A method for screening compounds that interfere with the phase separation of interfering RNA

Abstract

The present invention relates to the field of antimicrobial drug screening and discloses a method for screening compounds that interfere with RNA phase separation. The method comprises: 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 the aggregates using a fluorescence microscope to determine whether ordered cavitation occurs; and d. selecting the test compound as a potential antimicrobial agent if the cavitation parameter Rn significantly increases, where Rn is the ratio of the hollow radius to the aggregate radius. The present invention utilizes a DEAE-dextran / RNA aggregate model to optimize the weight ratio of RNA to DEAE-dextran (e.g., R = 0.4) to form stable aggregates. The interference effect of the test compound is then observed using a fluorescence microscope, thus filling a gap in the prior art for screening compounds that interfere with RNA phase separation.

Description

Technical Field

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

[0002] Antibiotic resistance has become a major challenge in global health. Traditional antimicrobial agents mainly target bacterial cell walls, membranes or protein synthesis, but with the increase of drug-resistant strains, the development of antimicrobial 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 extensively studied in eukaryotic cells, for example, in the formation of P bodies and stress granules, regulating RNA processing and storage. In bacteria, although less studied, there is evidence that the interaction between RNA and specific proteins may form LLPS-like condensates, participating in the regulation of gene expression. For example, the study by Brangwynne et al. (Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution / Condensation) revealed the role of LLPS in cellular compartmentalization, indirectly supporting the possibility of a similar mechanism in bacteria.

[0004] ε-Poly-lysine hydrochloride (PLL) possesses a moderate positive charge and exhibits broad-spectrum antibacterial activity. Studies by Shima et al. (Production of ε-poly-L-lysine by Fermentation) demonstrated that PLL is effective against both Gram-positive and Gram-negative bacteria. This mechanism is generally thought to involve interactions with the negatively charged bacterial membrane, disrupting membrane integrity. However, recent studies suggest that PLL may also act by interfering with RNA phase separation within bacteria, specifically by inducing the ordered hollowing out of RNA aggregates, thereby disrupting RNA compartmentalization. Despite the known antibacterial potential of PLL, systematic methods are lacking for screening other compounds that similarly interfere with RNA phase separation. DEAE-dextran (diethylaminoethyl dextran) is a commonly used molecular biology reagent used for DNA / RNA precipitation and transfection. Its positive charge enables it to form complexes with RNA, mimicking the electrostatic interactions between RNA and positively charged proteins in bacteria. The study 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 interactions and provided a theoretical basis for its use as a model system.

[0005] In view of this, we propose a method to screen 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, so as to solve the problems raised in the above background technology.

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

[0008] A method for screening a compound that interferes with RNA phase separation, the method comprising:

[0009] a. RNA and DEAE-dextran are mixed in a weight ratio R of 0.2 to 1.2 to form aggregates;

[0010] b. adding a test compound to the aggregate;

[0011] c. Use fluorescence microscopy to observe whether the aggregates show ordered cavitation;

[0012] d. If the cavitation parameter Rn is significantly increased, the test compound is selected as a potential antibacterial agent, where Rn is the ratio of the hollow center radius to the aggregate radius.

[0013] Preferably, the concentration of DEAE-dextran is about 2.5 mg / mL.

[0014] Preferably, the RNA is labeled with propidium iodide (PI).

[0015] Preferably, the test compound is labeled with a fluorescent dye different from that of the RNA.

[0016] Preferably, a significantly increased Rn is determined by comparing the average Rn values of aggregates treated with the test compound to the average Rn values of a control group (comprising DEAE-dextran / RNA aggregates but without the addition of the test compound) using statistical analysis.

[0017] Preferably, the test compound is added and incubated for about 15 minutes before observation.

