Responsive DNA tetrahedral lysosome targeting chimera as well as construction method and application thereof

Abstract

The invention relates to a response type DNA tetrahedral lysosome targeting chimera as well as a construction method and application thereof, and belongs to the technical field of biology. The response type DNA tetrahedron lysosome targeting chimera is obtained by self-assembling a tetrahedron I and a tetrahedron II; a sticky tail end H1 is arranged on the tetrahedron I, and a sticky tail end H2 is arranged on the tetrahedron II; three tyrosine kinase receptor aptamers are anchored on the first tetrahedron, three lysosome shuttle receptor aptamers are anchored on the second tetrahedron, stimuli-responsive factor aptamers are hybridized on the cohesive end H2, and the binding capacity with cells is obviously enhanced based on a trivalent aptamer modification strategy of a tetrahedral framework. According to the present invention, the VEGF is adopted as the stimulation response factor to construct the responsive DNA tetrahedral lysosome targeting chimera, and the responsive DNA tetrahedral lysosome targeting chimera carries the HER2 protein to the lysosome under the mediation of the lysosome shuttle receptor IGF2R so as to degrade, such that the HER2 protein expression on the cell membrane is reduced, and the degradation specificity and the degradation effect are enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a responsive DNA tetrahedral lysosome-targeting chimera, its construction method, and its application. Background Technology

[0002] Cancer is a major public health problem worldwide, with lung cancer being the leading cause of cancer death globally. Lung cancer is mainly divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with NSCLC being the predominant type, accounting for approximately 80-85% of all lung cancer cases. Studies have shown that tyrosine kinases are extensively dysregulated in NSCLC. This dysregulation leads to abnormal phosphorylation in kinase-mediated cell signaling pathways, often initiating oncogenic transformation and resulting in uncontrolled cell proliferation. The main targeted therapy strategy against tyrosine kinases is the use of tyrosine kinase inhibitors (TKIs), but in some cases, its therapeutic effect is not ideal, often exhibiting drug resistance. Further research suggests that the reason for drug resistance may be that tyrosine kinase receptors form heterodimers with different kinase receptors, thereby inducing resistance to tyrosine kinase inhibitors (TKIs) by inhibiting apoptosis. Lysosomal targeted degradation technology (LYTAC) constructs conjugates between lysosomal shuttle receptors (LTRs) and target proteins (POIs) to achieve targeted degradation of extracellular proteins. In LYTAC technology, the selection of LTR ligands and POI ligands is crucial, 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 great flexibility, making them a focus of attention in areas such as lysosomal targeted degradation. In recent years, self-assembled DNA nanostructures have garnered significant attention due to their inherent biocompatibility, programmability, and multifunctionality. tFNAs possess efficient addressability and programmability; by modifying different functional groups or ligands (e.g., nucleic acid aptamers, fluorescent dyes) at the four vertices, side lengths, or internal gaps, specific functions can be assigned to them. Furthermore, the structural sequence of tFNAs can be autonomously designed according to experimental needs and respond to environmental changes. More notably, tFNAs, using bases as assembly raw materials, exhibit better biocompatibility than other nanomaterials, along with good cell membrane permeability, low immunogenicity, and low toxicity to non-target cells. Related studies have shown that overexpression of vascular endothelial growth factor (VEGF) is associated with the occurrence, development, and metastasis of NSCLC, and its expression level is relatively higher around cancer cells than in normal cells. Therefore, using VEGF as a stimulus-response factor and combining the unique advantages of DNA tetrahedrons and nucleic acid aptamers, constructing a novel DNA tetrahedral lysosomal targeted protein degradation strategy will be of great significance for the degradation of tumor cell surface proteins (such as HER2).Chinese patent application CN116904556A discloses a composition, a composite DNA nanostructure, a kit, and its application in the detection and targeted degradation of HER2 homodimers. The composition targeting HER2 protein comprises two DNA tetrahedra, HAT1 and HAT2, modified with HER2 aptamers. HAT1 is formed by the self-assembly of a first probe S1-T1, a second HER2 aptamer S2-Apt, a third nucleic acid chain S3, and a fourth nucleic acid chain S4. HAT2 is formed by the self-assembly of the first aptamer S1-Apt, the second probe S2-T2, the third nucleic acid chain S3, and the fourth nucleic acid chain S4. This composition targeting HER2 protein requires the formed composite DNA nanostructure to be recognized and encapsulated by endosomes for degradation by lysosomes, preventing the use of the cell's original endocytic pathway for lysosomal degradation. Summary of the Invention

