Mechanically sensitive nanomachines and methods of making and using the same

Abstract

The application discloses a mechanically sensitive nanomachine and a preparation method and application thereof. The nanomachine comprises a sensing module and a membrane tension response module. The sensing module is a nucleic acid nanopore structure. The membrane tension response module is connected to the sensing module. When the nanomachine is embedded in a membrane, the nanopore structure deforms when the membrane tension changes, and the membrane tension response module responds to the deformation and emits fluorescence corresponding to the membrane tension. The nanomachine has the advantages of high sensitivity, high time resolution, good biocompatibility and the like, and has a significant advantage in membrane tension fluorescence detection.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, and in particular relates to a biomimetic mechanosensitive nanomachine based on nucleic acid nanostructures and its method for fluorescence detection of membrane tension. Background Technology

[0002] Membrane tension plays a crucial role in numerous cellular processes. At the cellular level, it regulates processes such as cell migration, phagocytosis, and cell division. A typical example is that increased membrane tension affects the stability between daughter cells, thus delaying their separation. At the subcellular level, membrane tension also plays a vital role. For instance, increasing membrane tension can inhibit clathrin polymerization and activate membrane fusion, thereby suppressing endocytosis. Furthermore, membrane tension can regulate the opening and closing of mechanosensitive ion channels, thus influencing cellular metabolic processes. Therefore, membrane tension detection technology has wide applications in cell migration, cell recognition, and other fields.

[0003] Existing membrane tension detection methods can be divided into two categories: physical detection methods (such as micropipette technology, atomic force microscopy, and optical tweezers) and fluorescence detection methods. Physical detection methods can directly measure the magnitude of membrane tension by stretching the cell membrane, offering high sensitivity. However, these methods suffer from low spatial resolution, complex experimental procedures, and difficulty in real-time detection of membrane tension changes. Fluorescence detection methods utilize mechanosensitive fluorescent probes, reflecting changes in membrane tension through the fluorescence lifetime of molecules. This method enables in-situ detection of membrane tension and offers high spatial resolution. However, limited by the long data acquisition time of fluorescence lifetime imaging (typically requiring tens of seconds to generate one image) and the constraints of the molecular's cross-sectional area, this method has low temporal resolution, and the sensitivity of the probe needs further improvement.

[0004] Therefore, there is a lack of a highly sensitive membrane tension detection device and method with high spatiotemporal resolution in this field, which is of great significance to the development of biomechanics. Summary of the Invention

[0005] The purpose of this invention is to provide a mechanically sensitive nanomachine and a method for testing membrane tension using it. The nanomachine of this invention possesses advantages such as high sensitivity, high temporal resolution, and good biocompatibility, exhibiting significant advantages in membrane tension fluorescence detection and opening new avenues for the development of cell mechanics research.

[0006] In a first aspect of the invention, a mechanically sensitive nanomachine is provided, the nanomachine comprising: a sensing module, the sensing module being a nucleic acid nanopore structure; and a membrane tension response module connected to the sensing module; wherein, when the nanomachine is embedded in a membrane, the nanopore structure deforms when the membrane tension changes, causing the membrane tension response module to emit fluorescence corresponding to the membrane tension in response to the deformation.

[0007] In another preferred embodiment, the nucleic acid nanopore structure is an enclosed structure, and the center of the enclosed structure is a nanopore.

[0008] In another preferred embodiment, the diameter of the nanopore is 3-15 nm; preferably, 4-10 nm; more preferably, 5-8 nm; and most preferably, about 6 nm.

[0009] In another preferred embodiment, the sensing module is scalable at least radially.

[0010] In another preferred embodiment, in response to a change in membrane tension, the membrane tension response module changes from a nonpolar state to a polar state, thereby emitting fluorescence (or fluorescence intensity in response to the membrane tension) in response to the membrane tension.

[0011] In another preferred embodiment, the sensing module is a hollow polygonal prism formed by combining multiple nucleic acid prisms.

[0012] In another preferred embodiment, the polygon is an N-prism, where N is an integer from 3 to 20; preferably, 4 to 10; more preferably, 5 to 8.

[0013] In another preferred embodiment, the polygon is a triangular prism, a quadrangular prism, a pentagonal prism, a hexagonal prism, a heptagonal prism, an octagonal prism, etc.

