Biomolecule aggregate freezing soft X-ray imaging method based on synchrotron radiation device and application of biomolecule aggregate freezing soft X-ray imaging method

Abstract

The invention provides a freezing soft X-ray imaging method of a biomolecule aggregate based on a synchrotron radiation device and application of the freezing soft X-ray imaging method. The method comprises the following steps: adding a biomolecule aggregate solution on a carrier net, carrying out natural sedimentation, removing redundant liquid, carrying out rapid freezing, and carrying out low-temperature soft X-ray imaging based on a synchrotron radiation imaging line station after freezing, the biomolecules are biomolecules capable of generating a phase separation behavior; the biomolecules are selected from any one or a combination of at least two of protein, nucleic acid molecules or polysaccharide molecules; and the energy range of the soft X rays is 280 to 520 eV. According to the method disclosed by the invention, the morphology imaging of the complete biomolecular aggregate can be realized under the conditions of no marking and no slicing. Furthermore, the instantaneous structure of the aggregate can be more truly preserved by a freezing immobilization method in which the solution / cell containing the aggregate is rapidly frozen in liquid nitrogen compared to chemical immobilization.

Description

Technical Field

[0001] The present invention belongs to the field of biological imaging, and specifically relates to a method for cryo-soft X-ray imaging of biomolecular aggregates based on a synchrotron radiation device and its application, in particular to a method for cryo-microscopic imaging of protein aggregates, and a method for microscopic imaging of the regulatory effect of metal ions on the three-dimensional morphology of protein aggregates. Background Art

[0002] Through multivalent interactions between multiple amino acid molecules / nucleotide molecules, proteins / nucleic acid molecules in cells can undergo a liquid-liquid phase separation process to form membraneless biomolecular condensates. Condensates are usually associated with certain life activities, such as the storage and removal of specific biomolecules, the activation of enzymatic reactions, and the blocking of specific physiological activities. The morphological changes and structural variations of condensates are usually related to the occurrence of diseases, such as neurodegenerative diseases, cancer, autoimmune diseases, etc.

[0003] Studies have found that metal ions can affect the structure and physicochemical properties of biomolecular aggregates by regulating multivalent interactions between biomolecules. In fact, various metal ions in cells can activate / inhibit specific physiological pathways by regulating the phase separation process of protein / nucleic acid molecules. Studying the effects of metal ions on the aggregation behavior of biomolecules, including changes in the morphology and structure of aggregates after the addition of metal ions, is of great significance for understanding the regulatory mechanism of metal ions on physiological activities.

[0004] Studying the effect of metal ions on the structure and morphology of condensates has the following challenges: First, the fixation and capture of dynamic structures. Unlike membrane-containing organelles, biomolecular condensates usually exhibit relatively dynamic structural characteristics, which is also a major difficulty in structural imaging: the condensate itself is in a metastable physical state, especially in the early stages of condensate formation. At the beginning, condensates generally have droplet properties, the internal components are constantly flowing, and they are constantly exchanging substances with the external environment. Over time, droplet-shaped condensates usually transform into states with stronger structural rigidity and weaker internal fluidity, such as gel, fiber, glass, etc., and the morphology will also change greatly. This change in physical properties is usually related to life processes.

[0005] Traditional aldehyde-based chemical fixation methods are difficult to capture the transient state of the dynamic changes of condensates. At the same time, aldehyde fixatives promote cross-linking between biological molecules and change the internal structure of condensates. Compared with chemical fixation, the cryofixation method of rapidly freezing the solution / cells containing condensates in liquid nitrogen can more realistically preserve the transient structure of condensates. Existing imaging methods also have technical defects. Since the size of condensates is in the micron and submicron levels, the observation of their internal structure requires imaging technology with nanoscale resolution. However, traditional nano-imaging technology, mainly super-resolution fluorescence imaging technology and electron microscopy imaging technology, has shown certain limitations in condensate imaging research: fluorescence imaging depends on labeled molecules and cannot observe the spatial distribution of unlabeled molecules. Some super-resolution imaging technologies (such as STED and PALM) have strict requirements on the performance of labeled molecules. The introduction of larger fluorophores (such as green fluorescent protein GFP) may affect the formation process of protein phase separation and the final assembly structure. Although electron microscopy can achieve label-free nano-resolution imaging of condensates, the penetration of electron beams is weak and the penetration depth is only a few hundred nanometers. It is impossible to use cryo-electron microscopy tomography (Cryo-ET) to achieve three-dimensional structural observation of complete condensates of micrometer size. Therefore, samples usually need to be sliced ​​before electron microscopy imaging, and the sample preparation process is complicated and easy to introduce artifacts. Therefore, developing a new three-dimensional imaging method for condensate morphology and structural changes to capture the instantaneous state of condensates during dynamic changes has important application value in biomolecular research. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device and its application. The present invention can achieve three-dimensional high-resolution and large depth-of-field imaging of the instantaneous structure of condensates without a complicated slicing process, and can achieve three-dimensional imaging of the morphology and structural changes of condensates after the addition of metal ions.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] First invention, the present invention provides a method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device, the method comprising: adding a biomolecular condensate solution to a grid, allowing natural sedimentation, removing excess liquid, and rapidly freezing, and after freezing, performing low-temperature soft X-ray imaging based on a synchrotron radiation imaging beamline;

