A FUS protein hydrolysis targeting chimera, its preparation method and application

Abstract

The present invention belongs to the field of protein targeted degradation chimera preparation and biomedical technology, and specifically relates to a FUS protein hydrolysis targeted chimera and its preparation method and application. Specifically, the present invention is based on the fact that the RNA motif "GGUG" can specifically bind to FUS, designs a ligand that specifically binds to FUS, and develops a FUS protein hydrolysis targeted chimera that specifically degrades wild-type and mutant FUS by connecting a CRBN ligand at its 5' end. At the same time, in order to ensure that the FUS protein hydrolysis targeted chimera effectively exerts its effect in the brain, the present invention designs a DNA nanoflower based on a transferrin receptor nucleic acid aptamer, which can effectively load the FUS protein hydrolysis targeted chimera and deliver it to the brain to exert its effect, thereby realizing precision medicine for patients with amyotrophic lateral sclerosis caused by FUS mutations.

Description

Technical Field

[0001] The invention belongs to the technical field of protein targeted degradation chimera preparation and biomedicine, and specifically relates to a FUS protein hydrolysis targeted chimera and a preparation method and application thereof. Background Art

[0002] Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease characterized by premature degeneration of motor neurons, leading to progressive, fatal paralysis, usually with death within 3 years of symptom onset. Mutations in the FUS gene cause 4% of familial ALS and 1% of sporadic ALS. FUS is a multifunctional DNA / RNA binding protein composed of 526 amino acids that is involved in RNA metabolism, including RNA transcription, splicing, transport, translation, and degradation. Under physiological conditions, FUS is mainly localized in the nucleus, and mutant FUS accumulates in the cytoplasm in granular, filamentous, or dense forms. Most mutations found in ALS patients are clustered in glycine-rich regions and the extreme C-terminal region of the protein, including P525L, R521C, R521G, R521H, etc. Mutations in ALS have also been reported in ALS patients. FUS Mutations occur in the 3' untranslated region of the gene. FUS The abundant accumulation of FUS found in the nucleus and cytoplasm of fibroblasts suggests a mechanism of translational dysregulation. FUS Mutations can induce disease through gain of function in the cytoplasm or loss of function in the nucleus. The treatment of ALS is mainly supportive, including physical therapy, nutritional support and artificial ventilation in the later stage. The only available drug treatment is riluzole, but it can only moderately increase the survival rate in clinical trials. Therefore, a new drug for the treatment of ALS is urgently needed.

[0003] Proteolysis targeting chimera (PROTAC) is a bifunctional small molecule, including two functional ligands and an intermediate linker: one ligand is responsible for binding to the target protein (POI), and the other ligand is responsible for binding to the ubiquitin ligase (E3). When both ends of PROTAC bind to the target protein and E3 at the same time, the target protein is ubiquitinated and then degraded by the ubiquitin-proteasome system. However, the inventors found that there is no PROTAC reported for the targeted degradation of FUS protein.

[0004] At the same time, the brain delivery of drugs for central nervous system diseases is usually restricted by the blood-brain barrier (BBB). The receptors highly expressed on the surface of endothelial cells of the blood-brain barrier include transferrin receptor (TfR), insulin receptor and low-density lipoprotein receptor. Using these surface receptors to mediate the trans-blood-brain barrier transport of drugs is a promising strategy. Studies have shown that the use of nucleic acid aptamers targeting transferrin receptors as carriers can improve the blood-brain barrier penetration ability of nucleic acid drugs. Nucleic acid nanomaterials have good biocompatibility, biodegradability and programmability, and have important applications in the biomedical field. Rolling circle amplification is an isothermal enzymatic nucleic acid amplification technology used to mass-produce nucleic acid nanomaterials with pre-designed desired structures and functions, such as DNA nanoflowers, DNA origami, nanotubes, etc. However, the inventors found that there is currently no report on the use of nucleic acid aptamers targeting transferrin receptors to develop DNA nanoflower carriers for brain delivery. Summary of the invention

[0005] In view of the problems existing in the above-mentioned prior art, the inventors have provided a FUS proteolysis targeted chimera and its preparation method and application after long-term technical and practical exploration. Specifically, the present invention designs, synthesizes and screens chimeras that efficiently degrade wild-type FUS and its mutant proteins. In addition, the present invention also constructs a DNA nanoflower delivery vector based on transferrin receptor nucleic acid aptamers to achieve brain delivery of FUS chimera degradation agents. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a FUS protein hydrolysis targeting chimera, wherein the FUS protein hydrolysis targeting chimera has a general structural formula: FE;

[0008] Wherein, F is a nucleic acid aptamer that specifically binds to FUS protein; and E is a ligand of E3 ubiquitin ligase.