[0014] In another preferred embodiment, the sensing module is composed of multiple nucleic acid prisms spliced ​​together.

[0015] In another preferred embodiment, each nucleic acid prism is independently connected to a membrane tension response module, an anchoring module, a reference module, or a combination thereof.

[0016] In another preferred embodiment, one or more nucleic acid prisms in the polygon are not connected to the above-mentioned modules.

[0017] In another preferred embodiment, the membrane tension response module includes a membrane tension-responsive fluorescent reporter molecule.

[0018] In another preferred embodiment, the membrane tension-responsive fluorescent reporter molecule includes, but is not limited to, spiropyran molecules, Flipper, TPE, Laurdano, ASP-PE, etc.

[0019] In another preferred embodiment, the nanomachine is a membrane tension sensor.

[0020] In another preferred embodiment, the nanomachine further includes an anchoring module, one end of which is connected to the sensing module, and the other end of which is connected to a test membrane to anchor the sensing module to the test membrane.

[0021] In another preferred embodiment, the anchoring module includes a hydrophobic anchoring group.

[0022] In another preferred embodiment, the anchoring group includes, but is not limited to, cholesterol groups, alkyl chain groups, vitamin E groups, and porphyrin groups.

[0023] In another preferred embodiment, the anchoring module includes one or more anchoring groups.

[0024] In another preferred embodiment, the number of anchoring groups is an integer greater than or equal to 2; preferably, it is 2-20; more preferably, it is 3-10.

[0025] In another preferred embodiment, a plurality of the anchoring modules are circumferentially spaced around the outer periphery of the sensing module.

[0026] In another preferred embodiment, the interval is uniform or substantially uniform.

[0027] In another preferred embodiment, the nanomachine further includes a reference module configured to provide a reference signal.

[0028] In another preferred embodiment, the reference signal is a fluorescence signal.

[0029] In another preferred embodiment, the reference signal does not respond to or is substantially unresponsive to changes in membrane tension.

[0030] In another preferred embodiment, the reference module is connected to the membrane tension response module.

[0031] In another preferred embodiment, the reference signal and the fluorescence spectrum of the membrane tension response module are independent of each other or distinguishable.

[0032] In another preferred embodiment, the reference module includes, but is not limited to, Alexa Fluor 488, Alexa Fluor 647, Atto 488, Cy2, Cy5, etc.

[0033] In another preferred embodiment, the reference module provides a detectable signal that does not respond to or substantially does not respond to changes in membrane tension.

[0034] In another preferred embodiment, the membrane comprises a cell membrane or a similar lipid bilayer membrane.

[0035] In another preferred embodiment, the membrane has a lipid bilayer structure, such as a phospholipid bilayer structure.

[0036] In another preferred embodiment, the membrane further includes cholesterol compounds located between lipid bilayers (such as phospholipid bilayers).

[0037] In a second aspect of the invention, a method for preparing the nanomachines described above is provided, the method comprising the steps of:

[0038] (a) A plurality of nucleic acid prisms are provided, wherein at least one of the nucleic acid prisms is coupled to the membrane tension response module;

[0039] (b) The plurality of nucleic acid prisms are mixed to assemble the nanomachines described in the first aspect of the present invention.

[0040] In another preferred embodiment, the membrane tension response module, the anchoring module, and the reference module are respectively disposed on the same or different prism surfaces of the sensing module.

[0041] In another preferred embodiment, each nucleic acid prism is independently connected to a membrane tension response module, an anchoring module, a reference module, or a combination thereof; or is not connected to any of the above modules.

[0042] In a third aspect of the invention, a method for measuring membrane tension is provided, the method comprising:

[0043] S1. Provide the nanomachines and test membranes described in the first aspect of the present invention;

[0044] S2. Mix the nanomachine with the test membrane, and embed the nanomachine into the test membrane;

[0045] S3. Under predetermined membrane tension test conditions, measure the fluorescence emitted by the membrane tension response module in response to the membrane tension; and

[0046] S4. Based on the fluorescence in response to the membrane tension, the membrane tension of the test membrane is measured.

[0047] In another preferred embodiment, the membrane tension of the test membrane is determined based on the fluorescence intensity.

