Method for screening compounds interfering RNA phase separation

Abstract

The invention relates to the field of antibacterial drug screening, and discloses a method for screening compounds interfering RNA phase separation, and the method comprises the following steps: a, mixing RNA and DEAE-glucan according to a weight ratio R of 0.2-1.2 to form an aggregate; b, adding a test compound into the aggregate; c, observing whether the aggregate has ordered cavitation or not by using a fluorescence microscope; d, if the cavitation parameter Rn is remarkably increased, the test compound is selected as the potential antibacterial agent, and Rn is the ratio of the hollow radius to the aggregate radius. A DEAE-glucan / RNA aggregate model is utilized, the weight ratio (such as R = 0.4) of RNA to DEAE-glucan is optimized, a stable aggregate is formed, the interference effect of a tested compound is observed through a fluorescence microscope, and the blank of screening interference RNA phase separation compounds in the prior art is filled.

Description

Technical Field

[0001] The present invention relates to the field of antibacterial drug screening, and specifically to a method for screening compounds that interfere with RNA phase separation. Background Art

[0002] Antibiotic resistance has become a major challenge in the global health field. According to the World Health Organization (WHO) report in 2024, antimicrobial resistance causes millions of deaths every year, putting huge pressure on the healthcare system. Traditional antibacterial agents mainly target bacterial cell walls, membranes, or protein synthesis. However, with the increase in drug-resistant strains, the development of antibacterial agents with new mechanisms has become urgent.

[0003] Liquid-liquid phase separation (LLPS) is an important phenomenon in cell biology, involving the spontaneous formation of droplet-like compartments by biomolecules under membrane-free conditions. This process has been widely studied in eukaryotic cells, such as the formation of P bodies and stress granules, which regulate RNA processing and storage. In bacteria, although less studied, there is evidence that the interaction between RNA and specific proteins may form condensates similar to LLPS, which participate in gene expression regulation. For example, the research by Brangwynne et al. (Germline PGranules Are Liquid Droplets That Localize by Controlled Dissolution / Condensation) revealed the role of LLPS in cell compartmentalization, indirectly supporting the possible existence of a similar mechanism in bacteria.

[0004] ε-Polylysine hydrochloride (PLL) carries a certain positive charge and has broad-spectrum antibacterial activity. The research by Shima et al. (Production of ε-poly-L-lysine by Fermentation) shows that PLL is effective against both Gram-positive and Gram-negative bacteria, and its mechanism is generally considered to be through interaction with the negative charge of the bacterial cell membrane, disrupting membrane integrity. However, recent research has proposed that PLL may also act by interfering with RNA phase separation in bacteria, specifically manifested as inducing ordered cavitation (hollowing out) of RNA aggregates, thus disturbing the compartmentalization of RNA. Although the antibacterial potential of PLL is known, there is a lack of systematic methods to screen other compounds that can similarly interfere with RNA phase separation. In the prior art, DEAE-dextran (diethylaminoethyl dextran) is a commonly used molecular biology reagent for DNA / RNA precipitation and transfection. Its positively charged property enables it to form complexes with RNA, mimicking the electrostatic interaction between RNA and positively charged proteins in bacteria. The research by McCutchan et al. (Enhancement of the Infectivity of Simian Virus 40 Deoxyribonucleic Acid with Diethylamino ethyl-dextran) demonstrated the application of DEAE-dextran in nucleic acid interaction, providing a theoretical basis for its use as a model system.

[0005] In view of this, we propose a method for screening compounds that interfere with RNA phase separation. Summary of the Invention

[0006] The object of the present invention is to provide a method for screening compounds that interfere with RNA phase separation to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions.

[0008] A method for screening compounds that interfere with RNA phase separation, the method comprising: a. Mixing RNA and DEAE-dextran at a weight ratio R of 0.2 to 1.2 to form aggregates; b. Adding a test compound to the aggregates; c. Using a fluorescence microscope to observe whether ordered cavitation occurs in the aggregates; d. If the cavitation parameter Rn increases significantly, then selecting the test compound as a potential antibacterial agent, where Rn is the ratio of the radius of the hollow center to the radius of the aggregate.

[0009] Preferably, the concentration of the DEAE-dextran is about 2.5 mg / mL.

[0010] Preferably, the RNA is labeled with propidium iodide (PI).

[0011] Preferably, the test compound is labeled with a fluorescent dye different from that of the RNA.

[0012] Preferably, the significantly increased Rn is determined using statistical analysis by comparing the average Rn values of the aggregates after treatment with the test compound with those of the control group (containing DEAE-dextran / RNA aggregates but without the addition of the test compound).

[0013] Preferably, incubation is carried out for about 15 minutes after the addition of the test compound before observation.

