ATP-responsive DNA nanomachine for membrane protein degradation and application thereof in preparation of tumor treatment drugs

Abstract

The application belongs to the technical field of biomedical engineering, and discloses an ATP-responsive DNA nanomachine for membrane protein degradation and application thereof in preparation of tumor treatment drugs. The DNA nanomachine disclosed by the application is assembled by a lysosome-targeting chimera LYTAC and a frame nucleic acid FNA. The LYTAC is loaded into the nucleic acid frame FNA through an ATP aptamer connected to the end of the LYTAC; meanwhile, the surface of the nucleic acid frame FNA is modified with a ligand for targeting tumor cells, so as to endow the nanomachine with an active targeting ability. Based on the characteristics that ATP is accumulated in large amounts in a lesion microenvironment and the expression level of a specific receptor on the surface of target cells is significantly higher than that of normal cells, the DNA nanomachine can accurately identify target cells and release LYTAC in response to an ATP signal, so as to specifically target target membrane proteins to mediate degradation of the target membrane proteins through a lysosome pathway and play a biological function. As an example, the application provides a bispecific lysosome-targeting chimera targeting c-Met and EGFR and application thereof in treatment of non-small cell lung cancer. The nanomachine has no obvious toxic side effects on normal cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to an ATP-responsive DNA nanomachine for membrane protein degradation and its application in the preparation of tumor therapeutic drugs. In particular, this invention uses the degradation of c-Met and EGFR dual-target proteins as an example to verify the feasibility of this nanomachine platform, but is not limited thereto. Background Technology

[0002] Lysosomal targeted degradation (LYTAC) technology utilizes conjugates between lysosomal shuttle receptors and target proteins (POIs) to achieve targeted degradation of extracellular proteins. The selection of ligands for both the lysosomal shuttle receptor and the POI is crucial in LYTAC, often decisively influencing the specificity and selectivity of the entire method. Nucleic acid aptamers exhibit high affinity and specificity for POIs, and their sequence modification and design offer significant flexibility, making them a focus of attention in fields such as lysosomal targeted degradation.

[0003] In recent years, self-assembled DNA nanostructures have attracted widespread attention due to their inherent biocompatibility, programmability, and multifunctionality. Nucleic acid frameworks (FNAs) possess highly efficient addressability and programmability; by modifying different functional groups or ligands (such as nucleic acid aptamers, fluorescent dyes, and chemical groups) at the vertices, edges, or internal spaces of an FNA, specific functions can be assigned to it. Furthermore, the structural sequence of an FNA can be autonomously designed according to experimental needs and can respond to stimuli changes in the environment. More notably, FNAs use bases as assembly raw materials, exhibiting better biocompatibility than other nanomaterials, and possessing good cell membrane permeability, low immunogenicity, and stability, with low toxicity to non-target cells.

[0004] Related studies have shown that there are two major usable biological features in the tumor microenvironment: first, the extracellular ATP concentration in tumor tissue is hundreds to thousands of times higher than that in normal tissue due to metabolic abnormalities and necrosis; second, folate receptors, especially FRα, are highly overexpressed on the surface of tumor cells, while they are hardly expressed in normal tissues. Summary of the Invention

[0005] The first objective of this invention is to provide an ATP-responsive DNA nanomachine, which employs a universal modular design and can be used for the degradation of any target membrane protein, belonging to a class of intelligent degradation systems that respond to ATP signals. The ATP-responsive DNA nanomachine is assembled from a lysosome-targeting chimera LYTAC and a nucleic acid framework FNA.

[0006] The nucleic acid framework (FNA) is a three-dimensional cubic nanocage structure assembled from 13 single-stranded DNA molecules through stepwise annealing. One suspended single-stranded DNA molecule serves as the load linker, and one of these strands is modified with a targeting ligand. This structure forms six faces, eight vertices, and one internal cavity to accommodate the load linker. The vertices are corner structures formed by the folding of a single DNA strand at specific base positions. Each edge contains addressable sites for anchoring internal functional components. Each side of the nucleic acid framework FNA consists of 31 base pairs (31 bp) of double-stranded DNA as double-stranded arms. The double-stranded DNA backbone serves as the rigid domain of the FNA, giving the overall structure a precise nanoscale geometric shape and mechanical stability. Based on the helical parameter of 3.4 nm pitch, which is one rotation of the DNA double helix every 10.5 bp, the precise arm length of the double-stranded arm corresponding to each side of the nucleic acid framework FNA is 10.04 nm. The 31 bp length of each arm is designed to match the overall size of the FNA with the space requirements of the LYTAC (67 nt + 53 nt) molecule and the ATP aptamer chain (32 nt). At the same time, the larger structure of the FNA has a longer tumor retention time, making it suitable for tumor treatment applications.

[0007] The LYTAC is assembled from a target membrane protein binding element and a lysosomal shuttle receptor binding element with an ATP aptamer chain attached to its end through complementary base pairing of the binding element's complementary blocks, i.e., the linker chain. The ATP aptamer chain extending from the end of the lysosomal shuttle receptor binding element hybridizes with the load chain Linker through DNA-DNA complementary base pairing. This connection has a predetermined thermodynamic stability, ensuring that the ATP aptamer chain is stably suspended inside the cavity under physiological conditions. Simultaneously, when high ATP concentrations trigger conformational changes in the aptamer, a predetermined conformational unlocking reaction occurs. The LYTAC is suspended entirely within the internal cavity of the FNA by the pairing of the ATP aptamer chain with the load chain Linker, effectively "binding" the LYTAC within the FNA backbone, forming a "cage-in-a-warhead" structure to prevent non-specific exposure in normal tissue. Each FNA is connected to one ATP aptamer chain, serving as a single triggering element for the ATP response. The ATP aptamer is a known ATP-specific nucleic acid aptamer sequence, acting as a molecular switch in response to abnormally high ATP concentrations in the tumor microenvironment. Under low ATP conditions, such as in normal tissues, aptamers maintain a stable folded conformation, blocking the release pathway of internal loads. Under high ATP conditions, in tumor tissues where membrane proteins are degraded, aptamers undergo conformational changes, triggering the opening of the DNA strand structure and releasing downstream functional modules.

