A method for imaging biomolecular condensates by cryogenic soft X-ray microscopy based on a synchrotron radiation facility and applications thereof

Abstract

The application provides a kind of biological molecule condensate's frozen soft X-ray imaging method and its application based on synchrotron radiation device, the method comprises: biological molecule condensate solution is added on carrier net, and natural settlement is carried out, remove excess liquid, carry out quick freezing, after freezing, based on synchrotron radiation imaging line station, low-temperature soft X-ray imaging is carried out;The biological molecule is biological molecule capable of phase separation behavior;The biological molecule is selected from any one or combination of at least two in protein, nucleic acid molecule or polysaccharide molecule;The energy range of the soft X-ray is 280-520eV.The method of the application can realize the topographic imaging of complete biological molecule condensate under the condition of not being labeled, without sectioning.In addition, compared with chemical fixation, the instantaneous structure of condensate can be more truly preserved by the cryofixation method of rapidly freezing solution / cell containing condensate in liquid nitrogen.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological imaging, and particularly relates to a method for cryogenic soft X-ray imaging of biomolecular condensates based on a synchrotron radiation device and application thereof, in particular to a method for cryogenic microscopic imaging of protein aggregates, and a method for microscopic imaging of the regulating effect of metal ions on the three-dimensional morphology of protein aggregates. BACKGROUND

[0002] Through multivalent interactions between multiple amino acid molecules / nucleotide molecules, proteins / nucleic acid molecules in cells can undergo liquid-liquid phase separation (LLPS) to form membraneless biomolecular condensates. The condensates are usually associated with certain life activities, such as storage and exclusion of specific biomolecules, activation of enzymatic reactions, and blocking of specific physiological activities. Changes in the morphology and structure of condensates are usually associated with the occurrence of diseases, such as neurodegenerative diseases, cancer, and autoimmune diseases.

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

[0004] There are several challenges in studying the effects of metal ions on the structure and morphology of condensates: one is 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 condensates themselves are in a metastable physical state, especially in the early stages of condensate formation. At the beginning, the general condensates have the properties of droplets, with internal components constantly flowing and constantly exchanging materials with the external environment. With the passage of time, the droplet-like condensates usually transform into states with stronger structural rigidity and weaker internal flow, such as gel-like, fibrous, and glassy states, and the morphology also undergoes large changes. 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 in the process of condensate dynamic changes, and aldehyde fixatives promote cross-linking between biomolecules, changing the internal structure of condensate. Compared with chemical fixation, the cryofixation method by rapidly freezing the solution / cell containing condensate in liquid nitrogen can more truly save the transient structure of condensate. The existing imaging method also has technical defects. Since the size of the condensate is in microns and sub-microns, the observation of the internal structure requires nanoscale resolution imaging technology. However, traditional nanoscale imaging technology, mainly super-resolution fluorescence imaging technology and electron microscopy imaging technology, has 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 techniques (such as STED and PALM) have strict requirements for the performance of labeled molecules. The introduction of larger fluorescent groups (such as green fluorescent protein GFP) may affect the formation process and final assembly structure of protein phase separation. Although electron microscopy imaging can achieve label-free nanoscale resolution imaging of condensate, due to the weak penetration ability of the electron beam, the penetration depth is only a few hundred nanometers, and the complete condensate cannot be observed by Cryo-ET (Cryo-ET) three-dimensional structure observation of micron size. Therefore, the sample usually needs to be sliced before electron microscopy imaging, and the sample preparation process is complex and easy to introduce artifacts. Therefore, developing a new type of three-dimensional imaging method for the morphology and structure changes of condensate to capture the transient state in the process of condensate dynamic changes has important application value in biomolecular research. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide a frozen soft X-ray imaging method of biomolecular condensate based on a synchrotron radiation device and its application. The present application can realize three-dimensional high-resolution large-depth-of-field imaging of the transient structure of condensate without complex slicing process, and realize three-dimensional imaging of the morphology and structure changes of condensate after the addition of metal ions.

