A conditional protein targeted degradation chimera based on nucleic acid aptamers and its preparation method and application

Abstract

The present invention belongs to the field of protein targeted degradation chimera preparation and biomedicine technology, and specifically relates to a conditional protein targeted degradation chimera based on nucleic acid aptamers, and its preparation method and application. The present invention uses nucleic acid aptamers that specifically target nucleolin as ligands to develop a series of chimera degraders for nucleolin. Further, by introducing a photosensitive nucleic acid aptamer complementary chain, a chimera that conditionally degrades nucleolin is developed, thereby achieving spatiotemporal controllable degradation of nucleolin. The protein targeted degradation chimera prepared by the present invention has good degradation efficiency and anti-tumor cell effect; at the same time, the use of nucleotides as connecting molecules greatly simplifies the difficulty of synthesizing E3 ligands; in addition, regulating the chimera activity by regulating the conformation of nucleic acid aptamers provides a new method for specifically degrading pathogenic proteins in diseased tissues, greatly reducing the toxic and side effects of chimeras on normal tissues, and has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of protein targeted degradation chimera preparation and biomedicine technology, and specifically relates to a conditional protein targeted degradation chimera based on nucleic acid aptamers, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Protein targeted degradation chimeras (PROTACs) are heterobifunctional molecules that bind to target proteins and E3 ubiquitin ligases through two ligands to form a ternary complex, and then use the ubiquitin-proteasome system in the cell to specifically degrade the target protein. PROTACs can target previously "difficult to drug" proteins, such as scaffold proteins and transcription factors. However, it is difficult to find suitable small molecule compound ligands for many target proteins due to their smooth surfaces. In addition, normal tissues also express the E3 ligases and target proteins used by PROTACs, and systemic administration may lead to inevitable toxic side effects. Therefore, adding a switch to PROTACs to spatially activate PROTACs activity in diseased tissues will help eliminate adverse targeting effects in normal tissues.

[0004] Aptamers are short single-stranded oligonucleotides artificially identified through an in vitro iterative selection strategy called exponential enrichment ligand system evolution. Aptamers have many advantages, including high specificity and affinity, good stability, low immunogenicity and toxicity, rapid tissue penetration, and ease of chemical synthesis and modification, so they have been widely used in biosensor detection, clinical diagnosis and therapeutics. The unique secondary or tertiary conformation of aptamers is crucial for the binding of target proteins. In theory, all proteins can be bound by aptamers. Importantly, the higher-order structure of aptamers can be regulated by changing the pH value, chemical molecules or introducing complementary chains in the microenvironment, thus providing the possibility of regulating the interaction between aptamers and target proteins.

[0005] Nucleolin (NCL) is a multifunctional nucleolar protein that is also localized in the nucleoplasm, cytoplasm, and cell membrane. Nucleolin plays an important role in many cellular activities and has been considered a promising therapeutic target for cancer patients. However, the inventors found that there are currently no reports of PROTACs that conditionally target and degrade nucleolin. Summary of the invention

[0006] In view of the above-mentioned prior art, the inventors, after long-term technical and practical exploration, provide a conditional protein-targeted degradation chimera based on nucleic acid aptamers and its preparation method and application. The present invention uses nucleic acid aptamers that specifically target nucleolin as ligands to develop a series of chimeric degradation agents for nucleolin. Furthermore, the present invention develops a chimera that conditionally degrades nucleolin by introducing a photosensitive nucleic acid aptamer complementary chain, thereby achieving spatiotemporal controllable degradation of nucleolin. Based on the above research results, the present invention is completed.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a protein targeted degradation chimera, wherein the protein targeted degradation chimera has a general structural formula: TLE;

[0009] Wherein T is a nucleic acid aptamer that specifically binds to a target protein; L is a connecting molecule between the nucleic acid aptamer and the E3 ubiquitin ligase, and the connecting molecule may be a nucleotide; and E is a ligand of the E3 ubiquitin ligase.

