Biomacromolecule efficient packaging method and application thereof

Abstract

The invention discloses an efficient packaging method for biomacromolecules and application of the efficient packaging method. The packaging method comprises the following steps that S1, primary vesicles are manufactured, wherein amphoteric molecules are used for assembling the primary vesicles to package a biomacromolecule sample; s2, manufacturing working vesicles: breaking the primary vesicles and recombining the primary vesicles into the working vesicles, wherein the volume of the working vesicles is smaller than that of the primary vesicles; s3, removing residual samples: removing the free biomacromolecule samples which are not packaged by the vesicles; and S4, enriching the working vesicles: loading the working vesicles onto a carrier network. According to the method, the steps S1-S2 or S1-S3 are used for medicine or vaccine packaging; the steps S1 to S4 are used for preparing the cryoelectron microscope sample. The packaging efficiency of the biomacromolecule sample is improved, and the technical bottleneck that the low-stability compound biomacromolecule sample is prone to dissociation, damage or serious advantage orientation is broken through.

Description

Technical Field

[0001] The present invention relates to the technical field of biophysics, and particularly to a method for efficiently packaging biological macromolecules and its applications. Background Art

[0002] In the field of cryo-electron microscopy, in recent years, due to the development of software and hardware such as direct electron detectors and instrument stability in many aspects, a large number of single-particle samples of biological macromolecules have been resolved to near-atomic spatial resolution levels, making great contributions to the fields of basic and applied biomedicine.

[0003] However, sample preparation in this field remains a bottleneck restricting the progress of specific scientific research projects. Specifically, in the process of preparing cryo-EM samples, an ice layer with a thickness of the order of 100 nm needs to be prepared. The ice layer is still liquid within a few seconds before rapid freezing. During this period, most types of biological macromolecules (especially dynamic complexes with poor stability) are affected by various factors including the air-liquid interface, resulting in dissociation, destruction, or severe preferred orientation of biological macromolecules, thereby hindering the acquisition of near-atomic resolution structures and restricting the progress of the project. Figure 1a The schematic diagram of the most commonly used holy carbon grid is shown. Refer to Figure 1a , where 101 is the grid, 102 is the carbon film, 103 is the amorphous ice formed after rapid freezing, and 104 - 106 are biological macromolecules. It can be seen from the figure that due to influencing factors such as the air-liquid interface in this method, biological macromolecules in the pores without a carbon film almost all appear at the interface between the air and the ice layer, and most of the biological macromolecules among them undergo severe changes such as preferred orientation, complex dissociation, protein unfolding, or covalent bond cleavage, such as 104 - 106, affecting structure reconstruction. Currently, methods to solve this problem include using affinity grids, non-specific adsorption grids such as graphene or graphene oxide, etc. Figure 1b The schematic diagrams of these methods are shown. Refer to Figure 1b , 107 is the non-specific adsorption interface such as graphene or graphene oxide or the affinity layer of the affinity grid, and 104 - 106 are biological macromolecules. Although these methods can reduce the influence of the air-liquid interface on the sample, resulting in fewer biological macromolecules at the air-liquid interface, the problems of the air-liquid interface or non-bio-friendly interfaces have not been completely solved. Severe changes such as preferred orientation, complex dissociation, protein unfolding, or covalent bond cleavage of biological macromolecules can still be observed in 104 - 106. In addition, it should be emphasized that for a large number of biological macromolecule complexes with poor stability, the dissociation time of the complex is too short or the dissociation constant is too large, making it difficult to resolve the structure of the complex by the above methods or to determine whether the resolved structure is an artifact.

[0004] In summary, due to the above technical defects in the cryo-EM sample preparation method, the universality of its application is limited.

[0005] In addition, there are technical problems of low efficiency in the packaging method of drugs or vaccines: the packaging method requires the use of special formulations, which are costly and have limited material selection. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a high-efficiency packaging method for biological macromolecules and its application, improve the packaging efficiency, and break through the technical bottleneck that biological macromolecule samples with low-stability complexes are prone to dissociation, destruction or serious preferential orientation.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is: a high-efficiency packaging method for biological macromolecules, comprising the following steps:

[0008] S1. Produce primary vesicles: Use amphiphilic molecules to assemble primary vesicles to package biological macromolecule samples;

[0009] S2. Produce working vesicles: Mechanically transform the primary vesicles into working vesicles, and the volume of the working vesicles is smaller than that of the primary vesicles;

[0010] S3. Remove residual samples: Remove the free biological macromolecule samples that are not packaged by vesicles.

