Lysosome targeted degradation system based on DNA phase separation aggregate as well as preparation method and application of lysosome targeted degradation system

Abstract

The invention discloses a lysosome targeted degradation system based on a DNA phase separation aggregate as well as a preparation method and application of the lysosome targeted degradation system, and belongs to the technical field of biological medicine and nanotechnology. The lysosome targeted degradation system based on the DNA phase separation aggregate comprises an RNA-DNA tetrahedral framework with a cohesive end, the RNA-DNA tetrahedral framework is formed by self-assembly of a core chain and an edge chain through complementary base pairing, the edge chain is a DNA-RNA chimeric oligonucleotide chain, and the DNA-RNA chimeric oligonucleotide chain is a DNA-RNA chimeric oligonucleotide chain. One or more sections of RNA ribonucleotide sequences which can be specifically recognized and cut by RNase H enzyme are embedded in the sequence, and the tail end of the RNA ribonucleotide sequence is modified with a cohesive tail end for driving phase separation and a cell membrane anchoring group; the RNA-DNA tetrahedral framework is modified with an aptamer of a targeted membrane protein. After entering cells, the system is subjected to liquid-liquid phase separation in a lysosome acid environment through interaction of cohesive ends, a micron-sized large-size aggregate is formed in situ, the residence time of a nano-drug in the lysosome is prolonged, and efficient degradation of target membrane protein is realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanotechnology, specifically relating to a lysosomal targeted degradation system based on DNA phase separation condensates, its preparation method, and its application. Background Technology

[0002] Membrane proteins, as key hubs for intracellular and extracellular signal transduction, play a central role in the regulation of the tumor immune microenvironment. Among them, programmed death-ligand 1 (PD-L1) is a representative molecule mediating tumor immune escape. By binding to the PD-1 receptor on the surface of immune cells, PD-L1 can directly inhibit T cell proliferation and cytotoxicity, constructing an "immune exemption barrier" for tumor cells and becoming an important target for tumor therapy.

[0003] Currently, mainstream clinical PD-L1 antibody blockade therapy, while able to disrupt the PD-1 / PD-L1 signaling pathway and restore immune surveillance, only achieves signal interference and cannot clear PD-L1 protein from the membrane surface. Furthermore, tumor cells can continuously synthesize new PD-L1 to replenish membrane surface molecules, leading to problems such as insufficient antibody occupancy and efficacy attenuation with treatment cycles. The emergence of targeted protein degradation technologies (such as PROTAC and LYTAC) has provided new approaches to protein clearance, but their application has significant limitations: PROTAC mainly relies on the ubiquitin-proteasome system to degrade intracellular proteins, with limited ability to degrade membrane proteins; while LYTAC can target membrane proteins, it relies on endogenous lysosomes to sort receptors, and its degradation efficiency is limited by receptor expression levels.

[0004] DNA nanostructures, with their precise base pairing programmability and excellent biocompatibility, have become a research hotspot for novel drug carriers. However, conventional DNA nanocarriers, once inside cells, are easily encapsulated by endosomes and transported to lysosomes. The acidic environment of lysosomes and nucleases rapidly destroy their structure, while exocytosis expels undegraded carriers, resulting in a short retention time as "degraders" that severely limits degradation efficiency. Therefore, developing a novel DNA nanosystem that can prolong lysosomal retention time and resist acidic and enzymatic environments is of significant scientific and clinical value for overcoming the bottleneck of membrane protein degradation and improving the efficacy of tumor immunotherapy. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a lysosomal targeted degradation system based on DNA phase separation condensates, its preparation method and application, so as to solve the technical problem of low degradation capacity of existing lysosomal targeted degradation systems.

[0006] To achieve the above objectives, the present invention employs the following technical solution: The first aspect of the present invention discloses a lysosomal targeted degradation system based on DNA phase separation condensates, comprising an RNA-DNA tetrahedral framework with sticky ends, wherein the RNA-DNA tetrahedral framework is self-assembled by a core strand and an edge strand through complementary base pairing, and the RNA-DNA tetrahedral framework is modified with aptamers targeting membrane proteins. The edge chain is a DNA-RNA chimeric oligonucleotide chain, which contains one or more RNA ribonucleotide sequences that can be specifically recognized and cleaved by RNase H enzyme, and is modified with sticky ends for driving phase separation and cell membrane anchoring groups for membrane anchoring.

[0007] Preferably, the core strand is a single-stranded DNA with structural support function, and its 5' or 3' end extends with a linker sequence for connecting aptamers that target membrane proteins.

[0008] More preferably, the linker is 7 nucleotides in length.

[0009] Preferably, the nucleotide sequence of the sticky terminus is ATCGAT.

[0010] Preferably, the edge length of the RNA-DNA tetrahedral framework is 17 nucleotides.

[0011] Preferably, the aptamer targeting the membrane protein is a nucleic acid aptamer that specifically recognizes the PD-L1 protein.

[0012] Preferably, there is at least one sticky end and one cell membrane anchoring group.

[0013] More preferably, there is one sticky end and one cell membrane anchoring group.

[0014] Preferably, there are two sticky ends and two cell membrane anchoring groups.

[0015] Preferably, the cell membrane anchoring group is cholesterol.

[0016] Preferably, the nucleotide sequence of the core chain is as shown in SEQ ID NO.1, the edge chains include TH17-2-2SE-AF647-10T, TH17-3-2SE-10T and TH17-4-2SE, and the nucleotide sequences are shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively. The nucleotide sequence of the aptamer targeting the membrane protein is shown in SEQ ID NO.5, and the nucleotide sequence of the cell membrane anchoring group is shown in SEQ ID NO.6.

[0017] In a second aspect, the present invention discloses a method for preparing a lysosomal targeted degradation system based on DNA phase separation condensates. A buffer solution containing a core chain, an edge chain, an aptamer targeting membrane proteins, and a cell membrane anchoring group is kept at 90-98°C for 2-10 minutes, and then slowly cooled to 4-25°C at a rate of 0.7-0.8°C / min to assemble the lysosomal targeted degradation system based on DNA phase separation condensates.