[0009] The FUS protein is of human origin, and may be a wild-type FUS protein or a mutant FUS protein. The mutant FUS protein includes a FUS mutant protein that mediates human amyotrophic lateral sclerosis.

[0010] The nucleotide sequences of the nucleic acid aptamers that specifically bind to the FUS protein are shown in SEQ ID NOs: 1-5.

[0011] The second aspect of the present invention provides a method for preparing the above-mentioned FUS proteolysis targeting chimera, the preparation method comprising:

[0012] A single-stranded RNA containing an azide modification and an E3 ubiquitin ligase ligand with a terminal alkyne modification were synthesized and coupled by a click reaction.

[0013] Furthermore, the azide modification is on the 5' end of the single-stranded RNA, specifically by connecting the phosphoramidite monomer of the C6 amino group to the 5' end of the single-stranded RNA.

[0014] The third aspect of the present invention provides the use of FUS proteolysis targeting chimera in the preparation of DNA nanoflowers based on transferrin receptor nucleic acid aptamers.

[0015] The fourth aspect of the present invention provides a DNA nanoflower based on a transferrin receptor aptamer, wherein the DNA nanoflower is loaded with the above-mentioned FUS protein hydrolysis targeting chimera. Specifically, the DNA nanoflower utilizes a transferrin receptor-targeted nucleic acid aptamer and rolling circle amplification technology to penetrate the blood-brain barrier, and at the same time utilizes the base complementarity principle to load the FUS protein hydrolysis targeting chimera on the DNA nanoflower carrier, thereby delivering it to the brain, thereby degrading wild-type and mutant FUS proteins to alleviate and treat amyotrophic lateral sclerosis.

[0016] A fifth aspect of the present invention is the use of the above-mentioned FUS protein hydrolysis targeting chimera or transferrin receptor-based nucleic acid aptamer DNA nanoflower in the preparation of products.

[0017] The product has at least one or more of the following functions:

[0018] (a) Degradation of FUS protein;

[0019] (b) Treatment of amyotrophic lateral sclerosis.

[0020] The sixth aspect of the present invention provides a drug, wherein the active ingredient of the drug at least comprises the above-mentioned FUS protein hydrolysis targeting chimera or the DNA nanoflower based on the transferrin receptor nucleic acid aptamer. The drug can be used to degrade FUS protein and then treat amyotrophic lateral sclerosis.

[0021] The seventh aspect of the present invention provides a method for treating amyotrophic lateral sclerosis, which comprises administering to a subject a therapeutically effective dose of the above-mentioned FUS proteolysis targeting chimera, transferrin receptor-based nucleic acid aptamer DNA nanoflower or drug.

[0022] It should be noted that although the above technical solution is based on FUS proteolysis targeting chimeras, and further constructs DNA nanoflowers based on transferrin receptor nucleic acid aptamers, which can effectively cross the blood-brain barrier and be used to treat ALS. Therefore, based on the inventive concept of the present invention, the use of other nucleic acid-PROTAC chimeras, antisense oligonucleotides (ASOs) and siRNA drugs to achieve brain targeting and then be used to treat amyotrophic lateral sclerosis and other related diseases also falls within the scope of protection of this application.

[0023] Compared with the existing technical solutions, the above one or more technical solutions have the following beneficial effects:

[0024] The above technical solution is based on the "GGUG" RNA motif that can specifically bind to FUS, and designs a ligand that specifically binds to FUS, and develops PROTAC that specifically degrades wild-type and mutant FUS by connecting the CRBN ligand at its 5' end. At the same time, in order to ensure that PROTAC effectively exerts its effect in the brain, the above technical solution designs a DNA nanoflower based on a transferrin receptor nucleic acid aptamer. The DNA nanoflower can effectively load PROTAC and deliver it to the brain to exert its effect, realizing precision medicine for ALS patients caused by FUS mutations.

[0025] Experimental verification shows that the RNA-based PROTAC involved in the above technical solution has good degradation efficiency and alleviates neurotoxicity caused by FUS overexpression in vitro. In vivo, nanoflowers can effectively deliver PROTAC to the brain and degrade FUS protein, which has good practical application value in the treatment of ALS caused by FUS mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the mass spectrum of the RNA ligand N3-FUS-RNA#1 with azide modification at the 5' end synthesized in Example 1 of the present invention.

[0027] Figure 2 This is the mass spectrum of the RNA ligand N3-FUS-RNA#2 with azide modification at the 5' end synthesized in Example 1 of the present invention.

