Method for detecting ncl conformation and its g4 complex based on solid-state nanopore

Abstract

The application belongs to the technical field of molecular detection, and particularly relates to a method for detecting NCL conformation and G4 complex thereof based on a solid nanopore. The method is as follows: a solid nanopore is used, bias is applied on both sides of the nanopore, translocation signals of a sample to be tested through the nanopore are tested under the condition of the applied bias, and nucleolin in the sample to be tested is qualitatively and / or quantitatively analyzed through the translocation signals. By analyzing the blocking current amplitude and translocation time of NCL and G4 complex thereof entering the nanopore channel under bias, information such as the size and electrification of NCL and G4 complex thereof can be obtained. The application can detect NCL of different concentrations, greatly reduce the test cost, and at the same time, trace detection of NCL can be realized with high sensitivity and rapidness. The application provides a new research idea for targeted treatment of diseases such as cancer through detection of NCL and NCL-G4 complex.

Description

Technical Field

[0001] This invention belongs to the field of molecular detection technology, specifically relating to a method for detecting NCL conformation and its G4 complex based on solid nanopores. Background Technology

[0002] Nucleolin (NCL / C23) is one of the most abundant phosphoproteins in the nucleolus. NCLs can interact with other proteins in various ways, thus participating in many intracellular biological processes, such as DNA repair, remodeling, and genome stabilization. They can also control cellular homeostasis by regulating cell proliferation, survival, or apoptosis. The DNA / RNA quadruplex (G4) can be used to inhibit tumor proliferation and cell life cycle; simultaneously, G4 exhibits specific interactions with various proteins, which can be used to regulate the mechanisms of certain disease development. Therefore, studying the interactions between G4 and proteins has significant biological implications.

[0003] Existing research has shown that G4 AS1411 and pre-miRNA-149 (RNA149 for short) have been used to target NCL and have shown significant activity against cancers such as metastatic renal cell carcinoma. Furthermore, the G-quadruplex structure formed by these sequences has also been used as a biosensor to selectively detect NCL. Therefore, rapid and sensitive detection of NCL and its complex with G4 is of great significance for the early prevention or targeted treatment of certain cancers.

[0004] Traditional methods for detecting NCL and its G4 complex include nuclear magnetic resonance (NMR), ultraviolet spectroscopy, Western blotting, and circular dichroism spectroscopy. While these methods have been proven effective for NCL detection, they suffer from drawbacks such as complex procedures, high equipment costs, demanding operator skills, and large sample volumes. Nanopore detection technology, however, offers a novel approach due to its high sensitivity, low cost, rapid detection capabilities, and single-molecule assays. Nanopore detection technologies include biological nanopore detection and solid-state nanopore detection. Although biological nanopore detection ensures high reproducibility, its pore size limitation restricts the types and sizes of proteins it can detect. Solid-state nanopore detection, on the other hand, offers the inherent advantage of adjustable pore size. Therefore, by adjusting the pore size of solid-state nanopores, optimal conditions for detecting NCL and its ligand complexes can be achieved, enabling rapid and sensitive detection of NCL and its G4 complex.

[0005] In view of this, the present invention proposes a method for detecting the single-molecule conformation of NCL and its G4 complex using a solid nanopore system. This method has not been reported to date and there is no patent application for it. Summary of the Invention

[0006] One of the objectives of this invention is to provide a method for detecting nucleolin protein conformation based on solid nanopores, which enables sensitive detection of trace NCL.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for detecting nucleolin protein conformation based on solid nanopores utilizes solid nanopores to test the translocation signal of the sample passing through the nanopore under an applied bias voltage. The translocation signal is used to perform qualitative and / or quantitative analysis of nucleolin in the sample. The sample is a composite solution composed of nucleolin and a buffer solution.

[0009] Furthermore, the buffer solution is composed of 1M KCl, 10mM Tris, and 1mM EDTA, with a pH of 7.4.

[0010] Furthermore, the concentration of nucleolin in the sample to be tested is 1 nM to 10 nM.

[0011] Furthermore, the solid nanopores are supported by silicon nitride films; the diameter of the solid nanopores is 8.5 nm to 15.5 nm.

[0012] Furthermore, the method for preparing the solid nanopores is as follows: the silicon nitride film is cleaned with a piranha solution, the cleaned silicon-based silicon nitride film is assembled in a test cell flowcell, and a conductive solution is added to the flowcell; the solid nanopores are prepared by dielectric breakdown; the conductive solution is 1M KCl, 10mM Tris, 1mM EDTA, pH 8.

[0013] The purpose of using piranha solution to clean silicon nitride films is to remove impurities from the surface of the silicon nitride films and to improve the hydrophilicity of the silicon nitride films.