[0008] The target membrane protein described in this invention can be any target protein located on the cell membrane, possessing an extracellular domain, and associated with disease, including but not limited to growth factor receptors, immune checkpoint molecules, viral receptors, and other transmembrane proteins. Depending on the target membrane protein, ATP-responsive DNA nanomachines targeting different targets can be constructed simply by replacing the corresponding target membrane protein binding element in LYTAC.

[0009] The three-dimensional spacing between the FNA and the ATP aptamer chain is jointly controlled by the linker length of the load chain, the anchoring point position, and the cavity geometry. This DNA nanomachine is self-assembled from three functional modules, arranged in a logic-gated synergistic layout within the cavity of the FNA: Module 1 is a targeting / carrier platform, corresponding to the nucleic acid framework (FNA) modified with the targeting ligand; Module 2 is a trigger / control module, with the ATP aptamer chain suspended inside the cavity, responsible for sensing changes in ATP concentration penetrating into the cavity and serving as the "input signal"; Module 3 is a function / effect module, with the target membrane protein binding element and lysosomal shuttle receptor binding element of LYTAC suspended within the same cavity via the ATP aptamer chain, responsible for performing lysosomal targeted degradation and serving as the "output effect". In this process, the FNA cavity not only provides physical protection but also acts as a nanoscale reactor to achieve close functional coupling between the ATP aptamer chain and LYTAC.

[0010] As a specific embodiment of the ATP-responsive DNA nanomachine of the present invention, when the targeting ligand is folic acid, the FNA is a folic acid ligand-modified nucleic acid framework. Among the 13 DNA single strands used to assemble the folic acid ligand-modified nucleic acid framework, one DNA strand has a folic acid ligand stably modified at the C6-1-NH2-FA end via an amide bond, used to target the folic acid receptor highly expressed on the surface of tumor cells. The remaining DNA strands include the linker load strand, C1-1, C1-2, C2-1, C2-2, C3-1, C3-2, C4-1, C4-2, C5-1, C5-2, and C6-2. The nucleic acid sequences of C1-1, C1-2, C2-1, C2-2, C3-1, C3-2, C4-1, C4-2, C5-1, C5-2, C6-1-NH2-FA, C6-2, and the linker load strand are as shown in SEQ ID NO.1~SEQ ID NO.2. As shown in IDNO.13, the ATP-responsive DNA nanomachines constructed by encapsulating lysosomal targeting chimeras with ATP aptamers via a linker load chain to achieve subsequent functionalization can target cells with high expression of folate receptors on the cell membrane. Folate receptors, especially FRα, are highly overexpressed on the surface of tumor cells, while they are hardly expressed in normal tissues. The ATP-responsive DNA nanomachines can not only target the aforementioned tumor cells, but also effectively enter cells and have better biological stability due to their unique three-dimensional structure.

[0011] Preferably, the preparation steps of C6-1-NH2-FA include: dissolving folic acid in anhydrous DMSO, then adding EDC and NHS sequentially, and vortexing to assist dissolution, wherein the mass-to-volume ratio of folic acid to anhydrous DMSO is 4.4 mg:500 μL, and the molar ratio of folic acid, EDC, and NHS is 1:2:2; adding triethylamine, with a volume ratio of triethylamine to anhydrous DMSO of (2~3):500, and adjusting the pH to 7.0~8.5; incubating at 20~30℃ for 8~16 h under light-protected conditions, and the resulting reaction solution is a DMSO solution of folic acid-NHS ester with a concentration of 20 mM; mixing a single-stranded DNA with an amino (-NH2) modified 5′ end as shown in SEQ ID NO.11, designated C6-1-NH2, with the activated folic acid-NHS ester in a NaHCO3 / Na2CO3 buffer at pH 8.5, and incubating at 20~30℃ under light-protected conditions; subsequently adding 1 M, pH The reaction was terminated by mixing and incubating with 7.5% Tris-HCl at 20-30°C for 10 min. 3 M NaOAc at pH 5.2 and 20 mg / mL glycogen were added to the reaction product, mixed well, and then purified by ethanol precipitation to obtain the C6-1-NH2-FA.

[0012] In a more specific embodiment of the ATP-responsive DNA nanomachine of the present invention, the targeting ligand is folic acid and the target membrane protein is c-Met or EGFR; in this case, the target membrane protein binding element is a nucleic acid aptamer of c-Met or EGFR; and the lysosomal shuttle receptor binding element is an IGFIIR nucleic acid aptamer. This ATP-responsive DNA nanomachine can only mediate the degradation of cell membrane c-Met and EGFR via the lysosomal pathway in the presence of ATP, thereby enhancing the targeting and specificity of membrane protein degradation.

[0013] Preferably, when the target membrane protein is c-Met, the LYTAC of the ATP-responsive DNA nanomachine is formed by hybridization of the nucleic acid aptamer Met-13Y corresponding to the target protein c-Met and IGFIIR-13-ATP, denoted as c-Met target degradation element LYTAC-Met, and IGFIIR-13-ATP is composed of IGFIIR nucleic acid aptamer and ATP aptamer chains; when the target membrane protein is EGFR, the LYTAC of the ATP-responsive DNA nanomachine is formed by hybridization of the nucleic acid aptamer EGFR-13Y corresponding to the target protein EGFR and IGFIIR-13-ATP, denoted as EGFR target degradation element LYTAC-EGFR; the nucleic acid sequences of Met-13Y, EGFR-13Y, and IGFIIR-13-ATP are shown in SEQ ID NO.14~SEQ ID NO.16.

