DNA-based origami nanoscale rulers and their applications

Abstract

This invention provides a DNA origami-based nanoscale ruler and its application. The nanoscale ruler includes a substrate structure, fluorescent indicator chains, and spacing indicator chains. The substrate structure is a three-layer rectangular structure constructed using DNA origami technology to maintain the basic configuration of the nanoscale ruler. Several fluorescent indicator chains are distributed on the first surface of the substrate structure according to a predetermined rule. These fluorescent indicator chains emit fluorescent signals through their fluorescent groups under specific excitation light. Two spacing indicator chains are disposed on the first surface of the substrate structure, forming a predetermined distance between them. These spacing indicator chains are used to target and bind to receptor proteins on the cell membrane via ligand proteins. One technical advantage of this invention is its rational design, which not only enables rapid and accurate determination of the presence of EGFR mutations in tissue samples but also effectively shortens the time required to determine the presence of EGFR mutations.

Description

Technical Field

[0001] This invention belongs to the field of cell membrane surface protein distribution spacing measurement technology, specifically relating to a DNA origami-based nanoscale ruler and its application. Background Technology

[0002] Lung adenocarcinoma is the most common subtype of lung cancer and a leading cause of cancer-related deaths worldwide. Currently, 40-60% of lung adenocarcinoma patients have epidermal growth factor receptor (EGFR) mutations, and targeted therapy has become the standard first-line treatment for EGFR-mutant patients. For wild-type EGFR patients, chemotherapy remains the preferred treatment. In clinical case analyses of lung adenocarcinoma, the proportion of patients carrying EGFR-mutant genotypes and wild-type EGFR patients is roughly equal. Therefore, detecting the presence of EGFR mutations and further identifying the mutation type has become a routine clinical test, playing a crucial role in the treatment of lung adenocarcinoma patients. Immunohistochemistry is a routine detection method for almost all lung adenocarcinoma patient subtyping, and can detect tumor markers / potential target proteins in approximately 4-6 hours. However, because immunohistochemistry can only identify the presence of specific types of proteins and not gene mutations, traditional immunohistochemical methods cannot directly detect EGFR mutations.

[0003] Current cutting-edge biological and medical research has revealed an important principle:

[0004] The spatial distribution of EGFR on the cell membrane is strongly correlated with the EGFR mutation status, and new strategies for inferring related gene mutations by exploring the protein distribution on the cell membrane have become an attractive topic.

[0005] Currently, there are two main methods for identifying EGFR mutation types in lung adenocarcinoma patients.

[0006] Technology-like:

[0007] 1. Next-generation sequencing (NGS) is used to sequence tissue samples to identify EGFR mutation types. The drawbacks of this method are its high cost and long waiting period. Furthermore, for wild-type EGFR patients, the expensive sequencing cannot assist in drug selection. And the risk of cancer cell progression further increases during the long wait for NGS results.

[0008] 2. EGFR mutation types can be identified by detecting EGFR mutations in tissue samples using the amplification arrest mutation system polymerase chain reaction (ARMS-PCR) method. This method requires specially designed primers, cell lysis, and nucleic acid extraction from the sample, placing strict requirements on sample quality and concentration. Furthermore, samples with low gene abundance are highly susceptible to false negatives, making the identification of the presence of EGFR mutations inaccurate. Summary of the Invention

[0009] The present invention aims to solve at least one of the technical problems existing in the prior art, and to provide a new technical solution for a DNA origami-based nanoscale ruler and its application.

[0010] According to a first aspect of the present invention, a DNA-origami-based nanoscale ruler is provided, comprising:

[0011] The substrate structure is a three-layer rectangular structure constructed using DNA origami technology to maintain the basic configuration of the nanoscale ruler.

