Tumor markers for diagnosis of colorectal cancer and detection method based on nucleic acid aptamer

Abstract

The application discloses a tumor marker for colorectal cancer diagnosis and a nucleic acid aptamer-based detection method, relates to a nucleic acid aptamer sequence for specifically recognizing epithelial cell adhesion molecule (EpCAM) and prion protein (PRNP), EpCAM and PRNP proteins as colorectal cancer diagnosis markers and a method for detecting expression levels in exosomes. The nucleic acid aptamer sequence is obtained by screening a cell-SELEX (systematic evolution of ligands by exponential enrichment) technology with colorectal cancer cells as targets, can be chemically synthesized in vitro, is stable in structure, easy to modify and replace, low in cost and high in reproducibility; and the target protein is highly enriched in exosomes derived from colorectal cancer tumor cells and can be used as a colorectal cancer diagnosis marker. The nucleic acid aptamer obtained by the application can be applied to imaging of human colorectal cancer related malignant tumor cells and derived exosomes, preparation of coupled drugs, clinical diagnosis and development of related kits.

Description

Technical Field

[0001] This invention belongs to the field of biological diagnostics, specifically relating to the use of nucleic acid aptamer sequences that recognize specific target proteins as diagnostic biomarkers for colorectal cancer by detecting protein expression levels in exosomes. Background Technology

[0002] Colorectal cancer (CRC) is the second leading cause of cancer death worldwide and a major contributor to cancer mortality. Early detection of high-risk, advanced colonic adenomas and early-stage CRC is an effective way to reduce CRC mortality. Achieving personalized precision treatment, reducing treatment costs, and improving quality of life are crucial issues that urgently need to be addressed to improve the prognosis of CRC patients.

[0003] Nucleic acid aptamers are single-stranded nucleotides (DNA or RNA molecules) that can specifically bind to targets. They are small in molecular weight, easy to design and synthesize, and can fold into specific conformations to specifically bind to biological targets, thus earning them the reputation of "chemical antibodies." Cell-SELEX, an in vitro exponential enrichment ligand system evolution technique using live cells as targets, can effectively screen nucleic acid aptamers that specifically recognize targets from large random libraries. Nucleic acid aptamers possess advantages such as simple synthesis, easy modification, chemical stability, low immunogenicity, and low cost, making them promising novel, highly efficient, and stable molecular probes with great potential in areas such as early cancer diagnosis and precision targeted therapy, drug therapy, and biochemical sensing.

[0004] Liquid biopsy refers to the analysis and diagnosis of diseases such as cancer by examining blood or other bodily fluids. It has advantages such as rapid results, minimal invasiveness, and good patient compliance. In recent years, molecular markers carried by exosomes have gradually become one of the main components of tumor liquid biopsy. As an important component of the tumor microenvironment, exosomes in the bodily fluids of cancer patients are rich in nucleic acids, proteins, and lipids, which can promote tumor angiogenesis, immune escape, cellular drug resistance, and distant metastasis through multiple pathways. Exosomes are rich in protein components, especially specific proteins distributed on the surface or in the lumen of tumor-derived exosomes, which also facilitates the detection of nucleic acid aptamers. Summary of the Invention

[0005] This invention relates to obtaining the nucleic acid aptamer WHY-1c, which specifically recognizes epithelial cell adhesion molecule (EpCAM), and the nucleic acid aptamer WHY-3e, which specifically recognizes prion protein (PRNP), via CELL-SELEX. A method for detecting the expression levels of aptamer target proteins in exosomes using nucleic acid aptamers was established, which can be used in conjunction with exosomes derived from clinical samples (tissues, various body fluids) for tumor diagnosis and analysis. Results showed that the target proteins PRNP and EpCAM, bound by the nucleic acid aptamers, were highly enriched in exosomes derived from CRC tumor cells. Further detection of their expression levels in plasma exosomes from CRC patients using nucleic acid aptamers can effectively differentiate between colorectal cancer patients and healthy individuals. These results demonstrate the potential of EpCAM and PRNP expression levels in exosomes as diagnostic biomarkers for colorectal cancer. The use of EpCAM and PRNP as potential diagnostic biomarkers for CRC, and the method for detecting exosomes using nucleic acid aptamers, are of great significance for developing diagnostic and therapeutic strategies for CRC.

[0006] Specific aspects of the present invention include:

[0007] The first aspect of the present invention provides the use of nucleic acid aptamers or derivatives thereof in recognizing and binding or assisting in recognizing and binding EpCAM in a sample; or the use of nucleic acid aptamers or derivatives thereof in preparing products that recognize and bind or assist in recognizing and binding EpCAM in a sample; wherein the nucleic acid aptamer is a single-stranded DNA molecule as shown in Seq ID No: 1, 3-5; preferably a single-stranded DNA molecule as shown in Seq ID No: 5.

