WO2022095141A1 - 一种gpc1 dna适配体及其应用 - Google Patents

一种gpc1 dna适配体及其应用 Download PDF

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WO2022095141A1
WO2022095141A1 PCT/CN2020/130517 CN2020130517W WO2022095141A1 WO 2022095141 A1 WO2022095141 A1 WO 2022095141A1 CN 2020130517 W CN2020130517 W CN 2020130517W WO 2022095141 A1 WO2022095141 A1 WO 2022095141A1
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gpc1
sequence
stranded dna
aptamer
nucleotide sequence
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French (fr)
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段维
张佩琢
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苏州吉玛基因股份有限公司
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    • 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
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/16—Aptamers
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the present invention relates to a GPC1 DNA aptamer and its application.
  • Glypican-1 (GPC1, phosphatidylglycan-1 antigen) is a membrane-anchored protein that is highly expressed in pancreatic and breast cancers.
  • PDAC pancreatic ductal adenocarcinoma
  • pancreatic cancer patients improved with the reduction of the GPC1-positive exosome population, suggesting that GPC1-positive exosomes can be used as a prognostic indicator for pancreatic cancer patients (Nature. 2015Jul 9;523(7559):177-82 .).)
  • pancreatic cancer patients are diagnosed with advanced pancreatic cancer (IIb, III and IV), while only 11-30% of patients have pancreatic cancer at the time of diagnosis.
  • I, IIa Early (I, IIa). Although the use of ultracentrifugation to isolate exosomes is the most commonly used method in research, this method cannot be used as a screening method in clinical testing.
  • nucleic acid aptamers Compared with traditional antibodies, aptamers have emerged as a unique and effective diagnostic tool. They have better detection sensitivity than antibodies, which are 25 times smaller than conventional antibodies. Therefore, compared with antibodies, nucleic acid aptamers have the advantage of greatly reduced steric hindrance. Because of this, nucleic acid aptamers are more likely to enter tumor cells and have greater random diffusion ability in the reaction system. In the area corresponding to one antibody, 5-15 nucleic acid aptamers can be easily bound. Aptamer-based cancer diagnostics are very sensitive and require only a small number of targets to generate a signal. Based on their unique features of high stability, affinity, and ease of chemical manipulation, different types of aptamers have been developed.
  • aptamers After binding to the target, aptamers change their conformation, a property that has also been used to develop aptamer-based sensors to detect any specific target. Another attractive feature of aptamers is that no animal experiments are required for aptamer screening. Thus, aptamers can be synthesized very economically. Antibodies against all cancer markers are sometimes not available, and the synthesis process is indeed very expensive and cumbersome, instead, aptamers can be developed relatively cheaply against any cancer biomarker, making it an attractive means of cancer diagnosis .
  • aptamers have all these attractive features, in the applicant's research, the applicant focused on aptamer-based liquid biopsy cancer detection system, and subsequently, the applicant developed an aptamer targeting pancreatic cancer-specific GPC1 markers ligands to detect GPC1-positive exosomes in different biological fluids.
  • Aptamer-exosome-based liquid biopsy has attracted great attention in the diagnosis of early-stage cancer, and thus it is assisting patients in obtaining appropriate treatment.
  • Cancer cell-derived exosomes contain a large number of cancer-specific markers, proteins and nucleic acids. Exosome-based liquid biopsy methods have achieved great success.
  • Early diagnosis of cancer is essential to implement effective and precise treatment and subsequently improve the survival rate of cancer patients.
  • the technical problem to be solved by the present invention is how to develop a novel and effective aptamer-exosome-based liquid biopsy method for early detection of cancer.
  • the present invention provides a DNA molecule (aptamer), the DNA molecule is the single-stranded DNA of any one of the following A1-A40:
  • the nucleotide sequence of A1 is the single-stranded DNA of sequence 28;
  • the A2 nucleotide sequence is the single-stranded DNA of sequence 25;
  • A3 nucleotide sequence is the single-stranded DNA of sequence 26;
  • the A4 nucleotide sequence is the single-stranded DNA of sequence 27;
  • the A5 nucleotide sequence is the single-stranded DNA of sequence 29;
  • A6 nucleotide sequence is single-stranded DNA of sequence 30;
  • A7 nucleotide sequence is the single-stranded DNA of sequence 31;
  • the A8 nucleotide sequence is the single-stranded DNA of sequence 32;
  • A9 nucleotide sequence is the single-stranded DNA of sequence 33;
  • the A10 nucleotide sequence is the single-stranded DNA of sequence 34;
  • the nucleotide sequence of A11 is the single-stranded DNA of sequence 35;
  • the A13 nucleotide sequence is the single-stranded DNA of sequence 37;
  • the A14 nucleotide sequence is the single-stranded DNA of sequence 38;
  • the A15 nucleotide sequence is the single-stranded DNA of sequence 39;
  • the A16 nucleotide sequence is the single-stranded DNA of sequence 40;
  • the A17 nucleotide sequence is the single-stranded DNA of sequence 1;
  • the A18 nucleotide sequence is the single-stranded DNA of sequence 2;
  • the A19 nucleotide sequence is the single-stranded DNA of sequence 3;
  • A20 nucleotide sequence is the single-stranded DNA of sequence 4.
  • the nucleotide sequence of A21 is the single-stranded DNA of sequence 5;
  • the A22 nucleotide sequence is the single-stranded DNA of sequence 6;
  • the A23 nucleotide sequence is the single-stranded DNA of sequence 7;
  • the A24 nucleotide sequence is the single-stranded DNA of sequence 8;
  • the A25 nucleotide sequence is the single-stranded DNA of sequence 9;
  • A26 nucleotide sequence is single-stranded DNA of sequence 10;
  • the A27 nucleotide sequence is the single-stranded DNA of sequence 11;
  • the A28 nucleotide sequence is the single-stranded DNA of sequence 12;
  • A29 nucleotide sequence is the single-stranded DNA of sequence 13;
  • the A30 nucleotide sequence is the single-stranded DNA of sequence 14;
  • the nucleotide sequence of A31 is the single-stranded DNA of sequence 15;
  • the A32 nucleotide sequence is the single-stranded DNA of sequence 16;
  • the A33 nucleotide sequence is the single-stranded DNA of sequence 17;
  • the A34 nucleotide sequence is the single-stranded DNA of sequence 18;
  • the A35 nucleotide sequence is the single-stranded DNA of sequence 19;
  • A36 nucleotide sequence is the single-stranded DNA of sequence 20;
  • A37 nucleotide sequence is the single-stranded DNA of sequence 21;
  • the A38 nucleotide sequence is the single-stranded DNA of sequence 22;
  • A39 nucleotide sequence is the single-stranded DNA of sequence 23;
  • the A40 nucleotide sequence is the single-stranded DNA of sequence 24.
  • the DNA molecule is a compound that specifically binds to GPC1 protein.
  • Said DNA molecule is preferably nucleotide sequence 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, The single-stranded DNA of any one of SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40. More preferably, the DNA molecule is a single-stranded DNA whose nucleotide sequence is any one of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34. Most preferred is a single-stranded DNA whose nucleotide sequence is SEQ ID NO: 28.
  • the present invention also provides a probe, which is a substance obtained by labeling the above-mentioned DNA molecule with a marker.
  • the label refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect and that can be attached to a nucleic acid.
  • Labels include, but are not limited to, dyes; radiolabels, such as32P ; conjugated coupling groups, such as biotin; haptens, such as digoxin (DIG); chemiluminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can suppress or shift the emission spectrum by fluorescence resonance energy transfer (FRET).
  • Labeling can be provided by fluorescence, radioactivity, colorimetric, gravimetric, quantum dot, electrochemical, X-ray diffraction or absorption, magnetic, immunoenzyme labeling, filter paper-based immunoassays, affinity precipitation, affinity chromatography, enzymes Viability, microprojection or scanning imaging, super-resolution imaging, cell tracking, in vivo nanoparticle tracking imaging in animals or humans, nanofluidics, tunable resistive pulse induction, fluorescence correlation spectroscopy, surface plasmon resonance, fluorescence polarization , Surface-enhanced Raman spectroscopy, electrochemical sensing, microfluidics or microfluidics, chip analysis, proteomics, genomics, metabolomics, microbiomics, RNA (mRNA, lnRNA, snRNA), miRNA and other detection signals .
  • Labels can be charged moieties (positive or negative) or can be selected as desired and can be charge neutral. Labels can include nucleic acid or protein sequences or a combination thereof, so long as the sequences comprising the label are detectable. In some embodiments, the nucleic acid is detected directly without labeling (eg, by reading the sequence directly). The markers can also be used for targeted drug delivery.
  • the labels are fluorophores, colorimetric labels, quantum dots, biotin, and other labeling molecules that can be used for detection (eg, alkyne groups for Raman imaging, rings for click reactions) Alkenes, initiating groups for polymer labeling), can also be selected from polypeptide/protein molecules, LNA/PNA, unnatural amino acids and their analogs (such as peptidomimetics), unnatural nucleic acids and their analogs (nucleoside acids) and nanostructures (including inorganic nanoparticles, NV-centers, aggregation/assembly-induced luminescent molecules, rare earth ion ligand molecules, polyoxometalate clusters, etc.).
  • the fluorescein dyes include standard fluorescein and derivatives thereof, such as fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), cy5, cy3 , Quasar 670, Alexa Fluor 488/647, etc.
  • FITC fluorescein isothiocyanate
  • FAM hydroxyfluorescein
  • TET tetrachlorofluorescein
  • cy5 cy3
  • Quasar 670 Alexa Fluor 488/647
  • the fluorophore is usually labeled at the 5' end of the probe sequence, but it can also be placed at the 3' end by changing a modifier bond (eg -OH or -NH bond).
  • a modifier bond eg -OH or -NH bond.
  • the present invention also provides a sensor containing the DNA molecule or the probe.
  • the present invention also provides a diagnostic reagent for pancreatic cancer and/or breast cancer and/or prostate cancer and/or colorectal cancer and/or glioma, which contains the DNA molecule.
  • the above-mentioned diagnostic reagents for cancers such as pancreatic cancer and/or breast cancer and/or prostate cancer and/or colorectal cancer and/or brain glioma can also contain other diagnosable pancreatic cancer and/or other than the DNA molecule.
  • Substances for cancers such as breast and/or prostate cancer and/or colorectal cancer and/or glioma.
  • the diagnostic reagents for cancers such as pancreatic cancer and/or breast cancer and/or prostate cancer and/or colorectal cancer and/or glioma further contain a substance that specifically binds to CD81.
  • the substance specifically binding to CD81 may be an aptamer that specifically binds to CD81 or a magnetic bead conjugated to an aptamer that specifically binds to CD81.
  • the aptamer that specifically binds to CD81 is a single-stranded DNA (5'-CATTTGACCATCCGGGTCTATG-3') whose nucleotide sequence is sequence 41.
  • the substance specifically binding to CD81 can be an antibody against the aptamer CD81 or a magnetic bead conjugated with the antibody against CD81.
  • the present invention also provides a reagent for detecting exosome GPC1 protein, the reagent contains the DNA molecule and a substance that specifically binds to CD81.
  • the substance specifically binding to CD81 may be an aptamer that specifically binds to CD81 or a magnetic bead conjugated to an aptamer that specifically binds to CD81.
  • the aptamer that specifically binds to CD81 can be a CD81-2J-6 aptamer.
  • the substance specifically binding to CD81 can be an anti-CD81 antibody or a magnetic bead conjugated to an anti-CD81 antibody.
  • the present invention also provides the application of the DNA molecule as an aptamer for GPC1 protein.
  • the application may specifically be the following A1 and/or A2:
  • A1 The application of the DNA molecule in the preparation of diagnostic reagents for cancers such as pancreatic cancer and/or breast cancer and/or prostate cancer and/or colorectal cancer and/or glioma;
  • the GPC1-positive exosomes described in A2 may be GPC1-positive exosomes in body fluids.
  • the GPC1 protein can be derived from mammals, such as humans.
  • the exponentially enriched ligand system evolution technology Systematic Evolution of Ligands by Exponential Enrichment, SELEX
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • Figure 1 is a schematic diagram of two different but closely linked ligand phylogenetic evolution techniques used in the Examples for screening GPC1 nucleic acid aptamers for exponential enrichment, respectively targeting proteins and living cells.
  • FIG. 2 is a graph showing the experimental results of determining the optimal cycle number of PCR binding to target DNA recovered at the end of each round of exponentially enriched ligand phylogenetic evolution (SELEX) in Example 1.
  • Figure, the data shown is the average value ⁇ standard deviation, the number of replicates is 3, *** indicates that the result of significant analysis is P ⁇ 0.001.
  • FIG. 3 is a flow chart and a result picture of the separation of target single-stranded DNA sequences by urea-dPAGE in Example 1.
  • FIG. The left picture in Figure 3 is a flow chart of asymmetric PCR amplification of target aptamers, where dPAGE refers to 8M urea-12% denaturing polyacrylamide gel electrophoresis; the right picture in Figure 3 is the separation of target aptamers
  • the urea-dPAGE gel of the strand DNA sequence is imaged under blue light.
  • the 100-nucleotide DNA band in the picture is the coding or antisense strand, and the 80-nucleotide band below is the sense strand. Those are very faint
  • the DNA is a non-specific amplification product, and the electrophoretic migration positions of the downstream primer (40nt) and the upstream primer (20nt) are shown below the figure.
  • Figure 4 shows the ability of streptavidin-coated microplates to capture human GPC1 protein in Example 1, and the biotin-labeled anti-6X histidine antibody-coated plate was used to successfully capture histidine epitopes
  • Figure 5 shows the ability of streptavidin-coated microplates to capture human GPC1 protein in Example 1.
  • the biotin-labeled anti-6X histidine antibody-coated plates and groups were analyzed by western blotting. Results of binding ability of amino acid epitope-tagged human GPC1 protein.
  • FIG. 6 is a flow chart and a result diagram of the assay of the binding ability of the library in exponentially enriched ligand phylogenetic evolution (SELEX) aptamer to human GPC1 protein in Example 1.
  • FIG. The left panel in Figure 6 shows how a random DNA library labeled with fluorescein can be detected Stepwise enrichment of nucleic acid ligands bound to histidine epitope-tagged human GPC1 protein during exponentially enriched ligand phylogenetic evolution Schematic diagram of the experimental procedure.
  • the right picture in Figure 6 shows the results of enriching nucleic acid aptamers bound to histidine epitope-tagged human GPC1 protein based on the SELEX process, wherein the blank control is an anti-histidine antibody without biotin labeling , without the addition of GPC1 recombinant protein and DNA aptamer; the antibody control was without DNA aptamer, and the aptamer in the 0th round was the fluorescence that was not enriched at the beginning of the ligand phylogenetic evolution experiment for exponential enrichment
  • the aptamer added in the fifth round is the DNA aptamer pool after 5 rounds of SELEX enrichment
  • the aptamer added in the 6th round is the DNA after 6 rounds of SELEX enrichment aptamer pool.
  • FIG. 7 is a schematic diagram of the detection of the overexpression of recombinant human GPC1 protein in HEK293T cells in Example 1.
  • FIG. In Fig. 7, A is the structure diagram of the GPC1 overexpression plasmid, wherein, the open reading frame (is the protein-encoding complementary DNA of GPC1, and its length is 1722bp, and the length of the pCMV3 expression plasmid vector is 6164bp.
  • the open reading frame is the protein-encoding complementary DNA of GPC1 and its length is 1722bp
  • the length of the pCMV3 expression plasmid vector is 6164bp.
  • FIG. 7 B is the overexpression of GPC1
  • the electrophoresis images of the expression plasmid DNA after single (HindIII or XbaI) and double (HindIII and XbaI) restriction endonuclease digestion were performed on the expression plasmid respectively, and the left one is the 1kb DNA fragment as the size standard.
  • Figure 7 C is the result of Western blot analysis of HEK293T cell lysate after GPC1 overexpression plasmid transfection with anti-anti-histidine (6X His) antibody antibody, and the untransfected wild-type HEK293T lysate is used as a control.
  • Figure 7 is the semi-quantitative experimental results of the detection of GPC1 protein on the surface of HEK29T3 cells transfected with wild-type HEK293T and GPC1 overexpression plasmids by flow cytometry.
  • the data shown are expressed as mean ⁇ standard deviation, and the number of repetitions is 3 , **** indicates that the result of significant analysis is P ⁇ 0.0001.
  • FIG. 8 is the detection of enrichment of nucleic acid aptamer pools that bind to GPC1 protein in Example 1.
  • FIG. A in Figure 8 is a schematic diagram of the experiment using flow cytometry to detect the binding of nucleic acid aptamers to cell surface GPC1 in an exponentially enriched aptamer phylogenetic experiment using cells as a target.
  • B shows that the enriched nucleic acid aptamer pool bound to wild-type HEK293T or to GPC1 was detected by flow cytometry in the 11th round of the exponentially enriched aptamer phylogenetic experiment using cells as the target in Example 1 Graph of the analysis results of overexpressing cell binding.
  • the non-fluorescein-labeled anti-GPC1 nucleic acid ligand was used as the background control of the allophycocyanin-labeled anti-fluorescein antibody (200 nM), and 500,000 cells were used for each sample by flow cytometry.
  • the data shown are expressed as mean ⁇ standard deviation, the number of replicates is 3, and **** indicates that the result of significance analysis is P ⁇ 0.0001.
  • FIG. 9 is a flowchart of the next-generation sequencing analysis of the final enriched aptamer pool in the exponentially enriched aptamer phylogeny experiment with cells as the target for the next-generation sequencing analysis of the final enriched aptamer pool.
  • nucleic acid aptamers must reach more than 50 copies in the entire next-generation sequencing pool before they are included.
