Nucleic acid-small molecule non-covalent complex as well as preparation method and application thereof

Abstract

The invention discloses a nucleic acid-small molecule non-covalent compound as well as a preparation method and application thereof. The method for constructing the nucleic acid-small molecule non-covalent compound based on calculation virtual screening guidance is provided for the first time, and a natural active compound capable of forming the compound with framework nucleic acid with a stable three-dimensional structure through non-covalent binding is rapidly screened out through molecular docking; a trace thermophoresis technology verifies that the prediction method is high in accuracy, and a plurality of nucleic acid-small molecule non-covalent complexes including costunolide-framework nucleic acid complexes are successfully constructed. The method does not need chemical modification on drugs or nucleic acids, and is high in universality. The nucleic acid-small molecule non-covalent complex obtained according to the method disclosed by the invention, especially COS-FNA, shows excellent kidney targeting property and ultrahigh treatment efficacy in a kidney disease model, and provides a brand new universal platform for development of kidney targeting drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug delivery systems, and more particularly to a nucleic acid-small molecule non-covalent complex, a preparation method and application thereof. BACKGROUND

[0002] Natural bioactive compounds (NBCs) have become a treasure trove for new drug development due to their wide range of pharmacological activities (such as antioxidant, anti-inflammatory, etc.), and have shown great potential in the treatment of cancer, cardiovascular disease, diabetic nephropathy, acute kidney injury and other diseases. However, most NBCs face common challenges such as poor water solubility, low in vivo stability, non-specific biodistribution, and low bioavailability, which severely limit their clinical application.

[0003] To overcome these obstacles, nanodelivery systems (such as liposomes, polymer nanoparticles) have been widely studied. However, these systems generally have complex drug loading methods, and problems such as biocompatibility and safety of the carrier. In recent years, DNA nanotechnology, especially framework nucleic acid (FNA), DNA origami, etc., has shown great potential as a drug carrier due to its excellent biocompatibility, biodegradability, programmability, and biodistribution characteristics that can be precisely controlled by nanoscale size. Intravenous injection studies have shown that three-dimensional DNA structures such as FNA can rapidly accumulate in the kidneys, showing excellent organ targeting ability, which provides an ideal platform for targeted therapy of kidney diseases.

[0004] Currently, the main strategy for combining drugs with nucleic acids is covalent coupling, which usually requires complex chemical modifications to the drug or DNA. For example, in the construction of a nucleic acid-drug conjugate based on a phosphorothioate-modified nucleic acid (WO2020150915-A1, CN109568595-B), the nucleic acid-drug conjugate is formed by the reaction of the phosphorothioate group in the phosphorothioate-modified nucleic acid with the group on the drug molecule that can undergo electrophilic reaction with the phosphorothioate group. This process is complicated and may affect the activity of the drug during the drug modification process. More importantly, this strategy lacks universality and requires specific modification of each nucleic acid and drug molecule involved in the construction of the complex. In existing non-covalent nucleic acid-drug binding strategies, the loaded drugs are known to bind to DNA through intercalation, such as a small number of drugs (CN115944606A, CN111803511A) that can intercalate into DNA double strands through their planar and rigid structures. This method has very limited compatibility with drug structures and is not universally applicable in terms of strategy.

[0005] In summary, the prior art fails to provide a universal and predictable method to guide which drug can be efficiently loaded onto a DNA structure by non-covalent means. Specifically, it is still a blank to develop the phenomenon of "non-covalent binding of drugs and DNA grooves" into a universal platform technology without chemical modification, which is highly versatile and can achieve efficient targeted therapy. SUMMARY

[0006] The purpose of the present application is to provide a nucleic acid-small molecule non-covalent complex and its preparation method and application, so as to solve the problems of poor water solubility, low stability, and low bioavailability in the delivery of natural active compounds in the prior art, and the problems of complex drug loading, the need for complex chemical modification, and limited compatibility in the existing nano delivery system.

