A multivalent protein targeted degradation system based on programmable nucleic acid templates, its preparation method and applications

Abstract

This invention provides a multivalent protein-targeting degradation system based on a programmable nucleic acid template, its preparation method, and its applications, belonging to the fields of biomedicine and molecular engineering. The system includes monovalent nucleic acid degradation units and a nucleic acid template. The monovalent degradation unit is formed by hybridization of two nucleic acid chains coupled with a target protein ligand and an E3 ubiquitin ligase ligand. The nucleic acid template has n loading sites, which assemble into multivalent nucleic acid degraders through complementary base pairing. This invention achieves precise control over the spacing and configuration of monovalent units by regulating the template spacer sequence length and topology, resulting in a spatially ordered multivalent synergistic degradation effect and significantly improving the degradation efficiency of target proteins. This system is highly modular and programmable, providing a new technological platform for the development of targeted protein degradation drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and molecular engineering, specifically relating to a multivalent protein targeted degradation system based on a programmable nucleic acid template, its preparation method, and its uses. Background Technology

[0002] Proteolytic targeting chimeras (PROTACs) are a novel therapeutic strategy that utilizes the intracellular ubiquitin-proteasome system to degrade target proteins by simultaneously binding to both the target protein and an E3 ubiquitin ligase. Compared to traditional small molecule inhibitors, PROTACs offer advantages such as catalytic degradation mechanisms, high selectivity, and the ability to overcome drug resistance mutations, demonstrating promising applications in various refractory targets.

[0003] However, traditional small-molecule PROTACs often employ flexible chemical chains to connect target protein ligands and E3 ligase ligands, making precise spatial conformational control difficult, resulting in limited degradation efficiency and significant off-target effects. Furthermore, their synthetic routes are complex, structural optimization is time-consuming, and they lack versatility and programmability. Therefore, constructing novel PROTAC platforms with spatial controllability and modularity has become a current research hotspot.

[0004] In recent years, nucleic acid molecules have been incorporated into targeted degradation systems due to their programmable sequences, high spatial construction precision, and good biocompatibility. Template-based degradation systems achieve distance-dependent degradation and directional tunability by coupling target protein ligands with E3 ligase ligands to a controllable-length nucleic acid backbone, significantly improving the predictability and construction efficiency of degradation. The self-assembly properties of DNA also enable modular replacement capabilities in these systems, providing new insights for precise protein degradation.

[0005] Although nucleic acid degradation systems offer a degree of programmability compared to traditional small-molecule PROTACs, thus improving the controllability of degraders to some extent, most existing nucleic acid degradation systems are monovalent structures, containing only one set of target protein ligands and E3 ligase ligands. These monovalent systems often suffer from insufficient binding affinity, poor complex stability, and limited degradation efficiency in complex physiological environments, making it difficult to achieve efficient degradation of protein complexes or high-density membrane proteins. Summary of the Invention

[0006] The purpose of this invention is to overcome the technical defects of current small molecule PROTAC and monovalent nucleic acid degradation systems and achieve the following objectives: (1) improve the spatial organization accuracy of target protein ligands and E3 ubiquitin ligase ligands; (2) achieve multivalent synergistic degradation effect; and (3) construct a modular and scalable nucleic acid degradation platform.

[0007] To achieve the above objectives, the specific technical solution of the present invention is as follows: This invention provides a multivalent protein targeted degradation system based on a programmable nucleic acid template. The multivalent protein targeted degradation system is prepared using at least one monovalent nucleic acid degradation unit (also known as a "monovalent degrader") and a nucleic acid template as raw materials. The monovalent nucleic acid degradation unit is a double-stranded structure formed by a first nucleic acid chain and a second nucleic acid chain through complementary base pairing. The first nucleic acid chain is coupled with a target protein ligand, and the second nucleic acid chain is coupled with an E3 ubiquitin ligase ligand. The nucleic acid template is provided with n loading sites that are complementary to the anchoring sequence in the monovalent nucleic acid degradation unit, for loading the monovalent nucleic acid degradation unit to form a multivalent protein targeted degradation system (also known as "multivalent nucleic acid degrader" or "multivalent degrader"); n is an integer from 1 to 10.

[0008] Furthermore, the nucleic acid template is selected from linear nucleic acid templates or circular nucleic acid templates; n is an integer from 1 to 6; and the length of the spacer sequence between adjacent loading sites is greater than or equal to 0 bp.

