Method for analyzing interaction between biomolecular aggregate and drug
By monitoring the UV visible spectrum changes before and after the interaction between drugs and biocondensers, the problem of traditional methods destroying the structure of biocondensers or interfering with interactions is solved, and a rapid and high-throughput analysis of biocondensers and drug interactions is achieved.
Patent Information
- Application Number
- CN202510738348.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-04
- Publication Date
- 2025-08-19
AI Technical Summary
It is difficult to effectively analyze the interaction between biocondensers and drugs in the prior art. Traditional methods such as mass spectrometry and fluorescence methods can destroy the biocondenser structure or interfere with the interaction, resulting in impaired accuracy.
The signal reporting strategy based on the drug's own ultraviolet-visible light absorption characteristics is adopted, and the unique spatial confined effect and chemical microenvironment of biocondensate are used to monitor the color and ultraviolet-visible absorption spectrum changes before and after the interaction between drug and biomolecular condensate. The peak shift and intensity changes of characteristic absorption peaks are quantitatively analyzed, and the molecular interaction is determined by combining microscopic observation or centrifugal measurement.
Fast, high-throughput analysis without fluorescent labeling or centrifugation is achieved, enabling the analysis of multiple drugs and biocondensers within 5 minutes, with high accuracy and supporting the analysis of 96 or 384 drugs.
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Figure CN120507341A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomolecule-drug interaction analysis, and in particular to a method for analyzing the interaction between biomolecule condensates and drugs. Background Art
[0002] Biocondensates are supramolecular assemblies formed within cells through liquid-liquid phase separation. They dynamically recruit diverse molecules, and these interactions promote diverse biochemical processes, ranging from cell signaling and gene expression to stress responses. In particular, the selective recruitment of drug molecules by biocondensates is closely linked to drug targeting or resistance. Identifying the interactions between biocondensates and drug molecules is crucial for understanding their functional mechanisms, pharmacodynamics, and innovative drug development. Due to the dynamic nature of biocondensate-drug interactions and the lack of clear membrane boundaries, traditional methods based on physical separation and immunoprecipitation struggle to effectively capture interaction information. Current approaches for studying molecular interactions within biocondensates primarily include mass spectrometry coupled with centrifugation and fluorescence. While these methods are well-established in the field of bioanalysis, the unique membrane-less structure of biocondensates, which relies on weak interactions and presents a chemical microenvironment distinct from that of the aqueous phase, present significant challenges for these traditional approaches. For example, mass spectrometry often involves desolvation, ionization, or vacuum, which can disrupt the structural stability of biocondensates and the inherent weak interactions. Fluorescence methods rely on chemical modification of exogenous fluorophores, which interferes with the interaction, and the quantum yield of the fluorophore in the two phases is inconsistent, resulting in impaired method accuracy. Summary of the Invention
[0003] The technical problem to be solved by the present invention is to provide a method for analyzing the interaction between biomolecular condensates and drugs in response to the deficiencies in the above-mentioned prior art. The present invention provides a signal reporting strategy based on the ultraviolet-visible light absorption characteristics of the drug itself, utilizing the unique spatial confinement effect of biocondensates and the influence of a chemical microenvironment significantly different from the aqueous phase on the physicochemical properties of drug molecules, monitoring the color and ultraviolet-visible absorption spectrum changes before and after the interaction between the drug and the biomolecular condensate, and quantitatively analyzing the peak shift and intensity changes of its characteristic absorption peaks. At the same time, the molecular interaction is determined by microscopic observation or centrifugal measurement, and a corresponding relationship between the molecular interaction and the change in the absorption spectrum is established for the analysis of the interaction between biocondensates and drugs.
[0004] To achieve the above-mentioned object, the technical solution adopted by the present invention is: a method for analyzing the interaction between biomolecular condensates and drugs, the method comprising:
[0005] First, the naked eye color and UV-visible absorption spectrum of the drug molecule itself are recorded;
[0006] The drug molecules and PS condensates are then mixed under phase separation conditions to trigger the interaction between the PS condensates and the drug molecules. The naked eye color and UV-visible absorption spectrum of the product of the interaction between the drug molecules and the PS condensates are recorded.
[0007] The interaction between drug molecules and PS aggregates is determined by recording the naked eye color difference and / or UV-visible absorption spectrum difference before and after the interaction between drug molecules and PS aggregates.
