Nucleic acid aptamer and kit for detecting colorectal cancer

Abstract

The invention provides a nucleic acid aptamer and a kit for detecting colorectal cancer. The nucleotide sequence of the nucleic acid aptamer for detecting colorectal cancer is as shown in SEQ ID NO. 1. The nucleic acid aptamer is obtained by screening by taking a colorectal cancer tissue slice as a target through a tissue-SELEX (systematic evolution of ligands by exponential enrichment) technology, the secondary structure of the nucleic acid aptamer is predicted to show high stability, keeps strong affinity at 4 DEG C and 37 DEG C, specifically recognizes colorectal cancer cells and tissues, and is suitable for development of a detection kit and improvement of diagnosis precision and clinical transformation potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nucleic acid aptamer and kit for detecting colorectal cancer. Background Technology

[0002] Nucleic acid aptamers are single-stranded DNA or RNA molecules, typically composed of 15 to 80 nucleotides, screened using SELEX technology. They can bind to targets such as proteins or cells with high specificity. The main advantages of this technology include strong chemical stability, low immunogenicity, and flexible modifiability, making aptamers of significant value in molecular diagnostics. However, traditional SELEX technology has significant limitations: it relies on purified target molecules for screening, a lengthy process (requiring 10-20 rounds), and cannot simulate the in vivo microenvironment, leading to a decline in binding activity of aptamers during clinical translation. Improved technologies such as Cell-SELEX use live cells as targets to enhance affinity. However, making the screened nucleic acid aptamers more suitable for clinical diagnosis and treatment remains a significant challenge for the commercialization of nucleic acid aptamers.

[0003] In the field of colorectal cancer diagnosis, current technologies face serious shortcomings. First, the early diagnosis rate is low, with approximately 60% of patients diagnosed at an advanced stage, and a five-year survival rate of less than 15%. Second, commonly used screening methods have multiple drawbacks: colonoscopy is invasive, leading to poor patient compliance; serum biomarkers such as CEA and CA19-9 have insufficient sensitivity, with specificity only 40%-60%; and imaging techniques have limited detection rates for early, small lesions. Intraoperative rapid pathological diagnosis methods are limited, and molecular diagnosis is not possible. These shortcomings highlight the urgent need for highly specific molecular probes to achieve non-invasive or minimally invasive screening and improve the accuracy of pathological tissue identification. In summary, nucleic acid aptamers, as targeted recognition tools, possess unique potential in colorectal cancer diagnosis. Their matrix can be used to obtain specific diagnostic probes even in the absence of clear biomarkers / targets, providing a foundation for developing rapid tissue section detection technologies.

[0004] Therefore, it is necessary to develop a product that has high specificity and high sensitivity for detecting colorectal cancer. Summary of the Invention

[0005] The purpose of this invention is to provide a nucleic acid aptamer and reagent kit for detecting colorectal cancer. This kit has high sensitivity, good specificity, is simple to operate, and can rapidly detect colorectal cancer, showing promising application prospects.

[0006] In a first aspect of the invention, a nucleic acid aptamer for detecting colorectal cancer is provided, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Furthermore, the nucleic acid aptamer for detecting colorectal cancer further includes: a fluorescent substance, a nanoluminescent material, biotin, digoxigenin, and an enzyme labeling agent bound to the nucleotide sequence of the nucleic acid aptamer.

[0008] Furthermore, the fluorescent substance is a FAM fluorescent group or a Cy5 fluorescent group; the nanoluminescent material is a quantum dot or an upconversion nanoparticle; and the enzyme label is horseradish peroxidase or sucrase.

[0009] Furthermore, the nucleic acid aptamer for detecting colorectal cancer also includes a nucleic acid aptamer whose nucleotide sequence is phosphorylated, methylated, aminated, thiolated, or isotopically modified at a certain position.

