Preparation and application of membrane tension response type molecular sensor

Abstract

The invention relates to the technical field of biological nanotechnologies, and provides a preparation and application method of a membrane tension response type molecular sensor, the membrane tension response type molecular sensor comprises a nucleic acid frame structure and vibration-induced emission molecules, and the vibration-induced emission molecules are linked to the nucleic acid frame structure; the nucleic acid frame structure is hydrophilic, and the vibration-induced emission molecules are hydrophobic. The method has the advantages of high cell membrane targeting property, mechanical sensitivity and the like, and a new tool is provided for cell membrane tension detection.

Description

Technical Field

[0001] The invention belongs to the field of bio-nanotechnology, and in particular relates to a membrane tension responsive molecular sensor and a preparation and application method thereof. Background Art

[0002] Cells and organelles are enclosed in lipid membranes, and the mechanical properties of these membranes play crucial roles in various cellular activities, such as cell division, endocytosis, and cell migration. Among these properties, membrane tension is a major regulator of membrane deformation and plays an important role in regulating many cellular activities. For example, high membrane tension can effectively inhibit the metastasis and invasion of cancer cells by counteracting the curvature sensing / production of BAR family proteins. Traditional membrane tension measurement methods rely on traction force measurement techniques, which use optical tweezers or micropipette aspiration techniques to extract small membrane tubes from the lipid bilayer and measure their restoring forces. These techniques are limited by low spatiotemporal resolution and small test range, making it difficult to simultaneously detect membrane tension in a large field of view (e.g., the entire cell or multiple cells). Therefore, it is urgent to develop a visual, highly cell membrane-targeted and mechanically sensitive technology to monitor cell membrane tension. Summary of the invention

[0003] The purpose of the present invention is to provide a preparation and application method of a membrane tension-responsive molecular sensor, which has the advantages of high cell membrane targeting and mechanical sensitivity, and provides a new tool for cell membrane tension detection.

[0004] In a first aspect of the present invention, a membrane tension responsive molecular sensor is provided, comprising a nucleic acid framework structure and a vibration induced luminescent molecule, the vibration induced luminescent molecule is also called a VIE molecule, wherein the VIE molecule is linked to the nucleic acid framework structure;

[0005] Furthermore, the nucleic acid framework structure is hydrophilic, while the VIE molecule is hydrophobic.

[0006] In another preferred embodiment, the nucleic acid framework structure is formed by self-assembly of hydrophilic nucleic acid monomers.

[0007] In another preferred embodiment, the self-assembly is performed by complementary pairing of multiple nucleic acid monomers.

[0008] In another preferred embodiment, the nucleic acid monomer includes a nucleic acid monomer linked to the VIE molecule.

[0009] In another preferred embodiment, when the excitation light is irradiated, the VIE molecule is in an excited state, and its conformation is in the following conformations or undergoes conformational transitions in sequence: bent conformation, planar conformation and twisted conformation, and returns to the ground state by emitting fluorescence.

[0010] In another preferred embodiment, when the membrane tension changes, the degree to which the above-mentioned vibration-induced luminescent molecular conformational change process is suppressed also changes, ultimately leading to different fluorescence spectra.

[0011] In another preferred embodiment, the molecular sensor has one or more characteristics selected from the following group:

[0012] (a) The molecular sensor is fixed to or enriched in the cell membrane;

[0013] (b) The molecular sensor has a high cell membrane / intracellular distribution ratio R;

[0014] (c) The fluorescence emission wavelength of the molecular sensor is between 450nm and 700nm;

[0015] (d) The fluorescence emission wavelength B1 of the molecular sensor is different from the fluorescence emission wavelength B2 of the VIE molecule alone.

[0016] In another preferred embodiment, the distribution ratio R is the ratio C1 / C2 of the number of molecular sensors C1 located at the cell membrane to the number C2 of molecular sensors located inside the cell.

[0017] In another preferred embodiment, the distribution ratio R is the ratio Q1 / Q2 of the fluorescence signal intensity Q1 emitted by the molecular sensor located at the cell membrane to the fluorescence signal intensity Q2 emitted by the molecular sensor located inside the cell.

[0018] In another preferred embodiment, the distribution ratio R is ≥1, preferably ≥2, more preferably ≥3, such as 1.5-1.0 or 2-5.

[0019] In a preferred embodiment, it includes:

[0020] The nucleic acid monomer ssDNA-VIE linked to the VIE molecule has a structure of Formula I:

[0021] Z1-L-Z2 (I)

[0022] In the formula,

[0023] Z1 is a VIE molecule;

[0024] Z2 is a nucleic acid monomer;

[0025] L is a linking group located between Z1 and Z2.

[0026] In another preferred embodiment, the nucleic acid includes DNA, RNA, or a combination thereof.

[0027] In another preferred embodiment, the structure of formula I is formed by a click reaction between (i) an alkynyl- or alkyl-modified VIE molecule and (ii) an azide-modified single-stranded nucleic acid.

[0028] In another preferred embodiment, the alkynyl or alkyl modified VIE molecule has the following structure:

[0029]

[0030] In another preferred embodiment, L is -L1-L2-L3-, wherein L1 is -phenyl-O-; L2 is a C3-C7 substituted or unsubstituted alkylene group; and L3 is a linking group formed by a click reaction, such as

[0031] In another preferred embodiment, the L2 is a C3-C7 alkylene group.

[0032] In another preferred embodiment, the nucleic acid framework structure comprises at least one nucleic acid monomer of formula I.

[0033] In another preferred embodiment, the nucleic acid framework structure comprises n nucleic acid monomers of formula I, wherein n is a positive integer ≥1.