[0007] In order to achieve the purpose of the present application, the following technical solutions are adopted:

[0008] The first invention provides a frozen soft X-ray imaging method of biomolecular condensate based on a synchrotron radiation device, which comprises: adding a biomolecular condensate solution on a support net, performing natural sedimentation, removing excess liquid, rapidly freezing, and performing low-temperature soft X-ray imaging based on a synchrotron radiation imaging line station after freezing;

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

[0010] The biomolecule is selected from any one or a combination of at least two of the following: protein, nucleic acid molecule or polysaccharide molecule;

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

[0012] The soft X-ray imaging technology based on the synchrotron radiation device can effectively make up for the defects of the traditional imaging: the biological molecules rich in C and N elements exhibit higher absorption contrast than water molecules in the soft X-ray "water window" band (284-520 eV), so that the aggregates formed by the biological molecules can realize clear topographic imaging without labeling; in addition, the synchrotron X-ray imaging technology has the advantages of large depth of field and nanometer resolution, and has high penetration depth, so that the complete structure of the micron-sized aggregate can be three-dimensionally imaged. More importantly, compared with fluorescence imaging and electron microscope imaging, the development of dual-energy X-ray absorption contrast imaging or X-ray fluorescence imaging technology enables the X-ray imaging technology to have the function of element resolution, and in combination with the advantages of nanoscale resolution and high depth of field, the spatial distribution of metal ions in the aggregate can be directly observed. Therefore, the synchrotron X-ray imaging method has great application potential in the topographic imaging and structure analysis of the biological molecular aggregate. Based on this, the present application combines the freeze sample preparation technology with the synchrotron X-ray imaging technology, and designs a freeze soft X-ray imaging method for imaging the three-dimensional structure and topography of the biological molecular aggregate after the addition of metal ions.

[0013] Preferably, the biological molecular aggregate solution is prepared by a method comprising the following steps:

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

[0015] (S2) mixing a metal salt with the first solution to obtain a biological molecular aggregate solution.

[0016] In the present application, the biological molecule includes all proteins capable of phase separation behavior such as cGAS, STING and G3BP1, or non-protein biological molecules such as DNA, RNA and polysaccharide that can undergo phase separation.

[0017] Preferably, in step (S1), the composition of the phase separation buffer comprises 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 thereof.

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

[0021] In the present application, the PEG has a molecular weight ranging from 200 to 20000, for example, 200, 500, 1000, 5000, 10000, 15000 or 20000, etc.

[0022] In the present application, the sucrose polymer has a molecular weight ranging from 1000 to 20000, for example, 1000, 5000, 10000, 15000 or 20000, etc.

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

[0024] Preferably, the phase separation buffer includes a concentration agent including 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] For example, 5-1000 mM can be 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 500 mM, 800 mM or 1000 mM, etc. For example, 50-1000 mM can be 50 mM, 100 mM, 200 mM, 400 mM, 500 mM, 600 mM, 800 mM or 1000 mM, etc. For example, 0.1-10 mM can be 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM or 10 mM, etc. For example, 0.5-50 mg / mL can be 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL or 50 mg / mL, etc. For example, 1-10% can be 1%, 2%, 4%, 5%, 6%, 8% or 10%, etc. In the present application, the pH stabilizer functions to maintain the solution pH at neutral, preventing denaturation and inactivation of biological molecules under acidic or basic conditions.

[0026] In the present application, the ionic strength stabilizer functions to provide sufficient ionic strength in the solution to maintain the interaction between components within the biological molecule condensate, keeping the structure of the biological molecule condensate stable.

[0027] In the present application, the crowding agent functions to simulate the crowded environment created by the presence of various organelles and biological molecules in cells in vitro experiments using crowding agents of different molecular weights.

[0028] In the present application, the reducing agent functions to prevent self-aggregation of protein molecules due to cross-linking by disulfide bonds.