[0018] Preferably, the molar ratio of the core chain, the edge chain, the aptamer targeting the membrane protein, and the cell membrane anchoring group is 1:3:1:(1~2).

[0019] A third aspect of the present invention discloses the application of a lysosomal targeted degradation system based on DNA phase separation condensates in the preparation of antitumor drugs.

[0020] Preferably, the tumor is primary clear cell renal cell carcinoma, breast cancer, or cervical cancer.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a lysosomal targeted degradation system based on DNA phase-separation condensates. 1) An RNA nucleotide sequence specifically recognized and cleaved by RNase H enzyme is embedded in a single-stranded DNA cell, constructing an RNA-DNA tetrahedral framework. The RNA bases serve as enzyme response sites, providing the rigid DNA tetrahedron with the structural basis for specific enzyme recognition and cleavage. 2) Sticky ends enable the RNA-DNA tetrahedral framework to undergo phase separation or aggregation through intermolecular interactions after entering the cell, forming large-sized condensates and thus prolonging its residence time within the lysosome. 3) Aptamers targeting membrane proteins can specifically bind to target proteins on the cell surface. 4) Membrane anchoring groups promote cell membrane anchoring and endocytosis. Therefore, before entering the cell, the RNA-DNA tetrahedral framework maintains a rigid and intact structure. Although sticky ends are present, they cannot freely aggregate or undergo phase transitions due to the rigid topology of the tetrahedron. Once inside the cell, RNase H enzyme recognizes and cleaves the RNA sites in the framework, causing specific edges of the tetrahedron to break. Once the RNA site is cleaved, the DNA tetrahedron is "opened" or "activated," transforming from a rigid, three-dimensional tetrahedral structure into a flexible, planar triangular or other relaxed structure. This conformational change eliminates steric hindrance, fully exposing the sticky ends and greatly enhancing intermolecular interactions. It is this structural shift from rigid to flexible, triggered by enzymatic cleavage, that allows the originally dispersed nanostructures to recruit and aggregate, ultimately resulting in liquid-liquid phase separation and the in-situ formation of large, micron-sized DNA droplets. This phase transition significantly prolongs the residence time of nanomedicines within lysosomes, thus efficiently "locking" target membrane proteins within lysosomes for degradation. Gel electrophoresis confirmed the successful assembly of the system, while flow cytometry and confocal imaging demonstrated that the lysosome-targeted degradation system effectively reduced membrane protein levels on the surface of various tumor cells, including HeLa, 786-O, and MDA-MB-231. Furthermore, the degradation process relied on the lysosomal pathway rather than the proteasome pathway, indicating the excellent lysosomal accumulation capacity and protein degradation effect of this lysosome-targeted degradation system in various tumor cell lines.

[0022] Furthermore, the nucleotide sequence at the sticky end is ATCGAT, which enables the lysosomal targeted degradation system to form stable droplet-like aggregates at around 38°C.

[0023] Furthermore, the edge length is 17 nucleotides, which enables the framework to possess suitable liquid phase separation properties at physiological temperatures.