[0003] The first objective of this invention is to construct a trivalent aptamer-modified tetrahedron that can further enhance its binding affinity to target proteins and lysosomal targeted receptors.

[0004] The second objective of this invention is to provide a method for constructing a responsive DNA tetrahedral lysosome-targeting chimera, which can only mediate the degradation of cell membrane HER2 through the lysosomal pathway in the presence of VEGF, thereby enhancing the specificity of membrane protein degradation.

[0005] The third objective of this invention is to provide a responsive DNA tetrahedral lysosome-targeting chimera that significantly reduces the expression of HER2 protein on the cell surface and enhances the degradation effect compared to a single tetrahedron.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A responsive DNA tetrahedral lysosome-targeting chimera is formed by the self-assembly of tetrahedron 1 and tetrahedron 2. Tetrahedron 1 has a sticky end H1, and tetrahedron 2 has a sticky end H2 for hybridization with the sticky end H1. A tyrosine kinase receptor aptamer is anchored on tetrahedron 1, and a lysosome shuttle receptor aptamer is anchored on tetrahedron 2. A stimulus-response factor aptamer is hybridized on the sticky end H2 so that when a stimulus-response factor is present, the aptamer on the sticky end H2 binds to the stimulus-response factor, exposing the sticky end H2. The sticky end H2 then hybridizes with the sticky end H1 on tetrahedron 1 and self-assembles to form the responsive DNA tetrahedral lysosome-targeting chimera.

[0008] Furthermore, the tyrosine kinase receptor is HER2; the lysosomal shuttle receptor is IGF2R; the stimulation response factor is VEGF; the number of tyrosine kinase receptors anchored on tetrahedron 1 is 3, and the number of lysosomal shuttle receptor aptamers anchored on tetrahedron 2 is 3.

[0009] Furthermore, tetrahedron one is formed by the self-assembly of four nucleic acid chains sS1, sS2, sS3 and H1-S4, and the three tyrosine kinase receptors are anchored to nucleic acid chains sS1, sS2 and sS3, respectively; tetrahedron two is formed by the self-assembly of five nucleic acid chains sS1, sS2, sS3, H2-S4 and a stimulus response factor aptamer, and the three lysosomal shuttle receptor aptamers are anchored to nucleic acid chains sS1, sS2 and sS3, respectively.

[0010] Furthermore, the nucleic acid sequences of sS1, sS2, and sS3 are shown in SEQ ID NO 1-SEQ ID NO 3, the nucleic acid sequences of H1-S4 are shown in SEQ ID NO 4, the nucleic acid sequences of H2-S4 are shown in SEQ ID NO 5, the nucleic acid sequence of the stimulus response factor aptamer is shown in SEQ ID NO 6, the nucleic acid sequence of the tyrosine kinase receptor aptamer is shown in SEQ ID NO 7, and the nucleic acid sequence of the lysosomal shuttle receptor aptamer is shown in SEQ ID NO 8.

[0011] A method for constructing a responsive DNA tetrahedral lysosome-targeting chimera includes the following steps:

[0012] 1) Preparation of tFNA1-Apt: The four nucleic acid chains sS1, sS2, sS3, and H1-S4 constituting tetrahedron I were mixed in buffer, denatured, and incubated to obtain tFNA1. tFNA1 was then mixed with a tyrosine kinase receptor aptamer in buffer to obtain a mixed solution. The mixed solution was incubated in a constant temperature shaker to obtain tFNA1-Apt.