[0028] Figure 3 This is the mass spectrum of the RNA ligand N3-FUS-RNA#3 with azide modification at the 5' end synthesized in Example 1 of the present invention.

[0029] Figure 4 This is the mass spectrum of the RNA ligand N3-FUS-RNA#4 with azide modification at the 5' end synthesized in Example 1 of the present invention.

[0030] Figure 5This is the mass spectrum of the RNA ligand N3-FUS-RNA#5 with azide modification at the 5' end synthesized in Example 1 of the present invention.

[0031] Figure 6 This is the mass spectrum of the RNA ligand N3-FUS-RNA#NC with azide modification at the 5' end synthesized in Example 1 of the present invention.

[0032] Figure 7 The non-denaturing SDS-PAGE in Example 1 of the present invention shows the electrophoresis results before and after the connection of different azide-modified RNA ligands and CRBN ligands or VHL ligands, wherein A is the sequence information of different azide-modified RNAs, and B is the electrophoresis result diagram proving that different RNA sequences are successfully connected to CRBN or VHL ligands.

[0033] Figure 8 Figure 2 is a diagram showing the degradation effects of 10 PROTACs on FUS in PC12 cells in Example 2 of the present invention, A is an electrophoresis diagram of FUS degradation by 10 PROTACs, and B is a quantitative statistical diagram of A.

[0034] Fig. 9 The molecular simulation results of the binding of dFUS-C1 to FUS protein and CRBN ligand in Example 3 of the present invention, wherein A is a molecular simulation diagram of the binding of dFUS-C1 to FUS protein and CRBN ligand, and B is the specific amino acid site of the binding of dFUS-C1 to FUS and CRBN.

[0035] Fig.10 The figures are the concentration-dependent degradation effect diagram of FUS in PC12 and SH-SY5Y cells by dFUS-C1 in Example 3 of the present invention and the proteomic diagram before and after dFUS-C1 treatment, wherein A is the concentration-dependent degradation effect diagram of FUS in PC12 and SH-SY5Y cells by dFUS-C1, B is the quantitative statistical diagram of A, and C is the proteomic diagram before and after dFUS-C1 treatment.

[0036] Fig.11 The figure is a graph showing the degradation effect of dFUS-C1 in Example 4 of the present invention on mutant FUS in a concentration-dependent manner; wherein A is wild-type FUS (FUS-WT), B is mutant FUS-P525L, C is mutant FUS-R521C, D is mutant FUS-R521G, and E is mutant FUS-R521H.

[0037] Fig.12These are the results of dFUS-C1 in Example 5 of the present invention alleviating neurotoxicity caused by FUS overexpression, A is a graph for detecting the content of reactive oxygen species in SH-SY5Y cells, B is a graph for detecting the content of lactate dehydrogenase release in SH-SY5Y cells, and C is a graph for detecting the viability of SH-SY5Y cells.

[0038] Fig.13 This is the mass spectrum of dFUS-PS in Example 6 of the present invention.

[0039] Fig.14 Figures 1 and 2 are the serum stability test results of dFUS-C1 and dFUS-PS in Example 6 of the present invention and a diagram showing the effect of dFUS-PS on degradation of FUS in a dose-dependent manner. Figure 3 is a quantitative statistical diagram of dFUS-C1 and dFUS-PS at the indicated time. Figure 4 is a quantitative statistical diagram of A. Figure 5 is a diagram showing the effect of dFUS-PS on degradation of FUS in a dose-dependent manner. Figure 6 is a quantitative statistical diagram of C.

[0040] Fig.15 This is a schematic diagram of the synthesis and loading of dFUS-C1 of nanoflowers and their penetration of the blood-brain barrier in Example 7 of the present invention.

[0041] Fig.16 The results of the successful loading of dFUS-PS on the DNA nanoflowers in Example 7 of the present invention are shown in Figure 1, wherein A is a 620-700 nm fluorescence spectrum scan before and after loading the PROTAC nanoflowers, B is a particle size analysis diagram before and after loading the PROTAC nanoflowers, and C is a scanning electron microscope image before and after loading the PROTAC nanoflowers, with a scale of 300 nm.

[0042] Fig.17 These are the results of NF@Cy5-dFUS-PS penetrating the blood-brain barrier and entering the brain in Example 8 of the present invention. A is a picture of in vivo and in vitro photography at the indicated time after tail vein injection of NF@Cy5-dFUS-PS. B is a fluorescent staining picture of mouse brain slices after tail vein injection of NF@Cy5-dFUS-PS.

