A molecular dynamometer for analyzing the interaction between membrane proteins and membranes and its preparation method

Abstract

The present invention relates to a molecular dynamometer for analyzing the interaction between cell surface membrane proteins and cell membranes. It is a hollow silica microsphere modified with a phospholipid bilayer, and the surface is embedded with DNA1 modified with an alkane chain and an azide at both ends. The detection scheme first uses dibenzocyclooctyne, diazine and fluorescent dye FAM-labeled DNA2 to identify the membrane protein to be detected on the cell surface, and ultraviolet light causes the diazine to covalently link with the corresponding membrane protein. Then, buoyancy is used to make the cell and the molecular dynamometer contact and then separate. The competition between the alkane chain-phospholipid bilayer interaction and the membrane protein-cell membrane interaction will pull the membrane protein out of the cell membrane. Finally, fluorescence imaging is performed on cells treated with molecular dynamometers made of different alkane chain lengths to determine the alkane chain length on the molecular dynamometer used before and after the decrease in cell surface fluorescence intensity. The interaction force scale obtained by optical tweezers is substituted into the interaction force scale to achieve quantification of the interaction between membrane proteins and cell membranes.

Description

1. Technical Field

[0001] The invention relates to a molecular dynamometer for analyzing the interaction between membrane protein and cell membrane and a preparation method thereof. 2. Background Technology

[0002] The cell membrane is a supramolecular complex composed of amphipathic lipids and proteins. Membrane proteins, as key components of the cell membrane, participate in processes such as cell proliferation, growth, and signal transduction. Cells can modulate the interactions between membrane proteins and the lipids that form the cell membrane, altering their activity, aggregation, signal transduction, and other functions. Measuring the force required to pull a membrane protein out of the cell membrane can be used to characterize the interaction between membrane proteins and cell membranes, deepen our understanding of the mechanisms and functions of these interactions, and promote the exploration of related life processes and physiological mechanisms.

[0003] In order to analyze the interaction between membrane proteins and cell membranes, this patent prepared a molecular dynamometer based on hollow silica microspheres and established a visual analysis method for the interaction level between membrane proteins and cell membranes. 3. Summary of the Invention

[0004] The purpose of the present invention is to label DNA with an alkane chain of a specific length and azide (N3) to obtain DNA1, and then adsorb DNA1 onto a phospholipid monolayer on a hollow silica microsphere through the hydrophobic interaction between the alkane chain and the phospholipid, and finally hydrophobically self-assemble onto a second layer of phospholipid molecules to form a structure in which DNA1 is embedded in the phospholipid bilayer, thereby preparing a molecular force gauge ( Figure 1 The detection scheme first uses dibenzocyclooctyne (DBCO), diazine groups and the dye FAM to label the nucleic acid aptamer in DNA2 to recognize the membrane protein on the cell surface, and then covalently links the diazine group at the end of DNA2 to the corresponding membrane protein under ultraviolet light ( Figure 2 A), the molecular dynamometer was further covalently linked to the DNA2 of the membrane protein using the click chemistry reaction between azide and DBCO ( Figure 2 B) The buoyancy of the molecular force gauge stimulates the competition between the alkane chain-phospholipid bilayer interaction and the membrane protein-cell membrane interaction. By changing the length of the alkane chain, the strength of the alkane chain-phospholipid bilayer interaction is adjusted, and the membrane protein is pulled out of the cell membrane ( Figure 2 C), thereby measuring the interaction force between membrane proteins and cell membranes using the interaction force scale obtained by optical tweezers. Using the MUC1 mucin on the surface of the MCF-7 breast cancer cell line as a model, the prepared probe enables quantitative analysis of the interaction force between MUC1 mucin and cell membranes.

[0005] The present invention is achieved through the following technical solutions:

[0006] Hydroxylated hollow silica microspheres were used as carriers, and a layer of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE phospholipid) was covalently modified after epoxy silanization. Alkane chains of specific lengths and azide-labeled DNA1 were adsorbed on the phospholipid layer through hydrophobic interactions, and then a second layer of DSPE phospholipid was hydrophobically self-assembled to produce a molecular force gauge ( Figure 1 By varying the length of the alkane chain, a series of molecular dynamometers with different hydrophobic interaction forces can be obtained.