[0007] To solve the above technical problems, the present application adopts the following technical solutions:

[0008] According to a first aspect of the present application, a method for screening active small molecules capable of forming a complex with a nucleic acid carrier by non-covalent binding is provided, comprising the following steps: providing a library of natural active compounds to be screened; calculating the calculated binding energy of each natural active compound and a standard B-type double-stranded DNA structure by molecular docking simulation, wherein the DNA structure of PDB ID: 1BNA is used as the acceptor; comparing the calculated binding energy with a preset threshold value, and screening natural active compounds with-11.0 kcal / mol≤calculated binding energy≤-7.0 kcal / mol; verifying the binding activity of the screened natural active compounds and the nucleic acid carrier by micro-thermal convection technology, and when the experimental binding constant K d ≤100 μM, it is determined that the natural active compound can form a complex with the nucleic acid carrier by non-covalent binding.

[0009] According to a preferred embodiment of the present application, the molecular docking simulation is performed using AutoDock Vina software, and the pretreatment of natural active compounds includes: obtaining a three-dimensional structure from the PubMed database and converting it to mol2 format, adding Gasteiger charge through AutoDockTools software, detecting rotatable bonds, and saving it as PDBQT format; the pretreatment of the DNA structure of PDB ID: 1BNA includes: deleting water molecules, adding Kollman charge, merging hydrogen atoms, and saving it as PDBQT format after assigning AutoDock 4 atom types.

[0010] According to a preferred scheme of the present application, the molecular docking simulation takes the DNA structure of PDB ID: 1BNA as the receptor, and the grid box center coordinates are (14.888, 21.003, 9.579) with a size of 52Å × 60Å × 100Å. It should be understood that this grid box exactly covers a large groove and a small groove of the potential binding region of the receptor 1BNA and small molecules, providing sufficient space to accommodate ligand molecules and possible conformational changes, avoiding missing potential binding modes due to too small grid, while not causing computational redundancy and efficiency reduction due to too large grid.

[0011] According to a second aspect of the present application, a method for preparing a nucleic acid-small molecule non-covalent complex is provided, comprising the following steps: using the natural active compound screened by the above method; mixing the screened natural active compound with a pre-assembled nucleic acid with a stable three-dimensional structure in a buffer at a molar ratio of 100:1-200:1, and incubating at 4-37°C for 6-24 hours to form a nucleic acid-small molecule non-covalent complex.

[0012] Preferably, the buffer formula is: 10-20 mM Tris-HCl, 5-50 mM MgCl2, pH 7.5-8.0.

[0013] Preferably, the unbound natural active compound is removed by ultrafiltration purification.

[0014] According to a third aspect of the present application, a nucleic acid-small molecule non-covalent complex is provided, which is formed by non-covalent binding of a natural active compound and a nucleic acid carrier; wherein the calculated binding energy of the natural active compound to standard B-form double-stranded DNA (PDB ID: 1BNA) is between -11.0 kcal / mol and -7.0 kcal / mol, and the experimental binding constant K d of the natural active compound is ≤100 μM measured by microscale thermophoresis (MST) technology; and the action site of the non-covalent binding is a large groove or a small groove of the double-stranded DNA in the nucleic acid carrier.

[0015] Preferably, the natural active compound is selected from any one of the group consisting of costunolide, emodin, chrysophanol, rutin, baicalin, baicalein, berberine, quercetin, wogonin, curcumin, resveratrol, chrysin, epimedoside, andrographolide, scutellarin, puerarin, celastrol, paeoniflorin, acacetin, artemisinin, schisantherin B, luteolin, formononetin, geniposidic acid.

[0016] Preferably, the nucleic acid is a three-dimensional DNA structure with double-stranded DNA, including: framework nucleic acids (such as tetrahedral framework nucleic acids), DNA origami, and three-dimensional structures of double-stranded regions formed by classical B-type DNA double helix and its multi-chain assembly. It should be understood that as long as it is a three-dimensional DNA structure with double-stranded DNA structure, it can achieve the loading of the natural active compound screened by non-covalent binding.