[0009] In some specific embodiments, the nucleic acid template is a circular DNA strand.

[0010] Furthermore, the length of the interval sequence is 21 bp or 42 bp.

[0011] In some specific embodiments, the length of the interval sequence is 42 bp.

[0012] Furthermore, the preparation method of the monovalent nucleic acid degradation unit includes the following steps: selecting two oligonucleotide single-stranded nucleic acids with complementary sequences, introducing active groups that can be coupled to target protein ligands and E3 ubiquitin ligase ligands at their ends, and then coupling them to target protein ligands and E3 ubiquitin ligase ligands respectively to obtain a first nucleic acid chain and a second nucleic acid chain; and hybridizing the first nucleic acid chain and the second nucleic acid chain by base complementary pairing annealing to obtain the monovalent nucleic acid degradation unit.

[0013] Further, the oligonucleotide single-stranded nucleic acid is selected from DNA, RNA, peptide nucleic acid (PNA) or combinations thereof; the active group is selected from DBCO, cyclooctyne, alkynyl, thiol, amino or carboxyl; the coupling method is selected from copper-free click chemistry (SPAAC), copper-catalyzed azide-alkynyl cycloaddition reaction (CuAAC), NHS-ester-amine condensation reaction, thiol-maleimide addition reaction or amidation reaction.

[0014] Further, the nucleotide sequence of the first nucleic acid chain is shown in SEQ ID No. 1; the nucleotide sequence of the second nucleic acid chain is selected from SEQ ID No. 2 or SEQ ID No. 6; and the nucleotide sequence of the nucleic acid template is selected from SEQ ID No. 3~5 or SEQ ID No. 7~10.

[0015] In some specific embodiments, the nucleotide sequence of the first nucleic acid chain is shown in SEQ ID No. 1, the nucleotide sequence of the second nucleic acid chain is shown in SEQ ID No. 2, and the nucleotide sequence of the nucleic acid template is selected from SEQ ID No. 3 to 5; The nucleotide sequence of the first nucleic acid chain is shown in SEQ ID No. 1, the nucleotide sequence of the second nucleic acid chain is shown in SEQ ID No. 6, and the nucleotide sequence of the nucleic acid template is selected from SEQ ID No. 7 to 10.

[0016] Furthermore, the target protein is BRD4, and the E3 ubiquitin ligase ligand is AHPC; the coupling method is a copper-free click chemistry reaction.

[0017] In some specific embodiments, the target protein ligand is JQ1.

[0018] The present invention also provides a method for preparing the above-mentioned multivalent protein targeted degradation system, the method comprising the following steps: hybridizing the monovalent nucleic acid degradation unit with the nucleic acid template by base complementary pairing annealing to obtain a linear or cyclic multivalent protein targeted degradation system.

[0019] Furthermore, the molar ratio of the monovalent nucleic acid degradation unit to the nucleic acid template is 1:(2n-1) to 1:2n.

[0020] In some specific embodiments, when the number of loading sites n on the nucleic acid template is 1 (mixed multivalent), the molar ratio of the monovalent nucleic acid degradation unit to the nucleic acid template is 1:1; When the number of loading sites n is 2 (bivalent), the molar ratio is 1:3; When the number of loading sites n is 3 (trivalent), the molar ratio is 1:5; When the number of loading sites n is 6 (hexavalent), the molar ratio is 1:10.

[0021] This invention also provides the use of the above-mentioned multivalent protein targeted degradation system in the preparation of target protein degrading agents.

[0022] The present invention also provides a target protein degrader, wherein the active ingredient of the target protein degrader is the above-mentioned multivalent protein targeted degradation system, wherein the concentration of the multivalent protein targeted degradation system is 25~125 nM.

[0023] In some specific embodiments, the concentration of the multivalent protein targeted degradation system is 75 nM.

[0024] The present invention has achieved the following beneficial effects: The multivalent nucleic acid degradation system provided by this invention achieves precise spatial organization and multivalent synergistic recognition of target protein ligands and E3 ubiquitin ligases by periodically arranging multiple monovalent nucleic acid degradation units on a single nucleic acid template. This system can utilize efficient coupling reactions to construct modular and scalable nucleic acid degradation platforms. Simultaneously, the multivalent nucleic acid degradation system can enhance the local concentration and binding probability of target proteins and E3 ubiquitin ligases at the molecular level, significantly promoting ubiquitination reactions and degradation efficiency, achieving the goal of efficiently clearing target proteins. This system not only overcomes the shortcomings of traditional small-molecule PROTAC structures, such as high flexibility and uncontrollable spatial arrangement, but also compensates for the problems of low binding efficiency and insufficient synergy of monovalent nucleic acid degraders.