[0011] As a preferred technical solution of the present invention, the amphiphilic molecules in step S1 are phospholipid molecules. The method for producing primary vesicles is to place the biological macromolecule samples and phospholipid molecules in a container under temperature conditions that are conducive to maintaining the activity of biological macromolecules and higher than the phase transition temperature of phospholipid molecules, and package the biological macromolecule samples inside through the hydration reaction of phospholipid molecules to form primary vesicles with a diameter larger than that of the working vesicles.

[0012] As a preferred technical solution of the present invention, the primary vesicles are phospholipid bilayer vesicles with a diameter ranging from nanometers to micrometers.

[0013] As a preferred technical solution of the present invention, in step S2, the primary vesicles are repeatedly passed through a filter membrane with a pore size equal to the diameter of the target working vesicles by means of reciprocating extrusion, and the primary vesicles are ruptured and reorganized into working vesicles with a uniform diameter and good monolayer property.

[0014] As a preferred technical solution of the present invention, the diameter of the working vesicles is 50-100 nm (allowing for fluctuations up and down according to the material, sample characteristics and test requirements)

[0015] As a preferred technical solution of the present invention, there is no affinity between the biological macromolecule samples and the amphiphilic molecules.

[0016] As a preferred technical solution of the present invention, the biomacromolecule sample has an affinity with the amphiphilic molecule.

[0017] As a preferred technical solution of the present invention, the removal method in step S3 is biochemical degradation, filtration or centrifugation. For example, proteases or nucleases are used to hydrolyze proteins or nucleic acids.

[0018] As a preferred technical solution of the present invention, it further includes the step: S4. Loading the working vesicles onto the grid: The loading methods include non-specific adsorption, specific affinity interface enrichment of the working vesicles, and directly loading the vesicles onto the electron microscope grid without interface adsorption.

[0019] As a preferred technical solution of the present invention, the loading method in step S4 is non-specific adsorption, and the grid is a non-specific adsorption grid such as graphene or graphene oxide.

[0020] As a preferred technical solution of the present invention, the loading method in step S4 is specific affinity adsorption, and the grid is an affinity grid that can specifically affinity with the outer surface of the vesicles.

[0021] As a preferred technical solution of the present invention, the loading method in step S4 is directly loading the vesicles onto the electron microscope grid without interface adsorption.

[0022] The present invention also discloses the application of the high-efficiency packaging method of biomacromolecules, which is used for drug packaging and the preparation of cryo-electron microscopy samples.

[0023] The beneficial effects produced by adopting the above technical solutions are as follows: The present invention improves the packaging efficiency of biomacromolecule samples, breaks through the technical bottleneck that biomacromolecule samples with low-stability complexes are prone to dissociation, destruction or serious preferential orientation. It has a high degree of freedom in material selection and reduces material costs. It can be applied to drug packaging and the preparation of cryo-electron microscopy samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described in detail below with reference to the drawings and specific embodiments.

[0025] Figure 1a Shows a schematic diagram of a cryo-electron microscopy sample prepared based on a holy carbon grid in the prior art;

[0026] Figure 1b Shows a schematic diagram of a cryo-electron microscopy sample prepared based on an affinity grid or a non-specific adsorption grid such as graphene or graphene oxide in the prior art;

[0027] Figure 2 Shows a schematic diagram of a cryo-electron microscopy sample prepared based on vesicle packaging in an embodiment of the present invention;

[0028] Figure 3 Shows a schematic diagram of the main process for preparing a cryo-EM sample using a vesicle packaging according to an embodiment of the present invention;

[0029] Figure 4a Shows a schematic diagram of extrusion where the biomacromolecule has no affinity with the vesicle in an embodiment of the present invention;

[0030] Figure 4b Shows a schematic diagram of extrusion where the biomacromolecule has a certain affinity with the vesicle in an embodiment of the present invention.