[0014] The present invention also provides a method for constructing ATP-responsive DNA nanomachines in the more specific embodiments described above, comprising the following steps:

[0015] The 13 DNA strands constituting the framework, C1-1, C1-2, C2-1, C2-2, C3-1, C3-2, C4-1, C4-2, C5-1, C5-2, C6-1-NH2-FA, C6-2, and the linker support strand were mixed in buffer, denatured, and slowly cooled and annealed to 25°C. The mixture was then incubated in a constant-temperature shaker to obtain the nucleic acid framework FNA. The nucleic acid aptamer Met-13Y or EGFR-13Y was mixed with IGFIIR-13-ATP in buffer, denatured, and slowly cooled and annealed to 25°C. The mixture was then incubated in a constant-temperature shaker to obtain LYTAC-Met or LYTAC-EGFR. The FNA was then mixed with LYTAC-Met or LYTAC-EGFR in buffer and incubated at 20-30°C for 30 min to obtain the DNA nanomachines.

[0016] More preferably, the molar amounts of C1-1, C1-2, C2-1, C2-2, C3-1, C3-2, C4-1, C4-2, C5-1, C5-2, C6-1-NH2-FA, C6-2, and the linker are equal, the buffer solution is phosphate-buffered saline (PBS) buffer, the denaturation temperature is 95 °C, the denaturation time is 10 min, and the incubation temperature is 25 °C for 30 min.

[0017] More preferably, the molar ratio of Met-13Y or EGFR-13Y to IGFIIR-13-ATP is 1:1, the buffer is phosphate-PBS buffer, the denaturation temperature is 95 °C, the denaturation time is 10 min, and the incubation temperature is 25 °C for 30 min.

[0018] More preferably, the molar ratio of FNA to LYTAC-Met or LYTAC-EGFR is 1:1, the buffer solution is pH 7.4 phosphate PBS buffer, and the composition is 2.0 mM KH2PO4, 137 mM NaCl, 10.0 mM Na2HPO4, and 2.7 mM KCl; the incubation temperature is 25 °C, and the incubation time is 30 min; the final concentration of DNA nanomachines (Met / EGFR) in the mixed solution is 1 μM.

[0019] Secondly, this invention also provides the application of the ATP-responsive DNA nanomachine in the preparation of therapeutic drugs for non-small cell lung cancer. The ATP-responsive DNA nanomachine is any one or a combination of two of the aforementioned DNA Nanomachine Met or DNA Nanomachine EGFR. The combined use of the two ATP-responsive DNA nanomachines can be used to address the abnormal activation of the c-Met pathway and the interactive communication of the EGFR signaling network in non-small cell lung cancer (NSCLC). Combining the unique advantages of lysosomal targeting chimeras and nucleic acid frameworks, tumor-specific enrichment is achieved through folic acid targeting, and structural transitions are triggered by high concentrations of ATP. Simultaneously, c-Met and EGFR target degradation elements are exposed, achieving synchronous lysosomal targeted degradation of the two pathogenic factors. This provides a novel solution for overcoming drug resistance in NSCLC patients and improving treatment efficacy.

[0020] The advantages of this invention can be summarized in the following three aspects:

[0021] 1) Dual precise targeting and ATP-responsive release with minimal damage to normal cells: By utilizing the high concentration of ATP and high expression of folic acid receptors in the tumor microenvironment, dual precise recognition of tumor cells is achieved; LYTAC is "locked" within the framework by the ATP aptamer, triggering "on-demand drug release" only in the high ATP environment of the tumor, avoiding premature leakage in normal tissues, thus minimizing damage to normal cells.

[0022] 2) Overcoming drug resistance by utilizing endogenous lysosomal degradation pathways: After release, LYTAC is transported to lysosomes via lysosomal shuttle receptors such as IGFIIR for irreversible degradation of membrane proteins such as c-Met and EGFR. This directly utilizes the cell's own protein degradation mechanism, effectively overcoming the drug resistance problem of traditional small molecule inhibitors.

[0023] 3) Modular assembly with strong platform versatility: The modular design of “LYTAC + ATP aptamer + FNA modified with target ligand” is adopted. Each functional unit can be independently optimized or replaced, such as by changing the target ligand or response element, which is convenient for expansion to other tumor markers or disease research models. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the module assembly process of the two ATP-responsive DNA nanomachines constructed in this invention.

[0025] Figure 2 This is a schematic diagram illustrating the principle of the combined action of two ATP-responsive DNA nanomachines constructed in this invention on HCC827 / GR drug-resistant lung cancer cells.

[0026] Figure 3 Schematic diagrams of the structures of two different conformations of the chimera encapsulated in ATP-responsive DNA nanomachines, LYTAC-X and LYTAC-Y, and the results of polyacrylamide gel electrophoresis verification of LYTAC-X and LYTAC-Y;

[0027] Figure 4 Western blot characterization of the degradation efficiency of LYTAC in different configurations at the cellular level;

[0028] Figure 5 Flow cytometry characterization of LYTAC degradation efficiency of different LYTAC configurations at the cellular level;

[0029] Figure 6 This is a polyacrylamide gel electrophoresis characterization image of the ATP-responsive DNA nanomachines constructed in Example 1;

[0030] Figure 7 The stability characterization diagrams for LYTAC and DNA nanomachines are shown, with the left diagram representing nuclease resistance and the right diagram representing serum stability.

[0031] Figure 8 Selective characterization of the response of DNA nanomachines to ATP, CTP, GTP, UTP, FBS and Exo I;

[0032] Figure 9 Characterization of the dependence of DNA nanomachines on ATP concentration;

[0033] Figure 10 The time-dynamic characteristics of the response of DNA nanomachines to ATP in the control group without ATP were characterized.

[0034] Figure 11 The time-dynamic characteristics of the DNA nanomachines in the ATP-containing experimental group were characterized in response to ATP.

[0035] Figure 12 Flow cytometry characterization of the targeting performance and binding affinity of ATP-responsive DNA nanomachines;

[0036] Figure 13 Western blot characterization of the degradation performance of ATP-responsive DNA nanomachines;

[0037] Figure 14 Flow cytometry characterization of the degradation performance of ATP-responsive DNA nanomachines;

[0038] Figure 15 The diagram shows the effect of ATP-responsive DNA nanomachines on the cell viability of HCC827 / GR. The left and right diagrams compare the inhibitory effects of single-target and dual-target nanomachines, respectively.

