An antisense oligonucleotide-polypeptide conjugate, its preparation method and application

Abstract

The present invention discloses a conjugate of an antisense oligonucleotide and a polypeptide, a preparation method thereof and an application thereof, belonging to the technical field of biomedicine. Provided is a conjugate of an antisense oligonucleotide and a polypeptide, the general structural formula of which is shown in formula (I), wherein O is an antisense oligonucleotide, L is a linker, and P is a polypeptide; the antisense oligonucleotide is a nucleotide sequence obtained by phosphorothioate and 2'-O-methyl modification based on the nucleotide sequence of SEQ ID NO.1 or the nucleotide sequence shown in SEQ ID NO.2; the amino acid sequence of the polypeptide is shown in SEQ ID NO.3. In the conjugate of the antisense oligonucleotide and the polypeptide of the present invention, the polypeptide P catalyzes the cleavage of the phosphodiester bond of RNA, the antisense oligonucleotide O is complementary paired with the frameshift element, brings the polypeptide P to the vicinity of the frameshift element, and performs targeted cleavage. The linker L is responsible for coupling, specifically targeting and degrading pathogenic RNA in a sequence-specific manner, and disrupting the expression of RNA in cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a conjugate of an antisense oligonucleotide and a polypeptide, a preparation method thereof, and an application thereof. Background Art

[0002] Abnormalities in RNA function are closely related to the occurrence and development of many diseases. Irreversible sequence-selective cleavage of pathogenic RNA is an attractive therapeutic strategy that can be used for many targets and diseases that are currently untreatable by drugs. However, traditional small molecule drugs may cause off-target effects and toxicity due to their poor target selectivity and limited number of chemical functional groups. The method of targeting and interfering with RNA can target a variety of RNAs, including messenger ribonucleic acid, non-coding RNA, and viral ribonucleic acid. In addition to intracellular endogenous RNA, RNA plays a key role in viruses and bacteria. Functional elements of the viral or bacterial transcriptome / genome make these RNA structures excellent targets for anti-infective drugs. Antisense oligonucleotides (ASOs) can effectively inhibit RNA activity. They are oligonucleotides with a length of 12 - 25 mer that bind to RNA in cells through Watson-Crick base complementary pairing, preventing its translation, thereby inhibiting the expression of disease-related genes. Currently, drugs based on antisense oligonucleotides have been marketed.

[0003] A polypeptide is a chain-like biological macromolecule composed of multiple amino acid residues. It has the advantages of easy synthesis, easy chemical modification, and low immunogenicity. The types and sequences of amino acids in a polypeptide determine its properties. The polypeptide [(LeuArg)2Gly]2 composed of alternating leucine and arginine residues has been proven to be a relatively effective catalytic peptide, which can catalyze the cleavage of phosphodiester bonds in RNA. Summary of the Invention

[0004] To solve the above technical problems, the present invention proposes a conjugate of an antisense oligonucleotide and a polypeptide, a preparation method thereof, and an application thereof. It is composed of three parts: an antisense oligonucleotide O, a linker L, and a polypeptide P. The polypeptide P can catalyze the cleavage of phosphodiester bonds in RNA; the antisense oligonucleotide O is complementary to the frameshift element of SARS-CoV-2, bringing the catalytic polypeptide P to the vicinity of the frameshift element, thereby targeting and cleaving it. The linker L is responsible for coupling the two, achieving sequence-specific targeted degradation of pathogenic RNA and disrupting the expression of this RNA in cells.

[0005] To achieve the above object, the present invention provides a conjugate of an antisense oligonucleotide and a polypeptide. The structural general formula of the conjugate of the antisense oligonucleotide and the polypeptide is shown in formula (Ⅰ):

[0006]

[0007] Among them, O is an antisense oligonucleotide, L is a linker, and P is a polypeptide;

[0008] The antisense oligonucleotide is based on the nucleotide sequence of SEQ ID NO.1, with phosphorothioate backbone modifications at the 1st, 2nd, 14th, and 15th positions of the nucleotide sequence shown in SEQ ID NO.1, and 2'-O-methyl modifications of the ribose of all nucleotides in the nucleotide sequence shown in SEQ ID NO.1, or the nucleotide sequence shown in SEQ ID NO.2; the amino acid sequence of the polypeptide is as shown in SEQ ID NO.3.

