DNA methylation modified nucleic acid molecule for target gene expression silencing of mammals and application of DNA methylation modified nucleic acid molecule

Abstract

The invention discloses a DNA methylation modified nucleic acid molecule for mammal target gene expression silencing and application thereof, the DNA methylation modified nucleic acid molecule comprises two nucleic acid sequences, the first nucleic acid sequence comprises a target sequence part of a reverse complementary sequence of a target DNA fragment; the second segment of nucleic acid sequence comprises a DNA methylation regulation and control complex binding part, and the DNA methylation regulation and control complex is a protein complex containing DNA methyltransferase. The nucleic acid molecule can specifically induce DNA methylation formation in eukaryotic cells of mammals, so that DNA apparent level gene targeting silencing is realized. DNA methylation and downstream gene expression regulation are carried out by applying a drug containing the DNA methylation modified nucleic acid molecule to a mammal subject, DNA methylation targeted regulation treatment in a mammal body is achieved, reversibility is achieved, side reactions caused by off-target are controllable, and exogenous protein does not need to be introduced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to DNA methylation-modified nucleic acid molecules for silencing target gene expression in mammals and applications thereof. Background Art

[0002] DNA methylation is a common epigenetic modification that regulates gene expression by adding methyl groups to DNA molecules. Abnormal DNA methylation patterns are associated with the development of a variety of diseases, including cancer. CRISPR is a gene editing technology. CRISPR-based DNA methylation gene targeted silencing technology has made progress. Combined with CRISPR-associated protein (Cas) enzymes, it can achieve site-specific editing and regulation of specific gene sequences. Using CRISPR technology, Cas enzymes can be accurately guided to the target DNA sequence to achieve specific targeting of DNA methylation modification. By modifying the structure of the Cas enzyme or introducing other auxiliary proteins, methylation modifications can be removed or added, thereby enabling more precise control of gene expression. At present, researchers have used CRISPR technology to develop several gene targeted silencing methods related to DNA methylation. For example, using CRISPR-dCas9 (functionally inactivated Cas9) fused with DNA methyltransferase can achieve targeted induction of DNA methylation modification targeting specific genes.

[0003] However, there is no natural CRISPR / Cas9 system in mammals, and DNA methylation regulation technology based on the CRISPR / Cas9 system is difficult to apply to in vivo treatment. At the same time, the high off-target rate and irreversibility of the CRISPR / Cas9 system make this technology difficult to apply in clinical treatment. Therefore, it is particularly important to develop a DNA methylation targeted regulation technology that is safer and easier to apply in vivo.

[0004] In the prior art, for example, patent document CN101346393A discloses a chemically modified siRNA molecule and a method for silencing target gene expression using such siRNA molecules, wherein minimal chemical modification, such as 2'-O-methyl (2'OMe) modification, is performed at selected positions within one or both strands of the siRNA duplex, and the modified siRNA is less immunostimulatory than its unmodified siRNA sequence, wherein the nucleic acid-lipid particle includes the modified siRNA, cationic lipids and non-cationic lipids, and further includes conjugated lipids that inhibit particle aggregation, and gene expression is silenced by administering the modified siRNA to a mammalian subject, thereby eliminating the immunostimulatory activity of the siRNA and retaining complete RNAi activity. Patent document CN107916255A discloses a PDL-1 gene-targeted specific methylation epigenetic modification anti-tumor molecule and its use. According to the principles of bioinformatics, epigenetic engineering and immunology, a new protein molecule PPLA is designed for epigenetic methylation modification of the human PD-L1 gene promoter sequence. It is introduced into human cells through an adenovirus vector, and can efficiently express the protein molecule PPLA specific to the human PD-L1 gene promoter sequence. The expressed gene can effectively inhibit the PD-L1 gene. The molecule can produce a methylation chemical modification effect on the PDL-1 gene. In vitro cell experiments have confirmed that it has the function of efficiently blocking the expression of the PDL-1 gene, thereby breaking immune tolerance, and ultimately achieving the effect of treating chronic viral infections and tumors, and realizing anti-tumor and anti-viral immunotherapy effects. It has broad application prospects in the preparation of epigenetic engineering tumor and anti-viral drugs. Summary of the invention

[0005] The main purpose of the present invention is to provide a DNA methylation-modified nucleic acid molecule for silencing target gene expression in mammals, which can specifically induce DNA methylation formation in eukaryotic cells of mammals, thereby achieving targeted gene silencing at the DNA epigenetic level.

