A DNA tetrahedral probe for detecting lung cancer gene methylation and its application

Abstract

This invention relates to the field of molecular detection technology, specifically to a DNA tetrahedral probe for detecting lung cancer gene methylation. The probe is formed by the self-assembly of four single-stranded DNA strands S1-S4, with S1 extending into a recognition arm targeting the methylated fragment of the PTGER4 promoter region. This invention immobilizes the tetrahedral probe on the inner wall of a nuclear pore membrane, preparing it as the core electrochemical detection component of a reagent kit. Utilizing the specific recognition capabilities of the nuclear pore membrane and the probe, combined with electrochemical detection technology, rapid detection of PTGER4 gene methylation is achieved without the need for PCR nucleic acid amplification. This detection system is simple to operate, low in cost, and can be widely applied to screening high-risk groups for lung cancer.

Description

Technical Field

[0001] The invention relates to the fields of molecular biology detection and electrochemical analysis, and in particular to a probe for identifying methylation of the PTGER4 gene in lung cancer, and the application of an electrochemical detection kit based on the probe and a porous membrane with carboxyl groups on its surface. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related morbidity and mortality worldwide. According to the International Agency for Research on Cancer (IARC) of the World Health Organization, in 2022, there were over 2.2 million new cases of lung cancer globally, with approximately 1.8 million deaths. More than 70% of these patients were diagnosed at an advanced stage, with a 5-year survival rate of less than 20%. However, with standardized treatment, the 5-year survival rate for early-stage lung cancer patients can increase to over 70%. Therefore, early screening and diagnosis of lung cancer are crucial for reducing mortality. DNA methylation is an important aspect of epigenetic regulation. Hypermethylation of CpG islands in promoter regions can lead to transcriptional silencing of tumor suppressor genes, an early event in tumor development. In lung cancer, abnormal promoter methylation of multiple genes has been proven to be closely related to the carcinogenesis process.

[0003] The PTGER4 gene encodes a specific G protein-coupled receptor for prostaglandin E2. Recent studies have shown that abnormal CpG island methylation in the PTGER4 gene promoter region is associated with the development and progression of various malignant tumors. In lung cancer tissues, hypermethylation of specific segments in the PTGER4 promoter region can be detected in the early stages of carcinogenesis, and this methylation signal remains stable in circulating cell-free DNA (cfDNA), unaffected by tumor stage or pathological type, thus possessing clinical value as a molecular marker for early diagnosis of lung cancer and screening of high-risk populations.

[0004] Current methods for detecting PTGER4 methylation mainly rely on PCR techniques, including real-time quantitative PCR and digital PCR. While these techniques can meet detection needs to some extent, they have several inherent drawbacks. First, they require complex nucleic acid amplification steps, resulting in cumbersome procedures and detection cycles lasting several hours, making them unsuitable for rapid detection. Therefore, developing a PTGER4 gene methylation recognition probe with a simpler detection process is of great significance for advancing early lung cancer diagnostic technology. Summary of the Invention

[0005] To address the problems of complex operation, long detection cycle, and susceptibility to contamination in existing PTGER4 gene methylation detection methods, the primary objective of this invention is to provide a recognition probe for detecting PTGER4 gene methylation in lung cancer. Another objective of this invention is to provide the application of this probe in the electrochemical detection component of a kit, as well as a lung cancer PTGER4 gene methylation detection kit containing this probe, to achieve rapid and specific detection of PTGER4 gene methylation and meet the needs of early screening, diagnosis, and efficacy monitoring.

[0006] The complete genome sequence of the PTGER4 gene (prostaglandin E2 receptor 4 gene), which is targeted in this invention, was obtained from the NCBIGenBank database, accession number NC_000005.10, corresponding to human chromosome 5 (Chr5). To screen for methylation biomarkers suitable for early lung cancer detection, this invention first extracted the promoter region sequence of the PTGER4 gene. The Chr5 region (40681592-40681835) showed a high enrichment of CpG sites, strong sequence conservation, and a high rate of abnormal methylation in lung cancer tissue, thus meeting the criteria for a specific detection target.

[0007] Since DNA methylation detection probes are typically designed for sequences after bisulfite conversion, this invention simulates bisulfite methylation conversion on the wild-type Chr5:40681592-40681835 sequence: unmethylated cytosine in the sequence is converted to thymine, while the cytosine in the methylated CpG nucleotide remains unchanged, ultimately obtaining a specific methylation target sequence for the PTGER4 gene promoter region. This sequence is the target sequence recognized by the probe of this invention, and its nucleotide sequence is shown in SEQ ID NO.6, with a length of 244 bp.