[0008] A second aspect of this invention provides the use of nucleic acid aptamers or derivatives thereof in recognizing and binding to or assisting in recognizing and binding to PRNPs in a sample; and the use of nucleic acid aptamers or derivatives thereof in the preparation of products that recognize and bind to or assist in recognizing and binding to PRNPs in a sample.

[0009] The nucleic acid aptamer is a single-stranded DNA molecule as shown in Seq ID No: 2, 6-10; preferably, it is a single-stranded DNA molecule as shown in Seq ID No: 10.

[0010] The third aspect of the present invention provides the use of nucleic acid aptamers or derivatives thereof in recognizing and binding or assisting in recognizing and binding the combination of EpCAM and PRNP in a sample; or the use of nucleic acid aptamers or derivatives thereof in the preparation of products that recognize and bind or assist in recognizing and binding the combination of EpCAM and PRNP in a sample;

[0011] The nucleic acid aptamer that recognizes and binds to or assists in recognizing and binding to EpCAM is a single-stranded DNA molecule as shown in Seq ID No: 1, 3-5; preferably, it is a single-stranded DNA molecule as shown in Seq ID No: 5.

[0012] The nucleic acid aptamer that recognizes and binds to or assists in recognizing and binding to the PRNP is a single-stranded DNA molecule as shown in Seq ID No: 2, 6-10; preferably, it is a single-stranded DNA molecule as shown in Seq ID No: 10.

[0013] In a specific embodiment of the present invention, the identification and combination or auxiliary identification and combination in EpCAM and / or PRNP is for non-diagnostic purposes.

[0014] In a specific embodiment of the present invention, the product is an imaging reagent, a tumor diagnostic reagent, or a therapeutic reagent such as a drug conjugate, and the tumor is preferably colorectal cancer (CRC).

[0015] In a specific embodiment of the present invention, the sample is a bodily fluid such as blood, a tissue such as cancerous tissue or adjacent tissue, or an excrement such as feces; and / or a cell or exosome.

[0016] The fourth aspect of this invention provides the use of a reagent for detecting the expression level of the PRNP gene or protein in a sample in the preparation of a colorectal cancer (CRC) diagnostic kit.

[0017] The fifth aspect of the present invention provides the use of a combination of reagents for detecting the expression levels of EpCAM and PRNP genes or proteins in a sample in the preparation of a colorectal cancer (CRC) diagnostic kit.

[0018] In a specific embodiment of the present invention, the sample is an exosome, such as a blood-derived exosome.

[0019] Preferably, the diagnostic kit is prepared in combination with exosome separation, enrichment and purification reagents;

[0020] Preferably, the diagnostic kit is prepared by combining exosome isolation, enrichment and purification reagents.

[0021] In a specific embodiment of the present invention, the sample source is bodily fluids such as blood, tissues such as cancerous tissue or adjacent tissue, or excrement such as feces.

[0022] In a specific embodiment of the present invention, the reagent for detecting the expression level of PRNP or EpCAM in a sample is a nucleic acid aptamer or its derivative.

[0023] Preferably, the nucleic acid aptamer for detecting epithelial cell adhesion molecule (EpCAM) is shown in the sequence Seq ID No: 1, 3-5, and more preferably Seq ID No: 5;

[0024] Preferably, the nucleic acid aptamer for detecting prions (PRNP) is shown in the sequence Seq ID No: 2, 6-10, and more preferably Seq ID No: 10;

[0025] Preferably, the nucleic acid aptamer derivative is:

[0026] (1) Delete or add one or more nucleotides to the nucleic acid aptamer to obtain a derivative of the nucleic acid aptamer that has the same function as the nucleic acid aptamer;

[0027] (2) The nucleic acid aptamer is substituted or modified with nucleotides to obtain a derivative of the nucleic acid aptamer that has the same function as the nucleic acid aptamer;

[0028] (3) Modify the backbone of the nucleic acid aptamer to a thiophosphate backbone to obtain a derivative of the nucleic acid aptamer with the same function as the nucleic acid aptamer;

[0029] (4) Obtain a derivative of the nucleic acid aptamer that has the same function as the nucleic acid aptamer from the RNA molecule encoded by the nucleic acid aptamer;

[0030] (5) Obtain a derivative of the nucleic acid aptamer that has the same function as the nucleic acid aptamer from the peptide nucleic acid encoded by the nucleic acid aptamer;

[0031] (6) Connect a signal molecule and / or an active molecule and / or a functional group to one end or the middle of the nucleic acid aptamer to obtain a derivative of the nucleic acid aptamer that has the same function as the nucleic acid aptamer.

