Antisense oligonucleotide and polypeptide conjugate as well as preparation method and application thereof

Abstract

The invention discloses a conjugate of antisense oligonucleotide and polypeptide as well as a preparation method and application of the conjugate, belongs to the technical field of biological medicine, and provides the conjugate of antisense oligonucleotide and polypeptide, the structural general formula of the conjugate of antisense oligonucleotide and polypeptide is as shown in formula (I), O is antisense oligonucleotide, L is a connexon, and P is polypeptide; the antisense oligonucleotide is a nucleotide sequence obtained by modifying thiophosphate and 2 '-O-methyl on the basis of a nucleotide sequence as shown in SEQ ID NO.1 or a nucleotide sequence as shown in SEQ ID NO.2; and the amino acid sequence of the polypeptide is as shown in SEQ ID NO.3. According to the conjugate of the antisense oligonucleotide and the polypeptide, a phosphodiester bond of RNA is catalyzed by the polypeptide P to break, the antisense oligonucleotide O is complementarily paired with a frame-shift element, the polypeptide P is brought to the vicinity of the frame-shift element for targeted cleavage, and a linker L is responsible for coupling, sequence-specific targeted degradation of pathogenic RNA and destruction of the expression condition of RNA in cells.

Description

Technical Field

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

[0002] Abnormal RNA function is 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. This method of targeting and interfering with RNA can target a variety of RNAs, including messenger RNA, noncoding RNA, and viral RNA. In addition to endogenous RNA in cells, RNA plays a key role in both viruses and bacteria. The functional elements of viral or bacterial transcriptomes / genomes 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 to 25mers. They bind to RNA in cells through Watson-Crick base complementary pairing, prevent its translation, and thus inhibit the expression of disease-related genes. Antisense oligonucleotide drugs are currently on the market.

[0003] Peptides are chain-like biomacromolecules composed of multiple amino acid residues. They have the advantages of easy synthesis, easy chemical modification, and low immunogenicity. The type and order of amino acids in a polypeptide determine its properties. The polypeptide [(LeuArg)2Gly]2 composed of alternating leucine and arginine residues has been shown to be a relatively effective catalytic peptide, which can catalyze the cleavage of phosphodiester bonds in RNA. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a conjugate of an antisense oligonucleotide and a polypeptide, and a preparation method and application thereof, which consists of three parts: an antisense oligonucleotide O, a linker L and a polypeptide P. The polypeptide P can catalyze the breakage of the phosphodiester bond in RNA; the antisense oligonucleotide O is complementary to the frameshift element of SARS-CoV-2, and the catalytic polypeptide P is brought to the vicinity of the frameshift element, thereby targeted cutting thereof, and the linker L is responsible for coupling the two, thereby achieving sequence-specific targeted degradation of pathogenic RNA and destroying the expression of the RNA in cells.

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

[0006]

[0007] Wherein, 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, wherein the 1st, 2nd, 14th and 15th positions of the nucleotide sequence shown in SEQ ID NO.1 are modified with phosphorothioate backbone, and the ribose of all nucleotides in the nucleotide sequence shown in SEQ ID NO.1 are modified with 2'-O-methyl to obtain the nucleotide sequence or the nucleotide sequence shown in SEQ ID NO.2; the amino acid sequence of the polypeptide is 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 the conjugate of the antisense oligonucleotide and polypeptide, comprising the following steps:

[0014] 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.

[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, 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.

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

[0020] The present invention proposes a conjugate of an antisense oligonucleotide and a polypeptide, which consists of three parts: a polypeptide P, a linker L and an antisense oligonucleotide O. The polypeptide P is a catalytic peptide composed of arginine and leucine, which can catalyze the breakage of the phosphodiester bond in RNA; the antisense oligonucleotide O is complementary to the frameshift element in the SARS-CoV-2 gene, and the catalytic polypeptide P can be brought to the vicinity of the frameshift element, so that it can be targeted for cutting; the linker L is responsible for coupling the two macromolecules together. The conjugate of the antisense oligonucleotide and the polypeptide is a very promising treatment method, which realizes sequence-specific targeted degradation of pathogenic RNA and destroys the expression of the RNA in cells.

[0021] The present invention encapsulates the conjugate of antisense oligonucleotide and polypeptide into AD293 cells expressing GFP plasmid containing frameshift element through liposomes, and detects the fluorescence intensity through laser confocal microscope, and finds that the conjugate can effectively reduce the expression of frameshift element RNA. The conjugate of antisense oligonucleotide and polypeptide has high degradation efficiency of frameshift element RNA, and 500nM of antisense oligonucleotide and polypeptide conjugate in cells can effectively degrade frameshift element RNA; the sequence of antisense oligonucleotide can be changed at will according to the RNA to be targeted, and it is expected that this technology can be applied 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 embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments 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.