[0006] Another object of the present invention is to provide the use of the DNA methylation-modified nucleic acid molecule in the preparation of a drug for silencing target gene expression in a mammal, and to achieve targeted regulation of DNA methylation in the mammal by administering a drug containing the DNA methylation-modified nucleic acid molecule to a mammalian subject for DNA methylation and downstream gene expression regulation.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention provides a DNA methylation-modified nucleic acid molecule for silencing target gene expression in mammals, which comprises two nucleic acid sequences, wherein the first nucleic acid sequence comprises a targeting sequence portion of a reverse complementary sequence of a target DNA fragment; the second nucleic acid sequence comprises a DNA methylation regulatory complex binding portion, and the DNA methylation regulatory complex is a protein complex containing a DNA methyltransferase.

[0009] Preferably, the target DNA is the EGFR gene.

[0010] Preferably, the DNA methyltransferase is DNMT1.

[0011] Preferably, the first nucleic acid sequence is as shown in SEQ ID NO: 1, specifically UAAUACUUACCUGUCGG.

[0012] Preferably, the second nucleic acid sequence is as shown in SEQ ID NO: 2, specifically UGAGCUGCGUGAUGA.

[0013] Preferably, the sequence of the DNA methylation modified nucleic acid molecule is as shown in SEQ ID NO: 3, specifically 5'-UAAUACUUACCUGUCGGUGAGCUGCGUGAUGA-3'.

[0014] The present invention also provides a vector comprising any of the aforementioned DNA methylation-modified nucleic acid molecules.

[0015] The present invention also provides a recombinant cell, comprising any of the aforementioned DNA methylation modified nucleic acid molecules, or the aforementioned vector.

[0016] The present invention also provides the use of the DNA methylation modified nucleic acid molecule, the vector, or the recombinant cell in the preparation of a drug for targeted gene silencing at the DNA epigenetic level in mammals.

[0017] The present invention also provides a nucleic acid-liposome particle, which comprises the DNA methylation-modified nucleic acid molecule, and the preparation method comprises the following steps:

[0018] (1) synthesizing a DNA methylation-modified nucleic acid molecule whose sequence is shown in SEQ ID NO: 3;

[0019] (2) first transfecting the DNA methylation-modified nucleic acid molecule synthesized in step (1) into A549 cells using the liposome transfection reagent Lipofectamine 2000, and then incubating the cells with DMEM medium containing 10% FBS for 14-18 hours;

[0020] (3) Centrifuge at 4°C and 1500 rpm for 10 min, discard the supernatant, and wash at least twice with pre-cooled PBS.

[0021] Compared with the prior art, the present invention proposes a DNA methylation-modified nucleic acid molecule for silencing target gene expression in mammals, systematically studies the distribution and regulatory mechanism of DNA methylation based on the pan-cancer TCGA methylation database, and proposes endogenous DNA methylation-specific regulation based on U1snRNP and DNA methyltransferase DNMT1, that is, by constructing a DNA methylation-modified nucleic acid molecule (mrRNA) combined with a targeting sequence (first nucleic acid sequence) containing the reverse complementary sequence of a target DNA fragment and a DNA methylation regulatory complex (a protein complex containing DNA methyltransferases such as DNMT1) (second nucleic acid sequence), and transfecting it into the cell nucleus to methylate the CpG sites of the genomic DNA around the target position, thereby regulating downstream gene expression, and the beneficial effects are:

[0022] (1) Target DNA methylation regulation and downstream gene expression regulation are achieved at the DNA epigenetic modification level, which is reversible and safer, and the side effects caused by off-target effects are controllable.

[0023] (2) Utilize endogenous DNA-specific methylation regulation without the need to introduce exogenous proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the DNA methylation level around the EGFR gene in the example (***p<0.001, Students' T test).

[0025] Figure 2 is the EGFR gene expression level in the examples (***p<0.01, Students' T test). DETAILED DESCRIPTION

[0026] In order to more fully understand and demonstrate the technical solutions, purposes and advantages of the present invention, the technical effects produced by the present invention are further described in detail and completely in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all. It should be pointed out that for those of ordinary skill in the art, other embodiments obtained without departing from the concept of the present invention all belong to the protection scope of the present invention. The present invention is described in detail in conjunction with the accompanying drawings and embodiments.