[0032] More preferably, the modification is phosphorylation, methylation, amylation, thiolation, or isotopization; the functional group is a fluorescent group or nanoluminescent material, a magnetic or paramagnetic group, an affinity group such as a biotin group or digoxigenin group, a radioactive substance, an X-ray responsive group, a therapeutic substance, or an enzyme label.

[0033] The sixth aspect of the present invention provides a method for detecting the expression level of tumor exosome surface targets, comprising the following steps: 1) separating tumor exosomes; 2) using aldehyde / sulfate latex microbeads to adsorb and immobilize exosomes to achieve exosome enrichment; 3) using nucleic acid aptamers or their derivatives that recognize exosome surface targets to label the microbeads immobilized with exosomes; 4) analyzing the labeled microbeads using flow cytometry.

[0034] In a specific embodiment of the present invention, the method can realize the quantitative expression level of multiple targets on the surface of exosomes, and more preferably, the targets are PRNP or EpCAM or a combination thereof.

[0035] In a specific embodiment of the present invention, the nucleic acid aptamer or its derivative that identifies the target on the surface of exosomes is as defined above.

[0036] A seventh aspect of the present invention provides a diagnostic reagent combination based on the quantitative expression level of exosome surface targets, comprising nucleic acid aptamers or derivatives thereof as defined above, and aldehyde / sulfate latex microbeads.

[0037] The eighth aspect of the present invention provides the use of the diagnostic reagent combination in the preparation of tumor diagnostic reagents, preferably the tumor being colorectal cancer (CRC).

[0038] Beneficial effects

[0039] The method provided by this invention has the advantage of utilizing the reaction of aldehyde groups with proteins on the surface of exosome membranes to achieve the adsorption and immobilization of exosomes, thus reducing the requirements for instruments used in exosome detection (flow cytometry). The method is technically simple, requires a small total sample volume (100-200 μL of plasma), and exhibits good reproducibility, meeting the needs of high-throughput detection. This invention also found that PRNP, EpCAM, and their nucleic acid aptamers have high accuracy as biomarkers for colorectal cancer diagnosis and possess extremely high diagnostic value. Attached Figure Description

[0040] Figure 1 Predicted structures of nucleic acid aptamers, identification and validation of binding target proteins;

[0041] Figure 2 The binding and affinity of nucleic acid aptamers and truncation-optimized cells;

[0042] Figure 3 Nucleic acid aptamers were used to detect the relative expression levels of EpCAM and PRNP on the membrane surfaces of various colorectal cancer tumor cells and normal colorectal epithelial cells by flow cytometry (combined with a heatmap of the positive rate of nucleic acid aptamer microbeads).

[0043] Figure 4 The relative expression levels of EpCAM and PRNP in exosomes secreted by various colorectal cancer tumor cells and normal colorectal epithelial cells were detected by flow cytometry using WHY-3e nucleic acid aptamers (combined with a heatmap of nucleic acid aptamer microbead positivity).

[0044] Figure 5 The aptamer binding positivity rate was found in samples from 80 colorectal cancer patients and 23 healthy individuals.

[0045] Figure 6 ROC curves of data from 80 colorectal cancer patients and 23 healthy samples. Detailed Implementation

[0046] Example

[0047] Example 1. Nucleic acid aptamers WHY-1c and WHY-3e for target protein capture and result validation

[0048] 1. Cell Culture

[0049] All cells used in the experiment were obtained from the American Type Culture Collection (ATCC). Specific culture conditions are shown in Table 1. All culture media contained 10% FBS and 100 U / mL penicillin and streptomycin, and were cultured in a CO2 incubator at 37°C with a CO2 concentration of 5%. The digestion solution used for cell passage was 0.25% Trypsin-EDTA, and the cryopreservation solution used was a commercially available serum-free cryopreservation solution.

[0050] Table 1 Cell Culture

[0051]

[0052] 2. Solution preparation

[0053] Washing buffer: DPBS buffer (pH=7.4), 5 mM MgCl2, 4.5 g / L glucose.

[0054] Binding buffer: composed of washing buffer with 1 mg / mL bovine serum albumin (BSA) and 0.1 mg / mL herring sperm DNA.

[0055] 3. Preparation of stable isotope-labeled cells

[0056] (1) Add isotopically labeled essential amino acids, 10% FBS and 100 U / mL penicillin and streptomycin to SILAC-specific PRMI 1640 medium (Thermo Scientific) to prepare SILAC-specific complete medium containing light and heavy isotopic amino acids.

