Protein targeted degradation agent based on circular nucleic acid

Abstract

The invention discloses a protein targeted degradation agent based on circular nucleic acid, and belongs to the technical field of biology. The protein targeted degradation agent not only contains RNA (Ribonucleic Acid) capable of recruiting E3 ubiquitin ligase, but also contains a nucleic acid aptamer component of a targeted transferrin receptor and / or Tau protein, so that the protein degradation agent can specifically degrade the Tau protein in positive cells of the transferrin receptor; therefore, normal physiological activities of other transferrin receptor negative cells are not affected, and a new treatment strategy is provided for treatment of Alzheimer's disease. In addition, the invention also discloses a preparation method of the protein targeted degradation agent, which comprises the following steps: covalently linking an N3 group modified at the 3'end of the nucleic acid aptamer and a DBCO group modified at the 5 'end of RNA, simultaneously linking a phosphate group positioned at the 5' end of the nucleic acid aptamer and a hydroxyl group positioned at the 3 'end of RNA through T4RNA ligase, and purifying the product through T5 exonuclease and RNase R.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a protein targeting degradation agent based on circular nucleic acid. Background Art

[0002] Protein homeostasis involves the fine regulation of multiple links such as protein synthesis, folding, modification, transport, aggregation and degradation, and is the key to maintaining the normal physiological function of the body, and is closely related to the occurrence and development of diseases. Among them, the ubiquitin-proteasome system is an important way for the body to maintain protein homeostasis, that is, through the synergistic action of E1 activating enzymes, E2 conjugating enzymes and E3 ligases, the ubiquitination modification of target proteins is achieved, and then the target proteins are degraded by proteasomes.

[0003] Protein degradation targeting chimeras (PROTAC) consist of three parts: target protein ligand, E3 ligase ligand and linker. In the cell, PROTAC molecules shorten the distance between the target protein and the E3 ligase, promote the ubiquitination modification of the target protein by the E3 ligase, and make the target protein recognized and degraded by the proteasome. So far, small molecule-based PROTACs have been developed one after another, but small molecule PROTACs lack cell selectivity and have off-target risks; at the same time, it is difficult to develop corresponding small molecule ligands for some proteins, resulting in the limitation of small molecule PROTAC degradable targets.

[0004] Aptamers have application potential in PROTAC. Aptamers are a class of single-stranded DNA / RNA sequences that form a unique three-dimensional structure by folding and bind to the target with high specificity and high affinity. They are known as "chemical antibodies". The application of aptamers has spread to many biomedical fields, such as biomarker detection, diagnosis, imaging and precision targeted therapy. On the one hand, aptamers as targeting elements can promote the enrichment of PROTAC in lesions and reduce the risk of off-target; on the other hand, compared with small molecule PROTACs, aptamers can target nucleic acid binding proteins such as G4 binding proteins, RNA binding proteins and transcription factors, which is expected to broaden the range of PROTAC degradable targets; on the other hand, related studies on long non-coding RNA and circular RNA have confirmed the existence of natural RNA aptamers that can be used as E3 ligase ligands, which are expected to be applied to PROTAC.

[0005] Alzheimer's disease (AD) is a common neurodegenerative disease, in which neurofibrillary tangles formed by abnormal aggregation of Tau protein are one of the main pathological features of AD. The development of PROTAC molecules or nucleic acid aptamers targeting Tau protein can achieve the purpose of effectively degrading Tau protein or inhibiting Tau protein aggregation, thereby controlling the progression of AD.

[0006] In summary, there is no PROTAC molecule that can both improve cell selectivity and efficiently mediate the degradation of Tau protein by the ubiquitin-proteasome system. Summary of the invention

[0007] The present invention discloses a protein-targeted degrader based on circular nucleic acid, which belongs to the field of biotechnology. The protein-targeted degrader contains both RNA capable of recruiting E3 ubiquitin ligase and nucleic acid aptamer components targeting transferrin receptor (TfR) and / or Tau protein. These components enable the protein degrader to specifically degrade Tau protein in TfR-positive cells, thereby not affecting the normal physiological activities of other negative cells, and providing a theoretical basis for the treatment of Alzheimer's disease. In addition, the present invention also discloses a method for preparing the protein-targeted degrader: the N3 group modified at the 3' end of the nucleic acid aptamer is covalently linked to the DBCO group modified at the 5' end of the RNA; at the same time, the phosphate group at the 5' end of the nucleic acid aptamer and the hydroxyl group at the 3' end of the RNA are linked by T4 RNA ligase, and the product is purified by T5 exonuclease and RNase R. Furthermore, the present invention also provides a strategy for degrading Tau protein based on circular nucleic acid and cell targeting (see for details). Figure 1 ): Aptamer 1 targets TfR to achieve cell selectivity, and aptamer 2 specifically binds to Tau protein; RNA is used to recruit E3 ubiquitin ligase; the above three components are assembled into a circular nucleic acid, so that under the TfR-positive cell targeting conferred by aptamer 1, aptamer 2 and the RNA shorten the spatial distance between Tau protein and E3 ubiquitin ligase, thereby achieving cell-selective targeted degradation of Tau protein.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] On the one hand, the present invention provides a protein targeted degrader based on circular nucleic acid, wherein the circular nucleic acid comprises an RNA sequence capable of recruiting E3 ubiquitin ligase and at least one nucleic acid aptamer, wherein the RNA sequence and the nucleic acid aptamer are chemically linked together.

[0010] PROTAC molecules consist of three parts: target protein ligand, E3 ubiquitin ligase ligand and linker. In cells, PROTAC molecules shorten the distance between target protein and E3 ubiquitin ligase, promote E3 ubiquitin ligase to ubiquitinate target protein, and make target protein recognized and degraded by proteasome. So far, the E3 ubiquitin ligase ligand and target protein ligand are mostly small molecules, but the development of small molecule ligands is difficult and the cycle is long, and some E3 ligases and target proteins lack clear small molecule binding pockets, making it difficult to develop small molecule ligands. Some studies use peptides or proteins to target E3 ligases and target proteins, but the preparation process of peptides or proteins is complicated and the production cost is high; and their molecular weight is large, which is easily recognized by the body as foreign antigens, and then causes immune response. These shortcomings limit the application prospects of PROTAC molecules in clinical practice. And nucleic acid aptamers can make up for the above defects for the following reasons: nucleic acid aptamers are a DNA / RNA oligonucleotide sequence with small molecular weight, simple synthesis, easy and precise structure programming, and good biocompatibility. The spatial structure of nucleic acid is highly variable, and can form secondary structures such as stem-loop structure, pseudo-node structure, and quadruplex structure, which makes nucleic acid aptamers have the potential to be used as various protein ligands. At the same time, some non-coding RNA sequences naturally exist in cells that can specifically bind to E3 ubiquitin ligases, which are natural E3 ligase nucleic acid aptamers, so the present invention selects RNA as a ligand of E3 ubiquitin ligases, and selects nucleic acid aptamers as target protein ligands. In theory, proteasomes can recognize and degrade proteins modified by polyubiquitination, which means that it is only necessary to screen nucleic acid aptamers for degradation objects, and then the nucleic acid aptamers are chemically linked to the RNA sequence that can recruit E3 ubiquitin ligases provided by the present invention, and the chemical substance connected is the protein targeted degradation agent, which can degrade any one or more proteins in the cell.

[0011] Furthermore, the present invention provides a circular nucleic acid sequence design capable of cyclization, wherein the nucleic acid aptamer sequences are connected by a stem-loop structure to make the sequence secondary structure more stable. The structure of the nucleic acid sequence is also predicted using the NUPACK website, and the prediction parameters are set as follows: the temperature is 25°C, the nucleic acid sequence is as shown in SEQ ID NO: 1-11, and the nucleic acid concentration is 1.0 μM.

[0012] Furthermore, the E3 ubiquitin ligase includes but is not limited to MDM2 protein, MEX3A protein, MEX3C protein, VHL protein, CRBN protein, Dzip3 protein, and cIAP protein.

[0013] In this example, corresponding RNA ligands (SEQ ID NO: 1 to SEQ ID NO: 3) were designed for MDM2 protein, MEX3A protein and MEX3C protein, respectively.

