Small interfering RNA (Ribonucleic Acid) for knocking down Zbtb21 gene of animal living body and application of small interfering RNA

Abstract

The invention provides a small interfering RNA for animal living body Zbtb21 gene knockdown and application thereof, the siRNA molecule comprises a positive-sense strand and an antisense strand which are complementary to form a double-strand region, the length of the positive-sense strand is 21nt, the length of the antisense strand is 23nt, and each nucleotide of the positive-sense strand and the antisense strand is modified; the positive-sense strand nucleotide sequence of the siRNA molecule is as shown in SEQ ID NO: 1, and the antisense strand nucleotide sequence of the siRNA molecule is as shown in SEQ ID NO: 2. The siRNA molecule has very high knock-down efficiency on the Zbtb21 gene, and the efficiency can be maintained for more than one week in continuously split cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a small interfering RNA for knocking down the Zbtb21 gene in living animals and an application thereof. Background Art

[0002] Mice are key model organisms for target validation in preclinical studies. The current 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 studies of diseases. Small interfering RNA (siRNA) has been used to treat human liver diseases. Therefore, it is feasible to use small interfering RNA to knock down the Zbtb21 gene in vivo. However, the efficiency and durability of RNA interference are affected by many factors. Ordinary small interfering RNA cannot continue to work in vivo and is prone to degradation. Summary of the invention

[0003] In order to solve the above problems, the present invention proposes a small interfering RNA for knocking down the Zbtb21 gene in living animals and its application, which has a high knockdown efficiency for the Zbtb21 gene, and this efficiency can be maintained for more than one week in continuously dividing cells.

[0004] In order to achieve the above-mentioned object, the present invention provides, in the first aspect, a siRNA molecule for knocking down the Zbtb21 gene in a living animal, wherein 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, the antisense strand is 23 nt long, and each nucleotide of the sense strand and the antisense strand is modified;

[0005] 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.

[0006] According to the present invention, a siRNA molecule for knocking down the Zbtb21 gene in living animals has a high knockdown efficiency for the Zbtb21 gene, with a knockdown efficiency of more than 90% 48 hours after transfection; after modification, the efficiency is still more than 90% 72 hours after transfection, and the knockdown efficiency is still 75% on the 7th day after transfection. This efficiency can be maintained for more than one week in cells that continue to divide, 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 modification (glycol nucleic acid, GNA), phosphorothioate backbone modification (phosphorothioate (PS) linkage), vinyl phosphate group modification (5'-(E)-vinylphosphonate, 5'-(E)-VP), 2'-16 alkyl oxygen modification (2'-O-C16).

[0009] Furthermore, 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);

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

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

[0012] Wherein, ma, mc, mu and mg represent 2'-O-methyl modified nucleotides a, c, u and g, respectively; fA, fU and fC represent 2'-fluoro modified nucleotides A, U and C, respectively; · represents that the two nucleotides before and after are connected by a thiophosphate backbone; represents nucleotide U modified with ethylene glycol; (C16a) represents nucleotide a modified with 2'-16 alkyl oxygen; VP represents modification of the 5' end with a vinyl phosphate group.

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

[0014]

[0015] Further, the chemical formula of the 2'-16 alkyloxy is Formula II;

[0016]

[0017] In a second aspect, the present invention provides an RNAi molecule mixture for knocking down the Zbtb21 gene in living animals, which comprises the above-mentioned siRNA molecules.

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

[0019] Optionally, the animal is a mouse.

[0020] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 2 This is a diagram showing the effect of the siRNA sequence of Example 2 of the present invention on knocking down the Zbtb21 gene. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0024] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although 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 by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

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

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

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

[0028] siRNA of this example:

[0029] siNC is a negative control sequence without specific targeting (provided by Guangzhou Ruibo Biotechnology Co., Ltd., product number si N0000001-1-5);

[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 CCAGAAATTCCGAGCTCAT);

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

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

[0034] HEK293T cells overexpressing mouse Zbtb21 were transfected with siRNA by JinBaoLuo-siRNAPlus (Xiamen JinBaoLuo Technology Co., Ltd., catalog number A003) for 48 hours, and then the cells were lysed to obtain protein lysate. 5 A mixture containing 3 μL JinBaoLuo-siRNAPlus transfection reagent and 4 μL 20 μM siRNA was added to each HEK293T cell. After transfection at room temperature, the cells were cultured in a constant temperature incubator at 37 degrees Celsius for 12 hours and then replaced with fresh DMEM culture medium. 48 hours after transfection, the culture medium was removed and 80 μL 2×SDS sample buffer was used to directly pipette and mix the cells in one well of the 12-well plate. The cells were collected to obtain protein lysate, which was collected into a 1.5 mL tube, boiled in a metal bath at 100 degrees Celsius for 10 minutes, then mixed and centrifuged. This sample can be used for subsequent western blot detection.

