Lysosome sorting signal-nucleic acid aptamer chimera as well as preparation method and application thereof
By using a lysosomal sorting signal-nucleic acid aptamer chimera to couple the nucleic acid aptamer of the target protein with the low-density lipoprotein receptor NPXY motif, the problem of complex and costly degradation of membrane and extracellular proteins in existing technologies is solved, achieving specific and efficient degradation of the target protein and exhibiting anti-tumor activity.
Patent Information
- Application Number
- CN202511717058.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-21
- Publication Date
- 2026-02-13
AI Technical Summary
Existing membrane and extracellular protein degradation technologies are complex and costly, and cannot effectively utilize cellular degradation systems to eliminate proteins related to pathogenesis, especially methods that rely on specific cell surface lysosomal targeting receptors and antibodies, which are inefficient.
The lysosomal sorting signal-nucleic acid aptamer chimera is used to couple the nucleic acid aptamer targeting the target protein with the polypeptide sequence of the low-density lipoprotein receptor NPXY motif through a click chemical reaction, thereby achieving the targeting of the target protein and its transport to the lysosome for degradation.
It achieves specific and efficient degradation of target proteins, simplifies the preparation process, reduces costs, and demonstrates highly efficient degradation activity against PTK7 and MET proteins, which can inhibit tumor growth and migration.
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Figure CN121518481A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a lysosome sorting signal-nucleic acid aptamer chimera, its preparation method, and its application. Background Technology
[0002] Extracellular proteins and cell membrane-associated proteins (accounting for 40% of the products of all protein-coding genes) are key factors in cancer, age-related diseases, and autoimmune diseases; therefore, selective degradation of these proteins has the potential to improve human health. Over the past two decades, targeted protein degradation (TPD) has developed into a promising therapeutic modality and a useful research tool, eliminating proteins associated with pathogenesis by utilizing cellular degradation systems. Proteolysis-targeting chimeras (PTOTACs) represent the greatest success in TPD to date. PROTACs, known as proteolysis-targeting chimeras, are bifunctional molecules composed of an E3 ligase binder, a linker, and a target protein binder. Specifically, one end of a PROTAC molecule binds to a target protein, and the other end binds to an E3 ubiquitin ligase. The E3 ubiquitin ligase marks the target protein as defective or damaged by attaching a small protein called ubiquitin to it; subsequently, the proteasome degrades the marked target protein. However, PROTAC's degradation targets are limited to intracellular proteins containing cytoplasmic domains, and it cannot target membrane proteins and extracellular proteins.
[0003] Current membrane protein degradation technologies rely on specific cell surface lysosomal targeting receptors to deliver membrane proteins to lysosomes via receptor internalization, such as lysosome-targeting chimeras (LYTACs) and antibody-based PROTACs (AbTACs). LYTACs bind to cell surface lysosomal shuttle receptors and the extracellular domains of target proteins, achieving targeted degradation of extracellular and membrane-associated proteins. However, LYTAC preparation typically involves the binding of antibodies and trivalent N-acetylgalactosamine (tri-GalNAc), a complex and time-consuming process, and the large molecular weight results in low internalization efficiency. AbTACs can recruit membrane-bound E3 ligases to degrade cell surface proteins, but their preparation usually requires complex chemical synthesis and purification processes, involving antibody production and oligonucleotide synthesis and conjugation; these processes require specialized equipment and techniques, increasing production costs. Therefore, there is an urgent need to develop a chimera suitable for the specific degradation of extracellular proteins and cell surface membrane proteins. Summary of the Invention
[0004] In view of the problems and shortcomings of the existing technology, the purpose of this invention is to provide a lysosome sorting signal-nucleic acid aptamer chimera, its preparation method and application.
[0005] To achieve the objectives of this invention, the technical solution adopted is as follows: The first aspect of the present invention provides a lysosomal sorting signal-nucleic acid aptamer chimera, comprising a first sequence and a second sequence, wherein the first sequence and the second sequence are coupled by a click chemistry reaction; wherein the first sequence contains a nucleic acid aptamer that selectively binds to a target protein (i.e., a nucleic acid aptamer targeting the target protein), and the second sequence is a polypeptide sequence containing a low-density lipoprotein receptor (LDLR) NPXY motif.
[0006] According to the above-mentioned lysosomal sorting signal-nucleic acid aptamer chimera, preferably, the first sequence is composed of a first flexible linker and a nucleic acid aptamer, the first flexible linker being connected to the 5' end of the nucleic acid aptamer; the second sequence is composed of a second flexible linker and a low-density lipoprotein receptor NPXY motif, the second flexible linker being connected to the amino terminus of the low-density lipoprotein receptor NPXY motif, the sequence of the low-density lipoprotein receptor NPXY motif being: NPGY.
[0007] Based on the above lysosomal sorting signal-nucleic acid aptamer chimera, preferably, the nucleotide sequence of the first flexible linker is TTT; and the amino acid sequence of the second flexible linker is GGG.
[0008] Based on the aforementioned lysosomal sorting signal-nucleic acid aptamer chimera, preferably, the target protein is a cell surface protein or an extracellular protein. More preferably, the cell surface protein includes cell surface membrane proteins.
[0009] Based on the above lysosomal sorting signal-nucleic acid aptamer chimera, preferably, the target protein is protein tyrosine kinase 7 (PTK7) or mesenchymal-epidermal transition factor (c-Met).
