Method for establishing programmable intercellular communication by DNA nano machine based on pH response
A nanomachine and cell technology, applied in instruments, scientific instruments, material analysis by optical means, etc., can solve the problem of not realizing the transmission of response information molecules, and achieve the effect of simple and fast operation, high sensitivity and low dosage.
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Embodiment 1
[0022] Example 1 Modification of pH-responsive triple-stranded DNA on Ramos cell membrane:
[0023] The modification method is as follows:
[0024] 1) The DNA powders of the two sequences in the following table were centrifuged at 4000 rpm for 1 minute, dissolved in appropriate ultrapure water, and the final quantification was 100 μM. The DNA sequences used are as follows:
[0025]
[0026] 2) 250,000 Ramos cells were mixed in 1 ml of 1640 medium, placed in a 24-well plate and cultured overnight for 12 hours, after digestion, centrifuged with 1 ml of PBS at 1200 rpm for 3 minutes, and washed 3 times.
[0027] 3) Use 100 μL of PBS to resuspend the cells, add GGT-chol at a final concentration of 500 nM, and incubate at room temperature for 30 minutes.
[0028] 4) Centrifuge with 1 ml of PBS at 1200 rpm for 3 minutes and wash 3 times. Each group of cells was resuspended in 100 μL of PBS with pH 6.5 and pH 7.5, added with ACC-DTN-BHQ-AL488 at a final concentration of 1 μM, a...
Embodiment 2
[0035] Triple-stranded DNA-mediated cellular assembly at pH = 7.5.
[0036] 1) Centrifuge the DNA powders of the three sequences in the following table at 4000rpm for 1 minute, dissolve them in appropriate ultrapure water, and finally quantify them at 100 μM. The DNA sequences used are as follows:
[0037]
[0038] 2) 250,000 Ramos cells were mixed in 1 ml of 1640 medium, placed in a 24-well plate and cultured overnight for 12 hours, after digestion, centrifuged with 1 ml of PBS at 1200 rpm for 3 minutes, and washed 3 times.
[0039] 3) Cells were resuspended in 100 μL PBS, and cytoplasmic staining was performed with Celltrake Green. Then GGT-chol with a final concentration of 500 nM was added and incubated at room temperature for 30 minutes.
[0040] 4) Centrifuge with 1 ml of PBS at 1200 rpm for 3 minutes and wash 3 times. Use 100 μL of PBS to resuspend the cells, add ACC-DTN at a final concentration of 1 μM, and incubate at room temperature for 30 minutes.
[0041] 5...
Embodiment 3
[0047] Triple-stranded DNA-mediated cellular assembly at pH = 6.5.
[0048] sequence name 5’–3’ chol-tri488 Cholesterol-TTAAGGAAGAAGTTTACTTCTTCCTT-Alexa Flour488 AAG-chol AAGGA AGAAG TT-cholesterol
[0049] 2) 250,000 Ramos cells were mixed in 1 ml of 1640 medium, placed in a 24-well plate and cultured overnight for 12 hours, after digestion, centrifuged with 1 ml of PBS at 1200 rpm for 3 minutes, and washed 3 times.
[0050] 3) Use 100 μL of PBS to resuspend the cells, add chol-tri488 at a final concentration of 500 nM, and incubate at room temperature for 30 minutes.
[0051] 4) The cytoplasm of another group of blank cells was stained with Celltrake Deep Red. Then AAG-chol with a final concentration of 500 nM was added and incubated at room temperature for 30 minutes.
[0052] 5) The two groups of cells were centrifuged with 1 ml DPBS at 1200 rpm for 3 minutes, and washed 3 times. After resuspending the cells in 100 μL of DPBS, mix well and i...
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