[0012] A fluorescent indicator chain, wherein a plurality of the fluorescent indicator chains are distributed on a first surface of the substrate structure according to a preset rule; the fluorescent indicator chain includes a first nucleic acid chain and a fluorescent group; one end of the first nucleic acid chain is connected to the first surface of the substrate structure, and the other end is connected to the fluorescent group; the fluorescent indicator chain is used to emit a fluorescent signal through the fluorescent group under specific excitation light irradiation;

[0013] A spacing indicator chain, wherein two spacing indicator chains are disposed on the first surface of the substrate structure and a predetermined distance is formed between the two spacing indicator chains; the spacing indicator chain includes a second nucleic acid chain and a ligand protein, one end of the second nucleic acid chain is connected to the first surface of the substrate structure and the other end is connected to the ligand protein, and the spacing indicator chain is used to target and recognize and bind to receptor proteins on the cell membrane through the ligand protein;

[0014] The spacing between receptor proteins on the cell membrane is determined by a preset distance, and the mutation of the epidermal growth factor receptor is confirmed by analyzing the intensity of the fluorescence signal on the cell surface.

[0015] Optionally, the substrate structure includes a long single-stranded DNA and multiple fixed short strands, wherein the long single-stranded DNA and the fixed short strands are base-complementary to form a three-layer rectangular structure.

[0016] Optionally, the three-layer rectangular structure has a length of 100nm, a width of 17nm, and a height of 20nm; and the first surface of the substrate structure has a length of 100nm and a width of 17nm.

[0017] Optionally, the first nucleic acid chain is complementary to and bound together with the fluorescently modified nucleic acid chain to form the fluorescent indicator chain.

[0018] Optionally, the second nucleic acid chain binds to the ligand protein via complementary base pairing to form the spacing indicator chain.

[0019] Optionally, the second nucleic acid chain can be extended by increasing the number of bases.

[0020] According to a second aspect of the present invention, an application of a DNA-based origami nanoscale ruler is provided, comprising the following steps:

[0021] After fixing the cells with 4% paraformaldehyde, the first cell group was incubated with a first nanometer scale and the second cell group was incubated with a second nanometer scale; wherein, there is a first preset distance between the two spacing indicator chains of the first nanometer scale and a second preset distance between the two spacing indicator chains of the second nanometer scale, and the first preset distance is less than the second preset distance.

[0022] By analyzing the ratio of the fluorescence signal intensity under the first nanometer scale to the fluorescence signal intensity under the second nanometer scale, it can be determined whether the epidermal growth factor receptor is mutated.

[0023] Optionally, the first preset distance is 25nm and the second preset distance is 100nm.

[0024] Optionally, the cells are cell lines, or the cells are derived from human tissue clinical samples.

[0025] Optionally, when the ratio of the first preset distance to the second preset distance is greater than or equal to 1, the cell expresses the EGFR mutant genotype; when the ratio is less than 1, the cell expresses wild-type EGFR.

[0026] One technical advantage of this invention is that:

[0027] In this embodiment, the substrate structure is a three-layer rectangular structure constructed using DNA origami technology to maintain the basic configuration of the nanoscale ruler. Several fluorescent indicator chains are distributed on the first surface of the substrate structure according to a preset rule, emitting fluorescent signals through fluorescent groups under specific excitation light. Two spacing indicator chains are disposed on the first surface of the substrate structure, forming a preset distance between them, for targeting and binding to receptor proteins on the cell membrane via ligand proteins. The preset distance determines the spacing between receptor proteins on the cell membrane, and the intensity analysis of the fluorescence signal on the cell surface confirms whether the epidermal growth factor receptor has mutated.

[0028] Because the substrate structure is a three-layered rectangular structure constructed using DNA origami technology, it possesses a certain degree of rigidity, thus maintaining the overall stability of the nanoscaler. The spacing indicator chains attached to the substrate structure can target and bind to receptor proteins on the cell membrane via ligand proteins, and these chains can emit fluorescent signals through fluorophores under specific excitation light. Therefore, by adjusting the preset distance between two spacing indicator chains, different nanoscalers can be formed. Furthermore, since the intensity of the fluorescence signal captured by cells under different nanoscalers varies, the presence of EGFR mutations can be rapidly and visually analyzed by examining the intensity of the fluorescence signal on the cell surface (e.g., cancer cells).