[0010] In a second aspect of the invention, a kit for detecting colorectal cancer is provided, comprising the aforementioned nucleic acid aptamer.

[0011] Furthermore, the kit for detecting colorectal cancer also includes: (A) Sealing liquid; (B) Rinse solution.

[0012] Furthermore, the blocking solution consists of PBS buffer containing 1 mg / mL BSA, 0.1 mg / mL yeast tRNA, and 0.5 mg / mL salmon sperm DNA.

[0013] Furthermore, the washing solution consists of PBS buffer containing 5 mM Mg 2+ .

[0014] In a fourth aspect of the invention, the use of the nucleic acid aptamer for detecting colorectal cancer and / or the detection kit is provided in the preparation of products for detecting colorectal cancer.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: 1. This invention provides a nucleic acid aptamer and kit for detecting colorectal cancer. The nucleic acid aptamer for colorectal cancer screened by this invention using SELEX technology has high specificity and affinity. The Kd values ​​of aptamer T1 at 4℃ and 37℃ are 223.62±17.53 nM and 350.88±22.36 nM, respectively. Figure 3 It is 1000 times more effective than conventional antibodies (micromolar level).

[0016] 2. This invention provides a nucleic acid aptamer for detecting colorectal cancer that exhibits wide temperature range stability, maintaining 60% binding activity at 37°C. Figure 2 This technology overcomes the limitations of low-temperature dependence and supports intraoperative operation at room temperature.

[0017] 3. The reagent kit of the present invention has high sensitivity, good specificity, wide measurement range, simple operation, rapid detection, and is suitable for large-scale promotion, and has good market prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a prediction diagram of the secondary structure of nucleic acid aptamer T1.

[0020] Figure 2 The recognition performance of T1 at different temperatures was examined.

[0021] Figure 3 The identification performance of different concentrations of T1 was investigated.

[0022] Figure 4 To investigate the recognition of different colorectal cancer cells by T1, the nucleic acid aptamer concentration was 250 nM and the cell number was 20,000.

[0023] Figure 5 To investigate the recognition of normal tissues and colorectal cancer tissues by nucleic acid aptamer T1. Detailed Implementation

[0024] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0025] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0026] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0027] The following will provide a detailed description of a nucleic acid aptamer and kit for detecting colorectal cancer, based on embodiments and experimental data.

[0028] Example 1: Obtaining nucleic acid aptamers from colorectal cancer tissues based on tissue-SELEX screening 1. Using paraffin-embedded colon cancer tissue sections from clinical patients as positive screening targets and adjacent / normal intestinal tissue as controls for negative screening, library enrichment was performed.

[0029] Filtered library: 5'-TCA AGT CAC AGG TTC CAG GT-(N)40-ATA GGC ACT GAC ACG ACACT-3'; Pre-primer: 5'-CY5-TCA AGT CAC AGG TTC CAG GT-3' (SEQ ID NO.2); Back primer: 5'-BIOTIN-AGT GTC GTG TCA GTG CCT AT-3' (SEQ ID NO.3); Blocking sequence BC-15: 5'-GCAATGGGTACGGTACTTCCTGTGGCGAGGTAGGTGGG GTGTGTGTGTATCCAAAAGTGCACGCTACTTTGCTAA-3' (SEQ ID NO. 4); 2. To ensure parallelism across different rounds of screening, all solutions used in the screening were prepared, aliquoted, and frozen before screening. The solutions used in the experiment are as follows: (1) Phosphate-buffered saline (PBS): 0.01 M phosphate-buffered saline with pH 7.4, containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4•12H2O, and 2 mM KH2PO4. Weigh 4.00 g NaCl, 0.10 g KCl, 1.79 g Na2HPO4•12H2O, and 0.17 g KH2PO4 respectively, dissolve them in 500 mL of ultrapure water, adjust the pH to 7.4, autoclave for half an hour, aliquot and freeze for storage.