[0057] (2) Take HCT-8 cells required for the identification target, pass them through no more than 10 generations, observe that the cells adhere well, discard the ordinary culture medium, wash with DPBS 2-3 times, and finally add SILAC special complete culture medium to the culture dish.

[0058] (3) Cultured in SILAC medium for at least 8 generations. At this time, the intracellular proteins are stably labeled with isotopes and collected for mass spectrometry identification.

[0059] 4. Nucleic acid aptamer target capture and mass spectrometry identification

[0060] (1) Cell acquisition: Take the HCT-8 cells required for identification and culture them for at least 8 generations in light and heavy culture SILAC special medium.

[0061] (2) Nucleic acid aptamer binding: Take cells cultured in SILAC medium containing light and heavy isotopes respectively, discard the old medium, add DPBS to the dish and wash three times, then add enzyme-free digestion solution to digest and dissociate the cells. Resuspend the cells in a centrifuge tube, wash twice with washing buffer, and count the cells to ensure that the number of cells in each sample is not less than 10. 8 Add the nucleic acid aptamer WHY-3e or control aptamer (200 nM) diluted with binding buffer, incubate on ice for 30 min, shaking 3-4 times during incubation, centrifuge after incubation, add washing buffer 2-3 times, and take a small amount of cell suspension to monitor binding by flow cytometry.

[0062] (3) Formaldehyde crosslinking: Add 3 mL of 2% formaldehyde solution (diluted with DPBS) to the cell suspension and incubate on ice for 15 minutes. After incubation, add 400 µL of 3M glycine solution to terminate the crosslinking.

[0063] (4) Cell lysis: Add freshly prepared cell lysis buffer to each sample (10 µL PMSF and 10 µL cocktail protease inhibitor in 1 mL of lysis buffer), and shake at 4 °C for 1 hour.

[0064] (5) Protein extraction: Place the centrifuge tube in a centrifuge pre-cooled to 4°C, 10000g, 10 minutes, and after centrifugation, take the supernatant and transfer it to a new centrifuge tube.

[0065] (6) Capturing target proteins: Take 20 µL of streptavidin agarose beads (Cytiva), wash three times with 1 mL DPBS by centrifugation, add to protein supernatant, and incubate at 4°C with shaking for 2 hours. After incubation, wash the agarose beads three times with cell lysis buffer and three times with DPBS.

[0066] (7) Light and heavy isotope mixing: Mix agarose beads incubated with light isotope-labeled cells and the aptamer of the test nucleic acid with agarose beads incubated with heavy isotope-labeled cells and the control nucleic acid aptamer. Conversely, mix agarose beads incubated with heavy isotope-labeled cells and the aptamer of the test nucleic acid with agarose beads incubated with light isotope-labeled cells and the control nucleic acid aptamer. Incubate overnight at 4°C.

[0067] (8) Electrophoretic separation: After overnight agarose beads were added to 2X SDS Loading solution (Bio-Rad) at a ratio of 1:1 and heated to 95°C for 1 hour to denature them. Separation was performed using 12% SDS-PAGE gel.

[0068] (9) Cut the gel: Cut off the gel 1 cm after the bromophenol blue stripe, chop it up and put it into a centrifuge tube.

[0069] (10) Decolorization: Wash twice with a 50% acetonitrile ammonium bicarbonate solution, 30 minutes each time. Wash again with a 100% acetonitrile solution. Remove the acetonitrile and air dry for later use.

[0070] (11) Denaturation: Add 300 µL of DTT solution (DTT: 1.5 mg / mL) to the centrifuge tube and incubate at 56°C for 45 minutes. After removing the DTT solution, wash twice with 50% acetonitrile ammonium bicarbonate solution for 30 minutes each time. Wash once more with 100% acetonitrile. Remove the acetonitrile and air dry. Add 300 µL of IAA solution (10 mg / mL) and incubate at room temperature for 30 minutes. After removing the IAA, wash twice with 50% acetonitrile ammonium bicarbonate solution for 30 minutes each time. Wash once more with 100% acetonitrile. Remove the acetonitrile and air dry.

[0071] (12) Enzymatic digestion: Add 100 µL of trypsin to the centrifuge tube (the solution should cover the gel), place it on ice for 30 minutes, and then place it at 37°C overnight.

[0072] (13) Collect peptide fragments: Add 100 µL of acetonitrile to a centrifuge tube, shake and centrifuge, and collect the supernatant into a new centrifuge tube. Add 200 µL of solution A (containing 1% formic acid, 50% acetonitrile, and 50% deionized water) to the collected solution and wash twice. Collect the supernatant and evaporate it under vacuum.