  • Figure 10 shows the original repeat times of the sense strand, antisense strand and all strands in the final enriched aptamer pool next-generation sequencing analysis in the exponentially enriched aptamer phylogenetic experiment using cells as the target in Example 1 Histogram.
  • FIG. 11 is a flow chart of the screening and further adaptation of GPC1 aptamers in Example 1.
  • FIG. GPC1-17, GPC1-24, and GPC1-35 were selected from 15 clones in exponentially enriched ligand phylogenetic experiments. In the first round, GPC1-17 was selected from the original 17th series, while GPC35-A was selected from the original 35th series, and in the second round, GPC1-17A1 and GPC1-35A1 were selected . Finally, after the optimization is changed, GPC1-17A-1d is obtained.
  • Figure 12 is the secondary structure of the representative full-length GPC1 aptamer in Example 1, wherein the secondary structure covers GPC1-1, GPC1-2, GPC1-3, GPC1-7, GPC1-10, GPC1- 12.
  • FIG. 13 is a graph showing the results of analyzing the binding ability of 15 representative full-length GPC1 aptamers by flow cytometry in Example 1.
  • the black bar represents the fluorescence intensity of the aptamer binding to wild-type HEK293T.
  • Grey bars represent the fluorescence intensity of aptamer binding to GPC1-overexpressed HEK293T.
  • the vertical axis is the median of fluorescence intensity.
  • the data shown are expressed as mean ⁇ standard deviation, the number of repetitions is 3, * indicates that the result of significant analysis is P ⁇ 0.05, and *** indicates that the result of significant analysis is P ⁇ 0.001.
  • FIG. 14 shows the results of measuring the expression levels of GPC1 protein in Example 1 in Panc1 cells after knockdown of GPC1 by small RNA interference and in wild-type Panc1 control cells, respectively.
  • a in Figure 14 is the result of Western blotting (Western) with anti-human GPC1 antibody
  • B in Figure 14 is flow cytometry analysis of the binding of GPC1 antibody to wild-type Panc1 cells or Panc1 cells after knockdown of GPC1 using small RNA interference Semi-quantitative analysis. , the data shown are mean ⁇ standard deviation, and the number of replicates is 2.
  • FIG. 15 is an illustration of the secondary structure of the aptamers involved in the first round of aptamer adaptation engineering in Example 1.
  • FIG. A in Figure 15 is a diagram of the secondary structure of the evolution process of GPC1-17 in the first round of aptamer adaptation engineering
  • B in Figure 15 is the evolutionary process of GPC1-24 in the first round of aptamer adaptation engineering Diagram of the secondary structure
  • C in Figure 15 is the diagram of the secondary structure of the evolution process of GPC1-35 in the first round of aptamer adaptation engineering, and the nucleotides in the box with "X" are truncated.
  • Figure 16 is a graph of the sequence of the first round of aptamers modified by engineering truncated aptamers in Example 1 and the results of the measurement of their binding activity to GPC1-positive cells.
  • a in Figure 16 is the sequence of the original full-length aptamer and the first round of engineering truncated aptamers;
  • B in Figure 16 is the first round of aptamers GPC1-17, GPC1-24 and GPC1-35 generated Flow cytometry-based analysis of the binding ability of engineered truncated aptamers to CPC1 protein.
  • the cells used were wild-type Panc1 control cells and GPC1-overexpressing Panc1 cells.
  • FIG. 17 is the apparent dissociation constant of GPC1-17A-1 to GPC1-overexpressing HEK293T and GPC1-knockdown Panc1 cells in Example 1.
  • FIG. A in Figure 17 is the apparent dissociation constant (Kd) of GPC1-17A-1 to GPC1 overexpressed HEK293T and GPC1 knockdown Panc1, respectively.
  • B in Figure 17 is the apparent dissociation constant determination curve of GPC1-17A-1 on GPC1 overexpressed HEK293T cells and GPC1 knockdown Panc1 cells, respectively. Data shown are presented as mean ⁇ standard deviation with 2 replicates. Flow cytometry assays used 500,000 cells per sample.
  • FIG. 18 is an illustration of the secondary structure of the aptamer in the second round of aptamer adaptation engineering in Example 1.
  • FIG. A in Figure 18 is the secondary from GPC1-17A-1 to five subclones (GPC1-17A-1a, GPC1-17A-1b, GPC1-17A-1c, GPC1-17A-1d and GPC1-17A-1e) Structural evolution diagram;
  • Figure 18 B is from GPC1-35A-1 to five subclones (GPC1-35A-1a, GPC1-35A-1b, GPC1-35A-1c, GPC1-35A-1d and GPC1-35A-1e ) diagram of the evolution of the secondary structure.
  • nucleotides in the box with "X” are truncated, and the nucleotides in the box without "X” are replaced from A-T base pairs to C-G base pairs.
  • FIG. 19 is a diagram showing the sequence of the aptamer in the second round of aptamer transformation engineering in Example 1 and the result of analysis of its binding ability based on flow cytometry.
  • a in Figure 19 is the sequence from GPC1-17A-1 to five aptamers (GPC1-17A-1a, GPC1-17A-1b, GPC1-17A-1c, GPC1-17A-1d and GPC1-17A-1e) ;
  • B in Figure 19 is from GPC1-35A-1 to five aptamers (GPC1-35A-1a, GPC1-35A-1b, GPC1-35A-1c, GPC1-35A-1d and GPC1-35A-1e) sequence.
  • C in Figure 19 shows the results of flow cytometry-based binding ability analysis of five aptamers derived from GPC1-17A-1 to wild-type HEK293T control cells and GPC1-overexpressing HEK29T cells; D in Figure 19 shows GPC1-35A-1
  • the data shown are mean ⁇ standard deviation, the number of repetitions is 3, **** indicates that the result of significant analysis is P ⁇ 0.0001, ** indicates that the result of significant analysis is P ⁇ 0.01, and * indicates that the result of significant analysis is P ⁇ 0.01 0.05; ns means not statistically significant.
  • the aptamer concentration is 500nM.
  • the vertical axis is the median of fluorescence intensity. Flow cytometry uses 500,000 cells for each sample.
  • FIG. 20 shows the apparent dissociation constants of GPC1-17A-1d on GPC1-overexpressing HEK293T cells and GPC1-knockdown Panc1 cells in Example 1.
  • a in Figure 20 is the apparent dissociation constant (Kd) of GPC1-17A-1d to GPC1-overexpressing HEK293T cells and GPC1-knockdown Panc1 cells, respectively.
  • B in Figure 20 is the apparent dissociation constant measurement curve of GPC1-17A-1d on GPC1-overexpressing HEK293T cells and GPC1-knockdown Panc1 cells, respectively.
  • the vertical axis is the median of fluorescence intensity. The data shown are expressed as mean ⁇ standard deviation, and the number of replicates is 2. Flow cytometry assays used 500,000 cells per sample.
  • Figure 21 is a diagram of the secondary structure of the aptamers involved in the third round of aptamer transformation in Example 1, and six new aptamers (GPC1-17A-1d) were derived from GPC1-17A-1d -1, GPC1-17A-1d-2, GPC1-17A-1d-3, GPC1-17A-1d-4, GPC1-17A-d-5 and GPC1-17A-d-6).
  • a box with a "+” means adding the nucleotide in the box, and a circle and an arrow means replacing the nucleotide in the circle with another nucleotide pointed by the arrow.
  • Figure 22 shows six aptamers GPC1-17A-1d-1, GPC1-17A-1d-2, GPC1-17A-1d-3, GPC1-17A-1d derived from GPC1-17A-1d in Example 1 -4. Sequences of GPC1-17A-1d-5, GPC1-17A-1d-6.
  • FIG. 23 shows the results of measuring the binding ability of aptamers to cell surface GPC1 protein in the third round of aptamer modification in Example 1.
  • FIG. Aptamer concentrations were all 500 nM.
  • the vertical axis was the median of fluorescence intensity. Data shown are presented as mean ⁇ standard deviation with 2 replicates. The fluorescence of the cells without aptamer was used as a blank background fluorescence control.
  • FIG. 24 shows the detection results of the specificity of the binding of the GPC1-17A-1d aptamer to GPC1 on the cell surface in Example 2.
  • FIG. The upper panel in FIG. 24 is the verification of the overexpression of the other five unrelated recombinant transmembrane proteins tagged with each histidine epitope in HEK293T cells after transfection. Shown are Western blots detected with anti-histidine epitope tag antibodies.
  • the lower panel in FIG. 24 is the analysis result of the binding of GPC1-17A-1d aptamer to HEK293T cells transfected with recombinant transmembrane protein overexpressed with histidine epitope tag.
  • Figure 25 shows the GPC1-17A-1d aptamer labeled with Quasar-670 (Quasar-670) after capturing exosomes from the cell culture supernatant with biotin-labeled anti-CD81 antibody in Example 3 Schematic of detection by flow cytometry.
  • FIG. 27 is a schematic diagram of using CD81 aptamer to capture exosomes from cell culture supernatant and detect GPC1-positive exosomes in Example 4.
  • FIG. Exosomes in cell culture supernatants were captured with biotin-labeled anti-CD81 aptamer and immobilized on streptavidin surface-activated magnetic beads, followed by addition of Quasar-670 (Quasar-670) The labeled GPC1-17A-1d aptamer was detected by flow cytometry.
  • Figure 28 shows the fluorescently labeled GPC1-17A-1d aptamer used after capturing exosomes from GPC1 knockdown Panc1 cells or wild-type Panc1 cells with CD81 aptamer from the cell culture supernatant in Example 4
  • the result chart of the test In this experiment, exosomes in 0.5 ml of cell culture supernatant were first captured with 400 nM biotin-labeled CD81 aptamer (4°C, 1 h) and immobilized on streptavidin surface-activated magnetic beads , washed three times with a washing solution containing 0.1% Tween-20 in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • a in Figure 28 is the flow-through of exosomes from GPC1 knockdown Panc1 cells or wild-type Panc1 cells bound to GPC1-17A-1d aptamer then labeled with Quasar-670 (Quasar-670). Fluorescence intensity results in cytometry are shown.
  • Figure 28, B is a bar graph of quantitative analysis of GPC1 fluorescence intensity on exosomes from GPC1 knockdown Panc1 and Panc1 cells captured by CD81 affinity chromatography.
  • Figure 28, C is the result of Western blotting (Western) with anti-human GPC1 antibody on exosomes from GPC1 knockdown Panc1 and wild-type Panc1 cells.
  • D in Figure 28 is the analysis of GPC1 expression on exosomes from different cells captured by these two CD81 affinity chromatography using GPC1 Western blotting. Data shown are presented as mean ⁇ standard deviation with 2 replicates. ** indicates that the result of significant analysis is P ⁇ 0.01.
  • Figure 29 is a schematic diagram and results of using anti-GPC1 antibody to detect the expression level of GPC1 on captured exosomes after capturing exosomes with CD81 aptamer in Example 4.
  • the CD81 aptamer as shown in Figure 28 captures exosomes and then uses GPC1 antibody (Abcam, cat. no. ab199343, 1:70 dilution) and fluorescent (Brilliant Violet 510 TM ) labeled anti-immunoglobulin (Biolegend, cat. No. ) :406419, 1:1600 dilution) schematic diagram of secondary antibody to detect GPC1 abundance.
  • Figure 29B shows that GPC1 expression in exosomes from wild-type Panc1 cells was about 3-fold higher than that in exosomes from GPC1 knockdown Panc1 cells.
  • Figure 30 is a graph showing the results of the GPC1 aptamer in Example 5 being able to detect 1 GGPC1-positive exosome in a background of 500 to 1000 GPC1-negative exosomes in a simulated liquid biopsy.
  • GPC1 -positive exosomes representative exosomes secreted by pancreatic cancer with high GPC1 expression.
  • GPC1- negative exosomes prepared exosomes with low or no GPC1 expression from the cell culture supernatant of GPC1 knockdown Panc1 cells to mimic GPC1-negative exosomes.
  • Exosomes were prepared using the gold standard step ultracentrifugation method and counted using a nanoparticle tracer (Nanosight NS300, Malvern Instrument, UK). Next, we mixed these two different exosomes at different ratios for limiting dilution to prepare a mixture of five different ratios of GPC1 -positive exosomes relative to GPC1- negative exosomes. The ratios of GPC1 -positive exosomes to GPC1- negative exosomes for these five samples were 1:500, 1:1000; 1:1000; 1:2000; 1:5000 and 1:8000, respectively.
  • the five samples were all formulated to a total concentration of 3 x 10 10 exosomes per ml.
  • the five mixed exosomes in different ratios were captured with biotin-labeled anti-CD81 aptamer and immobilized on streptavidin surface-activated magnetic beads, and then labeled with Quasar-670 (Quasar-670)
  • Quasar-670 Quasar-670
  • the GPC1-17A-1d aptamer was detected by flow cytometry.
  • the ratio of GPC1 -positive exosomes to GPC1- negative exosomes in the sample in the figure is indicated right above the flow cytometry result frame, and the total magnetic beads detected have the quasar fluorophore-670 The percentage of positive beads is indicated in the upper right of the flow cytometry results box.
  • the scrambled control is a random sequence of DNA aptamers labeled with the quasar fluorophore-670 used to represent the background fluorescence of the experimental system.
  • the experimental methods in the following verification experiments are conventional methods unless otherwise specified.
  • the materials and reagents used in the following verification experiments, unless otherwise specified, are conventional biochemical reagents and can be obtained from commercial sources.
  • HEK293T cells (Cat. No. CRL-3216) is the product of the American Type Culture Collection i company.
  • Panc1 knocked down by GPC1 is the product of (ED200001020) Suzhou Zima Gene Co., Ltd.
  • the GPC1 plasmid with His-tag (Catalog No. HG10576-CH) is a product of China Jingyiqiao Shenzhou Technology Co., Ltd.
  • Recombinant human GPC1 protein (Cat. No. ab215589) is a product of Abcam Company.
  • Anti-human GPC1 antibody (Cat. No. ab199343, monoclonal antibody) is a product of Abcam Company.
  • Biotin-labeled anti-6X His antibody (Cat. No. ab106261, mouse monoclonal antibody) is a product of Abcam Company.
  • Biotin-labeled CD81 antibody (Cat. No. 349514, mouse monoclonal antibody) is a product of BioLegend.
  • Anti- ⁇ -actin antibody (Cat. No. ab6276, mouse monoclonal antibody) is a product of Abcam Company.
  • Allophycocyanin-labeled anti-fluorescein antibody (Cat. No. 17-7691-82) is a product of eBioscience.
  • the FAM mark (Item No. E17001) is a product of Suzhou Gema Gene Co., Ltd.
  • Quasar 670 (Q670) mark (Item No. 11-4200-XX) is a product of Suzhou Gema Gene Co., Ltd.
  • Streptavidin-coated 2.7 micron diameter magnetic beads (Cat. No. PL6827-103) are a product of Agilent Technologies.
  • the applicant's purpose is to develop DNA aptamers against GPC1 (hereinafter referred to as GPC1 aptamers) using protein-based exponential enrichment aptamer phylogenetic experiments (SELEX) (protein-based SELEX) and cell-SELEX (cell-SELEX) methods.
  • SELEX protein-based exponential enrichment aptamer phylogenetic experiments
  • Cell-SELEX cell-SELEX
  • Ligands a schematic diagram of the two techniques is shown in Figure 1.
  • protein-based SELEX targets commercially available recombinant human GPC1 protein to enrich for GPC1 aptamer sequences.
  • a major disadvantage of protein-based SELEX is that the recombinant protein may not have the conformation of the native protein.
  • the applicant used recombinant GPC1-overexpressing HEK293T cells as target cells, and utilized cellular SELEX to promote further enrichment of GPC1 aptamer sequences. These enriched DNA sequences are able to bind to the GPC1 protein that retains its native conformation. Therefore, Applicants determined that the selected GPC1 aptamers can successfully bind to the native GPC1 protein expressed by cells or exosomes. The specific process is as follows:
  • the single-stranded DNA sequence bound to the target protein constitutes only a small fraction of the single-stranded DNA pool. If the recovered target single-stranded DNA is directly applied to the next round of SELEX, most of it may be lost due to the small amount. Therefore, DNA bound to the target protein recovered at the end of each round of SELEX must be amplified using specially designed PCR. The optimal number of PCR cycles for each round of SELEX needs to be determined by clearance experiments.
  • DNA was recovered and amplified by PCR with PCR conditions: denaturation at 94 °C for 30 s, annealing at 48.6 °C for 40 s, and extension at 72 °C for 60 s, the number of cycles was 12-18, with no Template DNA is a negative control.
  • the amplification of PCR products with different cycle numbers was analyzed by 4% agarose gel electrophoresis. The representative results are shown in Figure 2, A, and the analysis of the relative amplicon intensity of the gel electrophoresis results is shown in Figure 2. B of 2.
  • the gel was imaged under a blue light (470 nm wavelength) illuminator (Major Science, Cat. No. MBE-300) to reduce the chance of cross-linking of DNA with common UV light.
  • a blue light (470 nm wavelength) illuminator Major Science, Cat. No. MBE-300
  • the antisense strand migrates behind the sense strand and the lower band of the sense strand DNA (containing the FITC-bound aptamer 40nt) is easily located and excised from the gel.
  • the image of the polyacrylamide gel denaturing electrophoresis gel for separating the target single-stranded DNA sequence under blue light is shown in the right image in Figure 3.
  • the 5'-end of the sense strand DNA is labeled with fluorescein (FITC), so The sense strand DNA can be easily seen under blue light and recovered from the gel.
  • FITC fluorescein
  • the DNA in the gel fragments was recovered by electroelution and DNA precipitation, and the recovered aptamer sense strand was subjected to the next round of SELEX.