[0010] Wherein, the target protein may be nucleolin protein, and the nucleotide sequence of the nucleic acid aptamer targeting nucleolin protein may be 5′-GGTGGTGGTGGTTGTGGTGGTGGTGG-3′ (SEQ ID NO.1);

[0011] The protein targeted degradation chimera may further include a complementary chain of the nucleic acid aptamer, wherein the complementary chain contains a photosensitive group, specifically a photo-cleavable spacer.

[0012] The second aspect of the present invention provides a method for preparing the above-mentioned protein targeted degradation chimera, the preparation method comprising:

[0013] S1. synthesizing azide-modified single-stranded DNA, wherein the single-stranded DNA comprises the above-mentioned linker molecule and a nucleic acid aptamer;

[0014] S2. The azide-modified single-stranded DNA is coupled to the ligand of the alkyne-modified E3 ubiquitin ligase by click reaction.

[0015] The preparation method further comprises synthesizing a nucleic acid aptamer complementary chain, and hybridizing the nucleic acid aptamer complementary chain with the product obtained in step S2 to obtain a light-activated protein targeted degradation chimera.

[0016] The third aspect of the present invention provides the use of the above-mentioned protein targeted degradation chimera in degrading nucleolin or preparing drugs for diseases related to abnormal nucleolin expression.

[0017] The diseases associated with abnormal nucleolin expression include, but are not limited to, inflammation-related diseases, viral infection-related diseases and tumors.

[0018] A fourth aspect of the present invention provides an anti-tumor drug, wherein the active ingredient of the anti-tumor drug comprises the above-mentioned protein targeted degradation chimera.

[0019] According to the present invention, when the product is a medicine, the medicine further comprises at least one inactive pharmaceutical ingredient.

[0020] A fifth aspect of the present invention provides an anti-tumor system, comprising:

[0021] i) the above protein targeted degradation chimera or the above anti-tumor drug; and,

[0022] ii) Lighting device.

[0023] Wherein, the light source emitted by the illumination device is ultraviolet light.

[0024] It should be noted that although the above technical solution is to develop a chimera that conditionally degrades nucleolin by introducing a photosensitive nucleic acid aptamer complementary chain, the conformation of the nucleic acid aptamer is not only regulated by the complementary chain, but also affected by the pH, metabolic molecules and small molecules in the environment. Therefore, based on the inventive concept of the present invention, the use of pH, metabolic molecules and small molecules to regulate the conformation of the nucleic acid aptamer, and then regulate the activity of PROTACs based on the nucleic acid aptamer, also falls within the scope of protection of this application.

[0025] Compared with the existing technical solutions, the above one or more technical solutions have the following beneficial effects:

[0026] The above technical solution uses nucleic acid aptamers that specifically target nucleolin as ligands to develop a series of chimeric nucleolin degraders. And by introducing light-sensitive nucleic acid aptamer complementary chains, a chimera that conditionally degrades nucleolin is developed, thereby achieving spatiotemporal controllable degradation of nucleolin.

[0027] It has been verified by experiments that the aptamer-based targeted chimera involved in the above technical solution has good degradation efficiency and anti-tumor cell effect, among which 500nM chimera molecules can effectively and significantly inhibit tumor proliferation, and the inhibition efficiency is about twenty times better than the anti-tumor effect of simple aptamers; at the same time, the use of nucleotides as connecting molecules greatly simplifies the difficulty of synthesizing E3 ligands, providing convenience for subsequent large-scale screening; in addition, regulating the chimera activity by regulating the conformation of aptamers provides a new method for the specific degradation of pathogenic proteins in diseased tissues, greatly reducing the toxic side effects of chimeras on normal tissues, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0029] Figure 1 This is a graph of the mass spectrometry results of the nucleic acid aptamer AS1411 and its modified forms in Example 1 of the present invention, wherein A is AS1411, B is biotin-AS1411, C is N3-AS1411#0, D is N3-AS1411#1, E is N3-AS1411#2, F is N3-AS1411#3, G is N3-AS1411#4, and H is N3-AS1411#5.

[0030] Figure 2 This is the NMR spectrum of the CRBN ligand in Example 1 of the present invention.

