CN119424665A - A copper-based DNA nanomaterial and its application in tumor treatment - Google Patents

Abstract

The present invention belongs to the field of biomedicine technology, and particularly relates to a copper-based DNA nanomaterial and its application in tumor treatment, which precisely downregulates the BMAL1 gene through the CRISPR-Cas13d system, disrupts the cell metabolic pathway, and enhances the effect of copper death in tumor cells. By precisely regulating the expression of the BMAL1 gene and inducing copper death, efficient killing of tumor cells is achieved, and the selectivity and efficiency of treatment are increased through a dual mechanism; this strategy can not only overcome the non-specific problems of existing copper delivery systems, but also regulate the metabolic pathways of tumor cells, weaken their metabolic homeostasis, thereby increasing the sensitivity of cells to copper death and enhancing the therapeutic effect; combined with the precise gene editing capabilities of the CRISPR-Cas13d system, the present invention has a wide range of application potentials in personalized cancer treatment, and provides a new treatment idea for anticancer therapy, especially in the fields of copper death induction therapy and biological clock gene regulation.

Description

Copper-based DNA nano material and application thereof in tumor treatment

Technical Field

The invention belongs to the technical field of biological medicine, and in particular relates to a copper-based DNA nano material and application thereof in tumor treatment.

Background

Biological clock genes are core genes that regulate circadian rhythms in organisms, and they play a key role in controlling important physiological processes such as proliferation, metabolism, and apoptosis of cells. BMAL1 (Brain andMuscle ARNT-Like 1) is one of the core biological clock genes, which not only regulates circadian rhythm, but also is closely related to cell cycle, metabolic balance and cell survival and death. Studies have shown that BMAL1 plays an important role in the development and progression of a variety of cancers. The main action mechanism is to influence the growth and drug resistance of tumor cells by regulating the energy metabolism, redox balance and apoptosis pathway in the cells.

In normal cells, BMAL1 supports cell survival by maintaining cell cycle stability, promoting metabolic homeostasis, enhancing the antioxidant capacity of the cells. However, deregulation of BMAL1 in tumor cells may promote uncontrolled cell proliferation, inhibit apoptosis, and enhance the antioxidant capacity of tumor cells, thereby promoting tumor development and drug resistance. It was found that downregulation of BMAL1 in tumor cells can induce cell cycle arrest and promote apoptosis, and thus, BMAL1 becomes a promising cancer therapeutic target.

Copper death is a novel cell death mode discovered in recent years and is characterized by accumulation of copper ions in cells, thereby leading to aggregation of mitochondrial proteins and destabilization of iron-sulfur clusters, and thus triggering cell death. Unlike traditional cell death modes (e.g., apoptosis, necrosis, pyrodeath, etc.), copper death occurs primarily by excessive accumulation of intracellular copper ions, which break the stability of iron-sulfur clusters in the tricarboxylic acid cycle (TCA cycle) by binding to intra-mitochondrial Dihydrolipoyltransferase (DLAT), resulting in accumulation of protein toxicity and eventual initiation of cell death.

Copper, which is a trace element essential to the human body, plays an important role in various biological reactions of cells, particularly in cellular energy metabolism and antioxidant reactions. However, in tumor cells, excessive copper accumulation can cause cell death by triggering copper death. The study found that the copper death pathway provides a new therapeutic strategy for tumor treatment, especially for those tumor types that are resistant to traditional therapies. Researchers have successfully triggered tumor cell death by increasing copper accumulation within tumor cells or by modulating related pathways, showing the potential of copper as an anticancer therapy.

Currently, the use of copper-based substances to induce tumor cell death, especially in cases where copper ions are capable of activating intracellular oxidative stress through the Fenton reaction, has been investigated to trigger specific cell death pathways, such as copper death. However, current copper delivery systems still face challenges that limit the effectiveness and safety of their clinical application. The main problem consists in the general lack of adequate copper transport proteins in tumor cells, which limits the accumulation of copper ions in tumor cells, thus affecting the effectiveness of copper death. The existing copper carrier design often cannot overcome the problem, so that copper ions are not accumulated enough, and copper death cannot be activated sufficiently. The regulation of tumor cell metabolism is insufficient, and most of the existing treatment strategies are focused on increasing the bioaccumulation of copper, but neglecting the metabolic characteristics of tumor cells, particularly the metabolic pathways under the regulation of biological clock genes. Tumor cells often evade cell death by altering metabolic pathways such as aerobic glycolysis, lactic acid fermentation, and the like. Lack of regulation of these metabolic pathways has limited the effectiveness of existing therapeutic regimens.