[0029] Preferably, in step (S1), the concentration of the biomolecule in the first solution 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.

[0030] In the present application, the concentration of the biomolecule affects the size and number of the biomolecule aggregates. At a low concentration, the 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 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.

[0033] In the present application, 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 anion 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 application, the concentration of the metal salt affects the size, number and flowability of the aggregates. Generally speaking, a high concentration of metal salt will cause the biomolecules to form aggregates that are larger in size, more in number, and less flowable, and tend to be gels.

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

[0038] Preferably, the volume of the biomolecule aggregate solution added 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 application, the role of natural sedimentation is to deposit the aggregates in the solution on the support grid by gravity, and the settling time affects the density of the aggregates on the support grid. A high density of aggregates on the support grid will limit the three-dimensional CT collection angle and affect the three-dimensional imaging effect. A settling time of about 5-30 min is more appropriate.

[0040] Preferably, the step of rapid freezing comprises: rapidly immersing the support net into a low-temperature liquid, and storing the rapidly frozen sample at a liquid nitrogen temperature.

[0041] In the present application, the function of rapid freezing is to form glassy ice in the condensed body, so as to maximize the integrity and authenticity of the internal structure.

[0042] Preferably, the low-temperature liquid is a liquid below-100℃, including liquid nitrogen or liquid ethane (the two are the main commonly used refrigerants).

[0043] Preferably, the speed of immersing into the low-temperature liquid is not less than 1m / s.

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

[0045] The technical scheme of the present application can realize the topography imaging of the complete biomolecular condensed body without labeling and slicing. In addition, compared with chemical fixation, the rapid freezing fixation method of rapidly freezing the solution / cell containing the condensed body in liquid nitrogen can more truly preserve the transient structure of the condensed body. Since metal ions have a regulatory effect on biomolecular condensed bodies, thereby affecting the function of biomolecules in cells, the present application combines the freezing sample preparation technology with the synchrotron X-ray imaging technology, realizes the clear observation of the three-dimensional structure and topography of the biomolecular condensed body after the addition of metal ions, and provides an effective research method for studying the regulatory mechanism of metal ions on the aggregation behavior of biomolecules.

[0046] As a preferred technical scheme of the present application, the freezing soft X-ray imaging method of biomolecular condensed body based on a synchrotron device comprises the following steps:

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

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

[0049] (S2) mixing a metal salt with the first solution to obtain a biomolecular condensed body solution;

[0050] The metal salt is a transition metal salt; 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; the acid radical ion in the transition metal salt is selected from any one of chloride, 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) 1-20 μL of the biomolecule condensate solution is added to the support net, natural sedimentation is carried out, the excess liquid is removed, the support net is quickly immersed in a low-temperature liquid, and after quick freezing, it is stored at a liquid nitrogen temperature; after freezing, low-temperature soft X-ray imaging is carried out based on a synchrotron imaging line station;

[0052] The support net is selected from any one of an electron microscope copper net, a nickel net, a molybdenum net, a gold net or a silicon nitride window; the low-temperature liquid is a liquid below-100 ℃, including liquid nitrogen or liquid ethane; and the speed of immersing in the low-temperature liquid is not less than 1 m / s.

[0053] In a second aspect, the application provides an application of the biomolecule condensate freezing soft X-ray imaging method based on a synchrotron device in the first application in biomolecule imaging.

[0054] The application scenario of the application is that the formation and structural change of biomolecule condensates are usually accompanied by specific physiological processes, such as abnormal aggregation of some proteins leading to neurodegenerative diseases and occurrence of cancer; in normal cells, many functional proteins play a role by forming condensates, such as cGAS / STING proteins regulating natural immune processes by forming micron-scale condensates. By developing a new technology to realize three-dimensional spatial imaging of the real structure of condensates, on the one hand, it is helpful to further understand the mechanism of occurrence of some physiological processes, and on the other hand, it is also conducive to drug research and development for some physiological processes.