[0029] Furthermore, since the cell identification process using a nanoscale can be completed in approximately 4-6 hours, it significantly reduces the time required to determine the presence of EGFR mutations. In addition, the detection process is based on the intensity analysis of fluorescence signals from individual cells, eliminating the need for cell lysis and placing no special requirements on sample concentration, thus simplifying the procedure. Scientific statistical analysis further enhances the accuracy of the identification results. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of a DNA origami-based nanoscale ruler according to a first embodiment of the present invention;

[0031] Figure 2 This is a top view of a DNA origami-based nanoscale ruler according to a first embodiment of the present invention;

[0032] Figure 3 This is a schematic diagram of a DNA origami-based nanoscale ruler before it binds to a receptor protein, according to the first embodiment of the present invention.

[0033] Figure 4 This is a schematic diagram of a DNA origami-based nanoscale ruler after binding with a receptor protein, according to a first embodiment of the present invention.

[0034] Figure 5 This is a schematic diagram of a DNA origami-based nanoscale ruler according to a second embodiment of the present invention;

[0035] Figure 6 This is a top view of a DNA origami-based nanoscale ruler according to a second embodiment of the present invention;

[0036] Figure 7 This is a schematic diagram of a DNA origami-based nanoscale ruler before it binds to a receptor protein, according to a second embodiment of the present invention.

[0037] Figure 8 This is a schematic diagram of a DNA origami-based nanoscale ruler after binding with a receptor protein, according to a second embodiment of the present invention.

[0038] Figure 9 This is a transmission electron microscope image of a DNA origami-based nanoscale ruler according to the first embodiment of the present invention.

[0039] Figure 10 This is a transmission electron microscope image of a DNA origami-based nanoscale ruler according to the second embodiment of the present invention.

[0040] In the diagram: 1. Base structure; 2. Spacing indicator chain; 21. First nucleic acid chain; 22. Fluorescent group; 3. Fluorescent indicator chain; 31. Second nucleic acid chain; 22. Ligand protein; 4. Cytoplasm; 5. Cell membrane; 6. Receptor protein. Detailed Implementation

[0041] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the present application.

[0042] The embodiments of this application will now be described in detail. Examples of these embodiments are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0043] The terms "first" and "second" in the specification and claims of this application may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise stated, "multiple" means two or more. Furthermore, "and / or" in the specification and claims indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0044] In the description of this application, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc., indicating the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.

[0045] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0046] join Figures 1 to 10 According to a first aspect of the present invention, a DNA origami-based nanoscale ruler is provided, comprising a substrate structure 1, a fluorescent indicator chain 3, and a spacing indicator chain 2.

[0047] Specifically, see Figure 1 and Figure 5 The substrate structure 1 is a three-layer rectangular structure constructed using DNA origami technology to maintain the basic configuration of the nanoscale ruler. Substrate structure 1 is relatively stable, thus contributing to the structural stability of the nanoscale ruler. Substrate structure 1 also exhibits good biocompatibility, enabling its better application in biological tissues.

[0048] More specifically, a plurality of the fluorescent indicator chains 3 are distributed on the first surface of the substrate structure 1 according to a preset rule; the fluorescent indicator chain 3 includes a first nucleic acid chain 21 and a fluorescent group 22; one end of the first nucleic acid chain 21 is connected to the first surface of the substrate structure 1, and the other end is connected to the fluorescent group 22; the fluorescent indicator chain 3 is used to emit a fluorescent signal through the fluorescent group 22 under specific excitation light irradiation.

[0049] It should be noted that the fluorescent indicator chain 3 is the key part for achieving visualization in this invention. The fluorescent indicator chain 3 is attached to the first surface of the substrate structure 1. The ends of the substrate structure 1 form outwardly extending fixed short chains through some bases. The extended bases can complementarily pair with the first nucleic acid chain 21 modified by the fluorescent group 22, thereby emitting fluorescence under specific excitation light.

[0050] For example, such as Figure 2 and Figure 6 As shown, the fluorescent indicator chains 3 are arranged at a certain spacing. Because the substrate structure 1 is narrow, the fluorescent indicator chains 3 can be arranged in three rows along the width of the substrate structure 1, and the number of rows can be increased as needed. It is worth noting that when selecting short nucleic acid chains attached to the surface of the substrate structure 1 to connect with the fluorescent indicator chains 3, the direction of the short nucleic acid chains can extend outwards from their ends; otherwise, it is difficult to connect them with the second nucleic acid chain 31 modified by the fluorescent group 22.

