A kind of photosensitive crosslinking agent and its application
A cross-linking agent and photosensitive technology, applied in the protein field, can solve the problems of increasing the experimental workload and working time, low efficiency, unfavorable RNPs enrichment and identification, etc., to achieve the effect of increasing the workload and working time, and shortening the experimental period.
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Embodiment 1
[0096] Example 1. Synthesis and NMR characterization of photosensitive cross-linking agent
[0097] 1. Synthesis
[0098] according to figure 1 The photosensitive cross-linking agent is prepared by the indicated synthetic route, and the specific steps are as follows
[0099] 1) Weigh 10 mg of SH-PEG-NHS, dissolve it in 1 mL of dichloromethane, add Boc-D-lysine at a molar ratio of SH-PEG-NHS:Boc-D-lysine=1:2, and sonicate it. Boc-D-lysine became finer particles, and 50 μL of triethylamine was added to maintain an alkaline environment, and the reaction was rotated at room temperature overnight. After the reaction was completed, 500 μL of 1M hydrochloric acid was added for extraction to remove unreacted Boc-D-lysine, and the procedure was repeated twice. The final dichloromethane layer was dried with nitrogen to obtain SH-PEG-Lys-Boc.
[0100] 2) After mixing dichloromethane and trifluoroacetic acid in a volume ratio of 1:1, dissolve SH-PEG-Lys-Boc, and rotate at room temperat...
Embodiment 2
[0106] Example 2. Imaging analysis of intracellular nucleic acid-protein complexes labeled with photosensitive cross-linking agents
[0107] The nucleic acid-protein complexes in cells were chemically labeled and fluorescently imaged using the photosensitive cross-linking agent prepared in Example 1. Specific steps are as follows:
[0108] Take HeLa cells of appropriate density and inoculate them in a 20mm glass-bottomed petri dish, incubate at 37°C, wash with PBS when the density is 70% to 80%, then add 200μM photosensitive cross-linking agent and incubate at 4°C for 30min, 365nm UV-crosslinking. The cells were washed 3 times with PBS, fixed with 4% paraformaldehyde (diluted in PBS) for 30 min at room temperature, and permeabilized with 0.5% Triton X-100 (diluted with PBS) for 30 min at room temperature; then the cells were washed 3 times with PBS, and click chemistry reaction solution was added (10μM Cy5-N 3 , 0.5 mM copper sulfate, 5 mM THPTA and 5 mM sodium ascorbate), c...
Embodiment 3
[0109] Example 3. Evaluation of the labeling of the same RNA with a photosensitive cross-linking agent combined with a biotin-based 7H-uraldehyde [3,2-g]benzopyran-7-one probe
[0110] according to Figure 4 The flow chart shown in A uses the photosensitive cross-linking agent prepared in Example 1 and the biotin-based 7H-uraldehyde [3,2-g]benzopyran-7-one probe to label the same RNA. The specific steps are as follows :
[0111] (1) Extract RNA:
[0112] Use a commercial kit to extract RNA according to the instructions, specifically miRNeasy Micro Kit. Specific steps are as follows:
[0113] Add 700 μL of QIAzol Lysis to the cells in a 10 cm culture dish, pipet repeatedly, vortex for 1 min to fully lyse the cells, place at room temperature for 5 min, add 140 μL of chloroform, shake vigorously for 15 s and place at room temperature for 2 min; About 350 μL) was transferred to a new RNase-free centrifuge tube, 1.5 times the volume of 100% ethanol was added, and the resulting ...
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