[0008] Preferably, the PS aggregates are aggregates spontaneously formed by the amino acid sequence PS through a liquid-liquid phase separation process.
[0009] Preferably, the amino acid sequence of PS is GHGVYGHGVYGHGPYGHGPYGHGLYW.
[0010] Preferably, the difference in UV-visible absorption spectra before and after the drug molecules interact with the PS condensate includes:
[0011] Compared with the UV-visible absorption spectrum of the drug molecule itself before reacting with PS condensates, the shift of the characteristic absorption peak of the drug molecule on the UV-visible absorption spectrum after the drug molecule reacts with PS condensates and the change in the relative absorption value of the characteristic absorption peak.
[0012] Preferably, the method for analyzing the interaction between biomolecule condensates and drugs comprises the following steps:
[0013] S1. First, record the naked eye color C0 and UV-visible absorption spectrum F0 of the drug molecules after they are dissolved in the solvent;
[0014] S2. Then, the drug molecules and the PS condensates are added to the solvent and mixed under phase separation conditions to induce the interaction between the PS condensates and the drug molecules. The naked eye color C1 and the UV-visible absorption spectrum F1 of the product of the interaction between the drug molecules and the PS condensates are recorded.
[0015] S3. Compare the difference between the naked eye color C1 and the naked eye color C0, and / or the shift ΔS of the characteristic absorption peak of the drug molecule in F1 compared to that in F0 and the change ΔD of the relative absorption value of the characteristic absorption peak, so as to judge the interaction between the drug molecule and the PS aggregate.
[0016] Preferably, the method for analyzing the interaction between biomolecule condensates and drugs comprises the following steps:
[0017] S1, first record the UV-visible absorption spectrum F0 of the drug molecule after it is dissolved in the solvent;
[0018] S2, then adding the drug molecules and the PS condensate into the solvent and mixing them under phase separation conditions to induce the interaction between the PS condensate and the drug molecules, and recording the UV-visible absorption spectrum F1 of the product of the interaction between the drug molecules and the PS condensate;
[0019] S3. Determine the interaction between drug molecules and PS aggregates:
[0020] S3-1. Calculate the shift ΔS of the characteristic absorption peak of the drug molecule in F1 compared to that in F0 and the change ΔD of the relative absorption value of the characteristic absorption peak, ΔS = S1 - S0, ΔD = D1 - D0;
[0021] Wherein, S0 represents the abscissa position of the characteristic absorption peak of the drug molecule in F0, and D0 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F0; S1 represents the abscissa position of the characteristic absorption peak of the drug molecule in F1, and D1 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F1;
[0022] S3-2. Using the pre-established relationship diagram f that characterizes the difference in UV-visible absorption spectra and the interaction between drug molecules and PS condensates, ΔS and ΔD are used to determine the current interaction between drug molecules and PS condensates.
[0023] Preferably, the relationship diagram f in step S3-2 is a three-part diagram, the horizontal axis of the three-part diagram is the characteristic absorption peak displacement before and after the drug molecules interact with the PS condensate, and the vertical axis is the change value of the characteristic absorption peak intensity before and after the drug molecules interact with the PS condensate. The three-part diagram is divided into three areas: the left area represents that the drug molecules do not interact with the PS condensate, the upper right area represents that the drug molecules interact with the surface of the PS condensate, and the lower right area represents that the drug molecules interact with the inside of the PS condensate.
[0024] Preferably, step S3-2 is specifically as follows:
[0025] Take ΔS as the abscissa and ΔD as the ordinate to represent the characteristic data point O(ΔS, ΔD) of the current drug molecule. Plot the characteristic data point O into the three-part graph. The interaction between the current drug molecule and the PS aggregate can be determined based on the distribution position of the characteristic data point O in the three-part graph:
[0026] The characteristic data point O in the upper right area represents that the drug molecules act on the surface of the PS condensate, and the characteristic data point O in the lower right area represents that the drug molecules act inside the PS condensate.
[0027] Preferably, the solvent is PBS buffer.
[0028] Preferably, the method for analyzing the interaction between biomolecule condensates and drugs comprises the following steps:
[0029] S1. First, the drug molecule is dissolved in PBS buffer to prepare a drug molecule solution with a concentration of 5-20 μg / mL. The drug molecule solution is added to a transparent well plate, and the UV-visible absorption spectrum F0 of the drug molecule solution is measured and recorded.