[0017] According to a fourth aspect of the present application, the nucleic acid-small molecule non-covalent complex is used for the preparation of a medicament for preventing and / or treating kidney diseases, and the nucleic acid-small molecule non-covalent complex can be specifically enriched in the kidney after intravenous injection.

[0018] Preferably, the medicament is used for preventing and / or treating acute kidney injury.

[0019] Particularly preferably, the nucleic acid-small molecule non-covalent complex is a costunolide-tetrahedral framework nucleic acid complex. The administration dose of the costunolide-tetrahedral framework nucleic acid complex is 1 / 300~1 / 200 of the administration dose of free costunolide, which can improve the kidney function and the pathological damage of kidney tissue.

[0020] According to a fifth aspect of the present application, a pharmaceutical composition is provided, comprising the nucleic acid-small molecule non-covalent complex, and a pharmaceutically acceptable carrier or excipient.

[0021] In combination Figure 1 , the overall technical route of the present application is summarized as follows: first, the natural active compound library is screened, the binding energy of small molecules and DNA is calculated by molecular docking simulation (taking the B-type double-stranded DNA structure of PDB ID:1BNA as the acceptor), and the natural active compounds with-11.0 kcal / mol≤calculated binding energy≤-7.0 kcal / mol are screened, and then the micro-thermal mobility technique is used to verify the binding activity (experimental binding constant K d value≤100μM) of the screened active small molecules and the pre-assembled 3D nucleic acid structure with double-stranded DNA structure (such as framework nucleic acid, DNA origami); then the complex is used for the targeted treatment of kidney diseases (such as acute kidney injury), and the recovery of disease damage is achieved.

[0022] The key invention point of the application mainly lies in that the "non-covalent groove binding" screening strategy verified by molecular docking (binding energy threshold - 11.0 ~ -7.0 kcal / mol) and microthermal mobility (MST) can realize efficient non-covalent binding of natural active small molecules and 3D nucleic acid structure without any chemical modification of the small molecules and / or nucleic acid, which breaks through the limitation of the prior art relying on drug embedding or chemical modification and has stronger universality.

[0023] Secondly, the application also first uses the framework nucleic acid with double-stranded DNA structure and DNA origami as a delivery carrier of natural active small molecules, realizes non-covalent drug loading by using the groove site of the three-dimensional structure, retains the biocompatibility and programmability of the nucleic acid carrier, and avoids the damage of chemical modification to the drug activity.

[0024] It is worth noting that the application also realizes precise targeted treatment of the kidney (especially acute kidney injury), and the costunolide-tetrahedral framework nucleic acid complex can be specifically enriched in the kidney within 5 minutes after intravenous injection, the retention time is up to 12 hours or more, and the dosage is only 1 / 300~1 / 200 of the free small molecule, which significantly improves the treatment efficiency and safety, and solves the delivery problem of natural active small molecules with poor water solubility and low bioavailability. According to the application, a complete technical chain of small molecule screening → complex preparation → kidney targeted treatment is constructed, which solves the series of problems of "drug loading difficulty, poor targeting and low efficacy" in the delivery of natural active compounds from the root, and forms an innovative technical closed loop from method to application.

[0025] The application first provides a screening method, which quantifies the calculated binding energy threshold of small molecules that can be combined with DNA grooves by calculating virtual screening and in vitro binding experiments, and quickly narrows down the range of small molecules that can construct nucleic acid drug complexes. Secondly, a preparation method is provided, which mixes and incubates the screened molecules with the pre-designed nucleic acid carrier with a stable three-dimensional structure to form the final complex. Thirdly, a nucleic acid drug non-covalent complex prepared by the above method is provided. Finally, the medical use of the complex is provided.

[0026] Compared with the prior art, the application has the following beneficial effects:

[0027] 1) Methodological innovation: for the first time, a quantifiable "estimation evaluation index" (calculated binding energy) is established, and the non-covalent binding of small molecules and DNA is promoted from a phenomenon to a predictable and designable universal platform technology.