[0008] The probe is formed by the self-assembly of four single-stranded DNA strands, namely S1 to S4, and the sequences of each single-stranded DNA strand are shown in SEQ ID NO.1 to 4.

[0009] The four DNA strands constituting the DNA tetrahedral probe each contain three sequence modules, each 17 bp in length. Each module is complementary to the corresponding module of the other three single-stranded DNA strands. Each single-stranded DNA strand extends around one face of the tetrahedron. At each corner vertex of the tetrahedron, two unpaired flexible bases are provided as bending hinges, allowing the DNA strands to bend flexibly and self-assemble into a stable tetrahedral three-dimensional backbone structure.

[0010] Among them, the 5' ends of S2, S3, and S4 are all modified with amino groups to covalently couple with the inner wall of the activated porous membrane nanopores; S1 consists of a recognition arm for recognizing the target and a backbone A; the target gene of the recognition arm is a specific methylated sequence fragment in the PTGER4 promoter region, and the genomic location of the specific methylated sequence fragment is Chr5:40681642-40681662. The nucleotide sequence of the recognition arm is shown in SEQ ID NO.5 and has a length of 21 bp.

[0011] Preferably, the molar ratio of the four single-stranded DNA strands of the DNA tetrahedral probe is 1:1:1:1.

[0012] Preferably, the self-assembly conditions are as follows: four single-stranded DNA molecules are mixed in Tris-HCl buffer and heated at 95°C for 5–10 min, then slowly cooled to room temperature and kept at that temperature for 1–2 h to complete the self-assembly of the DNA tetrahedral probe.

[0013] Preferably, the Tris-HCl buffer solution has a concentration of 10–20 mM and a pH of 7.4–8.0, and further contains 50–100 mM NaCl and 1–5 mM MgCl2.

[0014] This invention also provides a lung cancer gene methylation detection kit containing the DNA tetrahedral probe. The kit includes a functionalized porous membrane, an H-type electrolytic cell, an electrode assembly, an electrolyte solution, a PTGER4 gene methylation standard, and PBS washing solution. The functionalized porous membrane is composed of a porous membrane substrate and the DNA tetrahedral probe. The inner walls of the nanopores of the porous membrane substrate are rich in carboxyl groups. After activation, the DNA tetrahedral probe is covalently anchored within the modified nanopores.

[0015] The raw materials for the porous membrane are typically polycarbonate (PC), polyethylene terephthalate (PET), or polypropylene (PP).

[0016] The present invention further provides a method for preparing the functionalized porous membrane, comprising the following steps:

[0017] (1) The porous membrane was ultrasonically cleaned with anhydrous ethanol and deionized water in sequence, dried, and then immersed in the activation solution for activation.

[0018] (2) The modified porous membrane was incubated in a DNA tetrahedral probe solution, so that the probe was connected to the inner wall of the nanopore through a three-point covalent bond. The unbound probe was washed away to obtain the probe-modified porous membrane.

[0019] (3) The porous membrane is placed in a glycine solution prepared with 1×TE for sealing. The pH of the glycine solution is 7.1 to 7.5. After shaking on a decolorizing shaker for 10 to 60 minutes, it is sealed at 2 to 8°C. After sealing, it is washed 2 to 3 times with a 1×TE cleaning solution containing NaCl and Tween-20.

[0020] (4) After cleaning, the porous membrane is taken out and dried at 30-50℃ for 0.5-2h, and the edge part is removed to obtain the functionalized porous membrane.

[0021] The specific steps for detecting PTGER4 gene methylation in lung cancer using the kit of the present invention are as follows:

[0022] Functionalized nucleopore membranes were cut into uniform squares. One of these squares was used as the diaphragm of a dual-cell electrolytic cell. 1×PBS solution was used as the electrolyte. Two Ag / AgCl electrodes were used as the anode and cathode of the electrolytic cell. The anode was connected to the working electrode clamp of the electrochemical workstation, and the cathode was connected to the counter and reference electrode clamps. The electrolytic cell was then assembled for testing. Cyclic voltammetry was used, with an upper limit voltage of 1V, a lower limit voltage of -1V, an initial voltage of 0V, and 5 cycles. The current value at 1V was recorded as I0.

[0023] Take the cut functionalized nuclear pore membrane, place it in a culture dish, add 0.8~3mL of 1×PBS solution to the culture dish, and then place the culture dish in a 55℃ water bath for heating.