[0039] Figure 16 Figure showing the effect of single-target ATP-responsive DNA nanomachines on the cell migration ability of HCC827 / GR.

[0040] Figure 17 The effect of dual-target ATP-responsive DNA nanomachines on the cell migration ability of HCC827 / GR cells is shown in the figure. Detailed Implementation

[0041] The following example, using c-Met or EGFR degradation as targets, illustrates the construction method and application effects of the ATP-responsive DNA nanomachines described in this invention. It should be understood that the scope of protection of this invention is not limited to this specific target.

[0042] The main raw materials, reagent sources, or preparation methods involved in the following examples and test cases are shown below:

[0043] Preparation of washing buffer: Mix 4.5 g / L glucose and 5 mM MgCl2 in DPBS to remove excess DNA.

[0044] Preparation of binding buffer: Add 0.1 mg / mL yeast tRNA and 1 mg / mL BSA to the washing buffer to reduce nonspecific binding.

[0045] The nucleic acids used in this invention were purchased from Shanghai Sangon Biotech Co., Ltd. The specific nucleic acid names and base sequences are shown in Tables 1 and 2.

[0046] Table 1

[0047]

[0048] Table 2

[0049]

[0050] Example 1: Construction of ATP-responsive DNA nanomachines

[0051] The method for constructing ATP-responsive DNA nanomachines includes the following steps:

[0052] 1) Preparation of DNA-folic acid: Dissolve 4.4 mg of folic acid (10 μmol) in 500 μL of anhydrous DMSO; add 3.8 mg of EDC·HCl (20 μmol) and 2.3 mg of NHS (20 μmol) sequentially, and vortex to assist dissolution; add 2 μL of triethylamine to adjust the pH to 7.0; incubate at 25 ℃ in a constant temperature shaker for 24 h under light-protected conditions. The resulting reaction solution is 20 mM folic acid-NHS ester (solvent: DMSO solution); design a single-stranded DNA with an amino (-NH2) modified at the 5′ end, named C6-1-NH2-FA; mix this DNA strand with the pre-activated folic acid-NHS ester in NaHCO3 / Na2CO3 buffer (pH 8.5), and incubate at 25 ℃ in a constant temperature shaker for 6 h under light-protected conditions; then add 1 M Tris-HCl (pH 7.5), and incubate at 600 r / min in a constant temperature shaker at 25 ℃ for 10 h. The reaction was terminated at min; 3 M NaOAc (pH 5.2) and 20 mg / mL glycogen were added to the reaction product, mixed well, and purified by ethanol precipitation to obtain C6-1-NH2-FA; wherein, the molar ratio of folic acid:EDC:NHS in the pre-activated folic acid-NHS ester was 1:2:2; the molar ratio of C6-1-NH2 to the pre-activated folic acid-NHS ester was 1:20; the incubation temperature was 25 ℃, and the incubation time was 24 h and 6 h respectively; the buffers were 1 M Tris-HCl buffer (pH 7.5), composed of Tris(hydroxymethyl)aminomethane and concentrated hydrochloric acid HCl; 0.1 M NaHCO3-Na2CO3 buffer (pH 8.5), composed of 0.1 M Na2CO3 solution and 0.1 M NaHCO3 solution; and 3 M sodium acetate NaOAc (pH 5.2), composed of sodium acetate trihydrate CH3COONa·3H2O and glacial acetic acid CH3COOH.

[0053] 2) Preparation of folic acid ligand-modified nucleic acid framework: The 13 DNA strands constituting the framework (C1-1, C1-2, C2-1, C2-2, C3-1, C3-2, C4-1, C4-2, C5-1, C5-2, C6-1-NH2-FA, C6-2, Linker) were mixed in PBS buffer in equimolar amounts; after denaturation at 95℃ for 10 min, the mixture was slowly cooled and annealed to 25℃, and then incubated at 600 r / min for 30 min in a 25℃ constant temperature shaker to obtain the folic acid-modified nucleic acid framework (FNA).

[0054] 3) Preparation of lysosome-targeting chimeras: The nucleic acid aptamer Met-13Y or EGFR-13Y corresponding to c-Met or EGFR and the nucleic acid aptamer IGFIIR-13-ATP (containing an ATP aptamer at its end) corresponding to the lysosome shuttle receptor were mixed in PBS buffer at a molar ratio of 1:1; after denaturation at 95°C for 10 min, the mixture was slowly cooled and annealed to 25°C, and then incubated at 600 r / min for 30 min in a 25°C constant temperature shaker to obtain the bispecific lysosome-targeting chimera LYTAC-Met or LYTAC-EGFR;

[0055] 4) Assembly of ATP-responsive DNA nanomachines: The prepared FNA was mixed with LYTAC-Met or LYTAC-EGFR at a molar ratio of 1:1 in PBS buffer and incubated at 600 r / min for 30 min in a 25 ℃ constant temperature shaker to obtain ATP-responsive DNA nanomachines, namely DNA Nanomachine Met or DNA Nanomachine EGFR. The buffer used was phosphate buffer (PBS, pH 7.4), with the following components: 2.0 mM KH2PO4, 137 mM NaCl, 10.0 mM Na2HPO4, and 2.7 mM KCl. The final concentration of DNA Nanomachine Met or DNA Nanomachine EGFR in the mixed solution was 1 μM.

[0056] like Figure 1 As shown in the figure, this is an ATP-responsive DNA nanomachine, a smart lysosome-targeting chimeric (LYTAC) nanomachine based on a DNA framework nucleic acid (FNA), and its anti-tumor mechanism of action: First, an aptamer capable of recognizing c-Met or EGFR target proteins, an IGFIIR aptamer binding to the lysosome transport receptor IGFIIR, and an ATP aptamer responding to ATP signals are assembled into a LYTAC. Then, through base complementarity pairing, it is loaded onto a folic acid ligand-modified DNA framework nucleic acid (FNA) to construct an L-FNA-Met or L-FNA-EGFR nanomachine; Figure 2As shown, this nanomachine utilizes the highly expressed folate receptor on the surface of tumor cells to achieve active targeted enrichment. Simultaneously, it leverages the abundant ATP accumulated in the tumor microenvironment to bind with ATP aptamers, triggering the responsive release of LYTAC from the FNA. The released LYTAC then binds to the c-Met / EGFR target protein and the lysosomal shuttle receptor IGFIIR on the surface of tumor cells. Through receptor-mediated endocytosis, the target protein is transported to lysosomes for degradation, thereby blocking the oncogenic signaling pathway and exerting an anti-tumor effect. Normal cells, due to low folate receptor expression and normal ATP concentration in their microenvironment, are not activated. Therefore, this system achieves precise identification and selective killing of tumor cells through a dual locking mechanism of "folate targeting" and "ATP response," effectively avoiding toxic side effects on normal cells.