[0014] Furthermore, the RNA sequence for recruiting E3 ubiquitin ligase has more than 80% homology with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence listing.

[0015] Furthermore, the E3 ubiquitin ligase is MDM2 protein.

[0016] In some specific embodiments, the RNA sequence for recruiting E3 ubiquitin ligase has more than 80% homology with the sequence shown in SEQ ID NO: 1 in the sequence listing.

[0017] The present invention extracts a portion of the sequence from a longer RNA sequence known to recruit MDM2 protein, the extracted sequence being SEQ ID NO: 1, and finds that circular nucleic acids (SEQID NO: 20 and SEQ ID NO: 27-28) comprising the sequence shown in the sequence table SEQ ID NO: 1 can efficiently degrade the target protein. In some specific embodiments, the target protein is Tau protein.

[0018] Furthermore, the nucleic acid aptamer targets intracellular proteins and / or membrane proteins.

[0019] It should be understood that the intracellular protein is the degradation target of the circular nucleic acid / protein targeted degrader, while the nucleic acid aptamer targeting the membrane protein plays the role of locating the target cell, making the circular nucleic acid / protein targeted degrader cell-selective.

[0020] Furthermore, the intracellular proteins include but are not limited to Tau protein, p65 protein, Ras protein, and MEK protein; the membrane proteins include but are not limited to TfR protein, PTK7 protein, c-MET protein, and DEC205 protein.

[0021] Furthermore, the nucleic acid aptamer targets Tau protein (intracellular protein) and / or TfR (membrane protein).

[0022] In some specific embodiments, the nucleic acid aptamer only targets Tau protein, and the nucleic acid aptamer comprises the sequence shown in SEQ ID NO:5 in the sequence table, and the corresponding circular nucleic acid is circTau-MDM2 (SEQ ID NO:20).

[0023] In some other specific embodiments, the nucleic acid aptamer can target both Tau protein and TfR protein, and the nucleic acid aptamer comprises the sequence shown in SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:10 or SEQ ID NO:11 in the sequence list, and the corresponding circular nucleic acids are circTfR-Tau-MDM2 (SEQ ID NO:27), circTau-TfR-MDM2 (SEQ ID NO:28), circF-TfR-Tau-MDM2 (SEQ ID NO:25) and circF-Tau-TfR-MDM2 (SEQID NO:26). Among them, the binding efficiency of circTfR-Tau-MDM2 circular nucleic acid to target cells and the effect of degrading target protein are better than those of circTau-TfR-MDM2, so circTfR-Tau-MDM2 is more preferred; the concentration of circTfR-Tau-MDM2 is 0-500nM, preferably 400-500nM.

[0024] Furthermore, the RNA sequence and / or the nucleic acid aptamer carries any one of the following groups: hydroxyl, phosphate, carboxylic acid, amino, sulfhydryl, maleimide, N-hydroxysuccinimide, alkyne, and azide.

[0025] The RNA sequence and the nucleic acid aptamer are connected together by chemical bonds, and the type of chemical bonds is determined by the groups carried by the RNA sequence and the nucleic acid aptamer. In some specific embodiments, the 5' end of the RNA carries an alkyne (DBCO) group, and the 3' end carries a hydroxyl (-OH); and the 5' end of the nucleic acid aptamer carries a phosphate group (-Pi), and the 3' end carries an azide group (-N3).

[0026] In another aspect, the present invention provides a method for preparing the above-mentioned protein targeted degradation agent, the method comprising the following steps:

[0027] A. Connect the N3 group on the 3' end of the aptamer and the DBCO group on the 5' end of the RNA sequence by covalent bonding;

[0028] B. Using T4 RNA ligase to connect the phosphate group on the 5' end of the nucleic acid aptamer to the hydroxyl group on the 3' end of the RNA sequence to prepare a crude protein targeted degrader;

[0029] C. Purify the crude protein degrader described in step B using T5 exonuclease and RNase R enzymes; it should be understood that the two enzymes added during purification will only degrade uncircularized nucleic acids (including auxiliary chains).

[0030] When the protein-targeted degrading agent comprises a circular nucleic acid RNA capable of recruiting MEX3A protein, an auxiliary chain is further added in step B, and the auxiliary chain comprises a sequence as shown in SEQ ID NO: 13 in the sequence list. In some specific embodiments, when the protein-targeted degrading agent is a circular nucleic acid (circTau-MEX3A, SEQ ID NO: 22) that can both target Tau protein and recruit MEX3A protein, an auxiliary chain is further added in step B, and the auxiliary chain comprises a sequence as shown in SEQ ID NO: 13 in the sequence list.

[0031] When the protein-targeted degrading agent comprises RNA that can recruit MDM2 protein, no auxiliary chain needs to be added in step B. In some specific embodiments, when the protein-targeted degrading agent also comprises a nucleic acid aptamer targeting a specific protein (preferably Tau protein), no auxiliary chain needs to be added; in other specific embodiments, when the protein-targeted degrading agent also comprises a nucleic acid aptamer targeting two or more specific proteins (preferably Tau protein and TfR), no auxiliary chain needs to be added, but a stem-loop structure needs to be introduced when designing the nucleic acid aptamer.

[0032] The auxiliary chain (also called complementary bridging fragment) is complementary paired with the 5' end of the aptamer sequence and the 3' end sequence of the RNA, respectively, and the distance between the 5' end phosphate group of the aptamer and the 3' end hydroxyl group of the RNA is shortened, thereby promoting the connection of the T4 RNA ligase to form a phosphodiester bond. In terms of sequence design, the auxiliary chain length is usually around 20nt, of which 10nt is complementary paired with the fragment at the end of the 3' end of the RNA, and 10nt is complementary paired with the fragment at the beginning of the 5' end of the aptamer. According to different cyclization sequence designs, the length of the auxiliary chain should be flexibly adjusted. The use of auxiliary chains is an alternative means, and auxiliary chains are usually used when cyclization cannot be achieved without auxiliary chains.

[0033] Furthermore, the molar ratio of the N3 group on the 3' end of the nucleic acid aptamer to the DBCO group on the 5' end of the RNA sequence is 1:1, and the nucleic acid aptamer and RNA are reacted at -40°C for 1 hour.

[0034] Furthermore, the molar ratio of the phosphate group on the 5' end of the nucleic acid aptamer to the 3' hydroxyl group of the RNA sequence is 1:1.

[0035] More specifically, the present invention provides a method for synthesizing circular nucleic acid without auxiliary chain, comprising the following steps:

[0036] (1) Covalent linkage of the N3 group at the 3' end of the aptamer and the DBCO group at the 5' end of the RNA sequence: 5 μL of the aptamer (40 μM) with N3 modification at the 3' end (its nucleic acid sequence is such as SEQ ID NO: 4-9) and 5 μL of the RNA (40 μM) with DBCO modification at the 5' end (its nucleic acid sequence is such as SEQ ID NO: 1) were mixed; the reaction system was reacted at -40°C for 1 hour, then placed on ice for 10 minutes, and placed at 4°C for 5 minutes to thaw the system;

[0037] (2) The phosphate group at the 5' end of the aptamer and the hydroxyl group at the 3' end of the RNA sequence were connected by T4 RNA ligase: 2 μL of the product in (1), 2 μL of 10×T4 RNA Ligase reaction buffer, 4 μL of PEG8000 (50%) and 10.5 μL of DEPC water were mixed, heated at 95°C for 5 min, cooled on ice for 10 min, and then 0.5 μL of ATP (100 mM) and 1 μL of T4 RNA ligase were added. The above system was left to react at room temperature for 12 h.

[0038] (3) Purification of the cyclized product: 45 μL of the product in (2), 5 μL of 10×NEBuffer4, 1 μL of T5 exonuclease and 0.5 μL of RNase R were mixed, incubated at 37°C for 30 min, and then heated at 70°C for 10 min. After the reaction was completed, the product was purified using RNACleanup Kit. The synthesized product was characterized by 10% urea denaturing polyacrylamide gel electrophoresis.