[0024] Place 20-100 μL of the analyte into a sample tube, add 20-100 μL of 10×Bst buffer and 160-800 μL of 1×PBS solution to the sample tube, and shake thoroughly to mix. Place the mixed sample tube in a 90-95℃ water bath and heat for 3-20 min. After heating, pour all the solution in the tube into a culture dish, sonicate the culture dish for 30 s, and then transfer the culture dish to a 55℃ water bath and continue heating for 10-30 min.

[0025] After the heating reaction is complete, remove the functionalized nucleopore membrane from the culture dish and wash it 1-3 times with 1×PBS solution. Lay the washed functionalized nucleopore membrane flat on a pad, using this pad as a diaphragm to fix it to the salt bridge of the dual-cell electrolytic cell, ensuring a seamless seal between the membrane and the inner wall of the electrolytic cell. Add equal volumes of electrolyte solution to the anode and cathode chambers of the electrolytic cell, insert the electrode assembly into the solution, connect it to the electrochemical workstation, and set the detection parameters: upper limit voltage 1V, lower limit voltage -1V, initial voltage 0V, and 5 cycles. Start the test program.

[0026] Record the current value corresponding to 1V voltage during the test as I1, calculate the relative change rate of current (I0-I1) / I0, substitute it into the pre-established standard curve, and calculate the target concentration of the substance to be tested.

[0027] The working principle of the technical solution is based on the synergistic effect of the nanopores of the nuclear pore membrane and electrochemical detection technology: the probe recognition arm of the present invention is designed for the transformed methylated target sequence and is complementary to the methylated target; during detection, the synthesized methylated target sequence specifically hybridizes with the DNA probe and is captured on the inner wall of the nanopore; when hybridizing with the non-methylated target, base mismatch occurs due to changes in key sites, thereby achieving methylation-specific recognition.

[0028] A tetrahedral DNA probe was formed by the self-assembly of three single-stranded DNA strands modified with amino groups at their 5' ends and one single-stranded DNA strand containing a recognition arm sequence at its 5' end. Three vertices of the tetrahedron contain amino groups for anchoring within the nanopores of a porous membrane, and a DNA recognition arm extends from the remaining vertex. The target sequence is complementary to this recognition arm. The PTGER4 gene methylated sequence in the target analyte diffuses into the nanopores of the nuclear pore membrane and binds specifically to the probe anchored within the pores through complementary base pairing. This reduces the effective pore size of the nanopores, hindering ion migration. When the probe binds to the target methylated sequence, the ion migration resistance of the nanopores increases, and the ion flux decreases significantly. PTGER4 gene methylation standards at different concentration gradients were used for detection. A standard curve was established with methylation sequence concentration on the x-axis and relative current change rate on the y-axis. The current change rate of the target analyte was substituted into the standard curve to calculate the concentration of PTGER4 gene methylation.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects:

[0030] Controllable configuration and reduced steric hindrance: This invention uses a DNA tetrahedral probe as a three-dimensional nanoframework. The recognition arm extends directionally from one end of the tetrahedron, which can keep the recognition arm away from the inner wall of the porous membrane nanopores, reduce the steric hindrance of hybridization caused by the pore wall, and improve the accessibility of the target sequence.

[0031] The fixation method is stable and the interface repeatability is good: the 5' ends of S2~S4 are all amino-modified, which can form a stable connection with the activated carboxylated porous membrane, increase the anchoring stability of the probe on the inner wall of the pore, and improve the consistency and repeatability of the detection platform.

[0032] Suitable for building a convenient methylation detection platform: The probe of this invention can be combined with H-type electrolytic cells, porous membranes, electrode components, etc. to form a kit, which is suitable for carrying out methylation detection based on ion transport, current, conductivity, impedance or other pore response signals, and has the potential to develop towards portability, low cost and rapid detection. Attached Figure Description

[0033] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0034] Figure 1 This is a schematic diagram illustrating the detection principle of a lung cancer PTGER4 gene methylation detection kit.

[0035] Figure 2 Standard curves for the relative changes in current in response to different concentrations of PTGER4 gene.