[0057] Test Example 1: Conformation Optimization and Characterization Screening of LYTAC Chimeras

[0058] like Figure 3The system demonstrates the conformational optimization and characterization screening process of LYTAC chimeras. First, it reveals the design dilemma of the traditional LYTAC-X. The assembly of LYTAC chimeras is achieved through the connection of complementary blocks (linker chains) between aptamers. While adjusting the number of base pairs (N) in the linker chains can balance degradation efficiency and assembly yield, decreasing N improves degradation efficiency but reduces assembly yield, making it difficult to achieve both simultaneously. Therefore, a novel LYTAC-Y was constructed by changing the hybridization connection method. Polyacrylamide gel electrophoresis confirmed that chimeras with different linker chain lengths and connection orientations can achieve efficient assembly. The LYTAC-X scheme is as follows: Lane 1: Marker (25~500 bp); Lane 2: IGFIIR-13X; Lane 3: Met-13X; Lane 4: IGFIIR-13X + Met-13X; Lane 5: IGFIIR-20X; Lane 6: Met-20X; Lane 7: IGFIIR-20X + Met-20X; Lane 8: Lane 9: Met-30X; Lane 10: IGFIIR-30X + Met-30X; The LYTAC-Y protocol is as follows: Lane 1: Marker (25~500 bp); Lane 2: IGFIIR-13Y; Lane 3: Met-13Y; Lane 4: IGFIIR-13Y + Met-13Y; Lane 5: IGFIIR-20Y; Lane 6: Met-20Y; Lane 7: IGFIIR-20Y + Met-20Y; Lane 8: IGFIIR-30Y; Lane 9: Met-30Y; Lane 10: IGFIIR-30Y + Met-30Y; Lane 11: Marker (25~500 bp). The above nucleic acid sequences are shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.17~SEQ ID NO.18. As shown in IDNO.24, whether it is an X-type or Y-type chimera with 13, 20, or 30 base pairs, its single-chain components and hybridization products all show clear molecular weight difference bands, indicating that chimeras with different linker chain lengths and different linker directions can achieve efficient assembly.

[0059] The degradation efficiency of each conformation was further compared at the cellular level using Western blot and flow cytometry. Figure 4 Western blot analysis showed that after incubation at 500 nM for 24 h, the c-Met protein band at 154 kDa was the weakest, with the lowest relative expression level (0.3) in the 13Y group. Figure 5Flow cytometry analysis showed that the Cy5-c-Met positivity rate of the 13Y group decreased to 7.18%, and the mean fluorescence intensity (MFI) was significantly lower than that of the NC group and other groups (p<<0.0001). The degradation effects of the 13X, 20X, 30X, 20Y, and 30Y groups were all inferior to those of the 13Y group. These results together confirm that the LYTAC-Y constructed by changing the hybridization method, especially the 13Y conformation with a length of 13 base pairs, achieved the optimal target protein degradation efficiency while maintaining efficient assembly.

[0060] Test Example 2: Fabrication and Characterization of ATP-Responsive DNA Nanomachines

[0061] like Figure 6 The images show polyacrylamide gel electrophoresis characterizations of different construction stages of the DNA Nanomachine Met constructed in Example 1, which is an ATP-responsive DNA nanomachine constructed according to the optimal configuration in Test Example 1.

[0062] Figure 6 The polyacrylamide gel electrophoresis characterization diagram shown below:

[0063] Lane 1: Marker;

[0064] Lane 2: C6-2;

[0065] Lane 3: C6-2 + C2-2 + C2-1;

[0066] Lane 4: C6-2+C2-2+C2-1+C6-1-NH2-FA +C4-1;

[0067] Lane 5: C6-2+C2-2+C2-1+C6-1-NH2-FA +C4-1+C4-2+C3-1;

[0068] Lane 6: C6-2+C2-2+C2-1+C6-1-NH2-FA +C4-1+C4-2+C3-1+C3-2+C1-2;

[0069] Lane 7: C6-2+C2-2+C2-1+C6-1-NH2-FA+C4-1+C4-2+C3-1+C3-2+C1-2+C1-1+C5-2;

[0070] Lane 8: C6-2+C2-2+C2-1+C6-1-NH2-FA+C4-1+C4-2+C3-1+C3-2+C1-2+C1-1+C5-2+C5-1;

[0071] Lane 9: C6-2+C2-2+C2-1+C6-1-NH2-FA+C4-1+C4-2+C3-1+C3-2+C1-2+C1-1+C5-2+C5-1+Linker;

[0072] Lane 10: C6-2+C2-2+C2-1+ C6-1-NH2-FA+C4-1+C4-2+C3-1+C3-2+C1-2+C1-1+C5-2+C5-1+Linker+IGFIIR-13-ATP+ Met-13Y;

[0073] Lane 11: C6-2+C2-2+C2-1+ C6-1-NH2-FA +C4-1+C4-2+C3-1+C3-2+C1-2+C1-1+C5-2+C5-1+Linker + IGFIIR-13-ATP+ Met-13Y +10 mM ATP;

[0074] Lane 12: IGFIIR-13-ATP + Met-13Y;

[0075] Lane 13: IGFIIR-13-ATP;

[0076] Lane 14: Met-13Y;

[0077] Lane 15: Marker;

[0078] The above nucleic acid sequences are shown in SEQ ID NO.1~SEQ ID NO.16;