[0039] The present invention provides a method for synthesizing a circular nucleic acid requiring an auxiliary chain, comprising the following steps:

[0040] (1) Covalent linkage of the N3 group at the 3' end of the aptamer and the DBCO group at the 5' end of the RNA sequence: 5 μL of the aptamer (40 μM) with N3 modification at the 3' end (its nucleic acid sequence is such as SEQ ID NO: 4-9) and 5 μL of the RNA (40 μM) with DBCO modification at the 5' end (its nucleic acid sequence is such as SEQ ID NO: 2-3) were mixed; the reaction system was reacted at -40°C for 1 h, then placed on ice for 10 min, and placed at 4°C for 5 min to thaw the system.

[0041] (2) The phosphate group at the 5' end of the aptamer and the hydroxyl group at the 3' end of the RNA sequence were connected by T4 RNA ligase in the presence of an auxiliary chain: 2 μL of the product in (1), 2 μL of 10×T4 RNA Ligase reaction buffer, 4 μL of PEG8000 (50%), 1 μL of the auxiliary sequence (40 μM) (whose nucleic acid sequence is shown in SEQ ID NO: 13-18) and 9.5 μL of DEPC water were mixed, heated at 95°C for 5 min, cooled on ice for 10 min, and then 0.5 μL of ATP (100 mM) and 1 μL of T4 RNA ligase were added, and the above system was left to react at room temperature for 12 h.

[0042] (3) Purification of the cyclized product: 45 μL of the product in (2), 5 μL of 10×NEBuffer4, 1 μL of T5 exonuclease and 0.5 μL of RNase R were mixed, incubated at 37°C for 30 min, and then heated at 70°C for 10 min. After the reaction was completed, the product was purified using RNACleanup Kit. The synthesized product was characterized by 10% urea denaturing polyacrylamide gel electrophoresis.

[0043] On the other hand, the present invention provides a use of a circular nucleic acid for preparing a reagent for improving the degradation efficiency of Tau protein, wherein the circular nucleic acid comprises an RNA sequence capable of recruiting E3 ubiquitin enzyme.

[0044] It should be understood that the circular nucleic acid is the above-mentioned protein targeted degradation agent.

[0045] Furthermore, the circular nucleic acid comprises any one of the sequences shown in SEQ ID NO: 19 to SEQ ID NO: 28 in the sequence listing.

[0046] In some preferred embodiments, the circular nucleic acid comprises the sequence shown in SEQ ID NO:27 or SEQ ID NO:28 in the sequence listing, more preferably, SEQ ID NO:27.

[0047] On the other hand, the present invention provides a use of RNA for preparing a reagent for improving protein degradation efficiency, wherein the sequence of the RNA has more than 80% homology with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence listing, more preferably, SEQ ID NO: 1.

[0048] On the other hand, the present invention provides a use of a nucleic acid sequence for preparing a reagent for improving protein degradation efficiency, wherein the nucleic acid sequence has more than 80% homology with the sequence shown in SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:10 or SEQ ID NO:11 in the sequence listing, more preferably, SEQ ID NO:6 or SEQ ID NO:7, and most preferably, SEQ ID NO:6.

[0049] The beneficial effects of the present invention include:

[0050] 1. The present invention provides a method for preparing a circular nucleic acid and a feasible sequence design, wherein the stem-loop structure of the nucleic acid sequence is conducive to cyclization;

[0051] 2. The present invention provides a variety of circular nucleic acid synthesis steps, of which 4 circular nucleic acids were successfully synthesized; although the synthesis of 3 nucleic acids failed, it also provides experience that can be used as a reference for professionals in the same field;

[0052] 3. The present invention verified by Western blotting that the circular nucleic acid circTau-MDM2 can degrade endogenous Tau protein in HT22 mouse hippocampal neuron cells 24 hours after being transfected with Lipofectamine3000;

[0053] 4. The circular nucleic acids (circTfR-Tau-MDM2 and circTau-TfR-MDM2) containing nucleic acid aptamers targeting TfR prepared by the present invention were verified by flow cytometry to specifically bind to TfR-positive cells (bEnd.3 cells and HT22 cells) and almost not bind to negative cells, and the binding affinity was better than that of linear nucleic acids (whose sequences are SEQ ID NOs: 8 to 9), among which circTfR-Tau-MDM2 had a better effect;

[0054] 4. circTau-MDM2, circTfR-Tau-MDM2 and circTau-TfR-MDM2 prepared by the circular nucleic acid preparation method provided by the present invention have been confirmed to be able to be used as PROTAC molecules to degrade endogenous Tau protein in cells. At the same time, circTfR-Tau-MDM2 and circTau-TfR-MDM2 contain nucleic acid aptamer sequences targeting TfR, which can achieve high-affinity binding to TfR-positive cells, among which circTfR-Tau-MDM2 has a better effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0056] Figure 1 : Schematic diagram of the circular nucleic acid structure provided by the present invention and its working principle;

[0057] Figure 2 :Schematic diagram of the synthesis of circular nucleic acids circp65-MDM2 and circTau-MDM2 ( Figure 2 A and Figure 2 D) Secondary structure prediction diagram ( Figure 2 B and Figure 2 E) and urea polyacrylamide gel electrophoresis results ( Figure 2 C and Figure 2 F); among which, Figure 2 C and Figure 2 The black frame in F is the target band;

[0058] Figure 3 :Schematic diagram of the synthesis of circular nucleic acids circp65-MEX3A and circTau-MEX3A ( Figure 3 A and Figure 3 D) Secondary structure prediction diagram ( Figure 3 B and Figure 3 E) and urea polyacrylamide gel electrophoresis results ( Figure 3 C and Figure 3 F); among which, Figure 3 C and Figure 3 The black frame in F is the target band;

[0059] Figure 4 :Schematic diagram of the synthesis of circular nucleic acids circp65-MEX3C and circTau-MEX3C ( Figure 4 A and Figure 4 D) Secondary structure prediction diagram ( Figure 4 B and Figure 4 E) and urea polyacrylamide gel electrophoresis results ( Figure 4 C and Figure 4 F);

[0060] Figure 5 :Schematic diagram of the synthesis of circular nucleic acids circF-TfR-Tau-MDM2 and circF-Tau-TfR-MDM2 lacking a stem-loop structure ( Figure 5 A and Figure 5 D) Secondary structure prediction diagram ( Figure 5 B and Figure 5 E) and urea polyacrylamide gel electrophoresis results ( Figure 5 C and Figure 5 F); among which, Figure 5 A and Figure 5 The dark green frame in the D structure diagram is the nucleic acid sequence targeting the TfR protein, and the dark purple frame is the nucleic acid sequence targeting the Tau protein;

[0061] Figure 6 :Schematic diagram of the synthesis of circular nucleic acids circTfR-Tau-MDM2 and circTau-TfR-MDM2 with stem-loop structure ( Figure 6 A and Figure 6 D) Secondary structure prediction diagram ( Figure 6 B and Figure 6 E) and urea polyacrylamide gel electrophoresis results ( Figure 6 C and Figure 6 F); among which, Figure 6 A and Figure 6 The light green frame in the D structure diagram is the nucleic acid sequence targeting the TfR protein, and the light pink frame is the nucleic acid sequence targeting the Tau protein; Figure 6 C and Figure 6 The black frame in F is the target band;

[0062] Figure 7 :Flow cytometry was used to analyze the binding of circular nucleic acids targeting TfR protein to bEnd.3 cells, HT22 cells and HEK293T cells; the numbers in the figure are the mean fluorescence intensity;

[0063] Figure 8 : Western blotting was used to analyze the effect of circular nucleic acids on the degradation of Tau protein in HT22 cells; Figure 8 A shows the degradation of Tau protein 24 hours after circTau-MDM2 transfection in HT22 cells. Figure 8 B shows the degradation of Tau protein 24 hours after circTfR-Tau-MDM2 transfection in HT22 cells. Figure 8 C is the degradation of Tau protein after circTfR-Tau-MDM2 was incubated with HT22 cells for 24 h. Figure 8 D shows the degradation of Tau protein after circTau-TfR-MDM2 was incubated with HT22 cells for 24 h. The numbers in the figure are the relative expression levels of Tau protein, which are used to quantify the degradation of Tau protein. DETAILED DESCRIPTION

[0064] The present invention is further elaborated in detail below in conjunction with the accompanying drawings and specific embodiments of the specification. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0065] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. It should be noted that in the exploration of the circular nucleic acid synthesis method (Examples 1 to 3), the p65 nucleic acid aptamer sequence was used for cyclization in order to verify the versatility of the synthesis method. In addition, the circular nucleic acid synthesized and purified and tested by urea polyacrylamide gel electrophoresis can be directly used in subsequent experiments (such as transfection / incubation of cells); the nucleic acid sequences used in the present invention are all synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the specific sequences are shown in Table 1; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels, and the specific reagents and consumables are shown in Table 2.