[0009] The biomolecule is a biomolecule capable of phase separation;

[0010] The biomolecule is selected from: any one or a combination of at least two of proteins, nucleic acid molecules or polysaccharide molecules;

[0011] The energy range of the soft X-ray is 280-520 eV.

[0012] The soft X-ray imaging technology based on synchrotron radiation devices can effectively make up for the defects of the above-mentioned traditional imaging: biological molecules rich in C and N elements show higher absorption contrast than water molecules in the soft X-ray "water window" band (284-520eV), so that the aggregates formed by biological molecules can achieve clear morphological imaging without labeling; in addition, synchrotron radiation X-ray imaging technology has the advantages of large depth of field and nano-resolution, high penetration depth, and can achieve three-dimensional imaging of the complete structure of micron-sized aggregates. More importantly, compared with fluorescence imaging and electron microscopy imaging, the development of dual-energy X-ray absorption contrast imaging or X-ray fluorescence imaging technology has enabled X-ray imaging technology to have the function of element resolution, combined with the advantages of nano-level resolution and high depth of field, it can achieve direct observation of the spatial distribution of metal ions in aggregates. Therefore, the synchrotron radiation X-ray imaging method has great application potential in the morphological imaging and structural analysis of biomolecular aggregates. Based on this, the present invention combines the freezing sample preparation technology with the synchrotron radiation X-ray imaging technology, and designs a freezing soft X-ray imaging method to image the three-dimensional structure and morphology of biomolecular aggregates after the addition of metal ions.

[0013] Preferably, the biomolecule condensate solution is prepared by a method comprising the following steps:

[0014] (S1) mixing the biomolecule with a phase separation buffer to obtain a first solution;

[0015] (S2) Mixing the metal salt with the first solution to obtain a biomolecule aggregate solution.

[0016] In the present invention, biological molecules include all proteins that can undergo phase separation, such as cGAS, STING, G3BP1, or non-protein biological molecules that can undergo phase separation, such as DNA, RNA, and polysaccharides.

[0017] Preferably, in step (S1), the components of the phase separation buffer include: a pH stabilizer, an ionic strength stabilizer, a crowding agent and a reducing agent.

[0018] Preferably, the pH stabilizer is selected from any one of Tris-HCl, HEPES or phosphate.

[0019] Preferably, the ionic strength stabilizer is selected from any one of sodium salt, potassium salt or magnesium salt, or a combination of at least two of them.

[0020] Preferably, the crowding agent is selected from any one of PEG, BSA, and sucrose polymer, or a combination of at least two thereof.

[0021] In the present invention, the molecular weight of PEG is in the range of 200-20000, for example, it can be 200, 500, 1000, 5000, 10000, 15000 or 20000.

[0022] In the present invention, the molecular weight of the sucrose polymer is in the range of 1000-20000, for example, it can be 1000, 5000, 10000, 15000 or 20000.

[0023] Preferably, the reducing agent is selected from: any one of dithiothreitol, GSH or TCEP, or a combination of at least two thereof.

[0024] Preferably, the phase separation buffer comprises, by concentration, 5-1000 mM pH stabilizer concentration, 50-1000 mM ionic strength stabilizer, 0.1-10 mM reducing agent, and 0.5-50 mg / mL BSA or 1-10% PEG or 1-10% sucrose polymer.

[0025] Among them, 5-1000mM can be, for example, 5mM, 10mM, 20mM, 50mM, 100mM, 500mM, 800mM or 1000mM, etc. 50-1000mM can be, for example, 50mM, 100mM, 200mM, 400mM, 500mM, 600mM, 800mM or 1000mM, etc. 0.1-10mM can be, for example, 0.1mM, 0.5mM, 1mM, 2mM, 4mM, 6mM, 8mM or 10mM, etc. 0.5-50mg / mL can be, for example, 0.5mg / mL, 1mg / mL, 5mg / mL, 10mg / mL, 20mg / mL, 30mg / mL, 40mg / mL or 50mg / mL, etc. 1-10% can be, for example, 1%, 2%, 4%, 5%, 6%, 8% or 10%, etc. In the present invention, the pH stabilizer has the function of maintaining the pH of the solution at a neutral level to prevent the denaturation and inactivation of biomolecules under acidic or alkaline conditions.

