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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Preparation of siRNA

[0067] 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); is To ensure the specificity of siRNA, use BLAST (Basic ...

Embodiment 2

[0079] Example 2. siRNA cell level inhibition test

[0080] The siRNAs corresponding to the KRAS target genes shown in Table 2 prepared in Example 1 were separately transfected into HeLa cells (derived from ATCC), as follows:

[0081] 1. LF2K transfection

[0082] 100μL LF2K transfection system: 1μL LF2K (Invitrogen, 11668019), 5μL siRNA (final concentration of 100nM) and 94μL Opti-MEM cell culture medium (Thermo Fisher Scientific, 31985070).

[0083] The above-mentioned siRNAs are respectively the siRNAs corresponding to the target genes of TP53, BIRC5, CTNNB1, COPS5, STAT3, VEGFA, and KRAS prepared in Example 1.

[0084] Culture HeLa cells on a cell culture plate, and then add the above-mentioned 100 μL transfection system to each well and transfect for 48 hours to obtain cells transfected with siRNA corresponding to different target genes.

[0085] 2. RT-PCR detection inhibition rate

[0086] After 48 hours of transfection, the cells transfected with different target genes correspondin...

Embodiment 3

[0101] Example 3. Preparation of a 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 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;

[0105] 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.

[0106] The target gene is shown in Table 1, and the siRNA corresponding to the target gene is shown in Table 2.

[0107] The set of siRNAs for target genes can include 5, 6, 7 or 10 siRNAs. The grouping situation is shown in Table 3.

[0108] Table 5 The composition of the target gene set of siRNA

[0109]

[0110] Ea...

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Abstract

The invention discloses an oligonucleotide molecule used for inhibiting mRNA expression of a KRAS 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 oligonucleotide molecule used for inhibiting the expression of KRAS target gene mRNA and a complete composition thereof. Background technique [0003] Since AndrewFire 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 application of RNAi A series of progress has been made in research. RNAi not only plays a key role in preventing viral infections, preventing transposon jumping and other body protection mechanisms (Hu...

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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