[0073] (14) Peptide desalting: Solution B (100% acetonitrile, 0.1% formic acid), solution C (50% acetonitrile, 50% deionized water, 0.1% formic acid), and solution D (deionized water, 0.1% formic acid) were added sequentially to the desalting column for activation equilibration. The peptide was dissolved in 150 μL of 0.1% formic acid solution. The desalting column was then sequentially agitated 20 times in a centrifuge tube containing the peptide solution to adsorb the peptide. The column was then agitated twice with solution D for desalting, and finally agitated 20 times with solution C to elute the peptide. The peptide solution was then concentrated under vacuum for later use.

[0074] (15) The peptide products were analyzed and identified using an LTQ-OrbitrapVelos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) to obtain the raw mass spectrometry data.

[0075] (16) Mass spectrometry analysis: The raw mass spectrometry data obtained in step (5) was retrieved from the uniprot protein database using the MaxQuant search engine. Some parameters for the database search are as follows: immobilization modification is alkylation on cysteine, variable modification is oxidation on methionine and acetylation at the N-terminus of the protein. Two missed cleavage sites are allowed, the parent ion tolerance is 20 ppm, and the MS / MS fragment ion mass error is 0.5 Da.

[0076] The results are shown in Table 3. In the ratio of protein intensity captured by nucleic acid WHY-3e to that captured by WHY-1c (WHY-3e / WHY-1c), the ratio of endogenous biotinylated protein is close to 1, while the ratio of captured EpCAM is less than 0.05 and the ratio of captured PRNP is greater than 20. This indicates that nucleic acid aptamer WHY-1c can capture EpCAM protein and nucleic acid aptamer WHY-3e can capture PRNP protein.

[0077] 5. Verification of aptamer binding to target proteins (aptamer pull-down assay)

[0078] (1) Cell acquisition: Take the same HCT-8 cells used for nucleic acid aptamer identification, dissociate them with enzyme-free digestion solution, collect the cells in centrifuge tubes, centrifuge, and wash 2-3 times with washing buffer.

[0079] (2) Nucleic acid aptamer binding: Add the nucleic acid aptamer to be verified or the control aptamer (200 nM) diluted with binding buffer to the cells, incubate on ice for 30 minutes, shaking 3-4 times during the incubation, centrifuge after incubation, add washing buffer 2-3 times, and take a small amount of cell suspension to monitor the binding by flow cytometry.

[0080] (3) Steps 3 to 6 in the identification of repeating nucleic acid aptamer targets.

[0081] (4) Electrophoretic separation: Add agarose beads to 2X SDS Loading solution (Bio-Rad) at a 1:1 ratio and heat to 95℃ for 10 minutes to denature. Prepare the protein blotting assay (Western blotting) in advance, including separating and stacking gels of appropriate concentrations. Add the denatured protein sample to the wells for separation.

[0082] (5) Transfer membrane: Cut a PVDF membrane of appropriate size and set it aside. The order from positive electrode to negative electrode is sponge, filter paper, PVDF membrane, separating gel, filter paper, sponge. After assembly, place it in the electro-transfer tank and add electro-transfer solution to transfer the membrane.

[0083] (6) Blocking and primary antibody incubation: Place 5% skim milk (diluted with TBST) on a shaker and incubate at room temperature for 1-2 hours. After incubation, wash once with TBST, cut a membrane of appropriate size containing the target fragment, add PRNP monoclonal antibody (Aifang Bio) or EPCAM monoclonal antibody (abcam) and incubate overnight. After incubation, wash the membrane with TBST on a shaker for 5-10 minutes at room temperature, repeating 3 times.

[0084] (7) Secondary antibody incubation and development: Dilute the same animal species IgG horseradish enzyme-labeled secondary antibody with 5% skim milk at a ratio of 1:10000 and incubate at room temperature for 1-2 hours. After incubation, wash the membrane with TBST on a shaker at room temperature for 5-10 minutes, repeating 3 times. Remove the membrane from the TBST, blot off excess water with filter paper, and place it in the pre-prepared ECL ultrasensitive luminescent solution for full contact. Finally, image the membrane.

[0085] The results are as follows Figure 1 As shown, Western blotting experiments using specific monoclonal antibodies further validated that aptamer WHY-1c can specifically capture EpCAM protein, and aptamer WHY-3e can specifically capture PRNP protein. Therefore, aptamers WHY-1c and WHY3e can be used for the capture, enrichment, and extraction of EpCAM and PRNP proteins from complex samples, respectively.