[0034] In another preferred embodiment, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0035] In another preferred embodiment, one nucleic acid framework structure links m VIE molecules, wherein m is a positive integer ≥1.

[0036] In another preferred embodiment, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0037] In a preferred embodiment, it includes:

[0038] The VIE molecule is located at a predetermined position in the nucleic acid framework structure.

[0039] In another preferred embodiment, a plurality of VIE molecules are dispersed at predetermined positions in the nucleic acid framework structure.

[0040] In another preferred embodiment, the VIE molecule is located at a fixed position in the nucleic acid framework structure.

[0041] In another preferred embodiment, the nucleic acid framework structure and the VIE molecule are covalently linked.

[0042] In another preferred embodiment, the nucleic acid framework structure controls the valence state and spatial position of the VIE molecule.

[0043] In another preferred embodiment, the molecular sensor is anchored or bound to the outer surface of the membrane through the VIE molecule.

[0044] In another preferred embodiment, one nucleic acid framework structure links m VIE molecules, wherein m is a positive integer ≥1.

[0045] In another preferred embodiment, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0046] In another preferred embodiment, the membrane in the membrane tension responsive type comprises a model membrane formed by extrusion or electrostimulation of phospholipids.

[0047] In another preferred embodiment, the membrane comprises a cell membrane of a living cell.

[0048] In a preferred embodiment, it includes:

[0049] The nucleic acid framework structure is a planar structure or a polyhedral structure.

[0050] In another preferred embodiment, the polyhedral structure includes: a tetrahedral structure, a hexahedral structure, and an octahedral structure.

[0051] In another preferred embodiment, the nucleic acid framework structure is rigid or substantially rigid.

[0052] In another preferred embodiment, the side length of the polyhedron is in the range of 5-6.5 nm.

[0053] In another preferred embodiment, the side length of the polyhedron ranges from 10 to 20 bp or nt.

[0054] In a preferred example, the nucleic acid nanoshell of the nucleic acid framework structure is a hollow tetrahedral structure, and the nucleic acid framework structure is prepared and composed of four complementary ssDNA chains.

[0055] In another preferred embodiment, the four complementary ssDNA chains include at least one nucleic acid monomer of the structure of Formula I.

[0056] In another preferred embodiment, one of the tetrahedral nucleic acid framework structures links one to three of the VIE molecules.

[0057] The second aspect of the present invention provides a method for preparing the above-mentioned molecular sensor, comprising:

[0058] (a) chemically linking the VIE molecule with ssDNA to form a nucleic acid monomer ssDNA-VIE linked to the VIE molecule as shown in Formula I:

[0059] Z1-L-Z2 (I)

[0060] In the formula,

[0061] Z1 is a VIE molecule;

[0062] Z2 is a nucleic acid monomer;

[0063] L is a linking group located between Z1 and Z2.

[0064] (b) The nucleic acid monomers represented by formula I described above form a nucleic acid framework structure through self-assembly, thereby forming the molecular sensor described in claim 1.

[0065] In another preferred embodiment, in step (b), the nucleic acid monomers to be self-assembled include: (i) the nucleic acid monomers shown in Formula I, and (ii) optionally one or more nucleic acid monomers not coupled to a VIE molecule.

[0066] (c) Heating and annealing are performed, wherein the number of VIE molecules is controlled by changing the relative ratio of ssDNA-VIE and unmodified ssDNA.

[0067] In a preferred embodiment, it includes:

[0068] The VIE molecule is chemically linked to the ssDNA via a copper-catalyzed click reaction;

[0069] In another preferred embodiment, a plurality of complementary ssDNA chains are heated and annealed by a PCR instrument to prepare a nucleic acid framework structure.

[0070] The third aspect of the present invention provides a use of the above-mentioned molecular sensor, which includes: being used to prepare a reagent for measuring cell membrane tension.

[0071] A fourth aspect of the present invention provides a method for measuring cell membrane tension, the method comprising:

[0072] (a) mixing the molecular sensor according to claim 1 and a cell membrane;

[0073] (b) observing the fluorescent signal emitted by the molecular sensor according to claim 1.

[0074] In another preferred embodiment, the incubation time at room temperature is 20 min.

[0075] In another preferred embodiment, a confocal scanning microscope is used to perform fluorescence imaging of the cell membrane.

[0076] In another preferred embodiment, the method further comprises:

[0077] (c) The membrane is monitored based on the change in fluorescence signal, thereby determining the tension of the cell membrane.

[0078] Compared with the prior art, the main differences and effects of the embodiments of the present invention are:

[0079] The invention solves the problems that the existing membrane tension probe has poor water solubility, is easily affected by the probe concentration, and has poor cell membrane targeting.

[0080] Furthermore, it has the advantages of efficient cell membrane targeting, non-concentration dependence, ordered differentiation of model membrane phospholipids, cell membrane tension responsiveness, good biocompatibility, etc. Compared with existing nano fluorescent probes, it has significant advantages and provides a new tool for biological imaging.

[0081] Furthermore, fluorescence ratio imaging of cell membrane tension was achieved, revealing the great potential of this multifunctional molecular sensor in living cell applications.

[0082] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Schematic diagram of the structure and working mechanism of the molecular sensor used for membrane tension response analysis.

[0084] Figure 1 (a) shows the illustration of TDF as a structural scaffold and VIE as a fluorescent molecular reporter.

[0085] Figure 1 (b) in the figure shows a schematic diagram of the VIE action mechanism.

[0086] Figure 1 (c) shows the fluorescence emission spectra of VIE molecules dissolved in solutions of different viscosities.