[0036] Figure 3 To evaluate the repeatability of the PTGER4 gene

[0037] Figure 4 To evaluate the specificity of the PTGER4 gene. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0039] The samples used in the following examples are all chemically synthesized nucleic acid sequences, artificially constructed nucleic acid fragments, or non-target nucleic acid sequences, used to illustrate the technical effects and detection logic of the present invention. The embodiments of the present invention do not use human-derived samples as the detection target. The chemically synthesized nucleic acid sequences, artificially constructed nucleic acid fragments, or non-target nucleic acid sequences used in the present invention were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0040] Example 1

[0041] Preparation of a lung cancer PTGER4 gene methylation detection kit

[0042] Probe preparation: The synthesized single-stranded DNA was dissolved in enzyme-free ultrapure water. Then, equimolar amounts of the S1, S2, S3, and S4 single-stranded DNA stock solutions were added to self-assembly buffer (15 mM Tris-HCl, 75 mM NaCl, 2.5 mM MgCl2) to prepare a single-stranded mixed solution with a final concentration of 1 μM. The total reaction volume was 500 μL. After thoroughly vortexing the above mixed solution, the solution was heated at 95 °C for 5 min, cooled to room temperature, and then incubated at 25 °C for 1.5 h.

[0043] Substrate pretreatment: When using nuclear pore membranes as porous membranes, the nuclear pore membranes were ultrasonically cleaned twice each with anhydrous ethanol and deionized water, each time for 10 minutes; after cleaning, they were placed in a 40℃ forced-air drying oven for 1.5 hours for later use.

[0044] Activation: Prepare an activation solution with a molar ratio of EDC to NHS of 2:1, immerse the pretreated nuclear pore membrane in the solution, and activate at room temperature for 30 min;

[0045] Probe modification: The activated nuclear pore membrane was immersed in tetrahedral probe solution and incubated for 2 hours. After incubation, it was washed three times with PBS solution at pH 7.4 to remove unbound free probes and obtain probe-modified nuclear pore membrane.

[0046] Blocking: Prepare a glycine solution (pH 7.4) using 1×TE buffer. Place the probe-modified nuclear pore membrane into the glycine solution, ensuring that the membrane is completely submerged and free of air bubbles. Place the culture dish on a decolorizing shaker and shake for 30 min, then place it in a 4°C refrigerator for 2 h to block. After blocking, pour in 100 mL of washing solution (prepared by adding NaCl and Tween-20 to 1×TE solution, pH 7.4), shake and wash for 3 min, and repeat the washing twice.

[0047] Drying and assembly: The cleaned nuclear pore membrane was taken out and placed in a 40℃ forced-air drying oven for 1 hour to remove irregular parts at the edges, thus obtaining a functionalized nuclear pore membrane.

[0048] Example 2 The application of a lung cancer PTGER4 gene methylation detection kit as described above includes the following steps:

[0049] First, the functionalized nuclear pore membrane was cut into squares of uniform size. One square was placed in a culture dish, and 2 mL of 1×PBS solution was added. The culture dish was then placed in a 55°C water bath for heating. 50 μL of the target analyte was placed in a sample tube, and 30 μL of 10×Bst buffer and 480 μL of 1×PBS solution were added. The mixture was thoroughly shaken and mixed. The sample tube was then placed in a 95°C water bath for 5 min. After heating, the entire solution in the tube was poured into a culture dish, and the culture dish was sonicated. The culture dish was then transferred to a 55°C water bath for further heating for 30 min.

[0050] After the heating reaction is complete, the functionalized nucleopore membrane is removed from the culture dish and washed 1-3 times with 1×PBS solution. The washed functionalized nucleopore membrane is then laid flat on a pad, which is used as a diaphragm to fix it to the salt bridge of the dual-cell electrolytic cell, ensuring a seamless seal between the membrane and the inner wall of the electrolytic cell. Equal volumes of electrolyte solution are added to the anode and cathode chambers of the electrolytic cell, respectively. The electrode assembly is inserted into the solution and connected to an electrochemical workstation. The detection parameters are set as follows: upper limit voltage 1V, lower limit voltage -1V, initial voltage 0V, and 5 cycles. The test program is started, and the current value corresponding to 1V voltage is recorded during the test.

[0051] 1. Standard Curve Plotting: PBS solutions containing different concentration gradients of PTGER4 gene methylated fragments were analyzed following the steps described above. During this process, it was ensured that the target gene fragment fully hybridized with the probe on the nuclear pore membrane surface. A standard curve was plotted with the logarithm of the PTGER4 methylated gene fragment concentration on the x-axis and the corresponding relative change in current on the y-axis.

[0052] 2. Linear relationship analysis, Figure 2 The display shows that in 10 1 ~10 6 Within the range of copies / μL, the relative change rate of current (I0-I1) / I0 showed a good linear relationship with the PTGER4 concentration, and the linear regression equation was y=0.04998lgx+0.03049(R0 / μL). 2 =0.998).