Disclosure of Invention

The invention solves the technical problems in the prior art, and provides a copper-based DNA nanomaterial and application thereof in tumor treatment, wherein the copper-based DNA nanomaterial can selectively down regulate the expression of BMAL1 genes in tumor cells. The nano material carries a CRISPR-Cas13d system, and the BMAL1 gene is precisely targeted by an RNA targeting technology, so that the sensitivity of tumor cells to copper death is increased.

The technical scheme of the invention is as follows:

a copper-based DNA nanomaterial, the copper-based DNA nanomaterial being Cu-dna@crispr-Cas13d/BMAL1sgRNA (abbreviated Cu-RNP);

The Cu-DNA is a copper-based delivery carrier, and copper ions and DNA form a complex through coordination reaction;

The CRISPR-Cas13d/BMAL1 sgRNA is a Cas13d protein that reacts with a sgRNA directed against BMAL1 to form a Cas13d RNP complex.

The Cu-DNA complex forms a Cu-RNP complex with the Cas13dRNP complex by self-assembly.

Preferably, the Cu-RNP complex is a spherical nanoparticle having a particle size of about 90-150 nm.

Preferably, the molar ratio of the Cu-DNA to the CRISPR-Cas13d/BMAL1 sgRNA is 1:1.

Preferably, the self-assembly condition is that the reaction is carried out for 30 minutes or more under the condition of 37 ℃ in a buffer system.

Preferably, in the Cu-DNA complex, the DNA includes BMAL1 targeting DNA and template DNA.

More preferably, the molar ratio of the copper ions, the BMAL1 targeting DNA and the template DNA is 33.7:1:1

Preferably, the BMAL1 targeting DNA sequence is:

AGCCTACTGAGTTCAGCACATGAAAACATTAAGA, shown as SEQ No. 1;

the template DNA sequence is as follows:

ATGTCATCACGCCCACCCGCTGTGGAAGTGGATG, shown as SEQ No. 2;

The sgRNA sequence for BMAL1 is:

GGCACUGGUGCAAAUUUGCACUAGUCUAAAACUCUUAAUGUUUUCAUGUGCUG AAC as shown in SEQ No. 3.

Preferably, the conditions for the complexing reaction of the copper ions with the DNA are such that after mixing the copper ions with the DNA in solution, the copper ions are incubated for 5 hours at 95 ℃.

The Cu-RNP serving as the copper-based DNA nano material is injected into an animal body through tail vein and accumulated at the tumor site of the xenograft MCF-7 cells. It enters MCF-7 cells by endocytosis, releasing copper ions and CRISPR-Cas13d RNP in the tumor environment. In one aspect, cu ²⁺ catalyzes the production of Cu ⁺ by glutathione, while depletion of FDX1 and LIAS results in DLAT heteromultimerization and causes mitochondrial damage, resulting in copper death of the cells. On the other hand, CRISPR-Cas13dRNP carries out gene editing, reduces the level of BMAL1, further leads to the reduction of WEE1 level and triggers apoptosis, meanwhile, the destruction of a biological clock and the up-regulation of p21 lead to the arrest of a cell cycle in G ₂/M phase, and in addition, the influence on the metabolism level of lactic acid and pyruvic acid prevents the normal metabolism of tricarboxylic acid circulation, and further enhances copper death. By integrating multiple cell death pathways, the therapeutic effect of the tumor is improved overall.

Thus, the first and second substrates are bonded together,

The copper-based DNA nano material can be used for preparing medicines for inhibiting tumors.

The copper-based DNA nano material can be used for preparing medicines for interfering biological clock genes to promote copper death of tumor cells.