[0087] Figure 2 This is a schematic diagram of the construction process of molecular sensors.

[0088] Figure 2 (a) shows the chemical reaction process of ssDNA-VIE.

[0089] Figure 2 (b) shows the composition of the TDF-VIE3 molecular sensor.

[0090] Figure 2 (c) in the figure shows the composition of three molecular sensors.

[0091] Figure 3 This is the construction and characterization result of ssDNA-VIE.

[0092] Figure 3(a) shows a high performance liquid chromatogram.

[0093] Figure 3 (b) shows the polyacrylamide gel electrophoresis image.

[0094] Figure 3 (c) shows the absorption spectra of ssDNA-VIE in solutions with different acetonitrile ratios.

[0095] Figure 3 (d) shows the fluorescence emission spectra of ssDNA-VIE in solutions with different acetonitrile ratios.

[0096] Figure 4 This is the HPLC result of the molecular sensor.

[0097] Figure 5 This is the polyacrylamide gel electrophoresis characterization result of the molecular sensor.

[0098] Figure 6 These are the atomic force microscopy characterization results of the molecular sensor.

[0099] Figure 7 It is a structure that characterizes the spectroscopic properties of molecular sensors.

[0100] Figure 7 (a) in FIG. 1 shows an absorption spectrum.

[0101] Figure 7 (b) shows the fluorescence emission spectrum.

[0102] Figure 7 (c) shows the time-resolved fluorescence decay spectrum.

[0103] Figure 8 This is the confocal laser scanning microscopy result of the model membrane after molecular sensor treatment.

[0104] Fig. 9 It is the fluorescence spectrum result of molecular sensor applied to distinguish the lipid order of model membrane.

[0105] Fig.10 It is the fluorescence imaging and statistical results of molecular sensors applied to cell membrane targeting.

[0106] Fig.11 It is the fluorescence imaging result of molecular sensors used to monitor changes in cell membrane tension. DETAILED DESCRIPTION

[0107] After extensive and in-depth research and a large number of screenings, the inventors have developed for the first time a method for preparing and applying a membrane tension-responsive molecular sensor. The present invention constructs an "atomic-level" precise amphiphilic DNA framework molecular sensor through DNA nanotechnology. Based on the DNA framework molecular sensor, the valence state and spatial position of VIE are controlled, thereby achieving the construction of a molecular sensor with high cell membrane targeting, membrane tension responsiveness and non-concentration dependence, and on this basis, the present invention is completed.

[0108] The terms used herein, the molecular sensor of the present invention, the membrane tension responsive sensor of the present invention, the sensor of the present invention, the mechanical sensitive molecular assembly of the present invention can be used interchangeably. The vibration induced luminescence molecule of the present invention, the VIE molecule of the present invention, and the VIE probe of the present invention can be used interchangeably.

[0109] Molecular Sensors

[0110] The present invention provides a membrane tension responsive molecular sensor, or a mechanosensitive molecular assembly, such as Figure 1 As shown in (a), it includes a nucleic acid framework structure and a vibration-induced luminescent molecule, the vibration-induced luminescent molecule is also called a VIE molecule, wherein the VIE molecule is linked to the nucleic acid framework structure;

[0111] Furthermore, the nucleic acid framework structure is hydrophilic, while the VIE molecule is hydrophobic.

[0112] In another embodiment, the nucleic acid framework structure is formed by self-assembly of hydrophilic nucleic acid monomers.

[0113] In another embodiment, self-assembly occurs through complementary pairing of multiple nucleic acid monomers.

[0114] In another embodiment, the nucleic acid monomer includes a nucleic acid monomer linked to a VIE molecule.

[0115] In another embodiment, if Figure 1 As shown in (b), when the excitation light is irradiated, the VIE molecule is in an excited state, and its conformation will be in the following conformations or undergo conformational transitions in sequence: bent conformation, planar conformation and twisted conformation, and return to the ground state by emitting fluorescence.

[0116] In another embodiment, when the membrane tension changes, the degree to which the above-mentioned vibration-induced luminescent molecular conformational change process is suppressed also changes, ultimately resulting in different fluorescence spectra.

[0117] More specifically, the mechanism of VIE molecules as indicators of cell membrane tension is that when the cell membrane tension is high, the lipid packing density is low, and the VIE molecules can undergo a complete allosteric process. The excited state of VIE changes from the vertically transmitted Franck-Condon state (R*, initial bending state) to the first intermediate emission state (I1*, bending state), and then to the second intermediate emission state (I2*, planar state), and relaxes to the final product state (P*, twisted state), and the red emission wavelength is 620nm (R*→I1*→I2*→P*, Figure 1 In contrast, under lower membrane tension, the transition of VIE from the I2* excited state to the P* excited state in ES is suppressed due to the steric hindrance of high lipid density, resulting in the R*→I1*→I2* at short wavelengths (460 and 510 nm, Figure 1 (b) in the figure has strong fluorescence emission. Therefore, in the applied method, the change in the fluorescence ratio of the VIE molecule between 620nm and 510nm can indicate the change in cell membrane tension.

[0118] In another embodiment, the molecular sensor has one or more properties selected from the group consisting of:

[0119] (a) Molecular sensors are fixed or enriched in the cell membrane;

[0120] (b) The molecular sensor has a high cell membrane / intracellular distribution ratio R;

[0121] (c) Figure 1 As shown in (c), the fluorescence emission wavelength of the molecular sensor is 450nm-700nm;

[0122] (d) The fluorescence emission wavelength B1 of the molecular sensor is different from the fluorescence emission wavelength B2 of the VIE molecule alone.