[0025] Therefore, this invention provides experimental evidence for constructing a programmable, efficient, and specific protein degradation platform, demonstrating significant application potential in the development of targeted protein degradation drugs. This system is expected to be used for the efficient degradation of multi-subunit complexes, membrane proteins, and drug resistance-related targets, providing new ideas and technical support for precision targeted therapy and scalable nucleic acid drug design, and has important scientific significance and application value in the fields of anti-tumor and molecular targeted therapy.

[0026] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0027] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the preparation principle of a multivalent protein targeted degradation system based on a programmable nucleic acid template.

[0029] Figure 2 The diagram shows the design principle of the monovalent nucleic acid degrader and the verification results by non-denaturing polyacrylamide gel electrophoresis.

[0030] Figure 3The figure shows the results of Western blotting assays and semi-quantitative analysis of BRD4 protein degradation induced by monovalent nucleic acid degraders at different concentrations.

[0031] Figure 4 The diagram shows the design principle of nucleic acid degraders with different valence states (monovalent, divalent, trivalent, and hexavalent) and the verification results by non-denaturing polyacrylamide gel electrophoresis.

[0032] Figure 5 The figure shows the results of Western blotting assays and semi-quantitative analysis of BRD4 protein degradation induced by nucleic acid degraders of different valence states (monovalent, divalent, trivalent, and hexavalent).

[0033] Figure 6 Figure 1 shows the results of Western blotting assays and semi-quantitative analysis of BRD4 protein degradation induced by monovalent and hexavalent nucleic acid degraders.

[0034] Figure 7 The figure shows the results of Western blotting and semi-quantitative analysis of BRD4 protein degradation induced by bivalent nucleic acid degraders with different monovalent nucleic acid degrader spacings (42 bp and 21 bp).

[0035] Figure 8 Figure 1 shows the results of Western blotting assays and semi-quantitative analysis of BRD4 protein degradation induced by hexavalent nucleic acid degraders of different conformations (linear and circular). Detailed Implementation

[0036] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0037] Example 1: Construction of a linear monovalent protein targeted degradation system (for complementary pairing with a long-chain template, with a spacing of 42 bp). This embodiment follows Figure 1 The principle shown is used to construct a linear monovalent protein targeted degradation system.

[0038] 1. Preparation method of functionalized DNA single strands Two oligonucleotide single-stranded nucleic acids with complementary sequences (S1, S2) were selected, and DBCO (dibenzocyclooctylene) active groups were introduced at their 5' and 3' ends. The specific steps are as follows: The 5'-terminal DBCO modification was achieved by coupling a DBCO phosphoramidite reagent (name: 5'-DBCO-TEG Phosphoramidite, supplier: Glen Research, CAS No.: 1527468-08-1) to the end of the solid-phase synthesis. The reaction solvent was anhydrous acetonitrile, the coupling temperature was room temperature, and the coupling time was 10 min. The oxidation step used a 0.5 M camphorsulfonyl azirconium solution in anhydrous acetonitrile for 3 min. The deprotection conditions were 30% ammonia solution at 65 °C for 2 h (or room temperature for 17 h). For the 3'-terminal DBCO modification, a corresponding DBCO-modified solid-phase support (name: 3'-DBCO-Serinol CPG solid-phase support, supplier: Glen Research, catalog number: 20-2998) could be used for synthesis. After the reaction, the solid-phase support loaded with oligonucleotides was added to 30% ammonia water and reacted at 65 °C for 2 h for cleavage and deprotection. After cooling to room temperature, the mixture was centrifuged, the supernatant was collected, and the solid-phase support was washed 1-2 times with an appropriate amount of 30% ammonia water. The supernatants were then combined. Subsequently, the ammonia water was removed under reduced pressure. The crude product was first desalted by NAP desalting column or ethanol precipitation, and then purified by reversed-phase or ion-exchange high-performance liquid chromatography (HPLC) to obtain DBCO-functionalized DNA single strands, which were designated as ssDNA-1-DBCO and ssDNA-2-DBCO, respectively.