[0028] 2) Strong platform universality: the effectiveness of the method is verified by a success rate of 30 / 45, and is successfully applied to framework nucleic acid, realizing the flexibility of "one method, multiple small molecules".

[0029] 3) Drug loading method advantage: This strategy does not require any chemical modification of small molecules and nucleic acid carriers, maximally retains the original activity of small molecules, and simplifies the preparation process.

[0030] 4) Excellent performance: represented by COS-FNA, it shows excellent kidney targeting (5-minute enrichment, 12-hour retention) and ultra-high efficiency (only 1 / 300 dose is needed to take effect in acute kidney injury model).

[0031] In summary, the present application discloses a method for constructing a nucleic acid-small molecule non-covalent complex based on computational virtual screening, as well as a nucleic acid-small molecule non-covalent complex and a preparation method and application thereof. The method first screens active small molecules that can form a complex with a framework nucleic acid with a stable three-dimensional structure through non-covalent binding through molecular docking. The prediction method has high accuracy verified by micro-thermal mobility technology, and successfully constructs a plurality of complexes including costunolide-framework nucleic acid complex. The method does not require chemical modification of drugs or nucleic acids, and has strong universality. The obtained complex, especially COS-FNA, shows excellent kidney targeting and ultra-high therapeutic efficacy in kidney disease models, providing a new universal platform for the development of kidney-targeted drugs. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a technical route schematic diagram of the present application;

[0033] Figure 2 shows the results of establishing the threshold value of the calculated binding energy of the nucleic acid structure that can load small molecules by combining computational virtual screening with MST experiments according to the method of the present application;

[0034] Figure 3 shows the anti-inflammatory and antioxidant activity results of 30 natural active small molecules;

[0035] Figure 4 shows the structure characterization of COS-FNA; A is the PAGE identification chart, B is the DLS particle size chart, and C is the AFM characterization chart;

[0036] Figure 5 shows the in vitro anti-inflammatory activity comparison diagram of tFNA, free COS and COS-FNA;

[0037] Figure 6The kidney targeting of COS-FNA is shown; the left figure shows the fluorescence detection results of major organs after COS-FNA was injected into the tail vein of mice for 5 minutes, 10 minutes, 15 minutes and 12 hours; the right figure shows the fluorescence detection results of major organs after ssDNA, tFNA and COS-FNA were injected into the tail vein of mice for 12 hours.

[0038] Figure 7 The diagram shows a comparison of the effects of tFNA, free COS, COS-FNA, and COS (intraperitoneal pre-administration) on the treatment of acute kidney injury. In the diagram, A shows the blood urea nitrogen content in the serum of mice in each treatment group, B shows the creatinine content in the serum of mice in each treatment group, C shows the superoxide dismutase content in the serum of mice in each treatment group, D shows the relative MDA content in the kidneys of mice in each treatment group, E shows the kidney index of mice in each treatment group, and F shows the HE staining necrosis score of the kidneys of mice in each treatment group. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0040] Example 1: Molecular docking method for evaluating the binding affinity of compounds to DNA

[0041] This embodiment provides a specific method for predicting the binding energy of small molecules to DNA using molecular docking software.

[0042] First, 35 natural bioactive compounds (NBCs) with reported anti-inflammatory activity were selected from the literature, namely: emodin, chrysophanol, rutin, baicalin, berberine, quercetin, wogonin, curcumin, resveratrol, strophanthidin, cynomol, andrographolide, costenyl lactone, scutellarin, silymarin, puerarin, celastrol, tanshinone IIA, sennain A, imperatorin, salvianolic acid B, geniposide, gastrodin, ligusticum lactone, matrine, codonopsis glycoside, gallic acid, nocacolone, trigonelline, ligustrazine, sulforaphane, limonene, oleic acid, and allicin.