  • This experiment used recombinant C-terminal-6X His-tagged human GPC1 protein.
  • the conjugate of His-tagged GPC1 protein and biotin-tagged anti-6X His antibody plays an important role in the streptavidin-coated plate capture system.
  • Western Blotting analysis was performed, and the specific process is shown in Figure 4.
  • the results of Western analysis are shown in Figure 5, indicating that the amount of human GPC1 protein captured by the streptavidin-coated plate was significantly higher in the eluate than in the supernatant. Therefore, the streptavidin-coated plate can effectively capture human GPC1 protein, thereby establishing a reliable and robust streptavidin-coated plate-based SELEX system for human GPC1 protein.
  • the human GPC1 protein was used to detect the enrichment of GPC1 nucleic acid ligands.
  • a detection system with His-tagged GPC1 protein as an immobilized target was formed on a microplate to capture GPC1 nucleic acid aptamers enriched by SELEX, and this system was used as a positive selection for GPC1 nucleic acid aptamers.
  • a negative selection was first performed with a pool of aptamers using microplates without the addition of His-GPC1 protein (streptavidin-coated microplates containing only biotin-labeled anti-6X His antibody).
  • a microplate-based enrichment-cum-assay for GPC1 nucleic acid aptamers as described above was used to evaluate the enrichment from Round 0 (library), The degree of enrichment of aptamers that can bind to human GPC1 protein in the aptamer pool obtained from Round-P5 (human GPC1 protein SELEX 5 rounds) and Round-P6 (human GPC1 protein SELEX 6 rounds).
  • a His-tagged GPC1 cDNA expression plasmid (Catalog No. HG10576-CH) was purchased from China Jingyiqiao Shenzhou Technology Co., Ltd. (A in Figure 7).
  • pCMV-GPC1 is a 1722-nucleotide full-length cDNA encoding GPC1 with a carboxy-terminal His tag (GenBank NCBI Reference Sequence :NM_002081.2),
  • the GPC1 overexpression plasmid pCMV-GPC1 was digested with one and two restriction enzymes, respectively, and electrophoresed on a 0.8% agarose gel.
  • the electrophoresis results are shown in Figure 7, B:
  • the GPC1 overexpression plasmid pCMV-GPC1 was cut into linear sequences by single restriction enzyme digestion (Hind III or Xba I), while the His-tagged GPC1 cDNA was inserted by double restriction enzyme digestion (Hind III + Xba I)
  • the double-stranded DNA of the fragment (His-GPC1) was released from the vector, and two bands appeared in the double digestion group.
  • the band that appeared at the bottom was the His-tagged GPC1 ORF region (1722 bp in total, see the sequence in the sequence table for details). 42; the amino acid sequence encoded by the His-tagged GPC1 ORF region is shown in sequence 43 in the sequence listing), and the upper band represents the pCMV3-C-His vector (6164bp).
  • the above results indicate that the GPC1 overexpression plasmid is the correct expression construct for downstream applications.
  • the membrane was first probed with anti-6X His antibody, and then resolved with 10% SDS-PAGE using anti- ⁇ -actin antibody as a loading control.
  • the His-tagged Human GPC1 protein was successfully overexpressed in HEK293T cells, HEK293T/pCMV-GPC1 also known as GPC1 overexpressed HEK293T.
  • GPC1-overexpressing HEK293T was used as a positive selection, while HEK293T cells (WT) were used as a negative selection for binding affinity assays for cellular SELEX-enriched GPC1 nucleic acid ligands.
  • WT HEK293T cells
  • GPC1-overexpressing HEK293T and wild-type HEK293T lines were incubated with anti-GPC1 antibody, and a secondary antibody conjugated with Leuvenide 510TM combined with goat anti-rabbit IgG was added, and then analyzed by flow cytometry. .
  • the median fluorescence intensity of GPC1-overexpressing HEK293T cells was significantly higher, and the expression of GPC1 in HEK293T cells transfected with GPC1-expressing plasmid was 10-20-fold higher than that of wild-type HEK293T .
  • the SELEX process enriches the aptamers that bind to GPC1-overexpressed HEK293T
  • FIG. 8A A schematic diagram of flow cytometry analysis of GPC1 aptamer binding to HEK293T cells is shown in Figure 8A.
  • the aptamer pool of As a negative control the GPC1-overexpressed HEK293T obtained above was used as a positive control, and an anti-FITC antibody was used as a background control.
  • a small part of the 200nm FITC-labeled aptamer pool at the 11th round was used to incubate and wash the cells, and flow cytometry Cytometry. The results are shown in B in Figure 8.
  • the binding of the aptamer pool from the 11th round of cell-SELEX enrichment to GPC1-overexpressing HEK293T cells was 4.5 times higher than that to wild-type HEK293T cells, which indicated that in cells -
  • the binding efficiency of the aptamer pool to GPC1 increased after the 11th round of SELEX. So taking these data together, after 11 rounds of cellular SELEX, GPC1 aptamers against native human GPC1 protein were successfully enriched.
  • next-generation sequencing of aptamer clones was performed.
  • the GPC1 aptamer finally utilizes the obtained aptamer sequences to deduce high binding sequences.
  • the DNA of the entire aptamer pool was analyzed by next-generation sequencing after 11 rounds of cellular SELEX procedure for GPC1 aptamers.
  • the GPC1 aptamer has only the initial random region (bit and center) other than the forward and reverse primers that the applicant is interested in.
  • FIG. 9 The specific process is shown in Figure 9: Starting from a double-stranded DNA copy containing the random aptamer sequence, removing the ends After the primer sequence, the reverse complement of the antisense strand, sequence insertion, screening and clustering, more than 50 copies of the aptamer sequence were recorded. Of the potential GPC1 aptamers thus obtained, only DNA copy numbers ⁇ 50 were selected for further study, while other remaining sequences were not evaluated.
  • Figure 10 shows NGS results representing 147,524 repeats, each repeat with a central region of 40 nucleotides of the same sequence is marked. These 40 nucleotide sequences in the GPC1 aptamer pool are the DNA insert sequences of interest to the applicant, ie, the pool of potential aptamer candidates.
  • GPC1 ectodomain specifically, a total of 15 aptamers targeting the GPC1 ectodomain of GPC1 DNA after SELEX were screened in GPC1-negative or near-negative and GPC1-positive human cell lines. See step 9 for the detailed screening process.
  • wild-type HEK293T with low expression of GPC1 and HEK293T with overexpression of GPC1 were used as negative and positive controls, respectively.
  • 3 aptamers that can bind to GPC1 were selected after identification. See step 10 for the screening process.
  • the two selected aptamers that can bind to GPC1 were subjected to multiple rounds of truncation engineering, and the binding capacity was tested to screen suitable candidate sequences. Finally, the optimized GPC1 aptamers were determined by site-directed mutagenesis. The detailed molecular modification engineering and binding force assay screening process are shown in step 11-step 16.
  • a total of 15 aptamers with potential binding to the natural GPC1 protein were selected from the highest ranked candidate aptamers, including GPC1-1, GPC1-2, GPC1-3, GPC1-7, GPC1-10, GPC1-12, GPC1-15, GPC1-17, GPC1-22, GPC1-23, GPC1-24, GPC1-25, GPC1-35, GPC1-100 and GPC1-300, the specific sequences are shown in Table 1, The secondary structure is shown in Figure 12. Whether these full-length GPC1 aptamers can actually bind to the native GPC1 protein will be tested by experiments in the next step.
  • GPC1 aptamers which are representative full-length GPC1 aptamers, to native GPC1 protein
  • 15 kinds of GPC1 aptamers labeled with 500 nM FAM (fluorescein) were detected by flow cytometry.
  • HEK293T with wild-type HEK293T and GPC1-overexpressing HEK293T were incubated with 500,000 cells for 1 h at 4°C. After washing to remove unbound aptamer sequences, the cells were analyzed for signal from fluorescein by flow cytometry. The results of flow cytometry analysis are shown in Figure 13.
  • Applicants generated two to three truncated aptamers for each of the full-length aptamers (GPC1-17, GPC1-24, and GPC1-35) to select the aptamers with the highest binding capacity and The shortest sequence of aptamers.
  • the truncation of aptamers is advantageous because it reduces production costs and most importantly, it improves binding affinity.
  • GPC1-17, GPC1-24 and GPC1-35 initially screened were all initially 40 nucleotides in length, which were reduced to 13-25 nucleotides by truncation.
  • GPC1-17, GPC1-24 and GPC1-35 have 4, 2 and 3 possible secondary structures, respectively, which are denoted as 17A, 17B, 17C, 17D (see A in Figure 15), 24A, 24B (see Figure 15, B), 35A, 35B and 35C (see Figure 15, C).
  • Applicants have attempted to verify that all truncated aptamers have the same circular structure. With this in mind, applicants have generated a series of truncated aptamers from the full-length aptamers, the sequences of the original full-length GPC1 aptamer and each truncated aptamer are reported in Table 2 and Figure 16, A.
  • GPC1-17A-1 After confirming that GPC1-17A-1 has good binding ability to native GPC1 protein, the applicant began to characterize the binding kinetics of the GPC1 aptamer. For details, please refer to the published paper of the inventor (PMID: 21281402).
  • Kd equilibrium dissociation constant
  • GPC1 knockdown Panc1 was used as a negative control and GPC1 overexpressed HEK293T was used as a positive control.
  • the apparent dissociation constant (Kd) of GPC1-17A-1 to GPC1-overexpressed HEK293T was 494.1 ⁇ 13.57 nM, while the corresponding Kd of GPC1-knockdown Panc1 was 1631 ⁇ 135.7 nM.
  • the (Kd) value of GPC1 aptamer 17A-1 of GPC1 knockdown Panc1 was about 3.3-fold higher than that of GPC1 overexpressed HEK293T.
  • the kinetics of the binding of GPC1 aptamer 17A-1 to GPC1 knockdown Panc1 cells is close to a straight line, suggesting that the binding of the two is non-specific. This indicates that GPC1-17A-1 is a promising binder of GPC1. Therefore, in order to improve the binding affinity and selectivity of the aptamer, the applicant decided to further alter and truncate GPC1-17A-1.
  • the applicant further designed the molecular modification of the aptamer. Since stem length, stem size and composition can all affect the binding affinity of aptamers, Applicants made some changes to the stem, such as truncation, or changing A and/or T to C and/or G, respectively, But keeping the functional loop unchanged, as shown in Figure 18, five new aptamers were generated from GPC1-17A-1 and GPC1-35A-1 respectively.
  • the specific nucleotide sequences of the aptamers are shown in Table 3 and Figure 19 A and B of Figure 19.
  • aptamer name length Nucleic acid aptamer sequence (5'to3') number in the sequence listing GPC1-17A-1 15 CAATCCCCTTTTTA sequence 16 GPC1-17A-1a 15 CACTCCCCCTTTGTA sequence 25 GPC1-17A-1b 15 CCCTCCCCCTTTGGA sequence 26 GPC1-17A-1c 13 AATCCCCCTTTTT sequence 27 GPC1-17A-1d 13 CATCCCCCTTTTG sequence 28 GPC1-17A-1e 13 ACTCCCCCTTTGT sequence 29
  • the C-G base pair contains 3 hydrogen bonds, which will be stronger than the A-T base pair bond; assuming that the addition of a C-G pair in the stem region can enhance the affinity and specificity of the adapted aptamer, Applicants put the stem's A-T Base pairs are converted to C-G base pairs.
  • the more C-G base pairs present in the stem the less flexibility the loop may have to adopt different 3-D conformations, and thus the final ratio of efficient 3-D loop structures in the overall aptamer population may be will dominate over the original, resulting in better target binding.
  • the length of the second round of aptamers obtained is about 15 nucleotides.
  • GPC1-17A-1 derived aptamers GPC1-17A-1a, GPC1-17A-1b, GPC1-17A-1c, GPC1-17A-1e that were run in the second round of engineering showed better performance than the original Lower binding capacity of aptamer GPC1-17A-1
  • all GPC1-35A-1 derived aptamers GPC1-35A-1a, GPC1-35A-1b, GPC1-35A-1c, GPC1-35A-1d GPC1
  • the ability of -35A-1e to bind to GPC1-overexpressing HEK293T cells was not significantly different from the original GPC1-35A-1. Therefore, the aptamer GPC1-17A-1d (SEQ ID NO: 28 of the Sequence Listing), which had shown a significant increase in fluorescence intensity over the original aptamer GPC1-17A-1, was selected for further experiments.
  • GPC1-17A-1d is a promising binder for the native GPC1 protein on the cell membrane
  • Applicants further conducted studies to characterize the binding kinetics of this GPC1 aptamer.
  • Kd equilibrium dissociation constant
  • concentrations 100 nM, 250 nM, 500 nM, 1000 nM, 2000 nM, 5000 nM
  • 5'-Quasar 670-labeled GPC1-17A-1d were prepared using GPC1 Knockdown Panc1 (as a negative control, HEK293T/pCMV-GPC1 from step 5 (also known as GPC1-overexpressed HEK293T) was used as a positive control (PMID: 21281402).
  • GPC1-17A-1d for GPC1-knockdown Panc1 cells was approximately 13.4-fold higher than that for GPC1-overexpressing HEK293T cells, suggesting that GPC1-17A obtained during molecular alteration -1d is a promising aptamer that binds more specifically to GPC1.
  • the applicant decided to further optimize the GPC1-17A-1d.
  • the applicant After selecting GPC1-17A-1d from the second round of engineering, in order to improve the binding affinity and selectivity of the aptamer to the native GPC1 protein, the applicant further designed a third round of aptamers. Briefly, Applicants hypothesized that some changes were made in the loops in the secondary structure of the GPC1-17A-1d aptamer, and some AT or CG base pairs were added to the stem, as shown in Figure 21. May result in significant 3D conformational changes and enhance the binding efficiency of aptamers to GPC1.
  • aptamer name length Nucleic acid aptamer sequence (5'to3') number in the sequence listing GPC1-17A-1d 13 CATCCCCCTTTTG sequence 28 GPC1-17A-1d-1 13 CAGCGCGGTTG sequence 35 GPC1-17A-1d-2 15 CATGGCCCGGTTCTG sequence 36 GPC1-17A-1d-3 15 GCATCCCCCCTTTTGC sequence 37 GPC1-17A-1d-4 17 GACATCCCCCTTTTGTC sequence 38 GPC1-17A-1d-5 17 GGCATCCCCCTTTTGCC sequence 39 GPC1-17A-1d-6 19 GGACATCCCCCTTTTTGTCC sequence 40
  • Applicants performed a binding assay using flow cytometry at 500 nM Quasar dye-670-labeled aptamer bound to cells to examine its binding efficacy.
  • all derived aptamers showed lower binding capacity to GPC1 compared to the aptamer GPC1-17A-1d, see Figure 23 for details.
  • This result suggests that loop changes or base additions in the stem, at least in the third fixed round of engineering, do not contribute to the improvement in binding capacity, and these results confirm that loop changes are most likely to alter the binding efficacy of the aptamer . Therefore, the aptamer GPC1-17A-1d was selected as the final selected GPC1 aptamer.
  • the applicant After confirming the binding affinity of GPC1-17A-1d, the applicant further determined the specificity and selectivity of the aptamer with a variety of His-tagged overexpressing recombinant transmembrane protein cells: In order to determine the applicant's aptamer For the specificity, Applicants randomly selected unrelated transmembrane proteins and specifically selected the other two members of Glypican, namely GPC4 and GPC6, to verify that the aptamer does not bind to other cell surface membrane proteins even if overexpressed. details as follows:
  • Wild-type HEK293T with His tag HEK293T cells were transfected with the blank pCMV3-C-His vector plasmid, and the transfected HEK293T cells were obtained after 24 hours, which were named HEK293T/pCMV3-C. As a negative control for wild-type HEK293T.
  • HEK293T overexpressed with His-tagged CD9 HEK293T cells were transfected with His-CD9/pCMV3, and the transfected HEK293T cells were obtained 24 hours later, which were named HEK293T/pCMV-CD9.
  • HEK293T/pCMV-CD9 was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (Item No. RG80778-CH).
  • HEK293T overexpressed with His-tagged CD81 HEK293T cells were transfected with pCMV-CD81, and the transfected HEK293T cells were obtained after 24 hours, which were named HEK293T/pCMV-CD81.
  • HEK293T/pCMV-CD81 was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (Item No. HG14244-CH).
  • HEK293T overexpressed with His-tagged TGF ⁇ R3 HEK293T cells were transfected with pCMV-TGF ⁇ R3, and the transfected HEK293T cells were obtained after 24 hours, which were named HEK293T/pCMV-TGF ⁇ R3.
  • HEK293T/pCMV-TGF ⁇ R3 was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (Item No. HG10778-CH).
  • HEK293T overexpressed with His-tagged GPC1 HEK293T cells were transfected with pCMV-GPC1, and the transfected HEK293T cells were obtained after 24 hours, which were named HEK293T/pCMV-GPC1.
  • HEK293T/pCMV-GPC1 is HEK293T overexpressed with His-tagged GPC1.
  • pCMV-GPC1 was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (Item No. HG10576-CH).
  • HEK293T overexpressed with His-tagged GPC4 HEK293T cells were transfected with pCMV-GPC4, and the transfected HEK293T cells were obtained after 24 hours, which were named HEK293T/pCMV-GPC4.
  • HEK293T/pCMV-GPC4 is HEK293T overexpressed with His-tagged GPC4.
  • pCMV-GPC4 was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (Item No. HG10090-CH).
  • the results of the binding assay are shown in the lower panel in Figure 24.
  • the binding of GPC1-17A-1d to wild-type HEK293T remained at background levels, and the aptamer GPC1-17A-1d bound to overexpressed CD9, CD81, TGF ⁇ R3, GPC4 and The binding of GPC6 to HEK293T cells was not significantly different from that of wild-type HEK293T.