[0048] It should be noted that the method described is neither a diagnostic nor a treatment method.

[0049] In another preferred embodiment, the nanomachine includes a reference module.

[0050] In another preferred embodiment, S3 further includes measuring the reference signal of the reference module.

[0051] In another preferred embodiment, the reference module provides a detectable signal that does not respond to or substantially does not respond to changes in membrane tension.

[0052] In another preferred embodiment, the reference signal is a fluorescence signal.

[0053] In another preferred embodiment, the reference signal does not respond to or is substantially unresponsive to changes in membrane tension.

[0054] In another preferred embodiment, the reference signal and the fluorescence spectrum of the membrane tension response module are independent of each other or distinguishable.

[0055] In another preferred embodiment, in S4, the measurement result of the membrane tension is obtained by comparing the fluorescence signal of the membrane tension response module and the reference signal of the reference module.

[0056] In another preferred embodiment, the membrane tension response module emits a first fluorescence, which is measured at a first wavelength of 488±2 nm.

[0057] In another preferred embodiment, the reference module emits a second fluorescence, which is measured at a second wavelength of 561±2 nm.

[0058] In another preferred embodiment, the R / G value is obtained by the first fluorescence of the membrane tension response module and the second fluorescence of the reference module, and the R / G value is linearly negatively correlated with the osmotic pressure.

[0059] In a fourth aspect of the invention, a membrane tension measuring device is provided, the device comprising: a composite membrane forming module, the composite membrane forming module being a composite structure formed by the nanomachines and the test membrane described in the first aspect of the invention; a measuring module for measuring a first fluorescence signal; a processing module for processing the first fluorescence signal to obtain a membrane tension result; and an optional output module for outputting the membrane tension result.

[0060] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0061] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. The drawings are schematic diagrams, therefore the device and equipment of the present invention are not limited by the size or scale of the schematic diagrams.

[0062] Figure 1 This is a schematic diagram of the structure of a single-stranded DNA 2-SP, and the results of polyacrylamide electrophoresis characterization of a single-stranded DNA modified with spiropyran.

[0063] Figure 2 These are the mass spectrometry characterization results of DNA single-strand modification before and after spiropyran group modification;

[0064] Figure 3 These are the results of polyacrylamide electrophoresis characterization of mechanosensitive DNA nanomachines with different numbers of constituent chains added.

[0065] Figure 4 These are cryo-electron microscopy characterization images and two-dimensional overlay images of single particles of mechanosensitive DNA nanomachines, where the black bars represent 3 nm and the white bars represent 50 nm.

[0066] Figure 5 This is a cryo-electron microscopy characterization of mechanosensitive DNA nanomachines inserted into small monolayer vesicles;

[0067] Figure 6 The fluorescence intensity of small monolayer vesicles encapsulated with sulfonylrhodamine B changes over time after the addition of mechanosensitive DNA nanomachines.

[0068] Figure 7 It is a confocal fluorescence imaging of giant monolayer vesicle membrane tension changes under different osmotic pressure conditions using mechanically sensitive DNA nanomachines, and the calculated R / G image results.

[0069] Figure 8 These are statistical results of pixel intensity in R / G images under different osmotic pressure conditions;

[0070] Figure 9 It is a confocal fluorescence imaging of cell membrane tension changes under different osmotic pressure conditions using mechanically sensitive DNA nanomachines, and the calculated R / G image results.

[0071] Figure 10 This is a schematic diagram of the structure of a mechanically sensitive nanomachine in one embodiment of the present invention;

[0072] Figure 11The design and operating principle of the mechanosensitive DNA nanomachines are illustrated, in which... Figure 11 a) shows a schematic diagram of the structural deformation of the nanomachine in response to changes in membrane tension (open and closed states); b) shows the chemical structure of solvatochromicspiropyran (SP), in which SP undergoes reversible SP-anthocyanin (MC) isomerization under polarity; c) shows a simplified reaction coordinate diagram of dissolution and color change in SP, in which the polar medium reduces the energy and thermal barriers to the polar MC form; d) shows the expected fluorescence spectra of the nanomachine under different membrane tensions. Detailed Implementation