[0030] S2. PS is added to water to prepare a PS coacervate solution with a concentration of 1.5-7.5 mM; 1-4 μL of the PS coacervate solution is added to 24-96 μL of the drug molecule solution and mixed for 15-60 minutes to induce the interaction between the PS coacervate and the drug molecule; the product of the reaction between the drug molecule and the PS coacervate is added to a transparent well plate, and the UV-visible absorption spectrum F1 of the product is measured and recorded;
[0031] S3. Determine the interaction between drug molecules and PS aggregates:
[0032] S3-1. Calculate the shift ΔS of the characteristic absorption peak of the drug molecule in F1 compared to that in F0 and the change ΔD of the relative absorption value of the characteristic absorption peak, ΔS = S1 - S0, ΔD = D1 - D0;
[0033] Wherein, S0 represents the abscissa position of the characteristic absorption peak of the drug molecule in F0, and D0 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F0; S1 represents the abscissa position of the characteristic absorption peak of the drug molecule in F1, and D1 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F1;
[0034] S3-2. Using the pre-established relationship diagram f that characterizes the difference in UV-visible absorption spectra and the interaction between drug molecules and PS condensates, ΔS and ΔD are used to determine the current interaction between drug molecules and PS condensates.
[0035] The beneficial effects of the present invention are:
[0036] The present invention provides an analytical method for the interaction between biomolecular condensates and drugs. The present invention provides a signal reporting strategy based on the ultraviolet-visible light absorption characteristics of the drug itself, utilizing the unique spatial confinement effect of biocondensates and the influence of a chemical microenvironment significantly different from the aqueous phase on the physicochemical properties of drug molecules, monitoring the color and ultraviolet-visible absorption spectrum changes before and after the interaction between the drug and the biomolecular condensate, and then quantitatively analyzing the peak shift and intensity change of its characteristic absorption peak to ultimately achieve the analysis of the interaction between the biocondensate and the drug.
[0037] The analytical method used in the present invention is simple to operate, does not require fluorescent labeling or centrifugation, and can control the analysis time of the interaction between a single drug and bioaggregates to within 5 minutes. It also supports high-throughput analysis and can analyze the interactions between 96 or 384 or more (depending on the microplate specifications) drugs and molecular aggregates within 5 minutes. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 The reaction mechanism of the method for analyzing the interaction between biomolecular condensates and drugs of the present invention;
[0039] Figure 2 is the absorption spectrum before and after the drug molecules are mixed with PS condensates;
[0040] Figure 3 A three-part diagram characterizing the differences in UV-visible absorption spectra and the interaction between drug molecules and PS aggregates. DETAILED DESCRIPTION
[0041] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.
[0042] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.
[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Materials and reagents used in the following examples are commercially available unless otherwise specified. In the following examples, where specific conditions are not specified, the experiments were conducted under conventional conditions or those recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available conventional products.
[0044] The present invention provides a method for analyzing the interaction between biomolecular condensates and drugs, the method comprising:
[0045] First, the naked eye color and UV-visible absorption spectrum of the drug molecule itself are recorded;
[0046] The drug molecules and PS condensates are then mixed under phase separation conditions to trigger the interaction between the PS condensates and the drug molecules. The naked eye color and UV-visible absorption spectrum of the product of the interaction between the drug molecules and the PS condensates are recorded.
[0047] The interaction between drug molecules and PS aggregates is determined by recording the naked eye color difference and / or UV-visible absorption spectrum difference before and after the interaction between drug molecules and PS aggregates.
[0048] Among them, PS aggregates are aggregates spontaneously formed by the amino acid sequence PS through the liquid-liquid phase separation process.
[0049] In a preferred embodiment, the amino acid sequence of PS is GHGVYGHGVYGHGPYGHGPYGHGLYW.
[0050] In a preferred embodiment, the difference in UV-visible absorption spectra before and after the drug molecules interact with PS aggregates includes:
[0051] Compared with the UV-visible absorption spectrum of the drug molecule itself before reacting with PS condensates, the shift of the characteristic absorption peak of the drug molecule on the UV-visible absorption spectrum after the drug molecule reacts with PS condensates and the change in the relative absorption value of the characteristic absorption peak.