[0053] 3. To verify the repeatability of the kit's detection system, the following experiment was conducted: Under the same conditions, the PTGER4 standard solution (10...) was measured five consecutive times. 4 The relative changes in current of copies / μL were 28.13%, 28.03%, 27.91%, 28.08%, and 27.99%, respectively, with relative standard deviations (RSD) < 5%, indicating that the detection system has good intra-batch repeatability. Figure 3 ).

[0054] 4. To investigate the specificity of PTGER4 gene methylation, SHOX2 and RASSF1A standard solutions were used as controls. The experiment was conducted according to the detection method described above, and the results are as follows: Figure 4 As shown, this detection method has high specificity for PTGER4 gene methylation.

[0055] The above description is merely the preferred embodiment of the present invention and is not intended to limit the spirit and principles of the present invention. Any modifications, equivalent substitutions, improvements, etc., made should be included within the protection scope of the present invention.

Claims

1. A DNA tetrahedron probe for detecting lung cancer gene methylation, characterized in that, The DNA tetrahedral probe is formed by the self-assembly of four single-stranded DNA molecules, designated as S1 to S4, and the sequences of each single-stranded DNA molecule are shown in SEQ ID NO. 1 to 4. In this configuration, the 5' ends of S2, S3, and S4 are all modified with amino groups; S1 consists of a recognition arm and a backbone A. The target gene of the recognition arm is a specific methylated sequence fragment in the PTGER4 promoter region, which corresponds to the genomic location Chr5: 40681642-40681662, and its nucleotide sequence is shown in SEQ ID NO.5, with a length of 21 bp; the nucleotide sequence of the backbone A is shown in SEQ ID NO.7; the backbone A is used to self-assemble with S2, S3, and S4 to form a DNA tetrahedral backbone structure. The DNA tetrahedral probe forms a covalent bond with the porous membrane through its 5' amino group, thereby anchoring itself within the nanopores of the porous membrane. The structure of the DNA tetrahedral probe allows the recognition arm to extend spatially in the central region of the pores, avoiding steric hindrance from the inner wall of the porous membrane nanopores on the binding of the recognition arm to the target gene, thus ensuring that the recognition arm can bind to the target gene efficiently and accurately. The molar ratio of the four single-stranded DNA strands is 1:1:1:

1. The self-assembly conditions are as follows: heating at 95°C for 5-10 min in Tris-HCl buffer, followed by slow cooling to room temperature to complete self-assembly. The concentration of the Tris-HCl buffer is 10-20 mM, the pH is 7.4-8.0, and the buffer also contains 50-100 mM NaCl and 1-5 mM MgCl2.

2. A lung cancer gene methylation detection kit comprising the DNA tetrahedral probe of claim 1, characterized in that, The kit includes a functionalized porous membrane, an H-type electrolytic cell, an electrode assembly, an electrolyte solution, a PTGER4 gene methylation standard, and PBS washing solution. The functionalized porous membrane consists of a porous membrane substrate and the DNA tetrahedral probe. The inner wall of the nanopores of the porous membrane substrate contains carboxyl groups. After activation, the tetrahedral probe is covalently anchored in the modified nanopores.

3. A lung cancer gene methylation detection kit as described in claim 2, characterized in that, The method for preparing the functionalized porous membrane includes the following steps: (1) Substrate pretreatment: The porous membrane was ultrasonically cleaned with anhydrous ethanol and deionized water in sequence, dried, and then immersed in a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) for activation. (2) Probe immobilization: The activated porous membrane is incubated in a DNA tetrahedral probe solution. The probe is covalently linked to the nanopores. The free probe is washed away to obtain the probe-modified porous membrane. (3) Sealing treatment: Place the probe-modified porous membrane in a culture dish containing a glycine solution prepared with 1×TE, pH 7.1~7.5, ensuring that the porous membrane is completely immersed in the solution, place it on a decolorizing shaker and shake, and then place the culture dish in a refrigerator at 4℃ to seal; after sealing, pour out all the solution, take 20~150mL of the washing solution and pour it into the culture dish, shake and wash for 1~5min, wash 2~3 times, the washing solution is prepared by adding NaCl and Tween-20 to 1×TE solution, pH 7.1~7.5; (4) Drying: Take out the porous membrane and place it in a forced-air drying oven at 30~50℃ to dry, and obtain the functionalized porous membrane.

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