[0066] Table 1 Nucleotide sequences involved in the present invention

[0067]

[0068]

[0069] Note: The content in brackets in the sequence is the modified group on the sequence; since circular nucleic acids have no 5' end and 3' end, no additional annotation is made

[0070] Table 2 Reagents and consumables involved in the present invention

[0071]

[0072]

[0073] Example 1: Preparation of circular nucleic acids circp65-MDM2 and circTau-MDM2

[0074] This example provides a method for preparing circular nucleic acids circp65-MDM2 (SEQ ID NO: 19) / circTau-MDM2 (SEQ ID NO: 20) that simultaneously target p65 (also known as RelA, a subunit of the NF-κB complex, which acts as a transcription factor to regulate the expression of inflammatory and immune-related genes in cells, NCBI Gene ID: 5970) / Tau and MDM2 (an E3 ubiquitin ligase that participates in the regulation of the tumor suppressor gene p53 and is an E3 ligase target commonly used in PROTAC molecule design, NCBI Gene ID: 4193). The synthetic schematic diagrams of the circular nucleic acids circp65-MDM2 and circTau-MDM2 are shown in FIG. Figure 2 A and Figure 2 D. As can be seen from the above structural diagram, circp65-MDM2 / circTau-MDM2 consists of a nucleic acid aptamer (SEQ ID NO: 4 to SEQ ID NO: 5) that specifically targets p65 / Tau and an RNA (SEQ ID NO: 1) that can recruit MDM2 protein. The nucleic acid aptamer is connected to the RNA through an N3-DBCO bond and a phosphodiester bond to form a nucleic acid with a ring structure. The secondary structure diagrams predicted by the NUPACK website (https: / / www.nupack.org / ) are shown in Figure 2 B and Figure 2 E, Figure 2 B and Figure 2 E are the predicted structures of the two nucleic acids involved in the cyclization, for example Figure 2 B / Figure 2 The upper half of the figure is the sequence structure of DNA targeting Tau / p65, and the lower half of the figure is the sequence structure of RNA targeting MDM2). From the prediction results, it can be seen that the free energy of the above nucleic acid sequences is less than -10 kcal / mol. The lower free energy means that the above nucleic acid sequences can form a more stable structure. The preparation method of the circular nucleic acid is as follows:

[0075] 1.1 5 μL 40 μM P-p65-N3 (i.e., the above-mentioned nucleic acid aptamer specifically targeting p65 protein, whose sequence is shown in SEQ ID NO:4) or 5 μL 40 μM P-Tau-N3 (i.e., the above-mentioned nucleic acid aptamer specifically targeting Tau protein, whose sequence is shown in SEQ ID NO:5) and 5 μL 40 μM DBCO-MDM2-OH (i.e., the above-mentioned RNA capable of recruiting MDM2 protein, whose nucleic acid sequence is shown in SEQ ID NO:1) were mixed evenly, and reacted at -40°C for 1 hour to obtain reaction solution 1.1 (Note: the specific structure of the reaction solution is related to the sequence of the reactants, that is, it depends on whether P-p65-N3 (SEQ ID NO:4) or P-Tau-N3 (SEQ ID NO:5) is selected), and then the reaction solution 1.1 was placed on ice for 10 minutes and at 4°C for 5 minutes to melt the reaction solution 1.1;

[0076] 1.2 Take 2 μL of reaction solution 1.1, 2 μL 10×T4 RNA Ligase reaction buffer, 4 μL 50% PEG8000 and 10.5 μL DEPC water, mix, heat at 95°C for 5 min, cool on ice for 10 min, then add 0.5 μL 100 mM ATP and 1 μL T4 RNA ligase, react at room temperature for 12 h to obtain a crude cyclization product (Note: the specific structure of the cyclization product depends on the sequence of the reactants);

[0077] 1.3 Take 45 μL of the crude cyclized product, 10×NEBuffer 45 μL, T5 exonuclease 1 μL and RNase R 0.5 μL and mix them evenly. After reacting at 37°C for 30 min, heat at 70°C for 10 min, and purify with RNA Cleanup Kit to obtain the final cyclized product (i.e., circular nucleic acids circp65-MDM2 and circTau-MDM2 targeting p65 / Tau and MDM2);

[0078] 1.4 The products in the synthesis and purification process were detected by urea polyacrylamide gel electrophoresis. The specific steps are as follows: Take 2.5mL of Acryl / Bis 30% solution, 1.5mL of 5×TBE buffer, 1mL of DEPC water, and 3.1g of urea, mix thoroughly, then quickly add 55μL of 10% ammonium persulfate solution and 5.5μL of TEMED in a fume hood, mix thoroughly, pour the mixture between the glass plates for gel making, insert a comb to avoid bubbles, let it stand for 30min, and after the gel is completely polymerized, gently pull out the comb and clean the gel holes. Take 10μL of 1μM sample and mix it evenly with 10μL of supersaturated urea solution and load it into the gel hole, use 1×TBE buffer as the electrophoresis buffer, and electrophoresed at 120V for 30min. After the electrophoresis is completed, place the gel in 1×GelRed aqueous solution to avoid light and soak for staining for 10min, and then use the fully automatic gel image analysis system (AmershamImageQuant800) for imaging.

[0079] Depend on Figure 2 C and Figure 2 The electrophoresis results of F show that although there are many miscellaneous bands in the lane of the crude cyclized product, the target band of the same size can be clearly distinguished, and after purification by T5 exonuclease and RNase R, almost only the target nucleic acid is retained, that is, most of the miscellaneous bands disappear or become lighter (see Figure 2 Lane 3 of C and Figure 2 F), and the target nucleic acid band is clear and dark in color, indicating that the purification effect is good and the purified circular nucleic acid can be used for subsequent experiments.

[0080] In summary, this example demonstrates that DBCO-MDM2-OH (whose nucleic acid sequence is shown in SEQ ID NO: 1) can be cyclized with P-p65-N3 (whose nucleic acid sequence is shown in SEQ ID NO: 4) and P-Tau-N3 (whose nucleic acid sequence is shown in SEQ ID NO: 5) respectively through N3-DBCO covalent cross-linking and T4 RNA ligase enzyme linkage in the absence of an auxiliary chain to generate the corresponding circularized nucleic acids, and T5 exonuclease and RNase R have better purification effects on the circularized nucleic acids.

[0081] Example 2: Preparation of circular nucleic acids circp65-MEX3A and circTau-MEX3A

[0082] This example provides a method for preparing a circular nucleic acid circp65-MEX3A (SEQ ID NO: 21) / circTau-MEX3A (SEQ ID NO: 22) that simultaneously targets p65 / Tau and MEX3A (belonging to the MEX3 protein family, an RNA binding protein and E3 ligase, NCBI Gene ID: 92312). The synthetic schematic diagrams of the circular nucleic acids circp65-MEX3A and circTau-MEX3A are shown in Figure 3 A and Figure 3 D. As can be seen from the above structural diagram, circp65-MEX3A and circTau-MEX3A are synthesized by nucleic acid aptamers (SEQ ID NO:4-SEQ ID NO:5) that specifically target p65 / Tau and RNA (SEQ ID NO:2) that can recruit MEX3A protein with the help of an auxiliary chain (SEQ ID NO:13). The nucleic acid aptamer is connected to the RNA through an N3-DBCO bond and a phosphodiester bond, wherein the auxiliary chain is complementary to the 5' end of the nucleic acid aptamer sequence and the 3' end sequence of the RNA, respectively, and by shortening the distance between the 5' end phosphate group of the nucleic acid aptamer and the 3' end hydroxyl group of the RNA, it promotes the T4 RNA ligase to connect and form a phosphodiester bond, thereby assisting the synthesis of the circular structure nucleic acid; the secondary structure diagrams predicted by the NUPACK website (https: / / www.nupack.org / ) are shown in Figure 3 B and Figure 3 E, Figure 3 B / Figure 3 The upper half of the figure in E is the structure prediction diagram of the DNA sequence targeting Tau / p65, and the lower half is the structure prediction diagram of the RNA sequence targeting MEX3A. From the structural prediction results, the free energy of the RNA (SEQ ID NO: 2) sequence that recruits the MEX3A protein is relatively large, which is -1.42 kcal / mol, indicating that the structural stability of the sequence is relatively poor. This is the possible reason why circp65-MEX3A and circTau-MEX3A can only be effectively synthesized in the presence of auxiliary chains. The preparation method of the circular nucleic acid is as follows:

[0083] 2.1 5 μL 40 μM P-p65-N3 (nucleic acid sequence as shown in SEQ ID NO:4) or 5 μL 40 μM P-Tau-N3 (nucleic acid sequence as shown in SEQ ID NO:5) and 5 μL 40 μM DBCO-MEX3A-OH (i.e., the above-mentioned RNA capable of recruiting MEX3A protein, nucleic acid sequence as shown in SEQ ID NO:2) were mixed evenly, and reacted at -40°C for 1 hour to obtain reaction solution 2.1 (Note: the specific structure of the reaction solution is related to the sequence of the reactants, that is, it depends on whether P-p65-N3 (SEQ ID NO:4) or P-Tau-N3 (SEQ ID NO:5) is selected), and then the reaction solution 2.1 is placed on ice for 10 minutes and at 4°C for 5 minutes to melt the reaction solution 2.1;

[0084] 2.2 Take 2 μL of reaction solution 2.1, 1 μL of 40 μM CS-MEX3A (i.e., the above-mentioned auxiliary chain, the nucleic acid sequence is such as SEQ ID NO: 13), 2 μL of 10×T4 RNA Ligase reaction buffer, 4 μL of 50% PEG8000 and 9.5 μL of DEPC water, mix, heat at 95°C for 5 min, cool on ice for 10 min, then add 0.5 μL of 100 mM ATP and 1 μL of T4 RNA Ligase, and react at room temperature for 12 h to obtain a crude cyclization product (Note: the specific structure of the cyclization product depends on the sequence of the reactants). At the same time, set a control, that is, no auxiliary chain is added, and the product is directly synthesized and purified;

[0085] 2.3 Take 45 μL of the crude cyclized product, 10×NEBuffer 45 μL, T5 exonuclease 1 μL and RNase R 0.5 μL, mix them evenly, react at 37°C for 30 min, heat at 70°C for 10 min, and purify with RNA Cleanup Kit to obtain the final cyclized product (i.e., circular nucleic acids circp65-MEX3A and circTau-MEX3A targeting p65 / Tau and MEX3A);

[0086] 2.4 The products in the synthesis and purification process were detected by urea polyacrylamide gel electrophoresis, and the electrophoresis steps were the same as those described in Example 1.

[0087] Depend on Figure 3 C and Figure 3 The electrophoresis results of F show that, without the addition of the auxiliary chain (SEQ ID NO: 13), although the ligation product can be generated, the target nucleic acid is degraded after purification by T5 exonuclease and RNase R (see Figure 3 Lanes 2 to 3 of C and Figure 3F, lanes 4-5), indicating that the ligation product generated in this case is not a circular nucleic acid and is not resistant to T5 exonuclease and RNase R purification; however, after adding the auxiliary chain, the synthesized ligation product can withstand T5 exonuclease and RNase R purification (see Figure 3 Lanes 4 to 5 of C and Figure 3 F, lanes 6-7), indicating that the circular nucleic acids circp65-MEX3A and circTau-MEX3A were successfully synthesized and purified in this case. This may be because the RNA sequence targeting the MEX3A protein (SEQ ID NO: 2) has poor structural stability (its secondary structure simulation free energy is large, -1.42 kcal / mol), which makes it difficult for T4 RNA ligase to catalyze the formation of a phosphodiester bond between the 5'-terminal phosphate group of the p65 / Tau nucleic acid sequence and the 3'-terminal hydroxyl group of the nucleic acid sequence targeting the MEX3A protein in the absence of an auxiliary chain; the addition of the auxiliary chain plays a role in bringing the 5'-terminal phosphate group of the nucleic acid aptamer and the 3'-terminal hydroxyl group of the RNA closer, making up for the poor structural stability of the RNA sequence targeting the MEX3A protein (SEQ ID NO: 2), so that T4 RNA ligase can effectively function, so the addition of auxiliary chains is required in the synthesis process of circp65-MEX3A and circTau-MEX3A circular nucleic acids.

[0088] In summary, in the presence of auxiliary chains, DBCO-MEX3A-OH (nucleic acid sequence as shown in SEQ ID NO: 2) was cyclized with P-p65-N3 (nucleic acid sequence as shown in SEQ ID NO: 4) and P-Tau-N3 (nucleic acid sequence as shown in SEQ ID NO: 5) by N3-DBCO covalent cross-linking and T4 RNA ligase enzyme-linked methods, and then purified by T5 exonuclease and RNaseR to obtain the corresponding circular nucleic acid circp65-MEX3A / circTau-MEX3A. The target circular nucleic acid cannot be synthesized without adding auxiliary chains, indicating that the RNA sequence affects the cyclization results.

[0089] Example 3: Circular nucleic acids circp65-MEX3C and circTau-MEX3C cannot be synthesized

[0090] This example attempts to synthesize the circular nucleic acid circp65-MEX3C (SEQ ID NO: 23) / circTau-MEX3C (SEQ ID NO: 24) that simultaneously targets p65 / Tau and MEX3C (belonging to the MEX3 protein family, an RNA binding protein and E3 ligase, NCBI Gene ID: 51320). The schematic diagrams of the synthesis of the circular nucleic acid circp65-MEX3C and circTau-MEX3C are shown in Figure 4 A and Figure 4 D. As can be seen from the above structural diagram, circp65-MEX3C and circTau-MEX3C are composed of nucleic acid aptamers (SEQ ID NO: 4 to SEQ ID NO: 5) that specifically target p65 / Tau, RNA (SEQ ID NO: 3) that can recruit MEX3C protein, and an auxiliary chain (SEQ ID NO: 14). The nucleic acid aptamer is connected to the RNA through an N3-DBCO bond and a phosphodiester bond. The auxiliary chain is complementary to the 5' end of the nucleic acid aptamer sequence and the 3' end sequence of the RNA, respectively. The role of the auxiliary chain is the same as that described in Example 2, and then a nucleic acid with a ring structure is formed; the secondary structure diagrams of the two nucleic acid sequences involved in the cyclization predicted by the NUPACK website (https: / / www.nupack.org / ) are shown in FIG. Figure 4 B and Figure 4 E, Figure 4 B / Figure 4 The upper half of the figure in E is the predicted structure of the DNA sequence targeting Tau / p65, and the lower half is the predicted structure of the RNA sequence targeting MEX3C. From the structural prediction results, the free energy of the RNA (SEQ ID NO: 3) sequence that recruits the MEX3C protein is relatively large, which is -1.55 kcal / mol, indicating that the structural stability of the sequence is relatively poor, which may be the reason why circp65-MEX3A and circTau-MEX3C cannot be synthesized in the presence or absence of auxiliary chains. This example attempts to synthesize circular nucleic acids circp65-MEX3C and circTau-MEX3C without or with the addition of auxiliary chains. The preparation method of the circular nucleic acid is the same as that described in Example 1 (without adding auxiliary chains) and Example 2 (adding auxiliary chains), except that the RNA in the reaction system is RNA (SEQ ID NO: 3) that can recruit MEX3C protein, and the auxiliary chain is CS-MEX3C (SEQ ID NO: 14).

[0091] Depend on Figure 4 C and Figure 4F shows that the yield of crude cyclization products obtained by T4 RNA ligase ligation is generally low regardless of the presence of auxiliary chain (see Figure 4 Lanes 3 and 5 of C and Figure 4 F, lanes 4 and 6), which means that T4 RNA ligase is not suitable for the synthesis of circular nucleic acids circp65-MEX3C and circTau-MEX3C; and after subsequent purification, the target band disappeared, indicating that for the synthesis of circular nucleic acids circp65-MEX3C and circTau-MEX3C, the auxiliary chain cannot assist T4 RNA ligase to play a role. This may be because the sequences of the nucleic acid aptamers (SEQ ID NO:4 to SEQ ID NO:5) that specifically target p65 / Tau and the RNA (SEQ ID NO:3) sequence that can recruit MEX3C protein are unstable in the new sequence structure formed after the N3-DBCO reaction, and the auxiliary chain cannot achieve the pulling of the phosphate group and the hydroxyl group, and thus T4 RNA ligase cannot effectively perform the ligation function.