[0010] According to the above lysosomal sorting signal-nucleic acid aptamer chimera, preferably, when the target protein is protein tyrosine kinase 7 (PTK7), the sequence of the nucleic acid aptamer is: 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3' (as shown in sequence 1 in the sequence listing).
[0011] Based on the above lysosomal sorting signal-nucleic acid aptamer chimera, preferably, when the target protein is mesenchymal-epidermal transition factor (c-Met), the sequence of the nucleic acid aptamer is: 5'-TTTTTTTTTATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTGAT-3' (as in sequence 2 of the sequence listing).
[0012] The second aspect of the present invention provides a method for preparing the lysosome sorting signal-nucleic acid aptamer chimera described in the first aspect above, comprising: modifying a first sequence with a first group to obtain a modified first sequence; modifying a second sequence with a second group to obtain a modified second sequence; and performing a click chemical reaction between the modified first sequence and the modified second sequence to obtain the lysosome sorting signal-nucleic acid aptamer chimera.
[0013] According to the above method for preparing lysosome sorting signal-nucleic acid aptamer chimera, preferably, the first group is a dibenzocyclooctyne (DBCO) group, a trans-cyclooctene group, or a bicyclic [6.1.0]nonyne group; the second group is an azide group.
[0014] According to the above method for preparing lysosome sorting signal-nucleic acid aptamer chimera, preferably, the first group is modified at the 5' end of the nucleic acid aptamer, and the second group is modified at the N-terminus of the second sequence.
[0015] A third aspect of the present invention provides the use of the lysosomal sorting signal-nucleic acid aptamer chimera 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) Application in the preparation of drugs for treating tumors.
[0016] According to the above application, preferably, the protein is protein tyrosine kinase 7 or mesenchymal-epidermal transition factor.
[0017] According to the above application, preferably, the tumor includes cervical cancer.
[0018] A fourth aspect of the present invention provides a medicament comprising the lysosomal sorting signal-nucleic acid aptamer chimera described in the first aspect above and a pharmaceutically acceptable carrier.
[0019] The specific principle of the lysosomal sorting signal-nucleic acid aptamer chimera used for target protein degradation in this invention is as follows (e.g. Figure 1As shown): Lysosomal sorting signal - nucleic acid aptamer chimera's nucleic acid aptamer can bind to the target protein to be degraded. Its polypeptide sequence utilizes the NPXY motif of LDLR to internalize the target protein and transport it to the lysosome, thereby achieving the degradation of the target protein.
[0020] Compared with the prior art, the positive and beneficial effects achieved by the present invention are as follows: (1) In this invention, a lysosome sorting signal-nucleic acid aptamer chimera is obtained by coupling a nucleic acid aptamer targeting a target protein with a polypeptide sequence containing the NPXY motif of a low-density lipoprotein receptor via a click chemistry reaction. This lysosome sorting signal-nucleic acid aptamer chimera has the characteristic of simultaneously targeting both the target protein and the lysosome, and can specifically bind to the target protein to be degraded and transport the target protein to the lysosome for degradation. Therefore, it can achieve specific and efficient degradation of the target protein, without being limited by the receptor expression level or endogenous ligands. In addition, the lysosome sorting signal-nucleic acid aptamer chimera of this invention can be prepared by a click chemistry reaction, which is simple, low-cost, and easy to implement.
[0021] (2) The polypeptide sequence of the chimera of the present invention contains the flexible linker GGG. The flexible linker helps to expose the NPXY motif of LDLR and enhances the function of the device targeting lysosomes.
[0022] (3) The lysosome sorting signal-nucleic acid aptamer chimera of the present invention exhibits highly efficient degradation activity against PTK7 and MET proteins and can inhibit tumor growth and migration. It can be used to prepare drugs that inhibit tumor cell migration or treat tumors. Attached Figure Description
[0023] Figure 1 This is a schematic diagram illustrating the principle of the lysosomal sorting signal-nucleic acid aptamer chimera used for protein targeted degradation in this invention. Figure 2 A schematic diagram of the construction of a lysosomal sorting signal-nucleic acid aptamer chimera for the degradation of the membrane protein PTK7; Figure 3 The results are obtained by non-denaturing polyacrylamide gel electrophoresis of Sgc8c-GGGNPGY; lane 1 is Sgc8c-DBCO and lane 2 is Sgc8c-GGGNPGY. Figure 4 The mass spectrometry analysis results of the Sgc8c-GGGNPGY sample are shown in the figure. Figure 5 The results show the binding ability of Sgc8c-GGGNPGY to PTK7 on the cell surface; the left figure is the flow cytometry peak diagram of the binding; the right figure is the statistical result of the fluorescence intensity of the flow cytometry peak of the binding. Figure 6To analyze the detection results of Sgc8c-GGGNPGY internalization colocalization using confocal microscopy; Figure 7 The graph shows the results of flow cytometry analysis of Sgc8c-GGGNPGY internalization; the left graph shows the peaks of flow cytometry analysis of endocytosis; the right graph shows the statistical results of the fluorescence intensity of the peaks of flow cytometry analysis of endocytosis. Figure 8 The results of flow cytometry analysis of Sgc8c-GGGNPGY endocytosis were presented. Figure a shows the detection results of Sgc8c-DBCO endocytosis, with the left panel showing the flow cytometry peaks of Sgc8c endocytosis inhibition and the right panel showing the statistical results of the fluorescence intensity of the Sgc8c endocytosis inhibition peaks. Figure b shows the detection results of Sgc8c-GGGNPGY endocytosis, with the left panel showing the flow cytometry peaks of Sgc8c-GGGNPGY endocytosis inhibition and the right panel showing the statistical results of the fluorescence intensity of the Sgc8c-GGGNPGY endocytosis inhibition peaks. Figure 9 The Western Blot results show the degradation effect of different concentrations of Sgc8c-GGGNPGY on PTK7 protein in HeLa cell membranes. The left figure shows the Western Blot bands of PTK7 protein after treatment with different concentrations of Sgc8c-GGGNPGY, and the right figure shows the statistical chart of the quantitative results of Western Blot detection. Figure 10 The Western Blot results show the degradation effect of PTK7 by 500 nM Sgc8c-GGGNPGY and various control groups; the right figure is a statistical chart of the quantitative results of Western Blot detection, showing the Western Blot bands of PTK7 protein after treatment with different control groups and Sgc8c-GGGNPGY. Figure 11 The flow cytometry results show the degradation effect of Sgc8c-GGGNPGY on PTK7. The left figure shows the flow cytometry peaks of PTK7 protein after treatment with different concentrations of Sgc8c-GGGNPGY, and the right figure shows the statistical results of the fluorescence intensity of the flow cytometry peaks. Figure 12 The results are obtained by non-denaturing polyacrylamide gel electrophoresis of Met-GGGNPGY; lane 1 is Met-GGGNPGY and lane 2 is Met-DBCO. Figure 13 The image shows the mass spectrometry analysis results of the Met-GGGNPGY sample. Figure 14 To analyze the detection results of Met-GGGNPGY endocytosis colocalization using confocal microscopy; Figure 15 The results of flow cytometry analysis of Met-GGGNPGY endocytosis are shown in Figure a. Figure a shows the detection results of Met-DBCO endocytosis, with the left panel showing the flow cytometry peaks of Met endocytosis inhibition and the right panel showing the statistical results of the fluorescence intensity of the Met endocytosis inhibition flow cytometry peaks. Figure b shows the detection results of Met-GGGNPGY endocytosis, with the left panel showing the flow cytometry peaks of Met-GGGNPGY endocytosis inhibition and the right panel showing the statistical results of the fluorescence intensity of the Met-GGGNPGY endocytosis inhibition flow cytometry peaks. Figure 16 The Western Blot results show the degradation effect of different concentrations of Met-GGGNPGY on MET protein in HeLa cell membranes; the left figure shows the Western Blot bands of MET protein after treatment with different concentrations of Met-GGGNPGY, and the right figure shows the statistical chart of the quantitative results of Western Blot detection. Figure 17 The Western Blot results show the effects of 500 nM Met-GGGNPGY and various control groups on Met degradation; the left figure shows the Western Blot bands of Met protein after treatment with different control groups and Met-GGGNPGY, and the right figure shows the statistical chart of the quantitative results of Western Blot detection. Figure 18 The results of the cell scratch assay show that Met-GGGNPGY inhibits cancer cell migration by degrading c-Met; the left figure shows the scratch healing results of HeLa cells, and the right figure shows the statistical graph of scratch healing rate of HeLa cells. Detailed Implementation
[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below.
[0025] 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.
[0026] Example 1: Preparation of a lysosomal sorting signal-nucleic acid aptamer chimera for degrading the membrane protein protein protein tyrosine kinase 7 (PTK7) A lysosomal sorting signal-nucleic acid aptamer chimera for degrading the membrane protein protein protein tyrosine kinase 7 (PTK7) (e.g.) Figure 2As shown, the sequence (Sgc8c) and the second sequence (GGGNPGY) are coupled via a click chemistry reaction. Sgc8c consists of a first flexible linker and a nucleic acid aptamer targeting the membrane protein PTK7. The first flexible linker is attached to the 5' end of the nucleic acid aptamer, and the nucleotide sequence of the first flexible linker is TTT. The nucleotide sequence of the nucleic acid aptamer is 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3' (as shown in Sequence 1 of the sequence listing). GGGNPGY consists of a second flexible linker and a low-density lipoprotein receptor (LDL-P) NPXY motif. The second flexible linker is attached to the amino terminus of the LDL-P receptor NPXY motif, and the amino acid sequence of the second flexible linker is GGG. The sequence of the LDL-P receptor NPXY motif is NPGY.
[0027] The preparation method of the lysosome sorting signal-nucleic acid aptamer chimera for degrading membrane protein PTK7 is as follows: the 5' end of the first sequence (Sgc8c) is modified with a DBCO group to obtain the modified first sequence (denoted as Sgc8c-DBCO); the second sequence (GGGNPGY) is modified with an azide group (N3) to obtain the modified second sequence (N3-GGGNPGY); Sgc8c-DBCO and N3-GGGNPGY are placed in a 200 μl single tube, shaken to mix, and then slightly centrifuged. The mixture is reacted at room temperature for 1 hour to obtain the lysosome sorting signal-nucleic acid aptamer chimera for degrading membrane protein PTK7.
[0028] Example 2: Preparation of a lysosomal sorting signal-nucleic acid aptamer chimera for degrading the membrane protein interstitial-epidermal transition factor (c-Met). A lysosomal sorting signal-nucleic acid aptamer chimera for degrading the membrane protein interstitial-epidermal transition factor (c-Met) comprises a first sequence (denoted as Met) and a second sequence (denoted as GGGNPGY) coupled via a click chemistry reaction. The Met consists of a first flexible linker and a nucleic acid aptamer targeting the membrane protein c-Met. The first flexible linker is attached to the 5' end of the nucleic acid aptamer, and the nucleotide sequence of the first flexible linker is TTT. The nucleotide sequence of the nucleic acid aptamer is: 5'-TTTTTTTTTATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGGTGGGTTGGCAAGTCTGAT-3' (as shown in sequence 2 in the sequence listing). The GGGNPGY consists of a second flexible linker and a low-density lipoprotein receptor (LDL-P) motif (NPXY). The second flexible linker is attached to the amino terminus of the LDL-P receptor (NPXY) motif, and the amino acid sequence of the second flexible linker is GGG. The sequence of the LDL-P receptor (NPXY) motif is NPGY.
[0029] The preparation method of the lysosomal sorting signal-nucleic acid aptamer chimera for degrading membrane protein c-Met is as follows: the 5' end of the first sequence (Met) is modified with a DBCO group to obtain the modified first sequence (denoted as Met-DBCO); the second sequence (GGGNPGY) is modified with an azide group (N3) to obtain the modified second sequence (N3-GGGNPGY); Met-DBCO and N3-GGGNPGY are placed in a 200 μl single tube, shaken to mix, and then slightly centrifuged. The mixture is reacted at room temperature for 1 hour to obtain the lysosomal sorting signal-nucleic acid aptamer chimera for degrading membrane protein c-Met.
[0030] (i) Characterization and functional verification of the lysosomal sorting signal-nucleic acid aptamer chimera (hereinafter referred to as Sgc8c-GGGNPGY) used for the degradation of the membrane protein PTK7. Taking the Sgc8c-GGGNPGY prepared in Example 1 as an example, characterization and functional verification were performed.
[0031] 1. Non-denaturing polyacrylamide gel electrophoresis detection of Sgc8c-GGGNPGY: The Sgc8c-GGGNPGY prepared in Example 1 was subjected to non-denaturing polyacrylamide gel electrophoresis to verify the crosslinking of Sgc8c-DBCO and N3-GGGNPGY. The detection results are as follows: Figure 3 As shown.
[0032] Depend on Figure 3 The presence of a displacement difference between the two lanes indicates that the nucleic acid aptamer Sgc8c has successfully coupled with GGGNPGY.
[0033] 2. Mass spectrometry detection of Sgc8c-GGGNPGY: Mass spectrometry was used to detect the Sgc8c-GGGNPGY prepared in Example 1. The mass spectrometry conditions were as follows: instrument name: LTQ liquid mass ion trap mass spectrometer, model: LTQ XL; basic parameter settings: ionization spray voltage: 4.5 kV, nebulization temperature: 350℃, ion mode: negative ion mode, sample volume: 25 μL, concentration: 5~10 μM. The detection results are as follows. Figure 4 As shown.
[0034] Depend on Figure 4 The high purity of the product peak indicates that the Sgc8c-GGGNPGY coupling was successful.
[0035] 3. Study on the binding ability of Sgc8c-GGGNPGY to cell surface membrane protein PTK7 Remove HeLa cells from a dish with a cell density of 70%-80%, wash three times with washing buffer, add 0.2% EDTA and digest at room temperature for 10 minutes. Resuspend the cells in washing buffer by pipetting and centrifuge at 4000 rpm for 5 minutes. Carefully aspirate the supernatant, resuspend in binding buffer to obtain a cell suspension, take a certain amount of the cell suspension into an EP tube, and add binding buffer to bring the total volume to 200 μl. EP tubes containing cell suspension were divided into five groups: Blank group, Control group, Control-GGGNPGY group, Sgc8c group, and Sgc8c-GGGNPGY group. 250 nM FAM-modified Sgc8c-GGGNPGY was added to the Sgc8c-GGGNPGY group, 250 nM Sgc8c-FAM was added to the Sgc8c group, 250 nM FAM-modified Control-GGGNPGY was added to the Control-GGGNPGY group, and 250 nM Control-FAM was added to the Control group. The Blank group received no treatment. All groups were gently mixed by pipetting and incubated on ice for 30 minutes in the dark. The EP tubes were then centrifuged at 4000 rpm for 5 minutes. After centrifugation, the culture medium was removed from the EP tubes, and 200 μl of DPBS was added. The tubes were then centrifuged again at 4000 rpm for 5 minutes. After centrifugation, the supernatant was aspirated, and then 300 μl of DPBS (ServiceBio, catalog number G4200-500 μl) was added to the EP tube. The mixture was gently pipetted and then analyzed using a flow cytometry system. Data were analyzed using FlowJo. Results are as follows: Figure 5 As shown.
[0036] Depend on Figure 5 It is known that Sgc8c and Sgc8c-GGGNPGY can recognize and bind to the membrane protein PTK7, indicating that it is feasible to use Sgc8c-GGGNPGY as a PTK7 targeted degrader.
[0037] 4. Research on the mechanism of Sgc8c-GGGNPGY endocytosis (1) Confocal microscopy analysis of Sgc8c-GGGNPGY endocytosis co-localization: HeLa cells were seeded in live cell culture dishes (2.2 × 10⁶ cells per dish). 5 (1 cell per dish) 500 μl of complete culture medium was added to each dish for 24 hours of culture. After culturing, the culture medium was aspirated and the dish was washed three times with DPBS. The dishes were then divided into three groups: Sgc8c-GGGNPGY, Sgc8c, and Control-GGGNPGY. 500 μl of fresh complete culture medium was added to each dish. 250 nM Sgc8c-GGGNPGY-Cy5 (Sgc8c-GGGNPGY-Cy5 is Cy5-modified Sgc8c-GGGNPGY, directly synthesized by the manufacturer) was added to the Sgc8c-GGGNPGY group dishes, 250 nM Control-GGGNPGY-Cy5 (Control-GGGNPGY-Cy5 is Cy5-modified GGGNPGY, directly synthesized by the manufacturer) was added to the Control-GGGNPGY group dishes, and 250 nM Control-GGGNPGY-Cy5 (Control-GGGNPGY-Cy5 is Cy5-modified GGGNPGY, directly synthesized by the manufacturer) was added to the Sgc8c group dishes. Sgc8c-Cy5 (Sgc8c-Cy5 is Sgc8c modified with Cy5, directly synthesized by the manufacturer); All culture dishes were incubated in an incubator for 4 hours. After incubation, the dishes were washed three times with DPBS. 500 μl of serum-free DMEM medium and LysoTracker Green (lysosomal fluorescent dye) were added to each group's culture dish, and the dishes were incubated for 30 minutes. After incubation, each group's culture dish was washed three times with DPBS, 500 μl of serum-free DMEM medium was added, followed by 3 μl of Hoechst (Beyotime, C1029), and incubated for 15 minutes. The dishes were then removed, washed three times with DPBS, and finally 500 μl of phenol red 1640-free medium was added. The culture dishes were placed on ice and imaged using a Nikon fast-resolution laser confocal microscope. Results are as follows: Figure 6 As shown.
[0038] Depend on Figure 6 It was found that Cy5 fluorescence and green fluorescence in Sgc8c-GGGNPGY were significantly co-localized, indicating that Sgc8c-GGGNPGY can enter cells and target lysosomes, further demonstrating the feasibility of Sgc8c-GGGNPGY as a targeted degradation agent.
[0039] (2) Flow cytometry analysis of Sgc8c-GGGNPGY internalization Prior to the experiment, HeLa cells for internalization assays were seeded into the wells of a 24-well plate (10 cells per well). 5 In a culture well containing 100 cells, 500 μl of complete culture medium was added to each well. After culturing for 24 hours, the culture medium was aspirated and the cells were washed three times with DPBS. The wells were then divided into three groups: Sgc8c-GGGNPGY, Sgc8c, and Control-GGGNPGY. 500 μl of fresh complete culture medium was added to each well. 250 nM Sgc8c-GGGNPGY-Cy5 (Sgc8c-GGGNPGY-Cy5 is Cy5-modified Sgc8c-GGGNPGY, directly synthesized by the manufacturer) was added to the wells of the Sgc8c-GGGNPGY group, 250 nM Control-GGGNPGY-Cy5 (Control-GGGNPGY-Cy5 is Cy5-modified GGGNPGY, directly synthesized by the manufacturer) was added to the wells of the Control-GGGNPGY group, and 250 nM Sgc8c-GGGNPGY-Cy5 (Control-GGGNPGY-Cy5 is Cy5-modified GGGNPGY, directly synthesized by the manufacturer) was added to the wells of the Sgc8c group. Sgc8c-Cy5 (Sgc8c-Cy5 is Sgc8c modified with Cy5, directly synthesized by the manufacturer); then the 24-well plates were incubated in an incubator for 4 hours. After incubation, the cells were washed three times with DPBS, digested with trypsin, and the digestion reaction was terminated with culture medium. Cells were then transferred to Eppendorf tubes, centrifuged at 4000 rpm for 5 min, the culture medium was removed, 200 μl of DPBS was added, and the cells were centrifuged again. After centrifugation, the supernatant was removed, 300 μl of DPBS was added, and the cells were gently mixed by pipetting. Flow cytometry was used for analysis, and the data were analyzed using FlowJo. Results are as follows: Figure 7 As shown.
[0040] Depend on Figure 7 It can be seen that Sgc8c-GGGNPGY has a strong internalization capability.
[0041] (3) Flow cytometry analysis of the endocytosis mechanism of Sgc8c-GGGNPGY Prior to the experiment, HeLa cells for the endocytosis assay were seeded into the wells of a 24-well plate (10 cells per well). 5In a 24-well plate containing 100 cells, 500 μl of complete culture medium was added to each well. After culturing for 24 hours, the medium was aspirated and the plate was washed three times with DPBS. The wells were then divided into Blank, DMSO, Poly I, Filipin, CPZ, and EIPA groups, with 500 μl of fresh complete culture medium added to each group. The Blank group received no treatment. DMSO was added to the wells of the DMSO group, 40 μM of the endocytosis inhibitor Poly I was added to the wells of the Poly I group, 4 μM of the endocytosis inhibitor Filipin was added to the wells of the Filipin group, 30 μM of the endocytosis inhibitor chlorpromazine was added to the wells of the CPZ group, and 100 μM of the endocytosis inhibitor EIPA was added to the wells of the EIPA group. The plates were incubated for 1 hour and then washed three times with DPBS. Finally, 300 μM of culture medium and 250 nM of DPBS were added to the wells of each group. Sgc8c-GGGNPGY-Cy5 or Sgc8c-DBCO-Cy5 cells were incubated in an incubator for 4 hours, washed three times with DPBS, and digested with trypsin. The digestion reaction was terminated with culture medium. Cells were then transferred to Eppendorf tubes, centrifuged at 4000 rpm for 5 min, the culture medium was removed, and 200 μL of DPBS was added. After centrifugation, the supernatant was removed, 300 μL of DPBS was added, and the mixture was gently pipetted to mix. Flow cytometry was used for analysis, and the data were analyzed using FlowJo. Results are as follows: Figure 8 As shown.
[0042] Depend on Figure 8 It is known that the endocytosis of single-chain Sgc8c is mainly mediated by clathrin and macropinocytosis pathways; while the endocytosis of Sgc8c-GGNPGY is mainly mediated by clathrin.
[0043] 5. Study on the ability of Sgc8c-GGGNPGY to degrade PTK7 (1) Study on the degradation ability of different concentrations of Sgc8c-GGGNPGY on PTK7 protein HeLa cells were seeded into the wells of a 12-well plate (1.5 × 10⁶ cells per well). 5Cells were then incubated for 48 hours in 1 mL of complete growth medium containing different concentrations (100 nM, 200 nM, 500 nM, 800 nM) of Sgc8c-GGGNPGY. Subsequently, the cells were washed three times with pre-chilled DPBS, and then lysed on ice for 30 minutes with RIPA buffer containing a protein inhibitor (cocktail). The cell lysate was centrifuged at 12000 rpm for 15 min, and the supernatant was collected. The protein concentration in the supernatant was determined using a BCA protein quantification kit, and protein degradation was analyzed by Western blot. Results are shown below. Figure 9 As shown.
[0044] The specific procedure for protein immunoblotting is as follows: take a certain amount of protein solution and add protein loading buffer to it, place it at 37°C and incubate for 1 hour, then load the protein sample, transfer it to a PVDF membrane after 10% SDS-PAGE gel electrophoresis, block it with 5% skim milk powder, add primary antibody (4°C overnight) and secondary antibody (room temperature for 1 hour) in sequence, finally add chemiluminescent reagent for imaging, and analyze the bands using ImageJ.
[0045] Depend on Figure 9 It can be seen that Sgc8c-GGGNPGY exhibits concentration-dependent degradation of the membrane protein PTK7.
[0046] (2) Comparison of the effects of Sgc8c-GGGNPGY on PTK7 degradation: HeLa cells were seeded into the wells of a 24-well plate (1.5 × 10⁶ cells per well). 5(Number of cells), the culture wells were divided into Blank group, Sgc8c-GGGNPGY group, Control-GGGNPGY group, Sgc8c group, Control group, and GGGNPGY group. 500 μL of complete growth medium was added to the culture wells of the Blank group, 500 μL of complete growth medium containing Sgc8c-GGGNPGY was added to the culture wells of the Sgc8c-GGGNPGY group, 500 μL of complete growth medium containing Control-GGGNPGY was added to the culture wells of the Control-GGGNPGY group, 500 μL of complete growth medium containing Sgc8c was added to the culture wells of the Sgc8c group, 500 μL of complete growth medium containing Control was added to the culture wells of the Control group, and 500 μL of complete growth medium containing GGGNPGY was added to the culture wells of the GGGNPGY group. The 24-well plates were then incubated in an incubator for 48 h. Cells were then washed three times with pre-chilled DPBS, followed by lysis on ice for 30 minutes with RIPA buffer containing a protein inhibitor (cocktail). The cell lysates were centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. Protein concentration in the supernatant was determined using a BCA protein quantification kit, and protein degradation was analyzed by Western blot. Results are shown below. Figure 10 As shown.
[0047] Depend on Figure 10 It is known that Sgc8c-GGGNPGY can specifically degrade the membrane protein PTK7.
[0048] (3) Flow cytometry detection of the degradation effect of Sgc8c-GGGNPGY on PTK7 Prior to the experiment, HeLa cells were seeded into the wells of a 24-well plate (1.0 × 10⁶ cells per well). 5Add 500 μl of complete culture medium to each well of cells and incubate for 24 hours. After incubation, aspirate the medium and wash three times with DPBS. Add 500 μl of fresh complete culture medium and add different concentrations (100 nM, 200 nM, 500 nM, 800 nM) of Sgc8c-GGGNPGY, respectively. Incubate for 48 hours. Then wash three times with washing buffer, add 0.2% EDTA for room temperature digestion, and resuspend the cells in washing buffer. Centrifuge at 4000 rpm for 5 minutes. After centrifugation, carefully aspirate the supernatant and add 200 μl of binding buffer, then add 250 nM Sgc8c-DBCO-FAM. Incubate on ice in the dark for 30 minutes. Centrifuge again, aspirate the supernatant, add 200 μl of washing buffer, resuspend by pipetting, and centrifuge again. Discard the supernatant, add 300 μl of washing buffer, and analyze using a flow cytometry system. Analyze the data using FlowJo. The results are as follows Figure 11 As shown.
[0049] Depend on Figure 11 As can be seen, the flow cytometry results are consistent with those of Western blotting, indicating that Sgc8c-GGGNPGY exhibits concentration-dependent degradation of the membrane protein PTK7.
[0050] (II) Characterization and functional verification of the lysosomal sorting signal-nucleic acid aptamer chimera (hereinafter referred to as Met-GGGNPGY) used for the degradation of membrane protein c-Met. Taking the Met-GGGNPGY prepared in Example 2 as an example, characterization and functional verification were performed.
[0051] 1. Non-denaturing polyacrylamide gel electrophoresis detection of Met-GGGNPGY: The Met-GGGNPGY prepared in Example 2 was subjected to non-denaturing polyacrylamide gel electrophoresis to verify the crosslinking of Met-DBCO and N3-GGGNPGY. The results are as follows: Figure 12 As shown.
[0052] Depend on Figure 12 The presence of a displacement difference between the two lanes indicates that the nucleic acid aptamer targeting the Met protein has successfully coupled with GGGNPGY.
[0053] 2. Mass spectrometry detection of Met-GGGNPGY: The Met-GGGNPGY prepared in Example 2 was analyzed by mass spectrometry. The mass spectrometry conditions were as follows: instrument name: LTQ liquid mass ion trap mass spectrometer, model: LTQ XL; basic parameter settings: ionization spray voltage: 4.5 kV, nebulization temperature: 350℃, ion mode: negative ion mode, sample volume: 25 μL, concentration: 5~10 μM. The detection results are as follows. Figure 13 As shown.
[0054] Depend on Figure 13 The high purity of the product peak indicates that the Met-GGGNPGY coupling was successful.
[0055] 3. Met-GGGNPGY endocytosis mechanism (1) Confocal microscopy analysis of Met-GGGNPGY endocytosis colocalization HeLa cells were seeded in live cell culture dishes (2.2 × 10⁶ cells per dish). 5 (100 cells), 500 μl of complete culture medium was added to each dish for 24 hours of culture. After culturing, the culture medium was aspirated and the dish was washed three times with DPBS. The culture dishes were then divided into Met-GGGNPGY5 group, Met group, and Ctrl-GGGNPGY group. 500 μl of fresh complete culture medium was added to each group of culture dishes. Then, 250 nM Met-GGGNPGY-Cy5 (Met-GGGNPGY-Cy5 is Met-GGGNPGY modified with Cy5 and directly synthesized by the manufacturer) was added to the culture dishes of the Met-GGGNPGY group, 250 nM Control-GGGNPGY-Cy5 (Control-GGGNPGY-Cy5 is GGGNPGY modified with Cy5 and directly synthesized by the manufacturer) was added to the culture dishes of the Ctrl-GGGNPGY group, and 250 nM Met-Cy5 (Met-Cy5 is Met modified with Cy5 and directly synthesized by the manufacturer) was added to the culture dishes of the Met-Cy5 group. All culture dishes were incubated in an incubator for 4 hours. After incubation, the dishes were washed three times with DPBS. 500 μl of serum-free DMEM medium and LysoTracker Green (lysosomal fluorescent dye) were added to each group's culture dishes, and the dishes were incubated for 30 minutes. After incubation, each group's culture dishes were washed three times with DPBS, then 500 μl of serum-free DMEM medium was added, followed by 3 μl of Hoechst (Beyotime, C1029). After incubation for 15 minutes, the dishes were removed, washed three times with DPBS, and finally 500 μl of phenol red-free 1640 medium was added. The culture dishes were placed on ice and imaged using a Nikon fast-resolution laser confocal microscope. Results are as follows: Figure 14 As shown.
[0056] Depend on Figure 14 It can be seen that Cy5 fluorescence and green fluorescence are significantly co-localized in Met-GGGNPGY, indicating that Met-GGGNPGY can enter cells and target lysosomes, further demonstrating the feasibility of Met-GGGNPGY as a targeted degradation agent.
[0057] (2) Flow cytometry analysis of the endocytosis mechanism of Met-GGGNPGY Prior to the experiment, HeLa cells for the endocytosis assay were seeded into the wells of a 24-well plate (10 cells per well). 5 In a 24-well plate containing 100 cells, 500 μl of complete culture medium was added to each well. After culturing for 24 hours, the medium was aspirated and the plate was washed three times with DPBS. The wells were then divided into Blank, DMSO, Poly I, Filipin, CPZ, and EIPA groups, with 500 μl of fresh complete culture medium added to each group. The Blank group received no treatment. DMSO was added to the wells of the DMSO group, 40 μM Poly I was added to the wells of the Poly I group, 4 μM Filipin was added to the wells of the Filipin group, 30 μM chlorpromazine was added to the wells of the CPZ group, and 100 μM EIPA was added to the wells of the EIPA group. The 24-well plates were incubated for 1 hour and then washed three times with DPBS. Finally, 300 μl of culture medium and 250 nM sodium bicarbonate were added to the wells of each group. Met-GGGNPGY-Cy5 or Met-DBCO-Cy5 cells were incubated in an incubator for 4 hours, washed three times with DPBS, and digested with trypsin. The digestion reaction was terminated with culture medium. Cells were then transferred to Eppendorf tubes, centrifuged at 4000 rpm for 5 min, the culture medium was removed, and 200 μL of DPBS was added. After centrifugation, the supernatant was removed, 300 μL of DPBS was added, and the mixture was gently pipetted and analyzed. The cells were then analyzed using a flow cytometry system, and the data were analyzed using FlowJo. Results are as follows: Figure 15 As shown.
[0058] Depend on Figure 15 It is known that the endocytosis pathway of single-chain Met is mainly mediated by clathrin, and the endocytosis of Met-GGNPGY is also mainly mediated by clathrin.
[0059] 4. Study on the ability of Met-GGGNPGY to degrade c-Met (1) Study on the degradation ability of different concentrations of Met-GGGNPGY on c-Met protein HeLa cells were seeded into the wells of a 12-well plate (1.5 × 10⁶ cells per well). 5Cells were collected and incubated for 48 hours with 1 mL of complete growth medium containing different concentrations (200 nM, 500 nM, 1 μM) of Met-GGGNPGY. Cells were then washed three times with pre-chilled DPBS and lysed on ice for 30 minutes with RIPA buffer containing a protein inhibitor (cocktail). The cell lysate was centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. The protein concentration in the supernatant was determined using a BCA protein quantification kit, and protein degradation was analyzed by Western blot. Results are shown below. Figure 16 As shown.
[0060] Depend on Figure 16 It can be seen that Met-GGGNPGY exhibits concentration-dependent degradation of the membrane protein MET.
[0061] (2) Comparison of the effects of Met-GGGNPGY on the degradation of c-Met: HeLa cells were seeded into the wells of a 24-well plate (1.5 × 10⁶ cells per well). 5 Cells were divided into four groups (Blank, Met-GGGNPGY, Control-GGGNPGY, Met, and GGGNPGY) in 24-well plates. 500 μL of complete growth medium was added to the wells of the Blank group, 500 μL of complete growth medium containing Met-GGGNPGY to the wells of the Met-GGGNPGY group, 500 μL of complete growth medium containing Control-GGGNPGY to the wells of the Control-GGGNPGY group, 500 μL of complete growth medium containing Met to the wells of the Met group, and 500 μL of complete growth medium containing GGGNPGY to the wells of the GGGNPGY group. The plates were then incubated for 48 h. Cells were then washed three times with pre-chilled DPBS and lysed on ice for 30 min with RIPA buffer containing a protein inhibitor (Cotail). The cell lysates were centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. The protein concentration in the supernatant was determined using the BCA protein quantification kit, and protein degradation was analyzed by Western blot. Results are as follows: Figure 17 As shown.
[0062] Depend on Figure 17 It is known that Met-GGGNPGY can specifically degrade the membrane protein Met.
[0063] 5. Study on the effect of Met-GGGNPGY on inhibiting cancer cell migration by degrading c-Met (1) Cell scratch test HeLa cells were seeded into the wells of 24-well plates, 1 mL of complete culture medium was added, and the plates were incubated. When the cell confluence reached approximately 80%-90%, a 10 μl pipette tip was used to gently draw a straight line on the cell monolayer, perpendicular to the bottom of the plate, ensuring the scratch width was as uniform as possible. The cells were gently washed 2-3 times with DPBS to remove cell debris. Serum-free culture medium was added, and Met-GGGNPGY, Control-GGGNPGY, Met, and GGGNPGY were added respectively. At different time points (0 h, 24 h, and 48 h) after scratching, the cells were observed and photographed using an inverted microscope to record the scratch width and cell migration. The width of the scratch or the distance of cell migration at different time points was measured using ImageJ software, and the cell migration rate was calculated and statistically analyzed. The results are shown in Figure 18.
[0064] Depend on Figure 18 It is known that degradation of the hela cell membrane protein MET can inhibit cell migration.
[0065] 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 lysosomal sorting signal-nucleic acid aptamer chimera, characterized in that, It includes a first sequence and a second sequence, which are coupled by a click chemistry reaction; wherein the first sequence contains a nucleic acid aptamer that selectively binds to a target protein, and the second sequence is a polypeptide sequence containing a low-density lipoprotein receptor NPXY motif.
2. The lysosomal sorting signal-nucleic acid aptamer chimera according to claim 1, characterized in that, The first sequence consists of a first flexible linker and a nucleic acid aptamer, wherein the first flexible linker is connected to the 5' end of the nucleic acid aptamer; the second sequence consists of a second flexible linker and a low-density lipoprotein receptor NPXY motif, wherein the second flexible linker is connected to the amino terminus of the low-density lipoprotein receptor NPXY motif, wherein the sequence of the low-density lipoprotein receptor NPXY motif is NPGY.
3. The lysosomal sorting signal-nucleic acid aptamer chimera according to claim 2, characterized in that, The nucleotide sequence of the first flexible linker is TTT; the amino acid sequence of the second flexible linker is GGG.
4. The lysosomal sorting signal-nucleic acid aptamer chimera according to claim 1, characterized in that, The target protein is a cell surface protein or an extracellular protein.
5. The lysosomal sorting signal-nucleic acid aptamer chimera according to claim 4, characterized in that, The target protein is protein tyrosine kinase 7 or mesenchymal-epidermal transition factor.
6. The lysosomal sorting signal-nucleic acid aptamer chimera according to claim 5, characterized in that, When the target protein is protein tyrosine kinase 7, the sequence of the nucleic acid aptamer is: 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3'; when the target protein is mesenchymal-epidermal transition factor, the sequence of the nucleic acid aptamer is: 5'-TTTTTTTTTATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTGAT-3'.
7. The method for preparing the lysosomal sorting signal-nucleic acid aptamer chimera according to any one of claims 1-6, characterized in that, include: The first sequence is modified with the first group to obtain the modified first sequence; The second sequence is modified with a second group to obtain the modified second sequence; the modified first sequence and the modified second sequence are subjected to a click chemical reaction to obtain the lysosome sorting signal-nucleic acid aptamer chimera.
8. The preparation method according to claim 7, characterized in that, The first group is a dibenzocyclooctynyl group; the second group is an azide group; the first group is modified at the 5' end of the nucleic acid aptamer, and the second group is modified at the N-terminus of the second sequence.
9. The use of the lysosomal sorting signal-nucleic acid aptamer chimera 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.
10. A drug, characterized in that, The drug comprises the lysosomal sorting signal-nucleic acid aptamer chimera of any one of claims 1-6 and a pharmaceutically acceptable carrier.