Novel method for controlling liquid-liquid phase change of biomolecules by using magnetic field

Abstract

The invention belongs to the technical field of liquid-liquid phase separation, and particularly relates to a novel method for controlling liquid-liquid phase change of biomolecules by using a magnetic field, which uses the magnetic field to control liquid-liquid phase change separation of the biomolecules without any additive. The method has non-invasive and remote specificity, does not need to add any additive, is especially used in living cells, does not damage the living cells, and provides a new tool for researching cell functions and disease mechanisms.

Description

Technical Field

[0001] The invention belongs to the technical field of liquid-liquid phase separation, and in particular relates to a new method for controlling liquid-liquid phase transition of biomolecules by using a magnetic field. Background Art

[0002] Liquid-liquid phase separation (LLPS) is a fundamental process whereby biomolecules assemble and condense into droplets within living cells, playing a key role in organizing intracellular components and regulating various cellular functions, such as gene expression, signaling, and stress responses. Disruption of LLPS has been implicated in many diseases, including neurodegenerative diseases and cancer. Therefore, the spatiotemporal control of LLPS is of great interest for both research and practical applications. Conventional approaches to modulate LLPS have focused on altering physicochemical properties, such as temperature, pH, molecular concentration, or even using surfactants. However, while these approaches have provided valuable insights into the underlying mechanisms of LLPS, they often disrupt intracellular homeostasis and are invasive to living cells. In contrast to conventional approaches, optical methods have been used to trigger intracellular LLPS of optogenetic biomolecules, but these techniques are limited to the expression of specific proteins and exogenous genes. Furthermore, optical approaches are largely limited to in vitro cultured cells, as tissues in vivo extensively absorb and scatter visible light.

[0003] Control of liquid-liquid phase separation (LLPS) is crucial in various biological and chemical processes. Magnetic fields provide a non-invasive and remote method to modulate cellular dynamics and behavior; however, magnetic fields are generally not considered to be effective in controlling LLPS because it is generally believed that magnetic interactions occur only in magnetic materials containing magnetic elements (such as iron) and the presence of magnetic materials in biomolecules is limited. Summary of the invention

[0004] The purpose of the present invention is to provide a new method for controlling the liquid-liquid phase change of biomolecules using a magnetic field. Without any additives, the external magnetic field can be used to effectively control the liquid-liquid phase separation to solve the above-mentioned defects in the current separation of biomolecules.

[0005] The present invention adopts the following technical solution: A new method uses magnetic field to control the liquid-liquid phase change of biomolecules. In the absence of any additives, a magnetic field is used to control the liquid-liquid phase change separation of biomolecules.

[0006] The present study shows that the condensation of peptide and RNA systems can be promoted by an external magnetic field in the absence of any additives, and the enhancement effect is positively correlated with the magnetic field strength. The magnetic field controls the liquid-liquid phase transition separation of biomolecules, probably due to the presence of intrinsic magnetic moments with considerable probability in biomolecules with polar groups. These molecules present a considerable probability of triplet states. The triplet state has spin and magnetic moment to align with the external magnetic field. Our findings provide a non-invasive and remote method to regulate the dynamics of LLPS, paving the way for new human therapeutics related to protein LLPS. In addition, the results show that the symmetry between electricity and magnetism, which is inextricably linked, is restored at the interface, opening up numerous possibilities for magnetic applications. This is particularly relevant given the ubiquity of polar materials in biomaterials, pharmaceutical materials, chemical raw materials, and even important hormones in agricultural production.

[0007] Preferably, the liquid-liquid phase change separation of biomolecules refers to the formation of coacervates by the liquid-liquid phase change separation of biomolecules.

[0008] Preferably, the degree of liquid-liquid phase transition separation of the biomolecules is positively correlated with the magnetic field strength.

[0009] More preferably, the biomolecule is any one or more of a protein, a polypeptide, an RNA molecule or a DNA molecule.

[0010] Preferably, the magnetic field is generated by a neodymium iron boron permanent magnet.