[0014] Furthermore, the piranha solution is a mixed solution of H2SO4 and H2O2, wherein the volume ratio of H2SO4 to H2O2 is 3:1.

[0015] Furthermore, the sample to be tested is added to the cis end of the solid nanopore, and a bias voltage of 100mV is applied to both sides of the solid nanopore by a patch clamp to obtain the translocation signal of the NCL through the nanopore channel.

[0016] The second objective of this invention is to provide a method for detecting NCL-G4 complexes. This method is based on a solid-state nanopore detection system to perform trace detection of nucleolin protein and its G-quadruplex complexes formed with different nucleic acids, DNA, and RNA. The detection is rapid and highly sensitive.

[0017] To achieve the above objectives, the present invention adopts the following technical solution:

[0018] The detection method for the NCL-G4 complex includes the following steps:

[0019] (1) Prepare NCL-G4 complex samples;

[0020] (2) Using the method described in Objective 1, the NCL-G4 composite sample is subjected to qualitative and / or quantitative analysis based on solid nanopores; the diameter of the solid nanopores is 13 nm to 15 nm.

[0021] Furthermore, the method for preparing the NCL-G4 complex sample is as follows: A buffer solution is used to prepare G-rich sequence AS1411 and RNA 149 samples; annealing and cooling at room temperature yield RNA 149-G4 and AS1411-G4 samples forming a G4 structure; the RNA 149-G4 and AS1411-G4 samples are incubated with NCL respectively to allow them to fully bind, resulting in a complex of NCL with AS1411-G4 and RNA 149-G4.

[0022] Furthermore, the molar ratio of NCL to AS1411-G4 or RNA 149-G4 is 1:5.

[0023] Furthermore, the concentration of the G-rich sequence AS1411 and RNA 149 samples was 100 nM; the concentration of NCL was 20 nM.

[0024] Furthermore, the buffer solution is composed of 1M KCl, 10mM Tris, and 1mM EDTA, with a pH of 7.4.

[0025] Furthermore, the annealing conditions are 95°C for 5 minutes.

[0026] Furthermore, the cooling time is 1 hour.

[0027] Furthermore, the incubation conditions were 37°C for 2 hours.

[0028] The third objective of this invention is to provide a method for simultaneously detecting the conformation of nucleolin protein and NCL-G4 complex based on solid nanopores.

[0029] To achieve the above objectives, the present invention adopts the following technical solution:

[0030] A method for detecting the conformation of nucleolin protein and NCL-G4 complex based on solid nanopores, using the methods described in Objective 1 and Objective 2 to simultaneously perform qualitative and / or quantitative analysis of nucleolin protein and NCL-G4 complex.

[0031] The fourth objective of this invention is to provide an application of the method described in objective one and / or the method described in objective two in the monitoring, symptom assessment and prevention of cancer.

[0032] Furthermore, the cancerous diseases include any one or more of gastric cancer, hepatocellular carcinoma, lung cancer, and pancreatic ductal adenocarcinoma.

[0033] The beneficial effects of this invention are as follows:

[0034] 1. This invention discloses a method for detecting nucleolin protein and its G-quadruplex complexes with different nucleic acids (DNA and RNA) based on a solid-state nanopore detection system. By applying a bias voltage across the nanopore, the method analyzes the amplitude of the blocking current and the translocation time generated when the nucleolin protein (NCL) and its G4 complex enter the nanopore channel under the bias voltage, thereby obtaining information such as the size and charge of the NCL and its G4 complex. Compared with traditional detection methods, this invention is simple to operate, low in cost, rapid in detection, and has higher sensitivity, showing great application potential.

[0035] 2. By exploring the detection concentration of NCL, this invention obtained the detection limit of NCL in solid nanopores, and realized sensitive detection of trace NCL.

[0036] 3. This invention provides a trace detection method based on solid-state nanopores to recognize the conformation of single-molecule NCL and its G4 conjugate. Clinically, quantitative detection of NCL content in human blood has significant guiding significance for monitoring, symptom assessment, and early prevention of diseases such as gastric cancer, hepatocellular carcinoma, lung cancer, and pancreatic ductal adenocarcinoma. By detecting complexes of G-quadruplexes with different nucleic acid folds and NCL, it provides an important theoretical research basis for the early prevention and targeted therapy of cancer. Attached Figure Description

[0037] Figure 1 A schematic diagram of the configuration of the NCL and AS1411 / pre-miRNA-149G4 complex;

[0038] Figure 2 This is a schematic diagram of nanopore detection.

[0039] Figure 3 The original translocation trajectories of NCL at different concentrations through nanoporous channels are shown.