The advantages of the present invention over the prior art are as follows,

The invention provides an innovative copper-based DNA nanodelivery system (Cu-RNP) whose application in tumor therapy has significant advantages, mainly in the following aspects:

(1) Targeting tumor cells precisely and inducing cell death by CRISPR-Cas13d system, the invention targets and cuts mRNA of BMAL1 gene in tumor cells, and down regulates expression of BMAL1 precisely. BMAL1 is a key gene for biological clock regulation and plays an important role in tumor cell proliferation, metabolism and cell cycle. Unlike traditional chemotherapy and radiotherapy, the present invention adopts gene editing technology to realize targeting treatment, and has effectively lowered tumor cell growth capacity and raised tumor cell stress sensitivity.

(2) Dual therapeutic mechanism the Cu-RNP system of the present invention induces "copper death" of tumor cells by catalysis of copper ions. By utilizing the reaction of copper ions and GSH in the Cu-DNA nano particles, cu-RNP can generate monovalent copper ions, trigger Fenton reaction to generate free radicals, and further lead to cell death mechanisms such as mitochondrial dysfunction, protein aggregation, lipid peroxidation and the like. In addition to targeting BMAL1 via the CRISPR-Cas13d system, the invention also disrupts metabolic homeostasis of tumor cells, affects metabolism of lactate, pyruvate, inhibits glycolysis and tricarboxylic acid cycle by down-regulating the BMAL1 gene. This dual effect (gene editing and metabolic interference) renders the tumor cells ineffective against oxidative stress, thereby promoting death of the tumor cells.

(3) The invention can inhibit the proliferation and growth of tumor cells at different cell layers through the synergistic effect of a plurality of cell death mechanisms such as copper death, cell cycle arrest, apoptosis and the like. Compared with single drug treatment, the Cu-RNP can obviously enhance the anti-tumor treatment effect and reduce the drug resistance of tumor cells to treatment through the synergistic effect of multiple mechanisms.

Drawings

FIG. 1 shows a preparation route of Cu-RNP;

FIG. 2 shows the mechanism of action of Cu-RNP in tumor cells;

FIG. 3 is the activity of CRISPR-Cas13d to cleave BMAL1 mRNA;

FIG. 4 is a transmission electron microscope image of Cu-RNP;

FIG. 5 is a high angle annular dark field-scanning transmission electron microscope image of Cu-RNP and corresponding elemental profiles (C, cu, S, N, P and O);

FIG. 6 shows cell uptake and lysosomal escape of Cu-RNP (confocal laser image);

FIG. 7 shows the real-time reverse transcription quantitative polymerase chain reaction for detecting the mRNA level of cells of different treatment groups, wherein A is BMAL1, B is FDX1, and C is LIAS;

FIG. 8 shows intracellular metabolite levels of different treatment groups, A being lactate and B being pyruvate;

FIG. 9 is a plot of mitochondrial membrane potential in cells of different treatment groups (confocal laser image);

FIG. 10 shows intracellular ATP levels from different treatment groups;

FIG. 11 shows the detection of WEE1 mRNA levels in cells from different treatment groups by real-time reverse transcription quantitative polymerase chain reaction;

FIG. 12 shows the flow cytometry detection of apoptosis in different treatment groups;

FIG. 13 shows the detection of intracellular p21 mRNA levels by real-time reverse transcription quantitative polymerase chain reaction in different treatment groups;

FIG. 14 is a flow cytometry detection of cell cycle status of different treatment groups;

FIG. 15 is a fluorescent image of staining of live dead cells from different treatment groups;

FIG. 16 is a flow chart of the construction and administration of a model of a human breast cancer MCF-7 tumor-bearing mouse;

FIG. 17 is a graph showing tumor growth curves of MCF-7 tumor-bearing mice after treatment, B is a photograph of the tumors in the mice of the different treatment groups after dissection, and C is the average weight of the dissected tumors after treatment of each group;

FIG. 18 shows Western blot detection of protein expression levels in tumor tissues of different treatment groups, A being BMAL1 and LIAS, B being FDX1, C being DLAT aggregation;

FIG. 19 shows HE staining for tumor tissue morphology, immunohistochemical detection of TUNEL, ki67 and BMAL1 expression changes.