  • GPC1 has been shown to be highly expressed in pancreatic cancer cell-derived exosomes. To ensure that GPC1 aptamers could capture GPC1-positive exosomes, Applicants performed binding assays using the promising GPC1-17A-1d aptamer. The process is as follows: the exosomes successfully captured by biotin-labeled CD81 antibody in cell culture supernatant were immobilized on streptavidin-coated magnetic beads, and then Q670-labeled GPC1-17A-1d aptamer was immobilized on the streptavidin-coated magnetic beads. Placed in GPC1-positive exosomes, washed, and analyzed by flow cytometry, as shown in Figure 25.
  • Panc1 cells (Cat. No. CRL-1469 TM ) is the product of the American Type Culture Collection i company.
  • Panc1 cell culture supernatant (CCM) and GPC1 knockdown Panc1 cell culture supernatant (CCM) were prepared as described in detail (Proc Natl Acad Sci U S A.113(8): E968-E977, 2016), the only change was the addition of 0.5% exosome-depleted calf serum when collecting exosome-containing cell culture supernatants.
  • the applicant put the biotin-labeled CD81 aptamer CD81 -2J-6 exosomes successfully captured in cell culture supernatant (CCM) were immobilized on streptavidin-coated magnetic beads, and then Q670-labeled GPC1-17A-1d aptamer was placed on GPC1 Among the positive exosomes, after washing, they were analyzed by flow cytometry, as shown in Figure 27. Specifically, Applicants have used the CD81-2J-6 aptamer to capture exosomes, as it has been established in Applicants' laboratory that this aptamer can effectively capture exosomes in CCM.
  • Quasar 670-labeled GPC1 aptamer was obtained by using Quasar 670-labeled aptamer GPC1-17A-1d.
  • Panc1 knocked down by GPC1 is a product of Suzhou Zima Gene Co., Ltd.
  • Streptavidin-coated 2.7 micron diameter magnetic beads (Cat. No. PL6827-103) are a product of Agilent Technologies.
  • exosomes immobilized with CD81-aptamers produced up to 2-3-fold higher signal compared to exosomes immobilized with CD81-antibody .
  • the monoclonal antibody (IgG) CD81 antibody is 150 kDa
  • the aptamer CD81-2J-6 is only 6.8 kDa.
  • up to 14 nucleic acid aptamers can be bound, because the volume of the latter is nearly 14 times smaller than that of the former.
  • CD81 protein monoclonal antibody covers the surface of exosomes, there are far fewer vacant regions available for subsequent binding of GPC1 aptamers.
  • CD81-2J-6 nucleic acid aptamers at least ten-fold smaller than the antibody bound to the surface of exosomes, these aptamers of CD81 that had bound to the CD81 protein on the surface of exosomes were less sensitive to subsequent The binding of the GPC1 aptamer is much less sterically hindered.
  • the main challenge of EV-based liquid biopsy is the detection of very small amounts of cancer cell-derived exosomes in the context of large amounts of blood- or normal epithelial cell-derived exosomes and extracellular vesicles).
  • Zhang et al. have observed that the concentration of extracellular vesicles in the lung cancer patients they studied was about (1.41 ⁇ 0.31) ⁇ 10 10 per milliliter, while for normal healthy individuals, the concentration of extracellular vesicles was about ( 3.37 ⁇ 0.39) ⁇ 109 /ml. Therefore, it is a great challenge to efficiently detect trace amounts of cancer-derived EVs in the presence of large amounts of normal healthy cells from blood (Lab Chip., 2019, 19, 1114-1140; Nanoscale, 2019, 11 , 10106-10113).
  • the applicant established a liquid biopsy model.
  • limiting dilution of exosomes from pancreatic cancer Panc1 cells was mixed with exosomes from GPC1-knockdown Panc1 cells in a specific ratio.
  • a mixed suspension of these two exosomes was used as a test sample to mimic the background of few GPC1-negative exosomes from pancreatic cancer cells interspersed with 4 ⁇ 10 9 /ml of extracellular vesicles from normal cells.
  • GPC1-positive Panc1EV and GPC1-negative Panc1EV were extracted by standard ultracentrifugation or ultrafiltration. Biomedicine and Biotechnology, 2018:8545347.”.
  • Streptavidin-coated magnetic beads (Cat. No. PL6827-1030) are products of AGILENT TECHNOLOGIES AUSTRALIA PTY LTD.
  • Biotin-labeled CD81 aptamer obtained by labeling the aptamer CD81-2J-6 at the 5'-end with biotin labeling.
  • Quasar 670-labeled GPC1 aptamer was obtained by using Quasar 670-labeled aptamer GPC1-17A-1d.
  • exosomes were prepared from culture supernatants of two different cells, wild-type Panc1 cells and GPC1-downregulated Panc1 cells containing very low GPC1 protein.
  • concentration of the exosome suspension was deliberately adjusted to 3 ⁇ 10 10 exosomes per milliliter to mimic the concentration of actual exosomes present in the blood of clinical patients.
  • the percentage of signal obtained from the Quasar 670-labeled GPC1 aptamer GPC1-17A-1d was 1.09% in the 1:500 and 1:1000 groups, respectively. he10.87%.
  • This data shows that in the applicant's current CD81-based aptamer detection system, the Quasar 670-labeled GPC1 aptamer GPC1-17A-1d can be detected in every 500 background exosomes from normal cells , a GPC1-positive exosome derived from cancer cells was detected. It is worth noting that the results shown in Figure 30 are preliminary detection sensitivities obtained under conditions that are not yet fully optimized.
  • the detection sensitivity based on the GPC1 aptamer of the present invention is expected to be further improved.
  • the fluorescence quantum yield of the fluorophore used in Figure 271, Quasar 670, is 0.28, similar to Cy5.
  • fluorophores with higher quantum yields such as Alexa Fluor 488 with a quantum yield of 0.92, the detection sensitivity could be greatly improved.

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Abstract

一种GPC1DNA适配体及其应用。使用SELEX方法，进行严格的分子改造工程、筛选和深入表征，开发了针对经典外泌体生物标记物CD81和胰腺癌特异性生物标记GPC1的DNA分子作为适配体，并验证了该适配体特异结合GPC1蛋白，在此适配体的基础上建立了一个基于CD81适配体的外泌体检测系统，用于检测GPC1阳性外泌体，可成功检测出源自胰腺癌细胞的GPC1阳性外泌体。

Description

一种GPC1 DNA适配体及其应用 技术领域
本发明涉及一种GPC1 DNA适配体及其应用。
背景技术
Glypican-1(GPC1,磷脂酰基醇蛋白聚糖-1抗原)是一种膜锚定蛋白，在胰腺癌和乳腺癌中高表达。有研究人员发现，GPC1对于胰腺导管腺癌(PDAC)的早期检测至关重要。他们发现，随着肿瘤负担的增加，循环系统中GPC1阳性外泌体的水平也随之增加。通过检测胰腺导管腺癌患者血液样本，可以在肿瘤恶变之前的早期PDAC中检测到高水平的GPC1阳性外泌体。此外，胰腺癌患者的生存率随着GPC1阳性外泌体群体的减少而提升，这表明GPC1阳性外泌体可以作为胰腺癌患者的预后指标(Nature.2015Jul 9；523(7559):177-82.)。不幸的是，有70-89％的胰腺癌患者在被诊断时胰腺癌已发展到晚期(IIb，III和IV)，而只有11-30％的患者在胰腺癌被发现时其胰腺癌尚为早期(I，IIa)。虽然使用超速离心分离外泌体是在科研时最常用的方法，但此方法无法应用在临床检验中用作筛选方法。
当前用于胰腺癌诊断的方法灵敏度和特异性不高。早期成功检测循环系统中的GPC1阳性外泌体生物标记物用于胰腺癌的诊断已引起人们的关注。近来有详尽的研究表明，循环系统中的GPC1阳性外泌体可能是临床环境中进行下一代诊断的突破性工具。因此，从癌症患者体液中检测GPC1阳性外泌体可能是早期诊断胰腺癌的一种有吸引力的方法。
与传统抗体相比，适配体已经成为一种独特而有效的诊断工具。与抗体相比，它们具有更好的检测敏感性，它们比常规抗体小25倍。故与抗体比较，核酸适配体具有大大降低了的空间位阻击的优越性。正因如此，核酸适配体更容易进入肿瘤细胞，且在反应体系中有更大的随机扩散的能力。在对应于一个抗体所占的面积上，5-15个核酸适配体可以轻易地结合上去。基于适配体的癌症诊断方法非常灵敏，只需要少量靶标就能产生信号。基于其高稳定性，亲和力，易于化学操作的独特特征，已开发出不同类型的适配体。与靶标结合后，适配体会改变其构象，该特性也已被用于开发基于适配体的传感器来检测任何特定靶标。适配体的另一个吸引人的特征是对于适配体的筛选，不需要进行动物实验。因此，适配体可以非常经济地合成。有时无法获得针对所有癌症标志物的抗体，并且合成过程确实非常昂贵且繁琐，相反，可以以比较便宜的方式开发针对任何癌症生物标志物的适配体，这使其成为癌症诊断的诱人手段。由于适配体具有所有这些吸引人的特征，因此在申请人的研究中，申请人专注于基于适配体的液体活检癌检测系统，随后，申请人开发了针对胰腺癌特异性GPC1标记的适配体，以检测不同生物体液中的GPC1阳性外泌体。
适配体-外泌体为基础的液体活检在早期癌症的诊断中引起了极大的关注，因此它正在协助患者获得适当的治疗。癌细胞衍生的外泌体包含大量的癌症特异性标志物，蛋白质和核酸。基于外泌体的液体活检法已经取得了巨大的成功。最近，在2019年6月，FDA批准了首个基于外泌体的液体活检产品(ExoDx Prostate IntelliScore test)。针对这种基于血液的癌症诊断的研究和开发工作利用外泌体生物学，并最终为癌症诊断和治疗打开新的窗口。因此，严格而谨慎的基于适配体-外泌体的检测系统将有助于早期诊断不同类型的癌症。早期诊断癌症对于实施有效精准治疗并随后提高癌症患者的生存率至关重要。
发明内容
本发明所要解决的技术问题是如何开发一种新颖且有效的基于适配体-外泌体的液体活检方法，用于早期检测癌症。
为了解决以上技术问题，本发明提供了一种DNA分子(适配体)，所述DNA分子是如下A1-A40中任一种的单链DNA：
A1核苷酸序列为序列28的单链DNA；
A2核苷酸序列为序列25的单链DNA；
A3核苷酸序列为序列26的单链DNA；
A4核苷酸序列为序列27的单链DNA；
A5核苷酸序列为序列29的单链DNA；
A6核苷酸序列为序列30的单链DNA；
A7核苷酸序列为序列31的单链DNA；
A8核苷酸序列为序列32的单链DNA；
A9核苷酸序列为序列33的单链DNA；
A10核苷酸序列为序列34的单链DNA；
A11核苷酸序列为序列35的单链DNA；
A12核苷酸序列为序列36的单链DNA；
A13核苷酸序列为序列37的单链DNA；
A14核苷酸序列为序列38的单链DNA；
A15核苷酸序列为序列39的单链DNA；
A16核苷酸序列为序列40的单链DNA；
A17核苷酸序列为序列1的单链DNA；
A18核苷酸序列为序列2的单链DNA；
A19核苷酸序列为序列3的单链DNA；
A20核苷酸序列为序列4的单链DNA；
A21核苷酸序列为序列5的单链DNA；
A22核苷酸序列为序列6的单链DNA；
A23核苷酸序列为序列7的单链DNA；
A24核苷酸序列为序列8的单链DNA；
A25核苷酸序列为序列9的单链DNA；
A26核苷酸序列为序列10的单链DNA；
