Small interfering rna for knocking down zbtb21 gene in vivo of animals and application thereof

Abstract

This invention provides a small interfering RNA (siRNA) for knocking down the Zbtb21 gene in vivo in animals and its application. The siRNA molecule comprises a sense strand and an antisense strand that complement each other to form a double-stranded region. The sense strand is 21 nt in length, and the antisense strand is 23 nt in length. Each nucleotide of both the sense and antisense strands is modified. The nucleotide sequence of the sense strand of the siRNA molecule is shown in SEQ ID NO: 1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 2. This siRNA molecule has a high knockdown efficiency for the Zbtb21 gene, and this efficiency can be maintained for more than one week in continuously dividing cells.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a small interfering RNA for knocking down the Zbtb21 gene in living animals and its applications. Background Technology

[0002] Mice are key model organisms for target validation in preclinical studies. Currently, methods for knocking down the zinc finger and BTB domain protein 21 (Zbtb21) gene in mice are limited to constructing Zbtb21 knockout mice, which is not optimal for preclinical disease studies. Small interfering RNA (siRNA) has been used to treat human liver diseases; therefore, using siRNA to knock down the Zbtb21 gene in vivo is feasible. However, the efficiency and persistence of RNA interference are affected by many factors; ordinary siRNA cannot maintain its effect in vivo and is prone to degradation. Summary of the Invention

[0003] To address the aforementioned issues, this invention proposes a small interfering RNA for knocking down the Zbtb21 gene in living animals and its application. This RNA exhibits high knockdown efficiency for the Zbtb21 gene, and this efficiency can be maintained for more than a week in continuously dividing cells.

[0004] To achieve the above objectives, the present invention provides, in a first aspect, a siRNA molecule for knocking down the Zbtb21 gene in living animals, the siRNA molecule comprising a sense strand and an antisense strand that complement each other to form a double-stranded region, the sense strand being 21 nt in length and the antisense strand being 23 nt in length, each nucleotide of the sense strand and the antisense strand being modified.

[0005] The siRNA molecule has the sense strand nucleotide sequence as shown in SEQ ID NO: 1 and the antisense strand nucleotide sequence as shown in SEQ ID NO: 2.

[0006] According to the present invention, a siRNA molecule for knocking down the Zbtb21 gene in live animals has a high knockdown efficiency for the Zbtb21 gene, with a knockdown efficiency of over 90% after 48 hours of transfection; after modification, the efficiency is still over 90% after 72 hours of transfection, and 75% knockdown efficiency is still maintained on the 7th day after transfection. This efficiency can be maintained for more than a week in continuously dividing cells, which is an effect that cannot be achieved by various current gene knockdown tools.

[0007] Optionally, the modification is selected from any one or more of the following:

[0008] 2'-Fluoro-modification (2'-fluoro, 2'-F), 2'-O-methyl-modification (2'-O-methyl, 2'-OMe), glycol nucleic acid (GNA), phosphorothioate (PS) linkage, vinyl phosphate group modification (5'-(E)-vinylphosphonate, 5'-(E)-VP), and 2'-16 alkyl oxygen modification (2'-O-C16).

[0009] Furthermore, the sense strand structure of the siRNA molecule is shown in (A1); the antisense strand structure of the siRNA molecule is shown in (A2);

[0010] (A1)ma·mg·mumgmc(C16a)fUmafAfCfCmamgmamamcmamamc·ma·ma(5'-3');

[0011] (A2) (5'-3');

[0012] Where ma, mc, mu, and mg represent nucleotides a, c, u, and g that have been modified with 2'-O-methyl, respectively; fA, fU, and fC represent nucleotides A, U, and C that have been modified with 2'-fluorine, respectively; · indicates that the two nucleotides are linked by a phosphate thiophosphate backbone; U represents ethylene glycol-modified nucleic acid nucleotides; (C16a) represents nucleotide a modified with 2'-16 alkyl oxygen; VP represents modification with a vinyl phosphate group at the 5' end.