Detailed Description

The technical scheme of the invention is further described below with reference to the accompanying drawings. The methods are conventional methods unless otherwise specified. The starting materials are available from published commercial sources unless otherwise specified.

EXAMPLE 1 preparation and characterization of Cu-RNP

As shown in fig. 1, the preparation method of Cu-RNP includes copper ion chelation, DNA self-assembly, and assembly of Cas13d system. The specific procedure was Cu-DNA self-assembly by adding 16. Mu.L of 20mM CuCl ₂·2H₂ O solution to 380. Mu.L of 50. Mu.M mixed DNA solution, wherein the molar ratio of BMAL1 targeting DNA to template DNA was 1:1.

The BMAL1 targeting DNA sequence is AGCCTACTGAGTTCAGCACATGAAAACATTAAGA (shown as SEQ No. 1)

The DNA sequence of the template is ATGTCATCACGCCCACCCGCTGTGGAAGTGGATG (shown as SEQ No. 2)

The solution was incubated at 95℃for 5 hours, and copper ions formed Cu-DNA complexes by complexation with DNA. After incubation, unreacted components were removed by centrifugation (8000 rpm,10 min) and the nanoparticles were washed with deionized water. The resulting Cu-DNA nanoparticles were resuspended with deionized water and prepared for subsequent processing.

Assembly of Cas13d RNP purified Cas13d protein was mixed with engineered sgrnas for BMAL1 (synthesized by T7 RNA synthesis kit) and incubated in 20mM Tris-HCl buffer to form Cas13d RNP complex.

The sgRNA sequence is:

GGCACUGGUGCAAAUUUGCACUAGUCUAAAACUCUUAAUGUUUUCAUGUGCUG AAC (shown as SEQ No. 3)

As shown in fig. 3, cas13d RNP was verified to be able to cleave BMAL1mRNA efficiently and specifically by NATIVE PAGE analysis, demonstrating its RNA targeting activity.

The BMAL1 mRNA sequence is:

UUAGAUAAACUUACUGUGCUAAGGAUGGCUGUUCAGCACAUGAAAACAUUAAG AGGUGCCACCAAUCCAU (shown as SEQ No. 4)

Synthesis and characterization of Cu-RNP the Cu-DNA was incubated with Cas13dRNP complex for 30min at 37 ℃, and the surface of the Cu-DNA was self-assembled by extension fragment with Cas13dRNP to form Cu-RNP complex.

As shown in FIG. 4, the morphology of Cu-RNP was observed using a Transmission Electron Microscope (TEM), and the image showed that Cu-RNP formed uniformly distributed spherical nanoparticles with a particle size of about 90-150nm.

As shown in fig. 5, the high angle annular dark field scanning transmission electron microscope was used to image and verify the presence and spatial distribution of the key elements (C, cu, S, N, P and O) observed. The profile of the carbon (C) element reveals different regions inside the structure, cu-DNA core region with lower carbon content, while Cas13d RNP shell region is rich in carbon, and Cas13dRNP shell layer is assembled on Cu-DNA surface. This hierarchical distribution demonstrates successful integration of its core and shell components.

EXAMPLE 2Cu-RNP entry into tumor cells

As shown in FIG. 2, cu-RNP acts in tumor cells by injecting Cu-RNP into the body through the tail vein and accumulating at the tumor site of xenograft MCF-7 cells. It enters MCF-7 cells by endocytosis, releasing copper ions and CRISPR-Cas13d RNP in the acidic environment of the tumor microenvironment. In one aspect, cu ²⁺ catalyzes the production of Cu ⁺ by Glutathione (GSH) and hydrogen peroxide (H ₂O₂), while the depletion of FDX1 and LIAS results in DLAT heterodimerization and causes mitochondrial damage, resulting in copper death of the cells. On the other hand, the gene editing reduces the level of BMAL1, thereby leading to the reduction of WEE1 level and triggering apoptosis, and meanwhile, the disruption of a biological clock and the up-regulation of p21 lead to the arrest of a cell cycle in the G ₂/M phase, and in addition, the influence on the metabolic level of lactic acid and pyruvic acid prevents the normal metabolism of tricarboxylic acid cycle, thereby further enhancing copper death. By integrating multiple cell death pathways, the therapeutic effect of human breast cancer is improved as a whole.