A27核苷酸序列为序列11的单链DNA；
A28核苷酸序列为序列12的单链DNA；
A29核苷酸序列为序列13的单链DNA；
A30核苷酸序列为序列14的单链DNA；
A31核苷酸序列为序列15的单链DNA；
A32核苷酸序列为序列16的单链DNA；
A33核苷酸序列为序列17的单链DNA；
A34核苷酸序列为序列18的单链DNA；
A35核苷酸序列为序列19的单链DNA；
A36核苷酸序列为序列20的单链DNA；
A37核苷酸序列为序列21的单链DNA；
A38核苷酸序列为序列22的单链DNA；
A39核苷酸序列为序列23的单链DNA；
A40核苷酸序列为序列24的单链DNA。
所述DNA分子为与GPC1蛋白质特异结合的化合物。
所述DNA分子优选为核苷酸序列为序列25、序列26、序列27、序列28、序列29、序列30、序列31、序列32、序列33、序列34、序列35、序列36、序列37、序列38、序列39、序列40中任一种的单链DNA。所述DNA分子更优选为核苷酸序列为序列25、序列26、序列27、序列28、序列29、序列30、序列31、序列32、序列33、序列34中任一种的单链DNA。最优选为核苷酸序列为序列28的单链DNA。
本发明还提供了一种探针，所述探针为上述DNA分子被标记物标记得到的物质。
所述标记物指可用于提供可检测的(优选可定量的)效果且可以连接至核酸的任何原子或分子。标记物包括但不限于染料；放射性标记，诸如 32P；共轭偶连基团，诸如生物素(biotin)；半抗原，诸如地高辛(DIG)；化学发光、发磷光或发荧光部分；和单独的荧光染料或与可以通过荧光共振能量转移(FRET)抑制或移动发射光谱的部分组合的荧光染料。标记可以提供可通过荧光、放射性、比色、重量测定、量子点，电化学，X射线衍射或吸收、磁性、免疫酶标反应，基于滤纸的免疫测定,亲和沉淀，亲和色谱法,酶活性，显微投射或扫描成像，超分辨力成像,细胞示踪，动物或人体的活体纳米颗粒追踪示踪成像，纳米流式，可调电阻脉冲感应，荧光相关光谱表面等离子体共振，荧光偏振,表面增强拉曼光谱,电化学感应,微流体或微流控，芯片分析，蛋白质组学，基因组学，代谢组学，微生物组学，RNA(mRNA,lnRNA,snRNA),miRNA等检测的信号。标记可以是带电荷的部分(正电荷或负电荷)或可按需选定，可以是电荷中性的。标记可以包括核酸或蛋白序列或由其组合，只要包含标记的序列是可检测的。在一些实施方案中，核酸在没有标记的情况下直接检测(例如，直接读取序列)。所述标记物还可用于靶向给药。
在一些实施方式中，所述标记是荧光团、比色标记、量子点、生物素以及其他可以用于探测的标签分子(如用于拉曼衍射成像的炔烃基团，用于click反应的环烯烃，用于聚合物标记的引发基团)，也可以选自多肽/蛋白分子，LNA/PNA，非天然氨基酸及其类似物(比如拟肽)，非天然核酸及其类似物(拟核苷酸)和纳米结构(包括无机纳米颗粒，NV-center，聚集/组装诱导发光分子，稀土离子配体分子，多金属氧簇等)。
在一些实施方式中，所述荧光团可选自荧光素类染料、罗丹明类染料以及菁染料。
在一些实施方式中，所述荧光素类染料包括标准荧光素及其衍生物，如异硫氰酸荧光素(FITC)、羟基荧光素(FAM)、四氯荧光素(TET)、cy5、cy3、Quasar 670、Alexa Fluor 488/647等。
在一些实施方式中，所述罗丹明类染料包括R101、四乙基罗丹明(RB200)和羧基四甲基罗丹明(TAMRA)等。
在一些实施方式中，所述菁染料主要选自两类，一类是噻唑橙(thiazole orange,TO)、 噁唑橙(oxazole orange,YO)系列及其二聚体染料，另一类是多甲川系列菁染料。
在一些实施方式中，荧光团还可以选择下述染料：二苯乙烯、萘酰亚胺、香豆素类、吖啶类、芘类等。
荧光团通常标记在探针序列的5'端，但通过改变修饰键(例如-OH或-NH键)也可以将其置于3'端。
为了解决以上技术问题，本发明还提供含有所述DNA分子或含有所述探针的传感器。
本发明还提供一种胰腺癌和/或乳腺癌和/或前列腺癌和/或结直肠癌和/或脑胶质瘤等癌症的诊断试剂，含有所述DNA分子。
上述胰腺癌和/或乳腺癌和/或前列腺癌和/或结直肠癌和/或脑胶质瘤等癌症的诊断试剂除含有所述DNA分子外，还可含有其它可诊断胰腺癌和/或乳腺癌和/或前列腺癌和/或结直肠癌和/或脑胶质瘤等癌症的物质。
上述胰腺癌和/或乳腺癌和/或前列腺癌和/或结直肠癌和/或脑胶质瘤等癌症的诊断试剂还含有与CD81特异结合的物质。
所述与CD81特异结合的物质可为与CD81特异结合的适配体或与CD81特异结合的适配体共轭的磁珠。
所述与CD81特异结合的适配体为核苷酸序列为序列41的单链DNA(5'-CATTTGACCATCCGGGTCTATG-3')。
所述与CD81特异结合的物质可为抗适配体CD81的抗体或与抗CD81的抗体共轭的磁珠。
本发明还提供一种用于检测外泌体GPC1蛋白质的试剂，所述试剂含有所述DNA分子和与CD81特异结合的物质。
所述与CD81特异结合的物质可为与CD81特异结合的适配体或与CD81特异结合的适配体共轭的磁珠。
所述与CD81特异结合的适配体可为CD81-2J-6适配体。
所述与CD81特异结合的物质可为抗CD81的抗体或与抗CD81的抗体共轭的磁珠。
本发明还提供所述DNA分子在作为GPC1蛋白质的适配体中的应用。
所述应用具体可为如下A1和/或A2：
A1、所述DNA分子在制备胰腺癌和/或乳腺癌和/或前列腺癌和/或结直肠癌和/或脑胶质瘤等癌症的诊断试剂中的应用；
A2、所述DNA分子在制备检测GPC1阳性外泌体试剂中的应用。
上述应用中，A2所述GPC1阳性外泌体可为体液中的GPC1阳性外泌体。
所述GPC1蛋白质可来源于哺乳动物，如人。
本发明中使用指数富集的配体系统进化技术(Systematic Evolution of Ligands by Exponential Enrichment，SELEX)方法开发了针对经典外泌体生物标记CD81和胰腺癌特异性生物标记GPC1的DNA分子作为适配体：选择具有高结合亲和力和特异性的潜在GPC1适配体，进行严格的工程筛选和深入表征，得到了适配体的DNA分子。接下来，申请人建立了一个基于CD81适配体共轭磁珠的系统，以捕获细胞培养上清液中的外泌体。最后，申请人开发了一种有效的CD81适配体介导的外泌体检测系统，可成功检测出源自胰腺癌细胞 的GPC1阳性外泌体。
附图说明
图1为实施例中所采用的分别用蛋白质的为靶标和用活细胞为靶标的二个不同但紧扣衔接的用于筛选GPC1核酸配适体的指数富集的配体系统进化技术示意图。
图2为实施例1中每一轮指数富集的配体系统进化(SELEX)结束时回收的结合靶DNA的PCR最佳循环数确定实验结果图。其中，图2中的A为琼脂糖凝胶电泳图，n为循环数；图2中的B为对凝胶电泳结果的DNA库的相对扩增程度的分析见图，所示数据为平均值±标准差，重复数为3，***表示显著性分析结果为P≤0.001。
图3为实施例1中以尿素-dPAGE分离目标单链DNA序列的流程和结果图片。图3中的左图为以不对称PCR扩增目标适配体的流程图，其中，dPAGE是指含8M尿素-12％变性聚丙烯酰胺凝胶电泳；图3中的右图为分离目标单链DNA序列的尿素-dPAGE凝胶在蓝光下成像图，图中100核苷酸的那条DNA带为编码链或者反义链,下面那条80核苷酸的带是正义链，那些非常淡的DNA是非特异放大产物，图下方显示下游引物(40nt)以及上游引物(20nt)的电泳迁移位置。
图4为实施例1中确认链霉亲和素包被的微孔板对人GPC1蛋白的捕获能力，使用生物素标记的抗6X组氨酸抗体包被的板成功捕获用组氨酸表位标记的人GPC1蛋白的过程示意图。
图5为实施例1中确认链霉亲和素包被的微孔板对人GPC1蛋白的捕获能力，通过蛋白质印迹法分析了使用生物素标记的抗6X组氨酸抗体包被的板与组氨酸表位标记的人GPC1蛋白的结合能力结果图。
图6为实施例1中文库在指数富集的配体系统进化(SELEX)适配体对人GPC1蛋白的结合能力测定的流程和结果图。图6中的左图为如何检测用荧光素标记的随机DNA文库在指数富集的配体系统进化过程中检测与组氨酸表位标记的人GPC1蛋白结合的核酸配适体的逐步富集的实验过程示意图。图6中的右图为基于SELEX过程中富集与组氨酸表位标记的人GPC1蛋白结合的核酸配适体的测定结果，其中，空白对照为不加生物素标记的抗组氨酸抗体，不加GPC1重组蛋白及DNA配适体；抗体对照为不加DNA配适体，第0轮中配适体为用于指数富集的配体系统进化实验起始时未经富集的荧光素标记的随机DNA文库，第5轮中加的配适体为经过SELEX 5轮富集后的DNA配适体池，第6轮中加的配适体为经过SELEX 6轮富集后的DNA配适体池。配适体的折叠程序如下：在含2.5nM氯化镁的磷酸缓冲液中95℃ 5分钟，0℃ 5分钟，37℃ 15分钟。所示数据表示为平均值±标准差，重复数为3，*表示显著性分析结果为P≤0.05。
图7为实施例1中检测重组人GPC1蛋白在HEK293T细胞中的过表达示意图。图7中A为GPC1过表达质粒的结构图，其中，开放读码框(为GPC1的蛋白编码互补DNA，其长度为1722bp，pCMV3表达质粒载体的长度为6164bp。图7中B为对GPC1过表达质粒分别进行单(HindⅢ或XbaⅠ)和双(HindⅢ和XbaⅠ)限制性内切酶切得到的表达质粒DNA切开后的电泳图，其中，左边的是1kb DNA片断作为尺寸标准。图7中C为以抗抗组氨酸(6X His)抗体抗体对GPC1过表达质粒转染后的HEK293T细胞裂解液进行蛋白质印迹分析的结果图，以未转染的野生型HEK293T裂解液为对照。图7中D为用流式细胞仪对 野生型HEK293T及GPC1过表达质粒转染后的HEK29T3细胞表面GPC1蛋白量的检测的半定量实验结果，所示数据表示为平均值±标准差，重复数为3，****表示显著性分析结果为P≤0.0001。
图8为实施例1中与GPC1蛋白结合核酸配适体池富集的检测。图8中A为在用细胞为靶标的指数富集的配适体系统进化实验中用流式细胞仪来检测核酸配适体与细胞表面GPC1结合的实验原理图。图8中B为实施例1中针对用细胞为靶标的指数富集的配适体系统进化实验第11轮中流式细胞术检测经富集的核酸配适体池与野生型HEK293T结合或与GPC1过表达细胞结合的分析结果图。其中，不加荧光素标记的抗GPC1核酸配适体作为别藻青蛋白标记的抗荧光素抗体(200nM)的背景对照，流式细胞术检测每个样品用了50万个细胞。所示数据表示为平均值±标准差，重复数为3，****表示显著性分析结果为P≤0.0001。
图9为最终富集适配体池的二代测序分析用细胞为靶标的指数富集的配适体系统进化实验中对最终富集的适配体池进行二代测序分析的流程图。在此过程里，核酸配适体在整个二代测序池中必须达到50个以上拷贝的才被收入。
图10为实施例1中用细胞为靶标的指数富集的配适体系统进化实验中的最终富集适配体池二代测序分析中正义链、反义链及所有链的原始重复次数的柱状图。
图11为实施例1中GPC1适配体的筛选和进一步改照的流程图。GPC1-17，GPC1-24，GPC1-35是从指数富集的配体系统进化实验中的15个克隆中选拔出来的。在第一轮中，GPC1-17是从原始的第17系列中选出，而GPC35-A是从原始的第35系列中选出,在第二轮中，选出GPC1-17A1和GPC1-35A1.最后改照优化后得到的是GPC1-17A-1d。
图12为实施例1中具有代表性的全长GPC1适配体的二级结构，其中的二级结构涵盖GPC1-1、GPC1-2、GPC1-3、GPC1-7、GPC1-10、GPC1-12、GPC1-15、GPC1-17、GPC1-22、GPC1-23、GPC1-24、GPC1-25、GPC1-35、GPC1-100和GPC1-300。二级结构是采用Mfold生成。
图13为实施例1中以流式细胞仪分析15种代表性全长GPC1适配体结合能力分析结果图，图中，黑色柱表示适配体与野生型HEK293T结合的荧光强度。灰色柱表示适配体与GPC1过表达的HEK293T结合的荧光强度。纵轴为荧光强度的中位数。所示数据表示为平均值±标准差，重复数为3，*表示显著性分析结果为P≤0.05，***表示显著性分析结果为P≤0.001。
图14为实施例1中GPC1蛋白质分别在使用小RNA干扰敲低GPC1后的Panc1细胞和野生型Panc1对照细胞中表达水平的测定结果。图14中A为用抗人GPC1抗体进行蛋白印迹(Western)的结果，图14中B为流式细胞术分析GPC1抗体与野生型Panc1细胞或使用小RNA干扰敲低GPC1后的Panc1细胞结合的半定量分析。，所示数据为平均值±标准差，重复数为2。
图15为实施例1中第一轮适配体改照工程中所涉及的适配体的二级结构的图解。图15中A为第一轮适配体改照工程中GPC1-17的进化过程的二级结构的图解，图15中B为第一轮适配体改照工程中GPC1-24的进化过程的二级结构的图解，图15中C为第一轮适配体改照工程中GPC1-35的进化过程的二级结构的图解，有“×”的框内的核苷酸被截除。
图16为实施例1中第一轮适配体改照工程截短的适配体的序列及与GPC1阳性细胞结 合活性的测定结果图。图16中A为原始全长适配体和第一轮工程截短适配体的序列；图16中B为对适配体GPC1-17、GPC1-24和G PC1-35产生的第一轮工程截短适配体与CPC1蛋白基于流式细胞术的结合能力分析结果。所用细胞为野生型Panc1对照细胞以及GPC1过表达的Panc1细胞。所示数据为平均值±标准差，重复数为3，***表示显著性分析结果为P≤0.001，**表示显著性分析结果为P≤0.01，*表示显著性分析结果为P≤0.05；ns表示统计上不显著。
图17为实施例1中GPC1-17A-1对GPC1过表达的HEK293T和GPC1敲低的Panc1细胞的表观解离常数。图17中A为GPC1-17A-1分别对GPC1过表达的HEK293T和GPC1敲低的Panc1的表观解离常数(Kd)。图17中B为GPC1-17A-1分别对GPC1过表达的HEK293T细胞和GPC1敲低的Panc1细胞的表观解离常数测定曲线。所示数据表示为平均值±标准差，重复数为2。流式细胞术检测每个样品用了50万个细胞。
图18为实施例1中第二轮适配体改照工程中适配体的二级结构的图解。图18中A为从GPC1-17A-1到五个亚克隆(GPC1-17A-1a、GPC1-17A-1b、GPC1-17A-1c、GPC1-17A-1d和GPC1-17A-1e)的二级结构进化图解；图18中B为从GPC1-35A-1到五个亚克隆(GPC1-35A-1a、GPC1-35A-1b、GPC1-35A-1c、GPC1-35A-1d和GPC1-35A-1e)的二级结构进化图解。在图中用大方框圈示的每一对配适体中有“×”的框内的核苷酸被截除，无“×”的框内的核苷酸从A-T碱基对被换成C-G碱基对。
图19为实施例1中第二轮适配体改照工程中适配体的序列及其基于流式细胞术的结合能力分析结果图。图19中A为从GPC1-17A-1到五个适配体(GPC1-17A-1a、GPC1-17A-1b、GPC1-17A-1c、GPC1-17A-1d和GPC1-17A-1e)的序列；图19中B为从GPC1-35A-1到五个适配体(GPC1-35A-1a、GPC1-35A-1b、GPC1-35A-1c、GPC1-35A-1d和GPC1-35A-1e)的序列。图19中C为GPC1-17A-1衍生的5种适配体与野生型HEK293T对照细胞以及GPC1过表达HEK29T细胞基于流式细胞术的结合能力分析结果；图19中D为GPC1-35A-1衍生的5种适配体与GPC1蛋白基于流式细胞术的结合能力分析结果。所示数据为平均值±标准差，重复数为3，****表示显著性分析结果为P≤0.0001，**表示显著性分析结果为P≤0.01，*表示显著性分析结果为P≤0.05；ns表示统计上不显著。适配体浓度均为500nM.纵轴为荧光强度的中位数.流式细胞术检测每个样品用了50万个细胞。
图20为实施例1中GPC1-17A-1d对GPC1过表达的HEK293T细胞和GPC1敲低的Panc1细胞的表观解离常数。其中，每个细胞种类GPC1适配体采用了五个不同的浓度(100nM,250nM,500nM,1000nM,2000nM,5000nM)与细胞孵育来计算表观解离常数。图20中A为GPC1-17A-1d分别对GPC1过表达的HEK293T细胞和GPC1敲低的Panc1细胞的表观解离常数(Kd)。图20中B为GPC1-17A-1d分别对GPC1过表达的HEK293T细胞和GPC1敲低的Panc1细胞的的表观解离常数测定曲线。纵轴为荧光强度的中位数.所示数据表示为平均值±标准差，重复数为2。流式细胞术检测每个样品用了50万个细胞。
图21为实施例1中第三轮适配体改照过程中涉及的适配体的二级结构的图解，从GPC1-17A-1d衍生得到六个新的适配体(GPC1-17A-1d-1、GPC1-17A-1d-2、GPC1-17A-1d-3、GPC1-17A-1d-4,GPC1-17A-d-5和GPC1-17A-d-6)。有“+”的框表示添加框内的核苷酸，圆圈和箭头表示将圆圈内的核苷酸替换为箭头指向的另一种核苷酸。
图22为实施例1中从GPC1-17A-1d衍生得到的六个适配体GPC1-17A-1d-1、 GPC1-17A-1d-2、GPC1-17A-1d-3、GPC1-17A-1d-4、GPC1-17A-1d-5、GPC1-17A-1d-6的序列。
图23为实施例1中第三轮适配体改照中的配适体与细胞表面GPC1蛋白结合能力测定结果。适配体浓度均为500nM.纵轴为荧光强度的中位数。所示数据表示为平均值±标准差，重复数为2。不加适配体的细胞本身的荧光用来当做空白本底荧光对照。
图24为实施例2中GPC1-17A-1d适配体与细胞表面的GPC1结合的特异性的检测结果。图24中上图为各组氨酸表位标记的其他五个无关重组跨膜蛋白经转染后在HEK293T细胞里过表达的验证。所示的是用抗组氨酸表位标签抗体来检测的蛋白质印迹图。图24中下图为GPC1-17A-1d适配体与过表达组氨酸表位标签标记的重组跨膜蛋白转染后的HEK293T细胞结合的分析结果。显示的是用流式细胞术来进行检测用400nM类星体荧光基团-670(Quasar-670)标记的GPC1-17A-1d适配体与各种不同的细胞表面过表达的重组蛋白结合的结果，其中空白表达质粒转染的HEK293T细胞的荧光信号被用来标定本底荧光。所示数据为平均值±标准差，重复数为2，纵轴为荧光强度的中位数。*表示显著性分析结果为P≤0.05。
图25为实施例3中用生物素标记的抗CD81抗体从细胞培养上清液中捕获外泌体后用类星体荧光基团-670(Quasar-670)标记的GPC1-17A-1d适配体经流式细胞术来进行检测的原理图。