[0051] Two spacing indicator chains 2 are disposed on the first surface of the substrate structure 1 and form a preset distance between the two spacing indicator chains 2; the spacing indicator chain 2 includes a second nucleic acid chain 31 and a ligand protein 22, one end of the second nucleic acid chain 31 is connected to the first surface of the substrate structure 1 and the other end is connected to the ligand protein 22, and the spacing indicator chain 2 is used to target and recognize and bind to the receptor protein 6 of the cell membrane 5 through the ligand protein 22.

[0052] The spacing indicator chain 2 is a key component in achieving the beneficial effects of this invention. The spacing indicator chain 2 is attached to the first surface of the substrate structure 1. The ends of the substrate structure 1 form outwardly extending fixed short chains through some bases. The extended bases can be complementary-paired with the second nucleic acid chain 31 that connects to the EGF ligand protein 22, thereby enabling the EGF ligand protein 22 to be loaded onto the substrate structure 1 and subsequently recognize the receptor EGFR protein on the cell membrane 5.

[0053] In one specific implementation, the lengths of both the first and second nucleic acids can be extended by increasing the number of bases.

[0054] The spacing between receptor proteins 6 on the cell membrane 5 is determined by a preset distance, and the mutation of the epidermal growth factor receptor is confirmed by analyzing the intensity of the fluorescence signal on the cell surface. Specifically, when the preset distance between the two spacing indicator chains 2 of the nanoscale is different, the nanoscale can bind to receptor proteins 6 at different spacings, resulting in different intensities of fluorescence signals on the cell surface. This allows for the confirmation of whether the epidermal growth factor receptor is mutated based on the intensity of the fluorescence signal on the cell surface. The operation is simple, and the analysis results are relatively accurate.

[0055] It's important to note that the formation of the nanoscale ruler relies on DNA origami technology. The advent of DNA origami has provided researchers with a method to manipulate nanoscale structures. Furthermore, the development of DNA origami has offered methods for constructing large numbers of precisely sized and shape-specific nanostructures. Based on DNA origami, the nanoscale ruler accurately identifies the spacing of EGFR protein distributions by precisely controlling the size and contour of its nanostructures, thus aiding in the determination of whether EGFR mutations exist.

[0056] In this embodiment, the substrate structure 1 is a three-layer rectangular structure constructed using DNA origami technology, possessing a certain rigidity, thus maintaining the overall stability of the nanoscale ruler. The spacing indicator chain 2 connected to the substrate structure 1 can target and bind to the receptor protein 6 of the cell membrane 5 via ligand protein 22. The spacing indicator chain 2 connected to the substrate structure 1 can emit a fluorescent signal through fluorescent group 22 under specific excitation light irradiation. Therefore, by adjusting the preset distance between the two spacing indicator chains 2, different nanoscale rulers can be formed. Furthermore, since the intensity of the fluorescence signal captured by the cells under different nanoscale rulers varies, the presence of EGFR mutations can be quickly and visually analyzed by analyzing the intensity of the fluorescence signal on the surface of cells (e.g., cancer cells).

[0057] Furthermore, since the cell identification process using a nanoscale can be completed in approximately 4-6 hours, it significantly reduces the time required to determine the presence of EGFR mutations. In addition, the detection process is based on the intensity analysis of fluorescence signals from individual cells, eliminating the need for cell lysis and placing no special requirements on sample concentration, thus simplifying the procedure. Scientific statistical analysis further enhances the accuracy of the identification results.

[0058] See Figure 9 and Figure 10 . Figure 9 The successful synthesis of the nanoscale (with a preset distance of 20 nm) was verified by transmission electron microscopy imaging of a nanoscale with a preset distance of 25 nm between two spacing indicator chains 2 (hereinafter referred to as the 25 nm nanoscale).