[0040] Figure 4 The graph shows the relationship between NCL concentration and capture rate.

[0041] Figure 5 The circular dichroism chromatograms of AS1411-G4 and RNA149-G4 are shown.

[0042] Figure 6 The original translocation trajectory diagrams for RNA 149-G4, NCL, and the NCL-RNA149-G4 complex were obtained.

[0043] Figure 7-8 Translocation time diagrams for RNA149-G4, NCL, and the NCL-RNA149-G4 complex;

[0044] Figure 9-10 Histograms of ion current blocking amplitudes for RNA 149-G4, NCL, and the NCL-RNA 149-G4 complex.

[0045] Figure 11 Original translocation trajectory diagrams of AS1411-G4, NCL, and NCL-AS1411-G4 complexes with different ratios.

[0046] Figure 12 The normalized current blocking amplitude histogram obtained from the 10nM NCL test;

[0047] Figure 13 The normalized current blocking amplitude histogram of the NCL-AS1411-G4 complex with a molar ratio of 5:1.

[0048] Figure 14 The normalized current blocking amplitude histogram of the NCL-AS1411-G4 complex with a molar ratio of 1:1.

[0049] Figure 15 The normalized current blocking amplitude histogram of the NCL-AS1411-G4 complex with a molar ratio of 1:5. Detailed Implementation

[0050] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0051] Example 1

[0052] 1. Preparation of solid-state nanopores

[0053] Silicon nitride thin films were subjected to piranha solution (V) H2SO4 :V H2O2=3:1) cleaning to remove impurities from the surface of the silicon nitride film and improve its hydrophilicity; then the cleaned silicon nitride film was assembled in a test cell flowcell, and nanopore channels with diameters of 8.5 nm to 15.5 nm were prepared by dielectric breakdown. The conductivity solution was 1 M KCl, 10 mM Tris, 1 mM EDTA, pH 8; the conductivity G of the nanopores was obtained by patch clamp testing IV curves, and finally the diameter of the nanopore channels was calculated by the pore size calculation formula.

[0054] 2. Testing of NCL via solid-state nanoporous channels

[0055] NCL samples of different concentrations were prepared (buffer solution: 1M KCl, 10mM Tris, 1mM EDTA, pH 7.4), and then the samples were loaded onto the cis end of an 8.5 nm solid-state nanopore. A 100 mV bias voltage was applied across the solid-state nanopore using a patch clamp to obtain the translocation signal of NCL through the nanopore channel. By processing and analyzing these signals, the single-molecule conformation information, detection limit, and optimal capture rate of NCL within the solid-state nanopore were obtained.

[0056] 3. Circular dichroism spectroscopy assay of G4 aptamer AS1411 / RNA 149

[0057] Prepare 30 μM samples of G-rich sequence AS1411 and pre-miRNA 149 (buffer solution: 1 M KCl, 10 mM Tris, 1 mM EDTA, pH 7.4), anneal them (95 °C, 5 min), and then cool them at room temperature for 1 h to form G4 structures. Then scan them three times with light in the 220 nm to 320 nm band and take the average value of the polarized light.

[0058] 4. Testing of the NCL-G4 composite through nanoporous channels

[0059] 100 nM of G-rich sequence AS1411 and RNA 149 samples (buffer solution: 1 M KCl, 10 mM Tris, 1 mM EDTA, pH 7.4) were prepared and annealed (95 °C, 5 min), then cooled at room temperature for 1 h to form G4 structures. The formed RNA149-G4 and AS1411-G4 samples were then incubated with 20 nM NCL (37 °C, 2 h) to allow for complete binding, resulting in NCL-AS1411-G4 and RNA149-G4 complexes at a molar ratio of 1:5. Finally, the obtained NCL-DNA / RNA G4 complexes were loaded onto the cis end (ground end) of 13-15 nm nanopores. A 100 mV bias voltage was applied across the solid nanopores using patch clamps to obtain the translocation signal of the NCL-G4 complex through the nanopore channel. A schematic diagram of NCL and its configuration with the AS1411 / pre-miRNA-149G4 complex and its nanopore detection is shown below. Figures 1-2 As shown.

[0060] Results: The test results are as follows Figure 3-15 As shown. Figure 3 This is a diagram showing the original translocation trajectory of NCL at different concentrations (1 nM to 10 nM) through nanoporous channels in buffer solutions of 1 M KCl, 10 mM Tris, and 1 mM EDTA (pH 7.4). Figure 3 As shown, with the gradual increase of NCL concentration, the frequency of signal occurrence per unit time gradually increases, but this also increases the probability of interaction between NCL and the nanopore channel walls, which may lead to adsorption or pore blockage. The relationship between NCL concentration and capture rate was obtained by processing and analyzing the original NCL translocation trajectory diagram, as detailed in [link to diagram]. Figure 4 As shown, through Figure 4 It can be seen that as the NCL concentration increases, the capture rate of NCL by the nanopores also gradually increases. When the NCL concentration is 10 nM, the capture rate is 4.4 events / s, which can achieve the purpose of rapid detection of NCL. When the NCL concentration is 1 nM, the translocation signal of NCL can still be detected by the solid nanopore test system, which can realize the sensitive detection of trace NCL.