图26为实施例3中使用CD81抗体捕获源自GPC1下调后的Panc1或从野生型的Panc1细胞的的外泌体后用荧光标记的GPC1适配体来检测分析结果。CD81抗体与细胞培养上清液中在4℃孵育1小时后把外泌体捕获固定在链酶亲和素表面活化的磁珠(Agilent)上。然后加400nM类星体荧光基团-670(Quasar-670)标记的GPC1-17A-1d适配体4℃孵育1小时后用含0.1％Tween-20的磷酸缓冲液洗5次，经流式细胞术来进行检测。所示数据表示为平均值±标准差，重复数为2。纵轴为荧光强度的中位数。
图27为实施例4中用CD81适配体从细胞培养上清液中捕获外泌体并检测GPC1阳性外泌体的原理图。细胞培养上清液中的外泌体用生物素标记的抗CD81适配体捕获并固定在链酶亲和素表面活化的磁珠上，然后加类星体荧光基团-670(Quasar-670)标记的GPC1-17A-1d适配体经流式细胞术来进行检测。
图28为实施例4中从细胞培养上清液中用CD81适配体捕获来自GPC1敲低的Panc1细胞或野生型Panc1细胞的外泌体后用荧光标记的GPC1-17A-1d适配体用来测的的结果图。在本实验中，0.5ml的细胞培养上清液中的外泌体先用400nM生物素标记的CD81适配体捕获(4℃,1小时)固定在链酶亲和素表面活化的磁珠上，用洗涤液含0.1％Tween-20的磷酸缓冲液PBS)洗三遍。然后加入400nM类星体荧光基团-670(Quasar-670)标记的GPC1-17A-1d适配体在4℃孵育1小时。洗涤三次后，经流式细胞术用来检测磁珠上捕获的外泌体上的GPC1。图28中A为来自GPC1敲低的Panc1细胞或野生型Panc1细胞的外泌体与然后用类星体荧光基团-670(Quasar-670)标记的GPC1-17A-1d适配体结合后在流式细胞术中的荧光强度结果显示。图28中B为用CD81亲和层析捕获的来自GPC1敲低的Panc1和Panc1细胞的外泌体上面的GPC1荧光强度定量分析条形图。图28中C为对来自GPC1敲低的Panc1和野生型Panc1细胞的外泌体以抗人GPC1抗体进行蛋白印迹 (Western)的结果。图28中D为这二种CD81亲和层析捕获的来自不同细胞的外泌体上用GPC1蛋白印迹达来进行GPC1表达量的分析。所示数据表示为平均值±标准差，重复数为2。**表示显著性分析结果为P≤0.01。
图29为实施例4中以CD81适配体捕获外泌体后用抗GPC1抗体来检测GPC1在捕获外泌体上表达量的示意图和结果。图29A中为CD81适配体如图28所示捕获外泌体然后用GPC1抗体(Abcam,货号ab199343,1:70稀释)及荧光(Brilliant Violet 510 TM)标记的抗免疫球蛋白(Biolegend,货号:406419,1:1600稀释)二级抗体来检测GPC1丰度的示意图。图29B中显示野生型Panc1细胞的外泌体的GPC1比来自GPC1敲低的Panc1细胞外泌体的GPC1表达量高3倍左右。
图30为实施例5中GPC1适配体在模拟液体活检中能够在500至1000个GPC1阴性外泌体的背景中检测到1个GGPC1阳性的外泌体的结果图。我们首先制备里来自与Panc1细胞培养上清液的外泌体作为胰腺癌分泌的GPC1高表达的代表性外泌体(GPC1 阳性外泌体)。我们又从GPC1敲低的Panc1细胞细胞培养上清液制备了GPC1低表达或不表达的外泌体来模拟GPC1阴性的外泌体(GPC1- 阴性外泌体)。外泌体是用金标准的分步超速离心方法制备，用纳米颗粒示踪仪(Nanosight NS300,Malvern Instrument,英国)计数。接下来，我们把这二种不同的外泌体以不同比例混合进行有限稀释，制备成五种不同比例的GPC1 阳性外泌体相对于GPC1- 阴性外泌体的混合物。这五个样品分别为GPC1 阳性外泌体相对于GPC1- 阴性外泌体的比列为1:500,1:1000；1:1000；1:2000；1:5000和1:8000。为了模仿癌症病人血液中外泌体的浓度，这五个样品都配成毎毫升3×10 10外泌体的总浓度。这五种不同比例混合外泌体用生物素标记的抗CD81适配体捕获并固定在链酶亲和素表面活化的磁珠上，然后加类星体荧光基团-670(Quasar-670)标记的GPC1-17A-1d适配体经流式细胞术来进行检测。在此实验中，我们把一个样品中的总磁珠里有1％的磁珠呈现Quasar-670荧光阳性定为检测到GPC1阳性外泌体的诊断阈值。图中样品的GPC1 阳性外泌体相对于GPC1- 阴性外泌体的比例是标明在流式细胞术结果图框外的正上方，检测到的总磁珠中有呈现类星体荧光基团-670阳性的磁珠的百分比则标明在流式细胞术结果图框内的右上方。乱序对照是一个类星体荧光基团-670标记的随机序列的DNA适配体用来代表实验系统的本底荧光。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的验证实验仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的验证实验可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述验证实验中的实验方法，如无特殊说明，均为常规方法。下述验证实验中所用的材料、试剂等，如无特殊说明，均为常规生化试剂，可从商业途径得到。
HEK293T细胞(货号
Figure PCTCN2020130517-appb-000001
CRL-3216)为the American Type Culture Collection i公司产品。
Panc1细胞(货号
Figure PCTCN2020130517-appb-000002
CRL-1469 TM)为the American Type Culture Collection i公司产品。
GPC1敲低的Panc1为(ED200001020)苏州吉玛基因股份有限公司产品。
带有His标签的GPC1质粒(货号HG10576-CH)为中国京义翘神州科技股份有限公 司产品。
重组人GPC1蛋白(货号ab215589)为Abcam公司产品。抗人GPC1抗体(货号ab199343，单抗)为Abcam公司产品。
生物素标记的抗6X His抗体(货号ab106261，小鼠单抗)为Abcam公司产品。
生物素标记的CD81抗体(货号349514，小鼠单抗)为BioLegend公司产品。
抗β-actin抗体(货号ab6276，小鼠单抗)为Abcam公司产品。
亮紫罗兰510TM结合羊抗兔IgG(货号406419)为Biolegend公司产品。
别藻蓝蛋白标记的抗荧光素抗体(货号17-7691-82)为eBioscience公司产品。
FAM标记(货号E17001)为苏州吉玛基因股份有限公司产品。
Quasar 670(Q670)标记(货号11-4200-XX)为苏州吉玛基因股份有限公司产品。
链霉亲和素包被的2.7微米直径的磁珠(货号PL6827-103)为Agilent Technologies公司的产品。
下述验证实验中的所有数据均采用GraphpadPrism 8.0.进行显著性分析。
实施例1、筛选针对GPC1的目标适配体
申请人的目的是采用基于蛋白质的指数富集的配适体系统进化实验(SELEX)(protein-based SELEX)和细胞SELEX(cell-SELEX)方法开发针对GPC1的DNA适配体(以下简称GPC1适配体)，两种技术的示意图见图1。首先，基于蛋白质的SELEX以市购的重组人GPC1蛋白为靶标，以富集GPC1适配体序列。基于蛋白质的SELEX的主要缺点是重组蛋白质可能不具有天然蛋白质的构象。考虑到这一点，申请人以重组的过量表达GPC1的HEK293T细胞为靶细胞，利用细胞SELEX促进GPC1适配体序列的进一步富集。这些富集的DNA序列能够与保持天然构象GPC1蛋白结合。因此，申请人确定所选的GPC1适配体能够成功结合到细胞或外泌体表达的天然GPC1蛋白上。具体过程如下：
1、建立SELEX期间有效扩增单链DNA的PCR系统
在每一轮SELEX期间，与靶蛋白结合的单链DNA序列仅占单链DNA库中的一小部分。如果将回收的靶标单链DNA直接应用于下一轮SELEX，由于数量少，大部分可能会丢失。因此，每一轮SELEX结束时回收的与靶蛋白结合的DNA必须使用专门设计的PCR进行扩增。每一轮SELEX的PCR最佳循环次数需要通关实验来确定。因此，在每一轮SELEX结束时，DNA以PCR回收并扩增，PCR条件：94℃下变性30s，在48.6℃下退火40s，在72℃下延长60s，循环数为12-18，以无模板DNA为阴性对照。不同循环数的PCR产物扩增的情况用4％琼脂糖凝胶凝胶电泳来分析，其代表性的结果见图2中的A，对凝胶电泳结果的相对扩增子强度的分析见图2中的B。结果表明：随着PCR循环数的增加，PCR产物产量逐渐增加，第18周期产生的产量相对较高且非特异性DNA的量最少，因此申请人选择了PCR产量最高的第18周期作为每一轮SELEX结束时回收的靶结合DNA的PCR最佳循环数。
2、建立用于分离扩增DNA正义链的尿素-dPAGE法
使用不同的正义链(sensestrand)和反义链(anti-sense strand)引物，通过不对称PCR扩增目标适配体，具体流程见图3中的左图：正义链合成的上游引物(20nt)用荧光素(FITC)标记，而反义链合成的下游引物(40nt)连接有多聚A(Poly A)尾巴(n＝20)，因此，长 度为86nt的PCR产物的正义链(被FITC标记)含有的适配体为40nt，而与Poly A连接的反义链为106nt。详细实验细节见发明人的方法学论文“Human Gene Therapy Methods,30(1):1-16,2019”。为了分离富集的40nt序列用含12％尿素的聚丙烯酰胺凝胶变性电泳分离预先变性的PCR产物。为此，我们将预先变性了的PCR产物在12％尿素-dPAGE凝胶上电泳，以分离出富集的86nt正义链。单链DNA(86nt)用作分子大小标记。用Gel Star(Lonza,货号50535)染色后，将凝胶在蓝光(470nm波长)照明器(Major Science,货号MBE-300)下成像，以减少普通紫外线灯与DNA发生交联的机会。当反义链与Poly A尾端合成时，它在正义链后面迁移，正义链DNA(包含FITC结合的适配体40nt)的较低条带很容易定位并从凝胶中切除。分离目标单链DNA序列的聚丙烯酰胺凝胶变性电泳凝胶在蓝光下成像图具体见图3中的右图，正义链DNA的5’-端是用用荧光素(FITC)标记的，所以正义链DNA可以很容易地在蓝光灯下被看到并从凝胶中回收。通过电洗脱和DNA沉淀回收凝胶片段中的DNA，并对回收的适配体正义链进行下一轮SELEX。
3、建立以链霉亲和素包被板测定对人GPC1蛋白的捕获能力的SELEX系统
本实验使用了重组的C末端-6X His标记的人GPC1蛋白。His标记的GPC1蛋白与生物素标记的抗6X His抗体的结合物在链霉亲和素包被的板捕获系统中发挥重要作用。为了确定链霉亲和素包被的板对人GPC1蛋白的捕获能力，进行了蛋白质印迹(Western Blotting)分析，具体流程见图4。Western分析结果见图5，表明链霉亲和素包被的板在洗脱液中捕获的人GPC1蛋白的量显著高于上清液。因此，链霉亲和素包被的板可有效捕获人GPC1蛋白，从而为人GPC1蛋白建立了可靠而强大的基于链霉亲和素包被的板的SELEX系统。
4、基于SELEX测定人GPC1蛋白结合的核酸配适体的富集度
在SELEX过程中，用人GPC1蛋白来检测GPC1核酸配适体的富集度的流程见图6的左图：在GPC1SELEX中，用生物素标记的抗6X His抗体固定在链霉亲和素包被微孔板上来形成一个用His标记的GPC1蛋白为固定靶标的检测系统，用以捕获通过SELEX富集的GPC1核酸配适体，这个系统被用作GPC1核酸配适体的阳性选择。在毎一轮SELEX开始时，首先用不加His-GPC1蛋白质的微孔板(链霉亲和素包被微孔板上仅含生物素标记的抗6X His抗体)适配体池进行阴性选择以去除与检测系统非特异性结合的适配体，然后将含有适配体池的上清液转移至阳性选择孔，与所需靶标一起温育后，加入HRP标记的抗FITC抗体，弃去上清液，结合在His-GPC1蛋白质的微孔板上的适配体池用尿素(8M)洗脱并通过PCR扩增，信号被放大，上述过程称为基于微孔板的GPC1核酸配适体的富集暨测定法。
为了检查针对人重组GPC1蛋白选择的适配体池的富集情况，如上所述的基于微孔板的GPC1核酸配适体的富集暨测定法被用来评估了从Round 0(文库)，Round-P5(人GPC1蛋白SELEX 5轮)，Round-P6(人GPC1蛋白SELEX 6轮)获得的适配体池中能与人GPC1蛋白结合的适配体富集的程度。我们PCR扩增和凝胶纯化的单链DNA池(100nmol)来进行结合试验，以加入不含GPC1蛋白的HRP标记的抗FITC抗体时的信号作为背景，用VICTOR X5平板阅读器(PerkinElmer Life and Analytical Sciences)检测荧光强度，测量结合能力，结果见图6中的右图。根据此分析，申请人可以阐明，对于Round-P5和Round-P6 衍生的单链DNA，结合能力分别比Round 0高2倍和10.2倍。Round-P6适配体的结合能力明显高于Round 0(P≤0.05)。该结果最终表明，初始的完全随机的适配体池在Round-P6之后成功富集，并能够与人GPC1蛋白结合。
5、构建GPC1过表达的SELEX细胞
为了获得过量表达GPC1的HEK293T细胞，从中国京义翘神州科技股份有限公司购买了带有His标签的GPC1 cDNA表达质粒(货号HG10576-CH)(图7中的A)。pCMV-GPC1是将pCMV3-C-His的Hind III和Xba I识别位点间的核苷酸替换为全长为1722核苷酸的带有羧基端His标签的编码GPC1的cDNA(GenBank NCBI Reference Sequence:NM_002081.2)，
为了进一步确认GPC1过表达质粒pCMV-GPC1的结构正确，分别对其进行了一次和两次限制性酶切消化，并使用0.8％琼脂糖凝胶电泳进行电泳，电泳结果见图7中的B：通过单限制性酶消化(Hind III或Xba I)，将GPC1过表达质粒pCMV-GPC1切成线性序列，同时通过双重限制性内切酶消化(Hind III+Xba I)，His标签的GPC1 cDNA插入片段的双链DNA(His-GPC1)从载体中释放出来，并且在双重消化组中出现了两个条带，出现在底部的带是His标记的GPC1ORF区域(共1722bp，具体见序列表的序列42；该His标记的GPC1ORF区域编码的氨基酸序列见序列表的序列43)，上面的带代表pCMV3-C-His载体(6164bp)。上述结果表明，GPC1过表达质粒是用于下游应用的正确表达构建体。
以上述的GPC1过表达质粒pCMV-GPC1转染HEK293T细胞。24小时后得到转染后的HEK293T细胞，将其命名为HEK293T/pCMV-GPC1。为了验证人GPC1蛋白在HEK293T细胞中的过表达，将HEK293T/pCMV-GPC1制备成细胞裂解物，以HEK293T(又称野生型HEK293T或HEK293T(WT，图7中的C使用抗6X His抗体进行了蛋白质印迹分析重组His-GPC1的表达。首先用抗6X His抗体探测膜，然后用抗β-actin抗体作为加载对照，用10％SDS-PAGE进行解析如图7中的C所示，His标签的人GPC1蛋白在HEK293T细胞中成功地过表达，HEK293T/pCMV-GPC1又称为GPC1过表达的HEK293T。
将GPC1过表达的HEK293T作为阳性选择，而将HEK293T细胞(WT)作为阴性选择进行细胞SELEX富集的GPC1核酸配适体的结合亲和力测定。为了进一步验证重组GPC1蛋白进转染后的表达，GPC1过表达的HEK293T和野生型HEK293T系均用抗GPC1抗体孵育，加入亮紫罗兰510TM结合羊抗兔IgG的二抗，然后用流式细胞术分析。如图7中的D所示，GPC1过表达的HEK293T细胞的荧光强度中位数明显较高，与野生型HEK293T相比，GPC1表达质粒转染后的HEK293T细胞中GPC1的表达高10-20倍。
6、SELEX过程富集与GPC1过表达的HEK293T结合的适配体
对与HEK293T细胞结合的GPC1适配体用流式细胞术检测分析的原理图见图8的A。为了确定SELEX富集的效率并确定第11轮细胞SELEX的适配体池具有良好的结合能力，但SELEX进行到第11轮时，的适配体池进行流式细胞术，以野生型HEK293T用作阴性对照，而上述获得的GPC1过表达的HEK293T用作阳性对照，抗FITC抗体作为背景对照，用一小部分200nmFITC标记的第11轮时适配体池于细胞孵育并洗涤后，行流式细胞术检测。结果如图8中B所示，来自经细胞-SELEX第11轮富集后的适配体池与GPC1过表达的 HEK293T细胞的结合是其与野生型HEK293T细胞结合的4.5倍，这表明在细胞-SELEX第11轮后，适配体池的与GPC1结合效率提高了。所以综合这些数据，经过11轮细胞SELEX之后，针对天然人GPC1蛋白的GPC1适配体被成功地富集了。
7、通过下一代测序证实来自细胞SELEX的富集适配体池
为了确定最终富集池中适配体的原始序列，进行了适配体克隆的二一代测序(NGS)。GPC1适配体最终利用获得的适配体序列来推断高结合序列。GPC1适配体经过11轮细胞SELEX程序后，对整个适配体池的DNA进行二代测序分析。GPC1适配体只有正向和反向引物以外的初始随机区域(位与正中)是申请人感兴趣的，具体流程见图9：从含有随机适配体序列的双链DNA拷贝开始，去除末端的引物序列、反义链的反向补体、序列插入、筛选和聚类后，记录50个拷贝以上的适配体序列。这样得到的潜在的GPC1适配体中，只有DNA拷贝数量≥50才被选入继续研究，而其他剩余序列则未评估。我们使用多序列比对工具ClustalX对于SELEX DNA库中相同的中央区域40个核苷酸序列进行了序列比对。图10显示了代表147524个重复的NGS结果，每个重复序列具有中央区域40个核苷酸相同序列被标出。GPC1适配体池中这40个核苷酸序列是申请人感兴趣的DNA插入序列，即潜在的适配体候选物库。
8、建立全长GPC1适配体细胞筛选-暨分子改照体系
GPC1适配体的筛选和分子改照工程的流程图见图11：
首先基于上述构建的细胞SELEX的系统，以针对天然构象中的靶标筛选全长GPC1适配体，筛选的过程使用流式细胞术分析。所用的天然构象中的靶标是GPC1胞外域，具体是以GPC1阴性或近似阴性和GPC1阳性的人类细胞系筛选SELEX后针对GPC1胞外域的GPC1DNA的适配体共15个。详细的筛选过程见步骤9。