[0073] Through extensive and in-depth research and screening, the inventors have developed for the first time a mechanosensitive nanomachine and a method for testing membrane tension using it. This invention achieves highly sensitive membrane tension detection by constructing a biomimetic mechanosensitive fluorescent probe. Based on the highly encodeable nature of DNA sequences, DNA nanotechnology can achieve the construction of nanostructures with "atomic-level" precision. The lever structure within the membrane tension response module significantly enhances its mechanosensitivity, enabling it to respond to weak membrane tension stimuli and undergo significant conformational changes. Utilizing computer-aided design combined with molecular dynamics simulations, DNA molecules spontaneously fold into the desired structure and perform specific functions, thereby realizing the design of a biomimetic mechanosensitive DNA nanomachine. This invention is based on this foundation.

[0074] the term

[0075] As used herein, the terms "mechanically sensitive nanomachine of the present invention", "nanomachine of the present invention", "membrane tension responsive nanomachine of the present invention", "membrane tension responsive nanosensor of the present invention", etc., are used interchangeably to refer to the mechanically sensitive nanomachine described in the first aspect of the present invention.

[0076] In the claims and description of this patent, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0077] This invention relates to mechanically sensitive nanomachines.

[0078] A schematic diagram of the mechanically sensitive nanomachine of the present invention is shown below. Figure 10 As shown. This nanomachine includes a sensing module, a membrane tension response module, an anchoring module, and a reference module.

[0079] The sensing module is composed of multiple nucleic acid chains connected together, forming a nucleic acid nanopore structure. The nanopore is radially expandable and contractible. Specifically, the sensing module presents a hollow prismatic structure. In this embodiment, the sensing module contains a hexagonal prism-shaped hollow DNA nanopore. This nanopore can respond to membrane tension in the cell membrane and undergo conformational changes. Under high membrane tension conditions, the DNA nanopore will be open, while under low membrane tension conditions, it will be closed.

[0080] It should be noted that the aforementioned prism structure can be not only hexagonal, but also triangular, square, pentagonal, heptagonal, octagonal, etc. The membrane tension response module is connected to the outer periphery of the sensing module and is used to present different fluorescence images based on the expansion and contraction state of the nanopore. The membrane tension response module includes a membrane tension-responsive fluorescent reporter molecule. In this embodiment, the membrane tension-responsive fluorescent reporter molecule is a spiropyran molecule. Spiropyran can respond to the conformational change of the DNA nanopore and undergo a conversion between the spiropyran conformation (low fluorescence) and the anthocyanin conformation (high fluorescence). This conversion greatly changes the fluorescence intensity of the spiropyran. Therefore, changes in membrane tension can be detected in real time by fluorescence intensity.

[0081] like Figure 11 As shown, mechanosensitive DNA nanomachines can respond to membrane tension and undergo conformational changes. Under low membrane tension, the DNA nanomachine structure contracts and closes; under high membrane tension, it stretches and opens. During this process, spiropyran responds to the polarity change in the phospholipid membrane surrounding the DNA nanomachine caused by membrane tension, undergoing a transition between the spiropyran conformation (SP, low fluorescence) and the anthocyanin conformation (MC, high fluorescence). The highly polar medium lowers the energy barrier between the spiropyran and anthocyanin conformations, thus stabilizing the anthocyanin conformation. Therefore, changes in phospholipid membrane tension can be monitored in real time by measuring the fluorescence intensity of the DNA nanomachines.

[0082] One end of the anchoring module is connected to the periphery of the sensing module, and the other end is used to connect to the testing module, which is a membrane, such as (but not limited to) a cell membrane or a similar lipid bilayer. Anchoring groups include, but are not limited to, cholesterol groups, alkyl chain groups, vitamin E groups, and porphyrin groups. The anchoring module contains N (N is a positive integer ≥2, preferably 2-20, more preferably 3-10) hydrophobic anchoring groups. This embodiment shows three anchoring groups, which are circumferentially and uniformly distributed around the DNA nanopore, allowing the structure (i.e., the DNA nanomachine) to be successfully inserted into a membrane (such as a cell membrane).