[0059] Figure 10 The successful synthesis of a nanoscale (with a preset distance of 100 nm) between two spacing indicator chains 2 was verified by transmission electron microscopy imaging.

[0060] The size of the nanoscale bar is designed according to actual needs, based on the principle that 10.67 bp is approximately equal to 3.6 nm. For example, for a 25 nm nanoscale bar, the 0 end is selected at the endpoint of substrate structure 1, and a 15-poly(A) nucleic acid sequence is extended to complementarily pair with the 15-poly(T) nucleic acid chain linking EGF ligand protein 22. Then, a short nucleic acid chain is extended at a position 25 nm away from it and complementarily pairs with the 15-poly(T) linking EGF ligand protein 22, thus synthesizing a 25 nm spacing nanoscale bar. When other spacings of nanoscale bars need to be synthesized, these spacings can be determined according to specific requirements.

[0061] Optionally, the substrate structure 1 includes a long single-stranded DNA and multiple fixed short strands, wherein the long single-stranded DNA and the fixed short strands are base-complementary to form a three-layer rectangular structure.

[0062] In the above embodiments, the long single-stranded DNA is specially designed and paired with fixed short strands through base complementarity, thereby forming a relatively stable and rigid three-layer rectangular structure.

[0063] Optionally, the length of the three-layer rectangular structure is 100nm, the width is 17nm, and the height is 20nm; and the length of the first surface of the substrate structure 1 is 100nm and the width is 17nm.

[0064] In the above embodiment, the entire nanoscale exhibits a configuration of 100 nm in length, 20 nm in width, and 17 nm in height during transmission electron microscopy measurements. The relatively small overall size of the nanoscale facilitates its integration with cells. The three-layer rectangular structure is the optimal configuration for maintaining stability and rigidity, thus giving the nanoscale good stability and high rigidity.

[0065] In practical applications, the structure of the nanoscale can be designed according to the actual situation, for example, by obtaining the required configuration through different folding methods.

[0066] In one embodiment, the peak hydrodynamic dimension of the nanoscale measured using a Malvern analyzer is 100 nm.

[0067] Optionally, the first nucleic acid chain 21 is complementary to and bonded to the nucleic acid chain modified by the fluorescent group 22 to form the fluorescent indicator chain 3. This makes the structure of the fluorescent indicator chain 3 reasonable and enables stable connection with the substrate structure 1. It can also emit a fluorescent signal through the fluorescent group 22 under specific excitation light irradiation, which is beneficial for detecting the spacing between receptor proteins 6 on the cell membrane 5 by detecting the intensity of the fluorescent signal, thereby helping to confirm whether the epidermal growth factor receptor is mutated.

[0068] Optionally, the second nucleic acid chain 31 is linked to a specially modified ligand protein 22 through base complementarity to form the spacing indicator chain 2. This makes the structural design of the spacing indicator chain 2 reasonable, enabling stable connection with the substrate structure 1. Specifically, the special modification involves linking epidermal growth factor (EGF) to sulfosuccinylated 4-cyclohexane-1-carboxylate (Sulfo-SMCC), which is then ultrafiltered and subsequently linked to the nucleic acid chain, thereby synthesizing a ligand protein linked to the nucleic acid chain.

[0069] Optionally, the second nucleic acid chain 31 can be lengthened by increasing the number of bases. This allows the length of the second nucleic acid chain 31 to be extended as needed, and the extension method is relatively simple.

[0070] In one embodiment, the present invention provides a system for identifying the presence of EGFR mutations in tissue samples, comprising a first nanoscale, a second nanoscale, a fluorescence microscope, and an image processing device. Cells are incubated using the first and second nanoscales, respectively, and then the presence of EGFR mutations in the cells is observed using a fluorescence microscope. The acquired images can also be processed using the image processing device, thereby aiding in the accurate determination of the presence of EGFR mutations in the cells.

[0071] According to a second aspect of the present invention, an application of a DNA-based origami nanoscale ruler is provided, comprising the following steps:

[0072] After the cells were fixed with 4% paraformaldehyde, the first cell group was incubated with a first nanometer scale and the second cell group was incubated with a second nanometer scale; wherein, there is a first preset distance between the two spacing indicator chains 2 of the first nanometer scale and a second preset distance between the two spacing indicator chains 2 of the second nanometer scale, and the first preset distance is less than the second preset distance.