[0092] In summary, the method provided in this example cannot obtain high-purity circular nucleic acids circp65-MEX3C and circTau-MEX3C.

[0093] The present invention explored the synthesis process of various circular nucleic acids, and the results are shown in Table 3:

[0094] Table 3 Nucleic acid sequences required for the synthesis of circular nucleic acids

[0095]

[0096] Example 4: Circular nucleic acids circF-TfR-Tau-MDM2 and circF-Tau-TfR-MDM2 lacking a stem-loop structure cannot be synthesized

[0097] This example attempts the method described in Example 2 to synthesize circular nucleic acids circF-TfR-Tau-MDM2 (SEQ ID NO: 25) / circF-Tau-TfR-MDM2 (SEQ ID NO: 26) that simultaneously target Tau, TfR and MDM2 proteins and relatively lack stem-loop structures (used to distinguish the circular nucleic acids circTfR-Tau-MDM2 and circTau-TfR-MDM2 described in Example 5). The synthetic schematic diagrams of the circular nucleic acids circF-TfR-Tau-MDM2 and circF-Tau-TfR-MDM2 are shown in Figure 5 A and Figure 5D. As can be seen from the above structural diagram, circF-TfR-Tau-MDM2 / circF-Tau-TfR-MDM2 consists of a nucleic acid aptamer (SEQ ID NO: 10 or SEQ ID NO: 11) that specifically targets Tau and TfR, an RNA (SEQ ID NO: 1) that can recruit MDM2 protein, and an auxiliary chain (SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18), wherein the nucleic acid aptamer is connected to the RNA through an N3-DBCO bond and a phosphodiester bond, and the auxiliary chain is complementary to the 5' end of the nucleic acid aptamer sequence and the 3' end sequence of the RNA, respectively. The role and mechanism of the auxiliary chain are the same as those described in Example 2, and then a nucleic acid with a ring structure is formed; the secondary structure diagrams of the two nucleic acids involved in the cyclization predicted by the NUPACK website (https: / / www.nupack.org / ) are shown in FIG. Figure 5 B and Figure 5 E, Figure 5 B / Figure 5 The upper half of Figure E is the predicted structure of the DNA sequence of SEQ ID NO:10 or SEQ ID NO:11, and the lower half of the figure is the predicted structure of the RNA sequence targeting MDM2. From the structural prediction results, the nucleic acid aptamer (SEQ ID NO:10 or SEQ ID NO:11) that specifically targets Tau and TfR lacks a stem-loop structure between the 5' end and the 3' end, and its free energy is relatively large (-15.21 kcal / mol, higher than the free energy of -33.27 kcal / mol of the sequence SEQ ID NO:6 or SEQID NO:7 used in Example 5), which may affect the results of the cyclization. The example attempts to synthesize circular nucleic acids circF-TfR-Tau-MDM2 and circF-Tau-TfR-MDM2 with the addition of different auxiliary chains. The method for preparing the circular nucleic acid is the same as that described in Example 2, except that the nucleic acid aptamer in the reaction system is a nucleic acid sequence that can simultaneously target Tau and TfR proteins (SEQ ID NO: 10 or SEQ ID NO: 11), and the auxiliary chain is CS-TfR-1 / CS-TfR-2 / CS-Tau-1 / CS-Tau-2 (SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18).

[0098] Depend on Figure 5 C and Figure 5F It can be seen that although different auxiliary chains were used in this example, no crude cyclization product could be obtained. This may be because the nucleic acid sequence (SEQ ID NO: 10 or SEQ ID NO: 11) that simultaneously targets Tau and TfR proteins lacks a stem-loop structure between the 5' end and the 3' end, and its free energy is relatively large (-15.21 kcal / mol), which is higher than the free energy of the sequence SEQ ID NO: 6 or SEQ ID NO: 7 used in Example 5 (-33.27 kcal / mol), resulting in the inability of the auxiliary chain to bring the phosphate group and the hydroxyl group closer, and thus the T4 RNA ligase cannot effectively perform the connection function.

[0099] Example 5: Synthesis of circular nucleic acid circTfR-Tau-MDM2 and circTau-TfR-MDM2 with a stem-loop structure

[0100] In order to obtain a high-purity circular nucleic acid that can simultaneously target TfR, Tau and MDM2 proteins, the present invention optimizes the structure of the circular nucleic acid. In this embodiment, based on the nucleic acid aptamer (SEQ ID NO: 10 or SEQ ID NO: 11) that specifically targets Tau and TfR described in Example 4 and does not affect the structure of the nucleic acid aptamer itself, a partial sequence is changed (see SEQ ID NO: 6 or SEQ ID NO: 7 for details). Specifically, a structure with 7bp base complementary pairing is designed in the connecting portion of the nucleic acid aptamer targeting Tau and TfR, so that the 5' end and 3' end of the circular nucleic acid generated in theory can form a new stem-loop structure. At the same time, the secondary structure prediction results show that the free energy of the optimized circular nucleic acid is low (-33.27kcal / mol). However, if the number of complementary pairing bases in the connecting portion stem-loop structure is too much, the nucleic acid sequence will be too long, which will increase the difficulty of synthesis on the one hand, and may destroy the nucleic acid aptamer structure on the other hand, resulting in a decrease in its affinity for binding to the target protein, so the size and number of the stem-loop introduced need to be carefully designed. In this example, the circular nucleic acids with stem-loop structures (used to distinguish from the circular nucleic acids circF-TfR-Tau-MDM2 and circF-Tau-TfR-MDM2 described in Example 4) and simultaneously targeting TfR, Tau and MDM2 proteins are named circTfR-Tau-MDM2 (SEQ ID NO: 27) and circTau-TfR-MDM2 (SEQ ID NO: 28), respectively. The synthetic schematic diagrams of the circular nucleic acids circTfR-Tau-MDM2 and circTau-TfR-MDM2 are shown in Figure 6 A and Figure 6D. As can be seen from the above structural diagram, circTfR-Tau-MDM2 / circTau-TfR-MDM2 consists of a nucleic acid aptamer (SEQ ID NO:6 or SEQ ID NO:7) that specifically targets Tau and TfR and an RNA (SEQ ID NO:1) that can recruit MDM2 protein. The nucleic acid aptamer is connected to the RNA through an N3-DBCO bond and a phosphodiester bond to form a ring-shaped nucleic acid. The secondary structure diagrams predicted by the NUPACK website (https: / / www.nupack.org / ) are shown in FIG. Figure 6 B and Figure 6 E. From the structural prediction results, circTfR-Tau-MDM2 / circTau-TfR-MDM2 not only has an additional base complementary pairing sequence to form a stem-loop structure compared to circF-TfR-Tau-MDM2 / circF-Tau-TfR-MDM2, but the free energy of the former is also significantly smaller than that of the latter, which may be because the addition of the stem-loop structure is conducive to the stability of the circular nucleic acid structure. The preparation method of circTfR-Tau-MDM2 / circTau-TfR-MDM2 is the same as that described in Example 1, except that the nucleic acid aptamer in the reaction system is a nucleic acid sequence (SEQ ID NO: 6 or SEQ ID NO: 7) that can simultaneously target Tau and TfR proteins.

[0101] Depend on Figure 6 C and Figure 6 From the electrophoresis results of F, it can be seen that although there are more miscellaneous bands in the swimming lane of the crude product of the cyclization, the target band of the size can be clearly distinguished, and after purification by T5 exonuclease and RNase R, almost only the target nucleic acid is retained, that is, most of the miscellaneous bands disappear or become lighter, and the target nucleic acid band is clear and darker, indicating that the purification effect is better, and the purified circular nucleic acid can be used for subsequent experiments. On the other hand, compared with the synthesis method in Example 5 (i.e., using different auxiliary chains), adding a stem-loop structure is more beneficial to the acquisition of the target product.