[0039] A copper-free click chemistry reaction (SPAAC) was used to couple ssDNA-1-DBCO with JQ1-azide and ssDNA-2-DBCO with AHPC-azide to obtain functionalized DNA single strands. The specific steps were as follows: ssDNA-1-DBCO and ssDNA-2-DBCO were dissolved in phosphate-buffered saline (PBS), preferably 1×PBS buffer (pH 7.4), to a final concentration of 100 μM; JQ1-azide and AHPC-azide were dissolved in DMSO to prepare 10 mM stock solutions, which were then diluted to 100 μM with PBS. Subsequently, the solutions were mixed separately according to a molar ratio of 1:1.2 for ssDNA-1-DBCO to JQ1-azide and ssDNA-2-DBCO to AHPC-azide. The mixtures were incubated at room temperature in the dark for 12–16 h. After the reaction was completed, unreacted small molecules were removed by high performance liquid chromatography or ultrafiltration desalting to obtain ssDNA1-JQ1 and ssDNA2-AHPC, respectively.

[0040] The sequence of S1 (SEQ ID No. 1) is as follows: 5'-AATACTCGGTGGTTAATTGTCATTGAAGTAGTGGAAGTAAGATA-3'; The sequence of S2 (SEQ ID No. 2) is as follows: 5'-TCTTTGAGAGCTTTATCGCATATGACAATTAACCACCGAGTAAA-3'; The specific structure of JQ1-azide is as follows: The specific structure of AHPC-azide is as follows: .

[0041] 2. Construction of monovalent nucleic acid degradation units ssDNA1-JQ1 and ssDNA2-AHPC were hybridized via base complementarity annealing to obtain a monovalent nucleic acid degrader, denoted as OligoTAC-AHPC. The specific steps were as follows: ssDNA1-JQ1 and ssDNA2-AHPC were dissolved in phosphate buffer to a final concentration of 100 μM, mixed at a 1:1 molar ratio, incubated in a 37°C water bath for 1 h, and then allowed to stand at room temperature for 1 h to obtain OligoTAC-AHPC.

[0042] Example 2: Construction of a linear mixed multivalent protein targeted degradation system (spaced 42 bp) OligoTAC-AHPC prepared in Example 1 was hybridized with linear nucleic acid template S3, whose sequence is shown in SEQ ID No. 3: 5'-ATGCGATAAAGCTCTCAAAGATATCTTACTTCCACTACTTCA-3', by base complementarity pairing annealing. The specific steps were as follows: OligoTAC-AHPC and S3 were mixed at a molar ratio of 1:1 and incubated in a water bath at 37°C for 1 hour, and then allowed to stand at room temperature for 1 hour to obtain a linear mixed multivalent nucleic acid degrader with a spacing of 42 bp, which was denoted as OligoTAC-AHPC-4 (abbreviated as A4).

[0043] Example 3: Construction of a linear bivalent protein targeted degradation system (spaced 42 bp) OligoTAC-AHPC prepared in Example 1 was hybridized with linear nucleic acid template S4, whose sequence is shown in SEQ ID No. 4: 5'-TATCTTACTTCCACTACTTCAATGCGATAAAGCTCTCAAAGATATCTTACTTCCACTACTTCAATGCGATAAAGCTCTCAAAGA-3', through base complementarity pairing annealing. The specific steps were as follows: OligoTAC-AHPC and S4 were mixed at a molar ratio of 1:3 and incubated in a water bath at 37°C for 1 h, and then allowed to stand at room temperature for 1 h to obtain a linear divalent nucleic acid degrader with a spacing of 42 bp, which was denoted as OligoTAC-AHPC-2 (abbreviated as A2).

[0044] Example 4: Construction of a linear trivalent protein targeted degradation system (spaced 42 bp) The OligoTAC-AHPC prepared in Example 1 was hybridized with the linear nucleic acid template S5, whose sequence is shown in SEQ ID No. 5: 5'-TATCTTACTTCCACTACTTCAATGCGATAAAGCTCTCAAAGATATCTTACTTCCACTACTTCAATGCGATAAAGCTCTCAAAGATATCTTACTTCCACTACTTCAATGCGATAAAGCTCTCAAAGA-3', through base complementarity pairing annealing. The specific steps were as follows: OligoTAC-AHPC and S5 were mixed at a molar ratio of 1:5, incubated in a 37°C water bath for 1 h, and then allowed to stand at room temperature for 1 h to obtain a linear trivalent nucleic acid degrader with a spacing of 42 bp, denoted as OligoTAC-AHPC-3 (abbreviated as A3).