[0086] Example 2. Nucleic acid aptamers WHY-1c and WHY-3e were used for flow cytometry detection of EpCAM and PRNP proteins on the cell surface, respectively.

[0087] 1. Library preparation: The prepared single-stranded DNA library or truncated nucleic acid aptamer candidate was denatured at 95°C for 5 minutes, cooled on ice for 5 minutes, and annealed at room temperature for 15 minutes before use.

[0088] 2. Cell suspension preparation: Prepare HCT-8, HCT116, SW480, SW620, and NCM460 cells. Remove the culture medium, wash the cells three times with DPBS, add 1 mL of commercially available enzyme-free digestion solution, and digest in a 37°C incubator for 5-10 minutes. Gently pipette the cells and collect them into 1.5 mL EP tubes. Centrifuge at 1000 rpm for 3 minutes, wash once with washing buffer, and add binding buffer for later use.

[0089] 3. Cell and Library Incubation: Divide the prepared cells into several aliquots and incubate each aliquot with the denatured library on ice for 30 minutes. The incubation volume is 100 μL, and the final concentration of the library used in each aliquot is 200 nM. After incubation, centrifuge to remove the supernatant, wash once with washing buffer, add 300-400 μL of washing buffer, filter through a 400-mesh sieve, and then load the cells. All cell processing procedures are performed on ice.

[0090] 4. Flow cytometry detection: Flow cytometer was used for detection, collecting data from 10,000 cells per sample. Experimental data were processed using FlowJo software, and the average fluorescence intensity was used for quantitative analysis.

[0091] 5. Affinity test:

[0092] Different concentrations of nucleic acid aptamers were respectively mixed with 2×10 5 HCT-8 cells were incubated on ice for 30 minutes. After centrifugation, washing, and resuspending, the cells were analyzed by flow cytometry. Flow cytometry data were processed using FlowJo (Version 10.4.0) software and plotted using GraphPad Prism 9 (Version 9.3.0) software. The x-axis represents single-stranded DNA concentration (nM), and the y-axis represents the average fluorescence intensity after subtracting the cell autofluorescence value. The formula Y=B was used to calculate the average fluorescence intensity. max X / (Kd+X) is used to calculate the binding dissociation constant of the nucleic acid aptamer.

[0093] Nucleic acid aptamer optimization and affinity assay results are as follows Figure 2 As shown in Table 2, truncated aptamer sequences WHY-3a, WHY-3b, WHY-3c, WHY-3d, and WHY-3e were synthesized based on the WHY-3 sequence; truncated sequences WHY-1a, WHY-1b, and WHY-1c were synthesized based on the WHY-1 sequence. Flow cytometry analysis revealed that WHY-1c and WHY-3e bound well. Figure 2 AB). Further affinity assays were performed on these two nucleic acid aptamers in cells, showing that the affinity of WHY-1c was 1.2 ± 0.4 nM, and the affinity of WHY-3e was 67.2 ± 19.2 nM. Figure 2 CD). Nucleic acid aptamers WHY-1c and WHY3e were used to detect EpCAM and PRNP proteins on the cell membrane surface, respectively.

[0094] Further flow cytometry characterization, such as Figure 3 As shown, the nucleic acid aptamers WHY-1c and WHY-3e bind to different degrees with colorectal cancer cell lines HCT-8, HCT116, SW480, and SW620, and bind weakly or not at all with the normal colorectal epithelial cell line NCM460. This indicates that the nucleic acid aptamers WHY-1c and WHY-3e can detect the expression levels of EpCAM and PRNP in colorectal cancer tumor cells. The above experimental results also show that EpCAM and / or PRNP are expressed at higher levels in various colorectal cancer cells compared to the normal colonic epithelial cell line NCM460. Therefore, EpCAM, PRNP, and their nucleic acid aptamers WHY-1c and WHY-3e have the potential to become biomarkers for colorectal cancer detection. Furthermore, the nucleic acid aptamers WHY-1c and WHY-3e can be coupled with fluorescent groups according to the needs of the detection experiment, and the expression levels of EpCAM and PRNP in live cell samples can be quantified using flow cytometry, confocal microscopy, etc., providing flexible and diverse detection methods.

[0095] Example 3. Nucleic acid aptamers WHY-1c and WHY-3e can be used for exosome sample detection.