[0102] In summary, this example demonstrates that DBCO-MDM2-OH (whose nucleic acid sequence is shown in SEQ ID NO: 1) can be cyclized with P-TfR-Tau-N3 (whose nucleic acid sequence is shown in SEQ ID NO: 6) and P-Tau-TfR-N3 (whose nucleic acid sequence is shown in SEQ ID NO: 7) by N3-DBCO covalent cross-linking and T4 RNA ligase enzyme linkage in the absence of an auxiliary chain to generate the corresponding circularized nucleic acids, and T5 exonuclease and RNase R have better purification effects on circularized nucleic acids.

[0103] Example 6: Circular nucleic acid circTfR-Tau-MDM2 / circTau-TfR-MDM2 specifically binds to TfR-positive cells

[0104] In order to explore the efficiency of circular nucleic acids circTfR-Tau-MDM2 and circTau-TfR-MDM2 in targeting positive TfR cells, the two nucleic acids were used to transfect TfR protein-positive cells (mouse brain microvascular endothelial cells (bEnd.3 cells) and mouse hippocampal neuronal cells (HT22 cells)), and human embryonic kidney cells (HEK293T cells) with negative TfR protein were used as negative controls. The specific operations are as follows:

[0105] 6.1 Using FAM-P-TfR-Tau-N3 (SEQ ID NO: 8) and FAM-P-Tau-TfR-N3 (SEQ ID NO: 9) labeled with Int6'-FAM as raw materials, and with other materials unchanged, FAM-circTfR-Tau-MDM2 and FAM-circTau-TfR-MDM2 were synthesized and purified according to the steps described in Example 5. It should be understood that Int6'-FAM is a fluorescent group for flow cytometry detection, and the sequence shown in SEQ ID NO: 8 / SEQ ID NO: 9 is the same as the sequence shown in SEQ ID NO: 8 / SEQ ID NO: 7, except for the difference in whether or not the Int6'-FAM fluorescent group is modified.

[0106] 6.2 Take 10 respectively 4 HT22 cells (donated by Dr. Xiao Run from Hangzhou Institute of Medical Sciences, Chinese Academy of Sciences) / bEnd.3 cells (purchased from Zhejiang Meisen Cell Technology Co., Ltd.) / HEK293T cells (from ATCC) were washed twice with 1×DPBS buffer, resuspended in 100 μL of Binding Buffer (DPBS solution containing 1 mg / mL BSA, 25 mM glucose, 0.1 mg / mL yeast tRNA and 5 mM MgCl2) containing 400 nM Int6'-FAM-labeled nucleic acids (SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:12 (i.e., control group), FAM-circTfR-Tau-MDM2 and FAM-circTau-TfR-MDM2), incubated on ice in the dark for 1 h, washed twice with 1×DPBS, and analyzed using Attune NxT flow cytometer (Thermo Fisher). In addition to using FAM-N50 (SEQ ID NO: 12) as a negative control, the above experiments also included a blank control (ie, cell group) without any nucleic acid but containing only cells.

[0107] The results of the above experiments are as follows Figure 7 As shown, in general, the nucleic acid containing the nucleic acid aptamer component targeting the TfR protein can specifically bind to TfR-positive cells (bEnd.3 cells and HT22 cells), and cyclization of the nucleic acid can further improve the binding ability of the nucleic acid, especially when the binding object is HT22 cells; at the same time, the binding of TfR-negative cells HEK293T cells to the above-mentioned nucleic acid is relatively limited, which means that the nucleic acid containing the nucleic acid aptamer component targeting the TfR protein does not affect the normal physiological activities of other cells in the body to a certain extent, that is, if it is used as a therapeutic drug, the side effects are relatively small. In addition, the binding ability of circTfR-Tau-MDM2 is generally higher than that of FAM-circTau-TfR-MDM2. Specifically, after FAM-circTfR-Tau-MDM2 was co-incubated with TfR-positive cells (HT22 cells and bEnd.3 cells), its FAM average fluorescence intensity was greater than that of FAM-circTau-TfR-MDM2. The reason for these differences may be that the structure of the nucleic acid aptamer component targeting TfR protein in circTfR-Tau-MDM2 is more independent in spatial position and easier to bind to the target protein TfR on the cell surface. Therefore, the circular nucleic acid circTfR-Tau-MDM2 is preferred to transfect TfR-positive cells (preferably HT22 cells).

[0108] Example 7: Effect of circular nucleic acid circTau-MDM2 / circTfR-Tau-MDM2 / circTau-TfR-MDM2 on degradation of endogenous Tau protein

[0109] Example 6 examined the efficiency of the specific binding of circular nucleic acids circTfR-Tau-MDM2 and circTau-TfR-MDM2 to target cells, but it is not clear whether these two have any effect on the degradation of endogenous Tau protein. Therefore, this example uses circular nucleic acids circTau-MDM2, circTfR-Tau-MDM2 and circTau-TfR-MDM2 and HT22 cells as research objects to explore this. The specific operations are as follows:

[0110] 7.1 Preparation of circular nucleic acids

[0111] High-purity circular nucleic acids circTau-MDM2, circTfR-Tau-MDM2 and circTau-TfR-MDM2 were prepared according to Example 1 and Example 5, respectively.

[0112] 7.2 circTau-MDM2 / circTfR-Tau-MDM2 transfection into HT22 cells and degradation of Tau protein

[0113] One day before transfection, inoculate 10 5HT22 cells were placed in a 24-well plate to make the cell density at the time of transfection about 70%. On the day of transfection, on the one hand, different volumes (0, 0.625, 1.25, 2.5 and 5 μL) of circTau-MDM2 / circTfR-Tau-MDM2 at a concentration of 50 μM were mixed with 25 μL Opti-MEM medium, and then 3 μL Lipofectamine 3000 reagent was added to a total volume of 25 μL Opti-MEM medium, and the mixture was allowed to stand at room temperature for 15 min, and then serum-free DMEM medium was added to the system to 500 μL, so that the final concentration of circTau-MDM2 was 0, 62.5, 125, 250 and 500 nM, respectively. On the other hand, the medium in the 24-well plate was discarded, and the cells were washed twice with 1×DPBS buffer, and then 500 μL of the nucleic acid mixture was added to the 24-well plate containing HT22 cells and incubated for 24 h. After the incubation, the medium in the 24-well plate was discarded, 80 μL of RIPA lysis buffer containing 1× protease inhibitor was added to each well, and the 24-well plate was placed on ice for 30 minutes to lyse the cells. Subsequently, the lysate and cell mixture after lysis was collected into a 1.5 mL EP tube, and the mixture was centrifuged at 4°C, 13,000 rpm for 15 minutes using a centrifuge. The supernatant obtained after centrifugation was transferred to a new 1.5 mL EP tube, and 20 μL of 5× protein loading buffer was added to each tube to mix evenly, and the mixture was placed in a metal bath at 95°C for 10 minutes to fully denature the protein. After heating, the sample was placed at 4°C for 10 minutes to obtain the loading sample. 15 μL of each sample was loaded on a YoungPAGE (Bis-Tris) precast gel, and 5 μL of prestained protein molecular weight standards (i.e., MARKER) were loaded in the gel wells at both ends of the precast gel. Bis-Tris Gel MOPSSDS Running Buffer was used as the electrophoresis buffer, and electrophoresis was performed at a voltage of 100V for 90 minutes. Remove the gel and transfer the membrane using a PVDF membrane in the transfer buffer at 200 mA for 90 min. Remove the PVDF membrane and wash the membrane with 20 mL PBST (1×PBS solution containing 0.1% Tween-20) on a horizontal shaker at 70 rpm for 5 min. Then transfer the PVDF membrane to a PBST solution containing 5% skim milk powder and place it on a horizontal shaker for blocking at room temperature and 60 rpm for 1 h. After blocking, wash the PVDF membrane with 20 mL PBST on a horizontal shaker at 70 rpm for 5 min, and repeat three times.After washing, the PVDF membrane was cut in the middle of the 55kDa and 43kDa molecular weight standard bands, and the portion containing the larger molecular weight protein band was placed in a 5% skim milk powder PBST solution containing 0.1% Tau protein antibody, and the portion containing the smaller molecular weight protein band was placed in a 5% skim milk powder PBST solution containing 0.1% β-actin protein antibody, and placed in a horizontal shaker, incubated at 4°C, 70rpm for 16h. After incubation, 20mL PBST was used to wash the PVDF membrane at 70rpm for 5min on a horizontal shaker, and repeated three times. The PVDF membrane was then transferred to a 5% skim milk powder PBST solution containing 0.5% goat anti-rabbit IgG-HRP, and placed in a horizontal shaker, incubated at room temperature, 60rpm for 1h. After incubation, the PVDF membrane was washed with 20 mL PBST on a horizontal shaker at 70 rpm for 5 min, repeated three times, and then 100 μL of Super ECL Plus ultrasensitive luminescent liquid was added to the surface of the PVDF membrane, and imaging was performed using an automatic gel image analysis system (Amersham ImageQuant800). The imaging results were analyzed using ImageJ software.