[0031] In the figure: 101, grid; 102, carbon film; 103, amorphous ice after quick-freezing; 104 - 106, biomacromolecule; 107, affinity layer; 201, working vesicle; 301, container; 302, amphiphile; 303 - 304, syringe plug; 305, recombination chamber; 401, primary vesicle. Detailed implementation manners

[0032] Embodiment 1

[0033] Refer to Appendix Figure 3 、 4a And 4b, the method for highly efficient packaging of biomacromolecules of the present invention includes the following steps:

[0034] S1. Fabricate primary vesicles: Enclose and package the biomacromolecule sample with amphiphiles to form primary vesicles; the amphiphiles are phospholipid molecules. The method for fabricating primary vesicles is to place the biomacromolecule sample and phospholipid molecules in a container under temperature conditions that are conducive to maintaining the activity of the biomacromolecule and higher than the phase transition temperature of the phospholipid molecules, and package the biomacromolecule sample inside through the hydration reaction of the phospholipid molecules to form primary vesicles. The outer membrane of the primary vesicles is preferably a phospholipid bilayer at the micron scale. This step is as shown in the legend corresponding to S1 in Figure 3 the figure.

[0035] S2. Fabricate working vesicles: Mechanically transform the primary vesicles into working vesicles. The volume of the working vesicles is smaller than that of the primary vesicles. Specifically, it means that the primary vesicles are repeatedly passed through a filter membrane with a pore diameter equal to or ±10% of the diameter of the working vesicles by means of reciprocating extrusion. The primary vesicles are recombined into working vesicles with a uniform diameter and good monolayer property under the action of the mechanical force of the filter membrane; the diameter of the working vesicles is 50 - 100 nm (allowing for up and down fluctuations according to the material, sample characteristics, and experimental requirements). This step is as shown in Figure 3The legend corresponding to S2 in the figure. The working principle is as follows: Mechanical pressure is provided by pushing the piston of the syringe to push the primary vesicles through the filter membrane. The recombination chamber 305 between the two syringes is separated by the filter membrane. Pushing the pistons of the syringes at both ends can make the primary vesicle solution repeatedly pass through the filter membrane in the recombination chamber, thereby preparing working vesicles with good diameter uniformity and monolayer properties.

[0036] Step S2 is divided into two cases: The first case is that there is no specific affinity between the biological macromolecule sample and the amphiphile as shown in Figure 4a . This is applicable to the situation where the sample needs to maintain the complex assembly state under high-concentration conditions (such as greater than 1 mg / ml). To make the final sample suitable for cryo-EM data collection, it is only necessary to make the primary vesicles encapsulate high-concentration biological macromolecules ( Figure 4a ). The second case is that there is specific affinity between the biological macromolecule sample and the amphiphile as shown in Figure 4b . This is applicable to the situation where the sample does not need to maintain the complex assembly at a very high concentration. The biological macromolecules can be anchored to the vesicle phospholipid membrane through specific affinity, so as to achieve the effect of concentrating the biological macromolecules when the diameter of the primary vesicles shrinks ( Figure 4b ). For example: The two-dimensional phospholipid membrane is gradually hydrated in the biological macromolecule solution by gentle hydration method to prepare primary vesicles with an average diameter of 5 μm. During the formation of the primary vesicles, they will encapsulate the surrounding liquid, and the concentration of the biological macromolecules in the internal encapsulated liquid is of the same order of magnitude as that outside. After incubation, the biological macromolecules inside the primary vesicles will bind to the phospholipid membrane by affinity. At this time, the number of biological macromolecules anchored on the surface of the primary vesicles is proportional to the area of the phospholipid membrane. The primary vesicles are mechanically recombined by the extrusion method to prepare working vesicles with an average diameter of 100 nm. When the diameter is reduced by 50 times, the area of the vesicle phospholipid membrane and the number of biological macromolecules encapsulated in the vesicles are reduced to 1 / 50 2 , and the volume is reduced to 1 / 50 3 . As a result, the equivalent concentration of the biological macromolecules packaged in the vesicles is increased by 50 times during the process of the reduction of the vesicle diameter, achieving an ultra-high packaging efficiency. This method can prepare samples with high-concentration biological macromolecules packaged in vesicles from biological macromolecule samples with low concentrations, improving the utilization efficiency of biological macromolecules.