[0055] The numerical range in the application includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed, and due to the length and for the sake of simplicity, the application does not list the specific point values included in the range.

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

[0057] The application discloses a method for imaging biomolecular condensates by using a soft X-ray cryo-imaging device based on synchrotron radiation, which can realize the imaging of the morphology of complete biomolecular condensates without labeling and slicing. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 The bright field microscope imaging diagram of the protein-DNA molecular condensate prepared in the embodiment 1 of the application.

[0059] Figure 2 The Zn 2+ The confocal fluorescence microscope imaging diagram of the protein-DNA molecular condensate before adding.

[0060] Figure 3 The Zn 2+ The confocal fluorescence microscope imaging diagram of the protein-DNA molecular condensate after adding.

[0061] Figure 4 The Zn 2+ The TEM imaging diagram of the protein-DNA molecular condensate after adding.

[0062] Figure 5 The Zn 2+ The STEM-EDS imaging diagram of the protein-DNA molecular condensate after adding.

[0063] Figure 6 The Zn 2+ The synchrotron radiation cryo-soft X-ray imaging diagram of the protein-DNA molecular condensate after adding.

[0064] Figure 7 The Co 2+ The fluorescence imaging diagram of the RNA molecular condensate after adding.

[0065] Figure 8 The Co 2+ The synchrotron radiation cryo-soft X-ray imaging diagram of the RNA molecular condensate after adding.

[0066] Figure 9 The Fe 3+Synchronous radiation cryogenic soft X-ray imaging of polysaccharide molecule condensate after addition.

[0067] Figure 10 Flow chart of the method of synchronous radiation cryogenic soft X-ray imaging of biomolecule condensate. DETAILED DESCRIPTION

[0068] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0069] Unless otherwise specified in the embodiments, the techniques or conditions are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be commercially available through regular channels.

[0070] Example 1

[0071] Stimulated formation of protein-DNA condensate

[0072] A) Take 100 μL of 1M Tris-HCl (pH = 7.4), 150 μL of 5M NaCl solution, 250 μL of 20 mg / mL BSA solution and 50 μL of 100 mM DTT solution, and dilute to 5 mL with sterile enzyme-free water 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 in vitro purified human full-length cGAS with the phase separation buffer, and 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, and the final concentration of the DNA is 1 μM. Mix well at room temperature.

[0074] C) Weigh 68.15 mg of anhydrous zinc chloride, dissolve it with 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, and the final concentration of Zn 2+ is 50 μM. Mix well at room temperature and stand for 60 min. Drop the mixed solution on a glass slide and observe under a microscope.

[0076] The results are shown in Figure 1 , the cGAS protein in the dsDNA and Zn 2+under the action of the above-mentioned cationic polymer to produce micron-sized aggregates.

[0077] Example 2

[0078] Fluorescent imaging of protein-DNA aggregates

[0079] A) Take 100 μL of 1M Tris-HCl (pH=7.4), 150 μL of 5M NaCl solution, 250 μL of 20 mg / ml BSA solution and 50 μL of 100 mM DTT solution, and dilute to 5 mL with sterile enzyme-free water 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 human full-length cyclic guanosine monophosphate-guanosine synthetase (cGAS) with the phase separation buffer, and the final concentration of the protein solution is 10 μM, of which 3% is GFP-coupled cGAS protein, and the final volume is 100 μL, then add 1 μL of 100 μM 45 bp dsDNA (containing 3% TAMRA-dsDNA), and the final concentration of DNA is 1 μM. Mix well at room temperature.

[0081] C) Take 10 μL and drop on a glass slide for confocal imaging. The imaging results are shown in Figure 2 .

[0082] D) Weigh 68.15 mg of anhydrous zinc chloride, dissolve it with 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, and the final concentration of Zn 2+ is 50 μM. Mix well at room temperature and stand for 60 min. Drop the mixture on a glass slide for confocal imaging. The imaging results are shown in Figure 3 .