[0024] This invention provides the application of a lysosomal targeted degradation system based on DNA phase separation condensates in the preparation of antitumor drugs. By specifically binding to membrane proteins on the surface of tumor cells, it induces their endocytosis and transport to lysosomes, and uses phase separation condensates to block their escape, ultimately achieving the degradation of membrane proteins via the lysosomal pathway. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the working principle of the lysosomal targeted degradation system based on DNA phase separation condensates of the present invention. Figure 2 The diagram shows the structural parameter optimization characterization of the lysosomal targeted degradation system based on DNA phase separation condensates of the present invention; where A represents the effect of linkers of different lengths (3 nt, 5 nt and 7 nt, respectively) on the crosslinking behavior of the sticky ends of the RNA-DNA tetrahedral framework, and B represents the effect of different sticky end sequences (ATAT, CGCG, ATCGAT and CGATCG, respectively) on the morphology and distribution of the condensates. Figure 3 The images show the morphology and properties of the lysosomal targeted degradation system based on DNA phase separation condensates of the present invention; wherein: A is a non-denaturing polyacrylamide gel electrophoresis (Native PAGE) image, B is an atomic force microscopy (AFM) image and its corresponding schematic diagram, C is a confocal laser scanning microscopy image (a merged image of bright field and fluorescence field superposition), and D is a transmission electron microscopy (TEM) image. Figure 4 The figures represent the intracellular retention capacity and lysosomal accumulation characteristics of the DNA phase-separated condensate-based lysosomal targeted degradation system of the present invention. Among them, A is a confocal laser scanning microscope co-localization image and correlation scatter plot of DNA phase-separated condensates (orange) and lysosomes (green) at different time points (24 h and 48 h), B is a flow cytometry quantitative analysis image, and C is a high-resolution confocal image after transfection with LAMP-1-EGFP (lysosomal membrane-associated membrane protein, green). Figure 5This diagram illustrates the stepwise assembly of functional modules onto an RNA-DNA tetrahedral framework using non-denaturing polyacrylamide gel electrophoresis, as described in this invention. In the diagram, A represents the assembly process of a modified cholesterol degradation system (1Chol-6AT2se-PD-L1 aptamer): Lane S1: Contains only the core strand (TH17-1-linker); Lane S2: Contains the PD-L1 aptamer (clinker-PD-L1) added to the S1 layer. The first edge chain (TH17-2-2se-10T, containing sticky ends and cholesterol linkage sites) was added to S2 in lane S3, forming a core-aptamer complex. Lane S4 added a second edge chain (TH17-3-2se, containing sticky ends) to S3, forming a complete RNA-DNA tetrahedral framework without cholesterol linkage. Lane S5 added a cholesterol chain (10A-Chol) to S4, anchoring cholesterol to the framework through base pairing, forming the final single-cholesterol modified degradation system. Lane B represents the assembly process of the modified double-cholesterol degradation system (2Chol-6AT2se-PD-L1 aptamer): the assembly steps of lanes S1 to S3 are the same as in lane A. Figure 1 Lane S4: A modified second edge chain (TH17-3-2se-10T, with an additional cholesterol binding site) was added to S3 to form an RNA-DNA tetrahedral framework with two cholesterol binding sites; Lane S5: Sufficient cholesterol chain (10A-Chol) was added to S4 to form the final dual cholesterol-modified degradation system. Figure 6 This diagram illustrates the degradation mechanism and degradation effect verification of the lysosomal targeted degradation system based on DNA phase separation condensates of the present invention. A is a schematic diagram of the degradation mechanism, showing two different structures on the left: the phase separation droplet backbone (top) is a simple RNA-DNA tetrahedral framework without modified cholesterol or PD-L1 aptamers; the phase separation droplet protein degrader is a functionalized RNA-DNA tetrahedral framework (bottom) with cholesterol chains and PD-L1 aptamers connected to the backbone. The right side shows the workflow of this degradation system. B is a flow cytometry detection result. Figure 7 The diagram shows the spatiotemporal dynamic characterization of intracellular transport process and lysosome localization in the DNA phase separation condensate-based lysosome targeted degradation system of the present invention; wherein, A is a confocal laser scanning microscope image and corresponding intracellular transport schematic diagram at different incubation time points (4 h, 8 h and 16 h), and B is a quantitative statistical diagram of the corresponding co-localization coefficient. Figure 8The graph shows the degradation effect and sticky end valence dependence of the lysosomal targeted degradation system based on DNA phase separation condensates of the present invention in various tumor cell lines; where A is a confocal laser scanning microscope image in three different tumor cell lines (HeLa, 786-O and MDA-MB-231), and B is a quantitative statistical graph of the corresponding PD-L1 relative expression level (or average fluorescence intensity). Figure 9 This diagram serves as a verification of the protein degradation kinetics and degradation pathway mechanism of the lysosomal targeted degradation system based on DNA phase separation condensates of the present invention. A shows the changes in PD-L1 protein bands in cell lysates after co-incubation with the "phase separation droplet protein degrader" for different times (0 h, 4 h, 8 h, 16 h, and 24 h) in the presence of PD-L1 aptamers (GAPDH is used as an internal reference protein). B is a quantitative statistical line graph of the relative expression level of PD-L1 protein (normalized to GAPDH). C is a Western blotting experiment after treatment with different inhibitors. D is a bar chart of the quantitative statistical PD-L1 ratio in the corresponding inhibitor experiment. Figure 10 The diagram shows the effect of different functional modules of the DNA phase-separated condensate-based lysosomal targeted degradation system of the present invention on the degradation effect. Among them, A and C are flow cytometry results of the effect of different cholesterol modification numbers on PD-L1 degradation in 786-O cells (A) and MDA-MB-231 cells (C), respectively; B and D are flow cytometry results of the effect of different sticky ends on PD-L1 degradation in 786-O cells (B) and MDA-MB-231 cells (D), respectively. Detailed Implementation

[0026] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0027] This invention provides a lysosomal targeted degradation system based on DNA phase separation condensates (DFTCs), comprising an RNA-DNA chimeric tetrahedral framework with sticky ends (SE), wherein the RNA-DNA tetrahedral framework is self-assembled by a core strand and edge strands modified with specific functions through complementary base pairing; the RNA-DNA tetrahedral framework is modified with aptamers targeting membrane proteins and cell membrane anchoring groups. The core strand is single-stranded DNA (ssDNA) with structural support, and its 5' or 3' end extends with a spacer sequence for connecting aptamers targeting membrane proteins. The edge strand is a DNA / RNA chimeric oligonucleotide chain, which contains one or more RNA ribonucleotide sequences that can be specifically recognized and cleaved by RNase H enzyme, serving as RNase H cleavage response sites. Its ends are modified with sticky ends for driving phase separation and cell membrane anchoring groups for membrane anchoring. The RNA ribonucleotide sequences can induce a conformational change in the RNA-DNA tetrahedral framework and expose or activate the sticky ends to initiate phase separation and form condensates. The nucleotide sequence of the sticky ends is preferably ATCGAT, which allows the system to form stable droplet-like condensates at around 38°C. Preferably, the edge length of the RNA-DNA tetrahedral framework is 17 nucleotides. The aptamer targeting the membrane protein is a nucleic acid aptamer that specifically recognizes the PD-L1 protein (PD-L1Aptamer); the membrane anchoring group is preferably cholesterol, which promotes cell membrane anchoring and endocytosis, and the amount of cholesterol can regulate the degradation efficiency.

[0028] like Figure 1 As shown, in the lysosomal targeted degradation system based on DNA phase-separated condensates (DFTCs) described above, each single strand self-assembles into an RNA-DNA chimeric tetrahedral framework through complementary base pairing. At this point, due to the constraint of the rigid tetrahedral structure, the sticky ends are in a bound "locked" state, and the system exists as monodisperse nanoparticles in solution, without phase separation.

[0029] Subsequently, the lysosomal targeted degradation system based on DNA phase-separated condensates undergoes enzyme response activation and droplet formation in the following manner: 1. Initial Locked-in State: Before entering the cell or in an enzyme-free environment, the RNA-DNA tetrahedral framework maintains a complete rigid three-dimensional structure. Under this rigid topology, although the connectors extending from the framework have sticky ends, the interaction between the sticky ends is suppressed by the spatial constraint and topological limitation of the tetrahedral rigid framework. The system is monodisperse in solution and cannot undergo phase separation.

[0030] 2. Enzymatic cleavage response and structural opening: After the system enters the cell via membrane anchoring groups, the widely distributed intracellular RNase H enzyme specifically recognizes and cleaves the RNA-DNA hybridization region in the framework edge strand. The breakage of the RNA site causes the specific edges of the tetrahedral framework to break, inducing conformational collapse and rearrangement of the rigid three-dimensional tetrahedral structure, transforming it into a flexible, planar, or loosely open structure.