再验证所初选的15个适配体是否具有与天然构象GPC1蛋白的胞外域的特异性结合的能力，将低表达的GPC1的野生型HEK293T和GPC1过表达的HEK293T分别用作阴性和阳性对照，鉴定后选出3个能结合GPC1的适配体。筛选过程见步骤10。
对入选的二个优胜的能结合GPC1的适配体进行多轮的短截工程，并测结合力筛选合适的候选序列，最后通过定点突变确定优化后的GPC1适配体。详细的分子改照工程及结合力测定筛选过程见步骤11-步骤16。
9、在候选适配体中筛选具有能与CD81结合的结构独特的全长适配体
针对步骤7中通过下一代测序确定最终富集的SELEX库中全长GPC1候选适配体的序列，申请人通过使用在线DNA折叠工具mfold来鉴定具有具有能与天然GPC1蛋白结合的独特二级结构的全长GPC1适配体。从理论上讲，具有不同二级结构的适配体可能具有不同的三级结构，因此可能与GPC1蛋白的不同细胞外表位相互作用。
利用在线DNA折叠工具mfold从排名最高的候选适配体中挑选出具有潜在能与天然GPC1蛋白结合的适配体共15个，包括GPC1-1、GPC1-2、GPC1-3、GPC1-7、GPC1-10、GPC1-12、GPC1-15、GPC1-17、GPC1-22、GPC1-23、GPC1-24、GPC1-25、GPC1-35、GPC1-100和GPC1-300，具体序列见表1，二级结构如图12所示，这些全长GPC1适配体是否真正能够与天然GPC1蛋白结合，将在下一步用实验来检验。
表1 全长序列GPC1 DNA核酸配适体序列
Figure PCTCN2020130517-appb-000003
10、测定15个初筛候选全长GPC1适配体的结合能力
为了评估作为代表性全长GPC1适配体的15个GPC1适配体与天然GPC1蛋白质的结合能力，以流式细胞术检测，将500nM FAM(荧光素)标记了的15种GPC1适配体分别与野生型HEK293T和GPC1过表达的HEK293T在4℃下与500000个细胞孵育1小时。经洗涤除去未结合的适配体序列后，通过流式细胞术分析这些细胞上的来自荧光素的信号。流式细胞术分析结果见图13，GPC1-17适配体、GPC1-24适配体和GPC1-35适配体分别与GPC1过表达的HEK293T结合的荧光强度显著高于与野生型HEK293T结合的荧光强度。这些数据表明，GPC1-17、GPC1-24和GPC1-35是有希望的GPC1蛋白结合物，而其它代表性全长GPC1适配体存在大量的非特异性结合，表明其它代表性全长GPC1适配体可以结合细胞表面上的其它无关分子，而不是GPC1。因此，选择了GPC1-17(序列表的序列8)、GPC1-24(序列表的序列11)和GPC1-35(序列表的序列13)这三个适配体进行进一步分析。
11、以GPC1敲低的Panc1建立GPC1适配体特异性检测系统
为了更好的验证初步筛选到的GPC1适配体是否能够与质膜表面的GPC1特异性的结合，我们用小RNA干扰来敲低下调Panc1细胞株的GPC1的表达。通过流式细胞术和蛋白质印迹分析来证实并定量GPC1在Panc1细胞里被下调的程度，结果如图14所示。图14中A是用GPC1抗体在蛋白质印迹分析的结果。在GPC1被下调Panc1细胞裂解液里，几乎很难检测到GPC1蛋白质的信号，而野生型Panc1细胞裂解液里则出现了轻易可见的对应与GPC1分子量的条带。基于流式细胞术适合研究适配体与细胞表面有天然构象的跨膜蛋白的结合，我们通过流式细胞术再次证实并进一步定量估测GPC1下调的程度。根据流式细胞仪分析，与野生型Panc1相比，经RNA干扰下调的Panc1中GPC1的表达被下 调了约600％，具体见图14中B。这些数据表明，GPC1敲低的Panc1细胞株可以被用来检验候选的GPC1适配体与细胞或外泌体结合的特异性。
12、通过截短进行GPC1适配体序列的第一轮工程化
为了进一步改造适配体，申请人针对每一个全长适配体(GPC1-17、GPC1-24和GPC1-35)生成了两到三个截短的适配体，以选择具有最高结合能力和最短序列的适配体。适配体的截短是有利的，因为它降低了生产成本，最重要的是它可以提高结合亲和力。最初筛选的GPC1-17、GPC1-24和GPC1-35的最初长度均为40个核苷酸，通过截短减少到13-25个核苷酸。
在第一轮截短中，所有适配体的环保持完整，仅去除茎上的一些核苷酸。所有GPC1适配体的全长和截短形式也都被截断，以使所有截短的适配体都具有相同核苷酸序列的原始环区域，茎结构的截断希望能导致所有适配体的结合效率提高，一般来说，并不是原始全长适配体的所有核苷酸都是在与GPC1结合里是必不可缺的，多数情况下可能环区是主要参与了与GPC1的结合。对于每个显示出高结合能力的截短适配体，以Mfold程序生成具有不同热焓的2-4个可能的二级结构。例如，GPC1-17、GPC1-24和GPC1-35分别有4、2和3个可能的二级结构，分别表示为17A，17B，17C，17D(见图15中A)，24A，24B(见图15中B)，35A，35B和35C(见图15中C)。申请人试图验证所有截短适配体是否都具有相同的循环结构。考虑到这一点，申请人从全长适配体中产生了一系列截短适配体，表2和图16中A记载了原始全长GPC1适配体和各截短适配体的序列。
表2 第一轮GPC1 DNA核酸配适体改照后的序列
Figure PCTCN2020130517-appb-000004
Figure PCTCN2020130517-appb-000005
在合成类星体染料-670(Quasar 670)标记的第一轮适配体改照后的适配体后，使用野生型HEK293T和GPC1过表达的HEK293T进行了基于流式细胞术的适配体结合力分析，结果如图16中B所示，GPC1-17、GPC1-17A-1和GPC1-35、GPC1-35A-1已显示出与GPC1的显著结合能力，而GPC1-24中，与和野生型HEK293T相比，和GPC1过表达的HEK293T缺乏有统计学意义的新增结合力。因此，申请人选择GPC1-17A-1(序列表的序列16)和GPC1-35A-1(序列表的序列22)作为申请人的未来目标。
13、第一轮工程化后GPC1-17A-1的动力学研究
确认GPC1-17A-1与天然GPC1蛋白结合能力良好后，申请人开始表征该GPC1适配体的结合动力学,所用方法详见发明人已发表的论文(PMID:21281402)。为了确定了GPC1-17A-1的平衡解离常数(Kd)，将至少六种不同浓度(100nM，250nM，500nM，1000nM，2000nM，5000nM)的类星体染料-670标记的GPC1-17A-1用于结合测定，使用GPC1敲低的Panc1作为阴性对照，GPC1过表达的HEK293T作为阳性对照。如图17所示，GPC1-17A-1对GPC1过表达的HEK293T表观解离常数(Kd)是494.1±13.57nM，而GPC1敲低的Panc1的对应Kd为1631±135.7nM。GPC1敲低的Panc1的GPC1适配体17A-1的(Kd)值比GPC1过表达的HEK293T的(Kd)值高约3.3倍。再者GPC1适配体17A-1与GPC1敲低的Panc1细胞的结合的动力学图是接近一条直线，提示这二者的结合是非特异性结合。这表明GPC1-17A-1是GPC1的有希望的结合物。因此，为了提高适配体的结合亲和力和选择性，申请人决定对GPC1-17A-1进行进一步的改照和截短。
14、通过截短或替换进行GPC1适配体序列的第二轮改照
为了提高适配体的结合亲和力，申请人进一步设计了对适配体的分子改照。由于茎的长度，茎的大小和组成都可影响适配体的结合亲和力，因此申请人对茎进行了一些更改，例如截短，或者将A和/或T分别变成C和/或G，但是保持功能环不变，如图18所示从GPC1-17A-1和GPC1-35A-1分别产生了五个新的适配体，具体适配体的核苷酸序列见表3以及图19的A和图19的B。
表3 第2轮GPC1 DNA核酸配适体改照后的序列
配适体名称 长度 核酸配适体序列(5’to3’) 序列表中的编号
GPC1-17A-1 15 CAATCCCCCTTTTTA 序列16
GPC1-17A-1a 15 CACTCCCCCTTTGTA 序列25
GPC1-17A-1b 15 CCCTCCCCCTTTGGA 序列26
GPC1-17A-1c 13 AATCCCCCTTTTT 序列27
GPC1-17A-1d 13 CATCCCCCTTTTG 序列28
GPC1-17A-1e 13 ACTCCCCCTTTGT 序列29
GPC1-35A-1 30 ACAAAACACACACCCCCTTATCCCATTT 序列22
GPC1-35A-1a 25 AAACACACACCCCCTTATCCCATTT 序列30
GPC1-35A-1b 25 ACACACACACCCCCTTATCCCATGT 序列31
GPC1-35A-1c 25 CCACACACACCCCCTTATCCCATGG 序列32
GPC1-35A-1d 25 AACCACACACCCCCTTATCCCAGTT 序列33
GPC1-35A-1e 25 CGCCACACACCCCCTTATCCCAGCG 序列34
假定茎中明显不成对的碱基可能对适配体的结合功效产生不利影响，将其截除。C-G碱基对包含3个氢键，它将比A-T碱基对键更强；假定在茎区中添加C-G对可以增强改照后的适配体的亲和力和特异性，申请人将茎的A-T碱基对转换为C-G碱基对。另一方面，茎中存在的C-G碱基对越多，环采用不同的3-D构象的灵活性可能就会减少，因此，在整个适配体群体中形成有效的三维环结构的最终比例可能会比原来的占优势，导致更好的靶标结合。经过一系列分子改照工程后，得到的第二轮适配体的长度约为15核苷酸左右。
结合实验结果见图19的C和D，浓度为500nM时，与野生型HEK293T相比，GPC1-17A-1d与其未经改照过的原型GPC1-17A-1相比，对GPC1过表达的HEK293T具有更高的结合能力。不过，在第二轮工程中操作的所有其它GPC1-17A-1衍生的适配体GPC1-17A-1a、GPC1-17A-1b、GPC1-17A-1c、GPC1-17A-1e均显示出比原始适配体GPC1-17A-1更低的结合能力，所有GPC1-35A-1衍生的适配体GPC1-35A-1a、GPC1-35A-1b、GPC1-35A-1c、GPC1-35A-1d、GPC1-35A-1e与原始GPC1-35A-1结合GPC1过表达的HEK293T细胞的能力无显著性差异。因此，选择已显示出荧光强度比原始适配体GPC1-17A-1显著增加的适配体GPC1-17A-1d(序列表的序列28)用于进一步的实验。
15、GPC1-17A-1d的动力学研究
在确认GPC1-17A-1d是细胞膜上天然GPC1蛋白的有前途的结合物后，申请人进一步进行了表征该GPC1适配体的结合动力学的研究。为了确定GPC1-17A-1d的平衡解离常数(Kd)，首先，制备不同浓度(100nM，250nM，500nM，1000nM，2000nM，5000nM)的5'-Quasar 670标记的GPC1-17A-1d，使用GPC1敲低的Panc1(作为阴性对照，步骤5的HEK293T/pCMV-GPC1(又称为GPC1过表达的HEK293T)作为阳性对照(PMID:21281402)。如图20所示，GPC1-17A-1d对GPC1过表达的HEK293T的GPC1的表观解离常数(Kd)为383.7±66nM，而对GPC1敲低的Panc1相应的Kd为5141±39.6nM。在进行第二轮工程设计之前，针对GPC1过表达的HEK293T和GPC1敲低的Panc1的GPC1，GPC1-17A-1的Kd分别约为494.1±13.57nM和1631±135.7nM；经过第二轮轮工程设计后，GPC1-17A-1d对GPC1过表达的HEK293T的GPC1的Kd值降低了(383.7±66nM)，提示其与GPC1的亲和力提高了20％以上。而针对GPC1敲低的Panc1的Kd急剧增加(5141±39.6nM)并且其动力学图仍然是接近一条直线，表明其特异性提高了至少二倍。
值得注意的是，GPC1-17A-1d对GPC1敲低的Panc1细胞的Kd值比其与GPC1过 表达的HEK293T细胞结合的Kd高约13.4倍，这表明在分子改照过程里得到的GPC1-17A-1d是的一种能更特异性地与GPC1结合的有希望的适配体。为了提高适配体的结合亲和力和选择性，申请人决定对GPC1-17A-1d进行进一步的改照优化。
16、GPC1适配体序列的第三轮改照优化
从第二轮工程中选择GPC1-17A-1d后，为了提高适配体与天然GPC1蛋白的结合亲和力和选择性，申请人进一步设计了第三轮适配体。简要地说，申请人假设在GPC1-17A-1d适配体的二级结构中的环上进行一些更改，并在茎中添加了一些AT或CG碱基对，具体见图21，这些改照可能会导致显著的3D构象改变并增强适配体与GPC1的结合效率。
如图21所示，在进行工程设计后，申请人从以下位置产生了6个基于GPC1-17A-1d衍生得到的适配体，具体序列见表4和图22。
表4 第3轮GPC1 DNA核酸配适体改照后的序列
配适体名称 长度 核酸配适体序列(5’to3’) 序列表中的编号
GPC1-17A-1d 13 CATCCCCCTTTTG 序列28
GPC1-17A-1d-1 13 CAGCGCGCGGTTG 序列35
GPC1-17A-1d-2 15 CATGGCCCGGTTCTG 序列36
GPC1-17A-1d-3 15 GCATCCCCCTTTTGC 序列37
GPC1-17A-1d-4 17 GACATCCCCCTTTTGTC 序列38
GPC1-17A-1d-5 17 GGCATCCCCCTTTTGCC 序列39
GPC1-17A-1d-6 19 GGACATCCCCCTTTTGTCC 序列40
申请人在500nM类星体染料-670标记的适配体与细胞结合使用流式细胞仪进行了结合试验以检查其结合功效。不幸的是，与适配体GPC1-17A-1d相比，所有衍生的适配体均显示出与GPC1较低的结合能力，具体见图23。该结果表明，至少在第三定轮的工程中进行的环的改变或茎中碱基的添加不会有助于结合能力的提高，这些结果证实环的改变最可能改变适配体的结合功效。因此，将适配体GPC1-17A-1d作为最终选择的GPC1适配体。
实施例2、确认改照优化后的GPC1适配体GPC1-17A-1d的特异性
确认GPC1-17A-1d的结合亲和力后，申请人以多种带有His标签的过表达重组跨膜蛋白细胞进一步确定了该适配体的特异性和选择性：为了确定申请人的适配体的特异性，申请人随机选择了不相关的跨膜蛋白，特别选择了Glypican的其他两个成员，即GPC4和GPC6，以验证适配体即使过表达也不会与其他细胞表面膜蛋白结合。具体如下：
1材料与方法
带有His标签的野生型HEK293T：将而空白的pCMV3-C-His载体质粒转染HEK293T细胞，24小时后得到转染后的HEK293T细胞，将其命名为HEK293T/pCMV3-C。作为野生型HEK293T阴性对照。
带有His标签的CD9过表达的HEK293T：将His-CD9/pCMV3转染HEK293T细胞，24小时后得到转染后的HEK293T细胞，将其命名为HEK293T/pCMV-CD9。HEK293T/pCMV-CD9购于北京义翘神州科技股份有限公(货号RG80778-CH)。
带有His标签的CD81过表达的HEK293T：将pCMV-CD81转染HEK293T细胞，24小时后得到转染后的HEK293T细胞，将其命名为HEK293T/pCMV-CD81。HEK293T/pCMV-CD81购于北京义翘神州科技股份有限公(货号HG14244-CH)。
带有His标签的TGFβR3过表达的HEK293T：将pCMV-TGFβR3转染HEK293T细胞，24小时后得到转染后的HEK293T细胞，将其命名为HEK293T/pCMV-TGFβR3。HEK293T/pCMV-TGFβR3购于北京义翘神州科技股份有限公(货号HG10778-CH)。
带有His标签的GPC1过表达的HEK293T：将pCMV-GPC1转染HEK293T细胞，24小时后得到转染后的HEK293T细胞，将其命名为HEK293T/pCMV-GPC1。HEK293T/pCMV-GPC1即为带有His标签的GPC1过表达的HEK293T。pCMV-GPC1购于北京义翘神州科技股份有限公(货号HG10576-CH)。
带有His标签的GPC4过表达的HEK293T：将pCMV-GPC4转染HEK293T细胞，24小时后得到转染后的HEK293T细胞，将其命名为HEK293T/pCMV-GPC4。HEK293T/pCMV-GPC4即为带有His标签的GPC4过表达的HEK293T。pCMV-GPC4是购于北京义翘神州科技股份有限公(货号HG10090-CH)。
带有His标签的GPC6过表达的HEK293T：将pCMV-GPC6转染HEK293T细胞，24小时后得到转染后的HEK293T细胞，将其命名为HEK293T/pCMV-GPC6。HEK293T/pCMV-GPC6即为带有His标签的GPC6过表达的HEK293T。pCMV-GPC6购于北京义翘神州科技股份有限公(货号HG10102-CH)。
将野生型HEK293T、HEK293T/pCMV-GPC1(GPC1过表达的HEK293T)、pCMV-CD9(CD9过表达的HEK293T)、HEK293T/pCMV-CD81(CD81过表达的HEK293T)、pCMV-TGFβR3(TGFβR3过表达的HEK293T)、HEK293T/pCMV-GPC4(GPC4过表达的HEK293T)、HEK293T/pCMV-GPC6(GPC6过表达的HEK293T)分别制备成细胞裂解物，以抗6X His标签抗体进行蛋白质印迹分析，以野生型HEK293T为对照，用抗6X-His抗体对HEK293T细胞中His标记蛋白过度表达进行分析，结果见图24中的上图，证实了HEK293T中His标记蛋白的过表达，确认所有重组HEK293T细胞中蛋白的转染都是成功的，并且所有His标记的重组跨膜蛋白均在转染后成功表达。
以野生型HEK293T作为背景荧光对照，400nM类星体荧光基团-670(Quasar-670)标记的GPC1-17A-1d适配体与各种不同的细胞表面过表达的重组蛋白结合果用流式细胞术分析。
2结果