[0013] 2) Preparation of tFNA2-Apt: The five nucleic acid chains constituting tetrahedron II, sS1, sS2, sS3, H2-S4, and the stimulus response factor aptamer are mixed in buffer, denatured, and incubated to obtain tFNA2. tFNA2 is then mixed with the lysosomal shuttle receptor aptamer in buffer to obtain a mixed solution. The mixed solution is incubated in a constant temperature shaker to obtain tFNA2-Apt.

[0014] Further, in step 1), the molar ratio of sS1, sS2, sS3, and H1-S4 is 1:1:1:1; the denaturation temperature is 95°C and the denaturation time is 10 min; the incubation temperature is 25°C and the incubation time is 1 h; the final concentration of tFNA1 in the mixed solution is 1 μmol / L; and the molar ratio of tFNA1 to the tyrosine kinase receptor aptamer is 1:3.

[0015] Furthermore, in step 2), the molar ratio of sS1, sS2, sS3, H2-S4, and the stimuli-response factor aptamer nucleic acid chains is 1:1:1:1:1; the denaturation temperature is 95°C, and the denaturation time is 10 min; the incubation temperature is 25°C, and the incubation time is 1 h; the final concentration of tFNA2 in the mixed solution is 1 μmol / L; and the molar ratio of tFNA2 to the lysosomal shuttle receptor aptamer is 1:3.

[0016] Furthermore, the buffer solution in steps 1) and 2) is a TM Buffer, which has the following composition: 10 mmol / L Tris and 50 mmol / L MgCl2.

[0017] Application of responsive DNA tetrahedral lysosome-targeting chimeras in reducing HER2 protein expression.

[0018] The beneficial effects of this invention are:

[0019] This invention utilizes VEGF as a stimulus response factor to construct a responsive DNA tetrahedral lysosome-targeting chimera. Under the mediation of the lysosomal shuttle receptor IGF2R, the responsive DNA tetrahedral lysosome-targeting chimera carries HER2 protein for endocytosis and subsequent lysosomal degradation, significantly reducing the expression of HER2 protein on the cell surface, enhancing degradation specificity and degradation efficiency, and effectively inhibiting the expression of key downstream signaling pathway proteins p-Akt and p-Erk1 / 2, inducing tumor cell apoptosis, and providing an important pathway for the degradation of tumor cell surface proteins. Attached Figure Description

[0020] Figure 1 A schematic diagram of the responsive DNA tetrahedral lysosome-targeting chimera for enhancing lysosome-targeted degradation of HER2 protein;

[0021] Figure 2The image shows the polyacrylamide gel electrophoresis characterization of tFNA1-Apt in Example 1, where lane 1: Marker; lane 2: sS1; lane 3: sS1+sS2; lane 4: sS1+sS2+sS3; lane 5: sS1+sS2+sS3+H1-S4; lane 6: sS1+sS2+sS3+H1-S4+HER2 apt1; lane 7: sS1+sS2+sS3+H1-S4+HER2 apt2; lane 8: sS1+sS2+sS3+H1-S4+HER2 apt3.

[0022] Figure 3 The stability characterization diagrams for HER2 apt, tFNA1-Apt1, and tFNA1-Apt are shown, where A is the polyacrylamide gel electrophoresis characterization diagram of HER2 apt, tFNA1-Apt1, and tFNA1-Apt, and B is the quantitative trend diagram of the gray value of the bands of HER2 apt, tFNA1-Apt1, and tFNA1-Apt.

[0023] Figure 4 The polyacrylamide gel electrophoresis characterization of tFNA2-Apt in Example 1 is shown in the following lanes: Lane 1: Marker; Lane 2: sS1; Lane 3: sS1+sS2; Lane 4: sS1+sS2+sS3; Lane 5: sS1+sS2+sS3+H2-S4; Lane 6: sS1+sS2+sS3+H2-S4+VEGF apt; Lane 7: sS1+sS2+sS3+H2-S4+IGF2R apt1; Lane 8: sS1+sS2+sS3+H2-S4+IGF2R apt2; Lane 9: sS1+sS2+sS3+H2-S4+IGF2R apt3; Lane 10: sS1+sS2+sS3+H2-S4+VEGFapt+IGF2R apt3.

