CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands
Abstract
:1. Introduction
2. Results and Discussion
2.1. G4 Prediction and Selection in the SARS-CoV-2 Positive and Negative Genome
2.2. Confirmation That the Selected PQSs Fold In Vitro as G4
2.3. Cellular Nucleic Acids Binding Protein (CNBP) Interacts with Some SARS-CoV-2 G4s and Promotes Their Unfolding
2.4. Integration of Results with Putative Functions of G4 and CNBP in SARS-CoV-2 Replicative Cycle
3. Materials and Methods
3.1. Bioinformatics
3.2. Oligonucleotides
3.3. Circular Dichroism (CD) Spectroscopy
3.4. 1D 1H Nuclear Magnetic Resonance (NMR)
3.5. Thermal Difference Spectroscopy (TDS)
3.6. ThT Fluorescence Assays
3.7. CNBP Expression and Purification
3.8. Electrophoretic Mobility Shift Assay (EMSA)
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Betacoronavirus | Genome Accession Number | % G | Number of PQSs in QGRS Mapper | Selected PQSs | % of Selected over Predicted PQSs | |||
---|---|---|---|---|---|---|---|---|
Low Probability to Form G4 | Medium Probability to Form G4 | High Probability to Form G4 | Total | |||||
+gRNA | ||||||||
SARS-CoV-2 | NC_045512.2 | 19.6 | 37 | 1 | 1 | 1 | 3 | 8.1 |
RaTG13 | MN996532.2 | 19.6 | 37 | 1 | 2 | 1 | 4 | 10.8 |
Bat-SL-CoVZXC21 | MG772934.1 | 20.1 | 32 | 2 | 2 | 4 | 8 | 25.0 |
Bat-SL-CoVZC45 | MG772933.1 | 20.2 | 29 | 1 | 1 | 3 | 5 | 17.2 |
SARS-CoV | NC_004718.3 | 20.8 | 49 | 2 | 2 | 2 | 6 | 12.2 |
MERS-CoV | NC_019843.3 | 20.9 | 40 | 3 | 1 | 2 | 6 | 15.0 |
−gRNA | ||||||||
SARS-CoV-2 | NC_045512.2 | 18.4 | 19 | - | 3 | 2 | 5 | 26.3 |
RaTG13 | MN996532.2 | 18.4 | 20 | - | 1 | 3 | 4 | 20.0 |
Bat-SL-CoVZXC21 | MG772934.1 | 18.7 | 18 | 2 | 2 | - | 4 | 22.2 |
Bat-SL-CoVZC45 | MG772933.1 | 18.7 | 16 | 2 | 2 | - | 4 | 25.0 |
SARS-CoV | NC_004718.3 | 20.0 | 29 | 2 | - | 3 | 5 | 17.2 |
MERS-CoV | NC_019843.3 | 20.3 | 38 | 2 | 2 | 5 | 9 | 23.7 |
PQS Name | Genome | Length | Sequence | Prediction Scores | Reference of Previous Prediction | Reference of Experimental Evidence of G4 Formation | ||||
---|---|---|---|---|---|---|---|---|---|---|
G4RNA Screener | QGRS Mapper | PQS Finder | ||||||||
cGcC | G4H | G4NN | ||||||||
+644 | ORF1ab nsp1 | 20 | GGUAAUAAAGGAGCUGGUGG C G G C | 17 | 0.8 | 0.69 | 30 | 20 | [30,31,32,33,34,36] | [36] |
+3467 | ORF1ab nsp3 | 17 | GGAGGAGGUGUUGCAGGA A A G A A GAAG U U | 18 | 1 | 0.87 | 30 | 24 | [30,31,32,33,34,36] | [34] |
+28,903 | N | 15 | GGCUGGCAAUGGCGG UAU UU UU UUUAU AU ----- C | 5.33 | 0.87 | 0.57 | 33 | 27 | [30,31,32,33,34,36] | [34,35,36] |
−13,963 | ORF1ab nsp12 | 18 | GGAUCUGGGUAAGGAAGG G G G AA | 22 | 1.11 | 0.97 | 34 | 23 | [32,33,36] | - |
−23,877 | ORF1ab nsp3 | 17 | GGAUAUGGUUGGUUUGG AA C C A A | 160 | 0.94 | 0.02 | 34 | 24 | [32,33,36] | - |
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Bezzi, G.; Piga, E.J.; Binolfi, A.; Armas, P. CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands. Int. J. Mol. Sci. 2021, 22, 2614. https://doi.org/10.3390/ijms22052614
Bezzi G, Piga EJ, Binolfi A, Armas P. CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands. International Journal of Molecular Sciences. 2021; 22(5):2614. https://doi.org/10.3390/ijms22052614
Chicago/Turabian StyleBezzi, Georgina, Ernesto J. Piga, Andrés Binolfi, and Pablo Armas. 2021. "CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands" International Journal of Molecular Sciences 22, no. 5: 2614. https://doi.org/10.3390/ijms22052614