[0018] By means of the above technical solution, the present invention provides a method for screening compounds that interfere with RNA phase separation. It has at least the following beneficial effects:

[0019] The present invention utilizes the DEAE-dextran / RNA aggregate model to optimize the weight ratio of RNA to DEAE-dextran (e.g., R=0.4) to form stable aggregates. The interference effect of the test compound is observed under a fluorescence microscope, thus filling the gap in the prior art for screening compounds that interfere with RNA phase separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application:

[0021] Figure 1 Preparation Process Diagram (a) Flowchart for the preparation of DEAE-dextran / RNA aggregates. (b) Flowchart for the preparation of PLL / RNA aggregates (to verify whether the test compound interacts with the target RNA). (c) Schematic diagram of the test compound-induced RNA phase separation within the DEAE-dextran / RNA aggregates, confirming the test compound.

[0022] Figure 2 Figure 1 illustrates the preparation and characterization of PLL / RNA aggregates in the present invention. The particle size (a) and zeta potential (b) of the PLL / RNA aggregates were measured at various polyelectrolyte weight ratios (0.1 to 2.4, w / w). The PLL concentration was kept constant at 2.5 mg / mL, and the RNA concentration was adjusted. (c) Fluorescence micrographs show the formation of PLL / RNA aggregates at various R ratios. The green fluorescence is from the FITC labeling of the PLL / RNA.

[0023] Figure 3Figures show the preparation and characterization of DEAE-dextran / RNA aggregates according to the present invention. (a) When the final DEAE-dextran concentration is fixed at 2.5 mg / mL, varying the RNA concentration leads to aggregate formation at different R ratios (0.2 to 1.2, w / w). (b) Zeta potential measurement. This graph depicts the zeta potential of DEAE-dextran / RNA aggregates at different weight ratios. (c) Hydrodynamic size analysis. This graph shows the average hydrodynamic diameter of DEAE-dextran / RNA aggregates at different RNA:DEAE-dextran weight ratios. (d) Aggregate count analysis. A bar graph 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 fixed at 2.5 mg / mL for DEAE-dextran and varying the RNA concentration (RNA:DEAE-dextran = 0.2 to 1.2, w / w).

[0024] Figure 4 Schematic diagram of the PLL-driven transition of DEAE-dextran / RNA aggregates toward ordered hollowing. (a) After the addition of PLL, green fluorescence gradually appears in the DEAE-dextran / RNA aggregates. Simultaneously, RNA migrates toward the periphery of the coating, indicating structural rearrangement and vacuolation. The time point at which PLL is added to the DEAE-dextran / RNA (immediately after stabilization) is designated t0. (b) This schematic diagram depicts the changes in DEAE-dextran / RNA aggregates after cavitation, focusing on the ratio of the internal hollow radius to the total aggregate radius. (c) The graph shows the temporal evolution of the degree of internal cavitation in DEAE-dextran / RNA aggregates. (d) This graph shows the increase in the mean fluorescence intensity of Cy5-labeled PLL over time. (e) Zeta potential measurements, presented as a bar graph, show a shift from +5 ± 0.5 mV to +15 ± 1.5 mV with increasing PLL concentration, indicating an increase in surface charge. (f) Microscopic image showing the addition of DEAE-dextran / RNA aggregates to PLL after the system stabilized (15 min) and depicting the characteristics of internal cavitation.

[0025] Figure 5Figure 1 shows 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 assess the degree of ordered cavitation within the system. Time series images (from t0 to t0+84 s) demonstrate the dynamic changes in 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 exhibited a more ordered distribution, indicating that the degree of ordered cavitation increased at different time points. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] See also Figure 1-Figure 5 As shown, the present invention provides a method for screening compounds that interfere with RNA phase separation, the method comprising:

[0028] a. RNA and DEAE-dextran are mixed at a weight ratio R of 0.2 to 1.2 to form aggregates;

[0029] b. adding a test compound to the aggregate;

[0030] c. Use fluorescence microscopy to observe whether the aggregates show ordered cavitation;

[0031] d. If the cavitation parameter Rn is significantly increased, the test compound is selected as a potential antibacterial agent, where Rn is the ratio of the hollow center radius to the aggregate radius.