As shown in FIG. 6, the MCF-7 human breast cancer cell line was used and cultured in high-sugar DMEM medium containing 10% fetal bovine serum, at 37℃in an incubator containing 5% CO ₂, to the logarithmic growth phase. 24 hours prior to the experiment, cells were seeded into confocal microscope dishes at the bottom of the slide, with about 3X 10 ⁴ cells per well. When cell coverage reached 80%, cells were treated with medium containing 200. Mu.g/mL FITC-labeled Cu-RNP. This concentration was used to ensure adequate cellular uptake and lysosomal escape. After the treatment, the cells were removed at 1 hour, 4 hours and 8 hours, respectively, and washed 3 times with PBS to remove uninendocytic Cu-RNP. Staining with Hoechst 33342 dye (for nuclear staining) and LysoTracker Red (for lysosome labeling) was performed according to instructions. Observations were made using a laser confocal microscope (Leica SP 8). Co-localization of fluorescently labeled Cu-RNP and lysosomes can be quantified by Pearson correlation coefficient (Pearson's correlation coefficient, PCC), and uptake and lysosome escape of Cu-RNP can be analyzed. The fluorescence images were quantitatively analyzed and the overlap of the fluorescence intensity of the Cu-RNP and lysosome label was calculated by ImageJ software. The Pearson correlation coefficient was used to evaluate the degree of overlap of the two fluorescent signals. The images showed a higher overlap of the Cu-RNP with lysosomal fluorescence signal after 4 hours, indicating that Cu-RNP enters the cells by endocytosis and binds to lysosomes. After 6 hours, the overlap of Cu-RNP with lysosomes gradually decreased, indicating that Cu-RNP escapes into the cytoplasm through lysosomes, releasing copper ions and Cas13dRNP, thereby exerting its therapeutic effect.

EXAMPLE 3 mechanism of Cu-RNP exerting antitumor action

As shown in FIG. 7, MCF-7 cells in the logarithmic phase were inoculated into 24-well plates, 2.5X105 cells were inoculated per well, and after 16 hours of culture, the cells were treated with PBS, cu-DNA, cu-RNP, cu-RNP+UK5099 and the like. The final concentration of UK5099 was 100nM. After the treatment, the culture was continued for 24 hours. Total RNA from cells was extracted using UNlQ-10Column TRIZOL total RNA extraction kit, following the manufacturer's instructions. The concentration and quality of RNA were determined using a Nanodrop 2000 spectrophotometer. 1. Mu.g of RNA was used for cDNA synthesis, and cDNA was synthesized using HISCRIPT III RT SuperMix for qPCR (+ GDNA WIPER) kit according to the instructions. Using AceQ qPCRGREEN MASTER Mix (Without ROX) real-time quantitative PCR was performed. Beta-actin was selected as a reference gene, and the relative expression levels of the target genes (BMAL 1, FDX1, LIAS) were calculated using the 2- ΔΔCt method. The PCR results were analyzed, the relative amounts of mRNA of BMAL1, FDX1, LIAS in the different treatment groups were calculated, and the effect of Cu-RNP treatment on gene expression was evaluated. The results indicate that the mRNA levels of Cu-RNP groups BMAL1, FDX1, LIAS were significantly reduced, particularly downregulation of BMAL1, indicating that Cu-RNP effectively down-regulated the BMAL1 gene by the CRISPR-Cas13d system. Furthermore, down-regulation of the copper death gene FDX1 and LIAS also demonstrated a mechanism by which cells induced copper death, and copper death inhibitor UK5099 could attenuate down-regulation of the copper death gene.