[0026] In the present invention, the role of the ionic strength stabilizer is to provide sufficient ionic strength in the solution to maintain the interaction between components in the biomolecular aggregates and keep the structure of the biomolecular aggregates stable.

[0027] In the present invention, the role of the crowding agent is to use crowding agents of different molecular weights to simulate the crowded environment created in cells due to the presence of various organelles and biomolecules in in vitro experiments.

[0028] In the present invention, the role of the reducing agent is to prevent self-aggregation caused by disulfide cross-linking between protein molecules.

[0029] Preferably, in step (S1), the concentration of the biomolecules in the first solution is 1-1000 nM, for example, 1 nM, 5 nM, 10 nM, 100 nM, 200 nM, 400 nM, 500 nM, 600 nM, 800 nM or 1000 nM, etc.

[0030] In the present invention, the concentration of biomolecules affects the size and number of biomolecule aggregates. At low concentrations, biomolecule aggregates tend to be smaller in size and fewer in number.

[0031] Preferably, in step (S2), the metal salt is a transition metal salt.

[0032] Preferably, the transition metal ions in the transition metal salt are selected from any one or a combination of at least two of iron, zinc, manganese, nickel or cobalt ions.

[0033] In the present invention, the metals involved play an important role in cell life activities and can affect specific cell signaling pathways by regulating the aggregation behavior of key molecules.

[0034] Preferably, the acid ion in the transition metal salt is selected from any one of chloride, acetate, sulfate or nitrate.

[0035] Preferably, in step (S2), after the metal salt is mixed with the first solution, the final concentration of the metal salt is 1-1000 nM, for example, it can be 1 nM, 5 nM, 10 nM, 100 nM, 200 nM, 400 nM, 500 nM, 600 nM, 800 nM or 1000 nM, etc.

[0036] In the present invention, the concentration of the metal salt will affect the size, number and fluidity of the aggregates. Generally speaking, a high concentration of metal salt will cause the biomolecules to form aggregates with larger size, greater number, poorer fluidity and more gel-like.

[0037] Preferably, the carrier mesh is selected from any one of: a copper mesh, a nickel mesh, a molybdenum mesh, a gold mesh or a silicon nitride window.

[0038] Preferably, the dropwise addition volume of the biomolecular condensate solution is 1-20 μL, for example, it can be 1 μL, 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, 12 μL, 14 μL, 16 μL, 18 μL or 20 μL, etc.

[0039] In the present invention, the role of natural sedimentation is to deposit the coagulates in the solution on the grid by gravity. The sedimentation time will affect the density of the coagulates on the grid. If the density of the coagulates on the grid is too high, the 3D CT acquisition angle will be limited, affecting the 3D imaging effect. The sedimentation time is more suitable at about 5-30 minutes.

[0040] Preferably, the rapid freezing step comprises: rapidly immersing the carrier grid in a cryogenic liquid, rapidly freezing the grid and then storing the grid at liquid nitrogen temperature.

[0041] In the present invention, the role of rapid freezing is to form glassy ice in the agglomerate, thereby preserving the integrity and authenticity of the internal structure to the greatest extent.

[0042] Preferably, the cryogenic liquid is a liquid below -100°C, including liquid nitrogen or liquid ethane (these two are the main commonly used refrigerants).

[0043] Preferably, the speed of immersion in the cryogenic liquid is not less than 1 m / s.

[0044] In the present invention, the freezing speed will affect the imaging effect; if the freezing speed is too slow, crystalline ice tends to form in the agglomerate, and the crystalline ice will destroy the internal structure of the agglomerate, resulting in poor imaging effect.

[0045] The technical solution of the present invention can realize the morphological imaging of complete biomolecular condensates without labeling and sectioning. In addition, compared with chemical fixation, the cryofixation method of rapidly freezing the solution / cells containing condensates in liquid nitrogen can more realistically preserve the instantaneous structure of the condensates. In view of the fact that metal ions have a regulatory effect on biomolecular condensates, thereby affecting the function of biomolecules in cells, the present invention combines the freezing sample preparation technology with the synchrotron radiation X-ray imaging technology to achieve clear observation of the instantaneous three-dimensional structure and morphology of biomolecular condensates after the addition of metal ions, providing an effective research method for studying the regulatory mechanism of metal ions on the aggregation behavior of biomolecules.