[0096] 1. Exosome isolation

[0097] Exosome isolation: Supernatant from culture media containing HCT-8, HCT116, SW480, SW620, and NCM460 cells was collected after at least 24 hours of culture. Exosomes were isolated using ultracentrifugation. After centrifugation at 2000g for 10 minutes, the supernatant was collected and centrifuged at 10000g for 30 minutes. The supernatant was then centrifuged at 100000g for 90 minutes. The supernatant was discarded, and the precipitate was resuspended in DPBS. The precipitate was then centrifuged at 100000g for 90 minutes and resuspended in DPBS. The precipitate was stored at 4°C for later use.

[0098] Plasma exosome isolation: Fresh whole blood samples were collected in EDTA2K anticoagulant tubes and stored on ice for no more than 12 hours. The plasma was collected by centrifugation at 2000g for 15 minutes. The exosome precipitate was separated according to the instructions of the Total Exosome Isolation Kit (Invitrogen). The exosome precipitate was resuspended in DPBS and stored at 4°C for later use.

[0099] 2. Nucleic acid aptamer detection of EpCAM and PRNP protein enrichment levels in exosomes

[0100] Exosomes extracted from eight types of colorectal cancer tumor cells or clinical samples were analyzed according to the following steps:

[0101] Step 1: Obtain sufficient exosomes from the sample to be tested, with a total exosome count of not less than 30ug.

[0102] Step 2: Take 10 μL of aldehyde sulfate latex microbeads with a diameter of 4 µm and mix them with the exosome suspension to form a mixture. Incubate at room temperature with shaking for 15–30 minutes.

[0103] Step 3: Add DPBS to the above mixture to make up the volume to 1 mL, and incubate at room temperature with shaking for 15-30 minutes.

[0104] Step 4: Add 100 µL of 1M glycine and 200 µL of 10% bovine serum albumin solution to the above mixture to terminate the reaction, and incubate at room temperature with shaking for 1 to 2 hours.

[0105] Step 5: Centrifuge the above mixture at 15000g for 1–3 minutes. Discard the supernatant, retain the latex bead precipitate, add 1 mL of washing buffer to resuspend, centrifuge under the same conditions, and repeat twice.

[0106] Step 6: Add 800 µL of 10% bovine serum albumin solution and 200 µL of 5% herring sperm DNA solution to the above precipitate, and incubate with shaking at room temperature for 30 minutes.

[0107] Step 7: Repeat step 5.

[0108] Step 8: Add binding buffer to the precipitate to dilute the nucleic acid aptamer to 200 nM, and incubate at 37°C for 30 minutes.

[0109] Step 9: Repeat step 5.

[0110] Step 10: Add approximately 100–200 µL of washing buffer to the precipitate to resuspend the latex microbead precipitate. Analyze the precipitate using a flow cytometer, collect 10,000 particles, and record the percentage of microbeads that are positive for binding nucleic acid aptamers.

[0111] The applicant isolated exosomes generated during the culture of HCT-8, HCT116, SW480, SW620, and NCM460 cell lines and incubated them with candidate nucleic acid aptamers WHY-1c and WHY-3e. The test results are as follows: Figure 4 As shown, WHY-3e significantly bound to exosomes derived from tumor cell lines HCT-8, HCT116, SW480, and SW620, but bound weakly to exosomes derived from the normal colorectal epithelial cell line NCM460. This result suggests that the nucleic acid aptamers WHY-1c and WHY-3e can be used for exosome detection, and the expression levels of EpCAM and PRNP in exosomes can be quantitatively reflected by recording the percentage of positive results. The above experimental results indicate that EpCAM and / or PRNP are highly expressed in exosomes produced by various colorectal cancer cells and can be used as diagnostic markers for colorectal cancer.

[0112] Example 4. Nucleic acid aptamers WHY-1c and WHY-3e can serve as diagnostic markers for colorectal cancer.

[0113] 1. Detect the binding of exosomes and nucleic acid aptamers in the plasma of cancer patients.

[0114] The applicant had previously collected blood samples through the Ethics Review Committee of Zhejiang Cancer Hospital. Blood samples were processed by separating exosomes from plasma, and the exosome and nucleic acid aptamer detection process was as described above. A total of 80 colorectal cancer patient samples and 23 healthy samples were processed. The aptamer binding positivity rate is as follows: Figure 5 As shown, the positive rates of exosome binding to WHY-1c and WHY-3e in the plasma of cancer patients were higher than those in healthy samples. The positive rate of combining the two nucleic acid aptamers, WHY-1c & WHY-3e, was also higher than that in healthy samples. This suggests the feasibility, specificity, and accuracy of detecting WHY-3e in clinical samples.

[0115] 2. ROC curves were used to assess the accuracy of EpCAM, PRNP, and their nucleic acid aptamers WHY-1c and WHY-3e in predicting the diagnosis of colorectal cancer.