[0035] Western blot was used to detect protein expression levels: ACTIN was used as the internal reference protein, the primary antibody was from Affinity (Cat. No. T0022), the WB band size was about 43 kDa, and the secondary antibody was goat anti-mouse IgG (from Thermo Company, Cat. No. 31430); the size of HA-mZBTB21 was about 130 kDa, the primary antibody for HA was from Affinity (Cat. No. T0050), and the secondary antibody was goat anti-rabbit IgG (from Thermo Company, Cat. No. 31460); due to the large difference in molecular weight between HA-mZBTB21 and ACTIN, and different secondary antibodies were used, the applicant cut the same PVDF membrane horizontally: the upper half was the primary antibody rabbit anti-HA, the secondary antibody goat anti-rabbit; the lower half was the primary antibody mouse anti-ACTIN, the secondary antibody goat anti-mouse. The two membranes were then exposed together.

[0036] The results are as follows Figure 1 As shown, 48h after transfection, siZ1, siZ2 and siZ3 can effectively knock down the expression of Zbtb21, among which siZ2 has the most significant effect, with a knockdown efficiency of more than 90%. Therefore, siZ2 was selected to continue the subsequent optimization experiments.

[0037] Example 2

[0038] The siZ2 of Example 1 was modified, and the siZ2 sequence consisted of a sense strand and an antisense strand;

[0039] Sense strand of siZ2: 5′-agugcaUaACCagaacaacaa-3′ (SEQ ID NO: 1);

[0040] Antisense strand of siZ2: (SEQ ID NO: 2);

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

[0042]

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

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

[0045] Antisense strand of siZ2*: u·mc(antisense 5′-3′);

[0046] *Sense strand of V1: ma·mg·mumgmcmafUmafAfCfCmamgmamamcmamamc·ma·ma (sense5'-3');

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

[0048] *V2 positive chain: (C16a)·mg·mumgmcmafUmafAfCfCmamgmamamcmamamc·ma·ma

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

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

[0051] * V3's positive chain: ma·(C16g)·mumgmcmafUmafAfCfCmamgmamamcmamamc·ma·ma

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

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

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

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

[0056] * V5's positive chain: ma·mg·mumgmcmafUmafAfCfCmamgmama(C16c)mamamc·ma·ma

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

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

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

[0060] Similar to the cell transfection method of Example 1, HEK293T cells overexpressing HA-mZbtb21 were transfected with siZ2*, *V1, *V2, *V3, *V4 and *V5 72 h or 7 days later, the cells were lysed to obtain protein lysate, and the protein expression level was detected by western blot.

[0061] The results are as follows Figure 2As shown, 72h after transfection, with the division and passaging of cells, all siRNAs, whether modified or not, can knock down Zbtb21 with an efficiency of more than 90%; however, with continued passaging, on the 7th day after transfection, siZ2* still had a knockdown efficiency of 75%, *V1 retained a knockdown efficiency of about 40%, while most of the other siRNAs were ineffective. Therefore, the optimization of siZ2* effectively achieved a long-lasting knockdown of Zbtb21 expression to a great extent.

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

[0063] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0064] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A siRNA molecule for knocking down the Zbtb21 gene in living animals, 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, the antisense strand is 23 nt long, and each nucleotide of the sense strand and the antisense strand 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.

2. The siRNA molecule for knocking down the Zbtb21 gene in living animals according to claim 1, characterized in that: The modification is selected from any one or more of the following: 2'-Fluoro modification, 2'-O-methyl modification, ethylene glycol nucleic acid modification, thiophosphate backbone modification, vinyl phosphate group modification, 2'-16 alkyl oxygen modification.

3. The siRNA molecule for knocking down the Zbtb21 gene in living animals according to claim 2, characterized in that: The positive strand structure of the siRNA molecule is shown in (A1); the antisense strand structure 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'); Wherein, ma, mc, mu and mg represent 2'-O-methyl modified nucleotides a, c, u and g, respectively; fA, fU and fC represent 2'-fluoro modified nucleotides A, U and C, respectively; · represents that the two nucleotides before and after are connected by a thiophosphate backbone; U represents nucleotide U modified with ethylene glycol; (C16a) represents nucleotide a modified with 2'-16 alkyl oxygen; VP represents modification of the 5' end with a vinyl phosphate group.

4. The siRNA molecule for knocking down the Zbtb21 gene in living animals according to claim 3, characterized in that: The chemical formula of the vinyl phosphate group is Formula I; 5. The siRNA molecule for knocking down the Zbtb21 gene in living animals according to claim 3, characterized in that: The chemical formula of the 2'-16 alkyloxy is Formula II; 6. An RNAi molecule mixture for knocking down the Zbtb21 gene in living animals, characterized in that: The invention comprises the siRNA molecule according to any one of claims 1 to 5.

7. Use of the siRNA molecule according to any one of claims 1 to 5 or the RNAi molecule mixture according to claim 6 in preparing a biological material for knocking down the Zbtb21 gene in a living animal.

8. The use according to claim 7, characterized in that The animal is a mouse.

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