[0011] The present invention also provides novel uses of magnetic fields for modulating the dynamics of LLPS, and novel human therapeutics associated with protein LLPS.

[0012] By implementing the above technical solution, the present invention has the following beneficial effects: The present invention provides a new method for controlling liquid-liquid phase transition of biomolecules, which is non-invasive and remotely specific, and does not require any additives. In particular, it is non-destructive to living cells and provides a new tool for studying cell functions and disease mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1Magnetic field promotes peptide / RNA condensation; (A) Schematic diagram of the experimental setup, a 10 mm diameter chamber with a coverslip at the bottom contains TAMRA-labeled peptide and polyU solution (ddH2O), and a 5 mm diameter NdFeB permanent magnet is placed under the chamber. Spinning disk confocal microscopy images after 3 hours of incubation at room temperature: (B) Imaging analysis of the entire chamber under the conditions of 10 μM peptide and 0.05 μg / μL polyU RNA, the droplet density increases near the 5 mm diameter NdFeB magnet, while the number of droplets decreases at a distance. The number of droplets in the chamber without a magnet is uniform and very small, indicating that the magnetic field promotes droplet formation. (C) The intensity and diameter of a single droplet, as well as the statistical average of the number of droplets under the conditions of 100 μM peptide and 0.05μg / μL polyU RNA. (D) At 10 μM peptide and 0.05 μg / μL polyU RNA in the absence of a magnetic field, droplets formed were sparse due to the subcritical peptide concentration; (E) In contrast, in the presence of a magnetic field, the number of droplets increased significantly, indicating that the magnetic field promoted droplet formation. (F) At 100 μM peptide and 0.05 μg / μL polyU RNA, a bright, circular region of droplet aggregation formed at the magnet, in sharp contrast to the dark region in the absence of a magnetic field. The brightness of the circular region of droplet aggregation gradually decreased by systematically reducing the magnetic field strength by adding coverslips between the magnet and the sample, indicating that the reduction in magnetic field strength is associated with a decrease in the number of droplets formed by condensation. A panoramic image of the entire chamber was generated by stitching multiple confocal images. The magnetic field strength under each condition is: mag (+++), 253.2±6.6 mT; mag (++), 210.29±4.9 mT; mag (+), 168.7±5.6 mT, generated by the NdFeB permanent magnet at different distances shown in Figure (A). (G) Heat map shows the fluorescence intensity distribution corresponding to (F). (HJ) Statistics of the fluorescence intensity of a single droplet (G), the number of droplets (I), and the diameter of the droplet (I) on the horizontal axis of the entire chamber reveal a direct correlation between these parameters and the magnetic field strength, indicating that the magnetic field not only increases the size of the droplets, but also regulates the balance of LLPS. Error bars are standard deviations (SD).

[0014] Figure 2 Figure 2 is the response of LLPS to the magnetic field at different RNA concentrations, specifically the statistical results of the number of droplets on the horizontal axis of the entire chamber at different RNA concentrations. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0016] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

[0017] Example 1 Magnetic field-promoted peptide / RNA aggregation assay A peptide and polyU RNA with the sequence RRASLRRASLRRASL (triplet RRASL) were selected as an established in vitro LLPS system. To precisely observe the condensation process, the peptide was fluorescently labeled with TAMRA. A ddH2O solution containing 10 μM peptide and 0.05 μg / μL polyU RNA was prepared and introduced into a chamber with a coverslip bottom. A NdFeB permanent magnet was placed under the coverslip (Figure 1A). After incubation for 3 h at room temperature, the chamber was imaged using a spinning disk confocal microscope. In the absence of a magnetic field during the incubation, only a few droplets were formed ( Figure 1 D), which is consistent with previous reports, indicating that the peptide concentration used here is ten times lower than the critical coacervation concentration. However, in the presence of a magnet, a large number of droplets were observed (Figure 1E). To rigorously evaluate this phenomenon, we imaged the entire chamber and analyzed the distribution of droplets at different locations. The results showed that droplets were mainly formed in the area closest to the magnet, while the control chamber without a magnet had only few detectable droplets at all locations (Figure 1B). These observations are supported by control experiments and strongly suggest that the magnetic field induces the formation of droplets, possibly by changing the critical concentration required for LLPS.