[0007] Working principle of the present invention:

[0008] The preparation process of the molecular dynamometer proposed by the present invention for quantitative analysis of the interaction between membrane proteins and cell membranes is as follows: Figure 1 As shown in the figure, hydroxylated hollow silica microspheres were used as carriers. After epoxy silanization, a layer of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) was covalently modified. DNA1, modified with an alkane chain of specific length and an azide at each end, was adsorbed onto the DSPE phospholipid layer through hydrophobic interactions. Finally, the hydrophobic self-assembly of a second DSPE phospholipid layer formed a structure where DNA1 was embedded in the phospholipid bilayer, thus creating a molecular force gauge.

[0009] The working principle of the present invention is as follows Figure 2 As shown. The detection process firstly inoculates cells into a confocal dish for culture. The working principle of the present invention is as follows Figure 2 As shown. The detection process first inoculates cells in a confocal dish and cultures them. The aptamer in DNA2, which is labeled with a diazine group at one end and dibenzocyclooctyne (DBCO) at the other end, and with a dye FAM in the middle sequence, recognizes the membrane protein on the cell surface. Then, the diazine group modified at the end of the aptamer is covalently linked to the corresponding membrane protein under ultraviolet light ( Figure 2 A). Place the molecular dynamometer in the confocal dish and invert the dish. The molecular dynamometer will contact the cells through buoyancy, causing the DBCO groups and azide groups on the cells to undergo a click chemistry reaction, covalently binding them together ( Figure 2 B) The confocal dish is then returned to the right position, and the competition between the alkane chain-phospholipid bilayer interaction and the membrane protein-cell membrane interaction is stimulated by the buoyancy of the molecular force gauge, and the molecular force gauge is separated from the cell ( Figure 2C). The length of the alkane chain on the molecular force gauge affects the hydrophobic interaction between the alkane chain and the phospholipid bilayer. Longer alkane chains indicate stronger hydrophobic interactions. When the alkane chain-phospholipid bilayer interaction is weaker than the membrane protein-cell membrane interaction, the target protein will remain on the cell membrane; conversely, when the alkane chain-phospholipid bilayer interaction is weaker than the membrane protein-cell membrane interaction, the target protein will be pulled out of the cell membrane. Finally, fluorescence microscopy is used to monitor the fluorescence of cells treated with different molecular force gauges. The length of the alkane chain on the molecular force gauge used before and after the decrease in cell surface fluorescence intensity is determined. By comparing this with the interaction force scale obtained with optical tweezers, the interaction force between the membrane protein and the cell membrane can be quantified.

[0010] Compared with the prior art, the present invention has the following characteristics:

[0011] The silica microspheres used in this invention are close in size to cells, capable of rapid buoyancy separation and easy modification. The resulting molecular force gauge can efficiently and quickly interact with and separate from cells, enabling the measurement of the interaction force between target cell surface membrane proteins and the cell membrane.

[0012] Compared with existing methods for analyzing the interaction between membrane proteins and cell membranes, the present invention has the following advantages:

[0013] 1. The molecular dynamometer of the present invention is simple to prepare and can quantitatively analyze and calculate the interaction strength between specific membrane proteins and cell membranes on a large number of cells in a short period of time.

[0014] 2. The fluorescent signal of the present invention can indicate the amount of specific membrane proteins on the cell surface, making the analysis results visual. IV. Description of the Figures

[0015] Figure 1 Schematic diagram of molecular dynamometer preparation

[0016] Figure 2 .Using molecular force gauge to analyze the interaction between membrane proteins and cell membranes V. Specific Implementation Methods

[0017] Example 1: Combination Figure 1 , preparation and synthesis of molecular force gauges for analyzing the interaction between membrane proteins and cell membranes

[0018] Hollow silica microspheres (0.01 g) were first soaked in 5M sulfuric acid on a vertically rotating stirrer at room temperature overnight. The resulting hydroxylated silica microspheres were washed and dried, then added to 1 mL of dichloromethane containing 5% epoxy silanization reagent and stirred overnight to obtain epoxy-silanized silica microspheres. After washing three times with anhydrous ethanol, the microspheres were dispersed in an aqueous solution containing 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE phospholipid) (1 mg / mL) and sodium dodecyl sulfate (10 mg / mL). After stirring at room temperature for 6 hours, the microspheres were washed twice with water, once with anhydrous ethanol, and dried. Next, 2 μL of a 100 μM aqueous solution of DNA1, modified with an alkane chain of a specific length and an azide at each end, was added and thoroughly mixed. The microspheres were separated from the liquid by buoyancy and then dried. Finally, the obtained microspheres were dispersed in a decane / isobutanol (10:1, v / v) solution containing 1 mg / mL DSPE phospholipid. The microspheres and the liquid were separated and then dried and washed twice with water to obtain a molecular dynamometer.