After various treatments, cell culture supernatants were collected and the intracellular concentrations of lactic acid and pyruvic acid were determined, as shown in figure 8. Absorbance values were determined using a microplate reader (BioTek Synergy H1) and the concentration of lactic acid and pyruvic acid was calculated from a standard curve. Lactic acid and pyruvic acid levels were significantly lower in the Cu-RNP treated MCF-7 cells than in the control group, indicating that Cu-RNP inhibited energy metabolism by interfering with glycolysis and tricarboxylic acid cycle of tumor cells.

The intracellular mitochondrial membrane potential was measured using JC-1 dye. JC-1 dye was added to the treated cells and incubated at 37℃for 30 minutes. Red and green fluorescent signals within cells were observed using a laser confocal microscope. Healthy mitochondria fluoresce red, while mitochondria that lose membrane potential fluoresce green. As shown in FIG. 9, the mitochondrial membrane potential of the Cu-RNP treated group MCF-7 cells was measured using JC-1 dye. Cells of the Cu-RNP treated group showed weaker red fluorescence, increased green fluorescence, indicating significant loss of mitochondrial membrane potential, indicating that Cu-RNP caused impairment of mitochondrial function.

The intracellular ATP levels were measured and as shown in FIG. 10, the levels of ATP in the MCF-7 cells of the Cu-RNP treated group were significantly reduced, indicating that Cu-RNP inhibited ATP production and further exacerbated cell death by disrupting energy metabolism of tumor cells.

The relative expression levels of the WEE1 gene were calculated using beta-actin as a reference gene and apoptosis was detected in the different treatment groups using flow cytometry. As shown in fig. 11, the mRNA level of WEE1 in Cu-RNP treated cells was significantly decreased, and the apoptosis rate of Cu-RNP treated cells shown in fig. 12 was significantly increased to 42.1%, indicating that Cu-RNP down-regulates BMAL1, and apoptosis of tumor cells was effectively induced by inhibiting WEE 1.

The relative expression levels of the p21 gene were calculated using β -actin as a reference gene and the cell cycle status of the different treatment groups was examined using flow cytometry. As shown in FIG. 13, the mRNA level of p21 was significantly increased in the cells of the Cu-RNP treated group, the proportion of cells in the G ₂/M phase of the Cu-RNP group shown in FIG. 14 reached 45%, the proportion of cells in the G ₀/G₁ phase was decreased from 60% to 45% by 15% in comparison with 30% in the PBS group. The results indicate that Cu-RNP further promotes proliferation inhibition of tumor cells by down-regulating BMAL1 and interfering with cell cycle progression, resulting in cell cycle arrest.

As shown in fig. 15, the Cu-RNP treated group showed significant dead cell fluorescence after staining by Calcein-AM/PI, with significant enhancement of red fluorescence (PI-stained dead cells). Particularly in the Cu-RNP group, the red fluorescence ratio is 42.1 percent, and the red fluorescence ratio is obviously increased compared with the 15.3 percent of the PBS group, which indicates that the Cu-RNP can effectively induce the death of tumor cells.

Example 4 in vivo antitumor Effect evaluation

Female BALB/c-nu mice, 5-6 weeks old, were purchased from Venlhua laboratory animal technologies Inc. of Zhejiang, according to the in vivo protocol shown in FIG. 16. The human breast cancer tumor-bearing mouse model was established by subcutaneous injection of MCF-7 cells (5X 10 ⁶ cells per mouse). After 7 days, the volume was about 100mm ³. The mice were randomly divided into four groups (4 mice per group) of PBS group, cu-DNA group, cu-RNP group, and control group. Different doses (25 mg/kg) of Cu-RNP solution were administered by tail vein once every 3 days for a total of 3 doses. As shown in fig. 17, the body weight and tumor volume changes of mice were monitored, and the antitumor effect of Cu-RNP was evaluated. The change in tumor volume during treatment in each group of mice showed that the Cu-RNP treated group significantly inhibited tumor growth after dosing, with the Cu-RNP group having the slowest tumor volume increase rate compared to the control, PBS and Cu-DNA groups. At the end of treatment, the tumor volume of the mice in the Cu-RNP group was significantly smaller than that in the other groups. Specific data showed that the Cu-RNP group tumor volume was reduced by about 60% and TGI (tumor growth inhibition) was 87%. By determining the tumor weight, the tumor weight of the Cu-RNP group is obviously lower than that of the control group, and further proves that the Cu-RNP shows obvious anti-tumor effect in vivo.