[0123] More specifically, the molecular sensor emits fluorescence, which is excited by a 405nm laser and has a receiving wavelength of 450nm-700nm.

[0124] In another embodiment, the distribution ratio R is a ratio C1 / C2 of the number of molecular sensors C1 located at the cell membrane to the number of molecular sensors C2 located inside the cell.

[0125] In another embodiment, the distribution ratio R is a ratio Q1 / Q2 of the fluorescence signal intensity Q1 emitted by the molecular sensor located at the cell membrane to the fluorescence signal intensity Q2 emitted by the molecular sensor located inside the cell.

[0126] In another embodiment, the distribution ratio R is ≥1, preferably ≥2, more preferably ≥3, such as 1.5-10 or 2-5.

[0127] Nucleic acid monomer ssDNA-VIE

[0128] In one embodiment, the nucleic acid monomer ssDNA-VIE linked to the VIE molecule has the structure of Formula I:

[0129] Z1-L-Z2 (I)

[0130] In the formula,

[0131] Z1 is a VIE molecule;

[0132] Z2 is a nucleic acid monomer;

[0133] L is a linking group located between Z1 and Z2.

[0134] In another embodiment, the nucleic acid comprises DNA, RNA, or a combination thereof.

[0135] In another embodiment, the structure of Formula I is formed by (i) an alkynyl or alkyl modified VIE molecule and (ii) an azide group modified single strand of nucleic acid through a click reaction, and the reaction process is as follows: Figure 2 As shown in (a) in .

[0136] In another embodiment, the alkynyl or alkyl modified VIE molecule has the following structure:

[0137]

[0138] In another embodiment, L is -L1-L2-L3-, wherein L1 is -phenyl-O-; L2 is a C3-C7 substituted or unsubstituted alkylene; L3 is a linking group formed by a click reaction, such as

[0139] In another embodiment, L2 is C3-C7 alkylene.

[0140] In another embodiment, the nucleic acid framework structure comprises at least one nucleic acid monomer of the structure of Formula I.

[0141] In another embodiment, the nucleic acid framework structure comprises n nucleic acid monomers of formula I, wherein n is a positive integer ≥1.

[0142] In another embodiment, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0143] Positional relationship between VIE molecules and nucleic acid framework

[0144] In one embodiment, the VIE molecule is located at a predetermined position in the nucleic acid framework structure.

[0145] In another embodiment, a plurality of VIE molecules are dispersed at predetermined positions in the nucleic acid framework structure.

[0146] In another embodiment, the VIE molecule is located at a specific position in the nucleic acid framework structure.

[0147] In another embodiment, the nucleic acid framework structure and the VIE molecule are covalently linked.

[0148] In another embodiment, the nucleic acid framework structure controls the valency and spatial position of the VIE molecule.

[0149] In another embodiment, the molecular sensor is anchored or bound to the outer surface of the membrane via the VIE molecule.

[0150] In another embodiment, one nucleic acid framework structure links m VIE molecules, wherein m is a positive integer ≥1.

[0151] In another embodiment, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0152] In another embodiment, the membrane in the membrane tension responsive type includes a model membrane formed by extrusion or electroporation of phospholipids.

[0153] In another embodiment, the membrane comprises a cell membrane of a living cell.

[0154] Nucleic acid framework

[0155] In one embodiment, the nucleic acid framework structure is a planar structure or a polyhedral structure.

[0156] The DNA framework is highly programmable and addressable, making it ideal for precise molecular arrangement and studying cooperative interactions of multivalent molecular components within molecular assemblies.

[0157] In another embodiment, the polyhedral structure includes: a tetrahedral structure, a hexahedral structure, and an octahedral structure.

[0158] In another embodiment, the nucleic acid framework structure is rigid or substantially rigid.

[0159] In another embodiment, the polyhedron side length is in the range of 5-6.5 nm.

[0160] In another embodiment, the polyhedron side length ranges from 10-20 bp or nt.

[0161] In one embodiment, the nucleic acid nanoshell of the nucleic acid framework structure is a hollow tetrahedral structure (TDF), and the nucleic acid framework structure is prepared and composed of four complementary ssDNA chains.

[0162] In another embodiment, the four complementary ssDNA strands include at least one nucleic acid monomer of the structure of Formula I.

[0163] In another embodiment, one tetrahedral nucleic acid framework structure links one to three VIE molecules.

[0164] Preparation method of molecular sensor

[0165] In one embodiment, a method for preparing the above-mentioned molecular sensor is provided, comprising:

[0166] (a) chemically linking the VIE molecule with ssDNA to form a nucleic acid monomer ssDNA-VIE linked to the VIE molecule as shown in Formula I:

[0167] Z1-L-Z2 (I)

[0168] In the formula,

[0169] Z1 is a VIE molecule;

[0170] Z2 is a nucleic acid monomer;

[0171] L is a linking group located between Z1 and Z2.

[0172] (b) The nucleic acid monomers shown in Formula I form a nucleic acid framework structure through self-assembly, thereby forming the molecular sensor of claim 1.

[0173] In another embodiment, in step (b), the nucleic acid monomers to be self-assembled include: (i) the nucleic acid monomers of Formula I, and (ii) optionally one or more nucleic acid monomers not coupled to a VIE molecule.

[0174] (c) Heating and annealing are performed, wherein the number of VIE molecules is controlled by changing the relative ratio of ssDNA-VIE and unmodified ssDNA.

