Aptamer-based targeted protein degradation molecule and application thereof

Abstract

The invention provides a target protein degradation molecule based on a nucleic acid aptamer and application thereof, a target protein ligand is the nucleic acid aptamer capable of being combined with a target protein, specifically, BRD4 or IRAK4 is taken as the target protein, and the target protein degradation molecule based on the nucleic acid aptamer capable of degrading the target protein is provided. The molecules show the effect of effectively degrading the target protein in cellular level and in vivo experiments, and have small side effects, so that the molecules have good pharmaceutical activity for diseases caused by overexpression of the target protein.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a nucleic acid aptamer-based targeted protein degradation molecule and its use. Background Art

[0002] Proteins are the basic executors of life activities and also key factors in the occurrence and development of many diseases. Therefore, regulating the level and function of proteins is one of the important strategies for treating diseases. Traditionally, the development of protein-regulating drugs has mainly focused on small molecule inhibitors or antagonists, which block the interaction between the target protein and downstream signaling molecules by occupying the active site or binding site of the target protein, thereby inhibiting its function. However, the target proteins of small molecule inhibitors are usually enzymes or receptors with active sites, but undruggable proteins such as transcription factors, phosphatases, and intrinsically disordered proteins lack active sites for binding to small molecule inhibitors. Therefore, small molecule inhibitors cannot intervene in such proteins or the intervention effect is not good. In addition, small molecule inhibitors can only inhibit the enzymatic function of proteins and cannot inhibit their non-enzymatic functions, making it difficult to completely block the physiological functions of certain proteins. Therefore, small molecule inhibitors targeting undruggable targets have certain limitations in terms of drug efficacy and selectivity.

[0003] In recent years, Proteolysis Targeting Chimeras (PROTACs) have offered hope for breaking through this limitation. A PROTAC is a heterobifunctional molecule that utilizes the cell's natural protein degradation mechanism, namely the ubiquitin-proteasome system (UPS), to selectively target and degrade proteins relevant to the pathogenesis of diseases, thereby potentially modulating "undruggable" targets that are difficult to target with traditional small molecules. A classic PROTAC molecule consists of three parts: a ligand for the Protein of Interest (POI), a ligand for the E3 ubiquitin ligase, and a linker. The POI ligand is used to recruit and bind to the target protein, the E3 ubiquitin ligase ligand is used to recruit and bind to the E3 ubiquitin ligase, and the linker is used to connect the POI ligand and the E3 ubiquitin ligase ligand. When a PROTAC binds to both the POI and the E3 ubiquitin ligase simultaneously, a ternary complex of "target protein-PROTAC-E3 ubiquitin ligase" is formed, causing the target protein to be marked by ubiquitin molecules and completing the degradation of the POI through the ubiquitin-proteasome system (UPS). Moreover, after the ubiquitination of the target protein is completed, the PROTAC molecule dissociates from the ternary complex and is used to target and degrade the next target protein, thus entering an iterative cycle. Different from the occupancy-driven mode adopted by traditional inhibitors (where small drug molecules need to tightly bind to the active site of the target protein to inhibit the activity of the target protein and achieve a certain pharmacological effect), PROTAC is an event-driven mode that can bind to any position of the target protein and may induce the degradation of the target protein without requiring high affinity. Therefore, compared with traditional small molecule drugs, PROTACs have advantages such as a wider range of action, higher activity, better selectivity, higher safety, and lower drug resistance, and the PROTAC technology has shown great potential and prospects in the fields of cancer, immune disorders, neurodegenerative diseases, etc.

[0004] However, there are also difficulties and challenges in the research and development of PROTACs. One of them is the research and selection of POI ligands. The development of PROTACs relies on POI ligands that target the target protein (traditionally usually small molecule ligands), but suitable ligands have not been found for many key proteins. Therefore, it is difficult to develop corresponding PROTACs for these target proteins lacking corresponding ligands, which limits the target range of PROTACs. At the same time, optimizing the type and length of the linker is also a time-consuming, laborious and low-throughput process. These difficulties and challenges have largely hindered the development of PROTACs. To address these difficulties and challenges, some researchers have chosen to try other types of ligands besides small molecule POI ligands, such as peptide-, antibody- or oligonucleotide-based POI ligands, etc., and have studied aptamer-based targeted protein degradation molecules, providing a powerful tool for degrading some undruggable target proteins.

[0005] Nucleic acid aptamers, also known as aptamers, nucleic acid aptamers, nucleic acid aptazymes, are single-stranded oligonucleotide molecules that can specifically recognize targets (such as target proteins) screened from synthetic DNA / RNA libraries, including single-stranded DNA or single-stranded RNA. The sequence length of aptamers is mostly between 15 and 60 bases. The target types of aptamers are extensive, including ions, small molecules, peptides, proteins, and even whole living cells, viruses, bacteria, or tissues. In addition to high specificity and high affinity for targets, aptamers also have the advantages of simple synthesis, low molecular weight, high chemical stability, low toxicity, low immunogenicity, controllable procedures, and easy modification with various functional groups or materials. Since aptamers have the activity of binding proteins, they can replace small molecule POI ligands as part of the targeting of target proteins (also known as warheads or target heads). Developing aptamer-based targeted protein degradation molecules can solve the problem of target head design, and the synthesis and modification technologies of nucleic acids are relatively mature. The modifiable sites cover bases, riboses, and phosphates, so aptamers modified with E3 ubiquitin ligase ligands can be constructed. There have been reports on aptamer-based targeted protein degradation molecules. For example, Lin Zhang et al. reported the development of a molecule ZL216 that targets the degradation of nucleolin through the AS1411 aptamer (Zhang L. et al., Development of a novel PROTAC using the nucleic acid aptamer as a targeting ligand for tumor selective degradation of nucleolin. Mol Ther Nucleic Acids. 2022 Sep 19;30:66-79), and Lingping Kong et al. reported a molecule dp53m that targets the carcinogenic mutant p53-R175H based on a DNA aptamer (Kong L. et al., An engineered DNA aptamer-based PROTAC for precise therapy of p53-R175H hotspot mutant-driven cancer. Sci Bull (Beijing). 2024 Jul 15;69(13):2122-2135).

[0006] Traditional aptamer screening methods first identify affinity binders and enrich binding sequences through an in vitro process using a randomized initial library to find binders with high binding affinity. Then, these selected binders are subjected to functional screening to evaluate their therapeutic potential. However, traditional aptamer screening methods face difficulties when applied to the development of nucleic acid aptamer-based targeted protein degradation molecules because simply attaching an E3 ubiquitin ligase ligand to the selected binder does not ensure that the resulting molecule can effectuate efficient ubiquitination and degrade the target protein. To achieve maximum degradation efficiency, extensive optimization of aspects such as linker type, length, and ligand modification sites is required to achieve optimal ternary complex formation.