[0052] In a preferred embodiment, the method for analyzing the interaction between biomolecule condensates and drugs comprises the following steps:
[0053] S1. First, record the naked eye color C0 and UV-visible absorption spectrum F0 of the drug molecules after they are dissolved in the solvent;
[0054] S2. Then, the drug molecules and the PS condensates are added to the solvent and mixed under phase separation conditions to induce the interaction between the PS condensates and the drug molecules. The naked eye color C1 and the UV-visible absorption spectrum F1 of the product of the interaction between the drug molecules and the PS condensates are recorded.
[0055] S3. Compare the difference between the naked eye color C1 and the naked eye color C0, and / or the shift ΔS of the characteristic absorption peak of the drug molecule in F1 compared to that in F0 and the change ΔD of the relative absorption value of the characteristic absorption peak, so as to judge the interaction between the drug molecule and the PS aggregate.
[0056] In a preferred embodiment, the method for analyzing the interaction between biomolecule condensates and drugs comprises the following steps:
[0057] S1, first record the UV-visible absorption spectrum F0 of the drug molecule after it is dissolved in the solvent;
[0058] S2, then adding the drug molecules and the PS condensate into the solvent and mixing them under phase separation conditions to induce the interaction between the PS condensate and the drug molecules, and recording the UV-visible absorption spectrum F1 of the product of the interaction between the drug molecules and the PS condensate;
[0059] S3. Determine the interaction between drug molecules and PS aggregates:
[0060] S3-1. Calculate the shift ΔS of the characteristic absorption peak of the drug molecule in F1 compared to that in F0 and the change ΔD of the relative absorption value of the characteristic absorption peak, ΔS = S1 - S0, ΔD = D1 - D0;
[0061] Wherein, S0 represents the abscissa position of the characteristic absorption peak of the drug molecule in F0, and D0 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F0; S1 represents the abscissa position of the characteristic absorption peak of the drug molecule in F1, and D1 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F1;
[0062] S3-2. Based on the pre-established relationship diagram f between the difference in UV-visible absorption spectra and the interaction between drug molecules and PS aggregates, ΔS and ΔD are used to determine the interaction between the current drug molecules and PS aggregates. Specifically,
[0063] Take ΔS as the abscissa and ΔD as the ordinate to represent the characteristic data point O(ΔS, ΔD) of the current drug molecule. Plot the characteristic data point O into the three-part graph. The interaction between the current drug molecule and the PS aggregate can be determined based on the distribution position of the characteristic data point O in the three-part graph:
[0064] The characteristic data point O in the left area indicates that the drug molecules may not interact with the PS condensate. The characteristic data point O in the upper right area indicates that the drug molecules interact with the surface of the PS condensate. The characteristic data point O in the lower right area indicates that the drug molecules interact with the inside of the PS condensate.
[0065] In a preferred embodiment, the relationship diagram f in step S3-2 is a three-part diagram, wherein the horizontal axis of the three-part diagram is the characteristic absorption peak displacement before and after the drug molecules interact with the PS condensate, and the vertical axis is the change in the characteristic absorption peak intensity before and after the drug molecules interact with the PS condensate. The three-part diagram is divided into three regions: the left region, the upper right region, and the lower right region; the left region represents that the drug molecules may not interact with the PS condensate (data indicating no interaction will definitely fall on the left side of the graph; but points falling on the left side indicate that interaction or non-interaction is possible), the upper right region represents that the drug molecules interact on the surface of the PS condensate, and the lower right region represents that the drug molecules interact inside the PS condensate.
[0066] In a preferred embodiment, the method for analyzing the interaction between biomolecule condensates and drugs comprises the following steps:
[0067] S1. First, the drug molecule is dissolved in PBS buffer to prepare a drug molecule solution with a concentration of 5-20 μg / mL. The drug molecule solution is added to a transparent well plate, and the UV-visible absorption spectrum F0 of the drug molecule solution is measured and recorded.
[0068] S2. PS is added to water to prepare a solution with a concentration of 1.5-7.5 mM; 1-4 μL of the PS solution is added to 24-96 μL of the drug molecule solution and mixed for 15-60 minutes to induce the formation of PS aggregates and their interaction with the drug molecules. The product of the reaction between the drug molecules and the PS aggregates is added to a transparent well plate, and the UV-visible absorption spectrum F1 of the product is measured and recorded;
[0069] S3. Determine the interaction between drug molecules and PS aggregates:
[0070] S3-1. Calculate the shift ΔS of the characteristic absorption peak of the drug molecule in F1 compared to that in F0 and the change ΔD of the relative absorption value of the characteristic absorption peak, ΔS = S1 - S0, ΔD = D1 - D0;
[0071] Wherein, S0 represents the abscissa position of the characteristic absorption peak of the drug molecule in F0, and D0 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F0; S1 represents the abscissa position of the characteristic absorption peak of the drug molecule in F1, and D1 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F1;
[0072] S3-2. Using the pre-established relationship diagram f that characterizes the difference in UV-visible absorption spectra and the interaction between drug molecules and PS condensates, ΔS and ΔD are used to determine the current interaction between drug molecules and PS condensates.