[0045] Example 5: Construction of a linear monovalent protein targeted degradation system (complementary pairing with a long chain of 21 bp). Following the method described in Example 1, S1 and S2' were prepared as ssDNA-1 and ssDNA-2' modified with DBCO at 5' and 3' respectively. A copper-free click chemistry reaction was used to obtain ssDNA1-JQ1 and ssDNA2'-AHPC. These two were then subjected to base pairing annealing hybridization to obtain a monovalent protein-targeted degradation system OA with a 21 bp complementary pair to the long chain. The sequence of S2' is shown in SEQ ID No. 6: 5'-ATGACAATTAACCACCGAGTAAA-3'.

[0046] Example 6: Construction of a linear bivalent protein targeted degradation system (interval distance 21 bp) The OA prepared in Example 5 was hybridized with the linear nucleic acid template LC-2, whose sequence is shown in SEQ ID No. 7: 5'-TATCTTACTTCCACTACTTCATATCTTACTTCCACTACTTCA-3', via base complementarity annealing. Specifically, OA and LC-2 were mixed at a molar ratio of 1:3, incubated in a 37°C water bath for 1 hour, and then allowed to stand at room temperature for 1 hour to obtain a linear divalent nucleic acid degrader with a spacing of 21 bp, denoted as O2. Compared to Example 3, the only difference is that the two adjacent monovalent nucleic acid degraders are closer together on the long chain LC-2, only 21 bp apart, whereas the previous linear divalent degraders were spaced 42 bp apart on the long chain S4.

[0047] Example 7: Construction of a linear trivalent protein targeted degradation system (interval 21 bp) The OA prepared in Example 5 was hybridized with the linear nucleic acid template LC-3, whose sequence is shown in SEQ ID No. 8: 5'-TATCTTACTTCCACTACTTCATATCTTACTTCCACTACTTCATATCTTACTTCCACTACTTCA-3', via base complementarity pairing annealing. Specifically, OA and LC-3 were mixed at a molar ratio of 1:5, incubated in a 37°C water bath for 1 hour, and then allowed to stand at room temperature for 1 hour to obtain a linear trivalent nucleic acid degrader with a spacing of 21 bp, denoted as O3. Compared to Example 4, the only difference is that the two adjacent monovalent nucleic acid degraders are closer together on the long chain LC-3, only 21 bp, whereas the previous linear trivalent degraders were spaced 42 bp apart on the long chain S5.

[0048] Example 8: Construction of a linear hexavalent protein targeted degradation system (interval 21 bp) The OA prepared in Example 5 was hybridized with the linear nucleic acid template LC-6, whose sequence is shown in SEQ ID No. 9: 5'-TATCTTACTTCCACTACTTCATATCTTACTTCCACTACTTCATATCTTACTTCCACTACTTCATATCTTACTTCCACTACTTCATATCTTACTTCCACTACTTCATATCTTACTTCCACTACTTCA-3', by base complementary pairing annealing. The specific steps were as follows: OA and LC-6 were mixed at a molar ratio of 1:10, incubated in a 37°C water bath for 1 h, and then allowed to stand at room temperature for 1 h to obtain a linear hexavalent nucleic acid degrader with a spacing of 21 bp, denoted as O6.

[0049] Example 9: Construction of a cyclic hexavalent protein targeted degradation system (spaced 21 bp) The OA obtained in Example 5 was hybridized with the linear nucleic acid template Cir-6, whose sequence is shown in SEQ ID No. 10: 5'-CCACTACTTCAATGCGATAAAGCTCTCAAAGATATCTTACTTCCACTACTTCAATGCGATAAAGCTCTCAAAGATATCTTACTTCCACTACTTCAATGCGATAAAGCTCTCAAAGATATCTTACTT-3', via base complementarity pairing annealing. Specifically, OA and Cir-6 were mixed at a molar ratio of 1:10, incubated in a 37°C water bath for 1 hour, and then allowed to stand at room temperature for 1 hour to obtain a cyclic hexavalent nucleic acid degrader with a spacing of 21 bp, denoted as CO6. The only difference between CO6 and Example 8 is the template topology; Example 8 used a linear template to construct a linear hexavalent nucleic acid degrader, while Example 9 used a cyclic template to construct a cyclic hexavalent nucleic acid degrader.