[0083] In this invention, representative anchoring groups include (but are not limited to):

[0084]

[0085] A reference module is attached to the periphery of the membrane tension response module. In this embodiment, the reference module contains an Alexa Fluor 488 fluorophore. The spectrum of this fluorophore does not overlap with that of spiropyran and it maintains stable fluorescence in a variety of different solution environments. Therefore, this fluorophore can serve as an internal reference to correct for uneven distribution of DNA nanomachines on the phospholipid membrane during imaging. An R / G image of the reaction membrane tension is obtained by dividing the image of the spiropyran channel (R) by the image of the Alexa Fluor 488 channel (G). This reference module provides a reference detectable signal that does not respond to or is substantially unresponsive to changes in membrane tension.

[0086] Method for preparing nanomachines of the present invention

[0087] The present invention also provides a method for preparing nanomachines.

[0088] Taking DNA-based nanomachines as an example, the preparation method of the present invention includes the following steps:

[0089] (1) Preparation of spiropyran-modified single-stranded DNA. Spiopyran modified with an azide group and dibenzocyclooctyne-modified single-stranded DNA were used as raw materials. Spiopyran and DNA were coupled at room temperature via a click reaction, and excess free small molecules in the solution were removed by size exclusion chromatography.

[0090] (2) Preparation of DNA nanomachines. Six DNA single strands modified with spiropyran, cholesterol and Alexa Fluor488 fluorophores respectively were mixed and mechanically sensitive DNA nanomachines were obtained by high-temperature annealing.

[0091] Mechanically sensitive DNA nanomachines are hollow hexagonal prism-shaped DNA nanoporous structures formed by the hybridization of six single-stranded DNA molecules. It should be understood that other shapes of DNA nanoporous structures, such as tetragonal prisms, pentagonal prisms, and heptagonal prisms, can also be used in this system.

[0092] The buffer system for preparing the DNA nanomachines includes magnesium chloride and Tris-HCl, and the reaction system is maintained at pH 7.4 to ensure the formation of the DNA nanomachines.

[0093] Preferably, the spiropyran group, cholesterol group, and Alexa Fluor 488 group can be modified at the 5' or 3' end of the single-stranded DNA.

[0094] The reference group for the label is Aleax Fluor 488. It should be understood that other stable fluorescent molecules that do not spectrally overlap with spiropyrans can also be used in this system.

[0095] Application of the nanomachines of this invention

[0096] This invention also provides applications of nanomachines, particularly in the determination of membrane tension.

[0097] Typically, this invention provides the application of mechanosensitive nanomachines in membrane tension fluorescence detection, such as the detection of cell membrane tension.

[0098] In a preferred embodiment, the application includes incubating a mechanosensitive DNA nanomachine with a giant monolayer vesicle or cell sample to be tested. The osmotic pressure of the sample is adjusted to a target concentration using ultrapure water or a high-concentration sucrose solution, and the membrane tension of the sample can then be detected using a fluorescence confocal microscope. The preferred osmotic pressure concentration of the sample is 0-1000 mOsm, for example: 0 mOsm, 200 mOsm, 400 mOsm, 600 mOsm, 800 mOsm, 1000 mOsm, etc. The monolayer giant vesicles are composed of one or more combinations of 1-palmitoyl-2-oleoyl-lecithin, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and sphingomyelin. The cells are HepG2 cells. The fluorescence confocal microscope laser wavelengths used are 488 and 561 nm.

[0099] The main advantages of this invention include:

[0100] (a) The preparation of the biomimetic mechanically sensitive DNA nanomachine provided by the present invention and its application in membrane tension detection solves the problems of low sensitivity and long data acquisition time of existing membrane tension fluorescent probes.

[0101] (b) The mechanically sensitive DNA nanomachines prepared according to the present invention have the advantages of high sensitivity, high time resolution, good biocompatibility, and multifunctional module integration. They have significant advantages in membrane tension fluorescence detection and open up new avenues for the development of cell mechanics research.

[0102] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0103] Material

[0104] The sequence example is as follows:

[0105]

[0106]

[0107] Example 1

[0108] This embodiment provides a method for preparing a mechanically sensitive DNA nanomachine based on DNA nanotechnology, wherein a spiropyran-modified DNA single strand is synthesized.