[0073] Reference Figure 1 , the reaction principle of the present invention is:
[0074] First, the color or UV-visible absorption spectrum of the organic drug small molecule is recorded. This is then mixed with the PS amino acid sequence GHGVYGHGVYGHGPYGHGPYGHGLYW (which has the ability to spontaneously form condensates through a liquid-liquid phase separation process) under phase separation conditions to trigger the interaction between the PS condensate and the drug molecule. Under the unique spatial confinement effect of biocondensates and the chemical microenvironment that differs significantly from the aqueous phase, the physicochemical properties of the drug molecules change, triggering changes in color and absorption spectra. By comparing the spectra before and after molecular mixing, the interaction between the drug and condensate can be determined. Using 96-well or 384-well plates and a high-throughput microplate reader, efficient analysis of 96 or 384 or more drug-biocondensate interactions can be achieved in less than 5 minutes.
[0075] Reference Figure 2The black curve represents the UV-visible absorption spectrum of the drug molecule (sunitinib malate) measured alone. In contrast, the absorption spectrum (red curve) measured after the drug molecule is mixed with PS (i.e., PS aggregates) shows an increase in the baseline position, indicating an increase in solution turbidity, confirming the occurrence of phase separation and the formation of aggregates. It can be seen that the characteristic absorption peak position of the drug molecule interacting with the aggregates undergoes a red shift, while the relative absorbance value decreases. Therefore, changes in the UV-visible spectrum can be used to analyze the interaction between bioaggregates and drug molecules.
[0076] Using the above method, the present invention continues to measure the UV-visible absorption spectra of another 11 different molecules before and after mixing with PS condensates, and analyzes the changes in their characteristic absorption peak displacement (referred to as peak shift) and the change in the relative absorption value of the characteristic absorption peak, and finds that different molecules show different change trends. Through traditional means such as centrifugal determination of the molecular content in the precipitate and supernatant, microscopic observation, etc., it is found that the change in the absorption spectrum of the molecule is related to whether it is enriched by the condensate and the enrichment position: the occurrence of peak shift or a decrease in relative absorption value indicates that the molecule is enriched by the condensate; the simultaneous occurrence of peak shift and relative absorption value decrease indicates that the molecule is enriched inside the condensate; the occurrence of only peak shift and the unchanged relative absorption value indicates that the molecule is enriched in the surface layer at the edge of the condensate. This correspondence can be further simplified into an intuitive three-part diagram ( Figure 3 ), where the upper and lower parts indicate whether the relative absorbance value has changed (upper: unchanged; lower: decreased), respectively. The upper half of the unchanged relative absorbance value is further divided into the left and right parts based on whether a peak shift has occurred (left: unchanged; right: right shift). Therefore, this three-part plot can be used to intuitively, conveniently, and high-throughputly analyze the interaction between drug molecules and bioaggregates.
[0077] The above is the overall concept of the present invention. Detailed embodiments and comparative examples are provided below to further illustrate the present invention.
[0078] Example 1
[0079] A method for analyzing the interaction between biomolecular condensates and drugs, the method comprising the following steps:
[0080] S1. First, the drug molecule was dissolved in PBS buffer to prepare a drug molecule solution with a concentration of 10 μg / mL. The drug molecule solution was added to a 96-well plate (Corning, transparent flat bottom). Its color was observed with the naked eye. The UV-visible absorption spectrum F0 (200-800 nm, step size 1 nm) of the drug molecule solution was measured and recorded using a Spark multifunctional microplate reader (Tecan).
[0081] S2. PS (SEQ ID NO. 1: GHGVYGHGVYGHGPYGHGPYGHGLYW) was added to water to prepare a 3.75 mM PS solution. 2 μL of the PS solution was added to 48 μL of the drug molecule solution to induce the formation of PS aggregates and their interaction with the drug molecules. The mixture was allowed to react for 30 minutes. The product of the reaction between the drug molecules and the PS aggregates was added to a transparent well plate, and its color was observed with the naked eye. The UV-visible absorption spectrum F1 (200-800 nm, 1 nm step size) of the product was measured and recorded using a Spark multifunctional microplate reader (Tecan).