[0037] S3. Remove the residual sample: Remove the free biological macromolecule sample that has not been packaged by the vesicles. The removal method is biochemical degradation, filtration or centrifugation. For example, protease or nuclease is used to hydrolyze proteins or nucleic acids.

[0038] The packaging method of this embodiment is applied to the high-efficiency packaging of drugs and vaccines, and step S3 is a non-essential step.

[0039] The unexpected technical effects brought by this packaging method also include: this method can efficiently package biological macromolecules inside vesicles without strictly restricting the phospholipid formulation, with a high degree of freedom in material selection and reduced material costs. The phospholipid components can be widely selected according to needs. For example, phospholipids with a very low phase transition temperature can be selected to facilitate the stability of vesicles under low-temperature conditions, or components identical to those of biological cell membranes can be selected to improve the biocompatibility of vesicles.

[0040] Example Two

[0041] The feature of this example is that after step S3, step S4 is carried out: loading the working vesicles onto the grid. The loading methods include non-specific adsorption, specific affinity interface enrichment of the working vesicles, and directly loading the vesicles onto the electron microscope grid without passing through an interface.

[0042] The loading method in step S4 is divided into three types. The first loading method is non-specific adsorption, and the grid is a non-specific adsorption grid such as graphene or graphene oxide. It is applicable to the case where the sample amount is sufficient.

[0043] The second loading method is specific affinity adsorption, and the grid can specifically affinity the outer surface of the vesicles. It is applicable to the case where the sample is rare.

[0044] The third loading method is directly loading the vesicles onto the electron microscope grid without passing through an interface. The grid is a holycarbon grid, which is applicable to the case where the requirement for the sample density in the electron microscope photo is not high.

[0045] The packaging method of this example is applied to the preparation of cryo-electron microscopy samples.

[0046] The above description is only proposed as a technically feasible solution of the present invention and does not serve as a single limiting condition for its technical solution itself.

Claims

1. A method for efficient packaging of biological macromolecules, characterized in that, The following steps are involved: S1. Preparation of primary vesicles: Assemble primary vesicles with amphiphilic molecules to package biomacromolecule samples; S2. Making working vesicles: rupture the primary vesicles and reorganize them into working vesicles. The volume of the working vesicles is smaller than that of the primary vesicles.

2. The high-efficiency packaging method of biological macromolecules according to claim 1, wherein The step also includes: S3, removing residual samples: removing free biomacromolecule samples that are not packaged by vesicles; Removal methods are biochemical degradation, filtration or centrifugation.

3. The high-efficiency packaging method of biological macromolecules according to claim 1 or 2, characterized in that The amphiphilic molecules in step S1 are phospholipid molecules. The biomacromolecule sample and the phospholipid molecules are placed in a container, and the biomacromolecule sample is packaged inside through the hydration reaction of the phospholipid molecules to form primary vesicles with a diameter greater than or equal to 50-100nm; the primary vesicles are nano- to micron-sized phospholipid bilayers.

4. The high-efficiency packaging method of biological macromolecules according to claim 1 or 2, characterized in that, In step S2, the primary vesicles are repeatedly passed through the filter membrane by mechanical conversion and reciprocating extrusion, and the pore size range of the filter membrane is ±10% of the target diameter of the working vesicles. The primary vesicles are ruptured and reorganized into working vesicles with uniform diameter and good monolayer properties; The diameter of the working vesicle is 50-100 nm.

5. The method for highly efficient packaging of biomacromolecules according to claim 1 or 2, wherein The biomacromolecule sample has no affinity with the amphiphilic molecules.

6. The method for efficient packaging of biological macromolecules according to claim 1 or 2, characterized in that The biomacromolecule sample has affinity with the amphiphilic molecules.

7. The method for highly efficient packaging of biological macromolecules according to claim 2, wherein Also includes the steps: S4. Loading the working vesicles onto the grid: Loading methods include enriching the working vesicles using nonspecific adsorption, specific affinity interface, and directly loading the vesicles onto the electron microscope grid without going through the interface.

8. Use of the method for efficiently packaging biological macromolecules according to any one of claims 1-2, characterized in that: Used for drug or vaccine packaging.

9. Use of the method for efficiently packaging biological macromolecules according to claim 7, characterized in that: Used for preparation of cryo-electron microscopy samples.

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