[0073] By analyzing the ratio of the fluorescence signal intensity under the first nanometer scale to the fluorescence signal intensity under the second nanometer scale, it can be determined whether the epidermal growth factor receptor is mutated.

[0074] See Figure 3 and Figure 7 The cell membrane 5 encloses the cytoplasm 4, and receptor proteins attach to the surface of the cell membrane.

[0075] In the above embodiments, the application design of the DNA origami-based nanoscale ruler is reasonable. It can not only accurately confirm whether the cell epidermal growth factor receptor is mutated, but also help save detection time.

[0076] Optionally, the first preset distance is 25 nm, the second preset distance is 100 nm, and the first nanoscale and the second nanoscale can incubate the cell group respectively. Since the distance between the first nanoscale and the second nanoscale that target and bind to the receptor protein 6 of the cell membrane 5 is significantly different, it helps to analyze the intensity of the fluorescence signal of different nanoscales aggregated on the cell surface and quantitatively determine whether EGFR mutation exists, resulting in a more accurate detection result.

[0077] Optionally, the cells are cell lines or are derived from human tissue clinical samples, which makes the nanoscaler widely applicable and very convenient to use.

[0078] In one embodiment, the specific manufacturing method of the nanoscale ruler is as follows:

[0079] First, substrate structure 1, fluorescent indicator strand 3, and spacing indicator strand 2 were synthesized as follows: A mixture of 10 μg M 13mp18 scaffold long-chain single-stranded DNA and 10 μM custom primer pool was thoroughly dissolved in 100 μL reaction buffer (10 mM Tris, 1 mM EDTA, and 10 mM MgCl2). The primer pool contained all short strands. The mixture was annealed in a PCR instrument according to the following program: 95 °C for 30 seconds, then cooled to 20 °C at a rate of 10 min / °C to form the main structure of the nanoscale bar. The solution was filtered through PBS using a 0.5 ml 100 KD ultrafiltration tube and centrifuged three times at 2500 g for 2 minutes. Excess short fiber chains were removed, yielding purified nanostructures, known as Solution I, which were stored at 4 °C.

[0080] The procedure for ligating fluorescent indicator chain 3 to substrate structure 1 is as follows: Fluorescent indicator chain 3 is diluted to 10 μM using reaction buffer (10 mM Tris, 1 mM EDTA, and 10 mM MgCl2), and then mixed with solution I at a concentration ratio of 1:5. The mixture is annealed in a PCR instrument according to the following program: incubation at 45 °C, cooling to 20 °C at a rate of 10 min / °C, resulting in solution II, which is then stored at 4 °C.

[0081] The ligand protein EGF was ligated to substrate structure 1 using a specially modified method: 4 mM sulfosuccinylated 4-cyclohexane-1-carboxylate (Sulfo-SMCC) was ligated to 30 μg of EGF-Fc protein at 4 °C for 2 hours. The mixture was then filtered three times in PBS solution at 5000 g for 5 minutes using a 0.5 ml 30 kDa ultrafiltration tube. 100 μM polymer 15poly(T) was then added to the purified mixture and incubated at room temperature for 1 hour. The mixture was then centrifuged at 5000 g in reaction buffer (10 mM Tris, 1 mM EDTA, and 10 mM MgCl2) for 5 minutes using a 0.5 ml 30 kDa ultrafiltration tube. The purified ligand protein 22 with the liganded nucleic acid strand was obtained as Solution III and stored at 4 °C.

[0082] Synthesis steps of the nanoscaler: Finally, solution II and solution III were mixed at a concentration ratio of 1:10. The mixture was incubated at 37°C and cooled to 25°C at 1 h / °C. It was then centrifuged at 2500 g for 2 minutes using a 0.5 ml 100 KD ultrafiltration tube.