[0050] The following experimental examples demonstrate the beneficial effects of the present invention.

[0051] Example 1: Performance Verification of Protein-Targeted Degradation Systems with Different Valence States 1. Experimental Methods 1.1 Western blot assay Cells treated with each group of nucleic acid degraders were collected and lysed in a 100:1 (v / v) mixture of RIPA lysis buffer and PMSF, and incubated at 4 °C for 30 min. The cells were then centrifuged at 16,000 rpm for 15 min at 4 °C, and the supernatant was collected as total protein. A portion of the sample was analyzed for protein concentration using the BCA method. The remaining protein samples were mixed with 6× loading buffer at a 5:1 (v / v) ratio and denatured at 100 °C for 10 min. Electrophoresis was performed on a 7.5% SDS-PAGE separating gel based on the molecular weights of the target protein BRD4 and the internal reference protein GAPDH. The total protein loading volume in each lane was kept consistent according to the BCA results. Stacking gel electrophoresis was performed at 90 V initially, and the voltage was increased to 150 V after the samples entered the separating gel. After electrophoresis, the membrane was transferred at a constant current of 300 mA for 1 h under ice bath conditions. After transfer, the membrane was blocked with 5% skim milk powder at room temperature for 2 h, followed by incubation with primary protein antibody at 4 °C overnight. The next day, the membrane was washed 5 times with TBST, and then incubated with HRP-labeled goat anti-rabbit secondary antibody at room temperature for 1 h. After incubation, the membrane was washed again, and ECL chemiluminescence reagent was added for development. The results were recorded using a gel imaging system.

[0052] 1.2 Non-denaturing polyacrylamide gel electrophoresis (Native PAGE) Each nucleic acid component was dissolved and mixed in a suitable buffer solution at a predetermined molar ratio, and then annealed to obtain the nucleic acid degrader assembly product. The assembly product and corresponding raw material single strands were taken, added to non-denaturing DNA loading buffer, and loaded into a 10% non-denaturing polyacrylamide gel for electrophoresis. Electrophoresis was performed at a constant voltage of 100 V for approximately 1.5 h using 1×TBE buffer as the electrophoresis system. After electrophoresis, the gel was stained with nucleic acid dyes, and the band positions were observed and recorded using a gel imaging system. The success of the nucleic acid degrader assembly was determined by comparing the migration behavior of the assembly product and each raw material strand.

[0053] 2. Experimental Results 2.1 Native PAGE Validation of Nucleic Acid Degraders with Different Valence States Native PAGE results showed that the band positions of each designed product were consistent with the expected molecular weight, confirming the successful construction of monovalent, divalent, trivalent, mixed multivalent, linear hexavalent, and cyclic hexavalent nucleic acid degraders. Figure 2 , Figure 4 ).

[0054] 2.2 Results of BRD4 protein degradation induced by monovalent nucleic acid degraders at different concentrations Western blot results showed that ( Figure 3 At various concentrations, the monovalent nucleic acid degraders all exhibited varying degrees of degradation activity against the target protein BRD4, with the 75 nM concentration showing the most significant degradation effect, far superior to other concentrations.

[0055] 2.3 Results of BRD4 protein degradation induced by nucleic acid degraders of different valence states Western blot results showed that ( Figure 5 At a concentration of 75 nM, nucleic acid degraders of different valence states were able to degrade the target protein BRD4. Among them, the mixed multivalent degrader (OligoTAC-AHPC-4) showed significantly higher degradation efficiency for BRD4 protein than the trivalent (OligoTAC-AHPC-3), bivalent (OligoTAC-AHPC-2), and monovalent (OligoTAC-AHPC) nucleic acid degraders, indicating that the degradation efficiency increases with the increase of the valence state of the nucleic acid degrader.

[0056] 2.4 Results of BRD4 protein degradation induced by monovalent and hexavalent nucleic acid degraders Western blot results showed that ( Figure 6 At a concentration of 50 nM, both the monovalent nucleic acid degrader OA and the hexavalent nucleic acid degrader O6 could degrade the target protein BRD4, with O6 showing significantly higher degradation efficiency than OA.

[0057] 2.5 Results of BRD4 protein degradation induced by bivalent nucleic acid degraders with different spacing between monovalent nucleic acid degraders. Western blot results showed that ( Figure 7 At concentrations of 50 nM and 100 nM, the degradation efficiency of the bivalent nucleic acid degrader O2 with a spacing of 21 bp on BRD4 was significantly higher than that of the bivalent nucleic acid degrader A2 with a spacing of 42 bp.