[0175] In one embodiment, it includes:

[0176] The VIE molecules were chemically linked to ssDNA via a copper-catalyzed click reaction;

[0177] In another embodiment, a PCR instrument is used to heat and anneal multiple complementary ssDNA chains to prepare a nucleic acid framework structure.

[0178] In one embodiment, the method comprises the following steps:

[0179] (1) Preparation of VIE molecules. Alkyl-modified VIE molecules (alkynyl-VIE) were first synthesized. The successful synthesis of alkynyl-VIE was confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. The fluorescence emission spectra of alkynyl-VIE in RTM solutions of different viscosities (mineral oil rotational viscosity standard) were measured using an ELISA reader.

[0180] (2) Through copper-catalyzed click chemistry, alkynyl-VIE was chemically linked to single-stranded DNA (ssDNA) to form VIE-modified ssDNA (ssDNA-VIE, Figure 2 (a) in the above). CuBr (1 mg) was dissolved in 70 μL DMSO, and triphenyltriazolylamine (TBTA, 54 mg) was dissolved in 1 mL DMSO. In addition, sodium ascorbate (20 mg) was dissolved in 1 mL ultrapure water. Next, the present application mixed the CuBr and TBTA stock solutions in a volume ratio of 1:2 to form a ligand TBTA-Cu(I) solution. Then, the present application dissolved the alkynyl-VIE dye (0.2 mg, 0.13 μmol) in 90 μL DMSO and added an aqueous solution containing N3-DNA (50 μL, 10 OD). After brief stirring, 10 μL of the premixed ligand-Cu(I) solution was added to the reaction mixture. Subsequently, 50 μL of sodium ascorbate stock solution was added and shaken in a 40°C metal bath for 2 h. After the reaction was completed, the solvent was removed in vacuo, and the crude product was dissolved in ultrapure water and purified using high performance liquid chromatography. Finally, the present application uses lyophilization to remove the solvent, and the resulting product ssDNA-VIE is dissolved in ultrapure water and stored at 4°C for future use. The ssDNA-VIE molecules are then purified using high performance liquid chromatography, and the successful synthesis of ssDNA-VIE is verified by high performance liquid chromatography, polyacrylamide gel electrophoresis, and microplate reader spectral analysis. By measuring the absorbance and fluorescence emission spectra of ssDNA-VIE in solutions containing different proportions of acetonitrile, the present application verifies that the VIE probe maintains its triple emission properties when bound to ssDNA.

[0181] (3) The molecular sensor structure can be obtained by heating and annealing the four complementary ssDNA chains prepared by using a PCR instrument. By changing the relative ratio of ssDNA-VIE and unmodified ssDNA, the number of VIE molecules on the tetrahedral DNA framework can be quantitatively controlled ( Figure 2 (b) and (c)). These molecular sensors containing one, two, and three VIE molecules were named TDF-VIE1, ​​TDF-VIE2, and TDF-VIE3, respectively. The synthesized molecular combinations were purified by high-performance liquid chromatography, and the probes were characterized using polyacrylamide gel electrophoresis and atomic force microscopy.

[0182] Applications of Molecular Sensors

[0183] The invention also provides applications of the molecular sensor, especially applications for imaging the orderliness of model membrane phospholipids and monitoring changes in cell membrane tension.

[0184] In one embodiment, a method for distinguishing the ordered state of lipids in a model membrane based on the above-mentioned molecular sensor is provided, and the steps are as follows:

[0185] (a) The molecular sensor was mixed with the test model membrane and incubated at room temperature;

[0186] (b) using a microscope to perform fluorescence imaging on the test model membrane, and judging the targeting of the molecular sensor to the test model membrane based on the fluorescence imaging results;

[0187] (c) The fluorescence spectrum of the processed molecular sensor is measured using an ELISA instrument, and the ability of the molecular sensor to distinguish the ordered state of lipids in the test model membrane is determined by the fluorescence spectrum results.

[0188] In another embodiment, the incubation time at room temperature is 20 min.

[0189] In another embodiment, fluorescence imaging of cell membranes is performed using confocal scanning microscopy.

[0190] In one embodiment, a use of the above-mentioned molecular sensor is provided, and the use includes: being used for preparing a reagent for measuring cell membrane tension.

[0191] In one embodiment, a method for measuring cell membrane tension is provided, comprising the following steps:

[0192] (a) mixing the molecular sensor according to claim 1 and a cell membrane;

[0193] (b) observing the fluorescent signal emitted by the molecular sensor according to claim 1.

[0194] In one embodiment, a method for measuring cell membrane tension is provided, further comprising the steps of:

[0195] (c) The membrane is monitored based on the change in fluorescence signal, thereby determining the tension of the cell membrane.

[0196] In one embodiment, a method for measuring changes in cell membrane tension is provided, comprising the following steps:

[0197] (a) Synthesis of a tetrahedral nucleic acid framework structure and three VIE molecules to form the TDF-VIE3 molecular sensor;

[0198] (b) The TDF-VIE3 molecular sensor was mixed with the test cells and incubated at room temperature;

[0199] (c) using a microscope to perform fluorescence imaging of the cell membrane, and judging the targeting of the TDF-VIE3 molecular sensor to the cell membrane based on the fluorescence imaging results;

[0200] (d) Fluorescence imaging is performed through two channels, and the changes in fluorescence ratio are used to monitor the changes in cell membrane tension.

[0201] In another embodiment, the incubation time at room temperature is 20 min.

[0202] In another embodiment, a confocal scanning microscope is used to perform fluorescence imaging of the cell membrane, and the laser wavelength of the fluorescence confocal microscope used is 405 nm.

[0203] In another embodiment, the cell sample to be tested is washed twice using a cell culture medium. The cells are HeLa cells.