[0046] As a preferred technical solution of the present invention, a method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device comprises the following steps:

[0047] (S1) mixing the biomolecule with a phase separation buffer to obtain a first solution;

[0048] The phase separation buffer comprises, according to the concentration agent, 5-1000 mM pH stabilizer concentration, 50-1000 mM ionic strength stabilizer, 0.1-10 mM reducing agent, and 0.5-50 mg / mL BSA or 1-10% PEG or 1-10% sucrose polymer; the concentration of the biomolecule in the first solution is 1-1000 nM; the biomolecule is a biomolecule capable of phase separation, and the biomolecule is selected from: any one or a combination of at least two of a protein, a nucleic acid molecule or a polysaccharide molecule;

[0049] (S2) mixing the metal salt with the first solution to obtain a biomolecule condensate solution;

[0050] The metal salt is a transition metal salt; the transition metal ion in the transition metal salt is selected from: any one of iron, zinc, manganese, nickel or cobalt ions or a combination of at least two; the acid ion in the transition metal salt is selected from: any one of chloride ion, acetate, sulfate or nitrate; after the metal salt is mixed with the first solution, the final concentration of the metal salt is 1-1000 nM;

[0051] (S3) adding 1-20 μL of the biomolecular condensate solution onto the grid, allowing it to settle naturally, removing excess liquid, quickly immersing the grid into a cryogenic liquid, rapidly freezing it and storing it at liquid nitrogen temperature, and performing low-temperature soft X-ray imaging based on a synchrotron radiation imaging beamline after freezing;

[0052] The grid is selected from any one of an electron microscope copper grid, a nickel grid, a molybdenum grid, a gold grid or a silicon nitride window; the cryogenic liquid is a liquid below -100°C, including liquid nitrogen or liquid ethane; and the speed of immersion in the cryogenic liquid is not less than 1 m / s.

[0053] In a second aspect, the present invention provides the application of the cryo-soft X-ray imaging method of biomolecular condensates based on a synchrotron radiation device described in the first invention in biomolecular imaging.

[0054] The application scenario of the present invention is that the formation and structural changes of biomolecular condensates are usually accompanied by specific physiological processes, such as the abnormal aggregation of certain proteins can lead to neurodegenerative diseases and cancer; in normal cells, many functional proteins play a role by forming condensates, such as cGAS / STING proteins regulating the natural immune process by forming micrometer-scale condensates. By developing new technologies to achieve three-dimensional spatial imaging of the real structure of condensates, on the one hand, it helps to further understand the mechanism of occurrence of certain physiological processes, and on the other hand, it is also conducive to the development of drugs for certain physiological processes.

[0055] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] The present invention is a method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device, which can achieve morphological imaging of complete biomolecular condensates without labeling or slicing. Compared with chemical fixation, the cryo-fixation method of rapidly freezing the solution / cells containing condensates in liquid nitrogen can more realistically preserve the instantaneous structure of the condensates. Combining the cryo-sample preparation technology with the synchrotron radiation X-ray imaging technology, clear observation of the instantaneous three-dimensional structure and morphology of biomolecular condensates after the addition of metal ions can be achieved, providing an effective research method for studying the regulatory mechanism of metal ions on the aggregation behavior of biomolecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a bright field microscope image of the protein-DNA molecular condensate prepared in Example 1 of the present invention.

[0059] Figure 2 The Zn prepared in Example 2 of the present invention 2+ Confocal fluorescence microscopy image of protein-DNA condensates before addition.

[0060] Figure 3 The Zn prepared in Example 2 of the present invention 2+ Confocal fluorescence microscopy imaging of protein-DNA molecular condensates after addition.

[0061] Figure 4 The Zn prepared in Example 3 of the present invention 2+ TEM imaging of protein-DNA molecular condensates after addition.

[0062] Figure 5 The Zn prepared in Example 4 of the present invention 2+ STEM-EDS imaging of protein-DNA molecular condensates after addition.

[0063] Figure 6 Zn prepared in Example 5 of the present invention 2+ Synchrotron radiation cryo-soft X-ray imaging of protein-DNA molecular condensates after addition.

[0064] Figure 7 Co prepared in Example 6 of the present invention 2+ Fluorescence imaging of RNA molecule condensates after addition.

[0065] Figure 8 Co prepared in Example 6 of the present invention 2+ Synchrotron radiation cryo-soft X-ray imaging of RNA molecule condensates after addition.

[0066] Fig. 9 Fe prepared in Example 7 of the present invention 3+Synchrotron radiation cryo-soft X-ray imaging of polysaccharide molecular aggregates after addition.