[0007] Therefore, there is a need to develop new nucleic acid aptamer-based targeted protein degradation molecules with degradation effects for specific proteins. Among them, bromodomain protein 4 (BRD4) is a member of the bromodomain and extra-terminal (BET) family. It has been found that BRD4 is highly overexpressed in malignant tumors including liver cancer, breast cancer, non-small cell lung cancer, primary and metastatic melanoma, prostate cancer, etc. Targeted inhibitors targeting BRD4 such as CPI-0610, PLX-2853, GSK-2820151, etc. are in the clinical trial stage. Additionally, IRAK4 (interleukin-1 receptor-associated kinase 4) is one of the isoenzymes of the human IRAK kinase family and plays an important role in human inflammatory responses and tumors. The IRAK4 protein has both kinase activity and a scaffolding function. Therefore, degrading it using nucleic acid aptamer-based targeted protein degradation molecules can simultaneously block the kinase function and scaffolding function of IRAK4, thereby achieving complete inhibition of the signaling pathway and exerting good anti-inflammatory and anti-tumor activities. IRAK4 inhibition can be used for the treatment of various diseases such as solid tumors, hematological malignancies, myelodysplastic syndromes, diabetic nephropathy, and nerve damage. In December 2022, Kymera announced the phase 1 clinical data of its IRAK4 degrader KT-474, demonstrating good efficacy in patients with HS and AD.

[0008] Therefore, based on the need to develop degraders for overexpressed pathogenic proteins such as BRD4, IRAK4, etc., there is an urgent need in the art to provide new nucleic acid aptamer-based targeted protein degradation molecules. Summary of the Invention

[0009] Based on the unmet needs in the art described above, the technical solutions of the present invention are provided.

[0010] According to the first aspect of the present invention, there is provided an aptamer-based targeted protein degradation molecule, wherein the protein is BRD4, and the aptamer-based targeted protein degradation molecule is formed by linking an aptamer with an E3 ubiquitin ligase ligand, and the sequence of the aptamer is any one or more selected from SEQ ID NO: 5-8 or SEQ ID NO: 14.

[0011] The E3 ubiquitin ligase ligand is any type of ligand capable of recruiting and binding to an E3 ubiquitin ligase. Preferably, the E3 ubiquitin ligase ligand is selected from CRBN ligand, VHL ligand, IAP ligand, MDM2 ligand, DCAF ligand, RNF ligand, AhR ligand, FEM1B ligand, KEAP1 ligand. More preferably, the E3 ubiquitin ligase ligand is selected from thalidomide, pomalidomide, lenalidomide, VH032, VH285, VH298, VH101.

[0012] According to the second aspect of the present invention, there is provided an aptamer-based targeted protein degradation molecule, wherein the protein is BRD4, and the sequence of the aptamer-based targeted protein degradation molecule is SEQ ID NO: 20. The targeted protein degradation molecule is a bispecific RNA oligonucleotide capable of simultaneously recruiting and binding to BRD4 and an E3 ubiquitin ligase.

[0013] According to the third aspect of the present invention, there is provided the use of the aptamer-based targeted protein degradation molecule according to the first aspect or the second aspect of the present invention in the treatment of diseases, or the use of the aptamer-based targeted protein degradation molecule according to the first aspect or the second aspect of the present invention in the preparation of a drug for the treatment of diseases, wherein the disease is a disease caused by overexpression of the BRD4 protein.

[0014] Furthermore, the disease caused by overexpression of the BRD4 protein is a malignant tumor. Preferably, the malignant tumor is liver cancer, breast cancer, non-small cell lung cancer, melanoma (including primary and metastatic), prostate cancer. More preferably, the disease is liver cancer or melanoma.

[0015] According to the fourth aspect of the present invention, there is provided an aptamer-based targeted protein degradation molecule, wherein the protein is IRAK4, and the aptamer-based targeted protein degradation molecule is formed by linking an aptamer with an E3 ubiquitin ligase ligand, and the sequence of the aptamer is SEQ ID NO: 21.

[0016] Similarly, in the aptamer-based targeted protein degradation molecule provided in the fourth aspect of the present invention, the E3 ubiquitin ligase ligand is any type of ligand capable of recruiting and binding to an E3 ubiquitin ligase. Preferably, the E3 ubiquitin ligase ligand is selected from CRBN ligand, VHL ligand, IAP ligand, MDM2 ligand, DCAF ligand, RNF ligand, AhR ligand, FEM1B ligand, KEAP1 ligand. More preferably, the E3 ubiquitin ligase ligand is selected from thalidomide, pomalidomide, lenalidomide, VH032, VH285, VH298, VH101.

[0017] According to a fifth aspect of the present invention, there is provided the use of the aptamer-based targeted protein degradation molecule according to the fourth aspect of the present invention in the treatment of diseases, or the use of the aptamer-based targeted protein degradation molecule according to the fourth aspect of the present invention in the preparation of a medicament for the treatment of diseases, wherein the disease is a disease caused by overexpression of IRAK4 protein.

[0018] Furthermore, the diseases caused by overexpression of IRAK4 protein are solid tumors, hematological tumors, myelodysplastic syndromes, diabetic nephropathy and nerve damage.

[0019] According to the technical solution provided by the present invention, the present invention provides a new targeted protein degradation molecule with an aptamer as the targeting head, avoiding the design work of the targeting head of PROTAC molecules in the traditional technology, overcoming the difficulty of discovering small molecule POI ligands for specific target proteins when developing PROTAC molecules in the prior art, and solving the problem that it is difficult to develop PROTAC for some proteins due to the lack of small molecule POI ligands. At the same time, the present invention has established a rapid and high-throughput screening method for aptamer-based targeted protein degradation molecules by constructing a functionalized screening platform, avoiding the optimization work of the linker part, which will greatly accelerate the development process of such drugs and contribute to the application of such drugs to a wider variety of protein targeted degradation and related disease treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1To show the test result graphs of the cytotoxic effects and anti-tumor activities of the nucleic acid aptamer-based targeted protein degradation molecules PA2 and VIA2 on tumor cells provided in Embodiments 1-2 of the present invention. Among them, a: BRD4 protein levels in Hep3B cells treated with PA2 or VIA2 at different concentrations or for different times; b: Cell viability obtained by treating Hep3B cells with different concentrations of PA2 or VIA2 for 24, 48, and 72 hours respectively; c: Schematic diagram of testing the degradation effect of PA1, PA2, PA4, and PA6 on BRD4 protein in HEK293T cells by Western blotting.

[0022] Figure 2 To show the test result graphs of the cytotoxic effects and anti-tumor activities of the nucleic acid aptamer-based targeted protein degradation molecule BRA1 (bispecific RNA aptamer) on tumor cells provided in Embodiment 3 of the present invention. Among them, a: BRD4 protein levels in A375 cells treated with BRA1 at different concentrations or for different times; b: Cell viability obtained by treating A375 cells with different concentrations of BRA1 for 12, 24, and 36 hours respectively.

[0023] Figure 3 To show the in vivo anti-tumor activities of three nucleic acid aptamer-based targeted protein degradation molecules PA2, VIA2, and BRA1 provided in Embodiments 1-3 of the present invention. a: Administration scheme for testing the in vivo anti-tumor activities of PA2 and VIA2 using a subcutaneous tumor model of Hep3B cells; b: Curve graph of body weight and tumor volume changes after treatment with the experimental group (PA2, VIA2) and the control group (Vehicle); c: Tumor size graph after treatment with the experimental group (PA2, VIA2) and the control group (Vehicle); d: Column graph of tumor weights after treatment with the experimental group (PA2, VIA2) and the control group (Vehicle); e: Administration scheme for testing the in vivo anti-tumor activity of BRA1 using a subcutaneous tumor model of melanoma A375 cells; f: Curve graph of body weight and tumor volume changes after treatment with different doses of the experimental group (BRA1) and the control group (Vehicle); g: Tumor size graph after treatment with different doses of the experimental group (BRA1) and the control group (Vehicle); h: Column graph of tumor weights after treatment with different doses of the experimental group (BRA1) and the control group (Vehicle).