[0009] Preferably, the structural formula of the L linker is as shown in formula (Ⅱ):

[0010]

[0011] Preferably, the structural formula of the P polypeptide is as shown in formula (Ⅲ):

[0012]

[0013] The present invention also provides a method for preparing a conjugate of the antisense oligonucleotide and the polypeptide, comprising the following steps:

[0014] Synthesize an antisense oligonucleotide O with a thiol-modified end; prepare a polypeptide P with a maleimide-modified end by solid-phase peptide synthesis; couple the thiol-modified oligonucleotide O with the maleimide-modified polypeptide P, and separate and purify to obtain a conjugate of the antisense oligonucleotide and the polypeptide.

[0015] The present invention also provides the use of the conjugate of the antisense oligonucleotide and the polypeptide in the preparation of a drug for treating coronavirus infection and related diseases.

[0016] The present invention also provides the use of the conjugate of the antisense oligonucleotide and the polypeptide in the preparation of a drug for treating SARS-CoV-2 infection and related diseases.

[0017] Preferably, the conjugate of the antisense oligonucleotide and the polypeptide can degrade the SARS-CoV-2 frameshift element.

[0018] Preferably, the antisense oligonucleotide O in the conjugate of the antisense oligonucleotide and the polypeptide is complementary to the SARS-CoV-2 frameshift element. The antisense oligonucleotide O is connected to the polypeptide P through the linker L, and the polypeptide P is transferred to the vicinity of the SARS-CoV-2 frameshift element to target and cleave the SARS-CoV-2 frameshift element, degrade the pathogenic RNA, and disrupt the expression of RNA in cells.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] The present invention provides a conjugate of an antisense oligonucleotide and a polypeptide, which consists of three parts: polypeptide P, linker L, and antisense oligonucleotide O. Polypeptide P is a catalytic peptide composed of arginine and leucine, which can catalyze the cleavage of phosphodiester bonds in RNA. The antisense oligonucleotide O is complementary to the frameshift element in the SARS-CoV-2 gene and can bring the catalytic polypeptide P to the vicinity of the frameshift element, enabling targeted cleavage thereof. Linker L is responsible for coupling the two macromolecules together. The conjugate of the antisense oligonucleotide and the polypeptide is a promising therapeutic method that achieves sequence-specific targeted degradation of pathogenic RNA and disrupts the expression of this RNA in cells.

[0021] The conjugate of the antisense oligonucleotide and the polypeptide of the present invention is administered by liposome encapsulation into AD293 cells expressing a plasmid containing a frameshift element GFP. By detecting the fluorescence intensity through laser confocal microscopy, it is found that the conjugate can effectively reduce the expression of the frameshift element RNA. The conjugate of the antisense oligonucleotide and the polypeptide has a high efficiency of degrading frameshift element RNA. In cells, the conjugate of the antisense oligonucleotide and the polypeptide at 500 nM can effectively degrade the frameshift element RNA. The sequence of the antisense oligonucleotide can be arbitrarily changed according to the RNA to be targeted, and it is expected to apply this technology to the targeted degradation of other pathogenic viral RNAs, providing a reliable new idea for the treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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 use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0023] Figure 1 Mass spectra of conjugates of antisense oligonucleotides and polypeptides POC1, POC2, POC3, POC4, POC5, and POC6, where A is POC1, B is POC2, C is POC3, D is POC4, E is POC5, and F is POC6;

[0024] Figure 2 Mass spectra of conjugates of antisense oligonucleotides and polypeptides POC-4m, POC-4ms, m(POC-4), and m(POC4-s), where A is POC-4m, B is POC-4ms, C is m(POC-4), and D is m(POC-4s);