[0109] DNA single-stranded 2-DBCO was dissolved in ultrapure water and quantified to 100 μM using a UV spectrophotometer. Azide-modified spiropyran molecules were weighed and dissolved in dimethyl sulfoxide to a concentration of 10 mM. DNA single-stranded 2-DBCO and spiropyran were mixed at a molar ratio of 1:10 and incubated overnight at 37°C. Subsequently, the incubated sample was purified by size exclusion chromatography, and after removing excess solvent using a freeze dryer, it was dissolved in ultrapure water to prepare spiropyran-modified DNA single-stranded 2-SP( Figure 1 The DNA single-stranded 2-SP was then analyzed by polyacrylamide electrophoresis and mass spectrometry.

[0110] Results: Polyacrylamide gel electrophoresis ( Figure 1 The results showed that spiropyran-modified DNA single strands migrated more slowly than unmodified DNA single strands, which was attributed to the increased molecular weight of the DNA single strands after modification with the spiropyran group. Mass spectrometry results further showed that the molecular weight of the DNA single strands increased after modification with the spiropyran group, indicating that the preparation of spiropyran-modified DNA single strands was successful. Figure 2 ).

[0111] The DNA sequence is as follows:

[0112]

[0113] Example 2

[0114] The fabrication of mechanosensitive DNA nanomachines includes the following steps:

[0115] All DNA was dissolved in ultrapure water, and the absorbance of each single strand at 260 nm was measured using a UV spectrophotometer to bring the concentration of all DNA single strands to 100 μM. Equal volumes of 1-chol, 3-chol, 4,5-chol, 6-AF488, and 2-SP were mixed in TAE / Mg buffer (40 mM Tris-HCl, 20 mM CH3COOH, 2 mM EDTA, 12.5 mM MgCl2, pH 7.4) to bring the final concentration of each DNA single strand to 1 μM. The mixture was incubated at 95 °C for 5 min in a PCR instrument, and then cooled from 95 °C to 4 °C over 540 min to prepare mechanosensitive DNA nanomachines. The DNA nanomachines were then characterized by 12% polyacrylamide gel electrophoresis and cryo-electron microscopy. The electrophoresis run was performed at 110 V for 120 min. The morphology of the DNA nanomachines was characterized using cryo-electron microscopy combined with two-dimensional stacking of single particles.

[0116] Results: Polyacrylamide electrophoresis showed that the migration rate of the bands gradually slowed down with the increase of the number of DNA constituent strands, indicating that the DNA nanomachines were successfully prepared. Figure 3 Cryo-electron microscopy results clearly show hexagonal prism-shaped DNA nanostructures. Figure 4 ).

[0117] The DNA sequence is as follows:

[0118]

[0119] Example 3

[0120] The assessment of the mechanosensitivity of DNA nanomachines in phospholipid membranes includes the following steps:

[0121] Add 1 ml of chloroform solution containing 20 mg / ml phospholipid molecules to a round-bottom flask. Evaporate all solvent under vacuum using rotary evaporation to form a uniform phospholipid film at the bottom of the flask. Add 1 ml of hydration buffer (5 mM Tris-HCl, 300 mM sucrose, pH 7.4) to the flask and stir at room temperature for 1 hour using a magnetic stirrer to form giant monolayer vesicles. Extrude the prepared giant monolayer vesicles 21 times through a 50 nm pore size polycarbonate filter to prepare small monolayer vesicles. For fluorescently coated small monolayer vesicles, hydrate using a hydration buffer containing sulfonylrhodamine B (20 mM Tris-HCl, 200 mM sucrose, 50 mM sulfonylrhodamine B, pH 7.4). Purify the extruded vesicles using size exclusion chromatography to remove free fluorescent molecules from the solution, thus obtaining small monolayer vesicles coated with sulfonylrhodamine B.

[0122] Cryo-electron microscopy characterization: Mechanosensitive DNA nanomachines were mixed with small monolayer vesicles at a volume ratio of 3:1 and incubated at room temperature for 5 min. Cryo-electron microscopy samples were then prepared using a Vitrobot system. Subsequently, the state of the mechanosensitive DNA nanomachines on the surface of the small monolayer vesicles was observed using a 200 kV cryo-transmission electron microscope.

