CN116179554A - Conditional protein targeted degradation chimeric based on nucleic acid aptamer, and preparation method and application thereof - Google Patents

Conditional protein targeted degradation chimeric based on nucleic acid aptamer, and preparation method and application thereof Download PDF

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CN116179554A
CN116179554A CN202211530823.6A CN202211530823A CN116179554A CN 116179554 A CN116179554 A CN 116179554A CN 202211530823 A CN202211530823 A CN 202211530823A CN 116179554 A CN116179554 A CN 116179554A
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nucleic acid
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nucleolin
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周军
谢松波
刘敏
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Shandong Normal University
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Abstract

The invention belongs to the technical field of protein targeted degradation chimera preparation and biological medicine, and particularly relates to a conditional protein targeted degradation chimera based on a nucleic acid aptamer, and a preparation method and application thereof. The invention utilizes nucleic acid aptamer of specific targeting nucleolin as ligand to develop a series of nucleolin chimeric degradation agents. Further, by introducing a complementary strand of a photosensitive nucleic acid aptamer, a chimeric body for conditionally degrading nucleolin is developed, so that the time-space controllable degradation of nucleolin is realized. The protein targeted degradation chimera prepared by the invention has good degradation efficiency and anti-tumor cell effect; meanwhile, the nucleotide is used as a connecting molecule, so that the synthesis difficulty of the E3 ligand is greatly simplified; in addition, the activity of the chimera is regulated by regulating the conformation of the aptamer, so that a novel method is provided for specifically degrading pathogenic proteins in diseased tissues, the toxic and side effects of the chimera on normal tissues are greatly reduced, and the method has good practical application value.

Description

Conditional protein targeted degradation chimeric based on nucleic acid aptamer, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of protein targeted degradation chimera preparation and biological medicine, and particularly relates to a conditional protein targeted degradation chimera based on a nucleic acid aptamer, and a preparation method and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Protein targeted degradation chimeras (PROTACs) are heterobifunctional molecules that bind target proteins and E3 ubiquitin ligase, respectively, through two ligands to form a ternary complex, which in turn degrades target proteins specifically using the intracellular ubiquitin-proteasome system. The PROTACs are able to target proteins that were previously "hard to patent," such as scaffold proteins and transcription factors. However, many target proteins have difficulty finding suitable small molecule compound ligands due to surface smoothness and the like. In addition, normal tissues also express E3 ligase and target proteins for PROTACs, and systemic administration may lead to unavoidable toxic side effects. Thus, the addition of a switch to the PROTACs to spatially activate PROTACs activity in diseased tissue will help eliminate undesirable targeting effects in normal tissue.
A nucleic acid aptamer is a short single stranded oligonucleotide identified manually by an in vitro iterative selection strategy called exponential enrichment ligand systematic evolution. Nucleic acid aptamers have a number of advantages including high specificity and affinity, good stability, low immunogenicity and toxicity, rapid tissue penetration, and ease of chemical synthesis and modification, and thus they have been widely used in biosensor detection, clinical diagnosis and therapeutics. The unique secondary or tertiary conformation of the aptamer is critical for binding of the target protein, and in theory, all proteins can be bound by the aptamer. Importantly, the higher order structure of the aptamer can be modulated by changing the pH value in the microenvironment, chemical molecules or introducing complementary strands, thereby providing the possibility to modulate the interaction of the aptamer with the target protein.
Nucleolin (NCL) is a multifunctional nucleolin that also has a location on the nucleus, cytoplasm and cell membrane. Nucleolin has important functions in many cellular activities and has been considered as a promising therapeutic target for cancer patients, but the inventors have found that no PROTACs conditionally targeted to degrade nucleolin are currently reported.