[0031] Figure 3 It is the electrophoresis diagram of the connection between the nucleic acid aptamer and the E3 ubiquitin ligase ligand in Example 1 of the present invention; wherein A is the electrophoresis diagram after the nucleic acid aptamer is connected to the CRBN ligand, and B is the electrophoresis diagram after the nucleic acid aptamer is connected to the VHL ligand.

[0032] Figure 4 This is an electrophoresis diagram showing successful hybridization of the dNCL chimera with the complementary chain in Example 1 of the present invention.

[0033] Figure 5 This is a fluorescence resonance energy transfer test diagram showing successful hybridization of the dNCL chimera with the complementary chain in Example 1 of the present invention.

[0034] Figure 6 The ultraviolet radiation (365nm, 5mW / cm 2 ) Fluorescence resonance energy transfer diagram after different time periods.

[0035] Figure 7 The ultraviolet radiation (365nm, 5mW / cm 2 ) Live cell fluorescence imaging of MCF-7 cells after 5 minutes.

[0036] Figure 8 Schematic diagram of the synthesis of dNCL#1 in Example 2 of the present invention.

[0037] Fig. 9 Schematic diagram of the synthesis of opto-dNCL#1 in Example 2 of the present invention.

[0038] Fig.10 This is a graph showing the results of the test of the binding ability of dNCL#1 with nucleolin in Example 2 of the present invention.

[0039] Fig.11 This is a graph showing the results of the test of the binding ability of opto-dNCL#1 with nucleolin in Example 2 of the present invention.

[0040] Fig.12 This is a graph showing the results of verifying that opto-dNCL#1 has good serum stability in Example 3 of the present invention.

[0041] Fig.13 This is a graph showing the degradation results of nucleolin protein by dNCL#1 in Example 4 of the present invention.

[0042] Fig.14 This is a graph showing the degradation results of nucleolin protein by opto-dNCL#1 in Example 4 of the present invention.

[0043] Fig.15 The dNCL#1 in Example 5 of the present invention can significantly inhibit the growth rate of breast cancer cells.

[0044] Fig.16 In Example 5 of the present invention, ultraviolet irradiation can activate the activity of opto-dNCL#1, thereby inhibiting the growth rate of breast cancer cells. 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 above, nucleolin protein has important functions in many cellular activities and has been regarded as a promising therapeutic target for cancer patients, but there are currently no reports of PROTACs targeting the degradation of nucleolin.

[0048] In view of this, the present invention uses a nucleic acid aptamer that specifically targets nucleolin as a ligand to develop a series of chimeric nucleolin degraders. And by introducing a photosensitive nucleic acid aptamer complementary chain, a chimera that conditionally degrades nucleolin is developed, thereby achieving spatiotemporal controllable degradation of nucleolin.

[0049] Specifically, in a typical embodiment of the present invention, a protein targeted degradation chimera is provided, and the protein targeted degradation chimera has a general structural formula: TLE;

[0050] Wherein T is a nucleic acid aptamer that specifically binds to a target protein; L is a connecting molecule between the nucleic acid aptamer and the E3 ubiquitin ligase, and the connecting molecule may be a nucleotide; and E is a ligand of the E3 ubiquitin ligase, and may further be a CRBN ligand or a VHL ligand.

[0051] Wherein, the target protein may be nucleolin protein, and the nucleotide sequence of the nucleic acid aptamer targeting nucleolin protein may be 5′-GGTGGTGGTGGTTGTGGTGGTGGTGG-3′ (SEQ ID NO.1);

[0052] The present invention also performs a series of modifications on the nucleic acid aptamer, and the modification can be chemical modification, which is well known to those skilled in the art, such as 2'-OMe, 2'-F or PS modification, etc., which are not specifically limited here. By performing the above chemical modifications, the serum stability and affinity of the nucleic acid aptamer are effectively improved. The chemical modification can also include biotin modification.

[0053] The linker molecule may be n deoxythymidine nucleotides (T), where n=0-5. The linker molecule may be connected to the 5' end of the nucleic acid aptamer.