[0013] Furthermore, the chemical formula of the vinyl phosphate group is Formula I;

[0014]

[0015] Furthermore, the chemical formula of the 2'-16 alkyl oxygen is Formula II;

[0016]

[0017] In a second aspect, the present invention provides a mixture of RNAi molecules for knocking down the Zbtb21 gene in living animals, comprising the aforementioned siRNA molecules.

[0018] In a third aspect, the present invention proposes the application of the above-mentioned siRNA molecule or the above-mentioned RNAi molecule mixture in the preparation of biomaterials for knocking down the Zbtb21 gene in living animals.

[0019] Optionally, the animal is a mouse.

[0020] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0021] Figure 1 This is a diagram showing the effect of the siRNA sequence of Example 1 of the present invention on the knockdown of the Zbtb21 gene;

[0022] Figure 2 This is a diagram showing the effect of the siRNA sequence in Example 2 of the present invention on the knockdown of the Zbtb21 gene. Detailed Implementation

[0023] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0024] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0025] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0026] Example 1: In vitro screening of siRNA sequences targeting the Zbtb21 gene (cellular level)

[0027] In this embodiment, HEK293T cells overexpressing mouse Zbtb21 were selected for in vitro screening of target siRNA sequences.

[0028] The siRNA in this example:

[0029] siNC is a negative control sequence without specific targeting (provided by Guangzhou Ruibo Biotechnology Co., Ltd., product number siN0000001-1-5);

[0030] siG is a positive control sequence targeting the housekeeping gene Gapdh (provided by Guangzhou Ruibo Biotechnology Co., Ltd., product number siP0000001-1-5);

[0031] siZ1 is an unmodified Zbtb21 siRNA sequence (customized by the applicant from Guangzhou Ruibo Biotechnology Co., Ltd., product number siB2401150108560971, target sequence is CCAGAAATTCCGAGCTCAT);

[0032] siZ2 is an unmodified Zbtb21 siRNA sequence (customized by the applicant from Guangzhou Ruibo Biotechnology Co., Ltd., product number siB2401150108562063, target sequence is GTGCATAACCAGAACAACA);

[0033] siZ3 is an unmodified Zbtb21 siRNA (custom-made by the applicant from Guangzhou Ruibo Biotechnology Co., Ltd., product number siB2401150108563155, target sequence is AGTCAGATACCCTCTTCTA).

[0034] HEK293T cells overexpressing mouse Zbtb21 were transfected with siRNA via JinBaoLuo-siRNAPlus (Xiamen JinBaoLuo Technology Co., Ltd., catalog number A003) for 48 hours, and then the cells were lysed to obtain protein lysis buffer. Specifically, protein lysis buffer was obtained by transfecting each 5 × 10⁻⁶ cell line with siRNA. 5 HEK293T cells were transfected with a mixture containing 3 μL JinBaoLuo-siRNAPlus transfection reagent and 4 μL 20 μM siRNA. After transfection at room temperature, the cells were incubated at 37°C for 12 h, then replaced with fresh DMEM medium. 48 h after transfection, the medium was removed, and the cells in one well of a 12-well plate were directly mixed with 80 μL 2×SDS sample buffer. The cells were collected to obtain protein lysis buffer, which was then collected in a 1.5 mL tube, boiled in a metal bath at 100°C for 10 min, and then mixed and centrifuged. This sample is ready for subsequent Western blot analysis.

[0035] Western blotting was used to detect protein expression levels: ACTIN was used as the internal control protein, with the primary antibody from Affinity (catalog number T0022), and the band size was approximately 43 kDa. The secondary antibody was goat anti-mouse IgG (from Thermo Fisher Scientific, catalog number 31430). HA-mZBTB21 was approximately 130 kDa, with the primary antibody from Affinity (catalog number T0050) and the secondary antibody being goat anti-rabbit IgG (from Thermo Fisher Scientific, catalog number 31460). Due to the significant difference in molecular weight between HA-mZBTB21 and ACTIN, and the use of different secondary antibodies, the applicant horizontally cut the same PVDF membrane: the upper half used rabbit anti-HA as the primary antibody and goat anti-rabbit as the secondary antibody; the lower half used mouse anti-ACTIN as the primary antibody and goat anti-mouse as the secondary antibody. Both membranes were then exposed together.