[0030] (2) 0.5 M magnesium ion solution: Weigh 3.05 g MgCl2•6H2O and dissolve it in 30 mL of ultrapure water.

[0031] (3) Cell permeation solution: First, measure 3 mL of Triton X-100 and dissolve it in 27 mL of PBS to prepare a 10% Triton X-100 stock solution. Dilute it 100 times with PBS before use.

[0032] (4) Binding buffer (BB): The components are PBS -5 mM, Mg 2+-1 mg / mL BSA - 0.1 mg / mL yeast tRNA - 0.1 mg / mL salmon sperm DNA. To prepare 200 mL of BB, take 4 mL of 0.5 M magnesium ion solution, 0.2 g BSA, 20 mg yeast tRNA and 2 mL salmon sperm DNA solution (10 mg / mL), add PBS, stir and let stand until all components dissolve naturally, then filter sterilize, aliquot and store at -20 ℃.

[0033] (5) Washing buffer (WB): Each component is PBS-5 mM, Mg 2+ -1 mg / mL BSA. To prepare 400 mL of Western blotting (WB), take 8 mL of 0.5 M magnesium ion solution and 0.4 g of BSA, add PBS, stir, and let stand until all components dissolve naturally. Then filter to sterilize, aliquot, and store at -20 °C.

[0034] (6) Non-specific adsorption blocking solution: The components are PBS-1 mg / mL BSA-0.1 mg / mL yeast tRNA-0.5 mg / mL salmon sperm DNA. To prepare 200 mL of blocking solution, take 0.2 g BSA, 20 mg yeast tRNA and 10 mL salmon sperm DNA solution (10 mg / mL), add PBS and stir, let stand until all components dissolve naturally, then filter to sterilize, aliquot and store at -20 ℃.

[0035] (7) 5 × TBE: Weigh 27 g Tris-base and 13.75 g boric acid respectively, then take 10 mL of 0.5 mol / LEDTA solution (pH=8), dissolve in 100 mL of ultrapure water, add water to make up to 500 mL, and store at 4 ℃.

[0036] (8) Citrate buffer (pH=6): Prepare 0.1 mol / L citric acid solution (solution A) and 0.1 mol / L sodium citrate solution (solution B) separately and store them at 4 ℃. When using, take 9 mL of solution A and 41 mL of solution B and add water to 500 mL.

[0037] (9) Cell fixation solution: Weigh 4.8 g of paraformaldehyde solid and dissolve it in 60 mL of PBS solution to obtain a 4% formaldehyde solution. To accelerate dissolution, heat to 50-60 °C. Store at 4 °C after preparation.

[0038] 3. The cyclic screening of nucleic acid aptamers mainly consists of the following steps: a) Positive Screening: Before screening, paraffin-embedded tissue sections from colorectal cancer undergo dewaxing and antigen retrieval. The tissue sections are first baked in an oven at 50°C for 0.5 h, then soaked in xylene and different concentrations of ethanol. Excess ethanol is washed away with PBS, followed by microwave antigen retrieval using sodium citrate buffer. After washing the tissue twice with PBS, a blocking solution prepared from salmon sperm DNA and yeast tRNA is added and incubated in a humidified chamber to block non-specific DNA adsorption. After blocking, a library is added to the tissue surface, and after incubation for a certain period, the tissue surface is gently rinsed to remove unbound ssDNA. The tissue is scraped off the slide with a spatula, and the tissue-bound library is collected for amplification before being used in the next round of screening. The screening pressure is adjusted primarily by: firstly, decreasing the amount of library added in each round; secondly, decreasing the incubation time with the positive screening target in each round, while gradually increasing the incubation time with the negative screening material; and finally, gradually increasing the washing intensity.