[0019] Example 2: Combination Figure 2 , using molecular force gauge to detect the interaction force between membrane proteins and cell membranes

[0020] MCF-7 cells were seeded in a 10 mm single-well confocal dish and incubated overnight. PBS buffer containing 10% goat serum was added and blocked at 4°C for 30 minutes, and then washed three times with PBS buffer. 100 μL of 1 μM DNA2 with one end modified with DBCO, the other end modified with a diazine group of the aptamer sequence, and an intermediate marker dye FAM was added and incubated at 37°C for 30 minutes. DNA2 recognized the target protein through the aptamer sequence. After washing the MCF-7 cells with PBS buffer, the cells were irradiated with 365 nm ultraviolet light for 15 minutes. DNA2 was covalently linked to the target protein through the diazine group and ( Figure 2 A). Different types of molecular dynamometers (approximately 2.2×10 5 The confocal dish was placed upside down in a 37°C incubator and repeatedly flipped over to allow the molecular dynamometer to contact the cells using buoyancy. The cells were covalently linked to the molecular dynamometer through a click chemistry reaction between DBCO and azide. Figure 2B) The confocal dish is then straightened, and the buoyancy of the molecular force gauge is used to stimulate competition between the alkane chain-phospholipid bilayer interaction and the membrane protein-cell membrane interaction, separating the molecular force gauge from the cells. Finally, the cells are washed with PBS buffer, and fluorescence imaging of the cells treated with different molecular force gauges is performed using a fluorescence microscope. The fluorescence intensity of FAM on the cell surface is measured to determine the length of the alkane chain on the molecular force gauge used before and after the decrease in cell surface fluorescence intensity. As the hydrophobic interaction force provided by the different molecular force gauges gradually increases, the target protein is pulled out of the cell membrane, causing a sudden decrease in cell surface fluorescence intensity. At this point, it can be assumed that the interaction force between the membrane protein and the cell membrane is slightly weaker than the hydrophobic interaction force provided by the added molecular force gauge. By comparing this with the interaction force scale obtained by optical tweezers, the interaction force between the membrane protein and the cell membrane can be quantified.

Claims

1. A molecular dynamometer for analyzing the interaction force between membrane proteins and membranes, characterized in that: The molecular force gauge uses hydroxylated hollow silica microspheres as carriers, first assembling a layer of phospholipid molecules on the surface of the microspheres, then utilizing hydrophobic interactions to adsorb DNA1 modified with alkane chains and azide groups of specific lengths at both ends onto the phospholipid monolayer, and finally hydrophobically self-assembling a second layer of phospholipid molecules to form a DNA1-embedded phospholipid bilayer structure, thereby obtaining the molecular force gauge. The first assembling layer of phospholipid molecules refers to covalently modifying a layer of 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine after epoxy silanization on the carrier surface; the second layer of phospholipid molecules assembled on the hydrophobic self-assembly refers to assembling another layer of 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine through hydrophobic interactions; and the embedding of DNA1 is achieved by utilizing the hydrophobic interactions between the alkane chains at the ends of DNA1 and the phospholipid bilayer.

2. The method for detecting the interaction force between membrane protein and membrane using a molecular dynamometer according to claim 1, characterized in that The detection method first uses a nucleic acid aptamer in DNA2 labeled with dibenzocyclooctyne, a diazine group, and the dye FAM to identify a membrane protein to be detected on the cell surface, then covalently links the diazine group modified at the end of DNA2 to the corresponding membrane protein under ultraviolet light, further utilizes a click chemistry reaction between azide and dibenzocyclooctyne to covalently link a molecular force gauge to the DNA2 covalently linked to the membrane protein, then utilizes the buoyancy of the molecular force gauge to stimulate competition between the alkane chain-phospholipid bilayer interaction and the membrane protein-cell membrane interaction, and by changing the length of the alkane chain, adjusts the strength of the alkane chain-phospholipid bilayer interaction, causing the membrane protein to be pulled out of the cell membrane; after the membrane protein to be detected is pulled out of the cell membrane, fluorescence imaging is performed on cells treated with different molecular force gauges to determine the length of the alkane chain on the molecular force gauge used before and after the decrease in cell surface fluorescence intensity, and compares it with the interaction force scale obtained by optical tweezers to achieve quantification of the interaction force between the membrane protein and the cell membrane.

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