[0024] Figure 4 To show the test result graphs of the cytotoxic effects and activities of the nucleic acid aptamer-based targeted protein degradation molecule SHA1 on HEK293T cells provided in Embodiment 6 of the present invention. Among them, left: IRAK4 protein levels in HEK293T cells treated with different concentrations of SHA1; right: IRAK4 protein levels in Hep3B cells treated with SHA1 for different times. Detailed implementation manners

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0026] Generally speaking, when developing nucleic acid aptamer-based targeted protein degradation molecules, in order to solve the problem of how to develop corresponding binding ligands for specific POIs, the present invention provides a systematic evolution platform. This platform uses a fluorescence functional assay to evaluate the ability of each member in a randomized DNA library modified with an E3 ubiquitin ligase ligand to bind to the POI and simultaneously recruit the E3 ubiquitin ligase to achieve effective ubiquitination. Specifically, a 20-nucleotide randomized library surrounded by primer-binding regions was designed, and an E3 ubiquitin ligase ligand was attached to specific positions of each sequence in the DNA library. This library was displayed in monoclonal form on microbeads and incubated together with a red-fluorescently labeled POI, ubiquitin-fluorescein, and an E3 ubiquitin ligase system. Based on this platform, microbeads capable of binding to the POI and simultaneously recruiting the E3 ubiquitin ligase to achieve effective ubiquitination will exhibit a dual-fluorescent color and can be sorted and ranked using a fluorescence-activated cell sorter (FACS). Subsequently, the sequences on the screened microbeads were amplified. Further repeating this process can obtain aptamer chimeras capable of protein ubiquitination. After sequencing, aptamers were synthesized according to the sequencing information and further prepared as candidates for nucleic acid aptamer-based targeted protein degradation molecules, and their effects on degrading the target protein were tested.

[0027] In the specific implementation manners of the present invention, the sources of common materials or detection methods in each embodiment are as follows.

[0028] 1. Chemicals

[0029] In the embodiments of the present invention, the reagents used for chemical reactions, including propargylamine, N,N'-diisopropylcarbodiimide (DIC), 1-hydroxy-7-azabenzotriazole (HOAt), tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), and 3-(2-bromoethyl)indole, were all purchased from Beijing Yingnuo Kaisheng Technology Co., Ltd. (Innochem Technology). DMF was purchased from Shanghai Macklin Biochemical Co., Ltd. His-Tag magnetic affinity separation microbeads ( His-Tag Isolation and Pulldown beads) and MyOne TMCarboxylic acid magnetic beads are from Thermo Fisher Scientific. The 2'-deoxynucleotide triphosphate dNTP set (dATP, dTTP, dGTP, dCTP, 5-ethynyl-dUTP) is from Wuhu Huaren Science and Technology. The hot-start Taq DNA polymerase is from GenStar Biosolutions Co., Ltd, Beijing, while KOD-Plus-Neo is from Toyobo. λ exonuclease is purchased from New England Biolabs (Frankfurt, Germany). Human BRD4 (CR75) is from Novoprotein, and human IRAK4 (NM_016123) is from Abmart. The in vitro ubiquitination system, including UBE1 (E-305), UbcH3 (E2-611), VHL complex (E3-6505), CRBN complex (E3-650), and recombinant human ubiquitin N-terminal fluorescein (U-580), is from Bio-Techne. (S, R, S)-AHPC-PEG1-azide and pomalidomide-NH-CO-PEG1-N3 are purchased from Confluore Biotechnology. MG132 is purchased from MedChemExpress, and MLN4924 and 1× EDTA-free protease inhibitor mixture are purchased from Selleck. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 10 μg / mL streptomycin, and 100 units / mL penicillin are purchased from Thermo Fisher Scientific. DTT is purchased from Biosharp. TE buffer (B548106-0500) is purchased from Shanghai Sangon Biotech Co., Ltd.

[0030] The T7 High-Yield RNA Transcription Kit is purchased from Vazyme. Other reagents, including Klenow fragment, T7 RNA polymerase, DNase I, M-MLV, heat-stable RNase H, NTP mixture (25 mM each), and RNase inhibitor, are all purchased from Beyotime Biotech.

[0031] 2. Oligonucleotide Synthesis and Purification

[0032] In the examples of the present invention, oligonucleotides are synthesized on a DNA synthesizer (96-channel DNA synthesizer, Tsingke Biotechnology Co., Ltd, Beijing) at a 50 nanomole scale using the standard phosphoramidite protocol in the laboratory.

[0033] 3. Sequencing data analysis

[0034] Data processing and analysis were performed using Aptasuite49. Initially, the software counted the number of repeats for each sequence and calculated the edit distance between all sequences, which was then stored using locality-sensitive hashing. Subsequently, sequences were clustered into families based on an edit distance threshold of 5, and these clusters were sorted by the abundance of their members. The clusters that were enriched during the screening process and ranked highest in the final round were selected, and the highest-ranked sequence in each cluster was picked.

[0035] Example 1: Screening of DNA aptamer-pomalidomide-based targeted protein degradation molecules

[0036] This example provides a DNA aptamer-pomalidomide-based targeted protein degradation molecule and its screening method, including the following steps:

[0037] 1. Pre-enrichment of BRD4-binding sequences from the library:

[0038] An initial single-stranded DNA (ssDNA) library was constructed, and each sequence in this library had the sequence shown in SEQ ID NO: 1 (see Table 1), where the number of bases N was 20 and randomly selected from A, T, C, or G. This initial library was synthesized using a 192-channel oligonucleotide solid-phase synthesizer (Beijing Tsingke Biotechnology Co., Ltd.) in the inventor's laboratory. The full-length sequence was synthesized in one step. The specific steps were as follows: using the oligonucleotide synthesizer, the initial library sequence was synthesized by solid-phase synthesis; using an ammonia analyzer (Beijing Tsingke Biotechnology Co., Ltd.), the CPG synthesis column was deprotected at 95°C for 3 hours, and then the synthesis column was washed with 100% acetonitrile to remove short chains and desalinate. Purification was achieved by gel cutting and nucleic acid purification columns (Oligo Clean&Concentrator Kits, Zymo Research). Subsequently, the full-length DNA strand was eluted with DEPC water. The obtained DNA strand was then lyophilized and stored at -20°C.