Tumor tissue samples were taken and protein extraction was performed using RIPA lysate. As shown in FIG. 18, the expression of BMAL1 in tumor tissues of the Cu-RNP treatment group was significantly reduced, the expression of FDX1 and LIAS was also reduced, and the DLAT protein showed significant aggregation, by detecting the expression of BMAL1, FDX1, LIAS and the like by Western Blot. As shown in FIG. 19, after tissue sections and HE staining, the Cu-RNP group tumor tissue showed a distinct necrotic area, a reduced nucleus, and a different staining depth. The number of TUNEL positive cells in the Cu-RNP group tumor tissue is obviously increased, which proves that the Cu-RNP can effectively induce apoptosis of tumor cells. The proportion of Ki67 positive cells in the Cu-RNP group is significantly lower than that in the control group, indicating that the Cu-RNP inhibits proliferation of tumor cells. BMAL1 immunohistochemical analysis showed a significant decrease in the immune response of BMAL1 in tumor tissue of Cu-RNP group, further confirming that Cu-RNP exerts its anti-tumor effect by down-regulating BMAL 1.

Sequence listing

It should be noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and the equivalents or alternatives made on the basis of the above description are all included in the scope of the present invention.

Claims (10)

1. A copper-based DNA nanomaterial, characterized in that the copper-based DNA nanomaterial is Cu-DNA@CRISPR-Cas13d/BMAL1 sgRNA, abbreviated as Cu-RNP; The Cu-DNA is a copper-based delivery carrier, in which copper ions and DNA form a complex through coordination reaction; The CRISPR-Cas13d/BMAL1 sgRNA is a Cas13d protein that reacts with an sgRNA targeting BMAL1 to form a Cas13dRNP complex; The Cu-DNA complex and the Cas13dRNP complex form a Cu-RNP complex through self-assembly. 2. The copper-based DNA nanomaterial according to claim 1, wherein the Cu-RNP complex is a spherical nanoparticle with a particle size of 90-150 nm. 3. The copper-based DNA nanomaterial according to claim 1, wherein the Cu-DNA and the The molar ratio of CRISPR-Cas13d/BMAL1 sgRNA was 1:1. 4. The copper-based DNA nanomaterial according to claim 1, characterized in that the self-assembly condition is: reacting in a buffer system at 37°C for 30 minutes or more. 5. The copper-based DNA nanomaterial according to claim 1, characterized in that in the Cu-DNA complex, the DNA comprises BMAL1 targeting DNA and template DNA. 6. The copper-based DNA nanomaterial according to claim 5, characterized in that the molar ratio of the copper ions, BMAL1 targeting DNA, and template DNA is 33.7:1:1. 7. The copper-based DNA nanomaterial according to claim 5, wherein the BMAL1 targeting DNA sequence is: AGCCTACTGAGTTCAGCACATGAAAACATTAAGA, as shown in SEQ No. 1; The template DNA sequence is: ATGTCATCACGCCCACCCGCTGTGGAAGTGGATG, as shown in SEQ No. 2; The sgRNA sequence for BMAL1 is: GGCACUGGUGCAAAUUUGCACUAGUCUAAAACUCUUAAUGUUUUCAUGUGCUGAAC, as shown in SEQ No.3. 8. The copper-based DNA nanomaterial according to claim 1, characterized in that the conditions for the coordination reaction between the copper ions and DNA are: after the copper ions and DNA are mixed in a solution, they are incubated at 95°C for 5 hours. 9. Use of the copper-based DNA nanomaterial according to any one of claims 1 to 8 in the preparation of a drug for inhibiting tumors. 10. Use of the copper-based DNA nanomaterial according to any one of claims 1 to 8 in the preparation of a drug that interferes with biological clock genes and promotes copper cell death in tumor cells.