[0031] 3. Activation of sticky ends and aggregation formation: The "opening" of the structure eliminates the steric hindrance to the sticky ends, allowing them to swing freely and be fully exposed. The activated sticky ends then drive the loose DNA structural units to recruit and cross-link with each other through intermolecular multivalent complementary pairing, resulting in liquid-liquid phase separation (LLPS). This process occurs in situ in the acidic environment of the lysosome, rapidly transforming the dispersed nanomedicine into micron-sized large-scale DNA phase-separated aggregates (droplets), thereby achieving long-term drug retention and efficient degradation of target proteins within the lysosome.

[0032] This invention also provides a method for preparing the above-mentioned lysosomal targeted degradation system based on DNA phase separation condensates, the steps of which are as follows: 1. Preparation of the reaction system One core chain, three modified edge chains, a nucleic acid aptamer, and a cholesterol chain were mixed in equimolar ratio in a solution containing Mg. 2+ Prepare a homogeneous reaction mixture in the buffer solution.

[0033] 2. Thermal Annealing Self-Assembly The reaction mixture is heated to 90-98°C and held for 2-10 minutes to eliminate the secondary structure inside the single strand; then it is slowly cooled to 4-25°C at a rate of 0.7-0.8°C / min to assemble a lysosomal targeted degradation system based on DNA phase separation condensates. The preferred heating temperature is 95°C, and the preferred holding time is 5 minutes.

[0034] To construct a DNA nanosystem capable of efficient liquid-liquid phase separation and the formation of stable aggregates under cellular physiological conditions (approximately 37°C), this invention also systematically screened and optimized the key structures of the RNA-DNA tetrahedral framework of the aforementioned lysosomal targeted degradation system based on DNA phase separation aggregates. This mainly included optimizing the framework edge length, linker length, and sticky end sequences, as follows: 1. Selection of edge length The edge length of the RNA-DNA tetrahedral framework determines its structural rigidity and phase transition temperature. Therefore, experiments were conducted using lysosomal targeted degradation systems with different edge lengths. The results showed that when the edge length was 17 nucleotides (17-nt), the RNA-DNA tetrahedral framework exhibited suitable liquid phase separation properties within the temperature range of 40°C to 50°C, and possessed optimal assembly stability and dynamics at physiological temperatures. In contrast, shorter or longer edge lengths tended to result in phase transition temperatures that were either too low (failing to form droplets) or too high (forming solid gels). Therefore, this invention selected 17-nt as the edge length for the DNA tetrahedral framework.

[0035] 2. Optimization of connector length Linkers are double-stranded DNA regions that connect sticky ends to the vertices of the RNA-DNA tetrahedral framework. Their length directly affects the spatial degree of freedom and cross-linking efficiency of the sticky ends. Therefore, we constructed lysosomal targeted degradation systems with different linker lengths for experiments.

[0036] The experimental procedure was as follows: The core strand, edge strand 1, edge strand 2, edge strand 3, nucleic acid aptamers, and cholesterol were mixed in a molar ratio of 1:1:1:1:1:1 in a buffer solution (10 mM Tris-HCl, 10-20 mM MgCl2, pH 7.4) to prepare a homogeneous reaction mixture. The reaction mixture was heated to 95°C and held for 5 minutes; then slowly cooled to 4°C at a rate of 0.7°C / min to assemble a lysosomal targeted degradation system based on DNA phase separation condensates with different edge lengths. The core chain is TH17-1, with the nucleotide sequence shown in SEQ ID NO.1 of Table 1; the nucleic acid aptamer is clinker-PDL1aptamer, with the nucleotide sequence shown in SEQ ID NO.5 of Table 1; the cholesterol is 10A-cho, with the nucleotide sequence shown in SEQ ID NO.6 of Table 1; the edge chains 1, 2, and 3 corresponding to the 3-nt linker length are 3-nt-1, 3-nt-2, and 3-nt-3, respectively, with nucleotide sequences shown in SEQ ID NO.13~SEQ ID NO.15 of Table 1; the edge chains 1, 2, and 3 corresponding to the 5-nt linker length are 5-nt-1, 5-nt-2, and 5-nt-3, respectively, with nucleotide sequences shown in SEQ ID NO.13~SEQ ID NO.15 of Table 1; the edge chains 1, 2, and 3 corresponding to the 7-nt linker length are 7-nt-1, 7-nt-2, and 7-nt-3, respectively, with nucleotide sequences shown in SEQ ID NO.7~SEQ ID NO. Shown in NO.9.

[0037] The effects of different linker lengths (3-nt, 5-nt, and 7-nt, respectively) on the assembly morphology were compared using atomic force microscopy (AFM). 3-nt connector (17ntTD-3): Due to its short length and large steric hindrance, the sticky ends are difficult to contact effectively, and it mainly appears as a dispersed background in the field of view, failing to form an obvious network cross-linking structure; 5-nt linker (17ntTD-5): A small number of aggregates were observed, but they were unevenly distributed; 7-nt linker (17ntTD-7): exhibited the best crosslinking effect, and AFM images showed the formation of a dense and uniformly distributed network / granular structure.

[0038] The results are as follows Figure 2 As shown in Figure A, the 7-nt double-stranded connectors provide optimal conformational tension and flexibility, which is most conducive to inducing effective complementary pairing and aggregate assembly between viscous ends, forming a dense network structure.