[0204] In one embodiment, it includes:

[0205] The TDF-VIE3 molecular sensor obtains the efficiency of cell membrane targeting based on fluorescence intensity. More specifically, the change in the fluorescence ratio of the VIE molecule between 620nm and 510nm can reveal the change in cell membrane tension.

[0206] In another embodiment, the fluorescence emitted by the TDF-VIE3 molecular sensor is excited by a 405 nm laser, and dual-channel fluorescence imaging is performed, with emission collection channel 1: 450-560 nm, channel 2: 560-700 nm, and changes in cell membrane tension are monitored based on the fluorescence intensity ratio of channel 1 / channel 2.

[0207] In another embodiment, the test cells are subjected to osmotic pressure shock treatment using a high concentration sucrose solution to regulate the cell membrane tension.

[0208] The main advantages of the present invention include:

[0209] (a) The preparation and application of the membrane tension responsive molecular sensor provided by the present invention solves the problems of poor water solubility of existing membrane tension probes, susceptibility to probe concentration, and poor cell membrane targeting.

[0210] (b) The membrane tension-responsive molecular sensor prepared according to the present invention has the advantages of efficient cell membrane targeting, non-concentration dependence, ordered differentiation of model membrane phospholipids, cell membrane tension responsiveness, good biocompatibility, etc. Compared with existing nano fluorescent probes, it has significant advantages and provides a new tool for biological imaging.

[0211] (c) According to the membrane tension-responsive molecular sensor provided by the present invention, the present application realizes fluorescence ratio imaging of cell membrane tension, revealing the great potential of this multifunctional molecular sensor in living cell applications.

[0212] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0213] Example 1

[0214] The present invention provides a method for preparing a molecular sensor based on DNA nanotechnology. In this embodiment, ssDNA-VIE is synthesized and characterized. The method comprises the following steps:

[0215] First, alkynyl-modified VIE molecules (alkynyl-VIE) were synthesized. Subsequently, alkynyl-VIE was chemically linked to single-stranded DNA (ssDNA) through copper-catalyzed click chemistry to form VIE-modified ssDNA (ssDNA-VIE). CuBr (1 mg) was dissolved in 70 μL DMSO, triphenyltriazole methylamine (TBTA) (54 mg) was dissolved in 1 mL DMSO, and sodium ascorbate (20 mg) was dissolved in 1 ml ultrapure water. Next, the CuBr and TBTA stock solutions were premixed in a volume ratio of 1:2 to form a ligand TBTA-Cu(I) solution. Then, alkynyl-VIE dye (0.2 mg, 0.13 μmol) was dissolved in 90 μL DMSO, and an aqueous solution containing ssDNA-N3 (50 μL, 10 OD) was added. After brief stirring, 10 μL of the premixed TBTA-cu(I) solution was added to the reaction mixture. Subsequently, 50 μL of sodium ascorbate stock solution was added and shaken in a 40°C metal bath for 2 h. After the reaction was completed, the solvent was removed in vacuo, and the crude product was dissolved in ultrapure water and purified using high performance liquid chromatography. The solvent was removed by lyophilization, and the resulting product ssDNA-VIE was dissolved in ultrapure water and purified using high performance liquid chromatography. The successful synthesis of ssDNA-VIE was verified by high performance liquid chromatography, polyacrylamide gel electrophoresis, and microplate reader spectral analysis. The absorbance and fluorescence emission spectra of ssDNA-VIE in solutions containing different proportions of acetonitrile were measured to verify that the VIE probe maintained its triple emission properties when bound to ssDNA.

[0216] Results: HPLC results showed that the retention time of ssDNA-VIE increased by 6.82 min ( Figure 3 (a) in Figure 2). PAGE electrophoresis showed that the ssDNA-VIE band had fluorescence emission before adding DNA stain, while the ssDNA-N3 band had no fluorescence emission ( Figure 3(b) in the figure). These results confirm the successful construction of ssDNA-VIE. In pure aqueous solution, ssDNA-VIE emits green light with a wavelength of 510nm. This is because the amphiphilic ssDNA-VIE forms micelles at the test concentration (3μM), which inhibits the torsion process of VIE molecules in the excited state. As the acetonitrile content increases, the dispersibility of the ssDNA-VIE amphiphile increases, and the emission intensity at 620nm gradually increases ( Figure 3 (d) in the figure). Absorption spectrum ( Figure 3 Figure (c) further confirmed that the dispersibility of ssDNA-VIE increased in solutions with high acetonitrile content, and the absorption at 260 nm gradually increased, indicating that more DNA bases were exposed. These results confirmed that ssDNA-VIE retained the triple emission characteristics.

[0217] The DNA sequence is as follows:

[0218]

[0219]

[0220] Example 2

[0221] A method for preparing a membrane tension responsive molecular sensor comprises the following steps:

[0222] Next, TDF nanostructures were prepared by annealing four complementary ssDNA strands. By varying the relative ratio of ssDNA-VIE and unmodified ssDNA, the number of VIE molecules on the tetrahedral DNA framework can be quantitatively controlled ( Figure 2 (b) and (c) in the figure). These assemblies containing one, two, and three VIE molecules were named TDF-VIE1, ​​TDF-VIE2, and TDF-VIE3, respectively. The synthesized molecular sensors were purified by high performance liquid chromatography, and the probes were characterized by polyacrylamide gel electrophoresis and atomic force microscopy.