[0084] From Figure 2 it can be seen that after the cGAS protein binds to double-stranded dsDNA, regular spherical aggregates with a size of 1-2 μm are formed; and after the addition of Zn 2+ , the morphology of the aggregates becomes irregular and exhibits a certain heterogeneity: dsDNA is mainly distributed in the large-size spherical regions in the aggregates, and is less distributed in the connecting parts between the spherical regions.

[0085] Example 3

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

[0087] A) Take 100 μL of 1M Tris-HCl (pH = 7.4), 150 μL of 5M NaCl solution, 250 μL of 20 mg / mL BSA solution, and 50 μL of 100 mM DTT solution, and dilute to 5 mL with sterile enzyme-free water 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 human full-length cyclic guanosine monophosphate- guanylate synthetase (cGAS) with the phase separation buffer, and the final protein solution concentration is 10 μM in a final volume of 100 μL, then add 1 μL of 100 μM 45 bp dsDNA, and the final DNA concentration is 1 μM. Mix well at room temperature.

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

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

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

[0092] F) Add 10 μL of the reaction solution to the treated carbon film, and naturally deposit for 15 min, then absorb the excess liquid with filter paper, and then add 5 μL of 2.5% glutaraldehyde fixing solution, and fix at room temperature for 10 min.

[0093] G) Wash the carbon film in sterile enzyme-free water for 2-3 times, then air dry naturally, and perform TEM imaging.

[0094] The imaging results are shown in Figure 4 When cGAS is combined with dsDNA and then Zn 2+ is added, the condensate contains two parts of structure: regular spherical structures with a size greater than 500 nm; and adhesives of small size spherical structures of cGAS self-aggregation.

[0095] Example 4

[0096] Zn 2+ After treatment, the spatial distribution imaging of metal elements in protein-DNA condensate

[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 water 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 human full-length cyclic guanosine monophosphate-guanosine monophosphate synthetase (cGAS) with the phase separation buffer, and the final protein solution concentration is 10 μM with a final volume of 100 μL, then add 1 μL of 100 μM 45 bp dsDNA, and the final DNA concentration is 1 μM. Mix well at room temperature.

[0099] C) Weigh 68.15 mg of anhydrous zinc chloride, dissolve it in 50 mL of 20 mM Tris-HCl (pH=7.4) to prepare a 10 mM ZnCl2solution.

[0100] D) Add 0.5 μL of 10 mM ZnCl2solution to the cGAS protein and dsDNA mixed solution, and the final concentration of Zn 2+ is 50 μM. Mix well at room temperature and stand for 60 min.

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

[0102] F) Add 10 μL of reaction solution to the treated carbon film, and naturally deposit for 15 min, then absorb the excess liquid with filter paper, and then add 5 μL of 2.5% glutaraldehyde fixing solution, and fix at room temperature for 10 min.

[0103] G) Wash the carbon film in sterile water 2-3 times, then air dry naturally, and perform STEM-EDS element analysis.

[0104] Results are shown in Figure 5 , and the distribution of Zn elements in the condensate is not uniform: compared with the connection site, Zn elements are obviously enriched in the large-size spherical region.

[0105] Example 5

[0106] Zn 2+ After treatment, the protein-DNA condensate is imaged by synchrotron radiation cryogenic soft X-ray

[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 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 human full-length cyclic guanosine monophosphate-guanosine monophosphate synthetase (cGAS) with the phase separation buffer, the final concentration of the protein solution is 10 μM, 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, dissolve it with 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 cGAS protein and dsDNA mixed solution, the final concentration of Zn 2+ is 50 μM. Mix well at room temperature and stand for 60 min.

[0111] E) Before loading the grid, pre-treat the nickel grid with 20 mg / ml BSA solution for 30 min, then wash with water for 2-3 times, and absorb the excess water with filter paper.