[0024] Figure 5 The stability of IGF2R apt, tFNA2-Apt1 and tFNA2-Apt is characterized, where A is the polyacrylamide gel electrophoresis characterization of IGF2R apt, tFNA2-Apt1 and tFNA2-Apt, and B is the quantitative trend diagram of the gray value of IGF2R apt, tFNA2-Apt1 and tFNA2-Apt.

[0025] Figure 6 Characterization diagrams of the targeting performance of tFNA1-Apt1 and tFNA1-Apt;

[0026] Figure 7 Characterization diagrams of the targeting performance of tFNA2-Apt1 and tFNA2-Apt;

[0027] Figure 8 Flow cytometry plot of VEGF response;

[0028] Figure 9 Flow cytometry image of DNA tetrahedral lysosome-targeted chimera in response to VEGF;

[0029] Figure 10 Flow cytometry results for tFNA-LYTAC-mediated HER2 degradation;

[0030] Figure 11 The graphs show the effects of tFNA-LYTAC on the cell viability of A549 and BEAS-2B cells, where A represents the effect of tFNA-LYTAC on the cell viability of A549 cells and B represents the effect of tFNA-LYTAC on the cell viability of BEAS-2B cells.

[0031] Figure 12 The effect of tFNA-LYTAC on p-Akt and p-Erk1 / 2 levels in A549 cells under VEGF response. Detailed Implementation

[0032] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0033] Preparation of binding buffer 2: Accurately weigh 1.0000g BSA, 1.0165g MgCl2·6H2O and 5.5000g glucose using a balance, dissolve in PBS and bring the volume to 1000mL, mix well to obtain binding buffer 2 containing 1g / L BSA, 5mmol / L MgCl2 and 25mmol / L glucose.

[0034] The nucleic acids used in this invention were purchased from Shanghai Sangon Biotech Co., Ltd., as detailed in Table 1.

[0035] Table 1. Nucleic acid names and base sequences

[0036]

[0037]

[0038] Example 1

[0039] The method for constructing a responsive DNA tetrahedral lysosome-targeting chimera in Example 1 includes the following steps:

[0040] 1) Preparation of tFNA1-Apt: The nucleic acid strands sS1, sS2, sS3, and H1-S4 constituting the DNA tetrahedron were mixed in TM Buffer at a molar ratio of 1:1:1:1. The mixture was incubated in a 95°C water bath for 10 min, followed by incubation at 25°C with a metal shaker at 600 rpm for 1 h, to obtain a final concentration of 1 μmol / L tFNA1. tFNA1 and HER2 aptamer were then mixed in TM Buffer at a molar ratio of 1:3, with a final concentration of 1 μmol / L tFNA1. This mixture was then incubated at 37°C with a metal shaker at 500 rpm for 1 h to obtain tFNA1-Apt. The TM Buffer consisted of 10 mmol / L Tris and 50 mmol / L MgCl2.

[0041] 2) Preparation of tFNA2-Apt: The nucleic acid strands sS1, sS2, sS3, H2-S4, and VEGFapt constituting the DNA tetrahedron were mixed in a 1:1:1:1:1 molar ratio in TM Buffer. The mixture was then incubated in a 95°C water bath for 10 min, followed by incubation at 25°C for 1 h at 600 rpm in a metal shaker, to obtain a final concentration of 1 μmol / L tFNA2. tFNA2 was then mixed with IGF2R aptamer (IGF2R apt) in a 1:3 molar ratio in TM Buffer to obtain a mixed solution with a final tFNA2 concentration of 1 μmol / L. This mixed solution was then incubated at 37°C for 1 h at 500 rpm in a metal shaker to obtain tFNA2-Apt. The TM Buffer consisted of 10 mmol / L Tris, 50 mmol / L MgCl2, and pH 8.