[0039] 3. Screening of sticky terminal sequences The sequence of the sticky ends and their binding energy (enthalpy, ΔH) are the core factors determining phase separation behavior (droplet or gel formation). Therefore, lysosomal targeted degradation systems containing different sticky end sequences were constructed for experiments. The experimental procedures were the same as those for optimizing the linker length, except that the edge chains were different. For the sticky end sequence ATAT, edge chains 1, 2, and 3 are ATAT-1, ATAT-2, and ATAT-3, respectively, and their nucleotide sequences are shown in Table 1 (SEQ ID NO. 16~SEQ ID NO. 18). For the sticky end sequence CGCG, edge chains 1, 2, and 3 are CGCG-1, CGCG-2, and CGCG-3, respectively, and their nucleotide sequences are shown in Table 1 (SEQ ID NO. 19~SEQ ID NO. 21). For the sticky end sequence CGATCG, edge chains 1, 2, and 3 are CGATCG-1, CGATCG-2, and CGATCG-3, respectively, and their nucleotide sequences are shown in Table 1 (SEQ ID NO. 22~SEQ ID NO. 24). The edge chains 1, 2, and 3 corresponding to ATCGAT are TH17-2-2SE-AF647-10T, TH17-3-2SE-10T, and TH17-4-2SE, respectively, and their nucleotide sequences are shown in Table 1 as SEQ ID NO. 2~SEQ ID NO. 4. Then, the assembly behavior of four different sequences (ATAT, CGCG, and CGATCG) under physiological buffer conditions was compared using AFM images, and the results are as follows: Figure 2 As shown in B.

[0040] 4-nt sequences (ATAT, CGCG): Due to their weak binding force (small absolute value of ΔH), they are mainly dispersed or only form tiny aggregates under AFM, making it difficult to maintain stable phase-separated droplets at physiological temperatures; CGATCG (6-nt): Due to its high GC content and strong binding force, AFM images show that it induces the formation of large, irregular, over-crosslinked aggregates (Aggregates / Clumps). This state is close to a solid gel, which is not conducive to the dynamic exchange of molecules and biological applications, and does not meet the design requirements. Therefore, it was not selected in this invention. ATCGAT (6-nt): exhibits the most ideal assembly morphology. AFM images show that it forms a uniformly distributed, well-defined spherical aggregate structure. This sequence provides a moderate binding energy, enabling the system to form stable liquid-like droplets at around 38°C (close to human body temperature).

[0041] In summary, the optimal structural parameters finally determined by this invention are: DNA tetrahedral edge length of 17-nt, linker length of 7-nt, and sticky end sequence of ATCGAT.

[0042] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Conventional instruments and equipment in the art are used in the following embodiments. Experimental methods without specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. The nucleic acid chains used in the following embodiments were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and all other raw materials used, unless otherwise stated, are conventional commercially available products with specifications in the art.

[0043] I. Synthesis and Characterization of Lysosomal Targeted Degradation System Based on DNA Phase Separation Conglomerates 1. Synthesis of a lysosomal targeted degradation system based on DNA phase-separated condensates Example 1 A lysosomal targeted degradation system based on DNA phase-separated condensates modified with single cholesterol was synthesized using the following steps: The core chain (TH17-1, nucleotide sequence as shown in SEQ ID NO.1 in Table 1), edge chain 1 (TH17-2-2SE-AF647-10T, nucleotide sequence as shown in SEQ ID NO.2 in Table 1), edge chain 2 (TH17-3-2SE-10T, nucleotide sequence as shown in SEQ ID NO.3 in Table 1), edge chain 3 (TH17-4-2SE, nucleotide sequence as shown in SEQ ID NO.4 in Table 1), a clinker-PDL1 aptamer specifically recognizing PD-L1 protein, and cholesterol (10A-cho) were mixed in a molar ratio of 1:1:1:1:1:1 in a buffer solution containing 10 mM Tris-HCl and 5 mM... The mixture was heated at 95°C for 5 minutes in MgCl2 (pH 7.4), and then slowly cooled to 4°C at a rate of 0.7°C / min using a PCR instrument to obtain a single cholesterol-modified lysosomal targeted degradation system based on DNA phase separation condensates.

[0044] Table 1 Nucleotide sequence list

[0045] Note: The fluorescent insertion site is AF647 / iAF647dT / modified on the T base. The black hole quenching group BHQ3 / iBHQ3dT / modified on the T base. / rA / / rU / / rC / / rG / are RNA bases. Cholesteryl is the cholesterol binding site.

[0046] Example 2 The synthesis of a lysosomal targeted degradation system based on DNA phase-separated condensates modified with dual cholesterol was carried out as follows: The core chain (TH17-1), edge chain 1 (TH17-2-2SE-AF647-10T), edge chain 2 (TH17-3-2SE-10T), edge chain 3 (TH17-4-2SE), a clinker-PDL1 aptamer specifically recognizing PD-L1 protein, and cholesterol (10A-cho) were mixed in a molar ratio of 1:1:1:1:1:2 in a buffer solution (containing 10 mM Tris-HCl, 5 mM MgCl2, pH 7.4). The mixture was heated at 95°C for 5 minutes, and then slowly cooled to 4°C at a rate of 0.7°C / min using a PCR instrument to obtain the lysosomal targeted degradation system based on DNA phase-separated condensates modified with dual cholesterol.

[0047] The assembly process was monitored using native page adhesive, and the results were as follows: Figure 5 As shown, lanes S1 to S4 show the stepwise hysteresis migration of the core strand with the addition of the aptamer and edge strand; lane S5 shows the formation of the final product after the addition of the cholesterol chain; a second edge strand with a cholesterol linker was introduced in lane S4, and lane S5 shows the successful mounting of two cholesterol molecules, proving that all functional modules have been successfully assembled onto the RNA-DNA tetrahedral framework.

[0048] II. Structural Characterization of Lysosomal Targeted Degradation Systems Based on DNA Phase Separation Conglomerates The lysosome-targeted degradation system obtained in Example 1 was characterized by non-denaturing polyacrylamide gel electrophoresis (Native PAGE), atomic force microscopy (AFM), confocal laser scanning microscopy, and transmission electron microscopy (TEM).