[0032] It should be noted that aggregates were formed with a weight ratio R of RNA to DEAE-dextran of 0.2 to 1.2, and the test compound was added. Fluorescence microscopy was used to observe whether ordered cavitation (i.e., hollow center formation) occurred, and the cavitation parameter Rn (the ratio of the hollow center radius to the aggregate radius) was calculated. If Rn increased significantly, it was determined that the compound was a potential antibacterial agent. DEAE-dextran / RNA aggregates showed different morphologies and physical properties at different RNA to DEAE-dextran mass ratios (R). The aggregates were most stable when R=0.4.

[0033] Add the test compound to the preformed aggregates to the desired concentration and incubate for 15 min to allow the compound to interact with the aggregates;

[0034] Aggregates were imaged using a laser confocal fluorescence microscope, and 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.

[0035] The mean Rn of the test compound-treated group is compared with that of the control group (containing DEAE-dextran / RNA aggregates but without the test compound). If statistical analysis (e.g., t-test) shows a significant increase (p < 0.05), the compound is considered to interfere with RNA phase separation.

[0036] When PLL was added, the DEAE-dextran / RNA aggregates showed ordered cavitation, indicating that PLL may interfere with the phase separation of RNA through a charge-driven mechanism.

[0037] In contrast, DEAE-dextran had a minor effect on the structural changes of the PLL / RNA system and failed to induce similar cavitation.

[0038] This finding suggests that the high charge density of PLL may promote the transformation of aggregates from disordered structures to ordered hollow structures through strong electrostatic interactions with RNA, which may ultimately disrupt the transport and compartment stability of RNA inside bacteria, leading to cell death. This study provides theoretical support for the development of new antibacterial strategies, especially the design of new antibacterial agents by targeting RNA phase separation.

[0039] In this embodiment, the steps of forming aggregates with a weight ratio R of RNA to DEAE-dextran of 0.3 to 0.5 include:

[0040] 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 Biotechnology Co., Ltd.

[0041] DEAE-dextran was dissolved in Tris-HCl buffer (pH 7.4) to prepare a 50 mg / mL stock solution, which was diluted to a final concentration of 2.5 mg / mL;

[0042] Next, RNA was added to the stock solution so that the weight ratio of RNA to DEAE-dextran, R, was 0.4, and the volume was adjusted using Tris-HCl buffer (pH 7.4);

[0043] Finally, incubation at room temperature allowed aggregate formation.

[0044] In this example, RNA was labeled with propidium iodide (PI);

[0045] It should be noted that RNA is labeled with propidium iodide (PI) to exhibit red fluorescence, and the test compound can be optionally labeled with a different fluorescent dye such as Cy5 to exhibit green fluorescence.

[0046] 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 observed after incubation for 15 minutes. The results showed that PLL induced significant cavitation, and the Rn value increased with increasing concentration, verifying the effectiveness of the method.

[0047] Table 1 summarizes the key observations under different experimental conditions:

[0048]

[0049] It should be noted that, in this document, relational terms such as first and second, etc., are used only 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 terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0050] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the 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 comprises: a. RNA and DEAE-dextran are mixed at a weight ratio R of 0.2 to 1.2 to form aggregates; b. adding a test compound to the aggregate; c. Use fluorescence microscopy to observe whether the aggregates show ordered cavitation; d. If the cavitation parameter Rn is significantly increased, the test compound is selected as a potential antibacterial agent, where Rn is the ratio of the hollow center radius to the aggregate radius.

2. The method for screening a compound that interferes with RNA phase separation according to claim 1, wherein: The concentration of DEAE-dextran was 2.5 mg / mL.

3. The method for screening a compound that interferes with RNA phase separation according to claim 1, wherein: The RNA was labeled with propidium iodide (PI).

4. The method for screening a compound that interferes with RNA phase separation according to claim 1, wherein: The test compound is labeled with a different fluorescent dye than the RNA.

5. The method for screening a compound that interferes with RNA phase separation according to claim 1, characterized in that: Significantly increased Rn is determined by comparing the mean Rn values of aggregates treated with the test compound to those of the control group using statistical analysis.

6. The method for screening compounds that interfere with RNA phase separation according to claim 1, wherein: After adding the test compound, incubate for 15 minutes before observation.

Images