[0025] Figure 3Mass spectrometry diagrams of the SARS-CoV-2 frameshift element RNA, where A is *FSE35 and B is *FSE52;

[0026] Figure 4 Polyacrylamide gel electrophoresis diagrams of POC1, POC2, POC3, POC4, POC5, and POC6 binding to the SARS-CoV-2 frameshift element RNA in vitro;

[0027] Figure 5 Polyacrylamide gel electrophoresis diagrams of POC1, POC2, POC3, POC4, POC5, and POC6 cleaving the SARS-CoV-2 frameshift element RNA in vitro;

[0028] Figure 6 Polyacrylamide gel electrophoresis diagrams of the change in incubation time of m(POC4-S) in serum;

[0029] Figure 7 Laser confocal diagrams of the antisense oligonucleotide and polypeptide conjugate showing cleavage effect intracellularly, with the scale bar being 100 μm;

[0030] Figure 8 Sites where POC4 and POC5 cleave the SARS-CoV-2 frameshift element RNA;

[0031] Figure 9 The ligation process of linker L ligating antisense oligonucleotide O and polypeptide P. Detailed implementation modes

[0032] Now, various exemplary implementation modes of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing specific implementation modes and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0035] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the specification of the present invention, which will be apparent to those skilled in the art. Other embodiments obtained from the specification of the present invention will be apparent to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0036] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0037] Examples

[0038] I. Synthesis of Conjugates of Antisense Oligonucleotides and Polypeptides

[0039] (1) The antisense oligonucleotide O was synthesized by the solid-phase phosphoramidite triester method, and the synthesis direction was from the 3'-end to the 5'-end of DNA. First, 33 mg of universal CPG was weighed and loaded into a synthesis column with a sieve plate. ATCG and the thiol-modified C6 S-S phosphoramidite monomer were respectively dissolved in ultradry acetonitrile at a concentration of 0.1 g / mL, and the sulfurizing reagent xanthate was dissolved in ultradry pyridine at a concentration of 0.2 M. Next, the synthesis column was installed on a synthesizer for reaction. The first step was deprotection, and the protecting agent DMT on the 5'-end hydroxyl group was removed with trichloroacetic acid; the second step was activation coupling, and the hydroxyl group at the 5'-end reacted with the phosphoramidite monomer to form a phosphodiester bond; the third step was capping, and the unreacted 5'-end hydroxyl group reacted with acetic anhydride to make it not participate in the next reaction; the fourth step was oxidation, and the trivalent phosphorus of the phosphodiester bond was oxidized to pentavalent phosphorus to complete one cycle. After 16 cycles, the antisense oligonucleotide chain O was obtained. (In addition, for the thiol-modified C6 S-S phosphoramidite monomer, the coupling reaction time needs to be set to 10 min. When synthesizing nucleic acids with thiol modification on the phosphate chain, the thiol nucleoside needs to be replaced with a lowercase letter, and the synthesis sequence should be changed to deprotection, activation coupling, oxidation, capping.) The above synthesis process of the antisense oligonucleotide was automatically completed by a DNA synthesizer.