[0082] S3. Determine the interaction between drug molecules and PS aggregates:
[0083] S3-1. Calculate the shift ΔS of the characteristic absorption peak of the drug molecule in F1 compared to that in F0 and the change ΔD of the relative absorption value of the characteristic absorption peak, ΔS = S1 - S0, ΔD = D1 - D0;
[0084] Wherein, S0 represents the abscissa position of the characteristic absorption peak of the drug molecule in F0, and D0 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F0; S1 represents the abscissa position of the characteristic absorption peak of the drug molecule in F1, and D1 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F1;
[0085] S3-2. Using the pre-established relationship diagram f between the UV-visible absorption spectrum difference and the interaction between the drug molecule and the PS aggregate, ΔS and ΔD are used to determine the interaction between the current drug molecule and the PS aggregate:
[0086] Relationship diagram f is a three-part diagram. The horizontal axis of the three-part diagram represents the characteristic absorption peak shift before and after the drug molecules interact with the PS condensate, and the vertical axis represents the change in the characteristic absorption peak intensity before and after the drug molecules interact with the PS condensate. The three-part diagram is divided into three regions: the left region, the upper right region, and the lower right region. The left region represents the absence of interaction between the drug molecules and the PS condensate, the upper right region represents the interaction between the drug molecules and the PS condensate surface, and the lower right region represents the interaction between the drug molecules and the PS condensate interior.
[0087] Take ΔS as the abscissa and ΔD as the ordinate to represent the characteristic data point O(ΔS, ΔD) of the current drug molecule. Plot the characteristic data point O into the three-part graph. The interaction between the current drug molecule and the PS aggregate can be determined based on the distribution position of the characteristic data point O in the three-part graph:
[0088] The characteristic data point O in the left area indicates that the drug molecules may not interact with the PS condensate. The characteristic data point O in the upper right area indicates that the drug molecules interact with the surface of the PS condensate. The characteristic data point O in the lower right area indicates that the drug molecules interact with the inside of the PS condensate.
[0089] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.
Claims
1. A method for analyzing the interaction between biomolecular aggregates and drugs, characterized in that: The method includes: First, the naked eye color and UV-visible absorption spectrum of the drug molecule itself are recorded; The drug molecules and PS condensates are then mixed under phase separation conditions to trigger the interaction between the PS condensates and the drug molecules. The naked eye color and UV-visible absorption spectrum of the product of the interaction between the drug molecules and the PS condensates are recorded. The interaction between drug molecules and PS aggregates is determined by recording the naked eye color difference and / or UV-visible absorption spectrum difference before and after the interaction between drug molecules and PS aggregates.
2. The method for analyzing the interaction between biomolecule condensates and drugs according to claim 1, characterized in that: PS aggregates are aggregates spontaneously formed by the amino acid sequence PS through a liquid-liquid phase separation process.
3. The method for analyzing the interaction between biomolecule condensates and drugs according to claim 2, characterized in that: The amino acid sequence of PS is GHGVYGHGVYGHGPYGHGPYGHGLYW.
4. The method for analyzing the interaction between biomolecule condensates and drugs according to claim 3, characterized in that: The differences in UV-visible absorption spectra before and after the drug molecules interact with PS condensates include: Compared with the UV-visible absorption spectrum of the drug molecule itself before reacting with PS condensates, the shift of the characteristic absorption peak of the drug molecule on the UV-visible absorption spectrum after the drug molecule reacts with PS condensates and the change in the relative absorption value of the characteristic absorption peak.
5. The method for analyzing the interaction between biomolecule condensates and drugs according to claim 4, characterized in that: The method comprises the following steps: S1. First, record the naked eye color C0 and UV-visible absorption spectrum F0 of the drug molecules after they are dissolved in the solvent; S2. Then, the drug molecules and the PS condensates are added to the solvent and mixed under phase separation conditions to induce the interaction between the PS condensates and the drug molecules. The naked eye color C1 and the UV-visible absorption spectrum F1 of the product of the interaction between the drug molecules and the PS condensates are recorded. S3. Compare the difference between the naked eye color C1 and the naked eye color C0, and / or the shift ΔS of the characteristic absorption peak of the drug molecule in F1 compared to that in F0 and the change ΔD of the relative absorption value of the characteristic absorption peak, so as to judge the interaction between the drug molecule and the PS aggregate.