[0114] The results of the experiment are as follows Figure 8 As shown in A, the numbers in the figure represent the relative expression of Tau protein, which is used to quantify the degradation of Tau protein. The lower the relative expression of Tau protein, the more Tau protein is degraded in the cell. The calculation formula is as follows: The control group was the group with a drug concentration of 0 nM.

[0115] 7.3 CircTfR-Tau-MDM2 / circTau-TfR-MDM2 co-incubated with HT22 cells and degraded Tau protein

[0116] The day before the combination, inoculate 10 5 HT22 cells were plated in a 24-well plate so that the cell density was about 70% at the time of administration. On the day of administration, 0, 0.5, 1, 2, and 4 μL of circTfR-Tau-MDM2 or circTau-TfR-MDM2 at a concentration of 50 μM were taken respectively and the total volume of the system was supplemented to 150 μL with BindingBuffer (the ingredients are the same as described in step "6.2"); the cells were washed twice with 1×DPBS, and then the above 150 μL nucleic acid mixture was added to the 24-well plate and incubated with HT22 cells at 37°C for 2h, and then 350 μL of serum-free DMEM medium was added to each well and incubated at 37°C for another 22h. After the incubation, the cells were collected and the proteins were extracted, and protein immunoblotting was performed using Tau protein antibodies (the steps are the same as described above). The results of the above experiments are shown in Figure 8 C and Figure 8 As shown in D.

[0117] It should be understood that steps 7.2 and 7.3 are different because circTau-MDM2 does not have a cell recognition element (TfR targeting aptamer), and additional transfection agents need to be added to mediate the entry of the circular nucleic acid into the cell, but circTfR-Tau-MDM2 / circTau-TfR-MDM2 has a TfR targeting aptamer, so in theory, it can use the TfR-targeted nucleic acid aptamer-mediated endocytosis to enter the cell without the need for additional transfection reagents.

[0118] Depend on Figure 8 A~ Figure 8 B It can be seen that with the increase of circTau-MDM2 / circTfR-Tau-MDM2 transfection concentration (0-500nM), the degradation of Tau protein increased. Among them, when the circTau-MDM2 transfection concentration was 500nM, the degradation of Tau protein was the most, that is, 73% of Tau protein was degraded, and under this condition, circTfR-Tau-MDM2 could degrade 93% of Tau protein, indicating that circTau-MDM2 and circTfR-Tau-MDM2 can both efficiently degrade Tau protein, and circTfR-Tau-MDM2 has a better degradation effect on Tau protein, so circTfR-Tau-MDM2 is preferred. At the same time, this example also explores the effect of circTau-MEX3A on degrading target protein, but its effect is not as good as that of circTau-MDM2. Since circTau-MEX3C was not successfully synthesized, its effect on degrading target protein has not been explored for the time being.

[0119] On the other hand, circTfR-Tau-MDM2 and circTau-TfR-MDM2 were able to degrade Tau protein without the use of Lipofectamine3000 ( Figure 8 C~ Figure 8D), indicating that the presence of TfR-targeted aptamers enables these two circular nucleic acids to enter TfR protein-positive cells through endocytosis. Although the degradation effect of circTfR-Tau-MDM2 / circTau-TfR-MDM2 on Tau is slightly inferior to that of circTau-MDM2, which may be caused by different transfection / binding methods, when the concentration of circTfR-Tau-MDM2 is 400nM, its effect of degrading Tau is comparable to that of 125-250nMcircTau-MDM2. Moreover, circTfR-Tau-MDM2 has the function of efficiently targeting TfR-positive cells that circTau-MDM2 does not have, so it also has great application potential. Compared with circTfR-Tau-MDM2, the Tau protein degradation effect of circTau-TfR-MDM2 is generally poor, which may be caused by differences in binding ability (Example 6 proves that circTfR-Tau-MDM2 binds HT22 cells more efficiently), or it may be due to different circular nucleic acid sequences that lead to different conformations of binding to Tau protein, MDM2 protein and TfR protein. However, at a lower incubation concentration (50-100 nM), its effect is better than that of circTfR-Tau-MDM2 at the same concentration, especially at a concentration of 50 nM.

[0120] In summary, circular nucleic acids circTau-MDM2, circTfR-Tau-MDM2, and circTau-TfR-MDM2 can all effectively degrade endogenous Tau protein in cells, among which circTfR-Tau-MDM2 transfection with Lipofectamine 3000 has the best effect, followed by circTau-MDM2 transfection with Lipofectamine 3000, and then direct incubation of circTfR-Tau-MDM2 and direct incubation of circTfR-Tau-MDM2. That is, under the same incubation / binding conditions, circTfR-Tau-MDM2 has the best effect, and its use concentration is 0-500nM, preferably 400-500nM.

[0121] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description. Therefore, it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention, and any figure marks in the claims should not be regarded as limiting the claims involved.

Claims

1. A protein targeting degradation agent based on circular nucleic acid, characterized in that: The circular nucleic acid comprises an RNA sequence capable of recruiting an E3 ubiquitin ligase and at least one nucleic acid aptamer, wherein the RNA sequence and the nucleic acid aptamer are chemically linked together.

2. The protein targeted degradation agent according to claim 1, characterized in that The E3 ubiquitin ligase includes but is not limited to MDM2 protein, MEX3A protein, MEX3C protein, VHL protein, CRBN protein, Dzip3 protein, and cIAP protein.

3. The protein targeted degradation agent according to claim 1, characterized in that The RNA sequence for recruiting E3 ubiquitin ligase has more than 80% homology with the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence list.

4. The protein targeted degradation agent according to claim 1, characterized in that The nucleic acid aptamer targets intracellular proteins and / or membrane proteins.

5. The protein targeted degradation agent according to claim 4, characterized in that The intracellular proteins include but are not limited to Tau protein, p65 protein, Ras protein, and MEK protein; the membrane proteins include but are not limited to TfR protein, PTK7 protein, c-MET protein, and DEC205 protein.

6. The protein targeted degradation agent according to claim 1, characterized in that The RNA sequence and / or the nucleic acid aptamer carry any one of the following groups: hydroxyl, phosphate, carboxylic acid, amino, sulfhydryl, maleimide, N-hydroxysuccinimide, alkyne, and azide.

7. A method for preparing the protein targeted degradation agent according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: A. Connect the N3 group on the 3' end of the aptamer and the DBCO group on the 5' end of the RNA sequence by covalent bonding; B. Using T4 RNA ligase to connect the phosphate group on the 5' end of the nucleic acid aptamer to the hydroxyl group on the 3' end of the RNA sequence to prepare a crude protein targeted degrader; C. Purify the crude protein targeted degrader described in step B using T5 exonuclease and RNase R enzymes.

8. The method according to claim 7, characterized in that When the protein targeted degradation agent comprises RNA capable of recruiting MEX3A protein, step B further requires the addition of an auxiliary chain, wherein the auxiliary chain comprises a sequence as shown in SEQ ID NO: 13 in the sequence list; When the protein targeted degrader comprises RNA capable of recruiting MDM2 protein, step B does not require the addition of an auxiliary strand.

9. A use of RNA for preparing a protein targeting degradation agent, characterized in that: The sequence of the RNA has more than 80% homology with the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence list.

10. A use of a nucleic acid sequence for preparing a protein targeted degradation agent, characterized in that: The nucleic acid sequence has more than 80% homology with the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 10 or SEQ ID NO: 11 in the sequence list.

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