[0040] (2) The polypeptide P was synthesized by the solid-phase peptide synthesis (SPPS) method. 0.025 mmol of resin with 2,2,2-trichloroethyl chloroformate (2-CTC) (0.97 mmol / g) was swollen in 5 mL of dichloromethane (DCM) in an SPPS synthesis tube for 0.5 h. After removing the DCM, 0.1 mmol of Fmoc-AA-OH was added and dissolved in a small amount of dimethylformamide (DMF). Then, 0.25 mmol of N,N-diisopropylethylamine (DIPEA) and 0.25 mmol of HATU were added. Finally, the reaction mixture was shaken at room temperature for 0.5 h. After the reaction, the solvent was filtered off, and the resin was washed alternately with DCM (3 mL) and DMF (3 mL) three times. Then, the reaction was carried out by adding 3 mL of 20% piperidine / DMF solution and shaking for 5 min, and the solvent was removed by filtration. Then, 3 mL of 20% piperidine / DMF solution was added for 10 min. Then, the resin was washed alternately with DCM (3 mL) and DMF (3 mL) three times. Subsequently, the deprotection, washing, coupling, and washing steps were repeated until all the amino acids were sequentially linked. After all the amino acid residues were linked, 3-maleimidopropionic acid was linked to the N-terminus of the polypeptide in the same way, and thus the polypeptide modified with maleimidopropionic acid was obtained. After the synthesis was completed, 4 mL of a cleavage reagent (TFA:H2O:EDT:TIS volume ratio = 94:2.5:2.5:1.0) was added, and the reaction was shaken at room temperature for 2 h to cleave the peptide from the resin. Then, the filtrate was collected and dried by purging with nitrogen. The extract was precipitated with a certain amount of ice-cold diethyl ether, centrifuged at 3000 r / min for 3 min three times, the solvent was discarded, and finally the crude peptide was obtained. The crude peptide was purified by a conventional HPLC experiment to obtain polypeptide P. The amino acid sequence of the polypeptide P is shown in SEQ ID NO.3, SEQ ID NO.3: LRLRGLRLRG.

[0041] (3) After dissolving 10 μmol of the thiol-modified antisense oligonucleotide O in phosphate-buffered saline (100 μL) with a pH of 7, 3% (w / v) tris(2-carboxyethyl)phosphine (TCEP; 4 μL) was added, and the mixture was shaken for 4 h to reduce the disulfide bond in the thiol-modified antisense oligonucleotide. The maleimide-modified polypeptide P (0.1 mmol) was dissolved in the minimum volume of DMSO (1 μL), and then dropped into the antisense oligonucleotide solution. Then, the pH value was adjusted to 7 with NaOH (0.5 M). The mixture was mechanically shaken at room temperature for 12 h, and thus the polypeptide P and the antisense oligonucleotide O were successfully coupled (as Figure 9 shown). The reaction solution was diluted to 300 μL, filtered and purified, and according to the method for purifying the antisense oligonucleotide O, a conjugate of the antisense oligonucleotide and the polypeptide was obtained. The sequences of the conjugates of the antisense oligonucleotide and the polypeptide and the mass spectrometry results in the present invention are shown in Table 1 and Table 2. The mass spectrometry detection results are as Figure 1 ,Figure 2 and Figure 3 As shown, the experimental quality is consistent with the theoretical calculation.

[0042] Table 1 Conjugate sequences of antisense oligonucleotides and polypeptides and mass spectrometry results

[0043]

[0044]

[0045] Note: Peptide represents polypeptide P, m represents 2'-OMe modification, and * represents P-S modification.

[0046] Table 2 Sequence of antisense oligonucleotide O

[0047] Antisense oligonucleotide O Sequence (5’-3’) Sequence number O1 GTTTAAAAACGATTGT SEQ ID NO.6 O2 AAAAACGATTGTGCAT SEQ ID NO.7 O3 ACGATTGTGCATCAGC SEQ ID NO.8 O4 TTGTGCATCAGCTGAC SEQ ID NO.1 O5 GCATCAGCTGACTGAA SEQ ID NO.2 O6 TCAGCTGACTGAAGCA SEQ ID NO.9 O4-m m(TT)GTGCATCAGCTGm(AC) - O4-mS m(T*T*)GTGCATCAGCTGm(*A*C) - m(O4) m(TTGTGCATCAGCTGAC) - m(O4-S) m(T*T*GTGCATCAGCTG*A*C) -

[0048] Note: m represents 2'-OMe modification, and * represents P-S modification.

[0049] II. In vitro binding performance test of POC1, POC2, POC3, POC4, POC5 and POC6 with frameshift element RNA

[0050] First, NUPACK was used to simulate the energy after the binding of antisense oligonucleotides and frameshift element RNA. As shown in Table 3, the energies after the binding of O1 and O2 with frameshift element RNA are relatively high, indicating that the double-strands formed by them are relatively unstable. While the energies after the binding of O3, O4, O5 and O6 with frameshift element RNA are relatively low, indicating that the binding is more stable.