[0042] like Figure 1 As shown, tFNA1-Apt and tFNA2-Apt bind to the target protein HER2 and the lysosomal shuttle receptor IGF2R on the cell membrane, respectively, through receptor-ligand interactions. In the presence of the stimulus response factor VEGF, the VEGF aptamer on tFNA2-Apt specifically recognizes VEGF, causing tFNA2-Apt to expose its sticky end H2. This H2 hybridizes with the sticky end H1 originally present in tFNA1-Apt, thus self-assembling to form a responsive DNA tetrahedral lysosomal targeting chimera (tFNA-LYTAC). Subsequently, tFNA-LYTAC, mediated by the lysosomal shuttle receptor IGF2R, carries HER2 protein endocytosis and undergoes lysosomal degradation, effectively reducing the HER2 protein content on the cell membrane surface, thereby inhibiting downstream HER2 signaling and cell proliferation.

[0043] Comparative Examples 1-4

[0044] The construction methods of the responsive DNA tetrahedral lysosome-targeting chimeras in Comparative Examples 1-4 are largely the same as those in Example 1. The difference between the construction method of the responsive DNA tetrahedral lysosome-targeting chimera in Comparative Example 1 and Example 1 is that in Comparative Example 1, the molar ratio of tFNA1 to HER2 aptamer is 1:1 to obtain tFNA1-Apt1; in Comparative Example 2, the molar ratio of tFNA1 to HER2 aptamer is 1:2 to obtain tFNA1-Apt2; in Comparative Example 3, the molar ratio of tFNA2 to IGF2R aptamer is 1:1 to obtain tFNA2-Apt1; and in Comparative Example 4, the molar ratio of tFNA2 to IGF2R aptamer is 1:2 to obtain tFNA2-Apt2.

[0045] I. Characterization of tFNA1-Apt and tFNA2-Apt

[0046] tFNA1-Apt characterization: from Figure 2 As can be seen, in lane 2, single-stranded DNA sS1 presents a clear band. With the sequential addition of single-stranded DNA sS2, sS3, and H1-S4, the molecular weight gradually increases, and the migration rate slows down. In lane 5, a band with a delayed position and significantly increased brightness appears, indicating that single-stranded DNA sS1, sS2, sS3, and H1-S4 successfully self-assembled to form tFNA1. Lanes 6 to 8 show the formation process of different numbers of HER2 apt anchored on tFNA1. A band with a more delayed position in lane 8 compared to lane 7 indicates the formation of tFNA1-Apt. These experimental results prove that tFNA1 and tFNA1-Apt have been successfully prepared. The performance of single HER2 apt, tFNA1-Apt1, and tFNA1-Apt in a medium containing 10% FBS was compared by polyacrylamide gel electrophoresis (37℃). Figure 3 As shown, with increasing incubation time, the nucleic acid bands of tFNA1-Apt showed no significant change, while the single HER2 apt and tFNA1-Apt1 nucleic acid bands gradually faded. Quantitative results indicate that tFNA1-Apt has better stability than the other two. Dynamic light scattering (DLS) technology determined the particle size of tFNA1 to be 11.23 nm and that of tFNA1-Apt to be 20.53 nm. The potentials of tFNA1 and tFNA1-Apt were -6.545 mV and -10.93 mV, respectively, indicating the successful preparation of tFNA1-Apt.

[0047] Characterization of tFNA2-Apt: From Figure 4As can be seen, lanes 2 to 5 show the formation process of tFNA2. In lane 2, DNA single-strand S1 shows a clear band. With the addition of single-stranded sS2, sS3, and H2-S4, the molecular weight gradually increases, and the migration rate slows down. In lane 5, a band is shown that is lagging behind the binding of the three strands and is significantly brighter, indicating that single-stranded sS1, sS2, sS3, and H2-S4 undergo self-assembly hybridization to form a DNA tetrahedral structure. Due to the small molecular weight of VEGF apt, its addition results in a slight lag in the band (lane 6), indicating the synthesis of tFNA2. With the addition of IGF2R apt, a band that is even more lagging than in lane 9 appears in the upper part of lane 10, proving the successful preparation of tFNA2 and tFNA2-Apt. The stability of single IGF2R apt, tFNA2-Apt1, and tFNA2-Apt in a medium mixed with 10% FBS at 37°C was compared by polypropylene gel electrophoresis. Figure 5 As can be seen, the nucleic acid bands of the three gradually faded with increasing incubation time. After 8 hours, the single IGF2R apt band was the faintest, and the tFNA2-Apt band was slightly brighter than that of tFNA2-Apt1. Quantitative results show that tFNA2-Apt has better stability than the other two, indicating superior stability. Dynamic light scattering technology determined the particle size of tFNA2 to be 6.139 nm and the particle size of tFNA2-Apt to be 13.06 nm. The potential of tFNA2 was -5.733 mV and the potential of tFNA2-Apt was -9.983 mV, indicating the successful preparation of tFNA2-Apt.