[0039] 1 nmol of the initial ssDNA library was dissolved in 10 μl of binding buffer 1 (2.5 mM MgCl2, 1 mM CaCl2, 1 mg / ml BSA, 0.1 mg / ml salmon sperm DNA, 0.1% Tween 20, pH 7.4), heated to 95°C for 5 minutes, and then quickly cooled on ice for 5 minutes. At 25°C, 1 μg of recombinant BRD4 protein was combined with 1 μl His-Tag magnetic affinity separation beads were incubated in 10 μl of binding buffer 1 for 1 hour to obtain protein beads. Then, the initial ssDNA library treated as above was incubated with the protein beads in 10 μl of binding buffer 1 at 37 °C for 1 hour. The unbound ssDNA sequences were washed 3 times with binding buffer 1, and then the remaining bound ssDNA sequences were eluted by heating in water at 95 °C 3 times. The eluted sequences were amplified by PCR and digested into single-stranded DNA with λ exonuclease. Subsequently, the ssDNA obtained from the first-round pre-enrichment was used as the new library for the second-round screening, and a round of pre-enrichment was performed again according to the same steps. The ssDNA library obtained after 2 rounds of pre-enrichment was used as the candidate library for functional evolution screening.

[0040] 2. Functional evolution screening of aptamer-pomalidomide chimeric degrader:

[0041] Step 1: Emulsion PCR

[0042] Couple the forward primer (FP, SEQ ID NO: 2, see Table 1) to the beads to obtain FP-beads. The specific process is as follows: Wash 100 μl of MyOne TM Carboxylic acid magnetic beads (1 μm) 3 times with 100 μl of DMF, then add 50 μL of 2M propargylamine, 50 μL of 2.8M DIC, 100 μL of 1M HOAt (all dissolved in DMF) and mix; React the mixture at 50 °C and 1500 rpm for 3 hours; Then wash 3 times with DMF and resuspend with DMF; Incubate the beads in the CuAAC reaction (copper-catalyzed azide-alkyne cycloaddition reaction, one of the most commonly used classical reactions in the field of click chemistry) mixture at 37 °C and 1500 rpm for 3 hours. The composition of the CuAAC reaction mixture is 1.0 mM CuSO4, 1.2 mM THPTA, 6 mM ascorbic acid, 0.05% Tween 20, 100 μM 5′-N3-modified FP, 5% DMSO, 220 mM TEAA, pH = 7, 10×PBS, DEPC water, and the total volume is 100 μL.

[0043] Then, wash the FP-beads 3 times with TE buffer and resuspend in 100 μl of DEPC water.

[0044] The oil phase of emulsion PCR consists of 7% KF-6038 and 93% DMF-A-6CS and is rotated for 2 hours to mix evenly; the aqueous phase contains 6 μl dNTPs (dATP, dGTP, dCTP, dTTP, 2.5 mM for each nucleotide), 3 μl 100 μM reverse primer-1 (RP-1, SEQ ID NO: 3), 4.5 μl 25 mM MgCl2, 1 μl KOD-Plus-Neo, 5 pM template DNA, 12.5 μl PEG-8000, 5 μl FP-beads obtained through the above steps, and the total volume of water added is 75 μl. 300 μl of the oil phase is added to 75 μl of the aqueous phase and emulsified in a grinder at 40 Hz for 90 seconds to generate an emulsion. Then, the emulsion is aliquoted into PCR tubes at 50 μl each. PCR is carried out under the following cycling conditions: 95 °C for 3 minutes, followed by 30 cycles of 95 °C for 15 seconds and 50 °C for 15 seconds.

[0045] Step 2: Demulsification

[0046] After PCR, the emulsion is collected into a 2 ml PCR tube and vortexed for 30 seconds. The bead particles are collected after centrifugation at 3000 g for 5 minutes, and the clear oil phase is carefully removed. Then, 1 ml of demulsification buffer (containing 100 mM sodium chloride, 1% Triton X-100, 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA) is added to the bead particles. Magnetic separation is carried out after vortexing for 30 seconds and centrifugation at 15000 g for 90 seconds, and the buffer is carefully discarded. This step is repeated 2 times, and then magnetic separation washing is carried out 3 times with TE buffer.

[0047] Step 3: Generation of pomalidomide microbead particles

[0048] To generate ssDNA, the bead particles were incubated in 200 μl of 100 mM sodium hydroxide solution at 50 °C for 2 minutes. The supernatant was carefully removed by magnetic separation and repeated twice. To add an alkyne group to the ssDNA sequence, an EdUTP (ethynyl-modified dUTP) was added to the 3′ end of the sequence. The reaction system contained 20 μl of 100 μM reverse primer-2 (RP-2, SEQ ID NO: 4, with an additional A base at the 5′ end compared to RP-1, used to introduce EdU into ssDNA), 10 μl of 10 mM EdUTP, 10 μl of hot-start Taq polymerase, and water, with a total volume of 200 μl. Incubate at 95 °C for 3 minutes and at 72 °C at 1500 rpm for 25 minutes. To remove RP-2, the bead particles were placed in 200 μl of 90% formamide and 10% water, incubated at 50 °C for 2 minutes, and then the supernatant was carefully removed by magnetic separation. This step was repeated twice, and the bead particles were washed 3 times with water. Then, pomalidomide reacted with EdUTP through click chemistry. The specific conditions were: add 5 μl of 20 mM CuSO4, 5 μl of 24 mM THPTA, 5 μl of 120 mM ascorbic acid, 10 μl of 2.2 M TEAA, 10 μl of 1% Tween 20, 10 μl of 10×PBS, 5 μl of DMSO, and 5 μl of 10 mM pomalidomide-NH-CO-PEG1-N3 to initiate the reaction, add water to a final volume of 100 μl, and incubate the mixture at 37 °C at 1500 rpm. After the click chemistry reaction, the product was washed once with 1% Tween 20 and twice with water to obtain pomalidomide bead particles.

[0049] Step 4: Incubation of the in vitro UPS system

[0050] Pomalidomide microbeads were resuspended in TMC buffer (containing 5 mM MgCl2, 1 mM CaCl2, 50 mM Tris-HCl, pH 7.4), heated to 95 °C at 1500 rpm for 3 minutes, and then rapidly cooled on ice for 3 minutes. To obtain functional aptamer chimera degradants, the pomalidomide microbeads were incubated with the ubiquitination system at 37 °C for 3 hours. The ubiquitination system contained 100 nM BRD4, 50 nM UBE1, 500 nM UbcH3, 200 nM CRBN complex, 500 nM Ub-F, 5 mM ATP, 1 mg / ml BSA, 0.1 mg / ml salmon sperm DNA, 0.1% Tween 20, and 1 μM His-tagged peptide. Then, it was incubated with binding buffer 2 (containing 5 mM MgCl2, 1 mM CaCl2, 1 mg / ml BSA, 0.1 mg / ml salmon sperm DNA, 0.1% Tween 20, 50 mM Tris-HCl, pH 7.4) at 37 °C for 1 minute, and repeated 2 times. Subsequently, the pomalidomide microbeads were incubated with rabbit anti-FLGA antibody and 10 mM pomalidomide-NH-CO-PEG1-N3 in 200 μl of binding buffer 2 at 37 °C and 1500 rpm for 1 hour. The pomalidomide microbeads were washed with binding buffer 2 at 37 °C for 1 minute, and this step was repeated 2 times. Finally, the pomalidomide microbeads were incubated with goat anti-rabbit antibody AF647 in 200 μl of binding buffer 2 at 37 °C and 1500 rpm for 20 minutes, and then the pomalidomide microbeads were washed with binding buffer 2 at 37 °C for 1 minute, and this step was repeated 2 times.