[0051] Table 3 Binding energies of antisense oligonucleotides and frameshift element RNA

[0052]

[0053]

[0054] Next, subsequent binding experiments were carried out. The reaction mixtures (5 μL) of the conjugates of antisense oligonucleotides and polypeptides POC1, POC2, POC3, POC4, POC5 and POC6 with the complementary pairing of the target RNA contained 1 μM frameshift element RNA (as shown in Table 4), and the concentrations of the conjugates of antisense oligonucleotides and polypeptides were 1 - 20 μM. The whole system was subjected to complementary pairing in buffer 1 (50 mM Tris-HCl pH = 7.0, 0.2 M KCl, 1 mM EDTA) and incubated at 37 °C for 30 min. Then, polyacrylamide gel electrophoresis was used to check the binding efficiency of the conjugates of antisense oligonucleotides and polypeptides with the target RNA.

[0055] Take 2 μL of the incubated solution, then add 2 μL of 5×RNA loading buffer, and dilute the solution to 10 μL with Buffer 1. Thus, the loading solution for polyacrylamide is prepared. Add the loading solution into the wells of a 15% polyacrylamide gel with TBE (45 mM Tris-borate, 10 mM EDTA, pH = 8) as the running buffer. The results are as Figure 4 shown, the binding effect of the conjugate of the antisense oligonucleotide and the polypeptide; POC1 cannot bind to the frameshift element RNA at all concentrations. As the position of POC targeting *FSE35 changes, it is found that the conjugates of the antisense oligonucleotide and the polypeptide, POC2, POC3, POC4, POC5, and POC6, can all effectively bind to the frameshift element RNA *FSE35 in a concentration-dependent manner.

[0056] Table 4 SARS-CoV-2 frameshift element RNA sequences

[0057]

[0058] III. Cutting performance test of POC1, POC2, POC3, POC4, POC5, and POC6 on frameshift element RNA

[0059] The reaction mixtures (5 μL) for the conjugates of the antisense oligonucleotide and the polypeptide, POC1, POC2, POC3, POC4, POC5, and POC6, to cut the target RNA contain *FSE35 or *FSE52 at a concentration of 1 μM, the antisense oligonucleotide conjugate and the polypeptide conjugate at a concentration of 1 - 20 μM, and the whole system conducts the RNA cutting experiment in Buffer 1 (50 mM Tris-HCl, pH = 7.0, 0.2 M KCl, 1 mM EDTA).

[0060] For the experiment with RNase H present, Buffer 2 (20 mM Tris-HCl, pH = 7.8, 40 mM KCl, 8 mM MgCl2, and 1 mM DTT) is used as the buffer solution. The mixture is incubated at 37 °C and transferred to -80 °C to quench the reaction. Take 2 μL of the incubated solution, then add 5 μL of 2×RNA loading buffer (95% formamide, 10 mM NaOH, 10 mM EDTA), and dilute the solution to 10 μL with Buffer 1. Thus, the loading solution for polyacrylamide is prepared.

[0061] Add the loading solution into the wells of a 15% polyacrylamide gel with TBE (45 mM Tris-borate, 10 mM EDTA, pH = 8) as the running buffer, and the voltage is 150 V. To determine the cleavage sites, FSE-RNA is cleaved with 2 M imidazole to generate a marker that cleaves all sites. The results are as Figure 5As shown, the cutting effects of conjugates POC1, POC2, POC3, POC4, POC5, and POC6 of antisense oligonucleotides and polypeptides on the frameshift element RNA are presented. Among them, POC1 has no cutting effect on the frameshift element RNA, but POC2, POC3, POC4, POC5, and POC6 all have relatively obvious cutting and cleavage effects on the frameshift element RNA. Among them, the cleavage efficiencies of POC4 and POC5 are relatively high compared to others. The 72-hour cleavage rates of POC4 and POC5 on *FSE52 are 87% and 67% respectively, which are higher than those of other POCs, such as Figure 8 As shown, the sites where POC4 and POC5 cut the SARS-CoV-2 frameshift element RNA are presented.