[0039] b) Reverse screening: A reverse screening process is introduced after a certain number of rounds of forward screening using colon cancer tissue sections to enhance the specificity of the selected nucleic acid aptamers. Human normal intestinal / adjacent cancer tissue sections are dewaxed, antigen-retrievaled, and blocked for non-specific adsorption. The library is then added to the tissue surface and incubated in a humidified chamber. After a certain incubation time (which is gradually increased as the screening process progresses), the solution containing ssDNA that does not bind to human normal intestinal / adjacent cancer tissue is aspirated and used in the subsequent forward screening.

[0040] c) PCR Cycle Optimization and Amplification: ssDNA that can bind to the target tissue is amplified by PCR to meet the requirements of the next round of screening. Since the concentration of DNA template obtained after each screening is uncertain, the number of PCR cycles needs to be optimized to prevent non-specific amplification. After preparing the PCR reaction solution in the appropriate proportions, the number of PCR cycles is set according to an arithmetic progression, and the PCR products are verified by electrophoresis.

[0041] d) Single-streptavidin-modified agarose microspheres were placed in a 1.5 mL EP tube, centrifuged to remove the supernatant, and washed twice with PBS to capture biotin-containing double-streptated PCR products. After washing twice with PBS solution, 200 mM NaOH was added to collect ssDNA products. Subsequently, the ssDNA was added to a NAP-5 size-exclusion column to desalt and remove short-streptated DNA, and the ssDNA library was collected. After quantification, the library was dried for the next round of screening and library enrichment assessment.

[0042] Finally, the nucleic acid aptamer T1 was identified as binding to colorectal cancer tissue slices: 5'-TCA AGT CAC AGG TTC CAG GTC CGC ACC CCT CGC TGA ACT CCA TAA ACCAAC CTC TCA GGA AT A GGC ACT GAC ACG ACA CT-3' (SEQ ID NO. 1).

[0043] Example 2: Nucleic acid aptamers characterizing colorectal cancer tissue binding After several rounds of screening, the library was sequenced, and the sequence with the highest number of repeats was selected. The T1 aptamer was then used to synthesize and evaluate the sequence. 5'-CY5-TCA AGT CAC AGG TTC CAG GTC CGC ACC CCT CGC TGAACT CCA TAA ACC AAC CTC TCA GGA AT A GGC ACT GAC ACG ACA CT-3' The secondary structure of candidate sequences was predicted using the Mfold service. The simulation parameters were set as follows: temperature 25℃, Na+ concentration 100 mM, and Mg... 2+ Concentration 5 mM, secondary structure, sequence as follows Figure 1 As shown. Based on the sequence repeat number and nucleic acid aptamer secondary structure in the sequencing results, representative sequences were selected for synthesis.

[0044] We examined the recognition performance of nucleic acid aptamer T1 at different temperatures, such as... Figure 2 As shown, T1 cells effectively recognized the HCT116 colorectal cancer cell line at both 4°C and 37°C. However, the recognition strength at 37°C was weaker than that at 4°C, which is speculated to be because the metastable structure of T1 cells is more easily recognized by the target at low temperatures. Furthermore, although immobilized cells were used, the non-specific adsorption of some enzymes and proteins within the cells at 37°C also had some impact on the recognition performance.

[0045] We used a single-point adsorption model to determine the dissociation equilibrium constant (Kd value) between the nucleic acid aptamer T1 and immobilized HCT116 cells. A series of concentrations of T1 were bound to HCT116 cells (results are shown in...). Figure 3 After obtaining the mean fluorescence intensity of cells by flow cytometry, Sigma Plot software was used to plot the nucleic acid aptamer concentration and mean fluorescence intensity of cells on the x and y axes, respectively. Finally, the Kd value of nucleic acid aptamer T1 was simulated using the software's built-in single-point adsorption model (Y = BmaxX / (Kd + X)). The values ​​were in the nanomolar range at both 4℃ and 37℃, at 223.62 ± 17.53 nM and 350.88 ± 22.36 nM, respectively, indicating a strong affinity between the nucleic acid aptamer and cells.

