Method for detecting advanced structure of RNA (Ribonucleic Acid) virus based on proximity ligation
An RNA virus and advanced structure technology, applied in the field of detection of RNA virus advanced structure based on vicinal ligation, can solve problems such as difficulty in meeting, loss of structural details, difficulty in studying the structure of virus genome, etc., to improve ligation efficiency and reduce RNA loss. Effect
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Embodiment 1
[0066] Application of Hi-R technology to analysis of genome structure of novel coronavirus
[0067] 1. Experimental materials: cells and supernatant of Vero cells infected with novel coronavirus (SARS-CoV-2) Cross-linking agent: EZ-LinkPsoralen-PEG3-Biotin (Thermo Fisher Scientific) Permeabilization agent: digitonin (Sigma)
[0068] 2. Experimental steps
[0069] 2.1 Cross-linking
[0070] Will 9×10 7 Each / ml VeroE6 was infected with Wuhan-Hu-1 strain SARS-CoV-2 virus at MOI of 0.01 for 24 hours. Three of the replicate samples washed the cells three times with PBS, and collected the washed cells (denoted as C1, C2 and C3). The remaining infected samples were further cultured for 48 hours, and the virus culture supernatant was mixed with an equal volume of saturated sodium sulfate solution at 4° C. for 1 hour. The cells were washed three times with PBS, and the above virus pellets (denoted as V1, V2 and V3) and washed cells (denoted as L1, L2 and L3) were collected. Dilute...
Embodiment 2
[0107] Application of Hi-R technology to analysis of genome structure of Coxsackie virus
[0108] 1 Experimental materials
[0109] Virus particles in the supernatant of HeLa cells infected with Coxsackie virus (CVB-3);
[0110] Cross-linking agent: EZ-Link Psoralen-PEG3-Biotin (Thermo Fisher Scientific);
[0111] Permeabilizing agent: digitonin (Sigma).
[0112] 2. Experimental steps
[0113] 2.1 Cross-linking
[0114] 1×10 8 HeLa cells / ml were infected with MOI of 0.01CVB-3 virus strain for 24 hours. Concentrate virus by ultracentrifugation. Filter through a 0.6 μm microporous membrane, transfer to a 38 ml ultracentrifuge tube, carefully and gently add 5 ml of 35% sucrose solution filtered through a 0.2 μm microporous membrane to the bottom of the ultracentrifuge tube. Soldering iron seal. Centrifuge at 4°C and 100,000 g for 16 hours, centrifuge the virus particles to the bottom of the tube, carefully remove the upper culture medium, and collect the virus particles. ...
Embodiment 3
[0147] Use the dotplot method to judge the cross-linking efficiency of Coxsackie virus RNA. The specific method is as follows: use a certain concentration (1 μ M or 2 μ M) of EZ-Link Psoralen-PEG3-Biotin in PBS (containing 0.01% digitonin) and Coxsackie virus particles The samples were mixed and cross-linked for different times (0, 10 min, 20 min) under the irradiation of 365nm ultraviolet light. The biotin signal was detected in the samples. The denser the spot, the higher the cross-linking efficiency. The dotplot method can refer to prior art (Aw, J.G., Shen, Y., Wilm, A., Sun, M., Lim, X.N., Boon, K.L.,. Wan, Y. (2016).In Vivo Mapping of Eukaryotic RNA Interactomes Reveals Principles of Higher-Order Organization and Regulation. Mol Cell, 62(4), 603-617. doi:10.1016 / j.molcel.2016.04.028).
[0148] see results Figure 8 . Figure 8 The upper middle panel indicates the use of EZ-Link at a final concentration of 2 μM TM The biotin signal intensity of Psoralen-PEG3-Biotin cr...
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