[0079] from Figure 6 As can be seen, lanes 2 to 9 show that the bands gradually shift upwards after the DNA nanocages hybridize with the linker, indicating an increase in molecular weight and successful assembly. Lane 10 shows a new band at a higher molecular weight position, a band that lags behind and has significantly increased brightness, confirming that the DNA nanocages hybridize with LYTAC to form a larger, complete DNA nanomachine. In Lane 11, after the addition of 10 mM ATP, this high-molecular-weight band disappears, and a band appearing at the same location as in Lane 12 (the free LYTAC) appears. This indicates that a conformational change in the ATP aptamer leads to the dissociation and release of LYTAC from the nanocage, thus verifying that the DNA nanomachine can achieve controlled release of LYTAC under ATP stimulation. Lanes 13 and 14 are the IGFIIR-13-ATP and Met-13Y monomer controls, respectively, further confirming the identity and migration positions of each component. These experimental results demonstrate that the ATP-responsive DNA nanomachine has been successfully prepared.

[0080] Test Example 3: Stability Characterization of ATP-Responsive DNA Nanomachines

[0081] exist Figure 7 In this study, the nuclease resistance and biostability of the DNA Nanomachine Met constructed in Example 1 were verified by gel electrophoresis. After treatment with DNase I at 37 °C for 0 to 300 min, the DNA nanomachine band remained relatively intact and decayed slowly, exhibiting good nuclease resistance. In contrast, the band of free LYTAC rapidly weakened and diffused over time, indicating that it was easily degraded by nucleases. After incubation in 10% fetal bovine serum (FBS) at 37 °C for 0 to 48 h, the DNA nanomachine band remained discernible at 48 h, demonstrating excellent biostability. However, the band of free LYTAC disappeared rapidly in the serum, indicating poor biostability. These results demonstrate that the three-dimensional structure of the DNA framework nucleic acid can effectively encapsulate and protect the internal LYTAC from attack by nucleases and degradation components in serum, thereby significantly improving the stability of the material in the physiological environment and providing a guarantee for its in vivo application.

[0082] Test Example 4: Response Performance Analysis of ATP-Responsive DNA Nanomachines

[0083] Based on the principle of fluorescence resonance energy transfer (FRET), the response performance of DNA Nanomachine Met to ATP was systematically analyzed. When ATP was not bound, the FAM fluorescent group labeled on LYTAC was spatially adjacent to the BHQ-1 quencher on the DNA framework, effectively quenching the fluorescence. However, when ATP specifically bound to the aptamer, LYTAC dissociated from the nanoframework, and the FAM moved away from BHQ-1, restoring the fluorescence signal. Only ATP induced a strong fluorescence activation (approximately 100 au), while CTP, GTP, UTP, fetal bovine serum FBS, and the exonuclease Exo I caused almost no fluorescence change. Figure 8 The results confirmed that the system is highly selective for ATP; Figure 9 Concentration gradient experiments showed that significant fluorescence enhancement was observed even at a 0.5 mM ATP concentration, and the fluorescence intensity gradually increased with increasing ATP concentration from 0.5 mM to 5 mM; time-kinetic analysis further revealed that... Figure 10 In the control group without ATP, the fluorescence background remained at a very low level with almost no fluctuation from 0 to 60 minutes, while after the addition of ATP, Figure 11 The fluorescence signal rapidly increased with prolonged incubation time, and 90% signal recovery was achieved within 30 minutes, indicating that the DNA nanomachine can achieve rapid, sensitive and highly specific responsive drug release within the range of ATP concentrations relevant to the tumor microenvironment.

[0084] Test Example 5: Characterization of the targeting performance and binding ability of ATP-responsive DNA nanomachines

[0085] The 5′ end of the DNA strand Met-13Y was modified with the FAM fluorescent group. HCC827 / GR cells were incubated with Met-13Y, LYTAC, DNA NanomachineMet, and DNA NanomachineMet + 0.5 mM ATP groups at 4 °C for 30 min, respectively. The final concentration of each group was adjusted to 100 nM and the final volume to 400 μL. Fluorescence in the FAM channel was collected under 488 nm excitation. Figure 12 As can be seen, the negative control (NC) group showed almost no FAM green fluorescence signal, the Met-13Y single-strand group showed a weak fluorescence signal, the free LYTAC group showed a significantly enhanced fluorescence signal, while the DNA Nanomachine Met group showed a significantly weakened green fluorescence signal due to the FAM fluorescent group being encapsulated on the framework. Upon ATP treatment, LYTAC dissociated and released from the nanoframework, releasing the FAM group and restoring the fluorescence signal. Therefore, the DNA Nanomachine Met+ATP group exhibited a bright green fluorescence signal similar to that of free LYTAC. Flow cytometry quantitative analysis showed that the FAM positivity rate in the NC group was only 0.55%, in the Met-13Y group it was 19.7%, and in the LYTAC group it was 33.3%. The positivity rate in the assembled DNA Nanomachine Met group decreased to 8.68% due to the encapsulation of the fluorescent group, but rebounded significantly to 58.0% after ATP release. The corresponding mean fluorescence intensity (MFI) histogram showed that the NC group had the lowest MFI, with the Met-13Y and LYTAC groups increasing sequentially. The MFI in the Met group was significantly suppressed (**p<<0.01 compared with the LYTAC group), while the MFI increased sharply after ATP treatment (****p<<0.0001 compared with the DNA Nanomachine Met group). The above cellular-level quantitative fluorescence data confirm that the DNA nanomachine has a highly efficient ATP-responsive release capability and cell-to-cell binding affinity at the cellular level.