[0046] We then further utilized the nucleic acid aptamer T1 to identify different colorectal cancer cell lines, including NCM460 cells, which are immortalized normal human colon cells. Figure 4 It is evident that the nucleic acid aptamer T1 exhibits varying degrees of recognition efficacy against a variety of colorectal cancer cells. However, its non-specific cell adsorption on NCM460 cells is largely consistent with the non-specific adsorption of HCT116 cells and the control probe.

[0047] Based on the results of cell selectivity experiments, the CY5-labeled nucleic acid aptamer T1 was further used to distinguish between colorectal tissue sections and normal tissue sections. The results are shown in […]. Figure 5 As can be seen, the nucleic acid aptamer T1 exhibits a clear recognition signal for tumor tissue, with a significantly stronger binding affinity than normal tissue sections. However, we also observed significant non-specific adsorption in normal tissue sections, stronger than in adjacent tissues. Furthermore, in tumor tissue sections, besides the clearly identified tumor tissue, there were also locations where the signal was significantly lower than that of the tumor itself. This places higher demands on the experimental operator; when the sample size is small, it is difficult to select an appropriate microscopic observation area, and the determination of the recognition status can easily lead to false positives or false negatives. Example 3: Reagent kit for detecting colorectal cancer The kit for detecting colorectal cancer contains the following formulation: (A) Blocking solution: PBS + 1 mg / mL BSA + 0.1 mg / mL yeast tRNA + 0.5 mg / mL salmon sperm DNA; (B) Washing solution: PBS + 5 mM Mg 2+ ; (C) CY5-labeled nucleic acid aptamer (SEQ ID NO.1).

[0048] Example 4: Detection of colorectal cancer content in samples The kit for detecting colorectal cancer described in Example 3 was used to detect the content of colorectal cancer in infected samples, including: 1. Tissue sections were fixed with a protein-coated plate and incubated with blocking solution (A) for 30 minutes; 2. Add CY5-labeled nucleic acid aptamers (200 nM) and incubate at 37°C in the dark for 45 minutes; 3. Wash three times with rinsing solution (B) and observe under a fluorescence microscope: 4. Positive diagnosis: Focal strong fluorescence is observed in the tumor area ( Figure 5 superior); Negative control: Normal tissue showed only scattered background signals ( Figure 5(Below). The number of cells per unit area is counted by staining with nuclear dyes (DAPI or other cell dyes). If the proportion of cells with CY5-T1 probe fluorescence exceeds (≥) 70%, it is considered positive, that is, the tissue per unit area is considered to be colorectal cancer tumor tissue.

[0049] The method for determining colorectal cancer tissue based on nucleic acid aptamer T1 detection includes the following steps: (1) Using image analysis software (such as ImageJ / Fiji), under the same exposure and gain conditions, measure the average gray value and standard deviation of the tissue region to be tested, its adjacent normal tissue region and the background region respectively.

[0050] (2) Calculate the signal intensity ratio (SIR) of the tissue to be tested: SIR = average gray value of the area to be tested / average gray value of adjacent normal tissue.

[0051] (3) Judgment rule: If SIR ≥ [2.1] is satisfied at the same time, the tissue to be tested is judged to be positive for colorectal cancer. In the method, the target binding specificity can be further verified by calculating the Pearson correlation coefficient (R) between the nucleic acid aptamer dye (CY5) signal and the nuclear dye (DAPI) signal; preferably, for positive tissues, the R value is ≥ 0.4.

[0052] Example 5: Performance Determination of the Reagent Kit 1. Using the kit from Example 3 and the detection method from Example 4, 10 tissue samples with clinical pathological examination results were selected for comparative experiments. The detection results are as follows: Table 1

[0053] As shown in Table 1, the preliminary results demonstrate that the detection method is highly consistent with the clinical pathological results, proving that this method can be used as a testing method for clinical tumor tissue specimens.