[0043] Fig.18 This is a graph showing the non-toxicity of NF@dFUS-PS to various organs in Example 9 of the present invention.

[0044] Fig.19 This is a graph showing the results of NF@dFUS-PS degrading FUS protein in mouse brain in Example 10 of the present invention. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0046] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0047] As mentioned earlier, FUS Gene mutations cause 4% of familial ALS and 1% of sporadic ALS, and there is no good treatment so far. The blood-brain barrier prevents most drugs from taking effect in the brain. PROTAC has the function of specific protein degradation, so using nanoflowers loaded with PROTAC to penetrate the blood-brain barrier and degrade wild-type and mutant FUS to alleviate and treat ALS is a feasible way.

[0048] Based on the fact that the RNA motif "GGUG" can specifically bind to FUS, the present invention designs a ligand that specifically binds to FUS, and develops a PROTAC that specifically degrades wild-type and mutant FUS by connecting the CRBN ligand to its 5' end. In order to ensure that PROTAC effectively exerts its effect in the brain, the present invention designs a DNA nanoflower based on a transferrin receptor nucleic acid aptamer, which can effectively load PROTAC and deliver it to the brain to exert its effect, thus realizing precision medicine for ALS patients caused by FUS mutation.

[0049] In view of this, in a typical embodiment of the present invention, a FUS protein hydrolysis targeting chimera is provided, and the FUS protein hydrolysis targeting chimera has a general structural formula: EF;

[0050] E is the ligand of E3 ubiquitin ligase, and F is the nucleic acid aptamer that specifically binds to FUS protein;

[0051] Wherein, the ligand of the E3 ubiquitin ligase may be a CRBN ligand or a VHL ligand.

[0052] The FUS protein may be of human origin, and may be a wild-type FUS protein or a mutant FUS protein. The mutant FUS protein includes FUS mutant proteins that mediate amyotrophic lateral sclerosis (including FUS-P525L, FUS-R521C, FUS-R521G and FUS-R521H).

[0053] The nucleotide sequence of the nucleic acid aptamer that specifically binds to the FUS protein is shown in SEQ ID NOs: 1-5; more preferably, it is shown in SEQ ID NO: 1.

[0054] The present invention also performs a series of modifications on the above-mentioned nucleic acid aptamer, and the modification can be chemical modification, which is well known to those skilled in the art, such as phosphorothioate (PS), by which the serum stability and affinity of the nucleic acid aptamer are effectively improved. Further, the thio modification is full thio modification, and the full thio modification means that all the phosphorus oxygen bonds in the molecular structure of the nucleic acid aptamer are replaced by phosphorus sulfur bonds.

[0055] Furthermore, in order to achieve the connection between the nucleic acid aptamer and the ligand of the E3 ubiquitin ligase, the nucleic acid aptamer (5' end) is modified with an azide group, and the ligand of the E3 ubiquitin ligase is modified with an alkynyl group, and the two are coupled and connected through a click chemistry reaction.

[0056] Wherein, the ligand of the E3 ubiquitin ligase modified with an alkynyl group may be a CRBN ligand modified with an alkynyl group or a VHL ligand modified with an alkynyl group;

[0057] The CRBN ligand modified with an alkynyl group is a thalidomide-based CRBN ligand (thalidomide-O-amido-propargyl), and its structural formula is: The compound and its preparation method have been disclosed in Chinese patent CN 116179554 A, which will not be described in detail here.

[0058] The VHL ligand modified with an alkynyl group is VH032 modified with an alkynyl group (VH032-propargyl, CAS No.: 2098799-78-9), and its structural formula is:

[0059] The compound can be obtained by commercial means.

[0060] In another specific embodiment of the present invention, a method for preparing the above-mentioned FUS proteolysis targeting chimera is provided, and the preparation method comprises:

[0061] A single-stranded RNA containing an azide modification and an E3 ubiquitin ligase ligand with a terminal alkyne modification were synthesized and coupled by a click reaction.

[0062] Furthermore, the azide modification is on the 5' end of the single-stranded RNA, specifically by connecting the phosphoramidite monomer of the C6 amino group to the 5' end of the single-stranded RNA.

[0063] The specific reaction conditions of the click reaction coupling are: mixing the azide-modified single-stranded RNA and the terminal alkyne-modified E3 ubiquitin ligase ligand at a molar ratio of 1:1-10 (preferably 1:5), incubating at 30-40°C (preferably 37°C) for 1-10 hours (preferably 5 hours), and obtaining the product after purification.