Disclosure of Invention
Aiming at the prior art, the inventor provides a conditional protein targeting degradation chimeric body based on a nucleic acid aptamer, and a preparation method and application thereof through long-term technical and practical exploration. The invention utilizes nucleic acid aptamer of specific targeting nucleolin as ligand to develop a series of nucleolin chimeric degradation agents. Furthermore, the invention develops a chimeric body for conditionally degrading nucleolin by introducing a photosensitive nucleic acid aptamer complementary strand, thereby realizing the space-time controllable degradation of nucleolin. Based on the above results, the present invention has been completed.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a protein targeted degradation chimera having the structural formula: T-L-E;
wherein T is a nucleic acid aptamer that specifically binds to a target protein; l is a linker molecule between the aptamer and the E3 ubiquitin ligase, which may be a nucleotide; e is a ligand for E3 ubiquitin ligase.
Wherein the target protein can be nucleolin protein, and the nucleic acid aptamer of the target nucleolin protein can have a nucleotide sequence of 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3' (SEQ ID NO. 1);
the protein targeted degradation chimera may further comprise a complementary strand of the nucleic acid aptamer, the complementary strand comprising a photoactive group, which may be specifically a photocleavable spacer.
In a second aspect of the present invention, there is provided a method for preparing the above protein-targeted degradation chimera, the method comprising:
s1, synthesizing single-stranded DNA containing azide modification, wherein the single-stranded DNA comprises the connecting molecule and a nucleic acid aptamer;
s2, performing click reaction coupling on the azide-modified single-stranded DNA and the ligand of the alkynyl-modified E3 ubiquitin ligase.
The preparation method further comprises the steps of synthesizing a complementary strand of the nucleic acid aptamer, and hybridizing the complementary strand of the nucleic acid aptamer with the product prepared in the step S2 to obtain the light-operated activated protein targeted degradation chimera.
In a third aspect, the invention provides the use of the protein-targeted degradation chimera in degrading nucleolin or preparing a medicament for diseases associated with abnormal expression of nucleolin.
Such diseases associated with abnormal expression of nucleolin include, but are not limited to, inflammation-related diseases, viral infection-related diseases, and tumors.
In a fourth aspect of the present invention, there is provided an antitumor drug whose active ingredient comprises the protein-targeted degradation chimera described above.
According to the invention, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.
In a fifth aspect of the present invention, there is provided an anti-tumor system comprising:
i) The protein targeted degradation chimeric or the antitumor drug; the method comprises the steps of,
ii) an illumination device.
Wherein the light source emitted by the illumination device is ultraviolet light.
It should be noted that, although the above-mentioned technical solution is to develop a chimeric body for conditionally degrading nucleolin by introducing light into the complementary strand of a sensitive nucleic acid aptamer, the conformation of the nucleic acid aptamer is influenced by pH, metabolic molecules and small molecular compounds in the environment in addition to the complementary strand, so that, based on the inventive concept of the present invention, the conformation of the nucleic acid aptamer is regulated by using pH, metabolic molecules and small molecular compounds, etc., and further the activity of the protas based on the nucleic acid aptamer is regulated, which is also within the scope of protection of the present application.
Compared with the prior art, the one or more technical schemes have the following beneficial effects:
according to the technical scheme, a series of nucleolin chimera degrading agents are developed by using nucleic acid aptamer of the specific targeting nucleolin as a ligand. And by introducing a complementary strand of a photosensitive nucleic acid aptamer, a chimeric body for conditionally degrading nucleolin is developed, so that the time-space controllable degradation of nucleolin is realized.
Experiments prove that the targeting chimeric body based on the nucleic acid aptamer has good degradation efficiency and anti-tumor cell effect, wherein 500nM chimeric body molecules can effectively and obviously inhibit proliferation of tumors, and the inhibition efficiency is about twenty times better than that of a pure nucleic acid aptamer; meanwhile, the nucleotide is used as a connecting molecule, so that the synthesis difficulty of the E3 ligand is greatly simplified, and convenience is provided for subsequent large-scale screening; in addition, the activity of the chimera is regulated by regulating the conformation of the aptamer, so that a novel method is provided for specifically degrading pathogenic proteins in diseased tissues, and the toxic and side effects of the chimera on normal tissues are greatly reduced, thereby having good practical application value.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a mass spectrum of aptamer AS1411 and modified forms thereof in example 1 of the present invention, wherein A is AS1411, B is biotin-AS 1411, C is N 3 AS1411#0, D is N 3 AS1411#1, E is N 3 AS1411#2,F is N 3 AS1411#3, G is N 3 AS1411#4, H is N3-AS1411#5.
FIG. 2 is a nuclear magnetic resonance spectrum of CRBN ligand in example 1 of the present invention.
FIG. 3 is an electrophoretogram of the ligation of aptamer to E3 ubiquitin ligase ligand in example 1 according to the invention; wherein A is an electrophoresis pattern after the nucleic acid aptamer is connected with the CRBN ligand, and B is an electrophoresis pattern after the nucleic acid aptamer is connected with the VHL ligand.
FIG. 4 is an electrophoresis chart showing successful hybridization of dNCL chimeras with complementary strands in example 1 of the present invention.
FIG. 5 is a graph showing the fluorescence resonance energy transfer test of successful hybridization of dNCL chimera with complementary strand in example 1 of the present invention.
FIG. 6 shows the ultraviolet irradiation (365 nm,5 mW/cm) in example 1 of the present invention 2 ) Fluorescence resonance energy transfer patterns after different times.
FIG. 7 shows the ultraviolet irradiation (365 nm,5 mW/cm) in example 1 of the present invention 2 ) Live cell fluorescence imaging after 5 minutes of MCF-7 cells.
FIG. 8 is a schematic diagram showing dNCL#1 synthesis in example 2 of the present invention.
FIG. 9 is a schematic diagram of the synthesis of opto-dNCL#1 in example 2 of the invention.
FIG. 10 is a graph showing the results of the test for the binding capacity of dNCL#1 to nucleolin in example 2 of the present invention.
FIG. 11 is a graph showing the results of the binding ability test of opto-dNCL#1 to nucleolin in example 2 of the present invention.
FIG. 12 is a graph showing the results of verifying that opto-dNCL#1 has good serum stability in example 3 of the invention.
FIG. 13 is a graph showing the degradation results of nucleolin protein by dNCL#1 in example 4 of the present invention.
FIG. 14 is a graph showing the degradation results of nucleolin protein by opto-dNCL#1 in example 4 of the invention.
Fig. 15 shows that dncl#1 of example 5 of the present invention can significantly inhibit the growth rate of breast cancer cells.
FIG. 16 shows that ultraviolet irradiation activates the activity of opto-dNCL#1 in example 5 of the invention, thereby inhibiting the growth rate of breast cancer cells.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
As mentioned above, nucleolin proteins have important functions in many cellular activities and have been considered as a promising therapeutic target for cancer patients, but no PROTACs have been reported to target degradation of nucleolin.
In view of this, the present invention utilizes nucleic acid aptamers that specifically target nucleolin as ligands, developing a series of nucleolin chimera degradants. And a chimeric body for conditionally degrading nucleolin is developed by introducing a photosensitive nucleic acid aptamer complementary strand, so that the time-space controllable degradation of nucleolin is realized.
Specifically, in one exemplary embodiment of the present invention, a protein targeting degradation chimera is provided, and the structural general formula of the protein targeting degradation chimera is: T-L-E;
wherein T is a nucleic acid aptamer that specifically binds to a target protein; l is a linker molecule between the aptamer and the E3 ubiquitin ligase, which may be a nucleotide; e is a ligand for E3 ubiquitin ligase, further can be a CRBN ligand or a VHL ligand.
Wherein the target protein can be nucleolin protein, and the nucleic acid aptamer of the target nucleolin protein can have a nucleotide sequence of 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3' (SEQ ID NO. 1);
the nucleic acid aptamer is subjected to a series of modifications, wherein the modifications can be chemical modifications, such as 2'-OMe, 2' -F or PS modifications and the like which are well known to the person skilled in the art, and the chemical modifications are not particularly limited herein, so that the serum stability and affinity of the nucleic acid aptamer can be effectively improved, and the chemical modifications can also comprise biotin modifications.
The linker molecule may be n deoxythymidines (T), n=0-5. The linker molecule may be attached to the 5' end of the aptamer.
In order to realize the connection of the connecting molecule and the ligand of the E3 ubiquitin ligase, the connecting 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 click chemistry reaction.
In yet another embodiment of the present invention, the protein-targeted degradation chimera may further comprise a complementary strand of the nucleic acid aptamer, the complementary strand comprising a photoactive group, particularly a photocleavable spacer, more particularly a nucleotide sequence of the complementary strand may be: 5'-CCACCA// CCACCA// CAACCAC-3' (SEQ ID NO. 2); wherein "//" can be photocleavable 1- (2-nitrophenyl) ethyl, 2-nitrobenzyl, biotin phosphoramidite, phosphoramidite acrylate, diethylaminocoumarin, 1- (4, 5-dimethoxy-2-nitrophenyl) and the like, and in one embodiment of the present invention, the "//" is 1- (2-nitrophenyl) ethyl. By introducing the complementary oligonucleotide strand (CP) of the above-described nucleic acid aptamer containing a photoactive group, hybridization of the complementary strand CP with the nucleic acid aptamer results in a conformational change of the aptamer, thereby inactivating the nucleic acid aptamer-based PROTACs. Under ultraviolet light irradiation, the complementary strand degrades and the aptamer strand refoldes to form active PROTACs. Thus, in this way, the present invention creates a platform capable of spatiotemporal modulation of the activity of aptamer-based PROTACs.
In still another embodiment of the present invention, there is provided a method for preparing the above protein-targeted degradation chimera, the method comprising:
s1, synthesizing single-stranded DNA containing azide modification, wherein the single-stranded DNA comprises the connecting molecule and a nucleic acid aptamer;
s2, performing click reaction coupling on the azide-modified single-stranded DNA and the ligand of the alkynyl-modified E3 ubiquitin ligase.
In still another embodiment of the present invention, in the step S1, the above single-stranded DNA synthesis process may be performed by a DNA synthesizer, and the linker molecule is placed between the aptamer and the azide group;
in still another embodiment of the present invention, in the step S2, the alkynyl-modified E3 ligase ligand may be specifically an alkynyl-modified CRBN-containing ligand.
In another specific embodiment of the present invention, the preparation method further includes synthesizing a complementary strand of the aptamer, and hybridizing the complementary strand of the aptamer with the product obtained in step S2 to obtain the light-activated protein targeted degradation chimera. The product obtained in the step S2 may be purified in advance, and the purification step includes ultrafiltration, centrifugation, and the like, and is not particularly limited herein.
In yet another embodiment of the present invention, the complementary strand of the aptamer may likewise be synthesized using a DNA synthesizer.
In yet another embodiment of the present invention, there is provided the use of the protein-targeted degradation chimera described above for degrading nucleolin or for preparing a medicament for diseases associated with abnormal expression of nucleolin.
Such diseases associated with abnormal expression of nucleolin include, but are not limited to, diseases associated with inflammation, diseases associated with viral infection, and tumors, preferably tumors. Such tumors are used in the present invention as known to those skilled in the art, including benign tumors and/or malignant tumors. Benign tumors are defined as hyperproliferative cells that are unable to form aggressive, metastatic tumors in vivo. Conversely, a malignancy is defined as a cell with multiple cellular abnormalities and biochemical abnormalities that are capable of developing a systemic disease (e.g., tumor metastasis in a distant organ).
In yet another embodiment of the invention, the medicament of the invention is useful for the treatment of malignant tumors. Examples of malignant tumors that can be treated with the medicament of the invention include solid tumors and hematological tumors. The solid tumors may be tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (e.g., 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, testis, stomach, skin (e.g., melanoma), ureter, vagina and vulva. Malignant tumors include hereditary cancers such as retinoblastoma and nephroblastoma, and the like, and are not particularly limited herein.
In still another embodiment of the present invention, an antitumor drug is provided, the active ingredient of which comprises the protein-targeted degradation chimera described above.
According to the invention, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.
The pharmaceutically inactive ingredient may be a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a blending agent, a surfactant, a dispersing agent, or an antifoaming agent.
The pharmaceutically acceptable carrier may also be a virus, microcapsule, liposome, exosome, nanoparticle or polymer, and any combination thereof. The delivery vehicle for the pharmaceutically acceptable carrier may be a liposome, exosome, biocompatible polymer, lipoprotein, lipopolysaccharide, artificial viral envelope, inorganic particle, bacterial or viral, phage, cosmid or plasmid vector, and the like.
In yet another embodiment of the present invention, there is provided an anti-tumor system comprising:
i) The protein targeted degradation chimeric or the antitumor drug; the method comprises the steps of,
ii) an illumination device.
The light source emitted by the illumination device is ultraviolet light, and the wavelength of the ultraviolet light can be 365nm. The invention introduces the light-operated chimera into the cell expressing nucleolin protein in a liposome and exosome drug carrying mode, and ultraviolet light irradiation is carried out for 5-10 minutes to induce the degradation of complementary chains, and compared with a non-irradiated group, the ultraviolet light irradiation can obviously degrade the nucleolin protein in the cell through western blot detection.
The invention is further illustrated by the following examples, which are given for the purpose of illustration only and are not intended to be limiting. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the sales company; the present invention is not particularly limited and can be commercially available. Meanwhile, in the following examples, the nucleotide sequence of the complementary strand (or referred to as complementary photocleavable oligonucleotide) is as follows: 5'-CCACCA// caacca-3'; wherein "//" is 1- (2-nitrophenyl) ethyl.
Example 1: synthesis of chimeric molecules
(1) A series of single-stranded DNA with azide modification at the 5' end is designed and synthesized, and specifically comprises the following steps: 5' -N 3 -(T) n -GGTGGTGGTGGTTGTGGTGGTGGTGG-3′,(T) n Is a linker molecule, N 3 Azido modification of phosphoramidite monomers through the C6 amino group and (T) n Are connected. FIG. 1 shows the mass spectrum of a series of single stranded DNA molecules containing azide modifications.
(2) Synthesizing CRBN ligand molecules containing alkynyl modification, which are specifically as follows:
Figure BDA0003975822110000081
triethylamine (TEA, 1.2 mmol) was added to 3-aminopropyne (1 mmol) in dichloromethane (DCM, 10 mL) and mixed with stirring at 0deg.C. Subsequently, a solution of bromoacetyl bromide in DCM was added dropwise to the reaction solution. The mixture was stirred at room temperature for at least 2 hours. Finally, the residue was washed twice with petroleum ether and the product was purified by flash chromatography.
Figure BDA0003975822110000082
4-Hydroxyisobenzofuran (3 mmol) was mixed with 3-aminopiperidine (3 mmol) in dry toluene (20 mL) and TEA (3.36 mmol) was added to the mixture and stirred at 110℃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.
Figure BDA0003975822110000083
To K in DMF (5 mL) 2 CO 3 To a suspension of (6 mmol) and 2-bromo-N- (prop-2-yn-1-yl) acetamide (2 mmol) 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 twice with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. Purification by column chromatography (EtOH/DCM) gives a product as an off-white solid. FIG. 2 shows the nuclear magnetic pattern of CRBN ligands.
(3) Generation of chimeras based on nucleic acid aptamers: the nucleic acid aptamer and the E3 ligand are connected by utilizing click chemistry reaction, and the method specifically comprises the following steps: alkynyl-modified CRBN ligands were added to the azide-modified aptamer solution at a 3:1 ratio, reacted in reaction buffer (10 mM Tris-HCl, 10mM CuSO4, 10mM sodium isotoponate) at 37 ℃ for 6 hours, and then excess CRBN ligands were removed by ultrafiltration centrifuge tubes (0.5 ml,3 kda). FIG. 3 shows a polypropylene gel electrophoresis pattern of successful ligation.
(4) Generation of light-operated activation chimeras: optically activated chimeras were prepared by hybridizing Cy3 fluorescent-labeled chimeras to complementary photocleavable oligonucleotides labeled with BHQ2 quenchers, and FIG. 4 shows a polypropylene gel electrophoresis pattern of successful hybridization. FIG. 5 shows the result of fluorescence resonance energy transfer after successful hybridization. FIG. 6 is an illustration of ultraviolet radiation (365 nm,5 mW/cm) 2 ) The fluorescence resonance energy transfer results after various times are shown in FIG. 7, which shows ultraviolet irradiation (365 nm,5 mW/cm) 2 ) Live cell fluorescence imaging after 5 minutes of MCF-7 cells.
Example 2: dNCL#1 and opto-dNCL#1 binding Capacity test to nucleolin
After 3. Mu.g of pcDNA3.1-GFP-nucleolin plasmid was transfected with liposomes in HEK-293T cells for 24 hours, the cells were lysed with RIPA (50 mM Tris (pH 7.4), 150mM NaCl,1% NP-40,0.5%sodium deoxycholate). Irradiation with ultraviolet radiation (365 nm,5 mW/cm) 2 ) 1 μM biotin-modified opto-dNCL # 15 min, 1 μM biotin-modified dNCL #1 or UV-irradiated opto-dNCL #1 was incubated with 50 μL of cell lysate at 4deg.C for 3 hours, then 20 μL of streptomycin-labeled magnetic beads were added to the solution and incubated at 4deg.C for 2 hours, after 12000g, centrifuged at 4deg.C for 10 min, the supernatant was discarded, the pellet was washed 3 times with PBS solution, and finally 20 μL of 1 XSDS loading buffer was added to the pellet and boiled at 95deg.C for 5 min. dNCL#1 and opto-dNCL#1 naming convention is as follows: as shown in fig. 8, a different number of T's as a linker, when n=1, is named dncl#1. As shown in FIG. 9, dNCL#1 hybridizes with the complementary strand of the aptamer to form an opto-dNCL#1. Further, "-C" in dNCL#1-C represents that it is modified with a CRBN ligand, and "-V" in dNCL#1-V represents that it is modified with a VHL ligand. FIG. 10 shows the results of the binding ability test of dNCL#0 to nucleolin, and FIG. 11 shows the results of the binding ability test of dNCL#1 and opto-dNCL#1 to nucleolin.
Example 3: serum stability test of opto-dNCL#1
100ng of opto-dNCL #1 was dissolved in DMEM medium containing 15% serum, incubated at 37℃and 5. Mu.L of sample was removed at various time points and 5. Mu.L of loading solution was added, which was flash frozen with liquid nitrogen before freezing at-80 ℃. Then, 20% non-denaturing polyacrylamide gel electrophoresis was performed, and 10. Mu.L of the previously frozen sample was applied to each well after dissolution. The voltage is 150V, electrophoresis is carried out for about 1 hour, then SYBR gold nucleic acid gel dye is used for dyeing, and finally the gel is arranged on a Bio-RAD instrument for imaging, and the result is that the opto-dNCL#1 has good serum stability as shown in figure 12.
Example 4: dNCL#1 and opto-dNCL#1 test for degradation of nucleolin protein
dNCL#1 and opto-dNCL#1 solutions were prepared at concentration gradients of 62.5nM, 125nM, 250nM and 500nM, and were transfected into MCF-7 cells by liposome, respectively, after 4 hours of incubation, the opto-dNCL#1 treated cells were irradiated with ultraviolet light for 5 minutes, and then incubation was continued for 24 hours. After cell lysis, the change of the target nucleolin protein is detected by Western blot. FIGS. 13 and 14 are the degradation results of nucleolin protein by dNCL#1 and opto-dNCL#1, respectively.
Example 5: test of proliferation of breast cancer cells by dNCL#1 and opto-dNCL#1
The method utilizes an MTT kit (Biyunshan, #C0009S) to detect breast cancer cell proliferation, and the specific process is as follows: 1X 10 was added to a 96-well plate 4 MCF-7 cells (100. Mu.L/well) were placed at 37℃in 5% CO 2 Culturing in a cell incubator for 12 hours; dNCL#1 and opto-dNCL#1 were separately transfected into wells, wherein 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 at 37℃with 5% CO 2 Culturing in a cell incubator for a certain time; 50. Mu.L of 1 XMTT solution was added to each well, incubated at 37℃for 4 hours, the supernatant was aspirated, 150. Mu.L of DMSO was added to each well, and after shaking for 10 minutes, the optical density of each well was measured at 550nm using an ELISA reader. As a result, as shown in fig. 15, dncl#1 can significantly inhibit the growth rate of breast cancer cells; FIG. 16 shows that ultraviolet irradiation activates the activity of opto-dNCL#1, thereby inhibiting the growth rate of breast cancer cells.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims (10)

1. The protein targeting degradation chimeric body is characterized by having a structural general formula as follows: T-L-E; wherein T is a nucleic acid aptamer that specifically binds to a target protein; l is a connecting molecule between the aptamer and the E3 ubiquitin ligase, wherein the connecting molecule is a nucleotide; e is a ligand for E3 ubiquitin ligase.
2. The protein targeted degradation chimera according to claim 1, wherein the target protein is nucleolin protein, and the nucleic acid aptamer of the targeted nucleolin protein has a nucleotide sequence shown in SEQ ID NO. 1;
the ligand of the E3 ubiquitin ligase is CRBN ligand or VHL ligand.
3. The protein targeted degradation chimera of claim 1, wherein the nucleic acid aptamer is chemically modified, including 2'-OMe, 2' -F, and PS modifications;
the connecting molecules are n deoxythymidines, and n=0-5;
the connecting molecule is connected with the 5' end of the nucleic acid aptamer; furthermore, the connecting 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 click chemistry reaction.
4. The protein targeted degradation chimera of claim 1, further comprising a complementary strand of the nucleic acid aptamer, the complementary strand comprising a photoactive group, specifically a photocleavable spacer, more specifically a nucleotide sequence of the complementary strand: 5'-CCACCA// CCACCA// CAACCAC-3' (SEQ ID NO. 2); wherein "//" is any one of photocleavable 1- (2-nitrophenyl) ethyl, 2-nitrobenzyl, biotin phosphoramidite, acrylic phosphoramidite, diethylaminocoumarin and 1- (4, 5-dimethoxy-2-nitrophenyl).
5. The method for preparing the protein targeted degradation chimera according to any one of claims 1-4, wherein the method comprises the following steps:
s1, synthesizing single-stranded DNA containing azide modification, wherein the single-stranded DNA comprises the connecting molecule and a nucleic acid aptamer;
s2, performing click reaction coupling on the azide-modified single-stranded DNA and the ligand of the alkynyl-modified E3 ubiquitin ligase.
6. The method of claim 5, further comprising synthesizing a complementary strand of a nucleic acid aptamer, and hybridizing the complementary strand of a nucleic acid aptamer to the product obtained in step S2 to obtain the light-activated protein-targeted degradation chimera.
7. Use of the protein-targeted degradation chimera according to any one of claims 1-4 for degrading nucleolin or for the preparation of a medicament for diseases associated with abnormal expression of nucleolin;
further, the diseases associated with abnormal expression of nucleolin include inflammation-related diseases, viral infection-related diseases and tumors.
8. A medicament, characterized in that the active ingredient of the medicament comprises the protein targeted degradation chimera according to any one of claims 1-4.
9. A system, wherein the anti-tumor system comprises:
i) A protein targeted degradation chimera according to any one of claims 1-4 or a medicament according to claim 8; the method comprises the steps of,
ii) an illumination device.
10. The system of claim 9, wherein the light source emitted by the illumination device is ultraviolet light.
CN202211530823.6A 2022-12-01 2022-12-01 Conditional protein targeted degradation chimeric based on nucleic acid aptamer, and preparation method and application thereof Pending CN116179554A (en)

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