[0054] 2. Diagnostic efficacy analysis of nucleic acid aptamers T1 (AUC value, sensitivity, and specificity) To quantify the diagnostic accuracy of nucleic acid aptamer T1 for colorectal cancer, we performed receiver operating characteristic (ROC) curve analysis. To further quantify diagnostic efficacy, another group of 50 pathologically confirmed clinical tissue samples (25 colorectal cancer tissues and 25 normal or adjacent normal intestinal tissues, independent of Example 5) were selected and tested using the detection method described in Example 4. Using pathological diagnosis as the gold standard, statistical analysis was performed using the mean signal intensity ratio of each sample (defined as the mean gray value of the tumor region / the mean gray value of adjacent normal tissues) as the diagnostic indicator.

[0055] ROC curves were plotted using statistical software (such as SPSS 26.0 or the pROC package in R) and the area under the curve (AUC) was calculated. The analysis showed that the T1 aptamer significantly distinguished colorectal cancer tissue from normal tissue (AUC = 0.92, 95% confidence interval: 0.85–0.99). The optimal cut-off value determined by ROC curve analysis was a signal intensity ratio ≥ 2.1. At this optimal cut-off value, its sensitivity for diagnosing colorectal cancer was 96.0% (24 / 25), and its specificity was 84.0% (21 / 25).

[0056] These results demonstrate that the detection method based on nucleic acid aptamer T1 has excellent diagnostic accuracy and reliability, with an AUC value much higher than 0.5 (random guess), fully meeting the requirements of clinical diagnostic reagents for high precision and high reliability.

[0057] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0058] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0059] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A nucleic acid aptamer for detecting colorectal cancer, characterized in that, The nucleotide sequence of the nucleic acid aptamer used for detecting colorectal cancer is shown in SEQ ID NO.

1.

2. The nucleic acid aptamer according to claim 1, characterized in that, The nucleic acid aptamer for detecting colorectal cancer further includes one of the following: a fluorescent substance, a nanoluminescent material, biotin, digoxigenin, and an enzyme labeling, which is bound to the nucleotide sequence of the nucleic acid aptamer.

3. The nucleic acid aptamer according to claim 2, characterized in that, The fluorescent substance is a FAM fluorescent group or a Cy5 fluorescent group; the nanoluminescent material is a quantum dot or an upconversion nanoparticle; the enzyme label is horseradish peroxidase or sucrase.

4. The nucleic acid aptamer according to claim 1, characterized in that, The nucleic acid aptamer for detecting colorectal cancer further includes a nucleic acid aptamer whose nucleotide sequence is phosphorylated, methylated, aminated, thiolated, or isotopically modified at a certain position.

5. The nucleic acid aptamer according to claim 1, characterized in that, The 5' end of the nucleic acid aptamer T1 nucleotide sequence is labeled with an amino group -NH2, and the 5' end of the nucleic acid aptamer T1 nucleotide sequence is labeled with a thiol group -SH.

6. A reagent kit for detecting colorectal cancer, characterized in that, Includes the nucleic acid aptamer as described in any one of claims 1-5.

7. A reagent kit for detecting colorectal cancer according to claim 6, characterized in that, The kit for detecting colorectal cancer also includes: (A) Sealing liquid; (B) Rinse solution.

8. A reagent kit for detecting colorectal cancer according to claim 6, characterized in that, The blocking solution consists of PBS buffer containing 1 mg / mL BSA, 0.1 mg / mL yeast tRNA, and 0.5 mg / mL salmon sperm DNA; the rinsing solution consists of PBS buffer containing 5 mM Mg 2+ .

9. A reagent kit for detecting colorectal cancer according to claim 6, characterized in that, The concentration range for the nucleic acid aptamer is 100–500 nM.

10. The use of the nucleic acid aptamer according to any one of claims 1-5 and / or the detection kit according to any one of claims 6-9 in the preparation of products for the detection of colorectal cancer.

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