[0064] The purification includes steps such as ultrafiltration and centrifugation, such as removing excess CRBN ligand or VHL ligand through an ultrafiltration tube with a molecular weight cutoff of 3K, which is not specifically limited here.

[0065] In another specific embodiment of the present invention, there is provided the use of a FUS proteolysis targeting chimera in the preparation of a DNA nanoflower based on a transferrin receptor nucleic acid aptamer.

[0066] In another specific embodiment of the present invention, a DNA nanoflower based on a transferrin receptor aptamer is provided, wherein the DNA nanoflower is loaded with the above-mentioned FUS protein hydrolysis targeting chimera. Specifically, the DNA nanoflower utilizes a transferrin receptor-targeted nucleic acid aptamer and rolling circle amplification technology, thereby being able to penetrate the blood-brain barrier, and at the same time utilizes the base complementarity principle to load the FUS protein hydrolysis targeting chimera on the DNA nanoflower carrier, thereby delivering it to the brain, thereby degrading wild-type and mutant FUS proteins to alleviate and treat amyotrophic lateral sclerosis.

[0067] Specifically, the preparation method of the transferrin receptor-based aptamer DNA nanoflower includes:

[0068] DNA polymerase is added to a system containing a circularized DNA nanoflower template and primers for rolling circle amplification, and then the FUS protein hydrolysis targeting chimera is added thereto.

[0069] Wherein, the nucleotide sequence of the transferrin receptor nucleic acid aptamer is shown in SEQ ID NO: 7.

[0070] The circularized DNA nanoflower template at least comprises a nucleotide sequence complementary to the nucleic acid aptamer of the transferrin receptor and a nucleotide sequence of the FUS protein hydrolysis targeting chimera.

[0071] The primer is shown in SEQ ID NO: 9.

[0072] The DNA polymerase is preferably a DNA polymerase suitable for rolling circle amplification. In a specific embodiment of the present invention, the DNA polymerase is phi29 DNA polymerase.

[0073] Among them, the circularized DNA nanoflower template is obtained by phosphorylating the 5' end of the linear DNA nanoflower template and circularizing the linear DNA nanoflower template; specifically, T4 ligase can be used to circularize the linear DNA nanoflower template.

[0074] The nucleotide sequence of the linear DNA nanoflower template is shown in SEQ ID NO: 8.

[0075] In another specific embodiment of the present invention, there is provided the use of the above-mentioned FUS proteolysis targeting chimera or transferrin receptor-based nucleic acid aptamer DNA nanoflower in the preparation of products.

[0076] The product has at least one or more of the following functions:

[0077] (a) Degradation of FUS protein;

[0078] (b) Treatment of amyotrophic lateral sclerosis.

[0079] The FUS protein may be of human origin, and may be a wild-type FUS protein or a mutant FUS protein, wherein the mutant FUS protein includes a FUS mutant protein that mediates amyotrophic lateral sclerosis.

[0080] The (a) is specifically manifested as: penetrating the blood-brain barrier into the brain, achieving brain delivery, and thereby degrading brain FUS protein.

[0081] The product can be a drug or a test reagent. The test reagent is a common test reagent for non-medical purposes, and can be used for basic research related to the occurrence and development of amyotrophic lateral sclerosis.

[0082] In another specific embodiment of the present invention, a drug is provided, wherein the active ingredient of the drug at least comprises the above-mentioned FUS protein hydrolysis targeting chimera or the DNA nanoflower based on the transferrin receptor nucleic acid aptamer. The drug can be used to degrade FUS protein and then treat amyotrophic lateral sclerosis.

[0083] Specifically, the drug further comprises at least one inactive drug ingredient.

[0084] The inactive ingredients of the drug can be carriers, excipients and diluents commonly used in pharmacy, etc. Moreover, according to the usual method, it can be made into oral preparations, external preparations, suppositories and sterile injection solutions in the form of powders, granules, suspensions, emulsions, syrups, sprays, etc.

[0085] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0086] In another specific embodiment of the present invention, the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0087] In another specific embodiment of the present invention, the medicine of the present invention can be administered to the body by known means. For example, it can be delivered to the tissue of interest by intravenous systemic delivery or local injection. Such administration can be carried out via a single dose or multiple doses. It is understood by those skilled in the art that the actual dose to be administered in the present invention can vary depending on a variety of factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0088] In another specific embodiment of the present invention, the subjects of drug administration can be humans and non-human animals, especially non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.

[0089] In another specific embodiment of the present invention, a method for treating amyotrophic lateral sclerosis is provided, comprising administering to a subject a therapeutically effective amount of the above-mentioned FUS proteolysis targeting chimera, transferrin receptor-based nucleic acid aptamer DNA nanoflower or drug.