[0054] To achieve the connection between the linker molecule and the ligand of the E3 ubiquitin ligase, the linker molecule (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.

[0055] In another specific embodiment of the present invention, the protein-targeted degradation chimera may also include a complementary chain of the nucleic acid aptamer, the complementary chain contains a photosensitive group, specifically a photocleavage spacer, and more specifically, the nucleotide sequence of the complementary chain may be: 5′-CCACCA / / CCACCA / / CAACCAC-3′ (SEQ ID NO.2); wherein “ / / ” may be photocleaved 1-(2-nitrophenyl)ethyl, 2-nitrobenzyl, biotin phosphoramidite, acrylic acid phosphoramidite, diethylaminocoumarin, 1-(4,5-dimethoxy-2-nitrophenyl), etc. In a specific embodiment of the present invention, the “ / / ” is 1-(2-nitrophenyl)ethyl. By introducing the complementary oligonucleotide chain (CP) of the nucleic acid aptamer containing the above-mentioned photosensitive group, the complementary chain CP hybridizes with the nucleic acid aptamer, resulting in a conformational change of the aptamer, thereby inactivating the PROTACs based on the nucleic acid aptamer. Under ultraviolet light irradiation, the complementary chain degrades, and the nucleic acid aptamer chain refolds to form active PROTACs. In this way, the present invention thus creates a platform that can spatiotemporally regulate the activity of aptamer-based PROTACs.

[0056] In another specific embodiment of the present invention, a method for preparing the above-mentioned protein targeted degradation chimera is provided, and the preparation method comprises:

[0057] S1. synthesizing azide-modified single-stranded DNA, wherein the single-stranded DNA comprises the above-mentioned linker molecule and a nucleic acid aptamer;

[0058] S2. The azide-modified single-stranded DNA is coupled to the ligand of the alkyne-modified E3 ubiquitin ligase by click reaction.

[0059] In another specific embodiment of the present invention, in the step S1, the single-stranded DNA synthesis process can be completed by a DNA synthesizer, and the linker molecule is placed between the nucleic acid aptamer and the azide group;

[0060] In another specific embodiment of the present invention, in step S2, the alkyne-modified E3 ligase ligand can specifically be an alkyne-modified CRBN ligand.

[0061] In another specific embodiment of the present invention, the preparation method further comprises synthesizing a complementary strand of a nucleic acid aptamer, and hybridizing the complementary strand of the nucleic acid aptamer with the product obtained in step S2 to obtain a light-activated protein targeted degradation chimera. The product obtained in step S2 may be purified first, and the purification step includes ultrafiltration and centrifugation, etc., which are not specifically limited here.

[0062] In another specific embodiment of the present invention, the nucleic acid aptamer complementary chain can also be synthesized using a DNA synthesizer.

[0063] In another specific embodiment of the present invention, there is provided the use of the above-mentioned protein targeted degradation chimera in degrading nucleolin or preparing drugs for diseases related to abnormal nucleolin expression.

[0064] The diseases associated with abnormal expression of nucleolin include, but are not limited to, inflammation-related diseases, viral infection-related diseases and tumors, preferably tumors. The tumors are used in the present invention as known to those skilled in the art, and include benign tumors and / or malignant tumors. Benign tumors are defined as excessive proliferation of cells that cannot form aggressive, metastatic tumors in vivo. Conversely, malignant tumors are defined as cells with multiple cell abnormalities and biochemical abnormalities that can form systemic diseases (e.g., tumor metastasis in distal organs).

[0065] In another specific embodiment of the present invention, the medicine of the present invention can be used for treating malignant tumors. Examples of malignant tumors that can be treated with the medicine of the present invention include solid tumors and hematological tumors. Solid tumors can be tumors of the breast, bladder, bone, brain, central and peripheral nervous systems, colon, endocrine glands (such as thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testicle, stomach, skin (such as melanoma), ureter, vagina and vulva. Malignant tumors include hereditary cancers, such as retinoblastoma and Wilms' tumor, etc., which are not specifically limited here.