[0223] Results: HPLC data showed that the yields of the three probes were approximately 40.0±1.3%, 38.4±1.5% and 34.8±2.2%, respectively. Figure 4 ). Gel electrophoresis confirmed the successful assembly of the structure. The migration rates of the corresponding bands of the three structures were consistent, indicating that the synthesis was successful, and the electrophoretic migration rate was not affected by the modification of 1-3 VIE molecules ( Figure 5 ). Atomic force microscopy characterization further confirmed the uniformity of the prepared molecular assemblies ( Figure 6 ).

[0224] Example 3

[0225] A method for characterizing the optical properties of a membrane tension-responsive molecular sensor comprises the following steps:

[0226] The absorption spectrum, fluorescence emission spectrum and time-resolved fluorescence decay spectrum of the molecular sensor were measured using a UV-visible spectrophotometer, a microplate reader and a steady-state transient spectrometer, respectively.

[0227] Results: As the number of VIE molecules increased, the fluorescence emission spectra of the three molecular components shifted blue, which may be due to the different polarities of the three-vertex VIE molecules caused by different base microenvironments ( Figure 7 Interestingly, although the three prepared probes have good dispersibility in aqueous solution, none of them has a clear emission peak near 620nm ( Figure 7 (b) in the figure). This indicates that the DNA structure imposes certain restrictions on the torsion of the excited-state VIE molecule, probably due to the π-π stacking interaction between the VIE probe and the base, thereby inhibiting the torsion process of the excited-state VIE. To further analyze the luminescence mechanism, the fluorescence lifetimes of the three molecular assemblies were tested. The results showed that TDF-VIE1, ​​TDF-VIE2, and TDF-VIE3 had similar fluorescence lifetimes, indicating that the movement of the VIE molecule at the three vertices of TDF is uniformly restricted ( Figure 7 (c) in the figure.

[0228] Example 4

[0229] This example uses fluorescence confocal microscopy to study whether three molecular sensors can interact with lipid membranes and report the ordered state of lipids in the model membrane. The following steps are included:

[0230] First, two types of large unilamellar vesicles (LUVs) were prepared: liquid disordered (Ld) dioleylphosphatidylcholine (DOPC) LUVs and solid ordered (So) dipalmitoylphosphatidylcholine (DPPC) LUVs, both with an average size of 100 nm ( Fig. 9 ). Compared with So DPPC LUVs, the phospholipid chemical bonds in Ld DOPC LUVs are unsaturated, resulting in a lower packing density. This difference in phospholipid density results in different lateral pressures on the embedded VIE molecules, which theoretically greatly affects the fluorescence emission of the VIE molecules ( Fig. 9 ). The two LUVs were incubated with the three probes at the same concentration, and the changes in the fluorescence spectra after the probes bound to the two LUVs were measured. Larger single-component giant unilamellar vesicles (GUVs) (Ld DOPC GUV and So DPPC GUV) were synthesized, with a GUV size of approximately 10 μm, and imaged using confocal laser scanning microscopy to further verify the binding of the trivalent molecular assembly probes to the two model membranes ( Figure 8).

[0231] Results: Spectral data showed that after binding with So DPPC LUVs, the maximum emission spectra of the three probes shifted to around 525 nm and were significantly enhanced. In contrast, after incubation with Ld DOPC LUVs, the maximum emission of the three probes shifted to around 620 nm ( Fig. 9 ). As the number of VIE molecules in the molecular assembly increases, the fluorescence emission at 525nm and 620nm gradually increases ( Fig. 9 ). Both Ld DOPC GUV and So DPPC GUV treated with trivalent molecular assembly probes have strong fluorescence signals ( Figure 8 ). These results indicate that these molecular assemblies can bind to lipid membranes and report changes in lipid packing density.

[0232] Example 5

[0233] This example uses a fluorescence confocal microscope to study whether the molecular sensor has cell membrane targeting specificity. The following steps are included:

[0234] In this embodiment, HeLa cells were used as the research object. First, 3 μM TDF-VIE1, ​​1.5 μM TDF-VIE2 and 1 μM TDF-VIE3 were incubated with cells at room temperature for 20 min, and the cells were imaged using a confocal laser scanning microscope. In addition, 3 μM ssDNA-VIE probe and 3 μM free VIE dye were selected as the control group for comparative analysis. In order to eliminate the effect of dye concentration on imaging intensity, the concentration of VIE was kept consistent in all experiments. Subsequently, a 405 nm laser was used for excitation, and a fluorescence signal with a wavelength range of 450 nm-700 nm was collected.

[0235] Results: Fluorescence imaging results showed that free VIE dye was hydrophobic and easily aggregated into large particles in physiological buffer and was easily phagocytosed by cells ( Fig.10 On the other hand, ssDNA-VIE is evenly distributed in the cell without obvious bright spots, indicating that it is well dispersed in the cell culture medium. However, a single binding site cannot support the stable binding of ssDNA-VIE on the cell membrane, ultimately leading to cell phagocytosis ( Fig.10 (a) and (b) in the figure). The DNA framework can further improve the dispersion of the VIE probe. In the fluorescence images, no obvious aggregation of the three probes was observed ( Fig.10(a) and (b) in Figure 2). However, the number of VIE molecules on TDF resulted in different cellular fates for the three probes. TDF-VIE1 and TDF-VIE2 have one and two anchors on the cell membrane, respectively, and are easily engulfed by cells. In contrast, trivalent TDF-VIE3 exhibited the best membrane targeting ability ( Fig.10 (a) and (b) in the figure). This indicates that the valence state of VIE significantly affects the membrane targeting ability of the molecular assembly and the synergistic effect of the three VIE molecules in the molecular assembly plays an important role. This result shows that the invention can insert VIE molecules into the cell membrane.