[0112] F) Add 5 μL of the reaction solution to the treated grid, naturally deposit for 10 min, then absorb the excess liquid with filter paper to form a thin layer of water on the grid, then insert it into liquid ethane for rapid freezing, collect the projections of the aggregate at different angles (-65°-65°, interval 1°) under 520 eV energy X-ray, and perform three-dimensional reconstruction and rendering.

[0113] See Figure 6 , the three-dimensional imaging results show that the ends of the aggregate swell into spheres (according to the fluorescence results, the inside should be rich in DNA molecules). The spheres are connected to each other, corresponding to the results of electron microscopy ultra-microscopic imaging. The results of Example 5 are compared with the results of Examples 1-4, it is found that the synchrotron X-ray imaging technology used in the present application can obtain the three-dimensional spatial morphology of the aggregate without the need for fluorescent molecular labeling, which is not achieved by fluorescence microscopy and electron microscopy. The use of freezing technology avoids the use of chemical fixatives (such as paraformaldehyde), and maximizes the authenticity of the sample morphology and structure.

[0114] Example 6

[0115] Co 2+ Synchronous radiation cryogenic soft X-ray imaging of RNA molecule mediated condensate

[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 100 mM TCEP solution, add sterile enzyme-free water to 5 mL to obtain a phase separation buffer. In the buffer, the final concentration of HEPES is 20 mM, the final concentration of KCl is 135 mM, the final concentration of MgCl2 is 15 mM, the mass fraction of PEG8000 is 5%, and the final concentration of TCEP is 1 mM.

[0117] B) Dilute 45 bp dsRNA (3% FAM) with phase separation buffer to a final concentration of 1 mM and 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, and the final concentration of Co 2+ is 1 mM. Mix well at room temperature and stand for 120 min.

[0120] E) Pre-treatment of the grid: pre-treat the molybdenum grid with 20 mg / mL BSA solution for 30 min, then wash with water for 2-3 times, and absorb the excess water with filter paper.

[0121] F) Add 10 μL of the reaction solution to the treated grid, naturally deposit for 10 min, then perform fluorescence imaging, absorb the excess liquid with filter paper to form a thin layer of water film on the grid, then insert into liquid ethane for rapid freezing, collect the projections of the aggregate at different angles (-65°-65°, interval 1°) under 520 eV energy X-ray, and perform three-dimensional reconstruction and rendering.

[0122] See Figure 7 , the dsRNA molecules aggregate into irregular morphology condensates under the action of Co 2+ . Figure 8 The three-dimensional morphology map of the condensate obtained by using the synchronous radiation low-temperature soft X-ray three-dimensional imaging technology.

[0123] Example 7

[0124] Fe 3+ Synchronous radiation cryogenic soft X-ray imaging of polysaccharide molecule mediated condensate

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

[0126] B) Dilute the glycogen solution with the 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, and the final concentration of Fe2(SO4)3 is 2 mM. Mix uniformly at room temperature and stand for 6 h. 3+

[0129] E) Grid pretreatment: pretreat the silicon nitride window with a 20 mg / mL BSA solution for 30 min, then wash with water for 2-3 times, and absorb the excess water with filter paper.

[0130] F) Add 10 μL of the reaction solution to the treated silicon nitride window, naturally deposit for 15 min, then absorb the excess liquid with filter paper to form a thin layer of water film on the silicon nitride window, then insert into liquid ethane for rapid freezing, collect the projections of the aggregates at different angles (-65°-65°, interval 1°) under 520 eV energy X-ray, and perform three-dimensional reconstruction and rendering.