[0048] Targeting performance and binding ability of tFNA1-Apt and tFNA2-Apt

[0049] tFNA1-Apt1 and tFNA1-Apt were incubated with A549 cells at room temperature for 1 h. The final concentrations of tFNA1-Apt1 and tFNA1-Apt were adjusted to 200 nmol / L and the final volume to 300 μL. Real-time fluorescence imaging of the live cells was performed using a 40× objective lens of a laser confocal microscope. Figure 6 As can be seen, under 638nm excitation, collecting Cy5 channel fluorescence clearly reveals the cell outlines, but the fluorescence intensity of tFNA1-Apt is much higher than that of tFNA1-Apt1. Similarly, under 552nm excitation, collecting Cy3 channel fluorescence... Figure 7 It can be seen that the fluorescence intensity of tFNA2-Apt is much higher than that of tFNA2-Apt1. The above results indicate that the ability of DNA tetrahedrons to anchor three aptamers to target cells is better than that of anchoring a single aptamer.

[0050] The cell-binding affinity of HER2 apt, tFNA1-Apt1, and tFNA1-Apt was verified by flow cytometry: HER2 apt, tFNA1-Apt1, and tFNA1-Apt were incubated with A549 cells, and the final concentrations of HER2 apt, tFNA1-Apt1, and tFNA1-Apt were adjusted to 0 nmol / L, 10 nmol / L, 20 nmol / L, 50 nmol / L, 100 nmol / L, 150 nmol / L, 200 nmol / L, and 300 nmol / L, respectively. Cy5 channel fluorescence was collected under 638 nm excitation. Analysis of the fluorescence values ​​corresponding to different concentrations using Sigmaplot software showed that the Kd value of tFNA1-Apt was lower than that of HER2 apt and tFNA1-Apt1, indicating that the ability of DNA tetrahedral anchoring three aptamers to bind to cells is better than anchoring a single aptamer.

[0051] III. Construction and Characterization of VEGF-responsive DNA Tetrahedral Lysosome-Targeted Chimeras

[0052] 80 μL of A549 cell suspension was mixed thoroughly with Cy3-tFNA2-Apt, Cy3-tFNA2-Apt-BHQ2, and binding buffer 2, and incubated on ice with shaking for 20 min. Then, it was incubated with VEGF at 37°C for 500 rpm for 30 min. The final concentrations of the probe in the cell mixture were controlled at 250 nmol / L, the final concentration of VEGF protein was 10 μg / mL, and the final reaction volume was 200 μL. After incubation, the fluorescence intensity of each sample was measured by flow cytometry. Figure 8 As can be seen, under 552 nm excitation, fluorescence in the Cy3 channel was collected. In the presence of VEGF apt-BHQ2, the fluorescence of Cy3 in tFNA2-Apt was quenched. After the addition of VEGF, a significant fluorescence enhancement was observed in the Cy3 fluorescence collection channel, indicating that the added VEGF binds to VEGF apt-BHQ2 and restores the original fluorescence. These results show that tFNA2-Apt can specifically respond to the presence of VEGF, and tFNA2-Apt can be successfully anchored to the cell membrane. Further analysis by flow cytometry... Figure 9 It was confirmed that the responsive DNA tetrahedral lysosome-targeting chimera (tFNA-LYTAC) could be successfully constructed on the A549 cell membrane under VEGF stimulation.