[0036] The results are as follows Figure 1 As shown, siZ1, siZ2, and siZ3 all effectively knocked down Zbtb21 expression 48 hours after transfection, with siZ2 showing the most significant effect, achieving a knockdown efficiency of over 90%. Therefore, siZ2 was selected for further optimization experiments.

[0037] Example 2

[0038] The siZ2 of Example 1 is modified so that the siZ2 sequence consists of a sense chain and an antisense chain;

[0039] The justice chain of siZ2: 5'-agugcaUaACCagaacaacaa-3' (SEQ ID NO: 1);

[0040] The antisense chain of siZ2: (SEQ ID NO: 2);

[0041] The modification strategies are as follows: (1) The entire chain needs to be modified by ESC: uppercase letters are fluorinated (2'-F), lowercase letters are modified with methoxy (2'-OMe), and the bold underlined letter U on the antisense chain is ethylene glycol nucleic acid (GNA) modification; (2) A vinyl phosphate group 5'-(E)-VP needs to be added to the 5' end of the antisense chain, with the chemical formula shown in Formula I; (3) A 16-alkyl oxygen (2'-O-C16) needs to be added to N6 (position 6 from the 5' end) of the sense chain, with the chemical formula shown in Formula II; (4) PS linkage (phosphorothioate linkage) needs to be added between the three nucleotides at the 5' end of the sense and antisense chains and between the three nucleotides at the 3' end.

[0042]

[0043] The applicant modified siZ2 in different ways. The modified siZ2 were named siZ2*(#2-simZbtb21), *V1, *V2, *V3, *V4, and *V5, respectively, and their nucleotide compositions are as follows:

[0044] The justice chain of siZ2*: ma·mg·mumgmc(C16a)fUmafAfCfCmamgmamamcmamamc·ma·ma(sense 5'-3');

[0045] The antisense chain of siZ2*: u·mc(antisense 5'-3');

[0046] *V1's chain of justice: ma·mg·mumgmcmafUmafAfCfCmamgmamamcmamamc·ma·ma(sense5'-3');

[0047] *V1's antisense chain: VP-mu·fU·mgmumumg U mumcmumgmgmufUmafUmgmcmamcmu·mu·mc(antisense 5'-3');

[0048] *V2's Chain of Justice: (C16a)·mg·mumgmcmafUmafAfCfCmamgmamamcmamamc·ma·ma

[0049] (sense 5'-3');

[0050] *V2 antisense chain: VP-mu·fU·mgmumumg U mumcmumgmgmufUmafUmgmcmamcmu·mu·mc(antisense 5'-3');

[0051] *V3's Chain of Justice: ma·(C16g)·mumgmcmafUmafAfCfCmamgmamamcmamamc·ma·ma

[0052] (sense 5'-3');

[0053] *V3 antisense chain: VP-mu·fU·mgmumumg U mumcmumgmgmufUmafUmgmcmamcmu·mu·mc(antisense 5'-3');

[0054] *V4's chain of justice: ma·mg·mumgmcma(C16U)mafAfCfCmamgmamamcmamamc·ma·ma(sense 5'-3');

[0055] *V4 antisense chain: VP-mu·fU·mgmumumg U mumcmumgmgmufUmafUmgmcmamcmu·mu·mc(antisense 5'-3');

[0056] *V5's Chain of Justice: ma·mg·mumgmcmafUmafAfCfCmamgmama(C16c)mamamc·ma·ma

[0057] (sense 5'-3');

[0058] *V5 ansect: VP-mu·fU·mgmumumg U mumcmumgmgmufUmafUmgmcmamcmu·mu·mc(antisense 5'-3');

[0059] In the above, ma, mc, mu, and mg represent nucleotides a, c, u, and g modified by 2'-OMe, respectively; fA, fU, and fC represent nucleotides A, U, and C modified by 2'-F, respectively; · indicates that the two nucleotides are linked by PS linkage; (C16a), (C16g), (C16U), and (C16c) represent nucleotides a, g, u, and c modified by 2'-O-C16, respectively; VP indicates 5'-(E)-VP modification; and bold underline indicates GNA modification. The designed siZ2*, *V1, *V2, *V3, *V4, and *V5 were synthesized by Jima Gene Company.