[0014] By means of the above technical solution, the present invention provides a method for screening compounds interfering with RNA phase separation. It has at least the following beneficial effects: By using the DEAE-dextran / RNA aggregate model, the present invention optimizes the weight ratio of RNA to DEAE-dextran (such as R = 0.4) to form stable aggregates, and observes the interference effect of the test compound through a fluorescence microscope, filling the blank in the prior art for screening compounds interfering with RNA phase separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The drawings described herein are used to provide a further understanding of the present invention and form a part of this application: Figure 1 Process diagrams for preparation. (a) Flow chart for the preparation of DEAE-dextran / RNA aggregates. (b) Flow chart for the preparation of PLL / RNA aggregates (to verify whether the test compound acts on the target RNA). (c) Schematic diagram showing the RNA phase separation in DEAE-dextran / RNA aggregates induced by the test compound to verify the test compound.

[0016] Figure 2 It is the preparation and characterization diagram of PLL / RNA aggregates in the present invention; the particle size (a) and Zeta potential (b) of PLL / RNA aggregates were measured at different polyelectrolyte weight ratios (0.1 to 2.4, w / w), while the concentration of PLL was fixed at 2.5 mg / mL and the concentration of RNA was adjusted. (c) Fluorescence micrographs show the formation of PLL / RNA at different R ratios, and the green fluorescence comes from the FITC labeling of PLL / RNA.

[0017] Figure 3Preparation and characterization diagrams of DEAE-dextran / RNA aggregates in the present invention; (a) When the final concentration of DEAE-dextran is fixed at 2.5 mg / mL, changing the concentration of RNA will result in the formation of aggregates with different R ratios (0.2 to 1.2, w / w). (b) Zeta potential measurement. This diagram depicts the Zeta potential of DEAE-dextran / RNA aggregates at different weight ratios. (c) Hydrodynamic size analysis. This diagram shows the average hydrodynamic diameter of DEAE-dextran / RNA aggregates at different RNA and DEAE-dextran weight ratios. (d) Aggregate count analysis. The bar chart quantifies the number of DEAE-dextran / RNA aggregates at different weight ratios. (e) Bright-field and corresponding fluorescence 3D images of DEAE-dextran / RNA aggregates. The weight ratio is such that the concentration of DEAE-dextran is fixed at 2.5 mg / mL and the concentration of RNA is changed (RNA:DEAE-dextran = 0.2 to 1.2, w / w).

[0018] Figure 4 Diagram showing the transformation of PLL-driven DEAE-dextran / RNA aggregates towards an ordered hollowing direction in the present invention; (a) After adding PLL, green fluorescence gradually appears in the DEAE-dextran / RNA aggregates. At the same time, RNA migrates towards the periphery of the coating, indicating that the structure has rearranged and vacuoles have formed. The time point when PLL is added to DEAE-dextran / RNA (immediately after stabilization) is designated as t0. (b) This schematic diagram describes the changes in DEAE-dextran / RNA aggregates after cavitation, with an emphasis on the ratio of the internal hollow radius to the radius of the total aggregate. (c) The diagram shows the time variation of the degree of internal cavitation in DEAE-dextran / RNA aggregates. (d) This diagram shows that the average fluorescence intensity of Cy5-labeled PLL increases with time. (e) Zeta potential measurement values, presented as a bar chart, show that as the PLL concentration changes, the zeta potential changes from +5 ± 0.5 mV to +15 ± 1.5 mV, indicating an increase in surface charge. (f) Microscopic images show the situation of PLL added to DEAE-dextran / RNA aggregates after the system is stable (15 minutes) and describe the characteristics of internal cavitation.

[0019] Figure 5This is a graph showing the effect of different concentrations of PLL on the ordered hollow phase transition of DEAE-dextran / RNA aggregates in the present invention. After stabilizing the DEAE-dextran / RNA aggregates, different concentrations of PLL were introduced to evaluate the degree of ordered cavitation occurring within the system. Time-series images (from t0 to t0 + 84 s) showed the dynamic changes of the aggregates. Over time, the red fluorescence intensity of propidium iodide (PI-RNA) remained stable at concentrations of 0.5 mg / mL, 2.5 mg / mL, and 5 mg / mL, while the green fluorescence of Cy5-PLL gradually showed a more ordered distribution, indicating that the degree of ordered cavitation increased at different time points. Detailed implementation mode

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Please refer to Figures 1 - 5 As shown, the present invention provides a method for screening compounds that interfere with RNA phase separation. The method includes: a. Mixing RNA and DEAE-dextran at a weight ratio R of 0.2 to 1.2 to form aggregates; b. Adding a test compound to the aggregates; c. Using a fluorescence microscope to observe whether ordered cavitation occurs in the aggregates; d. If the cavitation parameter Rn increases significantly, then select the test compound as a potential antibacterial agent, where Rn is the ratio of the radius of the hollow center to the radius of the aggregate.