[0067] Fig.10 Flowchart of the synchrotron cryo-soft X-ray imaging method for biomolecular condensates. DETAILED DESCRIPTION

[0068] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0069] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0070] Example 1

[0071] Stimulated formation of protein-DNA condensates

[0072] A) Take 100 μL 1M Tris-HCl (pH=7.4), 150 μL 5M NaCl solution, 250 μL 20 mg / mL BSA solution and 50 μL 100 mM DTT solution, add sterile enzyme-free water to make up to 5 mL to obtain a phase separation buffer. In the buffer, the final concentration of Tris-HCl is 20 mM, the final concentration of NaCl is 150 mM, the final concentration of BSA is 1 mg / mL, and the final concentration of DTT is 1 mM.

[0073] B) Dilute the purified human full-length cGAS (cGAS) in vitro with phase separation buffer to a final concentration of 10 μM in a final volume of 100 μL, then add 1 μL of 100 μM 45 bp dsDNA to a final DNA concentration of 1 μM. Mix well at room temperature.

[0074] C) Weigh 68.15 mg of anhydrous zinc chloride and dissolve it in 50 mL of 20 mM Tris-HCl (pH=7.4) to prepare a 10 mM ZnCl2 solution.

[0075] D) Add 0.5 μL of 10 mM ZnCl2 solution to the mixed solution of cGAS protein and dsDNA. 2+ The final concentration is 50 μM. Mix well at room temperature and let stand for 60 minutes. Drop the mixture on a glass slide and observe under a microscope.

[0076] Results Figure 1 cGAS protein in dsDNA and Zn 2+Micron-sized aggregates are produced under the action of

[0077] Example 2

[0078] Fluorescence imaging of protein-DNA condensates

[0079] A) Take 100 μL 1M Tris-HCl (pH=7.4), 150 μL 5M NaCl solution, 250 μL 20 mg / ml BSA solution and 50 μL 100 mM DTT solution, add sterile enzyme-free water to make up to 5 mL to obtain a phase separation buffer. In the buffer, the final concentration of Tris-HCl is 20 mM, the final concentration of NaCl is 150 mM, the final concentration of BSA is 1 mg / mL, and the final concentration of DTT is 1 mM.

[0080] B) Dilute the in vitro purified full-length human cyclic AMP-GMP synthetase (cGAS) with phase separation buffer to a final concentration of 10 μM, of which 3% is GFP-coupled cGAS protein, and the final volume is 100 μL, followed by the addition of 1 μL 100 μM 45 bp dsDNA (containing 3% TAMRA-dsDNA), with a final DNA concentration of 1 μM. Mix well at room temperature.

[0081] C) Take 10 μL and drop it on a glass slide for confocal imaging. For imaging results, see Figure 2 .

[0082] D) Weigh 68.15 mg of anhydrous zinc chloride and dissolve it in 50 mL of 20 mM Tris-HCl (pH=7.4) to prepare a 10 mM ZnCl2 solution.

[0083] E) Add 10 mM ZnCl2 solution to the mixed solution of cGAS protein and dsDNA. 2+ The final concentration is 50 μM. Mix well at room temperature and let stand for 60 min. Add the mixture onto a glass slide and perform confocal imaging. The imaging results are shown in Figure 3 .

[0084] Depend on Figure 2 It can be seen that after cGAS protein binds to double-stranded dsDNA, it forms a spherical aggregate with a size of 1-2 μm and a regular morphology; 2+ After addition, the morphology of the aggregates became irregular and showed a certain heterogeneity: dsDNA was mainly distributed in the large-sized spherical regions in the aggregates and was less distributed in the connecting parts between the spherical regions.

[0085] Example 3

[0086] Zn 2+ Ultrastructural imaging of protein-DNA condensates after treatment (electron microscopy imaging)

[0087] A) Take 100 μL 1M Tris-HCl (pH=7.4), 150 μL 5M NaCl solution, 250 μL 20 mg / mL BSA solution and 50 μL 100 mM DTT solution, add sterile enzyme-free water to make up to 5 mL to obtain a phase separation buffer. In the buffer, the final concentration of Tris-HCl is 20 mM, the final concentration of NaCl is 150 mM, the final concentration of BSA is 1 mg / mL, and the final concentration of DTT is 1 mM.

[0088] B) Dilute the in vitro purified full-length human cyclic AMP-GMP synthetase (cGAS) with phase separation buffer, the final concentration of the protein solution is 10 μM and the final volume is 100 μL, then add 1 μL of 100 μM 45 bp dsDNA, the final concentration of DNA is 1 μM. Mix well at room temperature.

[0089] C) Weigh 109.5 mg of zinc acetate dihydrate and dissolve it in 50 mL of sterile enzyme-free water to prepare a 10 mM Zn(OAc)2 solution.

[0090] D) Add 0.5 μL of 10 mM Zn(OAc)2 solution to the mixed solution of cGAS protein and dsDNA. 2+ The final concentration is 50 μM. Mix well at room temperature and let stand for 60 minutes.