[0058] 2.6 Results of BRD4 protein degradation induced by hexavalent nucleic acid degraders of different conformations Western blot results showed that ( Figure 8 At concentrations of 50 nM and 100 nM, the cyclic hexavalent nucleic acid degrader CO6 showed significantly higher degradation efficiency for BRD4 than the linear hexavalent nucleic acid degrader O6.

[0059] In summary, this invention provides a multivalent protein-targeting degradation system based on a programmable nucleic acid template and its preparation method. This system achieves the construction of nucleic acid degraders with controllable valence, adjustable spacing, and variable topology through modular assembly, significantly improving the degradation efficiency of targeted proteins. It features programmable structure, simple preparation, and excellent degradation effect, providing a new technological platform for the development of targeted protein degradation drugs.

Claims

1. A multivalent protein targeted degradation system based on a programmable nucleic acid template, characterized in that: The multivalent protein targeted degradation system is prepared using at least one monovalent nucleic acid degradation unit and a nucleic acid template as raw materials; wherein, the monovalent nucleic acid degradation unit is a double-stranded structure formed by a first nucleic acid chain and a second nucleic acid chain through complementary base pairing, wherein the first nucleic acid chain is coupled with a target protein ligand, and the second nucleic acid chain is coupled with an E3 ubiquitin ligase ligand; The nucleic acid template is provided with n loading sites that are complementary to the anchoring sequence in the monovalent nucleic acid degradation unit, for loading the monovalent nucleic acid degradation unit to form a multivalent protein targeted degradation system; n is an integer from 1 to 10.

2. The multivalent protein targeted degradation system according to claim 1, characterized in that: The nucleic acid template is selected from linear nucleic acid templates or circular nucleic acid templates; n is an integer from 1 to 6; the length of the spacer sequence between adjacent loading sites is greater than or equal to 0 bp.

3. The multivalent protein targeted degradation system according to claim 2, characterized in that: The length of the interval sequence is 21 bp or 42 bp.

4. The multivalent protein targeted degradation system according to claim 1, characterized in that: The preparation method of the monovalent nucleic acid degradation unit includes the following steps: selecting two oligonucleotide single-stranded nucleic acids with complementary sequences, introducing active groups that can be coupled to target protein ligands and E3 ubiquitin ligase ligands at their ends, and then coupling them to target protein ligands and E3 ubiquitin ligase ligands respectively to obtain a first nucleic acid chain and a second nucleic acid chain; hybridizing the first nucleic acid chain and the second nucleic acid chain by base complementary pairing annealing to obtain the monovalent nucleic acid degradation unit.

5. The multivalent protein targeted degradation system according to claim 4, characterized in that: The oligonucleotide single-stranded nucleic acid is selected from DNA, RNA, peptide nucleic acid, or a combination thereof; the active group is selected from DBCO, cyclooctyne, alkynyl, thiol, amino, or carboxyl; the coupling method is selected from copper-free click chemistry, copper-catalyzed azide-alkynyl cycloaddition reaction, NHS-ester-amine condensation reaction, thiol-maleimide addition reaction, or amidation reaction.

6. The multivalent protein targeted degradation system according to claim 4, characterized in that: The nucleotide sequence of the first nucleic acid chain is shown in SEQ ID No. 1; the nucleotide sequence of the second nucleic acid chain is selected from SEQ ID No. 2 or SEQ ID No. 6; the nucleotide sequence of the nucleic acid template is selected from SEQ ID No. 3~5 or SEQ ID No. 7~10.

7. The multivalent protein targeted degradation system according to any one of claims 1 to 6, characterized in that: The target protein is BRD4, and the E3 ubiquitin ligase ligand is AHPC.

8. A method for preparing the multivalent protein targeted degradation system according to any one of claims 1 to 7, characterized in that: The method includes the following steps: hybridizing the monovalent nucleic acid degradation unit with the nucleic acid template through base complementary pairing annealing to obtain a linear or cyclic multivalent protein targeted degradation system.

9. The method according to claim 8, characterized in that: The molar ratio of the monovalent nucleic acid degradation unit to the nucleic acid template is 1:(2n-1) to 1:2n.

10. Use of the multivalent protein targeted degradation system according to any one of claims 1 to 7 in the preparation of target protein degrading agents.

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