6. The method for analyzing the interaction between biomolecule condensates and drugs according to claim 5, characterized in that: The method comprises the following steps: S1, first record the UV-visible absorption spectrum F0 of the drug molecule after it is dissolved in the solvent; S2, then adding the drug molecules and the PS condensate into the solvent and mixing them under phase separation conditions to induce the interaction between the PS condensate and the drug molecules, and recording the UV-visible absorption spectrum F1 of the product of the interaction between the drug molecules and the PS condensate; S3. Determine the interaction between drug molecules and PS aggregates: S3-1. Calculate the shift ΔS of the characteristic absorption peak of the drug molecule in F1 compared to that in F0 and the change ΔD of the relative absorption value of the characteristic absorption peak, ΔS = S1 - S0, ΔD = D1 - D0; Wherein, S0 represents the abscissa position of the characteristic absorption peak of the drug molecule in F0, and D0 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F0; S1 represents the abscissa position of the characteristic absorption peak of the drug molecule in F1, and D1 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F1; S3-2. Using the pre-established relationship diagram f that characterizes the difference in UV-visible absorption spectra and the interaction between drug molecules and PS condensates, ΔS and ΔD are used to determine the current interaction between drug molecules and PS condensates.
7. The method for analyzing the interaction between biomolecule condensates and drugs according to claim 6, characterized in that: The relationship graph f in step S3-2 is a three-part graph. The abscissa of the three-part graph represents the shift of the characteristic absorption peak before and after the drug molecules interact with the PS condensate, and the ordinate represents the change in the intensity of the characteristic absorption peak before and after the drug molecules interact with the PS condensate. The three-part graph is divided into three regions: the left region, the upper right region, and the lower right region. The upper right area represents the drug molecules acting on the surface of the PS condensate, and the lower right area represents the drug molecules acting inside the PS condensate.
8. The method for analyzing the interaction between biomolecule condensates and drugs according to claim 7, characterized in that: Step S3-2 is specifically as follows: Take ΔS as the abscissa and ΔD as the ordinate to represent the characteristic data point O(ΔS, ΔD) of the current drug molecule. Plot the characteristic data point O into the three-part graph. The interaction between the current drug molecule and the PS aggregate can be determined based on the distribution position of the characteristic data point O in the three-part graph: The characteristic data point O in the upper right area represents that the drug molecules act on the surface of the PS condensate, and the characteristic data point O in the lower right area represents that the drug molecules act inside the PS condensate.
9. The method for analyzing the interaction between biomolecule condensates and drugs according to claim 8, characterized in that: in, The solvent was PBS buffer.
10. The method for analyzing the interaction between biomolecule condensates and drugs according to claim 9, characterized in that: The method comprises the following steps: S1. First, the drug molecule is dissolved in PBS buffer to prepare a drug molecule solution with a concentration of 5-20 μg / mL. The drug molecule solution is added to a transparent well plate, and the UV-visible absorption spectrum F0 of the drug molecule solution is measured and recorded. S2. PS is added to water to prepare a PS solution with a concentration of 1.5-7.5 mM; 1-4 μL of the PS solution is added to 24-96 μL of the drug molecule solution and mixed for 15-60 minutes to induce the formation of PS aggregates and their interaction with the drug molecules. The product of the reaction between the drug molecules and the PS aggregates is added to a transparent well plate, and the UV-visible absorption spectrum F1 of the product is measured and recorded; S3. Determine the interaction between drug molecules and PS aggregates: S3-1. Calculate the shift ΔS of the characteristic absorption peak of the drug molecule in F1 compared to that in F0 and the change ΔD of the relative absorption value of the characteristic absorption peak, ΔS = S1 - S0, ΔD = D1 - D0; Wherein, S0 represents the abscissa position of the characteristic absorption peak of the drug molecule in F0, and D0 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F0; S1 represents the abscissa position of the characteristic absorption peak of the drug molecule in F1, and D1 represents the relative absorption value of the characteristic absorption peak of the drug molecule in F1; S3-2. Using the pre-established relationship diagram f that characterizes the difference in UV-visible absorption spectra and the interaction between drug molecules and PS condensates, ΔS and ΔD are used to determine the current interaction between drug molecules and PS condensates.