[0083] In the above fabrication method, the morphology of the nanosensor was determined using transmission electron microscopy. It exhibits a rectangular structure with a length of 100 nm, a width of 20 nm, and a height of 17 nm; the peak hydrodynamic size is 100 nm; and the nucleic acid sequence length determined by gel electrophoresis is approximately 2700 bp.

[0084] For example, nanoscales can be used to measure the spacing of the EGFR receptor protein on the cell membrane 5 surface. For instance, the presence of EGFR mutations in tissue samples can be identified based on the different intensities of fluorescence signals from different nanoscales that bind to the EGFR receptor protein on the cell membrane 5 surface.

[0085] Step 1: Using pure wild-type cell line A549 and cell lines containing two EGFR mutations, HCC827 and H1975, as standard models, the specific fluorescence signal intensity range was calculated using different nanoscale rulers. The specific operational steps are as follows:

[0086] Cells were cultured overnight in 6 cm culture dishes. After cell counting, the cells were evenly aliquoted into 1.5 ml centrifuge tubes. Cells in each tube were fixed with 4% paraformaldehyde for 15 minutes, and different nanoscalers (10 nM) were added to the cell suspension. The tubes were incubated at 4°C for 2 hours. All steps required washing three times with PBS. 50 μl of each treated cell sample was placed in a confocal culture dish. Confocal microscopy was then used for tomographic imaging. All images were analyzed using ImageJ software, and fluorescence intensity was statistically analyzed. Analysis of the ratio of 25nm to 100nm nanoscale bars observed in each cell type revealed that 25 / 100 ≥ 1 indicates a narrow distribution spacing of the cell membrane 5-cell surface receptor protein EGFR, which tends to be detected more frequently on the 25nm nanoscale bar, suggesting a higher probability of EGFR gene mutation. Conversely, 25 / 100 < 1 indicates a wide distribution spacing of the cell membrane 5-cell surface receptor protein EGFR, which tends to be detected more frequently on the 100nm DNA nanoscale bar, suggesting a higher probability of wild-type EGFR.

[0087] The second step is to identify the clinical samples according to the above standards:

[0088] Clinical samples were cut into small pieces in 10 cm culture dishes. Enzyme solution (1 ml type II collagenase, 500 μL type II protease, 20 μL primoin, 3.33 μL Y-27632 dissolved in 8.5 ml PBS) was added and the cells were lysed and digested at 37°C for 1 h to obtain a separated cell suspension. Digestion was terminated with 2 ml of serum. The resulting cell suspension was filtered through a 70 μm filter and centrifuged at 300 g for 5 min. To remove red blood cells, 1x red blood cell lysis buffer was added to the purified cell suspension, and the suspension was centrifuged at room temperature for 5 min at 300 g. The cell suspension was evenly distributed into different 1.5 ml centrifuge tubes for further experiments. Cell suspensions isolated from clinical lung cancer tissue were processed using the same procedure. 50 μl of each processed cell sample was placed in a confocal culture dish. Confocal microscopy was used for confocal imaging tomography. All images were analyzed using ImageJ software, and the fluorescence signal intensity was statistically analyzed. Calculate the ratio of the fluorescence signal intensity measured after treating cells with a 25 nm spacing nanoscale to that with a 100 nm spacing nanoscale. If 25 / 100 ≥ 1, the distribution spacing of EGFR receptor proteins on the cell membrane surface is narrow, tending to be detected more by the 25 nm spacing nanoscale, indicating a higher probability of EGFR gene mutation; conversely, if 25 / 100 < 1, the distribution spacing of EGFR receptor proteins on the cell membrane surface is wide, tending to be detected more by the 100 nm spacing nanoscale, indicating a higher probability of wild-type EGFR.

[0089] Optionally, when the ratio of the 25nm spacing nanoscale bar to the 100nm spacing nanoscale bar is greater than or equal to 1, the cell expresses the EGFR mutant genotype; when the ratio is less than 1, the cell expresses wild-type EGFR.