[0043] Then, molecular docking simulation was used to predict the binding ability and binding mode of 35 natural active compounds with DNA double helix structure. The three-dimensional structure of 35 natural active compounds was obtained from PubMed database and converted into mol2 file format. Subsequently, in the AutoDockTools software, Gasteiger charge was added, rotatable bonds were detected, and finally saved in PDBQT format. The classic B-type DNA double helix X-ray crystal structure (PDB ID: 1BNA) was selected as the receptor. The structure was downloaded from the Protein Data Bank (PDB, http: / / www.rcsb.org). In the AutoDockTools software, the receptor was pretreated, including: deleting all water molecules, adding Kollman charge, merging hydrogen atoms, and assigning AutoDock 4 atom types, and finally saved as a PDBQT format file.

[0044] The grid box for docking was defined using the AutoDockTools software. To ensure complete coverage of the main binding region of DNA (major groove and minor groove), the center coordinates of the grid box were set as (center_x = 14.888, center_y = 21.003, center_z = 9.579), and the box size was set to 52 Å × 60 Å × 100 Å. Molecular docking calculation was performed using AutoDock Vina software. After docking, the docking conformation with the lowest binding free energy in the analysis results was taken as the most likely binding mode, and its calculated binding energy was recorded.

[0045] The calculated binding energy is shown in Table 1, which is an indicator for evaluating the binding strength of 35 natural active compounds with DNA.

[0046] Table 1. Calculated binding strength of 35 natural active compounds with DNA.

[0047]

[0048] Example 2: Experimental verification of actual binding of compounds with DNA by microcalorimetric mobility shift technique

[0049] This example provides a method for quantitatively determining the affinity of small molecules with DNA framework structure using microcalorimetric mobility shift (MST) technology.

[0050] Four single-stranded DNAs (A, B, C, Cy5-D) shown in Table 2 were synthesized, one of which was labeled with a Cy5 fluorescent group. They were mixed in TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH 8.0) at a concentration of 1 µM and self-assembled into a tetrahedral framework nucleic acid structure by a thermal annealing program (95℃ for 10 minutes, cooling to 4℃).

[0051] Table 2. Framework nucleic acid sequence information

[0052]

[0053] The 35 NBCs to be tested (purchased from Shanghai Aladdin Biochem Technology Co., Ltd.) were prepared into 16 concentration gradients, starting at 250 µM and diluted by 15 times, with a final concentration of 7.63 nM. Then the Cy5-labeled DNA structure with a final concentration of 1 nM was mixed with 16 concentration gradients of the test compounds in equal volume, and incubated for 30 minutes.

[0054] The Monolith NT.115 system was used to detect the change of fluorescence signal, and the experimental binding constant (K d value) of each compound with the DNA structure was obtained by fitting the concentration-response curve. The K d values of all 35 compounds are shown in Table 3.

[0055] As shown in Table 3, a total of 22 NBCs (emodin, chrysophanol, rutin, baicalin, baicalein, berberine, quercetin, wogonoside, curcumin, resveratrol, chrysin, primulaverin, andrographolide, costunolide, scutellarin, silybin, puerarin, celastrol, tanshinone IIA, halleridin A, imperatorin, and salvianolic acid B) can successfully bind to the double-stranded nucleic acid structure to form a framework nucleic acid-small molecule complex. The remaining 13 NBCs cannot form a framework nucleic acid-small molecule complex.

[0056] Table 3. Experimental binding constants of 35 natural active compounds and DNA.

[0057]

[0058] N.D.: Not Detected.

[0059] Example 3: Establishing the calculation binding energy evaluation index of small molecule nucleic acid loading capacity

[0060] Based on the data of Examples 1 and 2, the core rule was found and established.

[0061] The calculated binding energies of the 35 compounds obtained in Example 1 were compared with the corresponding experimental binding constants (K) obtained in Example 2. d Perform correlation analysis.

