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

A target gene and a complete set of technologies, applied in the field of molecular biology, can solve problems such as time-consuming and labor-intensive

Inactive Publication Date: 2016-10-26
GUANGZHOU RIBOBIO
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] 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 expression of mRNA of target gene and composition set thereof
  • Oligonucleotide molecule used for inhibiting expression of mRNA of target gene and composition set thereof
  • Oligonucleotide molecule used for inhibiting expression of mRNA of target gene and composition set thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1, the preparation of siRNA

[0079] All single siRNAs designed can target all transcripts of the target gene, and the design method refers to the method of Elbashir et al.2002; Paddison et al.2002; Reynolds et al.2004; Ui-Tei et al. ,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 mammalian and chick RNAinterference. Nucleic Acids Res.32:936–948); T...

Embodiment 2

[0093] Embodiment 2, siRNA cell level inhibition test

[0094] The siRNAs corresponding to the TP53, BIRC5, CTNNB1, COPS5, STAT3, VEGFA, KRAS target genes shown in Table 2 prepared in Example 1 were separately transfected into HeLa cells (derived from ATCC), as follows:

[0095] 1. LF2K transfection

[0096] 100 μL LF2K transfection system: 1 μL LF2K (Invitrogen, 11668019), 5 μL siRNA (final concentration 100 nM) and 94 μL Opti-MEM cell culture medium (Thermo Fisher Scientific, 31985070).

[0097] The above siRNAs are siRNAs corresponding to the target genes of TP53, BIRC5, CTNNB1, COPS5, STAT3, VEGFA and KRAS prepared in Example 1, respectively.

[0098] HeLa cells were cultured on a cell culture plate, and then 100 μL of the above-mentioned transfection system was added to each well, and transfected for 48 hours to obtain cells transfected with siRNAs corresponding to different target genes.

[0099] 2. RT-PCR detection inhibition rate

[0100] After 48 hours of transfect...

Embodiment 3

[0114] Embodiment 3, the preparation of complete set of siRNA

[0115] 1. Design principles

[0116] A set of siRNA consists of 5-10 siRNA molecules;

[0117] Each siRNA is composed of 25 nucleotides 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;

[0118] The AS strand and the SS strand are completely reverse complementary, the AS strand is completely reverse complementary to the target sequence on the target gene, the 7 consecutive nucleotides from the 5' end and the 7 consecutive nucleotides from the 3' end of the SS are all After 2'-O-Me modification.

[0119] The target genes are shown in Table 1, and the siRNAs corresponding to the target genes are shown in Table 2.

[0120] The set of siRNAs for target genes may include 5, 6, 7 or 10 siRNAs, and the grouping conditions are shown in Table 5.

[0121] Table 5 Composition of a compl...

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Abstract

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

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an oligonucleotide molecule and a complete set of composition for inhibiting the expression of target gene mRNA. Background technique [0002] Since Andrew Fire and Craig Mello et al first discovered the phenomenon of RNAi in the nematode (Caenorhabditis elegans) in 1998, and Tuschl and Phil Sharp et al. confirmed the existence of RNAi in mammals in 2001, the mechanism of RNAi, gene function and clinical Applications and other research have made a series of progress. RNAi not only plays a key role in various body protection mechanisms such as defense against virus infection and anti-transposon jumping (Huntvagner et al, 2001; Tuschl, 2001; Waterhouse et al, 2001; Zamore 2001), and its related products are also very promising drug candidates. [0003] In 2001, Elbashir et al. found that siRNA inhibited the silencing of specific genes in mammalian cells. Studies have shown that s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/713A61P35/00A61P9/00A61P29/00A61P31/00
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 GUANGZHOU RIBOBIO
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