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

A target gene, a complete set of technologies, applied in the field of molecular biology, can solve problems such as time-consuming and labor-intensive, and achieve the effects of reducing off-target effects, facilitating storage and transportation, and improving the probability of silencing and the efficiency of 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 VEGFA target gene and set composition thereof
  • Oligonucleotide molecule used for inhibiting mRNA expression of VEGFA target gene and set composition thereof
  • Oligonucleotide molecule used for inhibiting mRNA expression of VEGFA target gene and set composition thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1, the preparation of siRNA

[0067] 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); for...

Embodiment 2

[0080] Embodiment 2, siRNA cell level inhibition test

[0081] The siRNA corresponding to the VEGFA target gene shown in Table 2 prepared in Example 1 was separately transfected into HeLa cells (derived from ATCC), as follows:

[0082] 1. LF2K transfection

[0083] 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).

[0084] The above siRNAs are siRNAs corresponding to the target genes of TP53, BIRC5, CTNNB1, COPS5, STAT3, VEGFA and KRAS prepared in Example 1, respectively.

[0085] 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.

[0086] 2. RT-PCR detection inhibition rate

[0087] After 48 hours of transfection, the cells transfected with siRNA corre...

Embodiment 3

[0101] Embodiment 3, the preparation of complete set of siRNA

[0102] 1. Design principles

[0103] A set of siRNA consists of 5-10 siRNA molecules;

[0104] 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;

[0105] 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.

[0106] The target genes are shown in Table 1, and the siRNAs corresponding to the target genes are shown in Table 2.

[0107] The set of siRNAs for target genes may include 5, 6, 7 or 10 siRNAs, and the grouping conditions are shown in Table 3.

[0108] Table 5 Composition of a compl...

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Abstract

The invention discloses an oligonucleotide molecule used for inhibiting mRNA expression of a VEGFA 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 with an application date of 2016-08-18, an application number of 201610687850.2, and an invention title of "an oligonucleotide molecule for inhibiting the expression of target gene mRNA and its complete composition". technical field [0002] 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 [0003] 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, gene function and clinical application of RNAi A series of progress has been made in the research. RNAi not only plays a key role in various body protection mechanisms such as defense against virus infection and a...

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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