[0086] Test Example 6: Characterization of the Degradation Performance of ATP-Responsive DNA Nanomachines

[0087] HCC827 / GR cells were incubated with Met-13Y, LYTAC, DNA Nanomachine Met, and DNA Nanomachine Met + 0.5 mM ATP at 37 ℃ for 24 h, with the final concentration adjusted to 100 nM and the final volume to 1 mL. Western blot and flow cytometry were used to verify at the protein level the function of the DNA nanomachines in releasing LYTAC in response to ATP and inducing the degradation of the target protein c-Met. Figure 13 Western blot results showed that the NC group had the highest c-Met protein expression level (154 kDa, with 36 kDa GAPDH as an internal control), the Met-13Y monoaptamer treatment group showed a slight decrease in c-Met expression, the free LYTAC group showed a significant downregulation of c-Met expression, while the DNA Nanomachine Met group had a c-Met level similar to the NC group under ATP-free conditions, indicating that the system was in a "closed" state and hardly degraded the target protein. When ATP was added, the DNA Nanomachine Met responded by releasing LYTAC, and the c-Met expression level dropped sharply to a low level comparable to that of free LYTAC. The quantitative bar chart showed that the relative expression levels of each group were statistically significant (p << 0.0001). Figure 14 The c-Met level on the cell surface was detected using Cy5 fluorescent dye labeling. Flow cytometry results showed that the c-Met positivity rate was 16.2% in the NC group, decreased to 8.05% in the Met-13Y group, and almost completely degraded c-Met in the LYTAC group (positive rate of only 0.82%). The DNA Nanomachine Met group still had a high positive rate of 11.9% without ATP, and the mean fluorescence intensity (MFI) was close to that of the NC group, indicating that the target protein remained stable when the nanomachine was not activated. However, after the addition of ATP, the positive rate dropped sharply to 2.7%, and the MFI also dropped significantly to a level close to that of the LYTAC group. The above results together prove that the DNA nanomachine can switch from a "closed" state to a "closed" state under ATP stimulation, accurately release LYTAC, and efficiently induce lysosomal degradation of the c-Met target protein, achieving ATP-controlled targeted protein degradation.

[0088] Test Example 7: Effects of ATP-responsive DNA nanomachines on the cell viability of HCC827 / GR

[0089] Figure 15The cytotoxic effect of DNA nanomachines on tumor cells and their ATP-dependent activation characteristics were evaluated using cell viability assays. First, HCC827 / GR cells were incubated at 37 °C for 48 h with Met-13Y, LYTAC, DNA Nanomachine Met, and DNA Nanomachine Met + 0.5 mM ATP groups, respectively. The final concentration and volume of each group were adjusted to 100 nM and 1 mL. Results showed that cell viability in the NC group and the ATP-treated group were close to 100% with no significant difference, indicating that ATP itself has no cytotoxicity. The viability of the Met-13Y monoaptamer group decreased to 65%, and the viability of the free LYTAC (Met) group further decreased to 40%. However, the viability of DNA Nanomachine Met remained at 90% even without ATP, with no significant difference from the NC group, indicating that the unactivated DNA Nanomachine Met was in a "closed" state. In a safe state, the cell viability of this group decreased sharply to 40% after the addition of ATP, which was comparable to that of the free LYTAC group and significantly lower than that of the group without ATP. Further comparison of the killing effects of single-target and dual-target nanomachines showed that the single-target DNA nanomachine (Met) + ATP group and the single-target (EGFR) + ATP group reduced cell viability to 50% and 40%, respectively. However, the dual-target DNA nanomachine (Met + EGFR) still had a viability close to 95% without ATP, but it decreased significantly to 30% after the addition of ATP. Moreover, its inhibitory effect was significantly better than that of any single-target group. The above results indicate that the dual-target DNA nanomachine is only activated and releases LYTAC in the presence of ATP, which significantly inhibits tumor cell proliferation at the functional level. Furthermore, the dual-target synergistic strategy exhibits stronger anti-tumor activity than the single-target strategy.

[0090] Test Example 8: Effect of ATP-responsive DNA nanomachines on the cell migration ability of HCC827 / GR cells

[0091] The inhibitory effect of DNA nanomachines on tumor cell migration and their ATP-dependent activation characteristics were evaluated using a wound healing assay. Figure 16 The results showed that the scratch widths were consistent across all groups at 0 h. After 24–72 h of culture, the scratches in the NC group and the ATP-treated group gradually healed and were almost completely closed by 72 h. The migration rate of the Met-13Y monoaptamer group was slightly slowed down, and the scratch healing of the LYTAC (Met) group was significantly inhibited. The scratch closure of the DNA nanomachine (DNANanomachine Met) group was similar to that of the NC group without ATP, indicating that the unactivated nanomachines did not affect cell migration. However, after the addition of ATP, the scratches in this group remained at their widest extent at 24 h, 48 h, and 72 h, and cell migration was significantly blocked. Figure 17Further comparison of the inhibitory effects of single-target and dual-target nanomachines revealed that scratches healed normally in the NC and ATP groups. The DNA nanomachines (Met) + ATP and (EGFR) + ATP groups effectively inhibited scratch healing. However, the dual-target DNA nanomachines (Met + EGFR) resulted in significantly narrower scratches without ATP, and almost no healing after the addition of ATP. Their inhibitory effect on migration was significantly better than either single-target group. These results collectively demonstrate that the DNA nanomachines are activated and release LYTAC only under ATP stimulation, effectively inhibiting tumor cell migration by degrading c-Met and / or EGFR target proteins. Moreover, the dual-target synergistic strategy exhibits stronger anti-metastasis potential.

Claims

1. An ATP-responsive DNA nanomachine for membrane protein degradation, characterized in that, The ATP-responsive DNA nanomachine is assembled from the lysosome-targeting chimera LYTAC and the nucleic acid framework FNA; The nucleic acid framework FNA is a three-dimensional cubic framework nanocage structure formed by assembling 13 single-stranded DNA strands through stepwise annealing. One suspended single-stranded DNA strand serves as the load linker, and one of these strands is modified with a targeting ligand, forming 6 faces, 8 vertices, and 1 internal cavity to accommodate the load linker. The vertices are corner structures formed by the folding of a single DNA strand at specific base positions. Each edge contains addressable sites for anchoring internal functional components. Each edge of the nucleic acid framework FNA consists of a 31-base-pair double-stranded DNA double-stranded arm. The LYTAC is assembled from a target membrane protein binding element and a lysosomal shuttle receptor binding element with an ATP aptamer chain attached to its end through complementary base pairing of the binding element and the linker chain. The ATP aptamer chain extending from the end of the lysosomal shuttle receptor binding element hybridizes with the linker chain through DNA-DNA complementary base pairing. When a high concentration of ATP binding triggers a conformational change in the aptamer, a preset conformational unlocking reaction can occur. The LYTAC is suspended as a whole in the internal cavity of the FNA by pairing the ATP aptamer chain with the linker chain. The target membrane protein is a target protein that is randomly located on the cell membrane, has an extracellular domain, and is associated with disease, including growth factor receptors, immune checkpoint molecules, viral receptors, and other transmembrane proteins.