[0066] In another specific embodiment of the present invention, an anti-tumor drug is provided, wherein the active ingredient of the anti-tumor drug comprises the above-mentioned protein targeted degradation chimera.

[0067] According to the present invention, when the product is a medicine, the medicine further comprises at least one inactive pharmaceutical ingredient.

[0068] The inactive drug ingredient may be a pharmaceutically acceptable carrier, which may be a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a conditioning agent, a surfactant, a dispersant or a defoaming agent.

[0069] The pharmaceutically acceptable carrier may also be a virus, a microcapsule, a liposome, an exosome, a nanoparticle or a polymer and any combination thereof. The delivery vehicle of the pharmaceutically acceptable carrier may be a liposome, an exosome, a biocompatible polymer, a lipoprotein, a lipopolysaccharide, an artificial viral envelope, an inorganic particle, and a bacteria or virus, a bacteriophage, a clay or plasmid vector, etc.

[0070] In another specific embodiment of the present invention, an anti-tumor system is provided, the anti-tumor system comprising:

[0071] i) the above protein targeted degradation chimera or the above anti-tumor drug; and,

[0072] ii) Lighting device.

[0073] The light source emitted by the illumination device is ultraviolet light, and the wavelength of the ultraviolet light can be 365nm. The present invention introduces the light-controlled chimera into cells expressing nucleolin protein through liposomes and exosomes, and ultraviolet light is irradiated for 5-10 minutes to induce degradation of the complementary chain. Western blot detection shows that compared with the non-irradiated group, ultraviolet light irradiation can significantly degrade the nucleolin protein in the cells.

[0074] The present invention is further described below in conjunction with specific examples. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the sales company; they are not particularly limited in the present invention and can be purchased through commercial channels. At the same time, in the following embodiments, the nucleotide sequence of the complementary chain (or complementary photocleavable oligonucleotide) is as follows: 5′-CCACCA / / CCACCA / / CAACCAC-3′; wherein, “ / / ” is 1-(2-nitrophenyl)ethyl.

[0075] Example 1: Synthesis of chimera molecules

[0076] (1) Design and synthesize a series of single-stranded DNA with 5' end modified by azide, specifically: 5'-N3-(T) n -GGTGGTGGTGGTTGTGGTGGTGGTGG-3′,(T) n It is a linker molecule, N3 azide modified through the phosphoramidite monomer of C6 amino group and (T) n Connected. Attached Figure 1 Shown are the mass spectrometry results of a series of azide-modified single-stranded DNA molecules.

[0077] (2) Synthesis of alkyne-modified CRBN ligand molecules, specifically:

[0078]

[0079] Triethylamine (TEA, 1.2mmol) is added to 3-aminopropyne (1mmol) in dichloromethane (DCM, 10mL), and stirred at 0°C. Subsequently, a DCM solution of bromoacetyl bromide is added dropwise to the reaction solution. The mixture is stirred at room temperature for at least 2 hours. Finally, the residue is washed twice with petroleum ether, and the product is purified by flash chromatography.

[0080]

[0081] 4-Hydroxyisobenzofuran (3 mmol) was mixed with 3-aminopiperidine (3 mmol) in anhydrous toluene (20 mL), and TEA (3.36 mmol) was added to the mixture and stirred at 110°C for 12 hours. The mixture was cooled to room temperature and filtered. The obtained filter cake was washed with flash chromatography (3:10) solution for 10 minutes, and then the crude product was purified by flash chromatography.

[0082]

[0083] To a suspension of K2CO3 (6 mmol) and 2-bromo-N-(prop-2-yn-1-yl)acetamide (2 mmol) dissolved in DMF (5 mL) was added 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindole-1,3-dione and reacted at room temperature. After two hours, the mixture was diluted with EtOAc, washed once with water, and then washed twice with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. Purification by column chromatography (EtOH / DCM) gave a beige solid product. Figure 2 Shown is the NMR spectrum of CRBN ligand.