[0131] Referring to Figure 9 , the polysaccharide molecules are aggregated into micron-sized condensates under the action of Fe 3+

[0132] In summary, the present application provides a freeze-soft X-ray imaging method of biomolecular condensates based on a synchrotron radiation device, and the method flow is as follows Figure 10 ​​The present application can realize the morphology imaging of complete biomolecule condensate under the condition of no labeling and no slicing. In addition, compared with chemical fixation, the instant structure of the condensate can be more truly preserved by the freezing fixation method of rapidly freezing the solution / cell containing the condensate in liquid nitrogen. In view of the regulatory effect of metal ions on biomolecule condensate, thereby affecting the function of biomolecules in cells, the present application combines the freezing sample preparation technology with the synchrotron X-ray imaging technology, realizes the clear observation of the three-dimensional structure and morphology of the instant biomolecule condensate after the addition of metal ions, and provides an effective research method for studying the regulation mechanism of metal ions on the aggregation behavior of biomolecules.

[0133] The applicant declares that the above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought out by any person skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. A cryogenic soft X-ray imaging method for biomolecular condensates based on a synchrotron radiation device, characterized in that, The method includes: adding a biomolecule condensate solution onto a carrier grid, allowing it to settle naturally, removing excess liquid, rapidly freezing it, and then performing low-temperature soft X-ray imaging based on a synchrotron radiation imaging beamline. The biomolecules mentioned are biomolecules capable of undergoing phase separation. The biomolecule is selected from any one or a combination of at least two of the following: protein, nucleic acid molecules, or polysaccharide molecules; The energy range of the soft X-rays is 280-520 eV; The biomolecule condensate solution was prepared using a method comprising the following steps: (S1) The biomolecules are mixed with the phase separation buffer to obtain the first solution; (S2) Mix the metal salt with the first solution to obtain a biomolecule aggregate solution; In step (S1), the phase separation buffer comprises: a pH stabilizer, an ionic strength stabilizer, a crowding agent, and a reducing agent; The pH stabilizer is selected from any one of Tris-HCl, HEPES, or phosphate. The ionic strength stabilizer is selected from any one or a combination of at least two of sodium salts, potassium salts, or magnesium salts. The crowding agent is selected from any one or a combination of at least two of PEG, BSA, and sucrose polymers; The reducing agent is selected from any one or a combination of at least two of the following: dithiothreitol, GSH, or TCEP; The phase separation buffer solution comprises, by concentration: 5-1000 mM pH stabilizer, 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.

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

3. The cryogenic soft X-ray imaging method for biomolecular condensates based on a synchrotron radiation device according to claim 1, characterized in that, In step (S2), the metal salt is a transition metal salt.

4. The cryogenic soft X-ray imaging method for biomolecular condensates based on a synchrotron radiation device according to claim 3, characterized in that, The transition metal ions in the transition metal salt are selected from any one or a combination of at least two of the following: iron, zinc, manganese, nickel, or cobalt ions. The anions in the transition metal salts are selected from any one of chloride, acetate, sulfate, or nitrate.

5. The cryogenic soft X-ray imaging method for biomolecular condensates based on a synchrotron radiation device according to claim 1, 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.

6. The cryogenic soft X-ray imaging method for biomolecular condensates based on a synchrotron radiation device according to claim 1, characterized in that, The screen is selected from any one of the following: copper screen, nickel screen, molybdenum screen, gold screen or silicon nitride window.

7. The cryogenic soft X-ray imaging method for biomolecular condensates based on a synchrotron radiation device according to claim 1, characterized in that, The volume of the biomolecule condensate solution added is 1-20 µL.

8. The cryogenic soft X-ray imaging method for biomolecular condensates based on a synchrotron radiation device according to any one of claims 1-7, characterized in that, The rapid freezing step includes: rapidly immersing the carrier net into a cryogenic liquid, rapidly freezing it, and then storing it at liquid nitrogen temperature; The cryogenic liquid is a liquid below -100°C, including liquid nitrogen or liquid ethane.

9. The cryogenic soft X-ray imaging method for biomolecular condensates based on a synchrotron radiation device according to claim 8, characterized in that, The immersion speed in the cryogenic liquid is not less than 1 m / s.

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

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