[0062] The results are as follows Figure 2 As shown, we discovered a clear pattern: when the calculated binding energy of natural small molecules to DNA double strands is ≤ -7.0 kcal / mol, the binding constant of the small molecules to tetrahedral framework nucleic acids can be determined by the MST experiment, and K... d The values ​​are generally better than 100 μM, indicating that it can be successfully loaded onto framework nucleic acid structures.

[0063] Based on this, we established "calculated binding energy ≤ -7.0 kcal / mol" as a reliable predictive criterion for predicting whether small molecules can be successfully loaded with framework nucleic acids.

[0064] To verify the reliability of this molecular docking system, we selected doxorubicin, a known DNA intercalating agent, as a strong positive control compound. Using the same docking parameters, the calculated binding energy between this compound and DNA (PDB: 1BNA) was predicted to be -10.8 kcal / mol. This value is significantly better than the -7.0 kcal / mol loading threshold established in this invention and is consistent with reported strong DNA binding characteristics in the literature, demonstrating the effectiveness of this docking system and the evaluation criteria.

[0065] Here, we further optimize the prediction index using the calculated binding energy of the DNA strong intercalating agent doxorubicin, and select "-11.0 kcal / mol ≤ calculated binding energy ≤ -7.0 kcal / mol" as a reliable prediction index to predict whether small molecules can be successfully loaded by framework nucleic acids.

[0066] Example 4: Verifying the universality of the predicted evaluation index

[0067] To verify the universality of the predicted evaluation index established in Example 3, we selected 10 new natural active products (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), including: berberine, paeoniflorin, acaciain, artemisinin, schisandrin B, luteolin, gentiopicrin, and genipin. First, we calculated their binding energies with DNA according to the method in Example 1. The results are shown in Table 4. Eight of these 10 small molecules had calculated binding energies ≤ -7.0 kcal / mol. Subsequently, we performed MST experiments according to the method in Example 2. The results confirmed that the binding constants of these eight small molecules with calculated binding energies ≤ -7.0 kcal / mol could all be measured at the micromolar level using MST (Table 4).

[0068] The results prove that the estimated evaluation index proposed in Example 3 has good universality and prediction accuracy, and can be used for efficient screening of small molecules capable of being loaded on nucleic acid structures.

[0069] Table 4. Calculated binding energy and experimental binding constant of 10 natural active compounds with DNA.

[0070]

[0071] N.D.: Not Detected.

[0072] Example 5: Characterization of a representative small molecule-frame nucleic acid complex

[0073] Comparison of anti-inflammatory and antioxidant activities of 30 small molecules capable of being loaded into frame nucleic acids verified in Examples 1-4.

[0074] RAW264.7 cells (purchased from the Chinese Academy of Sciences Typical Culture Collection Cell Bank) were plated in a 96-well plate and incubated with 4 μM small molecules for 2 hours after the cells adhered, and then LPS (1 μg / mL) was added for incubation for 24 hours. After that, the supernatant of each group was taken for determination of NO content.

[0075] RAW264.7 cells were plated in a 24-well plate and incubated with 4 μM small molecules for 2 hours after the cells adhered, and then LPS (1 μg / mL) was added for incubation for 24 hours. After that, the cells were washed with PBS, 5 μM DCFH-DA was added for incubation at 37°C for 30 minutes, and then the cells were collected after washing with PBS, and ROS was determined by flow cytometry.

[0076] The results are shown in Table 5. Figure 3 Among the 30 small molecules capable of being loaded into frame nucleic acids, costunolide (COS) has the best anti-inflammatory and antioxidant activities.

[0077] Based on this, the costunolide-frame nucleic acid complex was selected to verify the in vitro and in vivo activities and advantages of the nucleic acid small molecule complex system.

[0078] Example 6: Characterization of a costunolide-frame nucleic acid complex

[0079] A representative small molecule, costunolide, was incubated with frame nucleic acid in TM buffer at room temperature for 6 hours, and the molar feed ratio was 200:1. After incubation, 30 kDa molecular weight cutoff ultrafiltration centrifuge tubes were used for centrifugation at 3000 g for 10 minutes, and the operation was repeated three times to completely remove unbound COS molecules, and finally purified complexes were obtained.