[0084] (3) Generation of chimeras based on nucleic acid aptamers: Use click chemistry to connect nucleic acid aptamers and E3 ligands. Specifically, add the alkynyl-modified CRBN ligand to the azide-modified nucleic acid aptamer solution at a ratio of 3:1, react in a reaction buffer (10mM Tris-HCl, 10mM CuSO4, 10mM sodium isothiocarbate) at 37°C for 6 hours, and then remove the excess CRBN ligand through an ultrafiltration centrifuge tube (0.5mL, 3KDa). Figure 3 Shown is the polyacrylamide gel electrophoresis of a successfully connected cell.

[0085] (4) Generation of light-activated chimeras: Light-activated chimeras were prepared by hybridizing a Cy3 fluorescently labeled chimera with a complementary photocleavable oligonucleotide labeled with a BHQ2 quencher. Figure 4 The polyacrylamide gel electrophoresis diagram of successful hybridization is shown. Figure 5 is the fluorescence resonance energy transfer result after successful hybridization. Figure 6 UV irradiation (365nm, 5mW / cm 2 ) Fluorescence resonance energy transfer results after different time periods, attached Figure 7 UV irradiation (365nm, 5mW / cm 2 ) Live cell fluorescence imaging of MCF-7 cells after 5 minutes.

[0086] Example 2: Binding ability test of dNCL#1 and opto-dNCL#1 to nucleolin

[0087] HEK-293T cells were transfected with 3 μg of pcDNA3.1-GFP-nucleolin plasmid using liposomes for 24 hours, and then the cells were lysed with RIPA (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate). 2 ) 1μM biotin-modified opto-dNCL#1 for 5 minutes, incubate 1μM biotin-modified dNCL#1 or UV-irradiated opto-dNCL#1 with 50μL cell lysate at 4℃ for 3 hours, then add 20μL streptavidin-labeled magnetic beads to the solution and incubate at 4℃ for 2 hours, then centrifuge at 12000g, 4℃ for 10 minutes, discard the supernatant and wash the precipitate with PBS solution 3 times, finally add 20μL 1x SDS loading buffer to the precipitate and cook the sample at 95℃ for 5 minutes. The naming rules of dNCL#1 and opto-dNCL#1 are as follows: Figure 8 As shown in the figure, different numbers of T are used as linkers. When n=1, it is named dNCL#1. Fig. 9 As shown, dNCL#1 hybridizes with the complementary strand of the nucleic acid aptamer to form opto-dNCL#1. Furthermore, the "-C" in dNCL#1-C represents that it is modified with a CRBN ligand, and the "-V" in dNCL#1-V represents that it is modified with a VHL ligand. Fig.10 The results of the test of the binding ability of dNCL#0 to nucleolin are shown in the figure below. Fig.11 The results are the test results of the binding ability of dNCL#1 and opto-dNCL#1 with nucleolin.

[0088] Example 3: Serum stability test of opto-dNCL#1

[0089] Take 100ng of opto-dNCL#1 and dissolve it in DMEM medium containing 15% serum, incubate it at 37℃, and take out 5μL of sample at different time points, add 5uL of loading solution, first freeze it quickly with liquid nitrogen, and then freeze it at -80℃. Then perform 20% non-denaturing polyacrylamide gel electrophoresis, dissolve the previously frozen sample and load 10μL into each well. The voltage is 150V, and the electrophoresis is carried out for about 1 hour, then it is stained with SYBR gold nucleic acid gel dye, and finally placed in the Bio-RAD instrument for imaging. The results are shown in the attached figure. Fig.12 As shown, opto-dNCL#1 has good serum stability.

[0090] Example 4: Testing of the degradation performance of dNCL#1 and opto-dNCL#1 on nucleolin protein

[0091] dNCL#1 and opto-dNCL#1 solutions with concentration gradients of 62.5nM, 125nM, 250nM and 500nM were prepared and transfected into MCF-7 cells by liposomes. After incubation for 4 hours, the cells treated with opto-dNCL#1 were irradiated with UV for 5 minutes and then incubated for another 24 hours. After lysing the cells, the changes in the target nucleolin protein were detected by Western blot. Fig.13 and 14 These are the degradation results of nucleolin protein by dNCL#1 and opto-dNCL#1, respectively.