[0091] E) Grid pretreatment: Place the gold grid carbon support film in an eight-well confocal plate, add 500 μL 20 mg / mL BSA, block overnight at 37°C, then rinse with sterile enzyme-free water 2-3 times and air-dry naturally.

[0092] F) 10 μL of the reaction solution was added dropwise onto the treated carbon film, and after natural sedimentation for 15 min, the excess liquid was removed with filter paper, and then 5 μL of 2.5% glutaraldehyde fixative was added dropwise, and fixed at room temperature for 10 min.

[0093] G) The carbon film was washed 2-3 times in sterile enzyme-free water, then air-dried and subjected to TEM imaging.

[0094] Imaging results can be found in Figure 4 When cGAS binds to dsDNA, Zn 2+ The aggregates contain two structures: regular spherical structures with a size larger than 500nm; and adhesions of small spherical structures that are self-aggregated with cGAS.

[0095] Example 4

[0096] Zn 2+ After treatment, the spatial distribution of metal elements in protein-DNA condensates was imaged.

[0097] A) Take 100 μL 1M Tris-HCl (pH=7.4), 150 μL 5M NaCl solution, 250 μL 20 mg / mL BSA solution and 50 μL 100 mM DTT solution, add sterile enzyme-free water to make up to 5 mL to obtain a phase separation buffer. In the buffer, the final concentration of Tris-HCl is 20 mM, the final concentration of NaCl is 150 mM, the final concentration of BSA is 1 mg / mL, and the final concentration of DTT is 1 mM.

[0098] B) Dilute the in vitro purified full-length human cyclic AMP-GMP synthetase (cGAS) with phase separation buffer, the final concentration of the protein solution is 10 μM and the final volume is 100 μL, then add 1 μL of 100 μM 45 bp dsDNA, the final concentration of DNA is 1 μM. Mix well at room temperature.

[0099] C) Weigh 68.15 mg of anhydrous zinc chloride and dissolve it in 50 mL of 20 mM Tris-HCl (pH=7.4) to prepare a 10 mM ZnCl2 solution.

[0100] D) Add 0.5 μL of 10 mM ZnCl2 solution to the mixed solution of cGAS protein and dsDNA. 2+ The final concentration is 50 μM. Mix well at room temperature and let stand for 60 minutes.

[0101] E) Grid pretreatment: Place the copper grid carbon support membrane in an eight-well confocal plate, add 500 μL 20 mg / ml BSA, block overnight at 37°C, then rinse with sterile enzyme-free water 2-3 times and air-dry naturally.

[0102] F) 10 μL of the reaction solution was added dropwise onto the treated carbon film, and after natural sedimentation for 15 min, the excess liquid was removed with filter paper, and then 5 μL of 2.5% glutaraldehyde fixative was added dropwise, and fixed at room temperature for 10 min.

[0103] G) The carbon film was washed 2-3 times in sterile enzyme-free water, then air-dried, and subjected to STEM-EDS elemental analysis.

[0104] Results Figure 5 The distribution of Zn in the aggregate is not uniform: compared with the connecting parts, Zn is obviously enriched in the large-sized spherical areas.

[0105] Example 5

[0106] Zn 2+ Synchrotron radiation cryo-soft X-ray imaging of protein-DNA condensates after treatment

[0107] A) Take 100 μL 1M Tris-HCl (pH=7.4), 150 μL 5M NaCl solution, 250 μL 20 mg / mL BSA solution and 50 μL 100 mM DTT solution, add sterile enzyme-free water to make up to 5 mL to obtain a phase separation buffer. In the buffer, the final concentration of Tris-HCl is 20 mM, the final concentration of NaCl is 150 mM, the final concentration of BSA is 1 mg / mL, and the final concentration of DTT is 1 mM.

[0108] B) Dilute the in vitro purified full-length human cyclic AMP-GMP synthetase (cGAS) with phase separation buffer, the final concentration of the protein solution is 10 μM and the final volume is 100 μL, then add 1 μL of 100 μM 45 bp dsDNA, the final concentration of DNA is 1 μM. Mix well at room temperature.

[0109] C) Weigh 68.15 mg of anhydrous zinc chloride and dissolve it in 50 mL of 20 mM Tris-HCl (pH=7.4) to prepare a 10 mM ZnCl2 solution.

[0110] D) Add 0.5 μL of 10 mM ZnCl2 solution to the mixed solution of cGAS protein and dsDNA. 2+ The final concentration is 50 μM. Mix well at room temperature and let stand for 60 minutes.

[0111] E) Grid pretreatment: The nickel grid Fanghua membrane was pretreated with 20 mg / ml BSA solution for 30 min, then washed with water 2-3 times, and the excess water was absorbed by filter paper.

