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A gene silencing kit and method

A gene silencing and kit technology, which can be applied to other methods of inserting foreign genetic materials, DNA/RNA fragments, recombinant DNA technology, etc. Simple to use effects

Active Publication Date: 2018-01-05
江苏协合转化医学研究院有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are few reports on the use of DNAzyme as a tool for gene silencing for treatment. The main reason is that the content of divalent metal ions in cells is relatively low, even the relatively high content of Mg 2+ It is also far from meeting the requirements of 10-23DNAzyme to catalyze the cleavage of RNA

Method used

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  • A gene silencing kit and method
  • A gene silencing kit and method

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1 Flake MnO 2 Synthesis of nanocarriers

[0028] MnO 2 The synthesis refers to relevant literature (Deng, R.; Xie, X.; Vendrell, M.; Chang, Y.-T.; Liu, X., Intracellular glutathione detection using MnO 2 -nanosheet-modifiedupconversion nanopaRT-icles.Journal of the American Chemical Society 2011,133(50),20168-20171.), first prepare 10mL 0.3M MnCl 2 4H2O solution with 20 mL of 0.6M TMA OH and 3 wt% H 2 o 2 Mixture. Quickly mix the two solutions, the mixture immediately turns dark black. The mixture was stirred vigorously at room temperature overnight. The obtained precipitate was washed with water and methanol, centrifuged at 2000 r / min for 20 min, and vacuum-dried at 60°C. Then the obtained MnO 2 Disperse in 20mL aqueous solution, sonicate, and then centrifuge to remove the unstripped MnO 2 remove. Flake MnO 2 For analysis of nanocarrier properties, see figure 2 .

Embodiment 2

[0029] Synthesis of Example 2 Ce6-DNAzyme

[0030] The synthesis of chlorin e6-conjugated deoxynucleic acid consists of two parts: the synthesis of DNAzyme on a DNA synthesizer and the reaction of DNA with Ce6 after synthesis. The specific reaction steps are as follows: DNA was synthesized on an ABI3400 DNA synthesizer. After the synthesis, the DNA was dried and subjected to detrityl treatment: first dissolved, then placed in 200 μL of 80% acetic acid for 20 min, and centrifuged to discard the supernatant. The DNA on CPG was washed 3 times with DPBS and finally dissolved in 250 μL of 0.1 M NaHCO pH 7.5. 3 in solution.

[0031] There are three carboxyl groups attached to each Ce6 molecule, which can be conjugated to amino groups on synthetic DNA. 10 μM Ce6 was mixed with equal amounts of DCC and NHS, then dissolved in DMF and stirred for activation reaction. The DNA and activated Ce6 were then mixed and shaken vigorously overnight to allow coupling. The DNA conjugated with...

Embodiment 3

[0032] Example 3 Ce6-DNAzyme-MnO 2 Assembly of nanosystems

[0033] Combine Ce6-DNAzyme with prepared MnO 2 In HEPES buffer (20mM, pH 7.2, containing 150mM NaCl and 2mM MgCl 2 ) and mix it evenly, and then place it for about 10 minutes, that is, self-assemble into the desired sample.

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Abstract

The invention discloses a gene silencing kit and a method for inhibiting cell proliferation by silencing gene EGR‑1 based on the kit. The kit comprises a Ce6-DNAzyme-MnO2 nanometer system consisting of sheet-like MnO2 nanocarriers, 10-23DNAzyme and photosensitizer Chlorine6 (Ce6). The present invention first utilizes intracellular glutathione-activated Mn2+ to assist 10‑23DNAzyme to perform effective gene silencing, and overcomes the difficulty that 10‑23DNAzyme lacks auxiliary ions in cells. The simultaneous activation of fluorescent signals and nuclear magnetic signals in cells can monitor the efficiency of the system entering cells and verify each other. Moreover, the method is simple to operate, and has great significance for the application of DNAzyme in the field of gene therapy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a gene silencing kit and method. Background technique [0002] Deoxyribozyme (DNAzyme) is a single-stranded DNA fragment with catalytic function synthesized by in vitro molecular evolution technology, which has high catalytic activity and structure recognition ability. After the discovery of Ribozyme, an RNA molecule with biocatalytic activity and specific self-cleavage in 1982, it completely broke the traditional view that the chemical essence of enzymes is protein, and also brought a new perspective to antisense gene intervention therapy at the mRNA level. Vitality, creating a new idea of ​​gene therapy starting from the anti-mRNA strategy. DNAzyme, like general biological enzymes, contains binding sites and catalytic sites. DNAzyme has (1) RNA cleavage; (2) DNA cleavage; (3) metal chelation; (4) peroxidase activity; (5) DNA kinase activity (6) DNA ligase activity and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87C12N15/113C12N5/10
Inventor 张晓兵范换换谭蔚泓赵子龙
Owner 江苏协合转化医学研究院有限公司
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