[0116] This invention uses data from 80 colorectal cancer patients and 23 healthy individuals to perform ROC (Reactivity Index) assessment to represent the accuracy of its predictions. The results showed that the AUC (area under the curve) of PRNP was 0.964 (95% CI: 0.926-1). p<0.001 The AUC value of EpCAM was 0.826 (95% CI: 0.749-0.903). p<0.001 Furthermore, combining EpCAM and PRNP further increased the AUC value to 0.985 (95% CI: 0.961-1). p<0.001 )( Figure 6 In conclusion, EpCAM, PRNP, and their nucleic acid aptamers WHY-1c and WHY-3e have high accuracy as biomarkers for the diagnosis of colorectal cancer and possess extremely high diagnostic value.

[0117] Table 2 Nucleic Acid Aptamers

[0118]

[0119] (Note: Lowercase letters in nucleic acid sequences represent mutation base sites)

[0120] Table 3. Identification of WHY-1c and WHY-3e target proteins and their interacting proteins using SILAC.

[0121]

Claims

1. The application of nucleic acid aptamers or their derivatives in the preparation of products that recognize and bind to or assist in recognizing and binding to EpCAM in a sample; wherein the nucleic acid aptamer is a single-stranded DNA molecule as shown in Seq ID No: 5; wherein the derivative is a nucleic acid aptamer with a functional group attached to one end, wherein the functional group is a fluorescent group or nanoluminescent material, a magnetic or paramagnetic group, an affinity group, a radioactive substance, an X-ray responsive group, a therapeutic substance, or an enzyme label.

2. Application of nucleic acid aptamers or their derivatives in the preparation of products that recognize and bind to or assist in recognizing and binding to PRNPs in a sample; The nucleic acid aptamer is a single-stranded DNA molecule as shown in Seq ID No: 10; the derivative is a nucleic acid aptamer with a functional group attached to one end, and the functional group is a fluorescent group or nanoluminescent material, a magnetic or paramagnetic group, an affinity group, a radioactive substance, an X-ray responsive group, a therapeutic substance, or an enzyme label.

3. Application of nucleic acid aptamers or their derivatives in the preparation of products that recognize and bind to or assist in recognizing and binding to the combination of EpCAM and PRNP in a sample; The nucleic acid aptamer that recognizes and binds to or assists in recognizing and binding to EpCAM is the single-stranded DNA molecule shown in Seq ID No: 5; The nucleic acid aptamer that is recognized and bound or assisted in the recognition and binding of PRNP is the single-stranded DNA molecule shown in Seq ID No: 10; The derivative is formed by attaching a functional group to one end of a nucleic acid aptamer. The functional group can be a fluorescent group or nanoluminescent material, a magnetic or paramagnetic group, an affinity group, a radioactive substance, an X-ray responsive group, a therapeutic substance, or an enzyme label.

4. The application as described in any one of claims 1-3, wherein the product is an imaging reagent, a tumor diagnostic reagent, or a therapeutic reagent, and the tumor is colorectal cancer (CRC).

5. The application as described in any one of claims 1-3, wherein the sample source is exosomes derived from blood.

6. Use of the reagent for detecting the expression level of PRNP gene or protein in a sample in the preparation of a colorectal cancer (CRC) diagnostic kit, wherein the reagent for detecting the expression level of PRNP gene or protein in a sample is a single-stranded DNA molecule as shown in Seq ID No:

10.

7. The use of the reagent combination for detecting the expression levels of EpCAM and PRNP genes or proteins in a sample in the preparation of a colorectal cancer (CRC) diagnostic kit, wherein the reagent for detecting the expression level of PRNP genes or proteins in a sample is a single-stranded DNA molecule as shown in Seq ID No: 10, and the reagent for detecting the expression level of EpCAM genes or proteins in a sample is a single-stranded DNA molecule as shown in Seq ID No:

5.

8. The use as described in claim 6 or 7, wherein the sample is a blood-derived exosome.

9. The diagnostic kit is prepared by combining the detection reagent of claim 6 or the reagent combination of claim 7 with exosome separation, enrichment and purification reagents as described in claim 6.

10. The use as described in claim 6 or 7, wherein the diagnostic kit of claim 6 or 7 comprises exosome isolation, enrichment and purification reagents.

11. A diagnostic reagent kit for quantifying the expression level of exosome surface targets, comprising a single-stranded DNA molecule as shown in Seq ID No: 5 and / or Seq ID No: 10, and aldehyde / sulfate latex microbeads.

12. Use of the diagnostic reagent combination of claim 11 in the preparation of a tumor diagnostic reagent, wherein the tumor is colorectal cancer (CRC).

Images