[0049] The results are as follows Figure 3 As shown, this degradation system exhibits responsive characteristics under the action of RNaseH enzyme. Figure 3 (A); Atomic force microscopy results showed that the RNA-DNA tetrahedral framework transformed from a monodisperse tetrahedral state to a phase-separated aggregated state before and after enzyme cleavage activation. Figure 3 (B); Confocal laser scanning microscopy images show that the system can self-assemble in solution to form well-defined micron-sized spherical droplets (aggregates). Figure 3 The transmission electron microscope (TEM) images at higher resolution further reveal the tightly cross-linked micronetwork structure within the DNA phase-separated condensate. Figure 3 (D).

[0050] III. Performance Evaluation of Lysosomal Targeted Degradation System Based on DNA Phase Separation Conglomerates Using flow cytometry, Western blotting, and confocal microscopy, we systematically evaluated the degradation performance and mechanism of action of a lysosomal targeted degradation system based on DNA phase-separated condensates on the target membrane protein PD-L1 in various tumor cell lines.

[0051] 1. Evaluation of degradation effect 786-O (human clear cell renal cell carcinoma) cells were seeded in culture dishes and cultured in RPMI-1640 complete medium containing 10% fetal bovine serum. After cell attachment, the cells were co-incubated with the lysosome-targeted degradation system obtained in Example 1 at a concentration of 100 nM at different time points (3 h, 6 h, 12 h, 24 h, 48 h, and 72 h). Then, confocal laser scanning, flow cytometry quantitative analysis, and high-resolution confocal imaging were performed.

[0052] Confocal laser scanning results are as follows Figure 4 As shown in Figure A, at 24 h and 48 h, the DNA condensate signal and lysosomal signal still maintained a high degree of overlap (co-localization), indicating that the system can resist the lysosomal environment and exist stably within it. Flow cytometry quantitative analysis results are shown below. Figure 4 As shown in Figure B, a significant fluorescence signal was still detected even after 72 hours, demonstrating that the system can achieve long-term retention within cells. High-resolution confocal imaging results are shown below. Figure 4 As shown in Figure C, the red DNA phase-separated droplets are confined within the green lysosomal membrane structure, visually demonstrating the specific targeted accumulation capability of this degradation system within lysosomes.

[0053] 2. Investigation into the degradation mechanism 1) To elucidate the biological degradation pathway of PD-L1, 786-O cells were pretreated with different protein degradation pathway inhibitors, and four experimental groups were set up as follows: Control group: The old culture medium was removed, and an equal volume of fresh RPMI-1640 complete culture medium containing 10% fetal bovine serum was added for culture as a blank control.

[0054] Inhibitor-free group (normal degradation group): The lysosome-targeted degradation system obtained in Example 1 was added to RPMI-1640 complete medium containing 10% fetal bovine serum and co-incubated with 786-O cells at 37°C for 24 hours.

[0055] Chloroquine group (lysosomal function inhibitor): First, 50 μM chloroquine was added to RPMI-1640 complete medium containing 10% fetal bovine serum and pre-incubated for 2 hours to block lysosomal acidification; then the medium was removed and fresh medium containing 50 μM chloroquine and 100 nM of the lysosomal targeted degradation system obtained in Example 1 was added, and the cells were co-incubated with 786-O cells for 24 hours.

[0056] MG132 group (proteasome inhibitor): First, MG132 with a final concentration of 10 μM was added to RPMI-1640 complete medium containing 10% fetal bovine serum and pre-incubated for 2 hours to inhibit proteasome activity; then the medium was removed and fresh medium containing 10 μM MG132 and 100 nM of the lysosome-targeted degradation system obtained in Example 1 was added, and the cells were co-incubated with 786-O cells for 24 hours.

[0057] After incubation, immunoblotting and quantitative statistics of PD-L1 ratio were performed.

[0058] Immunoblotting results as follows Figure 9 As shown in Figure C, even after adding MG132 to inhibit the proteasome, PD-L1 was still effectively degraded (the protein band was relatively light, similar to the group without inhibitor), indicating that this degradation process does not depend on the ubiquitin-proteasome pathway. Conversely, after adding chloroquine to inhibit lysosomal function, the degradation process of PD-L1 was significantly blocked, and the protein level recovered to near the control group level. This result conclusively proves that the system of the present invention achieves targeted degradation of the membrane protein PD-L1 through the lysosomal pathway. The quantitative statistical results of PD-L1 ratio are shown below. Figure 9 As shown in Figure D, the PD-L1 degradation process mediated by this system mainly relies on the lysosomal pathway rather than the ubiquitin-proteasome pathway, further supporting the explanation of the mechanism of "lysosomal targeted degradation".

[0059] 2) Degradation pathway verification like Figure 6 As shown in Figure A, the workflow of the lysosomal targeted degradation system based on DNA phase-separation condensates is as follows: Aptamers specifically recognize and bind to the PD-L1 protein on the surface of tumor cells, using cholesterol-assisted anchoring to mediate endocytosis into the cell. Subsequently, phase separation occurs in the acidic environment of the lysosome, forming large-sized condensates (lysosomal accumulation), thereby blocking the recycling of PD-L1 and inducing its efficient degradation within the lysosome. The following experiments further validate this degradation pathway. Five experimental groups were set up as follows: Control group: without degradation system; Phase-separated droplet backbone assembly: It does not contain nucleic acid aptamers that specifically recognize PD-L1 protein and cholesterol, and is synthesized only from the core chain (TH17-1), edge chain 1 (TH17-2-2SE-AF647-10T), edge chain 2 (TH17-3-2SE-10T) and edge chain 3 (TH17-4-2SE) according to the method of Example 1; Phase-separated droplet backbone aptamer set: cholesterol-free, synthesized only from the core chain (TH17-1), edge chain 1 (TH17-2-2SE-AF647-10T), edge chain 2 (TH17-3-2SE-10T), edge chain 3 (TH17-4-2SE), and a nucleic acid aptamer that specifically recognizes PD-L1 protein according to the method of Example 1; A cholesterol degrader assembly containing one cholesterol unit: the lysosomal targeted degradation system obtained in Example 1; A cholesterol degrader assembly containing two cholesterol cells: the lysosomal targeted degradation system obtained in Example 2; After incubating each of the above systems at a concentration of 100 nM with adherent 786-O cells for 24 hours, the changes in the expression level of PD-L1 protein on the cell surface (PE fluorescence intensity) were detected.