[0051] Step 5: Sorting of aptamer-pomalidomide chimera degradants

[0052] The pomalidomide microbeads were resuspended in 4 ml of sorting buffer (containing 5 mM MgCl2, 1 mM CaCl2, 0.1% Tween 20, 50 mM Tris-HCl, pH 7.4). The fluorescence distribution of the pomalidomide microbeads was monitored, and a sorting gate containing approximately 0.4 - 0.5% of the APC channel and 5 - 6% of the FITC channel was selected. After collecting the pomalidomide microbeads with dual-fluorescent labels, the sequences were amplified by PCR to create an enrichment pool for the next round of aptamer chimera microbead synthesis. To increase the stringency of the second-round selection, we reduced the volume of the microbeads to 45 μl and decreased the concentration of BRD4 from 100 nM to 25 nM. Thus, the samples obtained by functional screening were obtained.

[0053] Step 6: Sequencing analysis and synthesis of nucleic acid aptamer-based targeted protein degradation molecules

[0054] For downstream analysis, samples obtained from pre-enrichment to two rounds of functional screening were sequenced using the Illumina Novaseq platform. Based on the sequencing results, DNA aptamer-pomalidomide chimeric degradants were synthesized, with the full-length sequence synthesized in one step: deprotection was carried out at 95 °C for 3 hours using an ammonia parser (Beijing Tsingke Biotechnology Co., Ltd.), and then the CPG synthesis column was washed with 100% acetonitrile to remove short chains and desalt. Purification was achieved by gel cutting and nucleic acid purification columns (Oligo Clean&Concentrator Kits, RNA Clean&Concentrator Kits, ZymoResearch). Subsequently, the full-length DNA strand was eluted with DEPC water. The obtained DNA strand was then lyophilized and stored at -20 °C.

[0055] According to the enrichment degree of the sequences in the sequencing results (for the specific operation process, refer to the aforementioned sequencing data analysis section), especially the top-ranked abundant clusters, four aptamer-based targeted protein degradation molecules designated PA1, PA2, PA4, and PA6 were finally selected for subsequent testing. The sequences of the aptamer parts were SEQ ID NO: 5-8 respectively (see Table 1).

[0056] Table 1 Sequences involved in Example 1

[0057]

[0058]

[0059] Example 2: Screening of aptamer-based targeted protein degradation molecules based on DNA aptamer-VH032

[0060] This example provides another aptamer-based targeted protein degradation molecule that has another E3 ubiquitin ligase ligand, namely the von Hippel-Lindau (VHL) ligand VH032, and is closer to the random region at the 3′ end of the forward primer. This adjustment aims to increase the possibility of forming a more favorable ternary complex conformation than the previous design (such as the aptamer-based targeted protein degradation molecule in Example 1, which used a fixed spacer between the aptamer and the E3 ubiquitin ligase ligand); in addition, to increase the possibility of obtaining aptamers with higher affinity, indole groups were added to the initial library. Therefore, Example 2 provides a screening method for aptamer-based targeted protein degradation molecules based on DNA aptamer-VH032, including the following steps:

[0061] 1. Library pre-enrichment:

[0062] Construct an indole-modified DNA library using solid-phase synthesis and "click chemistry" reactions to increase the nucleic acid structural diversity of the library. The sequence of the library is SEQ ID NO: 9 (see Table 2). The specific process is as follows: Synthesize a 40-nucleotide sequence containing several ethynyl-dU (EdU), and then introduce 3-(2-azidoethyl)-1H-indole into the alkyne-modified oligonucleotide sequence directly on a CPG solid-phase synthesis column through the CuAAC reaction. The 1 mM CuI catalyst solution is freshly prepared by adding 25 μL of freshly prepared 100 mM sodium ascorbate solution to 75 μL of a 1:4 CuSO4 / THPTA solution (final concentrations are 1 mM and 4 mM respectively). The click reaction is carried out using a thermal mixer in a final volume of 100 μL. The specific process of the click reaction is to mix 25 μL of a 500 mM 3-(2-azidoethyl)-1H-indole solution (in DMSO), 10 μL of 10×PBS buffer (pH 7), and 25 μL of the CuAAC catalyst solution with 40 μL of methanol (the synthesis method of 3-(2-azidoethyl)-1H-indole is reported in the prior art literature Hennessy, E.J. et al. (2017). Preparation of highly functionalized 1,5-disubstituted tetrazoles via palladium-catalyzed Suzuki coupling. Tetrahedron. Lett. 58, 1709-1713), and add the resulting mixture to the solid support; the suspension is incubated at 37 °C for 3 hours, and 100 mM sodium ascorbate solution is added every hour to ensure complete conversion; to ensure complete conversion, the click reaction and washing steps are repeated twice; next, synthesize the remaining 16 nucleotides, including one ethynyl-dU. Thus, the initial ssDNA library is obtained.

[0063] Dissolve 1 nmol of the constructed initial ssDNA library in 10 μl of binding buffer 1 (containing 2.5 mM MgCl2, 1 mM CaCl2, 1 mg / ml BSA, 0.1 mg / ml salmon sperm DNA, 0.1% Tween 20, pH 7.4). At 25 °C, mix 1 μg of recombinant BRD4 protein with 1 μl His-Tag magnetic affinity separation beads were incubated in 10 μl of binding buffer 1 for 1 hour to obtain protein beads. Then, the initial ssDNA library treated as above was incubated with the protein beads in 10 μl of binding buffer 1 at 37 °C for 1 hour. The unbound DNA sequences were washed three times with binding buffer 1. Subsequently, the remaining bound ssDNA sequences were eluted by heating in water at 95 °C three times. The eluted sequences were amplified by PCR, and a set of primers used were forward primer -1 (see SEQ ID NO: 10 in Table 2) and reverse primer (see SEQ ID NO: 11 in Table 2), and then digested with λ exonuclease into single-stranded DNA. Subsequently, the ssDNA obtained from the first-round pre-enrichment was used as the new library for the second-round screening, and a new round of pre-enrichment was carried out again according to the same steps. After two rounds of pre-enrichment, the obtained new ssDNA library was used as the library for functional evolution screening.

[0064] 2. Functional evolution screening of aptamer-VH032 chimera:

[0065] FP3Δ5 (i.e., forward primer -2, whose sequence is shown in SEQ ID NO: 12 in Table 2; compared with forward primer -1, forward primer -2 has five fewer bases at its 3′ end) was coupled with MyOne TM carboxylic acid magnetic beads in the same manner as the coupling process described in Example 1. The washed bead particles were incubated with 100 μM FPC 5Δ1 (SEQ ID NO: 13, see Table 2, which was formed by lacking a base A at the 5′ end of the fully complementary sequence of forward primer -1 and was used to introduce EdU at the end of forward primer -1), 10 mM dGTP, 10 mM dCTP, 10 mM EdUTP, and 5 μl of Klenow polymerase at 37 °C for 1 hour. Then, these bead particles were incubated in sodium hydroxide solution to remove the complementary strand. VH032 was then reacted with EdUTP through click chemistry. For emulsion PCR, the oil phase remained the same as the oil phase described in Example 1, while the aqueous phase contained 6 μl of dNTPs (dATP, dGTP, dCTP, EdUTP, 2.5 mM for each nucleotide), 3 μl of 100 μM reverse primer (SEQ ID NO: 11, see Table 2), 4.5 μl of 25 mM MgCl2, 1.5 μl of Easy Taq polymerase, 5 pM of template DNA, 12.5 μl of PEG-8000 (60 mM), and 5 μl of beads. The washed bead particles were incubated with 90% formamide and 10% water at 50 °C for 2 minutes to obtain ssDNA bead particles. Then, in the CuAAc reaction, indole groups were modified onto the library bead particles.