[0236] discuss

[0237] Mechanically sensitive fluorescent probes have a wide range of applications in sensing microenvironmental changes, especially in monitoring changes in viscosity and tension of cell membranes, which have been extensively studied. Currently developed membrane tension detection probes include molecular rotors, dynamic polyimides, molecular switches, and planar push-pull dyes. However, these molecules emit single-wavelength fluorescence, and their signal intensity is easily affected by differences in the concentration of fluorescent molecules. In order to eliminate the concentration effect, current membrane tension probes mainly rely on changes in fluorescence lifetime caused by changes in the ground state conformation of molecules under different tensions. For example, using Flipper probes, changes in cell membrane tension can induce the ground state planarization of Flipper probes to a non-planar conformation, resulting in changes in fluorescence lifetime. Matile et al. demonstrated that VIE can be embedded in phospholipid bilayers and used as a fluorescent ratio molecule for model membrane tension monitoring. Recently, this application applied VIE probes to monitor the viscosity inside micelles. However, due to the poor dispersibility of its polycyclic aromatic surface in aqueous solution and the large structural difference between its non-planar conformation and phospholipid molecules, free VIE dyes have a weak binding affinity to living cell membranes. In addition, current VIE probe research mainly focuses on the optical properties of single molecules, and the relatively low fluorescence intensity limits its application in visualizing cell membrane tension.

[0238] To achieve the targeting of mechanosensitive probes to cell membranes, specific membrane targeting components are usually required. However, combining free mechanosensitive molecules with targeting modules can reduce molecular performance and increase synthetic complexity. It remains challenging to improve cell membrane targeting and monodispersity in physiological environments using traditional chemical modification methods. It is very important to develop new methods to tune cell membrane targeting and the performance of mechanosensitive probes. Biological systems have evolved protein and polysaccharide frameworks that integrate various functional molecular components to achieve synergistic functions. The bacterial lipopolysaccharide structure is a typical example, in which a hydrophilic polysaccharide framework connects multiple hydrophobic phospholipid molecules through flexible bonds to achieve high-specific targeting to the bacterial outer membrane. Biopolymer frameworks provide a new approach for the development of novel molecular assembly structures. However, there are still challenges in achieving precise spatial arrangement of mechanosensitive molecular assemblies. Mechanosensitive fluorescent probes are valuable tools for detecting changes in cell mechanics and viscosity. However, the construction and use of mechanosensitive molecular assemblies for cell mechanics detection remain unexplored, probably due to the difficulty in designing molecular assemblies with synergistic and integrated functions.

[0239] In contrast to the membrane tension response strategy based on ground state conformational changes mentioned above, a mechanically sensitive vibration-induced emission (VIE) probe optimized in this application can respond to membrane tension in the excited state. After excitation, the VIE molecule can undergo continuous multi-step excited state structural transitions along the bent, planar, and twisted conformations, generating intrinsic dual or triple emission, and the ratio emission can be regulated by the environmental viscosity.

[0240] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A membrane tension responsive molecular sensor, characterized in that: comprising a nucleic acid framework structure and a vibration-induced luminescent molecule, wherein the vibration-induced luminescent molecule is linked to the nucleic acid framework structure; Furthermore, the nucleic acid framework structure is hydrophilic, while the vibration-induced luminescent molecule is hydrophobic.

2. The molecular sensor according to claim 1, characterized in that include: The nucleic acid monomer ssDNA-VIL linked to the VIL molecule has a structure of Formula I: Z1-L-Z2 (I) In the formula, Z1 is a vibration-induced luminescent molecule; Z2 is a nucleic acid monomer; L is a linking group located between Z1 and Z2.

3. The molecular sensor according to claim 1, characterized in that include: The vibration-induced luminescent molecule is located at a predetermined position in the nucleic acid framework structure.

4. The molecular sensor according to claim 1, characterized in that include: The nucleic acid framework structure is a planar structure or a polyhedral structure.

5. The molecular sensor according to claim 2, characterized in that The nucleic acid nanoshell of the nucleic acid framework structure is a hollow tetrahedral structure, and the nucleic acid framework structure is prepared and composed of four complementary ssDNA chains.

6. The method for preparing a membrane tension responsive molecular sensor according to claim 1, characterized in that: include: (a) chemically linking the vibration-induced luminescent molecule to ssDNA to form a nucleic acid monomer ssDNA-vibration-induced luminescence linked to the vibration-induced luminescent molecule as shown in Formula I: Z1-L-Z2 (I) In the formula, Z1 is a vibration-induced luminescent molecule; Z2 is a nucleic acid monomer; L is a linking group located between Z1 and Z2; (b) The nucleic acid monomers represented by formula I are self-assembled to form a nucleic acid framework structure, thereby forming the molecular sensor according to claim 1; (c) Heating and annealing are performed, wherein the number of VIL molecules is controlled by changing the relative ratio of ssDNA-VIL and unmodified ssDNA.

7. The method for preparing a molecular sensor according to claim 6, characterized in that: include: The VEL molecule was chemically linked to ssDNA via a copper-catalyzed click reaction.

8. The use of the molecular sensor according to claim 1, characterized in that: include: Used to prepare reagents for measuring cell membrane tension.

9. A method for measuring cell membrane tension, characterized in that: include: (a) mixing the molecular sensor according to claim 1 and a cell membrane; (b) observing the fluorescent signal emitted by the molecular sensor according to claim 1.

10. The method according to claim 9, characterized in that include: (c) The membrane is monitored based on the change in fluorescence signal, thereby determining the tension of the cell membrane.

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