[0060] The results are as follows Figure 6 As shown in Figure B, groups with only a backbone or only aptamers could not effectively reduce PD-L1 levels (MFI values ​​were between 95 and 98); while the cholesterol-modified degrader group could significantly reduce PD-L1 levels, and the dual cholesterol-modified group (Example 2) showed the best effect (MFI value dropped to 18), demonstrating the key role of multivalent cholesterol modification in enhancing endocytosis efficiency and final degradation effect.

[0061] 3. Degradation kinetics assessment To investigate the dynamic process of protein degradation, a time-dependent degradation experiment was conducted. The steps were as follows: The lysosome-targeted degradation system obtained in Example 1, with a concentration of 100 nM, was co-incubated with adherent 786-O cells. Cell samples were collected at 0, 4, 8, 16, and 24 hours for immunoblotting and flow cytometry analysis.

[0062] Immunoblotting results as follows Figure 9 As shown in Figure A, the color of the PD-L1 protein band gradually lightens with increasing incubation time, indicating a continuous decrease in protein abundance; the semi-quantitative analysis results are as follows. Figure 9 As shown in Figure B, the PD-L1 protein level showed a significant decreasing trend over time, especially at 16 h and 24 h, where the degradation effect was most obvious. After 16 hours of incubation, the PD-L1 protein level decreased by more than 60%, and the maximum degradation was reached at 24 hours, verifying that the system has a highly efficient dynamic degradation capability.

[0063] 4. Investigation into the impact of different functional modules on degradation efficiency 1) The impact of the presence or absence of functional modules and the amount of cholesterol on the degradation effect. To verify the contribution of each functional module (cholesterol, aptamer, and sticky ends) in the system to the degradation effect, the experimental groups were set up as in the "Degradation Pathway Verification" section: control group, phase-separated droplet backbone group, phase-separated droplet backbone aptamer group, degradation device group containing 1 cholesterol, and degradation device group containing 2 cholesterol. Each of the above groups, with a concentration of 100 nM, was co-incubated with adhered 786-O cells / MDA-MB-231 (human breast cancer cells) for 24 hours. Then, the cell culture supernatant was aspirated, and the cells were washed three times with sterile PBS buffer to thoroughly remove untaken DNA nanomaterials and residual free drugs. An appropriate amount of trypsin (preferably without EDTA to protect membrane proteins) was added to digest the cells. After the cells became rounded and detached, serum-containing medium was added to stop digestion. The cell suspension was collected into centrifuge tubes, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Cells from each group were incubated at 4°C in the dark for 30 minutes to allow the antibody to bind specifically and fully to the PD-L1 protein on the cell surface. After incubation, the cells were washed with 1 mL of pre-chilled PBS buffer, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The washing was repeated twice to remove unbound free antibody. Finally, the cells were resuspended in 300–500 µL of PBS to prepare a single-cell suspension. Flow cytometry was then performed immediately. Excitation and emission parameters were set (PE channel, Ex / Em≈488 / 575 nm), and at least 10,000 cellular events were collected per tube. The mean fluorescence intensity (MFI) of the cells was recorded to characterize the expression level of PD-L1 on the cell surface.

[0064] Flow cytometry results as follows Figure 10 As shown in Figures A and C, in both cell lines, groups containing only the cytoskeleton or only the aptamers could not effectively reduce PD-L1 levels on the cell surface (fluorescence intensity showed no significant difference from the control group). However, after introducing cholesterol modification, PD-L1 levels decreased, and the degradation effect of the dual-cholesterol modified group (2Chol) was significantly better than that of the single-cholesterol group. This indicates that multivalent cholesterol modification can significantly enhance the anchoring and endocytosis efficiency of nanosystems on the cell membrane surface, which is a prerequisite for achieving efficient degradation.

[0065] 2) The effect of the number of sticky ends on the degradation effect Construction of the reaction system (preparation of DNA frameworks with different valence states): To investigate the decisive influence of phase-separated condensate state on lysosomal retention time and protein degradation efficiency, three DNA nanoassemblies with different numbers of sticky ends but identical other functional modules (cholesterol, aptamers) were designed and prepared. 786-O cells and MDA-MB-231 cells were seeded in culture plates, and after the cells adhered and grew to a suitable density, the following four experimental groups were set up for treatment: Control group: Add an equal volume of fresh complete culture medium (containing no DNA material).

[0066] Non-sticky end group (0 SE): Lysosome-targeted degradation system with non-sticky ends added to a final concentration of 100 nM; the difference between this system and the lysosome-targeted degradation system of Example 1 is that edge chain 1, edge chain 2 and edge chain 3 are TH17-2, TH17-3 and TH17-4 respectively, and the nucleotide sequences are shown in Table 1 as SEQ ID NO.25~SEQ ID NO.27; Containing one set of sticky ends (1 SE): A lysosome-targeted degradation system containing one set of sticky ends was added to a final concentration of 100 nM; the difference between this system and the lysosome-targeted degradation system of Example 1 is that edge chain 1, edge chain 2 and edge chain 3 are TH17-2-1SE-AF647, TH17-3-1SE and TH17-4-1SE respectively, and the nucleotide sequences are shown in Table 1 as SEQ ID NO.28~SEQ ID NO.30; Contains two groups of sticky end groups (2 SE): the lysosomal targeted degradation system prepared in Example 1 with a final concentration of 100 nM.