[0090] The subject refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, and most preferably a human. The "therapeutically effective amount" refers to the amount of active compounds or agents, including the compounds of the present invention, which can cause the biological or medical response of the tissue system, animal or human sought by the researcher, veterinarian, doctor or other medical personnel, including the alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It must be recognized that the optimal dosage and interval of the active ingredients of the present invention are determined by their properties and external conditions such as the form, route and site of administration and the specific mammal being treated, and this optimal dosage can be determined using conventional techniques. It must also be recognized that the optimal course of treatment, that is, the daily dosage of the compound over a specified period of time, can be determined by methods known in the art.

[0091] The present invention is further described below in conjunction with specific examples, which are only for the purpose of explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the sales company; unless otherwise specified in the present invention, they can all be purchased through commercial channels.

[0092] Example 1: Synthesis of chimera molecules

[0093] (1) Design and synthesize RNA ligands with azide modification at the 5' end. The N3 azide modification is connected to the 5' end of the RNA ligand through the phosphoramidite monomer of the C6 amino group. The nucleotide sequences of the azide-modified RNA ligands (i.e., N3-FUS-RNA#1 to N3-FUS-RNA#5) are shown in SEQ ID NOs: 1-5. The nucleotide sequence of the control RNA ligand (N3-FUS-RNA#NC) is shown in SEQ ID NO: 6. The mass spectrometry results of the RNA ligands with different azide modifications are shown in Figures 1 to 6 shown.

[0094] (2) PROTAC ligation reaction, specifically: connecting the azide-modified RNA aptamer to the alkyne-containing CRBN ligand or VHL ligand The mixture was mixed at a molar ratio of 1:5 and incubated at 37°C for 5 hours. The excess alkynyl-containing CRBN ligand or VHL ligand was then removed by ultrafiltration with a molecular weight cutoff of 3K. The efficiency of the ligation reaction was finally detected by 20% non-denaturing SDS-PAGE gel. The results are shown in Figure 7 As shown in Figure B, dFUS-C1 migrates slower than FUS-1, indicating that the two are successfully connected. Figure 7 N3-FUS-RNA#1 shown in A is Figure 7 FUS-1 shown in B, and so on; dFUS-C1 represents the PROTAC obtained by coupling FUS-1 with CRBN ligand, dFUS-V1 represents the PROTAC obtained by coupling FUS-1 with VHL ligand, and so on, a total of 10 PROTACs are obtained.

[0095] Example 2: Degradation performance test of 10 PROTACs on FUS protein in PC12 cells

[0096] Ten PROTACs were transfected into PC12 cells via liposomes at a final concentration of 125 nM. The cells were lysed after 24 hours, and the changes in FUS protein were detected by Western blot. Figure 8 Figures A to B show the degradation of FUS in PC12 cells by different PROTACs. dFUS-C1 can stably and efficiently degrade FUS protein, so dFUS-C1 was selected as PROTAC for subsequent experiments.

[0097] Example 3: Degradation performance test of dFUS-C1 on FUS protein

[0098] dFUS-C1 solutions with concentration gradients of 9.4 nM, 18.7 nM, 37.5 nM, 62.5 nM, and 125 nM were prepared and transfected into PC12 or SH-SY5Y cells via liposomes. The cells were lysed after 24 hours, and the changes of FUS protein were detected by Western blot. Fig. 9 A to B in the figure are the molecular docking diagrams of dFUS-C1, FUS and CRBN, and the specific amino acid sites where dFUS-C1 binds to FUS and CRBN. The above results indicate that FUS-dFUS-C1-CRBN can form a stable ternary complex. Fig.10 Figures A to B show the degradation of FUS in PC12 and SH-SY5Y cells by dFUS-C1, indicating that dFUS-C1 can efficiently degrade FUS protein in different cell lines. Fig.10 The proteomic results of medium C showed that dFUS-C1 had a specific degradation effect on FUS protein.

[0099] Example 4: Degradation performance test of dFUS-C1 on mutant FUS protein

[0100] dFUS-C1 solutions with concentration gradients of 37.5 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1000 nM, and 2000 nM were prepared and transfected into 293T cells expressing different mutant FUS via liposomes. The cells were lysed after 24 hours and the changes in FUS protein were detected by Western blot. Fig.11 A to E show the degradation of different mutant FUS by dFUS-C1 in 293T cells. 1000 nM dFUS-C1 can efficiently degrade different forms of FUS protein (FUS-WT, FUS-P525L, FUS-R521C, FUS-R521G, FUS-R521H).