[0062] IV. Serum stability test of m(POC4-S)

[0063] 10% serum contains 20 μM of m(POC4-S). It is incubated at 37 °C, and 1 μL of the sample is taken at different time points and 5 μL of loading buffer is added. It is quickly frozen with liquid nitrogen first, and then stored at -80 °C. Finally, polyacrylamide gel electrophoresis is performed, and the results are as Figure 6 shown that m(POC4-S) has good serum stability.

[0064] V. Intracellular frameshift element RNA degradation performance test of m(POC4-S)

[0065] AD293 cells grow in DMEM medium at 37 °C and 5% CO2. This medium is supplemented with 10% fetal bovine serum, 100 unit / mL penicillin, and 100 μg / mL streptomycin. In the previous experiment, POC4 was selected as the sequence with the best cutting effect, so m(POC4-S) was chosen for the intracellular frameshift element RNA degradation performance test. The frameshift element RNA plasmid and different concentrations (0 nM, 100 nM, 250 nM, 500 nM) of m(POC4-S) were transfected using Lipo3000. After culturing for 6 h, the cells were replaced with DMEM antibiotic-free medium supplemented with 10% fetal bovine serum and cultured at 37 °C and 5% CO2 for another 24 h. The results are as Figure 7 shown that the mcherry red fluorescence as the negative control did not decrease significantly, while the green fluorescence as the frameshift element RNA reporter gene decreased significantly, indicating that m(POC4-S) can effectively catalyze the degradation of the frameshift element RNA intracellularly at a concentration of 500 nM.

[0066] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A conjugate of an antisense oligonucleotide and a polypeptide, characterized in that: The general structural formula of the conjugate of the antisense oligonucleotide and polypeptide is shown in formula (I): Wherein, O is an antisense oligonucleotide, L is a linker, and P is a polypeptide; The antisense oligonucleotide is based on the nucleotide sequence of SEQ ID NO.1, and the nucleotide sequence obtained by performing thiophosphate backbone modification on the 1st, 2nd, 14th and 15th positions of the nucleotide sequence shown in SEQ ID NO.1, and performing 2'-O-methyl modification on the ribose of all nucleotides in the nucleotide sequence shown in SEQ ID NO.1; the amino acid sequence of the polypeptide is shown in SEQ ID NO.

3.

2. The conjugate of antisense oligonucleotide and polypeptide according to claim 1, characterized in that: The structural formula of the L linker is shown in formula (II):

3. The conjugate of antisense oligonucleotide and polypeptide according to claim 1, characterized in that: The structural formula of the P polypeptide is shown in formula (III):

4. The method for preparing a conjugate of an antisense oligonucleotide and a polypeptide according to any one of claims 1 to 3, characterized in that: The following steps are involved: The method comprises the following steps: synthesizing an antisense oligonucleotide O with a thiol group modified at the end; preparing a polypeptide P with a maleimide group modified at the end by a polypeptide solid phase synthesis method; coupling the thiol group modified oligonucleotide O with the maleimide group modified polypeptide P, and separating and purifying them to obtain a conjugate of the antisense oligonucleotide and the polypeptide.

5. Use of the conjugate of the antisense oligonucleotide and polypeptide according to any one of claims 1 to 3 in the preparation of a drug for treating SARS-CoV-2 infection.

6. The use according to claim 5, characterized in that: The conjugate of the antisense oligonucleotide and the polypeptide can degrade the SARS-CoV-2 frameshift element.

7. The use according to claim 5, characterized in that: The antisense oligonucleotide O in the conjugate of the antisense oligonucleotide and the polypeptide is complementary to the SARS-CoV-2 frameshift element, and the antisense oligonucleotide O and the polypeptide P are connected by a linker L, and the polypeptide P is transferred to the vicinity of the SARS-CoV-2 frameshift element, and the SARS-CoV-2 frameshift element is targeted for cutting, thereby degrading pathogenic RNA and destroying RNA expression in cells.

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