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
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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
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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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