[0066] The obtained microbead particles were resuspended in TMC buffer (5 mM MgCl2, 1 mM CaCl2, 50 mM Tris-HCl, pH 7.4), heated to 95 °C at 1500 rpm for 3 minutes, and then rapidly cooled on ice for 3 minutes. The microbead particles were incubated with the ubiquitination system at 37 °C for 3 hours, which included 100 nM BRD4, 50 nM UBE1, 500 nM UbcH3, 200 nM VHL complex, 500 nM Ub-F, 5 mM ATP, 1 mg / ml BSA, 0.1 mg / ml salmon sperm DNA, 0.1% Tween 20, and 1 μM His-tag peptide. The microbead particles were washed with TMC buffer at 37 °C for 1 minute, and this step was repeated 2 times. Subsequently, the microbead particles and rabbit anti-FLGA antibody were placed in 200 μl of TMC buffer and incubated at 37 °C and 1500 rpm for 1 hour. The microbead particles were washed with binding buffer 2 (the components are shown in Example 1) at 37 °C for 1 minute, and this step was repeated 2 times. Finally, the microbead particles were incubated with goat anti-rabbit antibody AF647 in 200 μl of binding buffer 2 at 37 °C and 1500 rpm for 20 minutes, washed with binding buffer 2 at 37 °C for 1 minute, and this step was repeated 2 times. The microbead particles were resuspended in 4 ml of sorting buffer (containing 5 mM MgCl2, 1 mM CaCl2, 0.1% Tween 20, 50 mM Tris-HCl, pH 7.4). The fluorescence distribution of the microbead particles was monitored, and a population was selected in which approximately 0.4 - 0.5% of the APC channel and 5 - 6% of the FITC channel were present. After collecting the microbead particles with dual fluorescence labeling, the sequences were amplified by PCR to create an enrichment pool for the subsequent round of aptamer chimera microbead particle synthesis. To increase the selection pressure in the second round, the concentration of BRD4 was reduced to 25 nM. For downstream analysis, the samples from pre-enrichment to two rounds of functional selection were supplemented with adapter primers by PCR and then sequenced using the Illumina Novaseq platform.

[0067] Based on the enrichment degree of the sequences in the sequencing results (the specific operation process is shown in the aforementioned sequencing data analysis section), especially the abundance clusters with higher rankings, a nucleic acid aptamer-based targeted protein degradation molecule designated VIA2 was finally selected, and the sequence of its nucleic acid aptamer part is SEQ ID NO: 14 (see Table 2).

[0068] Table 2 Sequences involved in Example 2

[0069]

[0070]

[0071] Example 3: Screening of Targeted Protein Degradation Molecules Based on Bispecific RNA Aptamers

[0072] This example provides a screening of targeted protein degradation molecules based on bispecific RNA aptamers and its screening method, which specifically includes the following steps:

[0073] 1. Library Pre-enrichment:

[0074] The sequence of the initial library is SEQ ID NO: 15 (see Table 3), and this sequence was ordered from Beijing Tsingke Biotechnology Co., Ltd.

[0075] In the first round of screening, the sequences in the library were paired with a reverse primer (SEQ ID NO: 16, see Table 3), and a full-length double-stranded DNA was obtained through an extension experiment using Klenow enzyme (Shanghai Beyotime Biotechnology Co., Ltd.) according to the instructions. After purification, 10 nmol of the dsDNA library was obtained. An in vitro transcription kit (Nanjing Novozymes Biotech Co., Ltd.) was used for in vitro transcription to generate an ssRNA library. After purification, the ssRNA library was placed into binding buffer 3 (2.5 mM MgCl2, 1 mM CaCl2, 1 mg / ml BSA, 0.1 mg / ml yeast tRNA, 0.1% Tween 20, pH 7.4). The subsequent operations were the same as those in Example 1. Reverse transcription and PCR amplification were performed on the obtained single-stranded RNA library. The above steps were repeated for the second round of pre-enrichment.

[0076] 2. Functional Evolution Screening of Bispecific RNA Oligonucleotides for Degrading BRD4:

[0077] When performing subsequent functional screening, instead of using a reverse primer during amplification of RNA, primer 2 with 10 bases extended at the 5' end (see Table 3) was used. The 10 extended bases were used to be captured by complementary base pairs on MyOne TM carboxylic acid magnetic beads, so as to be immobilized on MyOne TM carboxylic acid magnetic beads. The specific process is as follows.

[0078] A capture primer with a length of 10 bases modified with N3 (SEQ ID NO: 17, used for sequence pairing and thus immobilized on magnetic beads) was paired with MyOne in the same manner as in Example 1 TMCoupling of carboxylic acid magnetic beads. Through extensive optimization of the reaction system, emulsion NASBA (eNASBA) met the requirements for the formation of monoclonal bead particles and amplification efficiency. The NASBA buffer (10×) contained 400 mM Tris, 700 mM potassium chloride, 120 mM MgCl2, 5 mM DTT, and 1 mg / mL BSA. In addition, the aqueous phase contained dNTPs (0.25 mM each), NTPs (0.5 mM each), primer 1 (4 μM, SEQ ID NO: 18, see Table 3), primer 2 (4 μM, SEQ ID NO: 19, see Table 3), template (0.5 pM), T7 polymerase (5 U / μL), MLV (1 U / μL), RNase H (0.1 U / μL), and PEG8000 (10 mM), while the oil phase was the same as in Example 1. Subsequent operations, including demulsification, incubation, and sorting, were the same as described in Example 1. To prevent RNA degradation, 1% (v / v) RNase inhibitor was added at each step. The post-treatment after sorting included reverse transcription and PCR amplification according to the kit instructions to complete the first round of screening. The above experiment was repeated to perform the second round of eNASBA, and the BRD4 concentration was reduced from 100 nM to 25 nM during incubation. Then, the samples of pre-enrichment, the first round of eNASBA, and the second round of eNASBA were sequenced using the Illumina Novaseq platform.

[0079] Based on the enrichment degree of the sequences in the sequencing results (see the aforementioned sequencing data analysis section for the specific operation process), especially the top-ranked abundance clusters, a bispecific RNA oligonucleotide-based targeted protein degradation molecule with the code name BRA1 was finally selected, and its sequence was SEQ ID NO: 20 (see Table 3).