[0022] It should be noted that aggregates are formed with a weight ratio R of RNA to DEAE-dextran of 0.2 to 1.2, a test compound is added, and a fluorescence microscope is used to observe whether ordered cavitation (i.e., the formation of a hollow center) occurs. By calculating the cavitation parameter Rn (the ratio of the radius of the hollow center to the radius of the aggregate), it is determined that if Rn increases significantly, then the compound is a potential antibacterial agent. The DEAE-dextran / RNA aggregates exhibit different morphologies and physical properties at different RNA to DEAE-dextran mass ratios (R), and the aggregates are most stable when R = 0.4; Add a test compound to the pre-formed aggregates to reach the required concentration, incubate for 15 minutes, and allow the compound to interact with the aggregates; Aggregates were imaged using a laser confocal fluorescence microscope. The hollow center radius (Rn') and total radius (Rn'') of each aggregate were measured using ImageJ software. Rn = Rn' / Rn'' was calculated, and the average Rn of multiple aggregates was calculated. The average Rn of the test compound treatment group was compared with that of the control group (containing DEAE-dextran / RNA aggregates without the addition of the test compound). If a significant increase (p < 0.05) was shown by statistical analysis (such as a t-test), the compound was considered to interfere with RNA phase separation.

[0023] When PLL was added, ordered cavitation occurred in the DEAE-dextran / RNA aggregates, indicating that PLL might interfere with the phase separation of RNA through a charge-driven mechanism.

[0024] In contrast, DEAE-dextran had less impact on the structural changes of the PLL / RNA system and failed to induce similar cavitation.

[0025] This finding suggests that the high charge density of PLL might promote the transformation of aggregates from a disordered structure to an ordered hollow structure through strong electrostatic interactions with RNA, ultimately potentially disrupting the transport and compartment stability of intracellular RNA and leading to cell death. This research provides theoretical support for the development of novel antibacterial strategies, especially for designing new antibacterial agents by targeting RNA phase separation.

[0026] In this example, the step of forming aggregates with a weight ratio R of RNA to DEAE-dextran ranging from 0.3 to 0.5 includes: First, the sources of the raw materials are described: ε-polylysine hydrochloride (PLL, MW < 5000) and diethylaminoethyl dextran (DEAE-dextran, analytical grade) were purchased from Sigma-Aldrich (Shanghai), and ribonucleic acid (RNA, yeast) was purchased from Dulai Biotech Co., Ltd.

[0027] DEAE-dextran was dissolved in Tris-HCl buffer (pH 7.4) to prepare a stock solution of 50 mg / mL and diluted to a final concentration of 2.5 mg / mL. Secondly, RNA was added to the stock solution to make the weight ratio R of RNA to DEAE-dextran 0.4, and the volume was adjusted using Tris-HCl buffer (pH 7.4). Finally, incubation was carried out at room temperature to allow aggregate formation.

[0028] In this example, RNA was labeled with propidium iodide (PI). It should be noted that RNA is labeled with propidium iodide (PI) to show red fluorescence, and different fluorescent dyes such as Cy5 can be selected to label the test compound to show green fluorescence.

[0029] Taking ε-polylysine hydrochloride (PLL) as an example, DEAE-dextran / RNA aggregates with R = 0.4 were prepared. PLL was added to concentrations of 0.5, 2.5, and 5 mg / mL, and after incubation for 15 minutes, observations were made. The results showed that PLL induced significant cavitation, and the Rn value increased with increasing concentration, verifying the effectiveness of the method.

[0030] Table 1 summarizes the key observations under different experimental conditions:

[0031] It should be noted that in this document, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variation thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0032] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for screening compounds that interfere with RNA phase separation, characterized in that: The method includes: a. Mixing RNA and DEAE-dextran at a weight ratio R of 0.2 to 1.2 to form aggregates; b. Adding a test compound to the aggregates; c. Observing whether ordered cavitation occurs in the aggregates using a fluorescence microscope; d. If the cavitation parameter Rn increases significantly, selecting the test compound as a potential antibacterial agent, where Rn is the ratio of the radius of the hollow center to the radius of the aggregate.

2. The method for screening a compound interfering with RNA phase separation according to claim 1, characterized in that: The concentration of the DEAE-dextran is 2.5 mg / mL.

3. A method for screening compounds interfering with RNA phase separation according to claim 1, characterized in that: The RNA is labeled with propidium iodide (PI).

4. A method for screening compounds that interfere with RNA phase separation according to claim 1, characterized in that: The test compound is labeled with a fluorescent dye different from that of the RNA.

5. A method for screening compounds that interfere with the phase separation of interfering RNA, characterized in that: The significantly increased Rn is determined using statistical analysis by comparing the average Rn values of the aggregates treated with the test compound and the control group.

6. The method for screening a compound interfering with RNA phase separation according to claim 1, characterized in that: Observation is carried out 15 minutes after adding the test compound.

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