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Specifically modified deoxynucleotide short chain and application thereof

A deoxynucleotide, specific technology, applied in the field of short deoxynucleotide chains, can solve problems such as lack, and achieve the effect of prolonging the action time and effectively anti-tumor prospects

Pending Publication Date: 2022-02-08
苏州冀玖生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is still a lack of relevant research on inhibiting the content of telomere RNA in cells and thus inhibiting telomerase activity.

Method used

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  • Specifically modified deoxynucleotide short chain and application thereof
  • Specifically modified deoxynucleotide short chain and application thereof
  • Specifically modified deoxynucleotide short chain and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0024] We used MGC-803 cells for preliminary research. Cells were seeded in 6-well plates, cultured in DMEM medium containing 10% FBS, transfected when the cells grew to a density of 30%-50%, and preheated at 37°C. - MEM I and medium. Dilute 3 μL Lipo 2000 into 250 μL opti-MEM I, gently pipette evenly, and let stand at room temperature for 5 minutes. Take 2 μL of 100 mM of one of the corresponding F1-F6 fragments, dilute to 250 μL of opti-MEM I, and gently pipette evenly; gently mix the diluted Lipo2000 and antisense nucleotide chains, and let stand at room temperature for 20 minutes. Aspirate the medium of the cells to be transfected in the six-well plate and replace with fresh prewarmed medium. After standing still for 20 min, the mixture was added dropwise into a six-well plate, and 1.5 ml of complete medium was added to make the final transfection concentration of antisense nucleotides 100 nM. The medium was changed after 6 hours, the supernatant was removed after 48 hou...

Embodiment 2

[0027] Select six tumor cells, hela (cervical cancer), SK-BR-3 (breast cancer), SMMC-7721 (liver cancer), HGC-27 (gastric cancer), A549 (lung cancer), Jurkat (leukemia), and transfect them with F5 Into the cells, the cells that were not transfected with any nucleotide short chain were used as the control, and the cells were collected after 48 hours of transfection, and Real-time PCR was used to detect the changes in the content of telomeric RNA in the cells, and the results were as follows: figure 2 as shown, figure 2 The control group is a white column, and the experimental group is a black column. It can be clearly observed that the content of telomeric RNA in each cell is significantly reduced, indicating that F5 has the effect of degrading telomeric RNA in different tumor cells.

[0028] Annexin V detection method of cell apoptosis: trypsinize the cells, do not discard any liquid, 300g, 4 ℃ horizontal centrifugation for 10min to collect the cells. Wash once with pre-co...

Embodiment 3

[0030] A549 (lung cancer) cells were selected, F5 was transfected into the cells, and cells not transfected with any short nucleotide chains were used as a control. Cells were collected 48 hours after transfection, and the key regulatory proteins Caspase 3, Caspase 3, and Expression of p53, Bax, Bcl-2. The result is as Figure 4shown. The results showed that when the telomere RNA in cells was significantly reduced, the expression of apoptosis regulatory proteins changed significantly, and the apoptosis signaling pathway was activated.

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Abstract

The invention discloses a specifically modified deoxynucleotide short chain. The deoxynucleotide short chain is characterized in that the nucleotide sequence of the deoxynucleotide short chain is 5'-CGGGGACTCGCTCCGTTCCTCTTC-3', the first three basic groups CGG at the 5'-end and the last three basic groups TTC at the 3'-end are subjected to sulfo-modification. After the deoxynucleotide short chain is specifically combined with telomerase RNA, RNase H in cells can recognize DNA-RNA heterochains and can degrade RNA chains in the DNA-RNA heterochains, therefore, telomere RNA in the cells can be degraded, thus the telomerase in the cells cannot play a role, and finally the apoptosis of the tumor cells is caused. The deoxynucleotide short chain can be successfully applied to knock-down of telomerase RNA of human gastric cancer cells, cervical cancer cells, liver cancer cells, breast cancer and JurkatT cells, and a remarkable cell apoptosis phenomenon is observed, so that the deoxynucleotide short chain has a broad-spectrum and effective anti-tumor prospect.

Description

technical field [0001] The invention relates to a specifically modified deoxynucleotide short chain and its application. Background technique [0002] Telomere (Telomere) is a small piece of DNA-protein complex that exists at the end of linear chromosomes in eukaryotic cells. It constitutes a special "cap" structure. The specific DNA sequence required. In normal cells, every time the cell divides, the telomere at the top of the chromosome is shortened once. As the number of cell divisions increases, the more the telomere wears out, the shorter the lifespan of the cell. When telomeres can no longer be shortened, cells cannot continue to divide, and then begin to move towards senescence or apoptosis. Therefore, telomeres are regarded as "life clock" by scientists. [0003] However, cancer cells have the ability to proliferate indefinitely. During the continuous replication of these cells, their telomeres do not shorten, and they do not enter the senescence or apoptosis path...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/7125A61P35/00
CPCC12N15/113A61K31/7125A61P35/00C12N2310/113C12N2310/334C12N2310/336C12N2310/335C12N2310/315
Inventor 杨耿李平舒妤钟欣妤刘兴柱
Owner 苏州冀玖生物科技有限公司
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