[0080] Table 3 Sequences involved in Example 3

[0081]

[0082] Example 4: Testing the cytotoxic effect and anti-tumor activity of the targeted protein degradation molecules in Examples 1 - 3 on tumor cells

[0083] In this embodiment, first, according to the sequencing information of Embodiments 1-3, PA1, PA2, PA4, PA6, VIA2, and BRA1 molecules were respectively prepared for testing: According to the sequencing information of the screening results of Embodiment 1, the corresponding nucleic acid aptamers of PA1, PA2, PA4, and PA6 were synthesized by solid-phase synthesis, and pomalidomide, a ligand of E3 ubiquitin ligase, was modified by "click chemistry" reaction to obtain targeted protein degradation molecules coded as PA1, PA2, PA4, and PA6; According to the sequencing information of the screening results of Embodiment 2, the corresponding nucleic acid aptamer of VIA2 was synthesized by solid-phase synthesis, and VH032, a ligand of E3 ubiquitin ligase, and an indole group were modified by "click chemistry" reaction to obtain a targeted protein degradation molecule coded as VIA2; According to the sequencing information of the screening results of Embodiment 3, a targeted protein degradation molecule based on bispecific RNA oligonucleotide coded as BRA1 was obtained by in vitro transcription. Subsequently, the following tests were carried out:

[0084] (1) Test the degradation effect of four targeted protein degradation molecules, PA1, PA2, PA4, and PA6, on BRD4 protein in HEK293T cells. The specific process was as follows: PA1, PA2, PA4, and PA6 were respectively transfected into HEK293T cells, and the cells were harvested after 24 h for Western Blot analysis. After analysis and comparison, the protein blots of BRD4 protein became lighter for all four targeted protein degradation molecules, PA1, PA2, PA4, and PA6, and showed a concentration dependence ( Figure 1 c), indicating that these four targeted protein degradation molecules all had a degradation effect on BRD4 protein; among them, the protein blot of PA2 was the lightest ( Figure 1 c), indicating that PA2 had the best degradation effect on BRD4. Therefore, in the subsequent steps, PA2, the targeted protein degradation molecule conjugated with pomalidomide, was selected for effect verification.

[0085] (2) Test the cytotoxicity and anti-tumor activity of the obtained PA2, VIA2, and BRA1 on tumor cells. Among them, Hep3B cells or A375 cells used were all cells with high expression of BRD4 protein.

[0086] First, in order to evaluate the ability of the two targeted protein degradation molecules, PA2 and VIA2, constructed in the present invention to degrade BRD4 protein in tumor cells, Hep3B cells were selected for the experiment. Different concentrations of PA2 and VIA2 were respectively transfected into Hep3B cells, and the cells were harvested after 24 h for Western Blot analysis. Moreover, in order to evaluate the anti-tumor activity of PA2 and VIA2, a cell proliferation experiment (CCK8) was carried out in Hep3B cells. After PA2 and VIA2 were transfected into the cells for 24, 48, and 72 hours, the inhibitory proliferation effects of PA2 and VIA2 on Hep3B cells were observed.

[0087] Secondly, to test the broad applicability of the screening method provided by the present invention, another cell line A375 with high expression of BRD4 protein was selected for testing. To evaluate the ability of the bispecific RNA aptamer BRA1 constructed by the present invention to degrade BRD4 protein in tumor cells, BRA1 was transfected into A375 cells by transfection. After 24 h, the cells were harvested for Western Blot analysis. Moreover, to evaluate the anti-tumor activity of BRA1, the effect of BRA1 on A375 cells was detected by cell proliferation assay (CCK8). After BRA1 was transfected into the cells for 12, 24, and 36 hours, the inhibitory effect of BRA1 on the proliferation of A375 cells was observed.

[0088] When conducting the above tests, the cell culture conditions were as follows: HEK-293T (ATCC CRL-3216), Hep3B (ATCC HB-8064), and A375 (ATCC CRL-1619) cells were all cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v / v) fetal bovine serum (FBS), 10 μg / mL streptomycin, and 100 units / mL penicillin at a culture temperature of 37 °C, a CO2 content of 5%, and a humidified incubator.

[0089] When conducting the above tests, the method of Western Blot (immunoblot analysis) was as follows: HEK-293T cells, Hep3B cells, or A375 cells were all seeded in 6-well plates at a density of 2×10 5 cells / well, and the culture medium was 2 ml (DMEM + 10% FBS). After 24 hours, (Polyplus) was used to transfect the aptamer-based degrader into the cells, while Transfect RNA oligonucleotides with the EL transfection reagent (TransGen Biotech). After incubation for 24 hours, collect the cells, wash them with PBS, and lyse them in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF, P0100, Solarbio) and 1× EDTA-free protease inhibitor mixture (B14001, Selleck) at 4°C for 30 minutes. Centrifuge the lysate at 14,000 g for 20 minutes to separate the soluble fraction. Quantify the protein concentration in the clarified lysate using the Pierce BCA Protein Assay Kit according to the manufacturer's protocol. Mix the clarified lysate with loading buffer containing 10 mM dithiothreitol and boil for 10 minutes. Perform gel electrophoresis on the boiled samples using polyacrylamide gels with an equal amount of total protein in each lane. Transfer the proteins to a PVDF membrane. After transfer, block the membrane in blocking buffer and then incubate it with primary antibodies in blocking buffer overnight at 4°C. Use the following primary antibodies for the indicated proteins at the reported dilutions: BRD4 long isoform (Cell Signaling, #13440; 1:1,000), GAPDH (Cell Signaling, #5174; 1:5,000), β-tubulin (Bioss, bsm-33034M; 1:5,000), BRD2 (Cell Signaling, #5848; 1:1,000), BRD3 (Abcam, ab50818; 1:500), c-Myc (Cell Signaling, #9402; 1:1,000), IRAK4 (Abcam, ab32511; 1:1,000). After overnight incubation, wash the membrane three times with TBST buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Tween 20, pH 7.4). Incubate the membrane with 1:10,000 goat anti-rabbit 680RD (LI-COR, 926-68071) and / or goat anti-mouse 800RD (LI-COR, 926-68070) in blocking buffer for 1 hour at room temperature. Wash the membrane three times with TBST buffer and visualize it on an Odyssey imaging system (LI-COR). Then normalize the values. In GraphPad Prism (v9.5.1), calculate the DC 50 values. Analyze significant differences by t-test or one-way ANOVA in Prism.

[0090] When performing the above tests, the method for cell viability assay used was as follows: Seed Hep3B cells or A375 cells into 96-well microplates at a density of 5×10 3Cells. After 24 hours, the cells were treated with the corresponding targeted protein degradation molecules three times, for 1 day, 2 days, and 3 days respectively. Cell viability was evaluated using the WST-8 reagent. After incubation at 37 °C for 3 hours, absorbance signals were obtained at 450 nm with a reference wavelength of 690 nm. These values were then normalized. In GraphPad Prism (v9.5.1), the IC 50 values were calculated using the log (inhibitor) vs. normalized response - variable slope model. Significant differences were analyzed by t-test or one-way ANOVA in Prism.

[0091] The results of the cytotoxicity and anti-tumor activity tests of PA2 and VIA2 against the tumor cell Hep3B are as Figure 1 shown. It can be seen that both PA2 and VIA2 showed good degradation effects on the BRD4 protein and showed time and concentration dependence ( Figure 1 a), indicating that the degradation of the BRD4 protein caused by PA2 and VIA2 occurred through the ubiquitin-proteasome system; moreover, it can be seen that they had anti-tumor cell proliferation activity ( Figure 1 b).

