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siRNA with silencing activity on PLK1 gene and high serum stability and application thereof

A stable and silent technology, applied in the field of genetic engineering, can solve the problems of reducing the gene silencing activity of siRNA, poor serum stability of siRNA, and low knockout activity, so as to prolong the half-life of biological activity, improve serum stability, and last for a long time. Effect

Inactive Publication Date: 2019-12-13
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although excessive or inappropriate chemical modifications can significantly enhance the nuclease resistance of siRNA, but also significantly reduce the gene silencing activity of siRNA, such as 2'-methoxy modification and 5-bromouracil modification, etc.
[0003] In addition, since RNA interference technology has been widely used, related studies have found that the gene knockout activity of chemically synthesized phosphorothioated siRNA is similar or lower than that of natural siRNA, and has obvious cytotoxicity; meanwhile, the serum stability of synthetic siRNA poor

Method used

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  • siRNA with silencing activity on PLK1 gene and high serum stability and application thereof
  • siRNA with silencing activity on PLK1 gene and high serum stability and application thereof
  • siRNA with silencing activity on PLK1 gene and high serum stability and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 In vitro transcription synthesis and purification of PLK1 siRNAs and PLK1 S-siRNAs

[0057] 1. Using the mRNA sequence of PLK1 as a template (GenBank serial number is NM_005030) (see Table 1), design an siRNA sequence for targeted knockout of PLK1 (see Table 2, where * represents the oxygen atom at the phosphate site of the nucleotide replaced by sulfur atoms).

[0058] like figure 1 As shown in A, the oxygen atoms at the phosphate sites of NTPs (adenosine triphosphate, cytidine triphosphate, and uridine triphosphate) were replaced with sulfur atoms (i.e., thio-NTPs (ATPαS, CTPαS, GTPαS, and UTPαS)) and polymerized using T7 Enzyme in vitro transcription synthesized nat-siRNAs and S-siRNAs, the specific sequences are shown in Table 2:

[0059] Table 2 siRNA sequence

[0060]

[0061]

[0062] * Indicates that the oxygen atom at the α-phosphate site of the nucleotide is replaced by a sulfur atom.

[0063] The specific synthesis process is as follows: ...

Embodiment 2

[0066] Example 2 Serum Stability Test of PLK1 siRNAs and PLK1 S-siRNAs

[0067] Take the 5 μM nat-siRNA or S-siRNA obtained in Example 1 and mix them with equal volumes of 50% fetal bovine serum (FBS), and place them in a water pot at 37°C to incubate for 0h, 1h, 2h, 4h, 6h, 8h, 12h After 24 hours and 24 hours, 2 μL of sample was taken and loaded, and the remaining amount of siRNA or S-siRNA was detected by 12.5% ​​non-denaturing polyacrylamide gel electrophoresis. The gray value of three independent experiments was analyzed by Image J software, and the graph was drawn by origin software, the results are shown in figure 2 .

[0068] in, figure 2 A is the serum stability test results of nat-siRNA1 and S-siRNA1, the band M in the figure represents marker, the left side of band M is nat-siRNA1, the right side is S-siRNA1, and the bands 1-7 on both sides Both represent 0h, 1h, 2h, 3h, 4h, 6h, 12h and 24h in turn;

[0069] figure 2 B is the serum stability test results of n...

Embodiment 3

[0072] Example 3 Cell Culture and Transfection

[0073] HepG2 cells (ATCC cell bank) were divided into 5×10 3 The density of cells / well was seeded in 96-well tissue culture plates, and the high glucose medium (DMEM, Hyclone) containing 10% fetal bovine serum (FBS, Gibco) was incubated at 37°C and 5% CO 2 Conditioned for 12h. According to the manufacturer's instructions of Lipofectamine 2000 (Lipo2000), Liposome 3000 (Lipo3000), Ribofect transfection reagent and Rfect small nucleic acid transfection reagent, siRNA with fluorescent FAM group (transfection final concentration is 10nmol / L) After mixing evenly with four different transfection reagents, incubate HepG2 cells for 4 hours, replace the medium, and observe the difference in transfection efficiency with a fluorescence microscope. The results are shown in image 3 .

[0074] image 3 Among them, A is the transfection result of Lipofectamine 2000 (Lipo2000); B is the transfection result of Liposome 3000 (Lipo3000); C is...

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Abstract

The invention discloses siRNA with silencing activity on a PLK1 gene and high serum stability and an application thereof. The lengths of a sense strand and an antisense strand of the siRNA are 21 nucleotides; the 3'ends of the sense strand and the antisense strand are two continuous uridines, 19 basic groups, except for the UU at the 3'end, of the two chains are complementary to form double chains, and the sense strand and an antisense strand are shown as any pair of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 or SEQ ID NO.5 and SEQ ID NO.6 respectively; and the sense strand and / or the antisense strand can be chemically modified, so that oxygen atoms of alpha phosphoric acid sites on all A, C, G and / or U bases in the sense strand and / or the antisense strand are substituted bya modifying group. The sulfur atoms are used for replacing oxygen atoms of the same main group of siRNAs phosphoric acid sites, so that the serum stability of siRNA can be remarkably improved under the condition that an RNA interference action mechanism is hardly influenced.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an siRNA with silencing activity on PLK1 gene and high serum stability and application thereof. Background technique [0002] On the 20th anniversary of the discovery of RNA interference, the first siRNA drug, Patisiran, was approved by the US FDA and officially launched. Functional nucleic acids have set off a wave of medical revolution in the fields of disease treatment, prevention and diagnosis. Although unmodified functional nucleic acid (such as siRNA)-mediated gene silencing has unlimited potential, chemical modifications are often used to improve the metabolic stability, in vivo delivery, and pharmacokinetic properties of siRNA. The main chemical modifications include 2'-methoxy modification (2'-O-Me), 2'-fluoro modification (2'-F), 2'-oxyallyl modification (2'-O-allyl ), 2,4-dinitrophenyl ether modification (2'-O-DNP), 4'-thio modification (4'-S),...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61K31/713A61P35/00
CPCA61K31/713A61P35/00C12N15/113C12N2310/14C12N2320/30C12N2320/32C12N2310/333C12N2310/334C12N2310/336C12N2310/335
Inventor 黄震
Owner SICHUAN UNIV
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