[0101] Example 5: Cytotoxicity test of dFUS-C1

[0102] In this example, ROS detection kit, LDH release detection kit and CCK8 kit were used to perform cytotoxicity detection. The specific process is as follows: 5×10 3 SH-SY5Y cells were placed in a 37°C, 5% cell culture incubator for 12 hours; FUS plasmids were transfected into the wells, and the cells were placed in a 37°C, 5% cell culture incubator for 24 hours. The indicated reagents were added to each well, and the corresponding indicators were detected according to the instructions of the kit. The results are shown in Figure 7 As shown in A to C, dFUS-C1 can significantly inhibit the neurotoxicity caused by FUS overexpression.

[0103] Example 6: Serum stability test of dFUS-C1 and dFUS-PS and FUS protein degradation performance test of dFUS-PS

[0104] For serum stability test, 5 μM dFUS-C1 or dFUS-PS (the dFUS-PS is obtained by full thiolation modification of dFUS-C1, Fig.13 The samples were dissolved in DNEM medium containing 10% serum and incubated at 37°C. The samples were taken out at the indicated time points and frozen at -80°C. Then, 15% non-denaturing polyacrylamide gel electrophoresis was performed at 120 V for about 70 min. The samples were then stained with SYBR gold nucleic acid gel dye and imaged on a Bio-RAD instrument. The results are shown in the figure. Fig.14 As shown in A to B, dFUS-PS has better serum stability than dFUS-C1. For the test of dFUS-PS on FUS protein degradation performance, dFUS-PS solutions with concentration gradients of 9.4 nM, 18.7 nM, 37.5 nM, 62.5 nM, and 125 nM were prepared and transfected into SH-SY5Y cells by liposomes. After 24 hours, the cells were lysed and the changes of FUS protein were detected by Western blot. The results are shown in Fig.14 As shown in C to D, dFUS-PS can still efficiently degrade FUS protein.

[0105] Example 7: Apparent performance test of DNA nanoflowers before and after loading dFUS-PS

[0106] The steps of preparing DNA nanoflowers based on transferrin receptor nucleic acid aptamers include:

[0107] (a): A linear DNA nanoflower template and primers were synthesized using a DNA synthesizer, and the 5′ end of the nanoflower template was phosphorylated. The sequence information used in the preparation process is shown in Table 1 below.

[0108] Table 1 Sequence information

[0109]

[0110] In Table 1, the phosphorylated-modified linear DNA nanoflower template includes a sequence complementary to the nucleic acid aptamer of the transferrin receptor (the bold letters) and a sequence of the FUS proteolysis targeting chimera (the underlined letters), and the two are connected by 16 A's. The nanoflower obtained by the subsequent reaction contains both the nucleic acid aptamer sequence of the transferrin receptor and the complementary sequence of the FUS proteolysis targeting chimera.

[0111] (b): Add 5 μL T4 ligase (20U / μL) to circularize the template.

[0112] (c): 0.6 μL Phi29 enzyme (10 U / μL) and 6 μL dNTPs (10 mM) were added to the system containing circularized template (5 μM) and primer (5 μM) and reacted at 30°C for 3 h, followed by heating at 65°C for 10 min to inactivate the enzyme.

[0113] (d): Add 500 μL ddH2O to resuspend the product from the previous step and centrifuge at 12000 rpm for 10 min to obtain nanoflower precipitate.

[0114] (e):200 μL TE / Mg 2+ The above precipitate was resuspended in buffer (10 mM Tris-HCl, 12.5 mM MgCl2; pH 8), and dFUS-PS labeled with cyanine dye Cy5 (Cy5-dFUS-PS) was added to the TE / Mg containing nanoflowers. 2+ The cells were heated at 95°C for 5 min in a buffer solution (10 mM Tris-HCl, 12.5 mM MgCl2; pH 8), cooled to room temperature, centrifuged at 12,000 rpm for 10 min, and resuspended in 200 μL ddH2O. Fig.15 shown.

[0115] The spectral scanning of 620-700 nm was performed by the enzyme marker, the particle size of the nanoflowers was measured by the nanoparticle size analyzer, and the scanning electron microscope was used to take pictures. Fig.16 The apparent performance of DNA nanoflowers before and after loading Cy5-dFUS-PS, among which, Fig.16 A in the middle is a 620-700 nm fluorescence spectrum scan before and after loading PROTAC nanoflowers. Fig.16 Middle B is the particle size analysis diagram before and after loading PROTAC nanoflowers. Fig.16 Middle C is a scanning electron microscope image of the nanoflower before and after loading PROTAC. The above results show that the particle size of DNA nanoflowers slightly increases after loading Cy5-dFUS-PS and does not affect the structure of the nanoflowers.