[0067] HeLa cells, 786-O cells, and MDA-MB-231 cells were seeded into culture dishes. After the cells adhered, the above system at a concentration of 100 nM was added to each cell and incubated for 4, 8, 16, and 24 hours, respectively. Then, flow cytometry, confocal laser scanning microscopy, and quantitative analysis of the relative expression level of PD-L1 were performed.

[0068] Flow cytometry tracking results as follows Figure 10As shown in Figures B and D, the intracellular fluorescence signal of RNA-DNA tetrahedral frameworks without sticky ends or containing only one sticky end (1SE) decays rapidly after 24 hours, indicating that they are easily exocytotic or rapidly metabolized, and have limited ability to clear PD-L1. In contrast, the system of this invention containing two sticky ends (2SE), due to the formation of large-sized phase-separated condensates, still exhibits significant intracellular fluorescence signal after 72 hours, demonstrating excellent long-term retention capacity. This confirms that liquid-liquid phase separation behavior driven by multivalent sticky ends is crucial for prolonging lysosomal retention time and achieving efficient protein degradation.

[0069] Confocal laser scanning microscopy imaging results are as follows Figure 7 and Figure 8 As shown, the control group showed strong PD-L1 signal on the cell surface; the group with one sticky end had limited degradation effect due to its inability to form stable phase-separated aggregates; while the PD-L1 red fluorescence signal on the cell surface of the two sticky end groups was significantly weakened or even almost disappeared, indicating that efficient protein degradation is highly dependent on phase separation behavior driven by multivalent sticky ends. Figure 8 (A) At 4 h, the targeted degradation system containing two groups of sticky ends mainly bound to and distributed on the cell membrane surface (as shown by the white dashed outline); as time progressed to 8 h and 16 h, the degraders were largely endocytosed into the cell and concentrated in the perinuclear region, showing a high degree of overlap with lysosomal signals (yellow spots in the Merge diagram, as shown by the white arrows), intuitively demonstrating that the system can be effectively transported and enriched in lysosomes via the endocytic pathway. Figure 7 (A)

[0070] Quantitative statistical results of PD-L1 relative expression level (or mean fluorescence intensity) are as follows: Figure 7 and Figure 8 As shown, in all three cell lines tested, the PD-L1 expression levels (column height) in the two sticky-end groups were significantly lower than those in the control group and one sticky-end group (P<0.001), demonstrating that this system has excellent potential for broad-spectrum anti-tumor applications, and only the 2SE structure with phase separation capability can achieve efficient lysosomal targeted degradation. Figure 8 (B) The colocalization coefficient between the degrader and lysosomes showed a significant increasing trend from 4 h to 8 h (P<0.001), and reached and remained at a high level at 16 h (close to 1.0, with no significant difference compared to 8 h). This quantitatively confirmed that the degradation system has excellent lysosomal targeting specificity and long-term retention capacity (e.g., ...). Figure 7 (B)

[0071] In summary, this embodiment demonstrates that the lysosomal targeted degradation system based on DNA phase separation condensates prepared in this invention can enter cells via multivalent cholesterol anchoring, utilize RNase H enzyme response to open the structure and undergo liquid-liquid phase separation, form condensates in situ within the lysosome that can remain for a long time, and ultimately efficiently and specifically degrade PD-L1 protein on the surface of tumor cells via the lysosomal pathway.

[0072] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A lysosomal targeted degradation system based on DNA phase-separated condensates, characterized in that, The RNA-DNA tetrahedral framework includes sticky ends, wherein the RNA-DNA tetrahedral framework is self-assembled from a core strand and an edge strand through complementary base pairing, and the RNA-DNA tetrahedral framework is modified with aptamers that target membrane proteins. The edge chain is a DNA-RNA chimeric oligonucleotide chain, which contains one or more RNA ribonucleotide sequences that can be specifically recognized and cleaved by RNaseH enzyme, and the ends of the sequence are modified with sticky ends for driving phase separation and cell membrane anchoring groups.

2. The lysosomal targeted degradation system based on DNA phase-separated condensates according to claim 1, characterized in that, The core strand is a single-stranded DNA with structural support function, and its 5' or 3' end extends with a linker sequence for connecting aptamers to target membrane proteins.

3. The lysosomal targeted degradation system based on DNA phase-separated condensates according to claim 1, characterized in that, The nucleotide sequence of the sticky terminus is ATCGAT.

4. The lysosomal targeted degradation system based on DNA phase-separated condensates according to claim 1, characterized in that, The edge length of the RNA-DNA tetrahedral framework is 17 nucleotides.

5. The lysosomal targeted degradation system based on DNA phase-separated condensates according to claim 1, characterized in that, The aptamer targeting the membrane protein is a nucleic acid aptamer that specifically recognizes the PD-L1 protein.

6. The lysosomal targeted degradation system based on DNA phase-separated condensates according to claim 1, characterized in that, The sticky end and the cell membrane anchoring group are both at least one.

7. The lysosomal targeted degradation system based on DNA phase-separated condensates according to claim 1, characterized in that, The nucleotide sequence of the core chain is shown in SEQ ID NO.

1. The edge chains include TH17-2-2SE-AF647-10T, TH17-3-2SE-10T, and TH17-4-2SE, with nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. The nucleotide sequence of the aptamer targeting the membrane protein is shown in SEQ ID NO.5, and the nucleotide sequence of the cell membrane anchoring group is shown in SEQ ID NO.

6.

8. A method for preparing a lysosomal targeted degradation system based on DNA phase separation condensates as described in any one of claims 1 to 7, characterized in that, A buffer solution containing a core chain, edge chain, aptamers targeting membrane proteins, and cell membrane anchoring groups is incubated at 90–98°C for 2–10 minutes, and then slowly cooled to 4–25°C at a rate of 0.7–0.8°C / min to assemble a lysosomal targeted degradation system based on DNA phase separation condensates.

9. The application of the lysosomal targeted degradation system based on DNA phase separation condensates as described in any one of claims 1 to 7 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The tumor is primary clear cell renal cell carcinoma, breast cancer, or cervical cancer.

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