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Locked nucleic acid ribozyme of targeted serine-threonine protein kinase aurora kinase A and application thereof

A technology of threonine protein and Aurora kinase, which is applied in the field of ribozyme targeting serine-threonine protein kinase Aurora kinase A, to achieve the effect of long serum half-life and strong inhibitory ability

Inactive Publication Date: 2010-12-15
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Prostate cancer is one of the most common malignant tumors. So far, there is no specific treatment. The molecular mechanism of prostate cancer has not been fully understood. However, recent studies have found that the occurrence and development of prostate cancer are closely related to the abnormality of Aurora A.

Method used

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  • Locked nucleic acid ribozyme of targeted serine-threonine protein kinase aurora kinase A and application thereof
  • Locked nucleic acid ribozyme of targeted serine-threonine protein kinase aurora kinase A and application thereof
  • Locked nucleic acid ribozyme of targeted serine-threonine protein kinase aurora kinase A and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: Aurora A-LNAzyme Design and Synthesis

[0015] Design of Aurora A-LNAzyme: introduce locked nucleic acid, modify the deoxyribozyme DNAzyme2 designed and synthesized by the inventor of this patent application, and design Aurora A-LNAzyme described in the present invention. The nucleotide sequence of the Aurora A-LNAzyme of the present invention is shown in SEQ ID NO: 1 in the sequence listing. In the nucleotide sequence, the first substrate recognition domain, the catalytic domain and the second substrate are composed The nucleotides of the recognition domain and their arrangement are as follows:

[0016] First substrate recognition domain Catalytic domain Second substrate recognition domain

[0017] 5't L t L aacagg ggctagctacaacga cct L gaaat L 3'

[0018] In order to verify the effect of Aurora A-LNAzyme described in the present invention, also designed contrast ribozyme (Control), the nucleotide sequence of contrast ribozyme and its arrangement are a...

Embodiment 2

[0023] Example 2: Aurora A-LNAzyme cleaves Aurora A in vitro

[0024] 1. Reagents and materials

[0025] RT-PCR kit: Qiagen LongRange 2-step RT-PCR Kit (Qiagen, Germany);

[0026] PC3, LNCaP and Du145 prostate cancer cells: purchased from ATCC, USA;

[0027] Plasmid pcDNA3.1(+): purchased from Invitrogen, USA;

[0028] EcoRI enzyme, XhoI enzyme and T4DNA ligase: purchased from Bao Biological Company;

[0029] Aurora A-LNAzyme: designed and synthesized in Example 1, prepared into a 50 μM / L solution with physiological saline for later use;

[0030] Control ribozyme: designed and synthesized in Example 1, prepared into a 50 μM / L solution with physiological saline for subsequent use;

[0031] T7 / SP6 in vitro transcription kit: purchased from Roche, USA;

[0032] Digoxigenin chemiluminescence detection kit: purchased from Roche, USA;

[0033] Formamide: purchased from Sigma, USA;

[0034] Bromophenol blue: purchased from Sigma, USA;

[0035] EDTA: purchased from Sigma, USA;...

Embodiment 3

[0046] Example 3: Transfection of prostate cancer cell PC3 with Aurora A-LNAzyme

[0047] 1. Reagents, materials and equipment

[0048] FuGENE6 was purchased from American Roche Company;

[0049] Aurora A-LNAzyme: designed and synthesized in Example 1, prepared into a 50 μM / L solution with physiological saline for later use;

[0050] Control ribozyme: designed and synthesized in Example 1, prepared into a 50 μM / L solution with physiological saline for subsequent use;

[0051] Prostate cancer cell PC3: purchased from ATCC, USA;

[0052] Coulter flow cytometer, purchased from Beckman company;

[0053] Data analysis software ModFitLT 2.0, product of Verity Software House, USA.

[0054] 2. Experimental method

[0055] Prostate cancer cell PC3 was routinely cultured. When the prostate cancer cell PC370% was fused, the Aurora A-LNAzyme solution with a concentration of 300nmol / L and the control ribozyme solution with a concentration of 300nmol / L were transfected into the prostat...

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Abstract

The invention relates to locked nucleic acid ribozyme of targeted serine-threonine protein kinase aurora kinase A, wherein the nucleotide sequence thereof is shown as SEQ IDNO:1 in a sequence table, 15 deoxynucleotides positioned at the middle part in the nucleotide sequence form a catalytic structure domain, 8 nucleotides positioned at the left side of the catalytic structure domain form a first substrate recognition structure domain, 8 nucleotides positioned at the right side of the catalytic structure domain form a second substrate recognition structure domain, and both the first substrate recognition structure domain and the second substrate recognition structure domain contain two locked nucleic acids tL. The locked nucleic acid ribozyme of the targeted serine-threonine protein kinase aurora kinase A can be applied to preparing medicines for treating prostatic cancer.

Description

technical field [0001] The present invention relates to a ribozyme targeting serine-threonine protein kinase Aurora kinase A (abbreviated as Aurora A, in the following contents of the specification, "Aurora A" means serine-threonine protein kinase Aurora kinase A) and its application . Background technique [0002] Prostate cancer is one of the most common malignant tumors. So far, there is no specific treatment. The molecular mechanism of prostate cancer has not been fully understood. However, recent studies have found that the occurrence and development of prostate cancer are closely related to the abnormality of Aurora A. The Aurora A gene is located on human chromosome 20q13, which is often amplified in human malignant tumors, so gene therapy is worth exploring. [0003] The inventors of this patent application have designed and synthesized the deoxyribozyme DNAzyme2 against Aurora A [see Cancer Gene Ther.200815(8):518 for the nucleotide sequence of DNAzyme2]. Experimen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P35/00A61P13/08
Inventor 母得志屈艺张林张莉赵凤艳伍金林王华石晶李熙鸿唐军
Owner SICHUAN UNIV
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