[0112] F) 5 μL of reaction solution was added to the treated Fanghua membrane. After natural sedimentation for 10 min, the excess liquid was absorbed with filter paper to form a thin layer of water film on the Fanghua membrane. The membrane was then inserted into liquid ethane for rapid freezing. Projections of the aggregates at different angles (-65°-65°, 1° interval) were collected under 520 eV energy X-rays, and three-dimensional reconstruction and rendering were performed.

[0113] See also Figure 6 , the three-dimensional imaging results show that the ends of the aggregates swell into spheres (according to the fluorescence results, the interior of the aggregates should be enriched with DNA molecules). The spheres are connected to each other, which corresponds to the electron microscope ultramicroscopic imaging results. The results of Example 5 are compared with the results of Examples 1-4 to find that the synchrotron radiation X-ray imaging technology used in the present invention can obtain the three-dimensional spatial morphology of the aggregates without the need for fluorescent molecule labeling, which is impossible with fluorescence microscopy and electron microscopy. The use of freezing technology avoids the use of chemical fixatives (such as paraformaldehyde) and retains the authenticity of the sample morphology and structure to the greatest extent.

[0114] Example 6

[0115] Co 2+ Synchrotron-radiation cryo-soft X-ray imaging of RNA molecular condensates

[0116] A) Take 100 μL 1M HEPES buffer (pH=7.4), 135 μL 5M KCl solution, 15 μL 5M MgCl2 solution, PEG8000 solution and 50 μL 100mM TCEP solution, add sterile enzyme-free water to make up to 5mL to obtain a phase separation buffer. In the buffer, the final concentration of HEPES is 20mM, the final concentration of KCl is 135mM, the final concentration of MgCl2 is 15mM, the mass fraction of PEG8000 is 5%, and the final concentration of TCEP is 1mM.

[0117] B) 45 bp dsRNA (3% FAM) was diluted with phase separation buffer to a final concentration of 1 mM in a final volume of 100 μL.

[0118] C) Weigh 145.5 mg of cobalt nitrate hexahydrate and dissolve it in 50 mL of sterile enzyme-free water to prepare a 10 mM Co(NO3)2 solution.

[0119] D) Add 10 μL of 10 mM Co(NO3)2 solution to the dsRNA solution. 2+ The final concentration is 1 mM. Mix well at room temperature and let stand for 120 minutes.

[0120] E) Grid pretreatment: The molybdenum grid Fanghua membrane was pretreated with 20 mg / mL BSA solution for 30 min, then washed with water 2-3 times, and the excess water was absorbed by filter paper.

[0121] F) 10 μL of reaction solution was added to the treated Fanghua membrane. After natural sedimentation for 10 min, fluorescence imaging was performed, and then the excess liquid was absorbed with filter paper to form a thin layer of water film on the Fanghua membrane. Then, it was inserted into liquid ethane for rapid freezing, and projection images of the aggregates at different angles (-65°-65°, 1° interval) were collected under 520 eV energy X-rays, and three-dimensional reconstruction and rendering were performed.

[0122] See also Figure 7 , in Co 2+ Under the action of dsRNA molecules, they aggregate into irregular condensates. Figure 8 This is the three-dimensional morphology of the condensate obtained using synchrotron radiation low-temperature soft X-ray three-dimensional imaging technology.

[0123] Example 7

[0124] Fe 3+ Synchrotron radiation cryo-soft X-ray imaging of polysaccharide molecular aggregates

[0125] A) Take 500 μL of 10×PBS buffer (pH=7.4), 135 μL of 5M NaCl solution, 15 μL of 5M MgCl2 solution, sucrose polymer (MW=8000) solution and 50 μL of 100mM GSH solution, add sterile enzyme-free water to make up to 5mL to obtain a phase separation buffer. In the buffer, the final concentration of NaCl is 135mM, the final concentration of MgCl2 is 15mM, the mass fraction of sucrose polymer is 5%, and the final concentration of GSH is 1mM.

[0126] B) Dilute the glycogen solution with phase separation buffer to a final concentration of 100 mM and a final volume of 100 μL.

[0127] C) Weigh 199.9 mg of ferric sulfate and dissolve it in 50 mL of sterile enzyme-free water to prepare a 10 mM Fe2(SO4)3 solution.

[0128] D) Add 10 μL of 10 mM Fe2(SO4)3 solution to the glycogen solution. 3+ The final concentration is 2 mM. Mix well and let stand for 6 hours at room temperature.

[0129] E) Grid pretreatment: The silicon nitride window was pretreated with 20 mg / mL BSA solution for 30 min, then washed with water 2-3 times, and excess water was absorbed with filter paper.

