data were then fitted using the Matlab 2019b (MathWorks) “Levenberg-Marquardt” algorithm to a standard binding isotherm (Slp2 was fitted using a constrained IC50 to 130 nM):
LNA treatment, caspase activation and cell viability assays: Cell-lines used for in cellulo experiments, Vero E6 and Huh-7, were maintained at 37°C in Dulbecco’s …
SciScore for 10.1101/2020.07.18.209270: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Frameshifting levels were calculated by taking the ratio: The resulting mean fold-change frameshifting data were then fitted using the Matlab 2019b (MathWorks) “Levenberg-Marquardt” algorithm to a standard binding isotherm (Slp2 was fitted using a constrained IC50 to 130 nM):
LNA treatment, caspase activation and cell viability assays: Cell-lines used for in cellulo experiments, Vero E6 and Huh-7, were maintained at 37°C in Dulbecco’s modified Eagle medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Invitrogen), penicillin and streptomycin (Gibco), and HEPES buffer (Gibco).
Huh-7suggested: NoneFor caspase activation and cell viability assays, one day prior to transfection, 10,000 cells per well of Huh-7 or Vero E6 cells were plated in growth media in black-walled, clear bottom 96-well plates (EK-25090, E&K Scientific) to be 50-70% confluent at time of transfection. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Frameshifting levels were calculated by taking the ratio: The resulting mean fold-change frameshifting data were then fitted using the Matlab 2019b (MathWorks) “Levenberg-Marquardt” algorithm to a standard binding isotherm (Slp2 was fitted using a constrained IC50 to 130 nM):
LNA treatment, caspase activation and cell viability assays: Cell-lines used for in cellulo experiments, Vero E6 and Huh-7, were maintained at 37°C in Dulbecco’s modified Eagle medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Invitrogen), penicillin and streptomycin (Gibco), and HEPES buffer (Gibco).
Matlabsuggested: (MATLAB, RRID:SCR_001622)Data were analyzed and graphed in Prism 8 by GraphPad. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)The initial models for both datasets were built in cryoSPARC using the ab-initio reconstruction option. cryoSPARCsuggested: (cryoSPARC, RRID:SCR_016501)For all other cases, 150,000 decoys were generated using the Stanford high performance computing cluster, Sherlock 2.0, and the Open Science Grid76. Sherlocksuggested: (Sherlock, RRID:SCR_001628)Results from OddPub: Thank you for sharing your code and data.
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:We note two caveats regarding our cryo-EM analysis. First, the 6.9 Å resolution of the cryo-EM map is not sufficient to directly resolve base interactions, much less atom positions. It is likely that hydrogen bonds and base pairs beyond our modeled secondary structure stabilize the FSE 3D structure, and higher resolution structures will be needed to resolve those details. Second, cryo-EM data processing can filter out alternative, less well-defined conformations while focusing on particle images that contribute to the most well-defined structures. De novo computer modeling, in addition to predicting the ring-threaded structure we observe, has suggested alternative conformations in which the 5′ end is not threaded through the ring (Extended Data Figure 9). Such alternative states of FSE may exist in our preparations but escape computational detection due to the extra conformational heterogeneity at the 5′ ends. Within our nanostructure-tagged map (Fig. 3b), alternative states may contribute to the lower resolution of the FSE segment compared to the nanostructure segment. Our cryo-EM-guided data and modeling, along with recent results from several groups, suggest explanations for why FSE targeting efforts by us and others have so far yielded only modest inhibition of frameshifting and viral replication4–6, 34. There is accumulating evidence that, in the full SARS-CoV-2 genome context, the FSE forms alternative structures involving genomic segments upstream of the element (Exten...
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: Please consider improving the rainbow (“jet”) colormap(s) used on page 49. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
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