[0092] The results of the cytotoxicity and anti-tumor activity tests of BRA1 against the tumor cell A375 are as Figure 2 shown. It can be seen that BRA1 showed good degradation effects on the BRD4 protein and showed time and concentration dependence ( Figure 2 a), and it also had anti-tumor cell proliferation activity ( Figure 2 b).

[0093] Example 5: Testing the in vivo anti-tumor activity of the targeted protein degradation molecules of Examples 1 - 3

[0094] 5.1 Evaluating the in vivo anti-tumor activity of PA2 and VIA2

[0095] A subcutaneous tumor model of Hep3B cells was used to evaluate the inhibitory effects of the targeted protein degradation molecules PA2 and VIA2 obtained in Examples 1 - 2 on the growth of subcutaneously inoculated tumors in nude mice.

[0096] To establish xenograft tumors, six-week-old female nude mice were obtained and cultured. Hep3B cells (1×10 6 cells per mouse) were inoculated subcutaneously into the right ventral side. When the tumor volume reached 100 mm 3When the tumor volume reached a certain value, the mice were randomly divided into three groups (6 mice in each group), and the drugs were administered according to the following regimens: The first group was administered with the vehicle (PEI) (Vehicle group), the second group was administered with VIA2 (6 mg / kg) (VIA2 group), and the third group was administered with PA2 (6 mg / kg, mixed with PEI according to the instruction manual) (PA2 group). The administration interval was once every two days. For the specific regimen, see Figure 3 a. The body weight, tumor length (L) and width (W) of the mice were measured every 2 days, and the tumor volume was calculated using the formula (L×W 2 ) / 2. After the administration was completed, the tumors were excised, photographed and weighed.

[0097] The results are shown in Figure 3 b-d. It can be seen that compared with the control group receiving the empty vehicle (Vehicle group), the tumor growth in the treatment groups receiving PA2 and VIA2 was significantly slower, and the tumor volume and weight in the treatment groups were also significantly lower. In addition, during the treatment period, no significant change in the body weight of the mice was observed, indicating that PA2 and VIA2 had no obvious side effects.

[0098] 5.2 Evaluation of the in vivo anti-tumor activity of BRA1

[0099] A subcutaneous tumor model of melanoma A375 cells (another type of tumor cell with high BRD4 expression) was used to evaluate the inhibitory effect of the dual-specific RNA oligonucleotide-based targeted protein degradation molecule BRA1 obtained in Example 3 on the growth of subcutaneously inoculated tumors in nude mice.

[0100] To establish xenograft tumors, six-week-old female nude mice were obtained and cultured. A375 cells (1×10 6 cells per mouse) were inoculated subcutaneously on the right side. When the tumor volume reached 100 mm 3 , the mice were randomly divided into three groups (6 mice in each group) according to the tumor volume, and the drugs were administered according to the following regimens: The first group was administered with the vehicle (Vehicle group), the second group was administered with BRA1 (3 mg / kg, mixed according to the instruction ), and the third group was administered with BRA1 (6 mg / kg, mixed according to the instruction ). The administration interval was once every two days. For the specific regimen, see Figure 3 e. The body weight, tumor length (L) and width (W) of the mice were measured every 2 days, and the tumor volume was calculated using the formula (L×W 2 ) / 2. After the administration was completed, the tumors were excised, photographed and weighed.

[0101] The results are shown in Figure 3As shown in f-h. It can be seen that in the subcutaneous tumor model of A375 melanoma cells, intratumoral injection of BRA1 resulted in significant tumor suppression, with both the volume and weight of the tumors decreasing significantly, and no significant change in the body weight of the mice, indicating that BRA1 has no obvious side effects.

[0102] From the above examples, it can be seen that the target protein degradation molecule provided by the present invention can achieve the degradation of the target protein BRD4 in different cells, showing time and concentration dependence, and having good cytotoxic effects on liver cancer cells, melanoma cells, etc., and also having good effects of inhibiting the proliferation of tumors subcutaneously inoculated in nude mice.

[0103] Example 6: Screening and activity verification of a target protein degradation molecule based on DNA aptamer-VH032 against IRAK4

[0104] To verify whether the method described in the present invention can be widely applied, according to the method described in Example 2, only the target protein was replaced from BRD4 to IRAK4, and the other conditions remained unchanged, and the screening of the target protein degradation molecule based on DNA aptamer-VH032 was carried out. Finally, the target protein degradation molecule SHA1 was obtained, and the sequence of its aptamer part is SEQ ID NO: 21 (see Table 4).

[0105] Table 4 Sequences involved in Example 6

[0106]

[0107] For SHA1, the method of testing VIA2 described in Example 4 was adopted, only the test cells were changed from Hep3B cells to HEK293T cells, and the other conditions remained unchanged.

[0108] The results are as Figure 4 shown. It can be seen that the degradation of IRAK4 mediated by SHA1 occurred in a concentration- and time-dependent manner in HEK293T cells.

[0109] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principles and spirits of the present invention, various changes, modifications, substitutions, and variations of these embodiments still fall within the protection scope of the present invention.

Claims

1. A targeted protein degradation molecule based on nucleic acid aptamers, wherein the protein is BRD4, the targeted protein degradation molecule based on nucleic acid aptamers is formed by connecting a nucleic acid aptamer with an E3 ubiquitin ligase ligand, and the sequence of the nucleic acid aptamer is any one or more selected from SEQ ID NO: 5-8 or SEQ ID NO:

14. 2 . The nucleic acid aptamer-based targeted protein degradation molecule according to claim 1 , wherein the E3 ubiquitin ligase ligand is any type of ligand that can recruit and bind to the E3 ubiquitin ligase.

3. According to the nucleic acid aptamer-based targeted protein degradation molecule of claim 2, the E3 ubiquitin ligase ligand is selected from CRBN ligand, VHL ligand, IAP ligand, MDM2 ligand, DCAF ligand, RNF ligand, AhR ligand, FEM1B ligand, KEAP1 ligand.

4. A nucleic acid aptamer-based targeted protein degradation molecule, wherein the protein is BRD4, and the sequence of the nucleic acid aptamer-based targeted protein degradation molecule is SEQ ID NO:

20.

5. Use of the nucleic acid aptamer-based targeted protein degradation molecule according to claim 1 or 4 in the preparation of a drug for treating a disease, wherein the disease is a disease caused by overexpression of BRD4 protein.

6. The use according to claim 5, wherein the disease caused by overexpression of BRD4 protein is a malignant tumor.

7. The use according to claim 6, wherein The malignant tumors are liver cancer, breast cancer, non-small cell lung cancer, melanoma, and prostate cancer.

8. A targeted protein degradation molecule based on nucleic acid aptamers, wherein the protein is IRAK4, the targeted protein degradation molecule based on nucleic acid aptamers is formed by connecting a nucleic acid aptamer with an E3 ubiquitin ligase ligand, and the sequence of the nucleic acid aptamer is SEQ ID NO:

21. 9 . Use of the nucleic acid aptamer-based targeted protein degradation molecule according to claim 8 in the preparation of a drug for treating a disease, wherein the disease is a disease caused by overexpression of IRAK4 protein.

10. The use according to claim 9, wherein the disease caused by overexpression of IRAK4 protein is solid tumor, blood tumor, myelodysplastic syndrome, diabetic nephropathy and nerve damage.

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