[0053] IV. Specificity and efficacy of VEGF-responsive tFNA-LYTAC-mediated degradation of cell surface HER2

[0054] The prepared tFNA1-Apt and tFNA2-Apt were added to A549 cells at a 1:1 molar ratio and incubated in a 37°C cell culture incubator for 20 min. Then, they were incubated with VEGF under the same conditions for 45 min to obtain tFNA-LYTAC. At this point, the prepared IGF2R apt, HER2 apt, tFNA-Apt1, and tFNA-Apt were added, and each group was incubated in a 37°C cell culture incubator for 6 h. The final concentration of each group was adjusted to 250 nmol / L and the final volume to 1 mL using culture medium without fetal bovine serum. After A549 cells were digested, they were transferred to 5 mL centrifuge tubes, 1 mL of cell suspension was added and mixed by pipetting. Then, 80 μL of the suspension was transferred to a 1.5 mL centrifuge tube. 80 μL of cell suspension was mixed thoroughly with HER2-Cy5 and binding buffer 2, maintaining a final chain concentration of 500 nmol / L in the cell mixture. The final reaction volume was 200 μL, and the mixture was incubated on ice with shaking for 20 min. Fluorescence intensity of each group of cells was detected by flow cytometry. Figure 10 As can be seen, when collecting fluorescence of the APC-H channel under 638 nm excitation, the fluorescence intensity of the experimental groups IGF2R apt and HER2 apt did not change significantly compared with the control group, indicating that they had no effect on HER2 expression on the cell surface. However, the fluorescence of the tFNA1-Apt and tFNA1-Apt-tFNA2-Apt groups was weakened. The fluorescence intensity of the VEGF-stimulated tFNA-LYTAC group was significantly lower than that of tFNA1-Apt alone, indicating that VEGF-responsive tFNA-LYTAC significantly reduced the expression of HER2 on the cell surface. These results demonstrate that VEGF-responsive tFNA-LYTAC enhances the specificity and effectiveness of cell membrane protein degradation.

[0055] V. Effects of tFNA-LYTAC on downstream signaling pathways and cell viability in A549 cells

[0056] Cell growth status was assessed using a CCK-8 assay kit, with BEAS-2B cells serving as a negative control. Figure 11 It can be seen that the IGF2R apt, HER2 apt, tFNA1-Apt1, tFNA1-Apt and tFNA-LYTAC experimental groups all showed inhibition of A549 cell viability. Among them, tFNA-LYTAC had the most significant effect on reducing A549 cell viability, while having a smaller effect on the viability of negative BEAS-2B cells.

[0057] Using p-Akt and p-Erk1 / 2 as research subjects, Western blotting was used to verify the effects of tFNA-LYTAC on downstream signaling pathways in A549 cells. Figure 12As shown in the figure, the first lane, representing the untreated group, shows HER2 expression after 6 hours of incubation with the culture medium in A549 cells. Lanes two through six, representing the experimental groups, show HER2 expression after 6 hours of incubation with IGF2R apt, HER2apt, tFNA1-Apt1, tFNA1-Apt, and tFNA-LYTAC, respectively. The figure shows that the inhibitory effect on phosphorylated proteins gradually increases with the increase of aptamer quantity, with the tFNA-LYTAC group showing the strongest inhibitory effect, indicating that inducing HER2 degradation can inhibit downstream HER2 signaling.

Claims

1. A responsive DNA tetrahedral lysosome-targeting chimera, characterized in that, It is obtained by self-assembly of tetrahedron 1 and tetrahedron 2; a sticky end H1 is provided on tetrahedron 1, and a sticky end H2 for hybridization with the sticky end H1 is provided on tetrahedron 2; a tyrosine kinase receptor aptamer is anchored on tetrahedron 1, a lysosome shuttle receptor aptamer is anchored on tetrahedron 2, and a stimulus response factor aptamer is hybridized on the sticky end H2 so that when a stimulus response factor is present, the stimulus response factor aptamer on the sticky end H2 binds to the stimulus response factor to expose the sticky end H2, and the sticky end H2 hybridizes with the sticky end H1 on tetrahedron 1 to form a responsive DNA tetrahedral lysosome-targeting chimera.