[0090] The DNA-based origami nanoscale ruler and its applications provided by this invention have the following technical effects:

[0091] (1) Visual analysis of the distribution spacing of proteins on the cell membrane surface was achieved, enabling the determination of the possibility of EGFR mutation from the image results. Specifically, by analyzing the intensity of the fluorescence signal of the nanoscale markers aggregated on the cell surface, the detection results were visualized through a fluorescence microscope, and combined with statistical analysis, the presence of EGFR mutation was quantitatively determined, resulting in relatively accurate detection results.

[0092] (2) It saves time in determining the presence of EGFR mutations, enabling rapid identification. Especially for wild-type patients, who cannot be assisted in drug selection, waiting for the expensive and lengthy sequencing process is unnecessary. Pre-analysis using this invention before sequencing to determine the presence of EGFR mutations can facilitate faster drug treatment for wild-type patients.

[0093] (3) Rapid sample processing is achieved, improving detection accuracy. When detecting cells using a nanoscale, cell lysis is unnecessary, and there are no special requirements regarding sample concentration, simplifying the operation. Furthermore, this invention analyzes the intensity of fluorescence signals for individual cells, further enhancing detection accuracy.

[0094] In summary, this invention utilizes a nanoscale ruler formed using DNA origami technology to achieve the visual identification of EGFR mutations in tissue samples. It provides a method different from existing publicly available techniques for identifying the presence of EGFR mutations in medical diagnostics, enabling more efficient, accurate, and rapid identification of EGFR mutations in tissue samples.

[0095] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of the present invention, and the present invention is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A nanoscale ruler based on DNA origami, characterized in that, include: The substrate structure is a three-layer rectangular structure constructed using DNA origami technology to maintain the basic configuration of the nanoscale ruler. A fluorescent indicator chain, wherein a plurality of the fluorescent indicator chains are distributed on a first surface of the substrate structure according to a preset rule; the fluorescent indicator chain includes a first nucleic acid chain and a fluorescent group; one end of the first nucleic acid chain is connected to the first surface of the substrate structure, and the other end is connected to the fluorescent group; the fluorescent indicator chain is used to emit a fluorescent signal through the fluorescent group under specific excitation light irradiation; A spacing indicator chain, wherein the spacing indicator chain is disposed on the first surface of the base structure and a preset distance is formed between two spacing indicator chains; The spacing indicator chain includes a second nucleic acid chain and a ligand protein. One end of the second nucleic acid chain is connected to the first surface of the substrate structure, and the other end is connected to the ligand protein. The spacing indicator chain is used to target and recognize and bind to a receptor protein on the cell membrane through the ligand protein. The ligand protein is epidermal growth factor (EGF), and the receptor protein is EGFR. By adjusting the preset distance between the two spacing indicator chains, a nanoscale bar with a preset distance of 25 nm and a nanoscale bar with a preset distance of 100 nm are formed. By incubating cell groups with a first nanoscale and a second nanoscale, respectively, and analyzing the intensity of fluorescence signals from different nanoscales aggregated on the cell surface, it was determined whether EGFR was mutated.

2. The DNA-based origami-based nanoscale ruler according to claim 1, characterized in that, The substrate structure includes a long single-stranded DNA and multiple fixed short strands, which form a three-layer rectangular structure through complementary base pairing.

3. The DNA-based origami-based nanoscale ruler according to claim 1, characterized in that, The three-layer rectangular structure has a length of 100nm, a width of 17nm, and a height of 20nm; and the first surface of the substrate structure has a length of 100nm and a width of 17nm.

4. The DNA-based origami-based nanoscale ruler according to claim 1, characterized in that, The first nucleic acid chain is complementary to and binds to the fluorescently modified nucleic acid chain to form the fluorescent indicator chain.

5. The DNA-based origami-based nanoscale ruler according to claim 1, characterized in that, The epidermal growth factor (EGF) is linked to sulfosuccinyl 4-cyclohexane-1-carboxylate, and then linked to a nucleic acid chain to synthesize a ligand protein linked to a nucleic acid chain. The second nucleic acid chain and the ligand protein are linked together through complementary base pairing to form the spacing indicator chain.

6. The DNA-based origami-based nanoscale ruler according to claim 1, characterized in that, The second nucleic acid chain can be extended by increasing the number of bases.

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