[0080] PAGE results Figure 4(A) shows that the tFNA was correctly assembled, and the electrophoretic mobility of the COS-FNA complex loaded with COS was lower than that of the support. DLS results ( Figure 4 (B) shows that the particle size of COS-FNA is approximately 15 nm, while that of tFNA is approximately 10 nm. The particle size of the complex obtained after loading COS onto tFNA increases. AFM results ( Figure 4 As shown in Figure C), the height of tFNA is 1.8 ± 0.5 nm, and the height of COS-FNA is 2.3 ± 0.4 nm. These results confirm the successful construction of the COS-FNA complex.

[0081] Example 7: The COS-FNA complex exhibits stronger anti-inflammatory and antioxidant activity than free COS.

[0082] RAW264.7 cells were seeded in 96-well plates and cultured. After cell attachment, LPS (1 μg / mL) was added. One hour later, equal amounts of free COS, tFNA, and COS-FNA were added, followed by incubation for 24 hours. Subsequently, the cell culture supernatant was collected from each group, and the levels of NO, IL-6, IL-1β, and TNF-α were measured.

[0083] The results are as follows Figure 5 As shown, the anti-inflammatory activity of COS-FNA is superior to that of COS and the complex carrier tFNA.

[0084] Example 8: Kidney targeting of the COS-FNA complex

[0085] Mice were injected via tail vein with ICG-labeled ssDNA, tFNA, and COS-FNA (2 μM, 150 μL), respectively. Twelve hours later, major organs were isolated and fluorescence detection was performed using a small animal in vivo imaging system.

[0086] In vivo imaging ( Figure 6 COS-FNA was specifically enriched in the kidneys within 5 minutes after tail vein injection and remained there for up to 12 hours. This indicates that the nucleic acid drug complex can rapidly reach the kidneys and has a certain degree of retention in the kidneys, which facilitates the exertion of the drug's efficacy.

[0087] Example 9: Superior therapeutic effect of COS-FNA complex in kidney injury

[0088] An acute kidney injury model in mice was established by intramuscular injection of glycerol. Healthy BALB / c mice were deprived of water for 15 hours, and then 50% glycerol (8 mL / kg) was injected into the muscles of both hind limbs. All mice were then allowed free access to water.

[0089] The acute kidney injury mice were randomly divided into 5 groups, namely, a model group, a tFNA administration group (2 μM, 200 μL), a COS-FNA administration group (2 μM, 200 μL), a COS administration group (40 μM, 200 μL), and an intraperitoneal pre-administration COS group (30 mg / kg, 2% DMSO, 40% PEG 400, 5% Tween 80). The intraperitoneal pre-administration COS group was modeled 2 hours after intraperitoneal administration, which was equivalent to 300 times the dosage of the COS-FNA group, and the rest of the groups were administered via tail vein injection 2 hours after modeling. Healthy mice were used as a blank control group. Serum was taken 24 hours after modeling to determine biochemical indicators (BUN, SCr, SOD), and the two sides of the kidney tissue were weighed to calculate the organ index, and part of the kidney tissue was homogenized to determine MDA. Kidney organ index = (kidney weight / mouse body weight) x 100%.

[0090] Results, as shown in Figure 7 In the mouse acute kidney injury model, the COS-FNA group (the dosage was only 1 / 300 of the high-dose COS intraperitoneal pre-administration group) could significantly improve kidney function and histopathological injury.

[0091] The above is only a preferred embodiment of the present application, and is not intended to limit the scope of the present application. The above-described embodiments of the present application can be variously changed. Any simple, equivalent changes and modifications made in accordance with the content of the claims and the specification of the present application fall within the scope of the present application. The present application is not described in detail, and is conventional technical content.

