Journal: bioRxiv

Article Title: Development of a simple in vitro assay to identify and evaluate nucleotide analogs against SARS-CoV-2 RNA-dependent RNA polymerase

doi: 10.1101/2020.07.16.205799

Figure Lengend Snippet: Nsp12mut contains an active site mutation (active site motif SDD→SAA). Nsp8-7 represents copurified nsp8 and nsp7. M, protein molecular weight markers. The sizes of protein markers (in kDa) are indicated on the left.

Article Snippet: SARS-CoV-2 nsp7 gene (nucleotide 11846-12091, GenBank: MN908947.3 ) was synthesized de novo by GenScript (Nanjing, China) and cloned into the pMal-c5X vector using the same way as nsp8.

Techniques: Mutagenesis, Molecular Weight

Journal: bioRxiv

Article Title: Development of a simple in vitro assay to identify and evaluate nucleotide analogs against SARS-CoV-2 RNA-dependent RNA polymerase

doi: 10.1101/2020.07.16.205799

Figure Lengend Snippet: (A) The RNA primer and template used in this assay. The 30-mer primer (top) contains a fluorescent label (Cy5.5) at the 5’ end and the arrow indicates the location and direction of primer extension to form a 40-mer product. (B) Analysis of the nsp12 polymerase activity in the presence of nsp7, nsp8 or nsp8-7 (co-purified nsp8 and nsp7). The enzymes used in each reaction are indicated at the bottom of the gel. The concentrations for nsp12, nsp12mut, nsp7, nsp8 and nsp8-7 (co-purified nsp8 and nsp7) are 50 nM, 50 nM, 10 μM, 2 μM, and 2 μM (2 μM nsp8, 10 μM nsp7), respectively. Different enzymes and P/T (5 nM) were incubated in reaction buffer and the reactions were initiated by the addition of 100 μM rNTPs, and then continued at 37 °C for 1 h, and then were stopped by adding stopping solution. The products were separated on denaturing polyacrylamide gels. (C) Primer extension activity using nsp12 and nsp8, nsp7 or nsp8-7 (copurified). Nsp12 (50 nM) was used in all the samples. Primer extension reaction was performed as described above. In lane 2-8, co-purified nsp8 and nsp7 was used, and in lane 9-15, nsp8 and nsp7 were added to the reaction separately. The final concentrations of nsp8, nsp7 in the reaction were labeled under each lane in micromolar.

Article Snippet: SARS-CoV-2 nsp7 gene (nucleotide 11846-12091, GenBank: MN908947.3 ) was synthesized de novo by GenScript (Nanjing, China) and cloned into the pMal-c5X vector using the same way as nsp8.

Techniques: Activity Assay, Purification, Incubation, Labeling

Journal: bioRxiv

Article Title: Development of a simple in vitro assay to identify and evaluate nucleotide analogs against SARS-CoV-2 RNA-dependent RNA polymerase

doi: 10.1101/2020.07.16.205799

Figure Lengend Snippet: (A) The RNA primers (Primer I, Primer II and Primer III) and RNA template used in the experiments whose results are shown in panel B. (B) Primer extension assay using the different P/Ts from panel A. Four different nsp12 concentrations (6.25 nM, 12.5 nM, 25 nM, 50 nM; indicated on top of the gel), 2 μM nsp8-7 (2 μM nsp8 and 10 μM nsp7), and 5 nM different P/Ts (formed by annealing Primer I, Primer II or Primer III with the template shown in panel A; indicated at the bottom of the gel) were used in the assay. The reactions were initiated by the addition of 100 μM rNTP and continued at 37 °C for 1 h, and the products were separated on denaturing polyacrylamide gels. Three P/Ts without nsp12 added were used as a negative control (left three lanes) and the length of the primers (12-mer, 20-mer, 30-mer) was shown on the left of the gel.

Article Snippet: SARS-CoV-2 nsp7 gene (nucleotide 11846-12091, GenBank: MN908947.3 ) was synthesized de novo by GenScript (Nanjing, China) and cloned into the pMal-c5X vector using the same way as nsp8.

Techniques: Primer Extension Assay, Negative Control

Journal: Molecular Biology and Evolution

Article Title: Robustness of Reconstructed Ancestral Protein Functions to Statistical Uncertainty

doi: 10.1093/molbev/msw223

Figure Lengend Snippet: Summary Statistics for Ancestral Reconstructions in this Study.

Article Snippet: DNA sequences encoding the AncSR1 ML, AltAll, and Bayes DBDs were codon-optimized for expression in mammalian cells, synthesized by GenScript, cloned into the mammalian expression vector pCMV-AD (Stratagene), and fused in-frame with the NF-κB activation domain.

Techniques:

Journal: Molecular Biology and Evolution

Article Title: Robustness of Reconstructed Ancestral Protein Functions to Statistical Uncertainty

doi: 10.1093/molbev/msw223

Figure Lengend Snippet: Number of Amino Acid Differences among Alternative Ancestral Reconstructions for Three Protein Domains.

Article Snippet: DNA sequences encoding the AncSR1 ML, AltAll, and Bayes DBDs were codon-optimized for expression in mammalian cells, synthesized by GenScript, cloned into the mammalian expression vector pCMV-AD (Stratagene), and fused in-frame with the NF-κB activation domain.

Techniques: