WO2021000598A1 - 一种双特异性核酸适体、衍生物、制备方法及其应用 - Google Patents

一种双特异性核酸适体、衍生物、制备方法及其应用 Download PDF

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WO2021000598A1
WO2021000598A1 PCT/CN2020/079139 CN2020079139W WO2021000598A1 WO 2021000598 A1 WO2021000598 A1 WO 2021000598A1 CN 2020079139 W CN2020079139 W CN 2020079139W WO 2021000598 A1 WO2021000598 A1 WO 2021000598A1
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nucleic acid
receptor
bispecific
acid aptamer
aptamer
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French (fr)
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李娟�
王丽萍
李婧影
杨黄浩
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福州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

Definitions

  • the present invention relates to the field of nucleic acid aptamers, nucleic acid aptamer drugs, and protein function regulation and control, and is specifically a bispecific nucleic acid aptamer, derivative, preparation method and application thereof, and in particular to the use of nucleic acid aptamers as drugs in tumor treatment application.
  • Receptor proteins on the cell membrane are involved in regulating most of the physiological and pathological processes of cells, such as cell cycle, cell communication, cell differentiation, immune response, cell apoptosis, cell proliferation, and cell migration. It is worth noting that in biological research, because receptor proteins on the cell membrane are involved in the occurrence and development of many diseases, for example, complement receptors are often associated with inflammation, and protein tyrosine kinases (PTKs) receptors are often associated with cancer. Relevant, therefore these membrane receptor proteins are often used as targets for drug action. By activating or inhibiting these targets, it helps to treat related diseases.
  • PTKs protein tyrosine kinases
  • the aptamer Since the aptamer interacts with the target in a similar manner to an antibody, it is also called a "chemical antibody". Studies have reported that the aptamer folds into a special three-dimensional structure and binds to the target protein with high affinity, thereby regulating the activity of the target protein. However, this single single-receptor protein regulation method is less efficient, and the over-expressed proteins on some diseased cells will also be expressed on normal cells, which is easy to cause toxic side effects of non-target cells. Therefore, there is an urgent need to develop a new strategy that can efficiently and specifically regulate the activity of receptor proteins.
  • the present invention proposes for the first time the use of bispecific nucleic acid aptamer probes to artificially induce protein pairing, thereby efficiently and specifically regulating the function of receptor proteins, and finally regulating the migration behavior of cells.
  • the selected paired receptor is a membrane protein highly expressed by tumor cells, the constructed bispecific aptamer will not cause functional effects on cells with low or no expression of the protein.
  • the bispecific aptamer has greatly improved the regulation efficiency and specificity of cell surface receptor proteins and cell behavior, reduced non-target cell toxicity, and is of great significance to the advancement of precision diagnosis and treatment, and has great significance Potential for inhibitors of protein function and cell behavior.
  • the technical idea of the present invention is to provide a bispecific nucleic acid aptamer.
  • the bispecific nucleic acid aptamer is composed of two traditional "nucleic acid aptamers” and forms a connecting structure through two "nucleic acid aptamers” , So that two "aptamers” can form a stable bispecific aptamer structure.
  • the technical problems to be solved by the present invention include, but are not limited to, any one or more of the following: how to realize the dual specificity of nucleic acid aptamers; how to realize the function regulation of the nucleic acid aptamers on target receptors or achieve the effect of interfering with receptor-related signal pathways ; How to realize the regulation and control of aptamers on receptors and cell functions; how to realize the treatment of tumor cells, etc.
  • the first aspect of the present invention provides a bispecific nucleic acid aptamer.
  • the nucleic acid aptamer includes a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence includes a target receptor-specific nucleic acid aptamer and a second nucleic acid sequence.
  • a linking part, the second nucleic acid sequence includes a paired receptor-specific nucleic acid aptamer and a second linking portion; the target receptor-specific nucleic acid aptamer can specifically bind to the target receptor, and the paired receptor is specific
  • the sex nucleic acid aptamer can specifically recognize the paired receptor; the first connecting portion and the second connecting portion form a connecting structure.
  • the target receptor is the first transmembrane receptor and the paired receptor is the second transmembrane receptor. body.
  • the receptor activation mode of the target receptor is receptor dimerization, oligomerization or multimerization.
  • the receptor activation mode of the target receptor is ligand-dependent receptor dimerization, oligomerization or multimerization.
  • the binding sites of the ligand, the target receptor specific nucleic acid aptamer and the target receptor are the same or close.
  • the first connecting part and the second connecting part form a connecting structure so that they can be paired with the target receptor-specific nucleic acid aptamer of the same bispecific nucleic acid aptamer.
  • the spatial distance between the target receptor and the paired receptor of the receptor-specific nucleic acid aptamer increases or decreases.
  • first linking part and the second linking part form a linking structure so that the target receptor specific nucleic acid aptamer and the paired receptor specific nucleic acid aptamer of the same bispecific nucleic acid aptamer can be formed respectively.
  • the spatial distance between the target receptor and the paired receptor of the body is reduced.
  • first linking part and the second linking part form a linking structure so that the target receptor specific nucleic acid aptamer and the paired receptor specific nucleic acid aptamer of the same bispecific nucleic acid aptamer can be formed respectively.
  • the spatial distance between the target receptor and the paired receptor of the body is reduced to the target receptor, and the paired receptor forms a steric hindrance that prevents the activation of the target receptor.
  • the first linking portion and the second linking portion form a linking structure so that they can be specific to the target receptor of the same bispecific nucleic acid aptamer.
  • the aptamer, the target receptor of the paired receptor-specific aptamer, and the paired receptor have reduced the spatial distance to the target receptor, and the paired receptor forms a heterodimer.
  • the manner of forming a connecting structure for the connecting portion includes the following: the first connecting portion and the second connecting portion form a double-stranded structure through mutual hybridization, a connecting structure formed by affinity, and a covalent structure. Bonding forms a connecting structure, or the first nucleic acid sequence and the second nucleic acid sequence are directly synthesized into one nucleic acid sequence so that the first connecting portion and the second connecting portion directly form a connecting structure.
  • the target receptor is a tumor cell highly expressed receptor.
  • the paired receptor is a tumor cell highly expressed receptor.
  • the second aspect of the present invention provides the application of the above-mentioned bispecific nucleic acid aptamer in the preparation of drugs or the preparation of protein function regulating molecules.
  • the third aspect of the present invention provides a derivative of the above-mentioned bispecific nucleic acid aptamer.
  • the “derivative” in this aspect refers to the modification of the bispecific nucleic acid aptamer through any chemical reaction without changing the specific recognition performance of the bispecific nucleic acid aptamer.
  • the fourth aspect of the present invention provides the application of the above-mentioned bispecific aptamer derivatives in the preparation of drugs or the regulation of protein functional molecules.
  • the fifth aspect of the present invention provides a method for preparing the above-mentioned bispecific nucleic acid aptamer, and the specific method steps are:
  • the sixth aspect of the present invention provides another method for preparing the above-mentioned bispecific nucleic acid aptamer, and the specific method steps are:
  • the tumor cells are human prostate cancer cell DU145, human cervical cancer cell Hela, human gastric cancer MKN-45 cell, human liver cancer cell HepG2, human non-small cell lung cancer A549, human breast cancer cell MCF-7, human acute lymphocytic leukemia cell (CCRF) -CEM) etc.; specifically, take human prostate cancer cell DU145 as an example.
  • the target receptors are highly expressed transmembrane receptors on the surface of tumor cells, mesenchymal epidermal transformation factor receptor (Met), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), vascular endothelial cells Growth factor receptor (VEGFR), transfer growth factor ⁇ receptor (TGF ⁇ R), tumor necrosis factor receptor (TNFR), etc.; specifically, take Mesenchymal Transforming Factor Receptor (Met) as an example.
  • Met mesenchymal epidermal transformation factor receptor
  • EGFR epidermal growth factor receptor
  • HER2 human epidermal growth factor receptor 2
  • VEGFR vascular endothelial cells Growth factor receptor
  • TGF ⁇ R transfer growth factor ⁇ receptor
  • TNFR tumor necrosis factor receptor
  • the receptor activation mode of the target receptor is ligand-dependent receptor dimerization, oligomerization or multimerization, wherein the ligand is hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular endothelial Cell growth factor (VEGF), transforming growth factor- ⁇ (TGF ⁇ ), tumor necrosis factor (TNF), etc.; specifically, take hepatocyte growth factor (HGF) as an example.
  • HGF hepatocyte growth factor
  • EGF epidermal growth factor
  • VEGF vascular endothelial Cell growth factor
  • TGF ⁇ tumor necrosis factor
  • TGF tumor necrosis factor
  • the paired receptors are highly expressed transmembrane receptors on the surface of tumor cells, transferrin receptor (TfR), nucleolin (Nucleolin), epithelial cell adhesion molecule (EpCAM), mucin (MUC1), protein tyrosine Acid kinase 7 (PTK7), epidermal growth factor receptor (EGFR), etc.; specifically, take transferrin receptor (TfR) as an example.
  • TfR transferrin receptor
  • Nucleolin nucleolin
  • EpCAM epithelial cell adhesion molecule
  • MUC1 mucin
  • PTK7 protein tyrosine Acid kinase 7
  • EGFR epidermal growth factor receptor
  • TfR transferrin receptor
  • the first nucleic acid sequence included in the bispecific nucleic acid aptamer is ATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTGATAAGTAGAACGTTATGACTAA (SEQ ID NO: 1)
  • the second nucleic acid sequence is GGATAGGGATTCTGTTGGTCGGCTGGTTGGTATCCTANO: SEQ ID NO:1 (SEQ ID NO:1)
  • the target receptor specific nucleic acid aptamer and the first connecting part included in the nucleic acid sequence are ATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTGAT (SEQ ID NO: 3) and GTAGAACGTTATGACTAA (SEQ ID NO: 4);
  • the second nucleic acid sequence includes the paired receptor specificity
  • the nucleic acid aptamer and the second connection part are GGATAGGGATTCTGTTGGTCGGCTGGTTGGTATCC (SEQ ID NO: 5) and TTAGTCAT
  • the first linking part and the second linking part hybridize to each other to form an 18bp double-stranded structure so that they can respectively interact with the target receptor Met and the paired receptor TfR of the same bispecific aptamer (Apt-Met-TfR).
  • the spatial distance between them is reduced, forming a heterodimer of Met and TfR, forming a steric hindrance around the Met receptor, hindering the binding of HGF and Met and inducing the formation of Met dimer.
  • the preparation method of the bispecific nucleic acid aptamer is:
  • the invention provides the application of the bispecific nucleic acid aptamer in the preparation of drugs, such as anti-tumor cell proliferation drugs, anti-tumor metastasis drugs, tumor cell apoptosis-inducing drugs and the like.
  • drugs such as anti-tumor cell proliferation drugs, anti-tumor metastasis drugs, tumor cell apoptosis-inducing drugs and the like.
  • the present invention provides derivatives of the bispecific aptamer, including replacing the bispecific aptamer sequence with artificial bases, replacing the bispecific aptamer backbone with phosphorothioate backbone, and replacing the bispecific aptamer sequence with artificial bases.
  • the specific nucleic acid aptamer sequence is transformed into a peptide nucleic acid, and the bispecific nucleic acid aptamer sequence is modified with polyethylene glycol to still have the same function as the derivative of the bispecific nucleic acid aptamer.
  • bispecific nucleic acid aptamers and derivatives are developed for the first time.
  • bispecific aptamer drugs are used to regulate the function of receptors highly expressed on the surface of tumor cells.
  • Bispecific aptamers can specifically recognize two tumor cell highly expressed receptors: target receptor and paired receptor.
  • the double-stranded DNA structure formed by the complementary sequence of the end of the nucleic acid aptamer will change the spatial distance between the two receptors. Due to the proximity of the paired receptors, an obvious steric hindrance is formed around the target receptor, which hinders the binding and activation of the ligand to the target receptor, thereby further affecting cell function.
  • the bispecific nucleic acid aptamer improves the regulation effect of the nucleic acid aptamer on receptors and cell functions. Furthermore, the bispecific nucleic acid aptamer is used to prepare antitumor drugs.
  • Figure 1 is a schematic diagram of the bispecific aptamer drug Apt-Met-TfR regulating receptor function
  • Figure 2 is a schematic diagram of the structure of (A) bispecific aptamer drugs; (B) PAGE gel imaging images of different aptamer drug structures; Lane 1: Apt-Me needle; lane 2: Apt-TfR; lane 3: Apt -Met-TfR; (C) Fluorescence spectra of different aptamer drugs;
  • Figure 3 is a flow cytometry analysis diagram of (A) DU145 cells incubated with different aptamer drugs; (B) laser confocal imaging diagrams of DU145 cells incubated with different aptamer drugs; ruler: 50 ⁇ m;
  • Figure 4 shows the Western blot analysis of Met and p-Met expression levels in DU145 cells
  • Figure 5 shows Western blot analysis of Met and p-Met expression levels after incubation of DU145 cells with different concentrations of the bispecific aptamer drug Apt-Met-TfR (0-50nM);
  • Figure 6 shows Western blot analysis of Met and p-Met expression levels after DU145 cells were incubated with different concentrations of Met aptamer (0-1000nM);
  • Figure 7 shows the Western blot analysis of Met and p-Met expression levels after incubating DU145 cells with different concentrations of Met inhibitor ARQ197 (0-3000nM);
  • Figure 8 shows Western blot analysis of the expression levels of Met, Akt, Erk, p-Met, p-Akt, and p-Erk on (A) DU145 cells and (B) MKN-45 cells;
  • Figure 9 shows Western blot analysis of Met and p-Met expression levels on DU145 cells
  • Figure 10 shows (A) Western blotting and (B) laser co-aggregation imaging analysis of Met protein and TfR protein expression levels on L02 cells and DU145 cells; ruler: 25 ⁇ m;
  • Figure 11 shows the Western blot analysis of Met and p-Met expression levels on L02 cells
  • Figure 12 is a scratch repair experiment to analyze the effect of nucleic acid aptamer drugs on the migration behavior of DU145 cells induced by HGF;
  • Figure 13 shows the effect of nuclear aptamer drugs on the migration behavior of DU145 cells induced by HGF by scattering experiments; ruler: 200 ⁇ m;
  • Figure 14 shows the effect of aptamer drugs on the migration behavior of DU145 cells induced by HGF in the Transwell cell migration experiment
  • Figure 15 is a microscope image of tracking the movement of DU145 cells; scale bar: 100 ⁇ m.
  • the Mesenchymal Epidermal Transformation Factor (Met) receptor protein involved in the occurrence and development of tumor cells is selected as the target receptor in the embodiment of the present invention (the left part in Figure 1).
  • Met receptor is the homologous receptor of hepatocyte growth factor (HGF). HGF activates Met receptor and promotes cell migration by binding to Met receptor and promoting Met receptor to form dimers.
  • HGF hepatocyte growth factor
  • TfR transferrin receptor
  • a bispecific aptamer drug was constructed and named Apt-Met-TfR.
  • the drug After the drug is incubated with the cells, it can simultaneously target the Met receptor and the TfR receptor, and draw the two receptors closer together to induce the two receptors to form an artificial receptor heterodimer. It is worth noting that the proximity of the paired receptor TfR forms an obvious steric hindrance near the target receptor Me, which prevents the binding of HGF ligand to the Met receptor and inhibits the activation of the Met receptor signaling pathway, thereby further Inhibit the migration behavior of cells, and the inhibitory effect is better than that of a single nucleic acid aptamer probe.
  • PBS Phosphate buffered saline
  • MEM MEM medium
  • DMEM DMEM medium
  • RPMI-1640 medium double antibodies (penicillin/streptomycin), fetal bovine serum (FBS), Lipofectamine 3000, Hoechst 33342, and ECL Plus Ultra Sensitive Luminescent Solution Purchased from Life Technologies, USA.
  • protease inhibitors and phosphatase inhibitors were purchased from Roche, USA. Crystal violet and 1% paraformaldehyde were purchased from Beijing Soleibao Technology Co., Ltd.
  • the Met receptor small molecule inhibitor ARQ197 was purchased from AdooQ Bioscience in the United States.
  • Recombinant human HGF factor was purchased from PeproTech, USA.
  • Anti-Met antibody, anti-phospho-Met antibody, anti-Erk antibody, anti-phospho-Erk antibody, anti-Akt antibody, anti-phospho-Akt antibody and anti-rabbit antibody labeled with HRP were all purchased from Cell Signaling Technology in the United States.
  • the human prostate cancer cell line DU145 and the human normal liver cell line L02 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the human gastric cancer MKN-45 cell line was purchased from the Wuhan University cell bank.
  • the mesenchymal epidermal transformation factor (Met) receptor protein involved in the occurrence and development of tumor cells is selected as the target receptor, and the transferrin receptor (TfR) highly expressed by tumor cells is selected as the paired receptor.
  • Complementary DNA sequences are added to the ends of Met aptamer and TfR aptamer, namely monospecific aptamers Apt-Met and Apt-TfR. The two are mixed at a ratio of 1:1, and incubated with shaking at room temperature for 15 minutes to form a double specific Sex aptamer drug Apt-Met-TfR.
  • the Apt-Met or Apt-TfR in Example 1 was directly used as the nucleic acid aptamer drug, and the other experimental conditions were the same.
  • the mesenchymal epidermal transformation factor (Met) receptor protein involved in the occurrence and development of tumor cells is selected as the target receptor, and the highly expressed nucleolin (Nucleolin) of tumor cells is selected as the paired receptor.
  • Complementary DNA sequences are added to the ends of Met's aptamer and Nucleolin's aptamer, namely monospecific aptamers Apt-Met and Apt-Nucleolin, which are mixed at 1:1, and incubated at room temperature for 15 minutes with shaking to form a double specific Sex aptamer drug Apt-Met-Nucleolin.
  • Example 2 The Apt-Met or Apt-Nucleolin in Example 2 was directly used as the nucleic acid aptamer drug, and the other experimental conditions were the same.
  • Example 1 The related nucleic acid aptamer drugs in Example 1, Comparative Example 1, Example 2, and Comparative Example 2 were subjected to the following related experiments:
  • 50nM Apt-Met-Cy5 and 50nM Apt-TfR-Cy3 were incubated with shaking at room temperature for 15 minutes, and the changes in Cy5 fluorescence were detected with a Hitachi F-4600 fluorescence spectrophotometer.
  • the excitation wavelength set by the instrument is 520nm, and the emission wavelength detected is 540-750nm.
  • Apt-Met and Apt-TfR were mixed 1:1, and incubated at room temperature for 15 minutes with shaking. Electrophoresis was performed with PAGE gel in 1 ⁇ TBE buffer. After electrophoresis, the gel was stained with GelRed nucleic acid staining solution, and the gel imager was used for imaging analysis.
  • DU145 cells were starved and cultured in MEM starvation solution containing 0.5% BSA for 24 hours in advance. After starvation, the culture medium was discarded, the cells were washed twice with PBS, and the cells were digested with 0.2% Na 2 EDTA and collected in a 1.5 mL centrifuge tube. The cell count was about 3 ⁇ 10 5 cells per sample. The cells were incubated with 50 nM Apt-Met-Cy5, Apt-TfR-Cy3 probes, Apt-Met-Cy5 and Apt-TfR-Cy3 for 30 minutes at room temperature with shaking. After incubation, the cells were washed 3 times with PBS and analyzed with a FACS CantoTM II flow cytometer. The experimental data was analyzed and processed with FlowJo 7.6 software.
  • the cells were seeded in a 35mm confocal culture dish in advance. After the cells adhered to the wall, the culture medium was replaced with 0.5% BSA-containing MEM medium and starved for 24 hours.
  • BSA-containing MEM medium 0.5% BSA-containing MEM medium and starved for 24 hours.
  • FRET FRET on cell surface DNA probes
  • After cell starvation culture incubate with 50nM Apt-Met-Cy5 probe, Apt-TfR-Cy3, Apt-Met-Cy5 and Apt-TfR-Cy3 for 30 minutes at room temperature. , Wash the cells 3 times with PBS, and use a laser scanning confocal microscope for imaging analysis.
  • the cells were seeded in a 6-well plate 24 hours in advance, and then starved and cultured in a starvation solution (MEM medium + 0.5% BSA) for 24 hours.
  • the cells were incubated with different concentrations of Apt-Met, Apt-TfR or Apt-Met-TfR for 15 minutes at room temperature, and then 30ng/mL HGF was added and incubated for 30 minutes at room temperature.
  • ARQ197 a small molecule inhibitor of Met receptor
  • cells were incubated with ARQ197 of different concentrations for 24 hours, and then 30ng/mL HGF was added and incubated for 30 minutes.
  • the cells were lysed with RIPA lysis buffer (containing protease inhibitor and phosphatase inhibitor).
  • RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor.
  • the protein extracted after cell lysis was separated with 8% SDS-polyacrylamide gel, and the separated protein was transferred to PVDF membrane, the corresponding strips were cut, and sealed with 5% milk powder/TBST for 2 hours.
  • the PVDF membrane was incubated with Met antibody, phosphorylated Met antibody, Erk antibody, phosphorylated Erk antibody, Akt antibody, and phosphorylated Akt antibody at 4°C overnight, and washed 3 times with 1 ⁇ TBST. Then incubate with HRP-labeled anti-rabbit antibody for 1 hour at room temperature, wash 3 times with 1 ⁇ TBST, add ECL Plus ultra-sensitive luminescent solution, and perform imaging analysis with a gel imager.
  • the cells were processed differently: 1) Cells were cultured in starvation solution; 2) Cells were incubated with 30ng/mL HGF for 12 hours; 3) Cells were incubated with 50nM Apt-Met for 15 minutes, and then 30ng/mL HGF was added to continue Incubate for 12 hours; 4) Incubate the cells with 50 nM Apt-Met-TfR for 15 minutes, then add 30 ng/mL HGF and incubate for 12 hours. During the incubation period, the cells were recorded with 0 hour, 4 hour and 12 hour scratch area change images, and the image analysis software Image J was used to analyze the scratch area.
  • DU145 cells were seeded in the upper chamber of Transwell in advance. After the cells adhered, the starvation solution was replaced and cultured for 24 hours. Then the cells were processed differently: 1) Cells were cultured in starvation solution; 2) Cells were incubated with 30ng/mL HGF for 12 hours; 3) Cells were incubated with 50nM Apt-Met for 15 minutes, and then 30ng/mL HGF was added to continue Incubate for 12 hours; 4) Incubate the cells with 50 nM Apt-Met-TfR for 15 minutes, then add 30 ng/mL HGF and incubate for 12 hours.
  • the DU145 cells were seeded in a 6-well plate. The number of cells in each well was about 1000 cells. After 3 days of culture, the complete culture medium was replaced with a starvation medium. After 24 hours of starvation culture, the cells are treated differently: 1) Cells are cultured continuously in starvation solution; 2) Cells are incubated with 30ng/mL HGF for 12 hours; 3) Cells are incubated with 50nM Apt-Met for 15 minutes, and then added 30ng/mL HGF continues to incubate for 12 hours; 4) Cells are incubated with 50nM Apt-Met-TfR for 15 minutes, and then 30ng/mL HGF is added to incubate for 12 hours. After the incubation, observe the distribution of cells with an inverted fluorescence microscope.
  • the DU145 cells were seeded in a 35mm confocal culture dish, and after the cells adhered to the wall, they were cultured under starvation for 24 hours. Stain the cell nucleus with Hoechst 33342. After washing twice with PBS, the cells were incubated with 50nM Apt-Met or 50nM Apt-Met-TfR for 15 minutes, and then 30ng/mL HGF was added to incubate for 2 hours, and real-time imaging was performed with a rotating disc confocal, imaging every 20 minutes . Statistics and analysis of single cell migration trajectory were performed with Nikon NIS-Elements software.
  • the bispecific aptamer drug Apt-Met-TfR is composed of aptamer (Apt-Met) targeting Met receptor and aptamer (Apt-TfR) targeting TfR. The ends of these two aptamers are extended respectively. A sequence of 18 bases that can be complementary to each other, so that Apt-Met and Apt-TfR can form a stable bispecific aptamer Apt-Met-TfR (Figure 2A). The formation of Apt-Met-TfR was investigated by polyacrylamide gel electrophoresis and fluorescence spectroscopy. When the two aptamers are incubated together, hybridization probes are formed, the DNA migration rate slows down (Figure 2B), and FRET signals can be generated ( Figure 2C).
  • Example 2 in order to further investigate the versatility of this strategy for other paired proteins, we selected nucleolin highly expressed by tumor cells instead of TfR as another paired receptor model, and designed the bispecific aptamer drug Apt-Met -Nucleolin. When Apt-Met-Nucleolin is incubated with DU145 cells, the nucleolin and Met protein are brought closer to form an artificial receptor heterodimer which inhibits the level of Met phosphorylation and Apt-Met-TfR on Met phosphorylation. The level of inhibitory effect is similar ( Figure 9), indicating the versatility of our proposed strategy for other paired receptors.
  • the IC50 value of the probe's inhibitory effect on L02 cells' Met phosphorylation level was 250 nM ( Figure 11), which was about the inhibition of Met phosphorylation level in DU145 cells with high TfR expression IC50 is 12 times, indicating that the probe only has a significant inhibitory effect on the Met signaling pathway of cells with high TfR expression, while it has less effect on the Met signaling pathway of cells with low TfR expression.
  • the needle can specifically act on the target cell, causing less toxic and side effects on other cells.
  • the HGF/Met signaling pathway is involved in the regulation of cell migration, and cell migration is often related to tumor cell metastasis.
  • Apt-Met-TfR the bispecific aptamer drug Apt-Met-TfR can inhibit cell migration caused by the activation of the Met signaling pathway.
  • Figure 12 we verify it through a scratch experiment. As shown in Figure 12, after DU145 cells are treated with Apt-Met-TfR, the scratch healing speed is significantly slower than that of cells treated with Apt-Met and without probe. At the same time, we also use the scattering experiment to investigate it. As shown in Figure 13, when DU145 cells are not stimulated, the cells form agglomerated communities; when the cells are stimulated with HGF, the cell migration ability increases, and the cell agglomerated communities disperse.
  • a bispecific aptamer drug to artificially induce receptor pairing to form an artificial receptor heterodimer, and regulate cell migration behavior by regulating the signaling pathway of Met receptor.
  • the probe not only inhibits the Met receptor signaling pathway more efficiently than monospecific aptamer drugs, but also acts more specifically on target cells, avoiding the impact on non-target cells.
  • DNA as a regulatory element in this study also makes the design of the regulatory strategy simpler and has broader versatility in application. It can be applied to various types of receptors by simply changing the sequence of the nucleic acid aptamer. Function regulation. Therefore, the bispecific aptamer drugs we constructed have broad application prospects in the regulation of cell signaling pathways and cell behavior, and are expected to become potential inhibitors of cell functions (such as cell growth, migration, and differentiation).

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Abstract

提供了一种双特异性核酸适体、衍生物、制备方法及其应用，该核酸适体包括第一核酸序列、第二核酸序列；第一核酸序列包括靶标受体特异性核酸适体和第一连接部分，第二核酸序列包括配对受体特异性核酸适体和第二连接部分；靶标受体特异性核酸适体可与靶标受体特异性结合，配对受体特异性核酸适体可与配对受体特异性识别；第一连接部分与第二连接部分形成连接结构。

Description

一种双特异性核酸适体、衍生物、制备方法及其应用
本申请要求于2019年07月03日提交中国专利局、申请号为201910596386.X、发明名称为“一种双特异性核酸适体、衍生物、制备方法及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及核酸适体领域、核酸适体药物、蛋白功能调控领域，具体为一种双特异性核酸适体、衍生物、制备方法及其应用，尤其涉及核酸适体作为药物在肿瘤治疗中的应用。
背景技术
细胞膜表面的受体蛋白参与调控细胞的大多数生理和病理过程，如细胞周期、细胞交流、细胞分化、免疫应答、细胞凋亡、细胞增殖以及细胞迁移等。值得注意的是，在生物学的研究中，由于细胞膜表面的受体蛋白参与了许多疾病的发生和发展，例如补体受体常与炎症有关，蛋白酪氨酸激酶(PTKs)受体常与癌症有关，因此这些膜受体蛋白也常被用作药物作用的靶点，通过激活或者抑制这些靶点有助于相关疾病的治疗。
目前，已发展了许多用于调控细胞表面受体蛋白活性的技术，如利用超声、磁、光等都可以调控细胞的信号通路。但这些物理的调控方式往往是作用于整个细胞，缺乏分子特异性和时空分辨率，对细胞的损伤较大。为了克服这些缺陷，许多研究报道了利用小分子或者单克隆抗体代替物理刺激调控蛋白功能。但许多合成的小分子发挥作用的靶点都不止一个，会同时影响多个信号通路，产生副作用；而单克隆抗体成本高、免疫原性高、热稳定性差等缺点，也大大限制了其在生物医学等研究领域的应用。
因核酸适体与靶标作用的形式与抗体类似，也被称为“化学抗体”。有研究报道，核酸适体通过折叠成特殊的三维结构与靶标蛋白进行高亲和性结合，从而调控靶标蛋白的活性。但是这种单一的单受体蛋白调控方式效率较低，且一些疾病细胞上过表达的蛋白在正常细胞上也会有表达，易造成非靶标细胞的毒副作用。因此迫切需要发展一种新的策略，能够高效并特异性地调控受体蛋白 的活性。
细胞表面会同时表达多种膜受体蛋白，因此同时靶向细胞表面多个受体蛋白能够提高对细胞功能调控的特异性，从而提高疾病诊断和治疗的准确性。本发明首次提出了利用双特异性核酸适体探针人工诱导蛋白配对，从而高效并特异性地调控受体蛋白的功能，最终调控细胞的迁移行为。此外，由于选用的配对受体是肿瘤细胞高表达的膜蛋白，因此所构建的双特异性核酸适体对于该蛋白表达量低或者不表达的细胞不会造成功能影响。总而言之，该双特异性核酸适体在细胞表面受体蛋白和细胞行为的调控效率和特异性方面有较大的提高，降低了非靶细胞毒性，对精准诊疗的推进具有重要意义，且具有作为蛋白功能和细胞行为抑制剂的潜能。
发明内容
本发明的技术构思在于：提供一种双特异性核酸适体，双特异性核酸适体由两个传统意义上的“核酸适体”复合而成，通过两个“核酸适体”形成连接结构，从而使两个“核酸适体”能够形成稳定的双特异性核酸适体结构。
本发明要解决的技术问题包括但不限于以下任一个或多个：如何实现核酸适体的双特异性；如何实现核酸适体对靶标受体的功能调控或达到干扰受体相关信号通路的效果；如何实现核酸适体对受体及细胞功能的调控作用；如何实现肿瘤细胞的治疗等。
为解决以上任一个或多个技术问题，本发明提供的具体技术方案如下。
本发明的第一方面提供了一种双特异性核酸适体，所述核酸适体包括第一核酸序列、第二核酸序列；所述第一核酸序列包括靶标受体特异性核酸适体和第一连接部分，所述第二核酸序列包括配对受体特异性核酸适体和第二连接部分；所述靶标受体特异性核酸适体可与靶标受体特异性结合，所述配对受体特异性核酸适体可与配对受体特异性识别；所述第一连接部分与所述第二连接部分形成连接结构。
对于靶标受体的分布，进一步的，本技术方案针对跨膜受体的作用更为明显和简易，所述靶标受体为第一跨膜受体、所述配对受体为第二跨膜受体。
对于靶标受体的激活方式，进一步的，所述靶标受体的受体激活方式为受 体二聚化、寡聚化或多聚化。
更进一步的，所述靶标受体的受体激活方式为配体依赖的受体二聚化、寡聚化或多聚化。
更进一步的，为了进一步提高作用效果，尤其是竞争性阻碍的效果，所述配体、所述靶标受体特异性核酸适体与所述靶标受体的结合位点相同或接近。
对于杂交部分的长度要求，进一步的，所述第一连接部分与所述第二连接部分形成连接结构可使得分别与同一所述双特异性核酸适体的靶标受体特异性核酸适体、配对受体特异性核酸适体的靶标受体、配对受体的空间距离增加或缩小。
更进一步的，所述第一连接部分与所述第二连接部分形成连接结构可使得分别与同一所述双特异性核酸适体的靶标受体特异性核酸适体、配对受体特异性核酸适体的靶标受体、配对受体的空间距离缩小。
更进一步的，所述第一连接部分与所述第二连接部分形成连接结构可使得分别与同一所述双特异性核酸适体的靶标受体特异性核酸适体、配对受体特异性核酸适体的靶标受体、配对受体的空间距离缩小至靶标受体、配对受体形成阻碍所述靶标受体激活的空间位阻。
更进一步的，为使空间位阻的阻碍效果最大化，所述第一连接部分与所述第二连接部分形成连接结构可使得分别与同一所述双特异性核酸适体的靶标受体特异性核酸适体、配对受体特异性核酸适体的靶标受体、配对受体的空间距离缩小至靶标受体、配对受体形成异二聚体。
可选择的，针对连接部分形成连接结构的方式包括以下几种：所述第一连接部分与所述第二连接部分通过相互杂交形成双链结构、通过亲和作用形成的连接结构、通过共价键连接形成连接结构，或者所述第一核酸序列、第二核酸序列直接合成为一条核酸序列使得所述第一连接部分与所述第二连接部分直接形成连接结构。
针对特定的应用时，更优的，所述靶标受体为肿瘤细胞高表达受体。
针对特定的应用时，为进一步提高技术效果，所述配对受体为肿瘤细胞高表达受体。
本发明的第二方面提供了上述的双特异性核酸适体在制备药物或制备蛋 白功能调控分子中的应用。
本发明的第三方面提供了上述的双特异性核酸适体的衍生物。
本方面中所谓的“衍生物”是指在不改变上述双特异性核酸适体的特异性识别性能的基础上，通过任意化学反应实现的对上述双特异性核酸适体的修饰。
本发明的第四方面提供了上述的双特异性核酸适体的衍生物在制备药物或制备蛋白功能分子调控中的应用。
本发明的第五方面提供了上述的双特异性核酸适体的制备方法，具体方法步骤为，
s1.合成所述第一核酸序列；
s2.合成所述第二核酸序列；
s3.使所述第一连接部分与所述第二连接部分相互杂交形成双链结构、通过亲和作用形成的连接结构或者通过共价键连接形成连接结构；
s4.得到所述双特异性核酸适体。
本发明的第六方面提供了上述的双特异性核酸适体的另一制备方法，具体方法步骤为，
s1.确定所述第一核酸序列；
s2.确定所述第二核酸序列；
s3.将所述第一核酸序列、第二核酸序列直接合成为一条核酸序列使得所述第一连接部分与所述第二连接部分直接形成连接结构；
s4.得到所述双特异性核酸适体。
尤其更进一步的，针对本发明提供的第一至第五方面的内容，列举相关更进一步的实例性内容如下：
所述肿瘤细胞为人前列腺癌细胞DU145、人宫颈癌细胞Hela、人胃癌MKN-45细胞、人肝癌细胞HepG2、人非小细胞肺癌A549、人乳腺癌细胞MCF-7、人急性淋巴白血病细胞(CCRF-CEM)等；具体的，以人前列腺癌细胞DU145为例。
所述靶标受体为肿瘤细胞表面高表达跨膜受体，间质表皮转化因子受体(Met)、表皮生长因子受体(EGFR)、人类表皮生长因子受体2(HER2)、 血管内皮细胞生长因子受体(VEGFR)、转移生长因子β受体(TGFβR)、肿瘤坏死因子受体(TNFR)等；具体的，以间质表皮转化因子受体(Met)为例。
所述靶标受体的受体激活方式为配体依赖的受体二聚化、寡聚化或多聚化，其中配体为肝细胞生长因子(HGF)、表皮生长因子(EGF)、血管内皮细胞生长因子(VEGF)、转化生长因子-β(TGFβ)、肿瘤坏死因子(TNF)等；具体的，以肝细胞生长因子(HGF)为例。
所述配对受体为肿瘤细胞表面高表达跨膜受体，转铁蛋白受体(TfR)、核仁素(Nucleolin)、上皮细胞粘附分子(EpCAM)、粘蛋白(MUC1)、蛋白质酪氨酸激酶7(PTK7)、表皮生长因子受体(EGFR)等；具体的，以转铁蛋白受体(TfR)为例。
所述双特异性核酸适体(Apt-Met-TfR)中包括的第一核酸序列为ATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTGATAAGTAGAACGTTATGACTAA(SEQ ID NO:1)，第二核酸序列为GGATAGGGATTCTGTTGGTCGGCTGGTTGGTATCCTTTAGTCATAACGTTCTAC(SEQ ID NO:2)；所述第一核酸序列包括的靶标受体特异性核酸适体和第一连接部分分别为ATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTGAT(SEQ ID NO:3)和GTAGAACGTTATGACTAA(SEQ ID NO:4)；所述第二核酸序列包括的配对受体特异性核酸适体和第二连接部分分别为GGATAGGGATTCTGTTGGTCGGCTGGTTGGTATCC(SEQ ID NO:5)和TTAGTCATAACGTTCTAC(SEQ ID NO:6)。
所述第一连接部分与所述第二连接部分相互杂交形成18bp双链结构可使得分别与同一所述双特异性核酸适体(Apt-Met-TfR)的靶标受体Met、配对受体TfR之间的空间距离缩小，形成Met与TfR的异二聚体，在Met受体周围形成空间位阻，阻碍HGF与Met结合并诱导形成Met二聚体。
所述双特异性核酸适体的制备方法为：
s1.合成所述第一核酸序列ATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTGATAAGTAGAACGTTATGACTAA(SEQ ID NO:1)；
s2.合成所述第二核酸序列GGATAGGGATTCTGTTGGTCGGCTGGTTGGTATCCTTTAGTCATAACGTTCTAC(SEQ ID NO:2)；
s3.在磷酸缓冲液中按1：1的摩尔浓度比例混合第一核酸序列与第二核酸序列；
s4.在25℃下反应15分钟得到所述双特异性核酸适体Apt-Met-TfR。
本发明提供了所述的双特异性核酸适体在制备药物中的应用，如抗肿瘤细胞增殖药物、抗肿瘤转移药物、诱导肿瘤细胞凋亡药物等。
本发提供了所述的双特异性核酸适体的衍生物，包括将双特异性核酸适体序列用人造碱基代替、将双特异性核酸适体骨架用硫代磷酸酯骨架代替、将双特异性核酸适体序列改造为肽核酸、将双特异性核酸适体序列用聚乙二醇修饰后仍具有与所述双特异性核酸适体相同功能的衍生物。
本发明的有益效果在于：首次开发双特异性核酸适体、衍生物的制备方法及应用。进一步的，将双特异性核酸适体药物用于调控肿瘤细胞表面高表达的受体功能。双特异性核酸适体能特异性识别两种肿瘤细胞高表达受体：靶标受体和配对受体。通过核酸适体末端互补序列形成的双链DNA结构等连接结构的形式将改变两个受体的空间距离。由于配对受体的靠近，在靶标受体周围形成明显的空间位阻，阻碍了配体对靶标受体的结合与激活，从而进一步影响细胞功能。该双特异性核酸适体提高了核酸适体对受体及细胞功能的调控作用。再进一步的，将双特异性核酸适体用于制备抗肿瘤药物。
附图说明
图1为双特异性核酸适体药物Apt-Met-TfR调控受体功能的示意图；
图2为(A)双特异性核酸适体药物结构示意图；(B)不同核酸适体药物结构的PAGE胶成像图；Lane 1：Apt-Me针；lane 2：Apt-TfR；lane 3：Apt-Met-TfR；(C)不同核酸适体药物的荧光光谱图；
图3为(A)DU145细胞与不同的核酸适体药物孵育的流式细胞术分析图；(B)DU145细胞与不同的核酸适体药物孵育的激光共聚焦成像图；标尺：50μm；
图4为蛋白质印迹分析DU145细胞Met和p-Met的表达水平；
图5为蛋白质印迹分析DU145细胞与不同浓度的双特异性核酸适体药物Apt-Met-TfR(0-50nM)孵育后的Met和p-Met的表达水平；
图6为蛋白质印迹分析DU145细胞与不同浓度的Met核酸适体(0-1000nM)孵育后的Met和p-Met的表达水平；
图7为蛋白质印迹分析DU145细胞与不同浓度的Met抑制剂ARQ197(0-3000nM)孵育后的Met和p-Met的表达水平；
图8为蛋白质印迹分析(A)DU145细胞和(B)MKN-45细胞上Met、Akt、Erk、p-Met、p-Akt、p-Erk的表达水平；
图9为蛋白质印迹分析DU145细胞上Met和p-Met的表达水平；
图10为(A)蛋白质印迹和(B)激光共聚集成像分析L02细胞和DU145细胞上Met蛋白和TfR蛋白的表达水平；标尺：25μm；
图11为蛋白质印迹分析L02细胞上Met和p-Met的表达水平；
图12为划痕修复实验分析核酸适体药物对HGF诱导的DU145细胞迁移行为的影响；
图13为散射实验分析核核酸适体药物对HGF诱导的DU145细胞迁移行为的影响；标尺：200μm；
图14为Transwell细胞迁移实验分析核酸适体药物对HGF诱导的DU145细胞迁移行为的影响；
图15为对DU145细胞运动情况追踪的显微镜成像图；标尺：100μm。
具体实施方式
以下，使用实施例和对比例更加详细地说明本发明的具体实施示例，本发明的技术范围不限于以下实施例。
为直观的表现本发明实施例与对比例的技术内容，参见图1做如下解释：
如图1所示，本发明实施例(图1中左侧部分)中选用参与肿瘤细胞发生发展过程的间质表皮转化因子(Met)受体蛋白作为靶标受体。Met受体是肝细胞生长因子(HGF)的同源受体，HGF通过与Met受体结合并促进Met受体形成二聚体从而激活Met受体，促进细胞发生迁移。此外，选用肿瘤细胞高表达的转铁蛋白受体(TfR)作为配对受体。构建双特异性核酸适体药物，命 名为Apt-Met-TfR。该药物与细胞孵育后能够同时靶向Met受体和TfR受体，并将这两个受体拉近，诱导这两个受体形成人工受体异二聚体。值得注意的是，配对受体TfR的靠近，在靶标受体Me附近形成一个明显的空间位阻，阻碍了HGF配体与Met受体的结合，抑制了Met受体信号通路的激活，从而进一步抑制细胞的迁移行为，且该抑制效果较单条核酸适配体探针的效果更好。
本具体实施方式中涉及的实验用品及相关验证方法如下：
化学试剂：实验用水均为Milli-Q Integarl纯水/超纯水一体化系统净化的超纯水(18.2MΩ·cm)。实验所用DNA均由生工生物工程(上海)股份有限公司合成并纯化。GelRed核酸染色液、RIPA裂解液和BCA蛋白定量试剂盒均购买于碧云天生物技术有限公司。磷酸缓冲液(PBS)、MEM培养基、DMEM培养基、RPMI-1640培养基、双抗(青霉素/链霉素)、胎牛血清(FBS)、Lipofectamine 3000、Hoechst 33342以及ECL Plus超敏发光液购买于美国Life Technologies公司。蛋白酶抑制剂与磷酸酶抑制剂均购买自美国Roche公司。结晶紫和1％多聚甲醛购买于北京索莱宝科技有限公司。Met受体小分子抑制剂ARQ197购买于美国AdooQ Bioscience公司。重组人HGF因子购买于美国PeproTech公司。抗Met抗体、抗磷酸化Met抗体、抗Erk抗体,抗磷酸化Erk抗体、抗Akt抗体、抗磷酸化Akt抗体以及标记HRP的抗兔抗体均购买于美国Cell Signaling Technology公司。人前列腺癌细胞系DU145和人正常肝细胞系L02购买自中科院上海细胞库，人胃癌MKN-45细胞系购买自武汉大学细胞库。
实施例1
选用参与肿瘤细胞发生发展过程的间质表皮转化因子(Met)受体蛋白作为靶标受体，选用肿瘤细胞高表达的转铁蛋白受体(TfR)作为配对受体。Met的核酸适体和TfR的核酸适体末端分别添加互补DNA序列，即单特异性核酸适体Apt-Met与Apt-TfR，二者按1：1混合，室温振荡孵育15分钟，形成双特异性核酸适体药物Apt-Met-TfR。
本实施例1中Apt-Met和Apt-TfR的序列参见表1。
对比例1
直接以实施例1中的Apt-Met或Apt-TfR单独作为核酸适体药物，其余实验条件均相同。
表1 实施例1与对比例1中所用的DNA序列
Figure PCTCN2020079139-appb-000001
实施例2
选用参与肿瘤细胞发生发展过程的间质表皮转化因子(Met)受体蛋白作为靶标受体，选用肿瘤细胞高表达的核仁素(Nucleolin)作为配对受体。Met的核酸适体和Nucleolin的核酸适体末端分别添加互补DNA序列，即单特异性核酸适体Apt-Met与Apt-Nucleolin，二者按1：1混合，室温振荡孵育15分钟，形成双特异性核酸适体药物Apt-Met-Nucleolin。
本实施例2中Apt-Met和Apt-Nucleolin的序列参见表2。
对比例2
直接以实施例2中的Apt-Met或Apt-Nucleolin单独作为核酸适体药物，其余实验条件均相同。
表2 实施例2和对比例2中采用的的DNA序列
Figure PCTCN2020079139-appb-000002
(一)相关实验过程
将实施例1、对比例1、实施例2、对比例2中的相关核酸适体药物进行以下相关实验：
A.荧光检测
50nM的Apt-Met-Cy5与50nM Apt-TfR-Cy3室温振荡孵育15分钟，用日立F-4600荧光分光光度计对Cy5荧光的变化情况进行检测。仪器设定的激发波长为520nm，检测的发射波长为540-750nm。
B.聚丙烯酰胺凝胶电泳(PAGE)分析：
Apt-Met与Apt-TfR按1：1混合，室温振荡孵育15分钟。在1×TBE缓冲液中用PAGE胶进行电泳。电泳后凝胶用GelRed核酸染色液染色，并用凝胶成像仪进行成像分析。
C.流式细胞术分析
DU145细胞提前在含有0.5％BSA的MEM饥饿液中饥饿培养24小时。饥饿后，弃除培养基，PBS洗涤细胞2次，用0.2％Na 2EDTA消化细胞，并收集于1.5mL离心管中，细胞计数，每个样细胞数约为3×10 5个细胞。细胞分别与50nM Apt-Met-Cy5，Apt-TfR-Cy3探针以及Apt-Met-Cy5和Apt-TfR-Cy3室温振荡孵育30分钟。孵育后，用PBS洗涤细胞3次，用FACS CantoTMⅡ流式细胞分析仪分析。实验数据用FlowJo 7.6软件进行分析处理。
D.共聚焦荧光成像
细胞提前接种于35mm的共聚焦培养皿中，待细胞贴壁后，培养液换成 含有0.5％BSA的MEM培养液饥饿培养24小时。对于细胞表面DNA探针发生FRET的成像分析实验，细胞饥饿培养后，分别与50nM Apt-Met-Cy5探针，Apt-TfR-Cy3以及Apt-Met-Cy5和Apt-TfR-Cy3室温孵育30分钟，PBS洗涤细胞3次，用激光扫描共聚焦显微镜进行成像分析。
E.蛋白质印迹分析
细胞提前24小时接种于6孔板中，随后在饥饿液(MEM培养基+0.5％BSA)中饥饿培养24小时。
对于核酸适体药物的抑制实验，细胞先与不同浓度的Apt-Met、Apt-TfR或者Apt-Met-TfR室温孵育15分钟，然后再加入30ng/mL HGF室温再孵育30分钟。
对于Met受体的小分子抑制剂ARQ 197的抑制实验，细胞先与不同浓度的ARQ 197孵育24小时，然后再加入30ng/mL HGF孵育30分钟。
孵育结束后，用RIPA裂解液(含蛋白酶抑制剂和磷酸酶抑制剂)裂解细胞。细胞裂解后所提取的蛋白用8％SDS-聚丙烯酰胺凝胶进行分离，并将分离后的蛋白转移到PVDF膜上，剪下相应的条带，用5％奶粉/TBST封闭2小时。封闭后，PVDF膜分别与Met的抗体、磷酸化Met的抗体、Erk的抗体、磷酸化Erk的抗体、Akt的抗体、磷酸化Akt的抗体在4℃中孵育过夜，1×TBST洗3次，再与标记HRP的抗兔抗体室温孵育1小时，1×TBST洗3次，加入ECL Plus超敏发光液，并用凝胶成像仪进行成像分析。
F.细胞划痕修复实验
提前24小时将DU145细胞接种于12孔板中，培养至细胞完全贴壁并铺满孔底。在饥饿液中培养24小时后，用无菌的20μL枪头尖端在孔底中央均匀地划一条直线，用D-PBS冲洗细胞3次，除去漂浮的细胞碎片，更换新鲜的饥饿液，并用倒置显微镜对划痕区域进行成像，该划痕面积即为t＝0时刻的划痕面积。随后对细胞进行不同的处理：1)细胞在饥饿液中继续培养；2)细胞与30ng/mL HGF孵育12小时；3)细胞先与50nM Apt-Met孵育15分钟，再加入30ng/mL HGF继续孵育12小时；4)细胞先与50nM Apt-Met-TfR孵育15分钟，再加入30ng/mL HGF继续孵育12小时。细胞在孵育期间分别记录0小时，4小时和12小时的划痕区域变化图像，并用Image J图像分析软件 进行划痕面积的分析。
G.Transwell细胞迁移实验
DU145细胞提前接种于Transwell的上室中，待细胞贴壁后，更换饥饿液，培养24小时。随后对细胞进行不同的处理：1)细胞在饥饿液中继续培养；2)细胞与30ng/mL HGF孵育12小时；3)细胞先与50nM Apt-Met孵育15分钟，再加入30ng/mL HGF继续孵育12小时；4)细胞先与50nM Apt-Met-TfR孵育15分钟，再加入30ng/mL HGF继续孵育12小时。孵育结束后，用湿棉签轻轻擦拭除去上室内的细胞，PBS洗涤2次，1％多聚甲醛固定，0.1％结晶紫染色，PBS洗涤，用倒置荧光显微镜观察并计数，以迁移至滤膜下表面的细胞数目差异来区别DU145细胞的迁移能力。
H.细胞散射实验
将DU145细胞接种于6孔板中，每个孔的细胞数约为1000个细胞，培养3天后，将完全培养液更换为饥饿液。饥饿培养24小时后，对细胞进行不同的处理：1)细胞在饥饿液中继续培养；2)细胞与30ng/mL HGF孵育12小时；3)细胞先与50nM Apt-Met孵育15分钟，再加入30ng/mL HGF继续孵育12小时；4)细胞先与50nM Apt-Met-TfR孵育15分钟，再加入30ng/mL HGF继续孵育12小时。孵育结束后，用倒置荧光显微镜观察细胞的分布情况。
J.单细胞迁移实验
将DU145细胞接种于35mm的共聚焦培养皿中，待细胞贴壁后，继续饥饿培养24小时。用Hoechst 33342对细胞核进行染色。PBS洗2次后，细胞与50nM Apt-Met或者50nM Apt-Met-TfR孵育15分钟后，再加入30ng/mL HGF继续孵育2小时，并用转盘式共聚焦进行实时成像，每隔20分钟成像一次。用尼康NIS-Elements软件对单细胞迁移轨迹进行统计和分析。
(二)相关实验结果
A.双特异性核酸适体药物Apt-Met-TfR的构建和表征：
双特异性核酸适体药物Apt-Met-TfR是由靶向Met受体的aptamer(Apt-Met)和靶向TfR的aptamer(Apt-TfR)组成，这两条核酸适体的末端各延长出一段18个碱基能够相互互补的序列，从而使Apt-Met和Apt-TfR能够形成稳定的双特异性核酸适体Apt-Met-TfR(图2A)。通过聚丙烯酰胺凝胶 电泳和荧光光谱考察了Apt-Met-TfR的形成。当两条核酸适体共同孵育后，形成杂交探针，DNA的迁移速率变慢(图2B)，且能产生FRET信号(图2C)，结果表明在溶液中Apt-Met和Apt-TfR能够相互杂交，形成稳定的Apt-Met-TfR，即双特异性核酸适体药物Apt-Met-TfR构建成功。为了考察构建的Apt-Met-TfR在细胞膜表面是否依然具有受体识别的能力，我们通过流式细胞术和共聚焦证实了当细胞与Apt-Met-TfR孵育时，在细胞上能够同时检测到Apt-Met的Cy5信号和Apt-TfR的Cy3信号(图3)，说明我们构建的Apt-Met-TfR在细胞膜表面依然具有受体识别的能力。以上实验结果说明了我们设计的Apt-Met和Apt-TfR能够高效地杂交成Apt-Met-TfR，并且在细胞膜表面依然具有高特异性的靶向识别能力。
B.双特异性核酸适体药物Apt-Met-TfR对Met受体信号通路的抑制作用：
我们假设双特异性核酸适体药物Apt-Met-TfR将Met受体和TfR受体拉近形成人工诱导的受体异二聚体，能够抑制HGF引起的Met受体二聚化，从而抑制Met受体信号通路的激活。为了验证这个假设，我们首先通过蛋白质印迹分析考察了不同处理条件下Met磷酸化水平的差异。如图4所示，当细胞与Apt-Met-TfR预孵育后，再与HGF孵育，Met磷酸化的水平明显降低。而当细胞与Apt-Met或者Apt-TfR孵育后，再与HGF孵育，Met磷酸化水平与只用HGF处理的相当，没有起到抑制Met磷酸化的作用。同时，我们也对比了不同处理对Met磷酸化抑制的IC50值的差异。双特异性核酸适体药物Apt-Met-TfR对Met磷酸化水平抑制的IC50值约为20nM(图5)，远远低于单特异性核酸适体药物Apt-Met(IC50约为750nM)(图6)和ARQ197(IC50约为1000nM)(图7)对Met磷酸化水平抑制的IC50值。此外，我们还考察了该策略在其他表达Met和高表达TfR的细胞系(如Hela细胞系)上的可行性。以上实验结果说明，双特异性核酸适体药物Apt-Met-TfR能够高效地将Met受体和TfR受体拉近形成人工诱导的受体异二聚体，抑制HGF引起的Met受体二聚化，从而抑制Met受体信号通路的激活，且该抑制效果明显优于单特异性核酸适体药物Apt-Met起到的抑制效果。
接下来我们考察了双特异性核酸适体药物Apt-Met-TfR对Met受体下游信号通路(如Akt和Erk)的抑制作用(图8A)。当DU145细胞与Apt-Met-TfR 孵育后，细胞内Akt和Erk分子的磷酸化水平明显降低，而与单特异性核酸适体药物Apt-Met或者Apt-TfR孵育后，细胞内Akt和Erk分子的磷酸化水平几乎没有改变。另外，值得注意的是，Apt-Met-TfR不能抑制配体不依赖性的受体激活。在本研究中，我们选了MKN-45细胞作为模型细胞，该细胞高表达Met受体，在无配体HGF存在时，该Met受体便可发生自二聚，形成有活性的二聚体。如图8B所示，当MKN-45细胞与Apt-Met-TfR探针孵育后，细胞内Akt和Erk分子的磷酸化水平与没有加入核酸适配体探针的对照组相比没有区别，说明Apt-Met-TfR对配体不依赖型的受体的激活无抑制作用。以上实验结果表明，Apt-Met-TfR只适于抑制配体依赖型的信号通路的激活，而不适于对配体不依赖型的信号通路激活的抑制。同时，该结果也进一步证实了我们的假说，即Apt-Met-TfR通过将TfR受体与Met受体拉近，提高了Met受体的空间位阻，阻遏了HGF配体与Met受体的作用，从而抑制了Met受体信号通路的激活，而对于配体不依赖的受体的激活，无需配体与受体的相互作用，因此空间位阻不影响受体的活性。
实施例2中，为了进一步考察该策略对于其他配对蛋白的通用性，我们选用肿瘤细胞高表达的核仁素代替TfR作为另一配对受体的模型，设计双特异性核酸适体药物Apt-Met-Nucleolin。当Apt-Met-Nucleolin与DU145细胞一起孵育后，将核仁素与Met蛋白拉近，形成的人工受体异二聚体对Met磷酸化水平的抑制效果与Apt-Met-TfR对Met磷酸化水平的抑制效果相似(图9)，说明了我们提出的策略对于其他配对受体的通用性。
C.双特异性核酸适体药物Apt-Met-TfR对Met受体信号通路抑制的细胞特异性：
为了进一步考察双特异性核酸适体药物AApt-Met-TfR对Met受体信号通路抑制作用的细胞特异性，我们选取了低表达TfR的人正常肝细胞系L02作为实验细胞。首先，我们利用蛋白质印迹法(图10A)和激光共聚焦成像(图10B)比较DU145细胞系和L02细胞系上Met和TfR表达量的差异，结果显示，L02细胞系TfR表达水平确实较DU145细胞系低。当Apt-Met-TfR探针与L02细胞孵育后，该探针对L02细胞Met磷酸化水平抑制作用的IC50值为250nM(图11)，约为高表达TfR的DU145细胞Met磷酸化水平抑制作用IC50 的12倍，表明该探针只对配对蛋白(TfR)高表达的细胞的Met信号通路有较明显的抑制作用，而对配对蛋白低表达的细胞的Met信号通路影响较小，即该探针能够特异性地作用于靶标细胞，对其他细胞造成的毒副作用较小。
D.双特异性核酸适体药物Apt-Met-TfR对DU145细胞迁移行为的抑制：
HGF/Met信号通路参与调控细胞的迁移行为，而细胞的迁移往往与肿瘤细胞的转移有关。我们考察了双特异性核酸适体药物Apt-Met-TfR是否能够抑制Met信号通路激活引起的细胞迁移。首先，我们通过划痕实验对其进行验证，如图12所示，DU145细胞用Apt-Met-TfR处理后，划痕愈合的速度明显慢于用Apt-Met和未经探针处理的细胞。同时我们还利用散射实验对其进行考察，如图13所示，DU145细胞不进行任何刺激时，细胞形成团聚的群落；当细胞用HGF刺激后，细胞迁移能力增加，细胞团聚的群落散开，呈散射状；当细胞与Apt-Met-TfR孵育后再用HGF刺激，细胞群落依然呈团聚状，而用Apt-Met处理的细胞群落则呈散射状。另外，在Transwell实验中(图14)，用Apt-Met-TfR处理后，迁移到膜另一侧的DU145细胞明显少于用Apt-Met处理处理的细胞。以上实验结果说明Apt-Met-TfR能够抑制HGF诱导的DU145细胞的迁移行为。为了进一步验证双特异性核酸适体药物Apt-Met-TfR对细胞迁移行为的抑制作用。我们利用转盘式共聚焦对单细胞的运动行为和迁移轨迹进行考察，如图15所示，相对于与孵育Apt-Met和未孵育核酸药物的细胞，与Apt-Met-TfR孵育后的细胞的运动距离明显更短。以上实验结果说明Apt-Met-TfR能够很好地抑制HGF诱导的细胞迁移行为，且具有作为新兴核酸抑制剂的潜能。
在发明中，我们构建了一种双特异性核酸适体药物，实现人为诱导受体配对，形成人工受体异二聚体，通过调控Met受体的信号通路进而调控细胞的迁移行为。该探针不仅比单特异性核酸适体药物更高效地抑制Met受体的信号通路，而且更特异性地作用于靶标细胞，避免对非靶标细胞的影响。且本研究用DNA作为调控元件，也使得该调控策略在设计上更简单，在应用上也具有更广泛的通用性，可以通过简单更换核酸适体的序列使其适用于各种类型的受体功能调控。因此，我们构建的双特异性核酸适体药物在细胞的信号通路和细胞行为的调控领域具有广泛的应用前景，有望成为细胞功能(如细胞生长、迁移和分化等)的潜在抑制剂。

Claims (17)

  1. 一种双特异性核酸适体，其特征在于，所述核酸适体包括SEQ ID NO:1所示的第一核酸序列、SEQ ID NO:2所示的第二核酸序列；所述第一核酸序列包括SEQ ID NO:3所示的靶标受体特异性核酸适体和SEQ ID NO:4所示的第一连接部分，所述第二核酸序列包括SEQ ID NO:5所示的配对受体特异性核酸适体和SEQ ID NO:6所示的第二连接部分；所述靶标受体特异性核酸适体与靶标受体特异性结合，所述配对受体特异性核酸适体与配对受体特异性识别；所述第一连接部分与所述第二连接部分形成连接结构。
  2. 根据权利要求1所述的一种双特异性核酸适体，其特征在于，所述靶标受体为第一跨膜受体；所述配对受体为第二跨膜受体。
  3. 根据权利要求1所述的一种双特异性核酸适体，其特征在于，所述靶标受体的受体激活方式为受体二聚化、寡聚化或多聚化。
  4. 根据权利要求3所述的一种双特异性核酸适体，其特征在于，所述靶标受体的受体激活方式为配体依赖的受体二聚化、寡聚化或多聚化。
  5. 根据权利要求4所述的一种双特异性核酸适体，其特征在于，所述配体、所述靶标受体特异性核酸适体与所述靶标受体的结合位点相同或接近。
  6. 根据权利要求1所述的一种双特异性核酸适体，其特征在于，所述第一连接部分与所述第二连接部分形成连接结构使得分别与同一所述双特异性核酸适体的靶标受体特异性核酸适体、配对受体特异性核酸适体的靶标受体、配对受体的空间距离增加或缩小。
  7. 根据权利要求6所述的一种双特异性核酸适体，其特征在于，所述第一连接部分与所述第二连接部分形成连接结构使得分别与同一所述双特异性核酸适体的靶标受体特异性核酸适体、配对受体特异性核酸适体的靶标受体、配对受体的空间距离缩小。
  8. 根据权利要求6所述的一种双特异性核酸适体，其特征在于，所述第一连接部分与所述第二连接部分形成连接结构使得分别与同一所述双特异性核酸适体的靶标受体特异性核酸适体、配对受体特异性核酸适体的靶标受体、配对受体的空间距离缩小至靶标受体、配对受体形成阻碍所述靶标受体激活的 空间位阻。
  9. 根据权利要求6所述的一种双特异性核酸适体，其特征在于，所述第一连接部分与所述第二连接部分形成连接结构使得分别与同一所述双特异性核酸适体的靶标受体特异性核酸适体、配对受体特异性核酸适体的靶标受体、配对受体的空间距离缩小至靶标受体、配对受体形成异二聚体。
  10. 根据权利要求1或6所述的一种双特异性核酸适体，其特征在于，所述第一连接部分与所述第二连接部分通过相互杂交形成双链结构、通过亲和作用形成的连接结构、通过共价键连接形成连接结构，或者所述第一核酸序列、第二核酸序列直接合成为一条核酸序列使得所述第一连接部分与所述第二连接部分直接形成连接结构。
  11. 根据权利要求1所述的一种双特异性核酸适体，其特征在于，所述所述靶标受体为肿瘤细胞高表达受体。
  12. 根据权利要求1所述的一种双特异性核酸适体，其特征在于，所述所述配对受体为肿瘤细胞高表达受体。
  13. 根据权利要求1-12任一项所述的双特异性核酸适体在制备药物或制备蛋白功能调控分子中的应用。
  14. 根据权利要求1-12任一项所述的双特异性核酸适体的衍生物。
  15. 根据权利要求14所述的双特异性核酸适体的衍生物在制备药物或制备蛋白功能调控分子中的应用。
  16. 一种如权利要求1-12任一项所述的双特异性核酸适体的制备方法，其特征在于，
    s1.合成所述第一核酸序列；
    s2.合成所述第二核酸序列；
    s3.使所述第一连接部分与所述第二连接部分相互杂交形成双链结构、通过亲和作用形成的连接结构或者通过共价键连接形成连接结构；
    s4.得到所述双特异性核酸适体。
  17. 一种如权利要求1-12任一项所述的双特异性核酸适体的制备方法，其特征在于，
    s1.确定所述第一核酸序列；
    s2.确定所述第二核酸序列；
    s3.将所述第一核酸序列、第二核酸序列直接合成为一条核酸序列使得所述第一连接部分与所述第二连接部分直接形成连接结构；
    s4.得到所述双特异性核酸适体。
PCT/CN2020/079139 2019-07-03 2020-03-13 一种双特异性核酸适体、衍生物、制备方法及其应用 WO2021000598A1 (zh)

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