[0061] Figure 5 The circular dichroism chromatograms of 30 μM AS1411-G4 and RNA 149-G4 in a buffer solution of 1 M KCl, 10 mM Tris, and 1 mM EDTA (pH 7.4) are shown. Figure 5 It can be seen that AS1411-G4 and RNA 149-G4 have obvious positive absorption peaks near 260 nm and obvious negative absorption peaks near 245 nm, indicating that both have formed parallel G4 conformations.

[0062] Figure 6 This is a diagram showing the original translocation trajectories of RNA149-G4, NCL, and the NCL-RNA149-G4 complex through a 13.5 nm nanopore channel under a bias voltage of 100 mV. Figure 6 It can be observed that the RNA149-G4 signal is almost undetectable. This is because, under large pore sizes, the G4 molecule, due to its small size and rapid translocation, is not easily captured. After processing the original translocation trajectory map, it was found that the translocation time of NCL is shorter than that of its translocation with the RNA149-G4 complex. (See details...) Figure 7 and Figure 9 This is because the composite has a large volume and a long residence time within the nanopores. Furthermore, the histogram of the ion current blocking amplitude of both... Figure 8 and Figure 10 It can be observed that due to the binding of NCL to RNA149-G4, a large ion current blocking amplitude is generated, and the complex shows a large peak value of 204.86pA.

[0063] Figure 11 The diagram shows the original translocation trajectories of AS1411-G4, NCL, and the NCL-AS1411-G4 complex through a 15.5 nm nanopore channel under a bias voltage of 100 mV. Figure 11 As shown, the capture rate of AS1411-G4 is extremely low. This is because, under large pore sizes, G4 molecules are difficult to capture due to their small size and rapid translocation speed. Further processing of the original translocation trajectory diagram revealed that the binding of NCL with AS1411-G4 generates a large ion current blocking amplitude. The normalized current blocking amplitude of the complexes with different proportions exhibits a value lower than that of the NCL monomer (ΔI / I0). o For a larger distribution (=0.025), see details. Figures 12-15 .

Claims

1. A method for detecting the conformation of nucleolin protein and / or the NCL-G4 complex based on solid-state nanopores, characterized in that, Using solid-state nanopores, the translocation signal of the sample passing through the nanopore is measured under an applied bias voltage. The translocation signal is used for qualitative and / or quantitative analysis of nucleolin and / or NCL-G4 complex in the sample. The sample is a composite solution of nucleolin and buffer solution and / or an NCL-G4 complex sample. The nucleolin is detected using solid-state nanopores with a diameter of 8.5 nm to 15.5 nm. The NCL-G4 complex sample is detected using solid-state nanopores with a diameter of 13 nm to 15 nm.

2. The method according to claim 1, characterized in that, The buffer solution consisted of 1 M KCl, 10 mM Tris, and 1 mM EDTA, with a pH of 7.

4.

3. The method according to claim 1, characterized in that, The concentration of nucleolin in the sample to be tested is 1 nM to 10 nM.

4. The method according to claim 1, characterized in that, The solid nanopores are supported by silicon nitride thin films.

5. The method according to claim 4, characterized in that, The solid nanopores are prepared by: cleaning the silicon nitride film with a piranha solution, assembling the cleaned silicon-based silicon nitride film in a test cell flowcell, adding a conductive solution to the flowcell, and preparing the solid nanopores by dielectric breakdown; the conductive solution is 1 MKCl, 10 mM Tris, 1 mM EDTA, pH 8.

6. The method according to claim 1, characterized in that, The method for preparing the NCL-G4 complex sample is as follows: A buffer solution is used to prepare G-rich sequence AS1411 and RNA 149 samples; annealing and cooling at room temperature yield RNA 149-G4 and AS1411-G4 samples forming a G4 structure; the RNA 149-G4 and AS1411-G4 samples are incubated with NCL to allow them to fully bind, resulting in a complex of NCL with AS1411-G4 and RNA 149-G4.

7. The method according to claim 6, characterized in that, The molar ratio of NCL to AS1411-G4 or RNA 149-G4 is 1:

5.

8. The method according to claim 6, characterized in that, The incubation conditions were 37 ℃ for 2 h.

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