2. The ATP-responsive DNA nanomachine as described in claim 1, characterized in that, The targeting ligand is folic acid, and the FNA is a folic acid ligand-modified nucleic acid framework.

3. The ATP-responsive DNA nanomachine as described in claim 1, characterized in that, Of the 13 DNA single strands used to assemble the folic acid ligand-modified nucleic acid framework, one DNA strand has a folic acid ligand stably modified at the C6-1-NH2-FA end via an amide bond for targeting the folic acid receptor highly expressed on the surface of tumor cells. The remaining DNA strands include the linker load strand, C1-1, C1-2, C2-1, C2-2, C3-1, C3-2, C4-1, C4-2, C5-1, C5-2, and C6-2; and the nucleic acid sequences of C1-1, C1-2, C2-1, C2-2, C3-1, C3-2, C4-1, C4-2, C5-1, C5-2, C6-1-NH2-FA, C6-2, and the linker load strand are as shown in SEQ ID NO.1~SEQ ID NO. As shown in NO.13, the ATP-responsive DNA nanomachines constructed by encapsulating the lysosomal targeting chimera LYTAC with an ATP aptamer via a linker load chain to achieve subsequent functionalization can target cells with high expression of folate receptors on the cell membrane.

4. The ATP-responsive DNA nanomachine as described in claim 3, characterized in that, The targeting ligand is folic acid and the target membrane protein is c-Met or EGFR; the target membrane protein binding element is a nucleic acid aptamer of c-Met or EGFR; the lysosomal shuttle receptor binding element is an IGFIIR nucleic acid aptamer. This ATP-responsive DNA nanomachine can only mediate the degradation of cell membrane c-Met and EGFR through the lysosomal pathway in the presence of ATP, thereby enhancing the targeting and specificity of membrane protein degradation.

5. The ATP-responsive DNA nanomachine as described in claim 4, characterized in that, When the target membrane protein is c-Met, the LYTAC of the ATP-responsive DNA nanomachine is formed by hybridization of the nucleic acid aptamer Met-13Y corresponding to the target protein c-Met and IGFIIR-13-ATP, denoted as c-Met target degradation element LYTAC-Met, and IGFIIR-13-ATP is composed of IGFIIR nucleic acid aptamer and ATP aptamer chains; when the target membrane protein is EGFR, the LYTAC of the ATP-responsive DNA nanomachine is formed by hybridization of the nucleic acid aptamer EGFR-13Y corresponding to the target protein EGFR and IGFIIR-13-ATP, denoted as EGFR target degradation element LYTAC-EGFR; the nucleic acid sequences of Met-13Y, EGFR-13Y, and IGFIIR-13-ATP are shown in SEQ ID NO.14~SEQ ID NO.

16.

6. The ATP-responsive DNA nanomachine as described in claim 4, characterized in that, The fabrication of ATP-responsive DNA nanomachines includes the following steps: The 13 DNA strands constituting the framework, C1-1, C1-2, C2-1, C2-2, C3-1, C3-2, C4-1, C4-2, C5-1, C5-2, C6-1-NH2-FA, C6-2, and the linker support strand were mixed in buffer, denatured, and slowly cooled and annealed to 25°C. The mixture was then incubated in a constant-temperature shaker to obtain the nucleic acid framework FNA. The nucleic acid aptamer Met-13Y or EGFR-13Y was mixed with IGFIIR-13-ATP in buffer, denatured, and slowly cooled and annealed to 25°C. The mixture was then incubated in a constant-temperature shaker to obtain LYTAC-Met or LYTAC-EGFR. The FNA was then mixed with LYTAC-Met or LYTAC-EGFR in buffer and incubated at 20-30°C for 30 min to obtain the DNA nanomachines.

7. The ATP-responsive DNA nanomachine as described in claim 6, characterized in that, The molar amounts of C1-1, C1-2, C2-1, C2-2, C3-1, C3-2, C4-1, C4-2, C5-1, C5-2, C6-1-NH2-FA, C6-2, and the linker supporting chain are equal. The buffer solution is phosphate-buffered saline (PBS) buffer. The denaturation temperature is 95 °C, and the denaturation time is 10 min. The incubation temperature is 25 °C, and the incubation time is 30 min.

8. The ATP-responsive DNA nanomachine as described in claim 7, characterized in that, The molar ratio of Met-13Y or EGFR-13Y to IGFIIR-13-ATP is 1:1, and the buffer is phosphate-buffered saline (PBS) buffer; the denaturation temperature is 95°C, and the denaturation time is 10 min; the incubation temperature is 25°C, and the incubation time is 30 min.

9. The ATP-responsive DNA nanomachine as described in claim 8, characterized in that, The molar ratio of FNA to LYTAC-Met or LYTAC-EGFR is 1:

1. The buffer solution is a phosphate-buffered saline (PBS) buffer with a pH of 7.4, consisting of 2.0 mM KH2PO4, 137 mM NaCl, 10.0 mM Na2HPO4, and 2.7 mM KCl. The incubation temperature is 25 °C, and the incubation time is 30 min. The final concentration of the DNA nanomachines in the mixed solution is 1 μM.

10. The use of the ATP-responsive DNA nanomachines according to any one of claims 1 to 9 in the preparation of therapeutic drugs for non-small cell lung cancer, characterized in that, The ATP-responsive DNA nanomachine is any one or a combination of two of DNA Nanomachine Met or DNA Nanomachine EGFR.

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