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Antisense oligonucleotide for specifically adjusting POKEMON gene expression and application thereof

An antisense oligonucleotide and gene expression technology, applied in the field of antisense oligonucleotides, can solve the problems that natural oligonucleotides cannot exist stably, natural oligonucleotides cannot be used as antisense therapy, etc., and achieve stability Sexual enhancement, less toxic and side effects

Inactive Publication Date: 2010-08-04
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Natural oligonucleotides are not stable in serum and will be completely degraded within a few minutes, therefore, natural oligonucleotides cannot be used as drugs for antisense therapy

Method used

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  • Antisense oligonucleotide for specifically adjusting POKEMON gene expression and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1. Obtainment of antisense oligonucleotides that specifically regulate the expression of POKEMON gene

[0020] 1. Prediction of the secondary structure of POKEMON gene and design of antisense oligonucleotides

[0021] The mRNA secondary structure of POKEMON gene (NM_015898) was predicted using workstation (www.bioinfo.rpi.edu.cn / applications / mfold). According to the reading frame of POKEMON gene, four 18-mer-containing thioantisense oligonucleotides AS1-AS4 (the following formulae I-formula IV) were designed in combination with its secondary structure. The 18-nucleotide sequence AS5, which has no homology to any human gene, was used as a control.

[0022] AS1: 5'CxCxAxCxGyCyCyGyCyCyGyGyCyCyAxTxCxTx 3' (Formula I);

[0023] AS2: 5'CxGxCxTxGyAyCyGyAyAyGyTyCyGyAxTxCxTx 3' (Formula II);

[0024] AS3: 5'GxGxCxCxCyGyGyCyCyCyAyTyAyGyAxAxGxTx 3' (formula III);

[0025] AS4: 5'TxTxCxAxGyGyTyCyGyTyAyGyTyTyGxTxGxGx 3' (Formula IV);

[0026] AS5: 5'AxTxGxTxAyGyTyCyGyTy...

Embodiment 2

[0032] Example 2. Experiment on the inhibitory effect of antisense oligonucleotides on POKEMON-pEGFP fusion protein

[0033] 1. Construction of the fusion plasmid of POKEMON and pEGFP

[0034] The reading frame fragment of POKEMON was cloned from human cervical cancer HeLa cells (purchased from American Type Culture Collection, ATCC), and subcloned into pEGFP-N 2 The reporter plasmid enables the expression of POKEMON-EGFP fusion protein in mammalian cells.

[0035] 1) Cell culture

[0036] Human uterine carcinoma (Hela) cells (purchased from American Type Culture Collection, ATCC number: CCL-2) were prepared with 10% fetal bovine serum, 100u / ml penicillin and 100u / ml streptomycin and 0.2% NaHCO 3 DMEM medium at 37°C, 5% CO 2 cultured under normal conditions.

[0037] DMEM (Dulbecco's Modified Eagle Medium) medium is very suitable for many mammalian cell culture. It is composed of inorganic salt components, amino acid components, vitamin components, etc. Its specific formu...

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Abstract

The invention discloses an antisense oligonucleotide for specifically adjusting POKEMON gene expression and application thereof. The antisense oligonucleotide is one of the substances expressed in the following chemical formulas: CxCxAxCxGyCyCyGyCyCyGyGyCyCyAxTxCxTx, CxGxCxTxGxAyCyGyAyAyGyTyCyGyAxTxCxTx, GxGxCxCxCyGyGyCyCyCyAyTyAyGyAxAxGxTx, TxTxCxAxGxGyTyCyGyTyAyGyTyTyGxTxGxGx, TxTxCxAxGyGyTyCyGyTyAyGyTyTyGxTxGxGx, wherein x refers to bonds between thiophosphate nucleosides. The antisense oligonucleotide can be used for preparing a gene therapy medicament for curing the disease caused by high POKEMON expression.

Description

[0001] This application is a divisional application with an application number of 200810089057.8, an application date of April 15, 2008, and the title of invention and creation "Antisense oligonucleotides that specifically regulate the expression of POKEMON genes and their applications". technical field [0002] The invention relates to an antisense oligonucleotide for specifically regulating the expression of POKEMON gene and its application. Background technique [0003] POKEMON is a newly discovered oncogene that can cause other oncogenes to attack and eventually lead to tumor formation. POKEMON can enable cancer cells to acquire the ability to resist aging and death, so it is called the "master switch" of cancer. A very important point is that the loss of intracellular Pokemon will not affect the normal growth of cells, which provides the possibility to design specific anticancer drugs with low side effects. [0004] Izant and Weintraub first proposed antisense technolo...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K48/00A61P35/00
Inventor 蒋宇扬张楠谢振华
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV