Bispecific nucleic acid aptamer based on LDLR lysosome shuttle pathway and preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a bispecific nucleic acid aptamer based on an LDLR lysosome shuttle path and a preparation method and application of the bispecific nucleic acid aptamer. The bispecific nucleic acid aptamer comprises a first sequence and a second sequence, the first sequence is composed of a first nucleic acid aptamer and a first connecting part, the second sequence is composed of a second nucleic acid aptamer and a second connecting part, and the first connecting part and the second connecting part are connected to form a connecting structure; the first nucleic acid aptamer specifically recognizes and is combined with a low-density lipoprotein receptor, and the second nucleic acid aptamer specifically recognizes and is combined with a target protein. The bispecific aptamer disclosed by the invention has bidirectional targeting accuracy, and can be combined with a target and a lysosome shuttle receptor on the surface of a cell at the same time, so that the specificity and efficient degradation of target protein are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a bispecific nucleic acid aptamer based on the LDLR lysosomal shuttle pathway, its preparation method, and its application. Background Technology

[0002] Membrane proteins are among the most important components of cells, playing a crucial role in cellular life processes. Many membrane proteins act as transport proteins, participating in the transport of substances between the cell and the extracellular space, such as carrier proteins and channel proteins; membrane receptor proteins recognize extracellular signaling molecules (such as hormones and neurotransmitters) and transmit these signals into the cell, triggering a series of physiological and biochemical reactions within the cell; some membrane proteins participate in maintaining cell morphology and structural stability, such as cytoskeleton-related membrane proteins; membrane proteins on the surface of immune cells (such as major histocompatibility complex proteins, MHC) play a key role in immune recognition. They can recognize foreign pathogens or antigens on the surface of abnormal cells in the body and initiate an immune response, protecting the body from pathogens; some membrane proteins possess enzymatic activity, catalyzing chemical reactions inside and outside the cell. Therefore, abnormalities in membrane proteins may lead to various diseases, and targeted degradation of membrane proteins is of great significance for the treatment of many diseases. Currently, a common approach to treating membrane proteins is protein degradation.

[0003] There are two main targeted protein degradation pathways in eukaryotic cells: the ubiquitin-proteasome pathway and the lysosomal pathway. The ubiquitin-proteasome system degrades target proteins by tagging them with ubiquitin; this pathway can eliminate short-lived proteins and soluble misfolded proteins. Lysosomes, on the other hand, are responsible for degrading long-lived proteins, insoluble protein aggregates, macromolecules, and even entire organelles through endocytosis, autophagy, and phagocytosis. Utilizing these two intracellular protein degradation pathways, targeted protein degradation technologies have been developed, primarily including the ubiquitin-proteasome-dependent proteolytic-targeting chimera (PROTAC) technology and the lysosomal-dependent lysosomal-targeting chimera (LYTAC) technology.

[0004] PROTAC molecules consist of three parts: a ligand that binds to the target protein, a ligand that binds to the E3 ubiquitin ligase, and a linker connecting the two. Like a "molecular glue," it brings the target protein and the E3 ubiquitin ligase closer, allowing the target protein to be labeled with ubiquitin, and subsequently recognized and degraded by the proteasome. However, many PROTAC molecules, due to their large molecular weight and relatively complex structure, have poor cell permeability, making it difficult to enter the cell and exert their effects, thus limiting their application in the degradation of intracellular proteins. Furthermore, optimizing drug properties by simultaneously considering the binding affinity to the target protein and the E3 ubiquitin ligase, as well as the appropriate length and physicochemical properties of the linker, is challenging. Finding a suitable E3 ubiquitin ligase ligand is also not easy; different target proteins may require specific E3 ligases for efficient degradation, making the development process complex. LYTACs, on the other hand, consist of a ligand that binds to the target protein, a part that binds to the lysosomal target receptor, and a linker. It can transport proteins from the cell surface or extracellular space into lysosomes for degradation via endocytosis, thereby expanding the range of degradable proteins and providing a new strategy for the degradation of membrane proteins. However, LYTACs have a complex structure, and their synthesis involves the connection and modification of multiple functional modules, requiring complex chemical synthesis processes. This results in high synthesis costs and limited yields, hindering large-scale research and application. Moreover, in applications such as tumor therapy, LYTACs have difficulty effectively penetrating into tumor tissues, making it difficult to fully contact and act on target proteins expressed by tumor cells, thus affecting their degradation of tumor-related proteins and therapeutic efficacy. Therefore, there is an urgent need to develop a technology with high specificity and protein degradation efficiency suitable for membrane protein degradation. Summary of the Invention

[0005] In view of the problems and shortcomings of the existing technology, the purpose of this invention is to provide a bispecific nucleic acid aptamer based on the LDLR lysosomal shuttle pathway, its preparation method and application.

[0006] To achieve the objectives of this invention, the technical solution adopted is as follows: The first aspect of the present invention provides a dual-specific nucleic acid aptamer based on the LDLR lysosomal shuttle pathway, comprising a first sequence and a second sequence, wherein the first sequence is composed of a first nucleic acid aptamer and a first linker, and the second sequence is composed of a second nucleic acid aptamer and a second linker, wherein the first linker and the second linker are connected to form a linker structure; the first nucleic acid aptamer specifically recognizes and binds to a low-density lipoprotein receptor, and the second nucleic acid aptamer specifically recognizes and binds to a target protein.

[0007] According to the above-described bispecific nucleic acid aptamer, preferably, the nucleotide sequence of the first nucleic acid aptamer is as follows: 5'-GGACAGGACCACACCCAGCGCGGTCGGCGGGTGGGCGGGGGAGAACGAGGTAGGGGTCAGGCTCCTGTGTGTCGCTTTGT-3' (as in sequence 1 of the sequence listing).

[0008] According to the above-mentioned bispecific nucleic acid aptamer, preferably, the target protein is a cell surface protein or an extracellular protein.

[0009] According to the above-mentioned bispecific nucleic acid aptamer, preferably, the target protein is protein tyrosine kinase 7 (PTK7), mesenchymal-epidermal transition factor (c-Met), PD-L1, epithelial cell adhesion molecule (EpCAM), nucleolin, mucin 1 (MUC1), human epidermal growth factor receptor 2 (HER2), or vascular endothelial growth factor (VEGF).

[0010] According to the above-mentioned bispecific nucleic acid aptamer, preferably, the target protein is protein tyrosine kinase 7 (PTK7), and the nucleotide sequence of the second nucleic acid aptamer is: 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3' (as shown in sequence 2 in the sequence listing).

[0011] According to the above-described bispecific nucleic acid aptamer, preferably, the first linker and the second linker are connected by chemical cross-linking through complementary base pairing or non-complementary base pairing. More preferably, the first linker and the second linker are connected through complementary base pairing; furthermore, the nucleotide sequence of the first linker is AAAAAAAAAAAA (as shown in Sequence 3 in the sequence listing); the nucleotide sequence of the second linker is TTTTTTTTTTT (as shown in Sequence 4 in the sequence listing).

[0012] The second aspect of the present invention provides a method for preparing the bispecific nucleic acid aptamer described in the first aspect above. The preparation method specifically involves connecting a first linker portion of a first sequence with a second linker portion of a second sequence to form a linker portion, thereby obtaining the bispecific nucleic acid aptamer.

[0013] A third aspect of the present invention provides the use of the bispecific nucleic acid aptamer described in the first aspect above in any of the following: (A) Applications in protein degradation; (B) Application in the preparation of protein degrading agents; (C) Application in the preparation of drugs that inhibit tumor cell migration; (D) Use in the preparation of medicines for the treatment of tumors, immune diseases, inflammatory diseases or age-related diseases.

[0014] According to the above applications, preferably, the protein is protein tyrosine kinase 7 (PTK7), mesenchymal-epidermal transition factor (c-Met), PD-L1, epithelial cell adhesion molecule (EpCAM), nucleolin, mucin 1 (MUC1), human epidermal growth factor receptor 2 (HER2), or vascular endothelial growth factor (VEGF).

[0015] According to the above applications, preferably, the tumors include cervical cancer and esophageal cancer; the immune diseases include systemic lupus erythematosus and rheumatoid arthritis; the inflammatory diseases include ankylosing spondylitis; and the aging diseases include Alzheimer's disease and Parkinson's disease.

[0016] A third aspect of the present invention provides a medicament, preferably comprising the bispecific nucleic acid aptamer described in the first aspect and a pharmaceutically acceptable carrier.

[0017] Compared with the prior art, the positive and beneficial effects achieved by the present invention are as follows: (1) In this invention, a nucleic acid aptamer targeting a target protein and a nucleic acid aptamer targeting a low-density lipoprotein receptor are linked through base complementary pairing to obtain a bispecific nucleic acid aptamer. One end of the bispecific nucleic acid aptamer targets the low-density lipoprotein receptor, and the other end targets a specific target protein. When the two ends of the bispecific nucleic acid aptamer bind to the LDLR and the target protein, respectively, LDLR-mediated endocytosis is triggered, thereby transporting the complex to the lysosome to achieve target protein degradation (see schematic diagram of degradation principle as shown in the figure). Figure 1 (As shown), while LDLR returns to the cell membrane to continue its function. Therefore, the bispecific nucleic acid aptamer of this invention has bidirectional targeting precision, and can simultaneously bind to the target and the lysosomal shuttle receptor on the cell surface, achieving specific and efficient degradation of the target protein.

[0018] (2) The method for preparing bispecific nucleic acid aptamers of the present invention is simple, low in cost and easy to implement; moreover, it has high immunogenicity, small molecular weight and good cell membrane permeability.

[0019] (3) Immunogenicity is an important consideration in tumor treatment. Many exogenous therapeutic proteins and antibodies may trigger the body's immune response, affecting the treatment effect and even bringing additional health risks. Bispecific nucleic acid aptamers are mainly composed of nucleic acids. Their structure is relatively simple and has certain similarities with the human body's own nucleic acid components. After entering the body, they are less likely to trigger immune rejection and can more safely and stably degrade target proteins, thereby achieving the purpose of treatment.

[0020] (4) The bispecific nucleic acid aptamer of the present invention utilizes the lysosomal shuttle receptor LDLR to degrade pathogenic proteins via the endocytosis-lysosomal pathway, which is a novel therapeutic approach. Most traditional drugs work by inhibiting protein function or blocking related signaling pathways, while the bispecific nucleic acid aptamer reduces the amount of pathogenic proteins at the source, allowing them to directly enter the cell for degradation. This quantitative regulation mechanism breaks the conventional functional inhibition pattern and provides a new direction for the intervention of various disease-related mechanisms.

[0021] (5) The bispecific nucleic acid aptamer of the present invention effectively inhibits tumor migration by degrading related proteins, and different aptamer adapters can be replaced for different diseases to achieve precision treatment. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the principle of target protein degradation achieved by the bispecific nucleic acid aptamer of the present invention; Figure 2 This is a schematic diagram of the structure of a bispecific nucleic acid aptamer used to degrade PTK7; Figure 3 The results are for non-denaturing polyacrylamide gel electrophoresis of LDLR-Sgc8c; lane 1 is Sgc8c-12T, lane 2 is LDLR-12A, lane 3 is Control, lane 4 is LDLR-Sgc8c, lane 5 is LDLR-Control, lane 6 is Sgc8c-12T, lane 7 is LDLR-12A, and lane 8 is Control. Figure 4 The results show the toxicity assays of different concentrations of aptamers on HeLa cells; where A represents the cell viability assay results after 48 h of treatment with different concentrations of LDLR-12A on HeLa cells, B represents the cell viability assay results after 48 h of treatment with different concentrations of Sgc8c-12T on HeLa cells, and C represents the cell viability assay results after 48 h of treatment with different concentrations of the bispecific aptamer LDLR-Sgc8c on HeLa cells. Figure 5 The results show the binding ability of the bispecific nucleic acid aptamer LDLR-Sgc8c to PTK7 on the surface of HeLa cells. Among them, A is the flow cytometry analysis of the binding ability of LDLR-12A to HeLa cells, B is the flow cytometry statistical graph of LDLR-12A binding, C is the confocal characterization graph of the binding ability of LDLR-12A to HeLa cells, D is the flow cytometry analysis of the binding ability of Sgc8c-12T to HeLa cells, E is the flow cytometry statistical graph of Sgc8c-12T binding, and F is the confocal characterization graph of the binding ability of Sgc8c-12T to HeLa cells. Figure 6This study validates the endocytosis performance of bispecific nucleic acid aptamers. Figure A shows the flow cytometry results of the endocytosis effect of the nucleic acid aptamers (LDLR-Sgc8c, LDLR-Control, Sgc8c, Control). The left figure shows the flow cytometry peak diagram of the endocytosis effect, and the right figure shows the fluorescence intensity statistics of the flow cytometry peak of the endocytosis effect. Figure B shows the confocal characterization results of the endocytosis. Figure 7 To investigate the endocytosis pathway of nucleic acid aptamers, the following experiments were conducted: A represents the flow cytometry results of Sgc8c-12T endocytosis inhibition, with the left image showing the peak data and the right image showing the fluorescence intensity statistics of the Sgc8c-12T endocytosis inhibition peaks; B represents the flow cytometry results of LDLR-12A endocytosis inhibition, with the left image showing the peak data and the right image showing the fluorescence intensity statistics of the LDLR-12A endocytosis inhibition peaks; C represents the flow cytometry results of LDLR-Sgc8c endocytosis inhibition, with the left image showing the peak data and the right image showing the fluorescence intensity statistics of the LDLR-Sgc8c endocytosis inhibition peaks. Figure 8 The following are the characterization results of the degradation of the target protein PTK7; where A is the Western Bolt detection band diagram of the concentration gradient degradation of the target protein PTK7, B is the statistical graph of the quantitative detection results of the concentration gradient degradation of the target protein PTK7, C is the Western Bolt detection band diagram of the degradation control of the target protein PTK7, and D is the statistical characterization graph of the immunofluorescence detection results of the degradation of the target protein PTK7. Figure 9 The results are experimental findings of the bispecific nucleic acid aptamer degradation pathway (CQ: lysosomal degradation pathway inhibitor). Figure 10 The results show the experimental findings of LDLR-Sgc8c inhibiting cell migration by degrading PTK7 protein; Figure A shows the results of HeLa cell scratch assay characterizing cell migration rate, and Figure B shows the results of Transwell assay characterizing HeLa cell migration rate. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below.

[0024] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. The following embodiments are intended to facilitate a better understanding of the present invention, but do not limit the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores. The quantitative experiments in the following embodiments were all performed in triplicate, and the results were averaged.

[0025] Example 1: Preparation of a bispecific nucleic acid aptamer for degrading protein tyrosine kinase 7 (PTK7) A dual-specific nucleic acid aptamer for degrading protein tyrosine kinase 7 (PTK7) (e.g.) Figure 2 As shown in the sequence listing), the first sequence (denoted as LDLR-12A) is prepared by chemical cross-linking of the first sequence and the second sequence through base complementary pairing or non-base complementary pairing. The first sequence (denoted as LDLR-12A) consists of a first linker and a first nucleic acid aptamer that specifically recognizes and binds to the low-density lipoprotein receptor. The nucleotide sequence of the first nucleic acid aptamer (denoted as LDLR) is: 5'-GGACAGGACCACACCCAGCGCGGTCGGCGGGTGGGCGGGGGAGAACGAGGTAGGGTCAGGCTCCTGTGTGTCGCTTTGT-3' (as shown in Sequence 1 of the sequence listing). The second sequence (denoted as Sgc8c-12T) consists of a second linker and a second nucleic acid aptamer that specifically recognizes and binds to PTK7. The nucleotide sequence of the second nucleic acid aptamer (denoted as Sgc8c) is: 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3' (as shown in Sequence 2 of the sequence listing). The first linker and the second linker are connected by chemical cross-linking through complementary base pairing or non-complementary base pairing to form a linker structure; preferably, the first linker and the second linker are connected by complementary base pairing to form a linker structure, the nucleotide sequence of the first linker is AAAAAAAAAAAA (as shown in sequence 3 in the sequence listing), and the nucleotide sequence of the second linker is TTTTTTTTTTTT (as shown in sequence 4 in the sequence listing).

[0026] The preparation method of the above-mentioned bispecific nucleic acid aptamer for degrading PTK7 is as follows: the first sequence and the second sequence are added to triple-distilled water at a molar ratio of 1:1, mixed evenly, reacted at 95°C for 10 min, and then quenched on ice to obtain the bispecific nucleic acid aptamer for degrading PTK7.

[0027] Example 2: Characterization and functional verification of the bispecific nucleic acid aptamer for PTK7 degradation prepared in Example 1 (hereinafter referred to as LDLR-Sgc8c). 1. Non-denaturing polyacrylamide gel electrophoresis detection of LDLR-Sgc8c The LDLR-Sgc8c prepared in Example 1 was subjected to non-denaturing polyacrylamide gel electrophoresis (ddH2O 5.3 ml, 10×TAE 1 ml, 30%AB 3.3 ml, 10%APS 73 μl, TEMED 7.3 μl) (detection conditions: 60V pre-run for 5 min, 110V run for 60 min) to verify the cross-linking of the first and second sequences used to prepare LDLR-Sgc8c; Control (the nucleotide sequence of Control is: 5'-TTTTTTTTTTTTCGCCGGGAAAATACTGTACGGTTAGA 3') was used as a negative control sequence, as Control did not bind to the target protein; LDLR-12A-Control (LDLR-12A cross-links with Control through AT complementary pairing to obtain LDLR-12A-Control) was used as a negative control bispecific aptamer. The detection results are as follows: Figure 3 As shown.

[0028] Depend on Figure 3 It can be seen that the two nucleic acid aptamers (LDLR-12A and Sgc8c-12T) can successfully cross-link.

[0029] 2. Detection of LDLR-Sgc8c cytotoxicity 8,000 HeLa cells were seeded in each well of a 96-well plate. The next day, the old culture medium was discarded, and the cells were washed three times with DPBS. Then, a concentration gradient of samples (LDLR-12A, Sgc8c-12T, LDLR-Sgc8c) diluted in DMEM, 10% FBS, and 1% PS was added. The cells were incubated at 37 °C for 48 h. The old culture medium was then discarded, and CCK-8 reagent (diluted 10-fold) was added. The cells were incubated at 37 °C for approximately 15 min, and the absorbance at 450 nm was measured. The results are as follows: Figure 4 As shown.

[0030] Depend on Figure 4 It can be seen that after treatment of HeLa cells with nucleic acid aptamers LDLR-12A, Sgc8c-12T and LDLR-Sgc8c for 48 h, the cell viability of HeLa cells was hardly affected, that is, the nucleic acid aptamers have almost no toxicity to cells.

[0031] 3. Study on the binding ability of LDLR-Sgc8c to PTK7 protein 24-well plates were seeded with 80,000 HeLa cells per well. The next day, the old culture medium was discarded, and the cells were washed three times with DPBS. 2 ml of 2% EDTA was added for digestion for 2 min, followed by centrifugation at 4000 rpm for 5 min. After washing three times with DPBS, the cells were incubated with FAM-modified strands (LDLR-12A, Control, Sgc8c-12T) (purchased from Sangon Biotech) (250 nM) in binding buffer. After binding on ice for 30 min, the cells were centrifuged at 4000 rpm for 5 min, washed three times with DPBS, and then analyzed by flow cytometry. The Control sequence (5'-TTTTTTTTTTTTCGCCGGGAAAATACTGTACGGTTAGA 3') was used as a negative control.

[0032] Confocal dishes were prepared, with 150,000 HeLa cells per well. On the second day, after washing three times with DPBS, FAM-modified strands (Control, Sgc8c-12T, LDLR-12A) (250 nM) were added and incubated in 500 μl binding buffer. After binding on ice for 30 min, the cells were washed three times with DPBS and stained with Hoechst (diluted in 500 μl DMEM, 10% FBS, 1% PS, 37 ℃, 15 min). After staining, the cells were washed three times with DPBS and then 1 ml of phenol red-free 1640 medium was added for imaging. The Control sequence (nucleotide sequence: 5'-TTTTTTTTTTTTCGCCGGGAAAATACTGTACGGTTAGA3') served as a negative control. The detection results are as follows: Figure 5 As shown.

[0033] Depend on Figure 5 It can be seen that, compared with the control aptamer, the fluorescence signal on HeLa cells treated with LDLR-12A and Sgc8c-12T is stronger, indicating that the two aptamers (LDLR-12A and Sgc8c-12T) can bind well to HeLa cells.

[0034] 4. Study on the endocytosis effect of LDLR-Sgc8c 24-well plates were seeded with 150,000 HeLa cells per well. The next day, the old culture medium was discarded, and the cells were washed three times with DPBS. Cy5-modified aptamers (purchased from Sangon Biotech) (250 nM, diluted in DMEM, 10% FBS, 1% PS) were added and incubated at 37°C for 4 h. The culture medium was then washed away, followed by three washes with DPBS. After trypsin digestion and centrifugation, the cells were resuspended in DMEM, 10% FBS, and 1% PS to terminate digestion. The cells were then centrifuged at 4000 rpm for 5 min, resuspended in DPBS, and analyzed by flow cytometry. Control (nucleotide sequence: 5'-TTTTTTTTTTTTCGCCGGGAAAATACTGTACGGTTAGA 3') was used as a negative control sequence, as Control does not bind to the target protein. LDLR-12A-Control (LDLR-12A cross-links with Control via AT complementary pairing to obtain LDLR-12A-Control) was used as a bispecific negative control aptamer.

[0035] Confocal dishes were prepared with 150,000 HeLa cells per well. On the second day, the cells were washed three times with DPBS and then incubated with Cy5-modified nucleic acid aptamers (LDLR-Sgc8c, LDLR-Control, Sgc8c-12T, Control, 250 nM) at 37 °C for 30 min in DMEM medium, 10% FBS, and 1% PS. After washing three times with DPBS, lysosomes were stained with lyso-tracker (37 °C, 30 min, DMEM incubation). After washing three times with DPBS, nuclei were stained with Hoechst (37 °C, 15 min, DMEM medium, 10% FBS, 1% PS incubation). After washing three times with DPBS, 1 ml of phenol red-free 1640 medium was added for imaging. Meanwhile, Control (with the nucleotide sequence: 5'-TTTTTTTTTTTTCGCCGGGAAAATACTGTACGGTTAGA 3') was used as a negative control sequence, as Control does not bind to the target protein; LDLR-12A-Control (LDLR-12A crosslinks with Control via AT complementary pairing to obtain LDLR-12A-Control) was used as a negative control bispecific aptamer. Results are as follows... Figure 6 As shown.

[0036] Depend on Figure 6 It can be seen that the bispecific nucleic acid aptamer LDLR-Sgc8c has good cell internalization ability. At the same time, it can be seen that the control nucleic acid aptamer Control has enhanced internalization ability after cross-linking with LDLR, indicating that LDLR-12A can bring the cross-linked nucleic acid aptamer into the cell through LDLR-mediated endocytosis.

[0037] 5. Study on the endocytosis pathway of LDLR-Sgc8c 24-well plates were seeded with 150,000 HeLa cells per well. The next day, the old culture medium was discarded, and the cells were washed three times with DPBS. Fresh culture medium (DMEM, 10% FBS, 1% PS) was added. Pretreatment with different inhibitors (CPZ 30 μM, EIPA 100 μM, PolyI (40 μg / ml), Filipin 4 μM) was performed at 37°C for 2 h. The culture medium was then discarded, and the cells were washed three times with DPBS. Cy5-modified aptamers (Sgc8c-12T, LDLR-Sgc8c, LDLR-12A, 250 nM) were added and incubated at 37°C for 4 h. The cells were washed three times with DPBS, and trypsin was added. Digestion was performed at room temperature for 3 min, followed by centrifugation at 4000 rpm for 5 min. The cells were resuspended in DPBS, and the centrifugation and washing were repeated twice before flow cytometry analysis. The results are shown below. Figure 7 As shown.

[0038] Depend on Figure 7 It is known that endocytosis of LDLR-12A depends on the caverin, clathrin, and macropinocytosis pathways, while Sgc8c-12T enters the cell through multiple pathways such as scavenger receptors, macropinocytosis, and clathrin. The bispecific nucleic acid aptamer LDLR-Sgc8c enters the cell through the caverin, scavenger receptors, clathrin, and macropinocytosis pathways.

[0039] 6. Study on the ability of LDLR-Sgc8c to degrade PTK7 Western blot assay: 12-well plates were seeded with 150,000 HeLa cells per well. Each well was co-incubated with aptamers (LDLR-Sgc8c, LDLR-Control, LDLR-12A, Sgc8c-12T) for 48 h. The old culture medium was discarded, and the cells were washed three times with DPBS. Lysis buffer was added, and the cells were lysed on ice for 10 min. The cell lysates were collected and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected for protein extraction, and BCA quantification was performed. Samples were loaded (5 μg) and run on electrophoresis. Pre-stirred at 80 V to the separating gel, then adjusted to 120 V for 60 min. Transfer was performed (300 mA for 60 min). Blocking with 5% skim milk powder for 1 h (40 rpm), washing three times with TBST (80 rpm for 5 min), incubating overnight at 4 °C with primary antibody (40 rpm), washing three times with TBST (80 rpm for 5 min), incubating with secondary antibody (40 rpm for 60 min), washing three times with TBST, and imaging was performed. Results are as follows: Figure 8 As shown.

[0040] Immunofluorescence assay: HeLa cells were seeded in confocal dishes at a density of 60,000 cells per dish. The next day, the old culture medium was washed away, followed by three washes with DPBS. 500 μl of fresh culture medium (DMEM, 10% FBS, 1% PS) was added, along with cross-linked nucleic acid aptamers (LDLR-Sgc8c and LDLR-Control, final concentration 1 μM). No sample was added to the control group. Cells were incubated for 48 h. The culture medium was discarded, and the cells were washed three times with DPBS, fixed with 4% paraformaldehyde on ice for 30 min, followed by three washes with DPBS. Blocking was performed with 5% BSA (prepared with DPBS) for 1 h. Primary antibody against the target protein (dilution ratio PBS:primary antibody = 1:200) was added, and incubation was performed overnight at 4 °C. The next day, the cells were washed three times with PBST, followed by incubation with secondary fluorescent antibody (37 °C for 1 h). After washing three times with PBST at 37 °C, Hoechst was added (30 °C, 30 min), followed by three more washes with PBST, and finally PBS was added for imaging. Results are shown below. Figure 8 As shown.

[0041] Depend on Figure 8 It was found that, compared with the negative control LDLR-Control, the bispecific nucleic acid aptamer LDLR-Sgc8c was able to achieve targeted degradation of the target protein PTK7. Furthermore, the degradation efficiency of the target protein PTK7 increased with increasing concentration of the bispecific nucleic acid aptamer LDLR-Sgc8c.

[0042] 7. Study on the pathway of LDLR-Sgc8c protein degradation 150,000 HeLa cells were seeded in each well of a 12-well plate. Pretreatment with the lysosomal inhibitor CQ (50 μM) at 37 °C for 2 h was followed by washing twice with DPBS. Fresh medium (DMEM, 10% FBS, 1% PS) was added, and the cells were incubated with the nucleic acid aptamers for 48 h. The old medium was discarded, and the cells were washed three times with DPBS. Lysis was performed on ice for 10 min, followed by centrifugation at 12,000 rpm for 15 min. The supernatant was collected for protein extraction, and after BCA quantification, 5 μg of the sample was loaded and run for electrophoresis. Pre-running at 80 V to the separating gel, then adjusting to 120 V for 60 min, followed by transfer to a membrane (300 mA, 60 min). Blocking with 5% skim milk powder for 1 h (40 rpm), washing three times with TBST (80 rpm, 5 min), incubating overnight at 4 °C with primary antibody (40 rpm), washing three times with TBST (80 rpm, 5 min), incubating with secondary antibody (40 rpm, 60 min), and washing three times with TBST before imaging. Meanwhile, Control (the nucleotide sequence of Control is: 5'-TTTTTTTTTTTTCGCCGGGAAAATACTGTACGGTTAGA3') was used as a negative control sequence, and Control did not bind to the target protein.

[0043] The results are as follows Figure 9 As shown.

[0044] Depend on Figure 9 It was found that after HeLa cells were pretreated with the lysosomal inhibitor CQ for 2 hours, the degradation effect of the bispecific nucleic acid aptamer LDLR-Sgc8c on PTK7 was weakened, indicating that the bispecific nucleic acid aptamer LDLR-Sgc8c mediates protein degradation through the lysosomal pathway. This is consistent with clathrin-mediated LDLR endocytosis.

[0045] 8. Study on the effect of LDLR-Sgc8c in inhibiting cell migration by degrading PTK7 protein Cell scratch assay: 204-well plates were seeded with 200,000 HeLa cells per well. When the cell density reached approximately 80%, a 20 μl pipette tip was used for streaking. After streaking, the cells were washed twice with DPBS, and then 300 μl of culture medium (DMEM, 10% FBS, 1% PS) was added. The cells were photographed, and after imaging, samples (LDLR-Sgc8c and LDLR-Control, 1 μM) were added. After 48 h, the old culture medium was discarded, the cells were washed three times with DPBS, and then fresh culture medium (DMEM, 10% FBS, 1% PS) was added. The cells were photographed again, and migration rates were calculated. LDLR-12A-Control (LDLR-12A cross-links with Control via AT complementary pairing to obtain LDLR-12A-Control) was used as a negative control bispecific aptamer. The nucleotide sequence of Control is: 5'-TTTTTTTTTTTTCGCCGGGAAAATACTGTACGGTTAGA-3'. Results are as follows: Figure 10 As shown.

[0046] Transwell assay: Transwell chambers were removed with forceps and placed in 24-well plates. 50 μL of serum-free medium was added to the upper chamber, and the plates were incubated at 37°C for 30 min to hydrate. 20,000 cells / well (DMEM) were evenly seeded in the upper chamber, and 500 μL of DMEM containing 10% FBS was added to the lower chamber. The plates were incubated overnight. The next day, the old medium was discarded. Fresh DMEM was added to the upper chamber, and cells were treated with drugs (LDLR-Sgc8c, LDLR-Control, 1 μM). Medium (DMEM, 10% FBS, 1% PS) was added to the lower chamber. After 48 h, the old medium was discarded, and the cells were washed three times with DPBS. 500 μL of 4% PFA was added to the lower chamber, and the cells were fixed at room temperature for 30 min. The fixative was discarded, and the cells were washed three times with DPBS. 500 μL of crystal violet dye was added to the lower chamber, and the cells were stained at room temperature for 1.5 h. After washing three times with DPBS, photographs were taken and the results recorded. Simultaneously, LDLR-12A-Control (LDLR-12A crosslinks with Control via AT complementary pairing to obtain LDLR-12A-Control) was used as a negative control bispecific aptamer. The nucleotide sequence of Control is: 5'-TTTTTTTTTTTTCGCCGGGAAAATACTGTACGGTTAGA-3'. The results are as follows: Figure 10 As shown.

[0047] Depend on Figure 10 It can be seen that after HeLa cells were treated with bispecific nucleic acid aptamers for 48 hours, compared with the control group LDLR-Control, the cell scratch width was wider and the number of cells migrating in the transwell chamber was less, indicating that cell migration was inhibited, and that the degradation of PTK7 inhibited the downstream pathways related to migration.

[0048] The above embodiments are specific implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any other combination, change, modification, substitution, or simplification that does not exceed the design concept of the present invention shall fall within the protection scope of the present invention.

Claims

1. A bispecific nucleic acid aptamer based on the LDLR lysosomal shuttle pathway, characterized in that, It includes a first sequence and a second sequence, wherein the first sequence is composed of a first nucleic acid aptamer and a first linker, and the second sequence is composed of a second nucleic acid aptamer and a second linker, wherein the first linker and the second linker are connected to form a linker structure; the first nucleic acid aptamer specifically recognizes and binds to the low-density lipoprotein receptor, and the second nucleic acid aptamer specifically recognizes and binds to the target protein.

2. The bispecific nucleic acid aptamer according to claim 1, characterized in that, The nucleotide sequence of the first nucleic acid aptamer is as follows: 5'-GGACAGGACCACACCCAGCGCGGTCGGCGGGTGGGCGGGGGGAACGAGGTAGGGGTCAGGCTCCTGTGTGTCGCTTTGT-3'.

3. The bispecific nucleic acid aptamer according to claim 1, characterized in that, The target protein is a cell surface protein or an extracellular protein.

4. The bispecific nucleic acid aptamer according to claim 3, characterized in that, The target proteins are protein tyrosine kinase 7, mesenchymal-epidermal transition factor, PD-L1, epithelial cell adhesion molecule, nucleolin, mucin 1, human epidermal growth factor receptor 2, or vascular endothelial growth factor.

5. The bispecific nucleic acid aptamer according to claim 4, characterized in that, The target protein is protein tyrosine kinase 7, and the nucleotide sequence of the second nucleic acid aptamer is: 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3'.

6. The bispecific nucleic acid aptamer according to any one of claims 1-5, characterized in that, The first connecting part and the second connecting part are connected by chemical cross-linking through base complementary pairing or non-base complementary pairing.

7. The method for preparing the bispecific nucleic acid aptamer according to any one of claims 1-6, characterized in that, By connecting the first linker of the first sequence to the second linker of the second sequence to form a linker, a bispecific nucleic acid aptamer is obtained.

8. The use of the bispecific nucleic acid aptamer according to any one of claims 1-6 in any of the following: (A) Applications in protein degradation; (B) Application in the preparation of protein degrading agents; (C) Application in the preparation of drugs that inhibit tumor cell migration; (D) Use in the preparation of medicines for the treatment of tumors, immune diseases, inflammatory diseases or age-related diseases.

9. The application according to claim 8, characterized in that, The tumors include cervical cancer and esophageal cancer; the immune diseases include systemic lupus erythematosus and rheumatoid arthritis; the inflammatory diseases include ankylosing spondylitis; and the age-related diseases include Alzheimer's disease and Parkinson's disease.

10. A drug, characterized in that, The drug comprises any one of the bispecific nucleic acid aptamers of claims 1-6 and a pharmaceutically acceptable carrier.

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