[0123] Liposome leakage characterization: The osmotic pressure of the prepared small monolayer vesicles containing sulfonylrhodamine B was adjusted to 0 mOsm, 300 mOsm, and 1000 mOsm using ultrapure water and a high-concentration sucrose solution. These solutions were then mixed with mechanosensitive DNA nanomachines at a volume ratio of 100:15. The fluorescence intensity of the solution over time was then measured using a microplate reader (excitation wavelength 550 nm, emission wavelength 580 nm).

[0124] Results: Cryo-electron microscopy revealed hexagonal prism-shaped nanostructures on the surface of small monolayer vesicles, indicating that mechanosensitive DNA nanomachines were successfully inserted into the small monolayer vesicles. Figure 5 The liposome leakage experiment showed that the rate of change in solution fluorescence intensity decreased with increasing osmotic pressure (decreased membrane tension). Figure 6 This indicates that mechanosensitive DNA nanomachines undergo structural shrinkage under hyperosmotic conditions, leading to a decrease in the leakage rate of sulfonylrhodamine B. These results demonstrate that mechanosensitive DNA nanomachines can respond to membrane tension and undergo conformational changes, exhibiting mechanosensitivity.

[0125] Example 4

[0126] This embodiment uses an artificial phospholipid membrane as an example to investigate whether mechanosensitive DNA nanomachines can be used for membrane tension detection. By adjusting the osmotic pressure in the solution, this embodiment can change the membrane tension in the artificial phospholipid membrane. The membrane tension detection capability is verified by comparing the fluorescence changes of DNA nanomachines inserted into the artificial phospholipid membrane under different osmotic pressure conditions. The steps include:

[0127] The giant monolayer vesicles prepared in Example 3 were mixed with mechanosensitive DNA nanomachines at a volume ratio of 10:1. After incubation at room temperature for 10 min, the osmotic pressure of the sample solution was adjusted to the target concentration using ultrapure water and a high-concentration sucrose solution. After incubation at room temperature for 30 min, the sample solution was added to a 14 mm confocal dish, and fluorescence images were directly obtained using a fluorescence confocal microscope at excitation wavelengths of 488 and 561 nm to obtain fluorescence images of the 488 nm channel (R) and the 561 nm channel (G). Using ImageJ and a self-made Python code, the R / G images were obtained by dividing the images of the two channels. The changes in pixel intensity (R / G value) in the R / G images under different osmotic pressure conditions were compared.

[0128] Results: Fluorescence imaging ( Figure 7 The results showed that as the osmotic pressure of the solution increased, the giant monolayer vesicles underwent significant shrinkage, indicating a decrease in membrane tension. Statistical analysis of pixel intensity in the R / G images (…) Figure 8 The results showed that the R / G value decreased with increasing solution osmotic pressure, and generally exhibited a linear correlation. This demonstrates that mechanosensitive nanomachines can detect changes in membrane tension within phospholipid membranes.

[0129] Example 5

[0130] The application of mechanosensitive DNA nanomachines in the detection of live cell membrane tension includes the following steps:

[0131] This study used HepG2 cells as the research subject. First, 0.4 μM mechanosensitive DNA nanomachines were incubated with live cells at room temperature for 15 min. Then, the cells were washed three times with DMEM culture medium to remove DNA nanomachines that had not intercalated into the cell membrane. The cells were then incubated for 15 min at room temperature in hypotonic (120 mOsm), isotonic (360 mOsm), and hypertonic (750 mOsm) solutions, respectively. Subsequently, fluorescence signals of the mechanosensitive DNA nanomachines in the 488 and 561 nm channels were acquired using fluorescence confocal microscopy, and R / G images were plotted using ImageJ and a self-made Python code.

[0132] Results: The intensity of pixels in the R / G image decreased with increasing osmotic pressure, indicating that the R / G value decreased with decreasing membrane tension. Figure 9This result indicates that mechanosensitive DNA nanomachines can be used to detect membrane tension in living cells.