[0092] Example 5: Testing of dNCL#1 and opto-dNCL#1 on breast cancer cell proliferation

[0093] This method uses the MTT kit (Biyuntian Biotechnology, #C0009S) to detect breast cancer cell proliferation. The specific process is as follows: 1×10 4 MCF-7 cells (100 μL / well) were placed in a 37°C, 5% CO2 cell culture incubator for 12 hours; dNCL#1 and opto-dNCL#1 were transfected into the wells respectively, and the wells transfected with opto-dNCL#1 were irradiated with ultraviolet light for 5 minutes 4 hours after transfection, and then the cells were placed in a 37°C, 5% CO2 cell culture incubator for a certain period of time; 50 μL 1×MTT solution was added to each well, incubated at 37°C for 4 hours, the supernatant was aspirated, 150 μL DMSO was added to each well, and after shaking for 10 minutes, the optical density of each well was detected at a wavelength of 550 nm using an enzyme marker. The results are shown in the attached Fig.15 , dNCL#1 can significantly inhibit the growth rate of breast cancer cells; Fig.16 It shows that ultraviolet irradiation can activate the activity of opto-dNCL#1, thereby inhibiting the growth rate of breast cancer cells.

[0094] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A protein targeted degradation chimera, characterized in that: The protein targeted degradation chimera has a general structural formula: TLE; wherein T is a nucleic acid aptamer that specifically binds to a target protein; L is a connecting molecule between the nucleic acid aptamer and the E3 ubiquitin ligase, and the connecting molecule is a nucleotide; and E is a ligand of the E3 ubiquitin ligase; The target protein is nucleolin protein, and the nucleotide sequence of the nucleic acid aptamer targeting nucleolin protein is shown in SEQ ID NO.1; The ligand of the E3 ubiquitin ligase is a CRBN ligand or a VHL ligand; The nucleic acid aptamer is chemically modified, including 2'-OMe, 2'-F and PS modifications; The linking molecule is n deoxythymidine nucleotides, n=0-5; The linker molecule is connected to the 5' end of the nucleic acid aptamer; the linker molecule 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; The protein targeted degradation chimera also includes a complementary chain of the nucleic acid aptamer, the complementary chain contains a photosensitive group and is a photo-cleavable spacer, and the nucleotide sequence of the complementary chain is: 5´-CCACCA / / CCACCA / / CAACCAC-3´ (SEQID NO.2); wherein " / / " is any one of photo-cleavable 1-(2-nitrophenyl)ethyl, 2-nitrobenzyl, biotin phosphoramidite, acrylic acid phosphoramidite, diethylaminocoumarin, and 1-(4,5-dimethoxy-2-nitrophenyl).

2. The method for preparing the protein targeted degradation chimera according to claim 1, characterized in that: The preparation method comprises: S1. synthesizing azide-modified single-stranded DNA, wherein the single-stranded DNA comprises the above-mentioned linker molecule and a nucleic acid aptamer; S2. The azide-modified single-stranded DNA is coupled to the ligand of the alkyne-modified E3 ubiquitin ligase by click reaction.

3. The preparation method according to claim 2, characterized in that: The preparation method further comprises synthesizing a nucleic acid aptamer complementary chain, and hybridizing the nucleic acid aptamer complementary chain with the product obtained in step S2 to obtain a light-activated protein targeted degradation chimera.

4. Use of the protein targeted degradation chimera according to claim 1 in the preparation of drugs for diseases related to abnormal nucleolin expression, wherein the disease related to abnormal nucleolin expression is breast cancer.

5. A drug, characterized in that The active ingredient of the drug comprises the protein targeted degradation chimera according to claim 1.

6. A system, characterized in that: The anti-tumor system comprises: i) the protein targeted degradation chimera according to claim 1 or the drug according to claim 5; and, ii) Lighting device.

7. The system according to claim 6, characterized in that: The light source emitted by the illumination device is ultraviolet light.

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