[0027] Unless otherwise specified in the following examples, all reagents and materials used were commercially available.

[0028] Example 1

[0029] 1. Methods

[0030] In this embodiment, a DNA methylation regulatory nucleic acid targeting the target DNA region is first synthesized, and then the methylation regulatory nucleic acid is delivered into the cell nucleus using liposome delivery, specifically:

[0031] 1. Synthesize targeted EGFR methylation regulatory nucleic acid (mrEGFR group): 5'-UAAUACUUACCUGUCGGUGAGCUGCGUGAUGA-3'.

[0032] 2. Synthetic control nucleic acid (Control group): containing only the first nucleic acid sequence 5'-UAAUACUUACCUGUCGG-3'.

[0033] 3. The target nucleic acid was transfected into A549 cells using the liposome transfection reagent Lipofectamine 2000 (Invitrogen) according to the instructions and incubated in DMEM medium containing 10% FBS for 16 h. Only Lipofectamine 2000 reagent was added to the Blank group.

[0034] 4. Centrifuge at 4°C, 1500 rpm for 10 min, discard the supernatant, and wash twice with pre-cooled PBS.

[0035] 5. Detection of DNA methylation levels around EGFR:

[0036] (1) Using Illumina The 2500System platform was used to detect the DNA methylation level of cells in each group according to the WGBS standard process.

[0037] (2) Use bismark and methylKit R software to extract DNA methylation around EGFR and perform data analysis.

[0038] 6. Quantitative PCR was used to determine the expression level of EGFR in each group.

[0039] (II) Results

[0040] (1) EGFR methylation level: The DNA methylation level around EGFR in cells treated with methylation regulatory nucleic acid targeting EGFR (mrEGFR group) was significantly increased compared with that in the Blank group and the Control group. Figure 1 shown.

[0041] (2) EGFR expression level: The DNA methylation level around EGFR in cells treated with methylation regulatory nucleic acid targeting EGFR (mrEGFR group) was significantly lower than that in the Blank group and the Control group. Figure 2 shown.

[0042] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A DNA methylation-modified nucleic acid molecule for silencing target gene expression in mammals, characterized in that: It contains two nucleic acid sequences, wherein: The first nucleic acid sequence comprises a targeting sequence portion of the reverse complementary sequence of a target DNA fragment; The second nucleic acid sequence contains a DNA methylation regulatory complex binding portion; The DNA methylation regulatory complex is a protein complex containing DNA methyltransferase.

2. The DNA methylation modified nucleic acid molecule according to claim 1, characterized in that The DNA methyltransferase is DNMT1.

3. The DNA methylation modified nucleic acid molecule according to claim 1, characterized in that The target DNA is the EGFR gene.

4. The DNA methylation modified nucleic acid molecule according to claim 1, characterized in that The first nucleic acid sequence is shown as SEQ ID NO:

1.

5. The DNA methylation modified nucleic acid molecule according to claim 1, characterized in that The second nucleic acid sequence is shown as SEQ ID NO:

2.

6. The DNA methylation modified nucleic acid molecule according to claim 1, characterized in that Its sequence is shown in SEQ ID NO:

3.

7. A carrier, characterized in that Comprising the DNA methylation-modified nucleic acid molecule according to any one of claims 1 to 6.

8. A recombinant cell, characterized in that Comprising the DNA methylation modified nucleic acid molecule according to any one of claims 1 to 6, or the vector according to claim 7.

9. Use of the DNA methylation modified nucleic acid molecule according to any one of claims 1 to 6, or the vector according to claim 7, or the recombinant cell according to claim 8 in the preparation of a drug for targeted gene silencing at the DNA epigenetic level in mammals.

10. A nucleic acid-liposome particle comprising the DNA methylation-modified nucleic acid molecule according to any one of claims 1 to 6; The preparation method of the nucleic acid-liposome particles comprises the following steps: (1) synthesizing a DNA methylation-modified nucleic acid molecule whose sequence is shown in SEQ ID NO: 3; (2) first transfecting the DNA methylation-modified nucleic acid molecule synthesized in step (1) into A549 cells using the liposome transfection reagent Lipofectamine 2000, and then incubating the cells with DMEM medium containing 10% FBS for 14-18 hours; (3) The product after incubation in step (2) was centrifuged at 4°C and 1500 rpm for 10 min, the supernatant was discarded, and the product was washed at least twice with pre-cooled PBS.

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