[0130] F) 10 μL of the reaction solution was added to the treated silicon nitride window. After natural sedimentation for 15 min, the excess liquid was absorbed with filter paper to form a thin layer of water film on the silicon nitride window. The window was then inserted into liquid ethane for rapid freezing. Projections of the aggregates at different angles (-65°-65°, 1° interval) were collected under 520 eV energy X-rays, and three-dimensional reconstruction and rendering were performed.

[0131] See also Fig. 9 , in Fe 3+ Under this action, polysaccharide molecules aggregate into aggregates with a size of micrometers.

[0132] In summary, the present invention provides a method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device, the method flow is as follows: Fig.10As shown; it can realize the morphological imaging of complete biomolecular condensates without labeling and sectioning. In addition, compared with chemical fixation, the cryofixation method of rapidly freezing the solution / cells containing condensates in liquid nitrogen can more realistically preserve the instantaneous structure of the condensates. In view of the regulatory effect of metal ions on biomolecular condensates, thereby affecting the function of biomolecules in cells, the present invention combines the freezing sample preparation technology with the synchrotron radiation X-ray imaging technology to achieve clear observation of the instantaneous three-dimensional structure and morphology of biomolecular condensates after the addition of metal ions, providing an effective research method for studying the regulatory mechanism of metal ions on the aggregation behavior of biomolecules.

[0133] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device, characterized in that: The method comprises: adding a biomolecule condensate solution onto a grid, allowing natural sedimentation, removing excess liquid, rapidly freezing, and performing low-temperature soft X-ray imaging based on a synchrotron radiation imaging beamline after freezing; The biomolecule is a biomolecule capable of phase separation; The biomolecule is selected from: any one or a combination of at least two of proteins, nucleic acid molecules or polysaccharide molecules; The energy range of the soft X-ray is 280-520 eV.

2. The method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device according to claim 1, characterized in that: The biomolecule condensate solution is prepared by a method comprising the following steps: (S1) mixing the biomolecule with a phase separation buffer to obtain a first solution; (S2) Mixing the metal salt with the first solution to obtain a biomolecule aggregate solution.

3. The method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device according to claim 2, characterized in that: In step (S1), the components of the phase separation buffer include: a pH stabilizer, an ionic strength stabilizer, a crowding agent and a reducing agent; Preferably, the pH stabilizer is selected from any one of Tris-HCl, HEPES or phosphate; Preferably, the ionic strength stabilizer is selected from: any one or a combination of at least two of a sodium salt, a potassium salt or a magnesium salt; Preferably, the crowding agent is selected from: any one or a combination of at least two of PEG, BSA, and sucrose polymer; Preferably, the reducing agent is selected from: any one or a combination of at least two of dithiothreitol, GSH or TCEP; Preferably, the phase separation buffer comprises, by concentration, 5-1000 mM pH stabilizer concentration, 50-1000 mM ionic strength stabilizer, 0.1-10 mM reducing agent, and 0.5-50 mg / mL BSA or 1-10% PEG or 1-10% sucrose polymer.

4. The method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device according to claim 2 or 3, characterized in that: In step (S1), the concentration of the biomolecules in the first solution is 1-1000 nM.

5. The method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device according to any one of claims 2 to 4, characterized in that: In step (S2), the metal salt is a transition metal salt; Preferably, the transition metal ion in the transition metal salt is selected from any one or a combination of at least two of iron, zinc, manganese, nickel or cobalt ions; Preferably, the acid ion in the transition metal salt is selected from any one of chloride, acetate, sulfate or nitrate.

6. The method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device according to any one of claims 2 to 5, characterized in that: In step (S2), after the metal salt is mixed with the first solution, the final concentration of the metal salt is 1-1000 nM.

7. The method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device according to any one of claims 1 to 6, characterized in that: The grid is selected from any one of an electron microscope copper grid, a nickel grid, a molybdenum grid, a gold grid or a silicon nitride window; Preferably, the dropwise addition volume of the biomolecule condensate solution is 1-20 μL.

8. The method for cryo-soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device according to any one of claims 1 to 7, characterized in that: The rapid freezing step includes: rapidly immersing the carrier grid in a cryogenic liquid, and storing the carrier grid at a liquid nitrogen temperature after rapid freezing.

9. The method for cryo-soft X-ray imaging of biomolecular aggregates based on a synchrotron radiation device according to claim 8, characterized in that: The cryogenic liquid is a liquid below -100°C, including liquid nitrogen or liquid ethane; Preferably, the speed of immersion in the cryogenic liquid is not less than 1 m / s.

10. Application of the cryo-soft X-ray imaging method of biomolecular condensates based on a synchrotron radiation device according to any one of claims 1 to 9 in biomolecular imaging.

Images