结合力分析的结果如图24中的下图所示，GPC1-17A-1d与野生型HEK293T的结合保持在背景水平，适配体GPC1-17A-1d与过表达CD9、CD81、TGFβR3、GPC4和GPC6的HEK293T细胞的结合与其野生型HEK293T的结合没有显着差异。与野生型HEK293T和过表达其他跨膜蛋白(包括过表达GPC4和GPC6的蛋白)相比，GPC1-17A-1d适配体与过表达GPC1的HEK293T细胞的结合明显要高出一倍，证明GPC1-17A-1d适配体与细 胞表面的他跨膜蛋白的结合是有很强的选择性，适配体GPC1-17A-1d不会因为一个无关的跨膜蛋白在膜上大量表达就与此结合。这些数据表明申请人的GPC1-17A-1d适配体与在质膜表面有天然构象的GPC1特异性地结合。
实施例3、用GPC1-17A-1d适配体检测GPC1阳性的外泌体
GPC1已显示在胰腺癌细胞衍生的外泌体中高表达。为确保GPC1适配体能够捕获GPC1阳性外泌体，申请人使用了有前途的GPC1-17A-1d适配体进行了结合测定。过程如下：将生物素标记的CD81抗体在细胞培养上清液中成功捕获的外泌体固定在链霉亲和素包被的磁珠上，然后将Q670标记的GPC1-17A-1d适配体置于GPC1阳性外泌体中，洗涤后，通过流式细胞术进行分析，具体如图25所示。
1材料与方法
1.1材料
Panc1细胞(货号
Figure PCTCN2020130517-appb-000006
CRL-1469 TM)为the American Type Culture Collection i公司产品。
GPC1敲低的Panc1为苏州吉玛基因股份有限公司产品。
Panc1的细胞培养上清液(CCM)以及GPC1敲低的Panc1的细胞培养上清液(CCM)按有详细描述的已经发表的了方法制备(Proc Natl Acad Sci U S A.113(8):E968-E977,2016),唯一有变动的是在收集含外泌体的细胞培养上清液时，加入了0.5％的去外泌体的小牛血清。
生物素标记的CD81抗体(货号349514，小鼠单抗)为BioLegend公司产品。
链霉亲和素包被的2.7微米直径的磁珠(货号PL6827-103)为Agilent Technologies公司的产品。
Quasar 670标记的GPC1适配体GPC1-17A-1d，是以Quasar 670在5'-端标记适配体GPC1-17A-1d得到的，GPC1-17A-1d的序列为5’-CATCCCCCTTTTG-3’(即序列表中的序列28)。
1.2方法
以GPC1敲低的Panc1和Panc1制备的两种细胞培养上清液(0.5毫升)分别在4℃下用生物素标记的CD81抗体孵育1h，让抗CD81抗体与外泌体结合，加入50微升的链霉亲和素包裹的磁珠(Agilent Technologies,货号PL6827-103)铺获固定，接下来申请人在4℃孵育Quasar 670标记的GPC1适配体GPC1-17A-1d 1h，洗涤后，最后进行流式细胞术。
2结果
结果如图26所示，来自野生型Panc1的外泌体的荧光强度比通过GPC1敲低的Panc1的外泌体的荧光强度高约2倍。该数据表明，GPC1-17A-1d适配体已成功检测到来自Panc1的GPC1阳性外泌体。因此可以预期GPC1适配体GPC1-17A-1d可以有效捕获存在于胰腺癌患者的不同体液中的GPC1阳性外泌体，最终将有助于开发基于外泌体的早期胰腺癌诊断方法。
实施例4、比较基于用GPC1抗体或GPC1核酸适配体在检测用CD81亲和捕获的胰腺癌细胞来源的外泌体上GPC1蛋白的效果
CD81是液体活检中外泌体的极好的标记蛋白，因为外周血中约有94％的细胞外囊泡 (EVs)是由血小板产生的，而这些来自血小板的EV不表达或表达极其低的CD81。为了评估CD81适配体CD81-2J-6(5'-CATTTGACCATCCGGGTCTATG-3'，国家知识产权局Provisional Patent专利申请号201911098597.7)的外泌体捕获能力，申请人将生物素标记的CD81适配体CD81-2J-6在细胞培养上清液(CCM)中成功捕获的外泌体固定在链霉亲和素包被的磁珠上，然后将Q670标记的GPC1-17A-1d适配体置于GPC1阳性外泌体中，洗涤后，通过流式细胞术进行分析，具体如图27所示。具体来说，申请人已经使用CD81-2J-6适配体来捕获外泌体，因为在申请人的实验室中已经确定该适配体可以有效捕获CCM中的外泌体。
1材料与方法
1.1材料
Quasar 670标记的GPC1适配体：以Quasar 670标记适配体GPC1-17A-1d得到Quasar 670标记的GPC1适配体GPC1-17A-1d。
5'-端生物素标记的CD81适配体。
Panc1细胞(货号
Figure PCTCN2020130517-appb-000007
CRL-1469 TM)为the American Type Culture Collection i公司产品。
GPC1敲低的Panc1为苏州吉玛基因股份有限公司产品。
Panc1的细胞培养上清液(CCM)以及GPC1敲低的Panc1的细胞培养上清液(CCM)按有详细描述的已经发表的了方法制备(Proc Natl Acad Sci U S A.113(8):E968-E977,2016),唯一有变动的是在收集含外泌体的细胞培养上清液时，加入了0.5％的去外泌体的小牛血清。
链霉亲和素包被的2.7微米直径的磁珠(货号PL6827-103)为Agilent Technologies公司的产品。
1.2方法
首先将800nM生物素标记的CD81-2J-6适配体与两种CCM(Panc1的细胞培养上清液和GPC1敲低的Panc1的细胞培养上清液)分别在4℃下孵育1小时后加入50微升的链霉亲和素包被的磁珠，孵育20分钟以捕获分离外泌体并将其固定到磁珠表面。接着加入500nM的Quasar 670标记的GPC1适配体GPC1-17A-1d在4℃下孵育1小时，最后进行分别用CD81和CD9抗体进行Western印迹(和流式细胞术分析。
2结果
结果如图28所示，图28中A显示来自Panc1的外泌体的荧光强度比通过GPC1敲低的Panc1的外泌体的荧光强度高约7倍。该数据表明，GPC1-17A-1d适配体已成功检测到来自Panc1的GPC1阳性外泌体。与使用CD81抗体提取外来体的结果比较，CD81适配体引导的外泌体捕获对于基于外来体的液体活检而言是一个更好的系统。令人惊奇的是，当使用相同的GPC1适配体检测时，与CD81-抗体固定的外泌体相比，与CD81-适配体固定的外泌体产生的信号最多高出2-3倍。这些数据可以用空间位阻来解释：单克隆抗体(IgG)CD81抗体为150k Da，而适配体CD81-2J-6仅为6.8kDa。对于一个蛋白质单克隆抗体覆盖的给定面积上，最多可以结合上14个核酸适配体，这是因为后者的体积比前者小近14倍。因此，当CD81蛋白质单克隆抗体覆盖外泌体的表面时，可用于随后结合GPC1 适配体的空位区域要少得多。相反，当比抗体小至少十几倍倍的CD81-2J-6核酸适配体结合到外泌体表面时，这些已经结合到外泌体表面的CD81蛋白质上的CD81的适配体对随后的GPC1适配体的结合的空间位阻就小得多。
如图28中C所示，用GPC1抗体进行的蛋白质印迹分析结果表明，如图28中A和B中所用的外泌体里，野生型Panc1细胞产生的外泌体中有较高的GPC1的表达，相反，经RNA干扰技术敲低GPC1后的Panc1产生的外泌体，GPC1的蛋白水平非常低。综上所述，本申请建立了一个基于CD81适配体的新型系统，不仅可以通过CD81适配体有效地捕获外泌体，而且还可以使用此专利申请中的GPC1适配体来灵敏地检测GPC1阳性的外泌体。因此，此处提供的数据为将来开发强大的液体活检系统以检测外泌体GPC1进行胰腺癌早期诊断铺平了道路。
以上在图28中，我们通过CD81适配体捕获Panc1产生的EV/外泌体后，使用CD81-2J-6核酸适配体来检测的捕获的外泌体上的GPC1蛋白。接下来，我们用CD81适配体捕获Panc1产生的EV/外泌体后，用抗人GPC1单抗体来检测捕获的外泌体上的GPC1蛋白。其实验流程如图29中A所示。应为用的是没有标记的单抗，为我们用里荧光标记的二抗来用流式细胞仪来检测分析捕获的外泌体上的GPC1蛋白。如图29中B所示来自野生型Panc1的外泌体的GPC1荧光强度比来自经RNA干扰技术敲低GPC1后的Panc1的外泌体的GPC1荧光强度只高出约3倍。与如图28在A和B中的7倍荧光强度的差别的结果对比，用GPC1适配体来检测用CD81亲和捕获的外泌体上的GPC1蛋白的水平的敏感性可提高至少一倍以上。综合图28和图29所示的实验结果，本发明的GPC1核酸配适体在检测在GPC1阳性的外泌体上，比传统的单克隆抗体有显著优势。
实施例5、探索GPC1适配体在模拟液体活检中的检测灵敏度
基于EV的液体活检的主要挑战是需在大量来源于血液或正常上皮细胞来源的外泌体和细胞外囊泡的背景情况下检测极少量的癌细胞衍生的外泌体)。如Zhang等人已观察到在其研究的肺癌患者中细胞外囊泡的浓度约为(每毫升1.41±0.31)×10 10个，而对于正常健康的个体，细胞外囊泡的浓度约为(3.37±0.39)×10 9个/ml。所以，在存在大量来自血液的正常健康细胞的背景情况下，有效第检测到微量的癌症来源的EV是一个很大的挑战(Lab Chip.,2019,19,1114-1140；Nanoscale,2019,11,10106-10113)。
为了探索GPC1适配体GPC1-17A-1d的细胞外囊泡检测系统的检测灵敏度(Cancer Cell.37(4):485-495,2020)，申请人建立了一个液体活检的模型。此模型把来自胰腺癌Panc1细胞的外泌体有限稀释后与来自GPC1敲低的Panc1细胞中产生的外泌体按特定的比例混合。用此二种外泌体的混合悬浮液来模仿来自胰腺癌细胞的少数GPC1阴性外泌体混匿于4×10 9/ml来自正常细胞的细胞外囊泡的背景下的情作为检验样品。
1材料与方法
1.1材料
GPC1阳性Panc1EV、GPC1阴性Panc1EV采用标准的超速离心或超滤方法提取外泌体，提取方法参见“Konoshenko MY,Lekchnov EA,Vlassov AV,Laktionov PP,Isolation of Extracellular Vesicles:General Methodologies and Latest Trends.Journal of Biomedicine and Biotechnology,2018:8545347.”。
抗生蛋白链菌素包被的磁珠(货号PL6827-1030)为AGILENT TECHNOLOGIES AUSTRALIA PTY LTD公司产品。
生物素标记的CD81适配体：以生物素标记在5'-端标记适配体CD81-2J-6得到。
Quasar 670标记的GPC1适配体：以Quasar 670标记适配体GPC1-17A-1d得到Quasar 670标记的GPC1适配体GPC1-17A-1d。
1.2方法
首先，从二种不同的细胞，即野生型Panc1细胞和GPC1下调的含极低GPC1蛋白的Panc1细胞培养上清液中制备了外泌体。在此实验中，外泌体悬浮液的浓度特意地调整为每毫升3×10 10外泌体，以模仿临床患者血液中存在的实际外泌体的浓度。然后，通过将GPC1阳性Panc1外泌体与GPC1低表达的Panc1外泌体仔细混合，以1：500至1：8000的比例(1:500,1:1000,1:2000,1:5000,1:8000)对GPC1阳性外泌体与GPC1低表达外泌体在保持总外泌体浓度不变的前提下进行了连续有限稀释。在这五种含不同GPC1高表达和GPC1低表达外泌体比例的连续稀释单但总外泌体浓度是相同的外泌体悬浮液样品中分别加入生物素标记的CD81适配体后捕获外泌体，然后把捕获的外泌体从悬浮液液中分离出来固定在链霉亲和素包被的磁珠上。最后加入用Quasar 670标记的GPC1适配体GPC1-17A-1d借助流式细胞术来检测外泌体后的总外泌体中有无GPC1阳性的来自胰腺癌细胞的外泌体。在被实验中，将荧光阳性的磁珠的百分比超过总珠子总数的1％作为检出GPC1阳性外泌体的阈值。
如图30所示，在1:500和1:1000组中，从Quasar 670标记的GPC1适配体GPC1-17A-1d获得的信号百分比分别为1.09％。he10.87％.该数据表明，在申请人当前基于CD81的适配体检测系统中，Quasar 670标记的GPC1适配体GPC1-17A-1d可在毎500个来自正常细胞的背景外泌体中，检测到一个来源于癌症细胞的GPC1阳性外泌体。值得注意的是，图30所展示的结果是在当前尚未完全优化的条件下得到的初步的检测敏感度。在进一步优化后，基于本发明的GPC1适配体的检测灵敏度可望进一步提高。譬如，图271中使用的使用的荧光团Quasar 670的荧光量子产率(fluorescence quantum yield)与Cy5相似，为0.28。将来，如果申请人使用具有更高量子产率的荧光团，例如量子产率为0.92的Alexa Fluor 488，则检测灵敏度会大大提高。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。

Claims (17)

  1. 一种DNA分子，其特征在于：所述DNA分子是如下A1-A40中任一种的单链DNA：
    A1核苷酸序列为序列28的单链DNA；
    A2核苷酸序列为序列25的单链DNA；
    A3核苷酸序列为序列26的单链DNA；
    A4核苷酸序列为序列27的单链DNA；
    A5核苷酸序列为序列29的单链DNA；
    A6核苷酸序列为序列30的单链DNA；
    A7核苷酸序列为序列31的单链DNA；
    A8核苷酸序列为序列32的单链DNA；
    A9核苷酸序列为序列33的单链DNA；
    A10核苷酸序列为序列34的单链DNA；
    A11核苷酸序列为序列35的单链DNA；
    A12核苷酸序列为序列36的单链DNA；
    A13核苷酸序列为序列37的单链DNA；
    A14核苷酸序列为序列38的单链DNA；
    A15核苷酸序列为序列39的单链DNA；
    A16核苷酸序列为序列40的单链DNA；
    A17核苷酸序列为序列1的单链DNA；
    A18核苷酸序列为序列2的单链DNA；
    A19核苷酸序列为序列3的单链DNA；
    A20核苷酸序列为序列4的单链DNA；
    A21核苷酸序列为序列5的单链DNA；
    A22核苷酸序列为序列6的单链DNA；
    A23核苷酸序列为序列7的单链DNA；
    A24核苷酸序列为序列8的单链DNA；
    A25核苷酸序列为序列9的单链DNA；
    A26核苷酸序列为序列10的单链DNA；
    A27核苷酸序列为序列11的单链DNA；
    A28核苷酸序列为序列12的单链DNA；
    A29核苷酸序列为序列13的单链DNA；
    A30核苷酸序列为序列14的单链DNA；
    A31核苷酸序列为序列15的单链DNA；
    A32核苷酸序列为序列16的单链DNA；
    A33核苷酸序列为序列17的单链DNA；
    A34核苷酸序列为序列18的单链DNA；
    A35核苷酸序列为序列19的单链DNA；
    A36核苷酸序列为序列20的单链DNA；
    A37核苷酸序列为序列21的单链DNA；
    A38核苷酸序列为序列22的单链DNA；
    A39核苷酸序列为序列23的单链DNA；
    A40核苷酸序列为序列24的单链DNA。
  2. 根据权利要求1所述的DNA分子，其特征在于：序列25、序列26、序列27、序列28、序列29、序列30、序列31、序列32、序列33、序列34、序列35、序列36、序列37、序列38、序列39、序列40中任一种的单链DNA。
  3. 根据权利要求2所述的DNA分子，其特征在于：序列25、序列26、序列27、序列28、序列29、序列30、序列31、序列32、序列33、序列34中任一种的单链DNA。
  4. 根据权利要求3所述的DNA分子，其特征在于：所述DNA分子是核苷酸序列为序列28的单链DNA。
  5. 一种探针，其特征在于：所述探针为权利要求1-4任一项所述DNA分子被标记物标记得到的物质。
  6. 一种传感器，其特征在于：所述传感器含有权利要求1-4任一项所述DNA分子或权利要求5所述探针。
  7. 一种胰腺癌和/或乳腺癌和/或前列腺癌和/或结直肠癌和/或脑胶质瘤的诊断试剂，其特征在于：含有权利要求1-4任一项所述DNA分子。
  8. 根据权利要求7所述的胰腺癌和/或乳腺癌和/或前列腺癌和/或结直肠癌和/或脑胶质瘤的诊断试剂，其特征在于：胰腺癌和/或乳腺癌和/或前列腺癌和/或结直肠癌和/或脑胶质瘤的诊断试剂还含有与CD81特异结合的物质。
  9. 根据权利要求8所述的胰腺癌和/或乳腺癌和/或前列腺癌和/或结直肠癌和/或脑胶质瘤的诊断试剂，其特征在于：所述与CD81特异结合的物质为与CD81特异结合的适配体或与CD81特异结合的适配体共轭的磁珠。
  10. 根据权利要求9所述的胰腺癌和/或乳腺癌和/或前列腺癌和/或结直肠癌和/或脑胶质瘤的诊断试剂，其特征在于：所述与CD81特异结合的适配体为核苷酸序列为序列41的单链DNA。
  11. 根据权利要求8所述的胰腺癌和/或乳腺癌和/或前列腺癌和/或结直肠癌和/或脑胶质瘤的诊断试剂，其特征在于：所述与CD81特异结合的物质为抗适配体CD81的抗体或与抗CD81的抗体共轭的磁珠。
  12. 一种用于检测外泌体GPC1蛋白质的试剂，其特征在于：含有权利要求1-4任一项所述DNA分子和与CD81特异结合的物质。
  13. 根据权利要求12所述的用于检测外泌体GPC1蛋白质的试剂，其特征在于：所述与CD81特异结合的物质为与CD81特异结合的适配体或与CD81特异结合的适配体共轭的磁珠。
  14. 根据权利要求13所述的用于检测外泌体GPC1蛋白质的试剂，其特征在于：所述与CD81特异结合的适配体为核苷酸序列为序列41的单链DNA。
  15. 根据权利要求12所述的用于检测外泌体GPC1蛋白质的试剂，其特征在于：所述与CD81特异结合的物质为抗适配体CD81的抗体或与抗CD81的抗体共轭的磁珠。
  16. 权利要求1-4任一项所述的DNA分子在作为GPC1蛋白质的适配体中的应用。
  17. 根据权利要求16所述的应用，其特征在于：所述应用为如下B1和/或B2：
    B1、所述DNA分子在制备胰腺癌和/或乳腺癌和/或前列腺癌和/或结直肠癌和/或脑胶质瘤的诊断试剂中的应用；
    B2、所述DNA分子在制备检测GPC1阳性外泌体试剂中的应用。
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