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Oligonucleotide molecule used for inhibiting mRNA expression of EIF4E target gene and set composition thereof

A target gene and a complete set of technologies, applied in the field of molecular biology, can solve time-consuming and labor-intensive problems, achieve the effects of reducing off-target effects, facilitating storage and transportation, and enhancing the effect of gene silencing

Active Publication Date: 2017-09-15
ARGORNA PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, since the effectiveness of siRNA is affected by various factors such as sequence specificity, target cell specificity, and target point, not all siRNAs obtained based on existing design principles can achieve effective silencing effects; generally designed siRNAs are about More than 50% of siRNAs have the effect of silencing target mRNA, and only 25% of siRNAs have a silencing effect of more than 75%. Therefore, subsequent experimental verification, screening or optimization of designed and synthesized siRNAs is required, which is time-consuming and labor-intensive; based on this, a general-purpose, Efficient and rapid RNAi technology and products need to be developed urgently

Method used

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  • Oligonucleotide molecule used for inhibiting mRNA expression of EIF4E target gene and set composition thereof
  • Oligonucleotide molecule used for inhibiting mRNA expression of EIF4E target gene and set composition thereof
  • Oligonucleotide molecule used for inhibiting mRNA expression of EIF4E target gene and set composition thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1. Preparation of siRNA

[0066] All single siRNA designed can target all transcripts of the target gene. The design method refers to Elbashir et al. 2002; Paddison et al. 2002; Reynoldset al. 2004; Ui-Tei et al. 2004 et al. (Elbashir, SM ,Harborth,J.,Weber,K.,and Tuschl,T.2002.Analysis of genefunction in somatic mammalian cells using small interfering RNAs.Methods 26:199–213; Paddison,PJ,Caudy,AA,Bernstein,E., Hannon,GJ,and Conklin,DS2002.Short hairpin RNAs(shRNAs)induce sequence-specific silencing inmammalian cells.Genes&Dev.16:948–958; Reynolds,A.,Leake,D.,Boese,Q.,Scaringe,S .,Marshall,WS,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 mammalian and chick RNAinterference.Nucleic Acids Res.32:936–948); To ensure the specificity of siRNA, BLAST (Basic Local A...

Embodiment 2

[0078] Example 2. Preparation of a complete set of siRNA

[0079] 1. Design principles

[0080] A set of siRNA consists of 5-10 siRNA molecules;

[0081] Each siRNA is composed of a 25-nucleotide SS sense strand and its reverse complementary antisense strand AS, and is blunt-ended; each siRNA is complementary to the target sequence on the target gene through its antisense strand;

[0082] The AS chain and the SS chain are completely reverse complementary, and the AS chain is completely reverse complementary to the target sequence on the target gene. The SS has 7 consecutive nucleotides from the 5'end and 7 consecutive nucleotides from the 3'end. After 2'-O-Me modification.

[0083] The target gene is shown in Table 1, and the siRNA corresponding to the target gene is shown in Table 2.

[0084] The set of siRNAs for target genes can include 5, 6, 7 or 10 siRNAs. The grouping situation is shown in Table 3.

[0085] Table 3 The composition of a complete set of siRNA for EIF4E target genes

...

Embodiment 5

[0128] Example 5. In vitro stability determination

[0129] Dilute each siRNA RB-KRA-D1, RB-TP5-D6, and RB-EIF-D3 to 5μM in RNase-free water and add an equal volume of fresh rat serum (product of Shanghai Yuanmu Biotechnology Co., Ltd.), then After incubating at 37°C for 6 hours, samples were taken for electrophoresis to observe the integrity of different siRNAs.

[0130] The result is figure 1 As shown, it can be seen that siRNA is stable in serum, and it is expected to have better efficacy in vivo.

[0131] The stability test results of other siRNAs are the same, and the specific figures are omitted.

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Abstract

The invention discloses an oligonucleotide molecule used for inhibiting mRNA expression of an EIF4E target gene and a set composition thereof. The invention provides a siRNA, which is composed of a positive sense strand including 19-27 nucleotides, and an antisense strand which is reverse-complementary therewith. In the positive sense strand, 5-9 continuous nucleotides beginning from a 5'-terminal and 5-9 continuous nucleotides beginning from a 3'-terminal are all 2'-ribose modified nucleotides. The siRNA molecule mixture can influence expression of the target gene in at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and 90% cells, inhibition ratio being at least 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and 95%.

Description

[0001] This application is a divisional application of an invention patent application whose application date is 2016-08-18, application number is 201610687850.2, and the title of the invention is "an oligonucleic acid molecule for inhibiting the expression of target gene mRNA and its complete composition". Technical field [0002] The present invention relates to the field of molecular biology, in particular to an oligonucleic acid molecule used for inhibiting the expression of EIF4E target gene mRNA and a complete set of composition thereof. Background technique [0003] Since Andrew Fire and Craig Mello et al. first discovered RNAi in Caenorhabditis elegans in 1998, and Tuschl and Phil Sharp et al. confirmed that RNAi also exists in mammals in 2001, the mechanism, gene function, and clinical aspects of RNAi A series of progress has been made in application and other research. RNAi not only plays a key role in preventing viral infections, preventing transposon jumping and other ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61K31/713A61P35/00A61P9/00A61P29/00A61P31/00
CPCA61K31/713C12N15/113C12N15/1135C12N15/1136C12N2310/14C12N2310/321C12N2310/3533C12N2310/3525C12N2310/3521
Inventor 张必良杨秀群丹米其·萨玛斯基克雷格·梅洛
Owner ARGORNA PHARM CO LTD
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