[0060] Similar to the cell transfection method in Example 1, HEK293T cells overexpressing HA-mZbtb21 were transfected with siZ2*, *V1, *V2, *V3, *V4 and *V5 for 72 h or 7 days. The cells were then lysed to obtain protein lysates, and the protein expression levels were detected by Western blot.

[0061] The results are as follows Figure 2As shown, 72 hours after transfection, with cell division and passage, all siRNAs, regardless of modification, could knock down Zbtb21 with an efficiency of over 90%. However, with continued passage, on day 7 after transfection, siZ2* still had a 75% knockdown efficiency, *V1 retained about 40% knockdown efficiency, while most other siRNAs were ineffective. Therefore, optimization of siZ2* has greatly and effectively achieved persistent knockdown of Zbtb21 expression.

[0062] In summary, according to embodiments of the present invention, siRNA modification achieves a high knockdown efficiency for the Zbtb21 gene, and this efficiency can be maintained for more than a week in continuously dividing cells, which is an effect that cannot be achieved by current gene knockdown tools.

[0063] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0064] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for animal in vivo Zbtb21 siRNA molecules for gene knockdown, characterized in that, The siRNA molecule comprises a sense strand and an antisense strand that complement each other to form a double-stranded region. The sense strand is 21 nt long and the antisense strand is 23 nt long. Each nucleotide of the sense strand and the antisense strand is modified. The siRNA molecule has the sense strand nucleotide sequence as shown in SEQ ID NO: 1 and the antisense strand nucleotide sequence as shown in SEQ ID NO:

2.

2. The method of claim 1, wherein the siRNA molecule is selected from the group consisting of SEQ ID NOs: 1- 12. Zbtb21 The siRNA molecule for gene knockdown, characterized in that, The structure of the sense strand of the siRNA molecule is shown in (A1); the structure of the antisense strand of the siRNA molecule is shown in (A2). (A1)ma·mg·mumgmc(C16a)fUmafAfCfCmamgmamamcmamamc·ma·ma (5'-3'); (A2)VP-mu·fU·mgmumumg U mumcmumgmgmufUmafUmgmcmamcmu·mu·mc (5'-3'); Where ma, mc, mu, and mg represent nucleotides a, c, u, and g that have been modified with 2'-O-methyl, respectively; fA, fU, and fC represent nucleotides A, U, and C that have been modified with 2'-fluorine, respectively; · indicates that the two nucleotides are linked by a phosphate thiophosphate backbone; U U represents ethylene glycol-modified nucleic acid nucleotides; (C16a) represents nucleotide a modified with 2'-16 alkyl oxygen; VP represents modification with a vinyl phosphate group at the 5' end.

3. The method for use on live animals as described in claim 2 Zbtb21 Gene knockdown siRNA molecules are characterized by, The chemical formula of the vinyl phosphate group is Formula I; Formula I.

4. The method for use on live animals as described in claim 2 Zbtb21 Gene knockdown siRNA molecules are characterized by, The chemical formula of the 2'-16 alkyl oxygen is Formula II; Formula II.

5. A device for use on live animals Zbtb21 A mixture of gene knockdown RNAi molecules, characterized in that, It contains the siRNA molecule according to any one of claims 1-4.

6. The siRNA molecule as described in any one of claims 1-4 or the RNAi molecule mixture as described in claim 5 in the preparation of knockdown animals in vivo. Zbtb21 Application of genes in biological materials, wherein the animal is a mouse.

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