[0116] Example 8: Test of blood-brain barrier penetration of DNA nanoflowers loaded with Cy5-dFUS-PS (NF@Cy5-dFUS-PS)

[0117] NF@Cy5-dFUS-PS was injected into BALB / C mice via the tail vein, and in vivo 3D imaging of the animals was performed at the indicated times. After imaging, the mouse brains were removed for fixation, dehydration, paraffin embedding, sectioning, and DAPI staining, and the slices were analyzed using a digital slice scanner. Fig.17 Figures A to B show how NF@Cy5-dFUS-PS penetrates the blood-brain barrier. The in vivo imaging results show that as time goes by, the Cy5-dFUS-PS signal in the brain increases.

[0118] Example 9: In vivo toxicity test of NF@dFUS-PS

[0119] NF@dFUS-PS was injected into BALB / C mice via the tail vein at a dose of 10 mg / kg. 24 hours later, the brain, heart, liver, spleen, lung, and kidney of the mice were removed for fixation, dehydration, paraffin embedding, sectioning, and HE staining. The slices were analyzed using a digital slice scanner. Fig.18 The in vivo toxicity of NF@dFUS-PS was investigated. HE staining results showed that NF@dFUS-PS had no obvious morphological damage to various organs.

[0120] Example 10: In vivo degradation test of FUS protein in mouse brain by NF@dFUS-PS

[0121] NF@dFUS-PS was injected into BALB / C mice via the tail vein at a dose of 10 mg / kg. 24 hours later, the mouse brains were removed for fixation, dehydration, paraffin embedding, sectioning, and FUS histochemical staining. The slices were analyzed using a digital slice scanner. Fig.19 The in vivo degradation of FUS protein in the mouse brain by NF@dFUS-PS was tested. The histochemical results showed that NF@dFUS-PS could efficiently degrade FUS protein in the hippocampus and cortex of the mouse brain.

[0122] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A FUS proteolysis targeting chimera, characterized in that The FUS proteolysis targeting chimera has a general structural formula: EF; E is the ligand of E3 ubiquitin ligase, and F is the nucleic acid aptamer that specifically binds to FUS protein; Wherein, the ligand of the E3 ubiquitin ligase is a CRBN ligand, and the CRBN ligand is a CRBN ligand based on thalidomide, and its structural formula is: ; The nucleotide sequence of the nucleic acid aptamer that specifically binds to the FUS protein is shown in SEQ ID NO:

1.

2. The method for preparing the FUS proteolysis targeting chimera according to claim 1, characterized in that: The preparation method comprises: A ligand containing an azide-modified single-stranded RNA and a terminal alkyne-modified E3 ubiquitin ligase was synthesized and coupled by a click reaction.

3. The preparation method according to claim 2, characterized in that: The specific reaction conditions of the click reaction coupling are: mixing the azide-modified single-stranded RNA and the terminal alkyne-modified E3 ubiquitin ligase ligand at a molar ratio of 1:1-10, incubating at 30-40°C for 1-10 hours, and obtaining the result after purification.

4. Use of the FUS proteolysis targeting chimera as claimed in claim 1 in preparing DNA nanoflowers based on transferrin receptor nucleic acid aptamers.

5. A DNA nanoflower based on transferrin receptor nucleic acid aptamer, characterized in that: The DNA nanoflower is loaded with the FUS proteolysis targeting chimera as described in claim 1.

6. The DNA nanoflower based on transferrin receptor nucleic acid aptamer as claimed in claim 5, characterized in that: The preparation method of the DNA nanoflower comprises: Adding DNA polymerase to a system containing a circularized DNA nanoflower template and primers for rolling circle amplification, and then adding the FUS protein hydrolysis targeting chimera thereto; Wherein, the nucleotide sequence of the transferrin receptor nucleic acid aptamer is shown in SEQ ID NO: 7; The circularized DNA nanoflower template comprises a nucleotide sequence complementary to a nucleic acid aptamer of a transferrin receptor and a nucleotide sequence of a FUS proteolysis targeting chimera; The primer is shown in SEQ ID NO: 9; The DNA polymerase is phi29 DNA polymerase.

7. A drug, characterized in that The active ingredient of the drug comprises the FUS proteolysis targeting chimera described in claim 1 or the transferrin receptor-based nucleic acid aptamer DNA nanoflower described in any one of claims 5-6.

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