2. The responsive DNA tetrahedral lysosome-targeting chimera according to claim 1, characterized in that, The tyrosine kinase receptor is HER2; the lysosomal shuttle receptor is IGF2R; the stimulation response factor is VEGF; the number of tyrosine kinase receptors anchored on tetrahedron 1 is 3, and the number of lysosomal shuttle receptor aptamers anchored on tetrahedron 2 is 3.

3. The responsive DNA tetrahedral lysosome-targeting chimera according to claim 2, characterized in that, The first tetrahedron is formed by the self-assembly of four nucleic acid chains sS1, sS2, sS3 and H1-S4, with the three tyrosine kinase receptors anchored to nucleic acid chains sS1, sS2 and sS3, respectively. The second tetrahedron is formed by the self-assembly of five nucleic acid chains sS1, sS2, sS3, H2-S4 and a stimulus-response aptamer, with the three lysosomal shuttle receptor aptamers anchored to nucleic acid chains sS1, sS2 and sS3, respectively.

4. The responsive DNA tetrahedral lysosome-targeting chimera according to claim 3, characterized in that, The nucleic acid sequences of sS1, sS2, and sS3 are shown in SEQ ID NO 1-SEQ ID NO 3, the nucleic acid sequences of H1-S4 are shown in SEQ ID NO 4, the nucleic acid sequences of H2-S4 are shown in SEQ ID NO 5, the nucleic acid sequence of the stimulus-response factor aptamer is shown in SEQ ID NO 6, the nucleic acid sequence of the tyrosine kinase receptor aptamer is shown in SEQ ID NO 7, and the nucleic acid sequence of the lysosomal shuttle receptor aptamer is shown in SEQ ID NO 8.

5. The method for constructing a responsive DNA tetrahedral lysosome-targeting chimera as described in claim 1, characterized in that, Includes the following steps: 1) Preparation of tFNA1-Apt: The four nucleic acid chains sS1, sS2, sS3, and H1-S4 constituting tetrahedron I were mixed in buffer, denatured, and incubated to obtain tFNA1. tFNA1 was then mixed with a tyrosine kinase receptor aptamer in buffer to obtain a mixed solution. The mixed solution was incubated in a constant temperature shaker to obtain tFNA1-Apt. 2) Preparation of tFNA2-Apt: The five nucleic acid chains constituting tetrahedron II, sS1, sS2, sS3, H2-S4, and the stimulus response factor aptamer are mixed in buffer, denatured, and incubated to obtain tFNA2. tFNA2 is then mixed with the lysosomal shuttle receptor aptamer in buffer to obtain a mixed solution. The mixed solution is incubated in a constant temperature shaker to obtain tFNA2-Apt.

6. The method for constructing a responsive DNA tetrahedral lysosome-targeting chimera according to claim 5, characterized in that, In step 1), the molar ratio of sS1, sS2, sS3, and H1-S4 is 1:1:1:1; the denaturation temperature is 95°C and the denaturation time is 10 min; the incubation temperature is 25°C and the incubation time is 1 h; the final concentration of tFNA1 in the mixed solution is 1 μmol / L; and the molar ratio of tFNA1 to the tyrosine kinase receptor aptamer is 1:

3.

7. The method for constructing a responsive DNA tetrahedral lysosome-targeting chimera according to claim 5, characterized in that, In step 2), the molar ratio of sS1, sS2, sS3, H2-S4, and the stimuli-response factor aptamer nucleic acid chains is 1:1:1:1:1; the denaturation temperature is 95℃ and the denaturation time is 10 min; the incubation temperature is 25℃ and the incubation time is 1 h; the final concentration of tFNA2 in the mixed solution is 1 μmol / L; and the molar ratio of tFNA2 to the lysosomal shuttle receptor aptamer is 1:

3.

8. The method for constructing a responsive DNA tetrahedral lysosome-targeting chimera according to claim 5, characterized in that, The buffer solution mentioned in steps 1) and 2) is a TM Buffer, which has the following composition: 10 mmol / L Tris and 50 mmol / L MgCl2.

9. The application of the responsive DNA tetrahedral lysosome-targeting chimera as described in claim 1 in reducing HER2 protein expression.

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