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A kind of complete set of siRNA for suppressing clusterin gene expression and application thereof

A suite of, gene-targeting techniques applied in the field of molecular biology to address issues such as increased response

Active Publication Date: 2020-06-26
GUANGZHOU RIBOBIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Blockade of EMT with oligonucleotides that interact with specific regions in sCLU leads to tumor growth inhibition and increased response to cytotoxic drugs

Method used

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  • A kind of complete set of siRNA for suppressing clusterin gene expression and application thereof
  • A kind of complete set of siRNA for suppressing clusterin gene expression and application thereof
  • A kind of complete set of siRNA for suppressing clusterin gene expression and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, the preparation of complete set of siRNA

[0078] 1. Design principles

[0079] All single siRNAs designed target the target gene CLU (as shown in Table 1), and the design method refers to Elbashir et al.2002; Paddison et al.2002; Reynolds et al.2004; Ui-Tei et al.2004 et al. S.M., Harborth, J., Weber, K., and Tuschl, T. 2002. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26:199–213; Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J., and Conklin, D.S. 2002. Short hairpin RNAs (shRNAs) induce sequence-specific silencing inmammalian cells. Genes & Dev. 16:948–958; Reynolds, A., Leake, D., Boese, Q., Scaringe, S ., Marshall, W.S., and Khvorova, A. 2004. Rational siRNA design for RNAinterference. Nat. Biotechnol. 22:326–330; Ui-Tei, K., Naito, Y., Takahashi, F., Haraguchi, T. , Ohki-Hamazaki, H., Juni, A., Ueda, R., and Saigo, K. 2004. Guidelines for the selection of highly effective siRNA sequences for mam...

Embodiment 2

[0095] Embodiment 2, the study of complete set of siRNA inhibiting target gene expression

[0096] 1. Comparison of the inhibitory effects of a complete set of siRNA RM-2 in different cell lines

[0097] Four different cell lines (293T, HeLa, A549 and HUVEC (ATCC) were inoculated in cell culture plates and cultured for 24 hours to observe the cells, and the cells were in good condition to start transfection.

[0098] Table 4 Source of Cell Lines

[0099] cell line name source 293T human embryonic kidney cells ATCC HeLa cervical cancer cells ATCC A549 Non-small cell lung cancer cells ATCC HUVECs human umbilical vein endothelial cells ATCC

[0100] 50μL riboFECT transfection system: 5μL riboFECT TM CP Reagent (Guangzhou Ruibo Biotechnology Co., Ltd., C10511-05), 5 μL of the complete siRNA group RM-2 prepared in Example 1 (the total concentration of all siRNAs is 100 nM) and 40 μL riboFECT TM CP Buffer (Guangzhou Ruibo Biotec...

Embodiment 3

[0131] Embodiment 3, in vitro stability determination

[0132] Dilute siRNA RB-CLU-D6 to 5 μM with RNase-free water, add an equal volume of fresh rat serum (product of Shanghai Yuanmu Biotechnology Co., Ltd.), and incubate at 37°C for 6 hours, take samples for electrophoresis to observe the siRNA integrity.

[0133] The result is as figure 1 As shown, the siRNAs of the present invention are stable in serum and are expected to have better potency in vivo.

[0134] For other RB-CLU-Dx (x is D1-D10), the experimental results are the same, and the specific figures are omitted.

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Abstract

The invention discloses a siRNA set for inhibiting cluster protein gene expression and application thereof. The siRNA set is composed of positive-sense strand and reverse-complementary antisense strand; the positive-sense strand is composed of 19-27 nucleotides, and 5-9 consecutive nucleotides from the 5' and3' ends of the positive-sense strand are both modified with 2'-ribose. Experiments show that the siRNA molecule mixtures improve the probability ofinhibiting gene expression by oligonucleotide, the inhibition efficiency is improved in general; the off-target effect is reduced; the complex design, screening, verification and optimization process of single RNAi molecule in the early stage is omitted in the application, easy to operate and low in cost, the siRNA set is a general, effective and fast RNAi tool.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a set of siRNA for inhibiting clusterin gene expression and application thereof. Background technique [0002] RNAi exists widely in species in nature. Since Andrew Fire and Craig Mello et al. first discovered RNAi phenomenon in nematode (Caenorhabditis elegans) in 1998, Tuschl and Phil Sharp et al. confirmed that RNAi also exists in mammals in 2001, about A series of progress has been made in the research on the mechanism, gene function and clinical application of RNAi; RNAi not only plays a key role in defense against virus infection, but also in various protective mechanisms of the body such as transposon jumping (Huntvagner et al, 2001; Tuschl, 2001; Waterhouse et al,2001; Zamore 2001), and their related products are also very promising drug candidates. [0003] Clusterin (Clusterin, CLU) is a heterodimer sulfate glycoprotein (sulfatealglycoprotein, SGP-2), which was first i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K48/00A61K31/713A61P35/00
CPCA61K31/713C12N15/113C12N2310/141C12N2310/321C12N2320/30C12N2310/322
Inventor 张必良杨秀群丹米其·萨玛斯基
Owner GUANGZHOU RIBOBIO
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