[0018] Next, the correlation between magnetic field strength and LLPS condensation was investigated. The peptide concentration was increased to 100 μM while the RNA concentration was kept at 0.05 μg / μL, close to the critical condensation concentration, so that a sufficient number of droplets could be observed even under weak magnetic fields. We observed that in the absence of a magnet, the field of view was uniform and dark, while when the magnet was introduced, a bright circular area appeared in the center of the chamber. Furthermore, the brightness of this circular area gradually decreased as more coverslip layers were added, i.e., the magnetic field strength at the chamber was gradually weakened ( Figure 1 F), the quantitative analysis results of brightness are shown in Figure 1 G.

[0019] To further quantify these observations, droplets in each sub-image were detected and counted as individual particles. The results show that along the horizontal axis of the chamber, the number of droplets increases from approximately 1 mm in the center to a plateau and then decreases from approximately 6 mm in the center (see Figure 1I). Notably, the width of the elevated plateau corresponds closely to the width of the magnet used. When the magnetic field strength is reduced, the increase in the number of droplets is correspondingly weakened. The droplet counting clearly demonstrates that the magnetic field promotes phase separation. In addition, we observed that the intensity of individual droplets in the region exposed to the magnetic field (see Figure 1I) is reduced. Figure 1 H) and diameter (see Figure 1 J) all increased and showed a similar dependence with the magnetic field strength. Figure 1 This dependence is further confirmed by the intensity and diameter of individual droplets within the magnet region in C, as well as the statistical average of the number of droplets. These results show that the magnetic field not only increases the size of the droplets, but also regulates the balance of LLPS. In summary, our results suggest that the magnetic field enhances the condensation of biomolecules, leading to the formation of more droplets.

[0020] Example 2 Test whether RNA concentration affects the response of LLPS to magnetic field This example investigates whether RNA concentration affects the LLPS response to a magnetic field. When the peptide concentration was kept at 100 μM and the RNA concentration was reduced from 5.00 μg / μL to 1.25 μg / μL, the effect of the magnetic field on LLPS was reduced. Nevertheless, a weak response to the magnetic field was still clearly observed at the lowest RNA concentration (see Figure 2 ).

Claims

1. A new method for controlling liquid-liquid phase transition of biomolecules using a magnetic field, characterized in that: In the absence of any additives, a magnetic field is used to control the liquid-liquid phase change separation of biomolecules.

2. A novel method for controlling liquid-liquid phase transition of biomolecules using a magnetic field according to claim 1, characterized in that: The liquid-liquid phase change separation of biomolecules refers to the formation of condensates by the separation of biomolecules through liquid-liquid phase change.

3. A novel method for controlling liquid-liquid phase transition of biomolecules using a magnetic field according to claim 1 or 2, characterized in that: The degree of liquid-liquid phase transition separation of the biomolecules is positively correlated with the magnetic field strength.

4. A novel method for controlling liquid-liquid phase transition of biomolecules using a magnetic field according to claim 1, characterized in that: The biological molecule is any one or more of a protein, a polypeptide, an RNA molecule or a DNA molecule.

5. The novel method of controlling liquid-liquid phase transition of biomolecules using magnetic field according to claim 1, characterized in that: The magnetic field is generated by a neodymium iron boron permanent magnet.

6. A new use of a magnetic field, characterized in that: Used to control the liquid-liquid phase change separation of biomolecules without any additives.

7. A new use of a magnetic field according to claim 6, characterized in that: The biological molecule is any one or more of a protein, a polypeptide, an RNA molecule or a DNA molecule.

8. A new use of a magnetic field according to claim 6, characterized in that: The magnetic field is generated by a neodymium iron boron permanent magnet.

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