[0133] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A mechanically sensitive nanomachine, characterized in that, The nanomachines include: The sensing module is composed of multiple nucleic acid chains connected together, forming a nucleic acid nanopore structure. The nanopores are radially expandable and contractible, exhibiting a hollow prismatic structure. A membrane tension response module is connected to the sensing module, and the membrane tension response module includes a membrane tension-responsive fluorescent reporter molecule, wherein the membrane tension-responsive fluorescent reporter molecule is a spiropyran molecule; An anchoring module, one end of which is connected to the sensing module, and the other end of which is connected to a test membrane for anchoring the sensing module to the test membrane, the anchoring module including a hydrophobic anchoring group; and A reference module is configured to provide a reference signal, which is a fluorescence signal. The reference signal does not respond to changes in membrane tension. The reference module is connected to the membrane tension response module, and the fluorescence spectrum of the reference signal and the membrane tension response module do not interfere with each other. When the nanomachine is embedded in the test membrane, the nanopore structure deforms when the membrane tension changes, causing the membrane tension response module to emit fluorescence corresponding to the membrane tension in response to the deformation.

2. The nanomachine as described in claim 1, characterized in that, The nucleic acid nanopore structure is an enclosed structure, with a nanopore at the center of the enclosed structure.

3. The nanomachine as described in claim 2, characterized in that, The diameter of the nanopore is 3-15 nm.

4. The nanomachine as described in claim 1, characterized in that, In response to the change in membrane tension, the membrane tension response module changes from a nonpolar state to a polar state, thereby emitting fluorescence in response to the membrane tension.

5. The nanomachine as described in claim 1, characterized in that, The anchoring groups include, but are not limited to, cholesterol groups, alkyl chain groups, vitamin E groups, and porphyrin groups.

6. The nanomachine as described in claim 1, characterized in that, Multiple anchoring modules are circumferentially spaced around the outer periphery of the sensing module.

7. The nanomachine as described in claim 1, characterized in that, The reference modules include, but are not limited to, Alexa Fluor 488, Alexa Fluor 647, Atto 488, Cy2, and Cy5.

8. A method for preparing the nanomachines as described in claim 1, characterized in that, The method includes the following steps: (a) Using azide-modified spiropyran and dibenzocyclooctylene-modified single-stranded DNA as raw materials, spiropyran and DNA were coupled by click reaction at room temperature, and excess free small molecules in the solution were removed by size exclusion chromatography column. (b) Six DNA single strands, each modified with spiropyran, cholesterol and Alexa Fluor 488 respectively, were mixed and annealed at high temperature to obtain the nanomachines as described in claim 1.

9. The method as described in claim 8, characterized in that, The membrane tension response module, anchoring module, and reference module are respectively disposed on the same or different prism surfaces of the sensing module.

10. The method as described in claim 8, characterized in that, Each nucleic acid strand may be independently connected to a membrane tension response module, an anchoring module, or a reference module; or it may not be connected to any of these modules.

11. A method for measuring membrane tension, characterized in that, The method includes: S1. Providing the nanomachines and test membrane as described in claim 1; S2. Mix the nanomachine with the test membrane, and embed the nanomachine into the test membrane; S3. Under predetermined membrane tension test conditions, measure the fluorescence emitted by the membrane tension response module in response to the membrane tension, and measure the reference signal of the reference module; and S4. Based on the fluorescence in response to the membrane tension, the membrane tension of the test membrane is measured.

12. The method as described in claim 11, characterized in that, The membrane tension of the test membrane was determined based on the fluorescence intensity.

13. The method as described in claim 11, characterized in that, In S4, the measurement result of the membrane tension is obtained by comparing the fluorescence signal of the membrane tension response module and the reference signal of the reference module.

14. The method as described in claim 11, characterized in that, The membrane tension response module emits a first fluorescence, which is measured at a first wavelength of 488±2nm.

15. The method as described in claim 14, characterized in that, The reference module emits a second fluorescence, which is measured at a second wavelength of 561±2 nm.

16. The method as described in claim 15, characterized in that, The R / G value is obtained by the first fluorescence of the membrane tension response module and the second fluorescence of the reference module, and the R / G value is linearly negatively correlated with the osmotic pressure.

17. A membrane tension measuring device, characterized in that, The device includes: A composite membrane forming module, wherein the composite membrane forming module is a composite structure formed from the nanomachines and the test membrane as described in claim 1; A measurement module is used to measure the fluorescence signal of the membrane tension response module and the reference signal of the reference module; The processing module is used to process the fluorescence signal from the membrane tension response module and the reference signal from the reference module to obtain the membrane tension result; and An output module is used to output the membrane tension result.

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