Claims

1. A method for screening active small molecules capable of non-covalently binding to nucleic acid carriers to form complexes, characterized in that, Includes the following steps: Provides a library of natural active compounds to be screened; The computational binding energy of each natural active compound to a standard B-type double-stranded DNA structure was calculated using molecular docking simulation, with the DNA structure of PDB ID: 1BNA as the docking acceptor. The calculated binding energy was compared with a preset threshold to screen out natural active compounds with a binding energy of -11.0 kcal / mol ≤ calculated binding energy ≤ -7.0 kcal / mol; The binding activity of the screened natural active compounds to the nucleic acid carrier was verified using micro-thermophoresis. The experimental binding constant was... K d When the value is ≤100μM, it is determined that the natural active compound can non-covalently bind with the nucleic acid carrier to form a complex.

2. The method as described in claim 1, characterized in that, The molecular docking simulation was performed using AutoDock Vina software. The preprocessing of the natural active compound included: obtaining the three-dimensional structure from the PubMed database and converting it to mol2 format, adding Gasteiger charges and detecting rotatable bonds using AutoDockTools software, and then saving it in PDBQT format. The preprocessing of the DNA structure with PDB ID: 1BNA included: deleting water molecules, adding Kollman charges, merging hydrogen atoms, assigning AutoDock 4 atom types, and then saving it in PDBQT format.

3. A method for preparing nucleic acid-small molecule non-covalent complexes, characterized in that, Includes the following steps: Natural active compounds screened using the method according to claim 1 or 2; The selected natural active compounds were mixed with pre-assembled nucleic acids with stable three-dimensional structures in a buffer solution at a molar ratio of 100:1 to 200:1 and incubated at 4 to 37°C for 6 to 24 hours to form nucleic acid-small molecule non-covalent complexes.

4. A nucleic acid-small molecule non-covalent complex, characterized in that, It is formed by non-covalent binding of a natural active compound and a nucleic acid carrier; wherein the calculated binding energy of the natural active compound with standard B-type double-stranded DNA-PDB ID: 1BNA is between -11.0 kcal / mol and -7.0 kcal / mol, and the experimental binding constant is measured by micro-thermophoresis. K d Value ≤100μM; the non-covalent binding site is the major or minor groove of the double-stranded DNA in the nucleic acid vector.

5. The nucleic acid-small molecule non-covalent complex as described in claim 4, characterized in that, The natural active compounds are selected from any one of the following groups: costunolide, emodin, chrysophanol, rutin, baicalin, berberine, quercetin, wogonin, curcumin, resveratrol, strophanthidin, crystallizing glycoside, andrographolide, silymarin, puerarin, celastrol, tanshinone IIA, sennain A, imperatorin, salvianolic acid B, berberine, paeoniflorin, acaciain, artemisinin, schisandrin B, luteolin, gentiopicrin, and genipin.

6. The nucleic acid-small molecule non-covalent complex as described in claim 4, characterized in that, The nucleic acid is a three-dimensional DNA structure with double-stranded DNA, including: framework nucleic acid, DNA origami, and a three-dimensional structure containing double-stranded regions formed by the assembly of a classic B-type DNA double helix and its multi-strands.

7. The use of a nucleic acid-small molecule non-covalent complex as described in any one of claims 4-6 in the preparation of a drug, characterized in that, The drug is used to prevent and / or treat kidney disease, and the nucleic acid-small molecule non-covalent complex can be specifically enriched in the kidneys after intravenous administration.

8. The application as described in claim 7, characterized in that, The nucleic acid-small molecule non-covalent complex is a costanone-tetrahedral framework nucleic acid complex.

9. The application as described in claim 8, characterized in that, The drug is used to prevent and / or treat acute kidney injury. When the dosage of the costunolide-tetrahedral framework nucleic acid complex is 1 / 300 to 1 / 200 of the dosage of free costunolide, it can improve renal function and renal tissue pathological damage.

10. A pharmaceutical composition, characterized in that, It comprises a nucleic acid-small molecule non-covalent complex as described in any one of claims 4-6, and a pharmaceutically acceptable carrier or excipient.

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