[0023] Figure 1 The mass spectra of antisense oligonucleotide and polypeptide conjugates POC1, POC2, POC3, POC4, POC5 and POC6, wherein 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 antisense oligonucleotide and peptide conjugates 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 3is the mass spectrum of the SARS-CoV-2 frameshift element RNA, where A is *FSE35 and B is *FSE52;

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

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

[0028] Figure 6 The polyacrylamide gel electrophoresis diagram of m(POC4-S) in serum with changing incubation time;

[0029] Figure 7 This is a laser confocal image showing the cleavage effect of antisense oligonucleotide and peptide conjugates in cells, with a scale of 100 μm;

[0030] Figure 8 the sites for POC4 and POC5 to cleave the SARS-CoV-2 frameshift element RNA;

[0031] Fig. 9 The linker L connects the antisense oligonucleotide O and the polypeptide P. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example

[0038] 1. Synthesis of antisense oligonucleotide and peptide conjugates

[0039] (1) The synthesis of antisense oligonucleotide O was carried out by solid-phase phosphoramidite triester method, and the direction of synthesis was from 3' end to 5' end of DNA. First, 33 mg of general CPG was weighed and loaded into the synthesis column with a sieve plate. ATCG and thiol-modified C6 SS phosphoramidite monomers were dissolved in ultra-dry acetonitrile at a concentration of 0.1 g / mL, and the thiolation reagent hydrogenated xanthine was dissolved in ultra-dry pyridine at a concentration of 0.2 M. Next, the synthesis column was installed on the synthesizer for reaction. The first step was deprotection, in which the protective agent DMT on the 5' terminal hydroxyl group was removed with trichloroacetic acid; the second step was activation coupling, in which the 5' terminal hydroxyl group reacted with the phosphoramidite monomer to form a phosphodiester bond; the third step was capping, in which the unreacted 5' terminal hydroxyl group was reacted with acetic anhydride to prevent it from participating in the next reaction; the fourth step was oxidation, in which the trivalent phosphoric acid of the phosphodiester bond was oxidized to pentavalent phosphoric acid to complete a cycle. After 16 cycles, the antisense oligonucleotide chain O was obtained. (In addition, the coupling reaction time of thiol-modified C6 SS phosphoramidite monomer needs to be set to 10 min. When synthesizing thio-modified nucleic acids on the phosphate chain, the thio nucleosides need to be replaced with lowercase letters, and the synthesis order should be changed to deprotection, activation coupling, oxidation, and capping.) The above antisense oligonucleotide synthesis process is automatically completed by a DNA synthesizer.

[0040] (2) Polypeptide P was synthesized by peptide solid phase synthesis (SPPS) method. 0.025mmol 2-2-chlorotrichloromethane (2-CTC) (0.97mmol / g) of the resin was swollen with 5mL of dichloromethane (DCM) in a SPPS synthesis tube for 0.5h. After removing DCM, 0.1mmol Fmoc-AA-OH was added and dissolved with a small amount of dimethylformamide (DMF). Then, 0.25mmol N,N-diisopropylethylamine (DIPEA) and 0.25mmol HATU were added. Finally, the reaction mixture was shaken at room temperature for 0.5h. After the reaction, the solvent was filtered out, and the resin was washed alternately with DCM (3mL) and DMF (3mL) for 3 times. Then, 3mL of 20% piperidine / DMF solution was added for 5min of shaking reaction, the solvent was removed by filtration, and then 3mL of 20% piperidine / DMF solution was added for 10min. Then, the resin was washed alternately with DCM (3mL) and DMF (3mL) for 3 times. Subsequently, the deprotection, washing, coupling and washing steps are repeated until all amino acids are connected in sequence. After all amino acid residues are linked, 3-maleimidopropionic acid is connected to the N-terminus of the polypeptide in the same way, so that a maleimidopropionic acid-modified polypeptide is obtained. After the synthesis is completed, 4 mL of a cleavage reagent (TFA:H2O:EDT:TIS volume ratio = 94:2.5:2.5:1.0) is added, and the reaction is shaken at room temperature for 2 hours to cut the peptide from the resin. The filtrate is then collected and dried by nitrogen purge. The extract is precipitated with a certain amount of ice ether, centrifuged at 3000r / min for 3 minutes, three times, and the solvent is discarded to finally obtain a crude peptide. The crude is purified by conventional HPLC experiments 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) Dissolve 10 μmol of thiol-modified antisense oligonucleotide O in phosphate buffered saline (100 μL) at pH 7, then add 3% (w / v) tris(2-carboxyethyl)phosphine (TCEP; 4 μL) and shake for 4 hours to reduce the disulfide bonds in the thiol-modified antisense oligonucleotide. Dissolve the maleimide-modified polypeptide P (0.1 mmol) in a minimum volume of DMSO (1 μL) and then add dropwise to the antisense oligonucleotide solution. Then adjust the pH to 7 with NaOH (0.5 M). Mechanically shake at room temperature for 12 hours, and the polypeptide P and antisense oligonucleotide O are successfully coupled (as shown in Figure 2). Fig. 9 The reaction solution was diluted to 300 μL, filtered and purified, and the antisense oligonucleotide and polypeptide conjugate was obtained according to the method for purifying antisense oligonucleotide O. The sequences of the antisense oligonucleotide and polypeptide conjugates in the present invention and the mass spectrometry results are shown in Tables 1 and 2. The mass spectrometry detection results are shown in Figure 1 , Figure 2 and Figure 3 As shown, the experimental quality is consistent with the theoretical calculation.

[0042] Table 1 Sequences of antisense oligonucleotides and peptide conjugates and mass spectrometry results

[0043]

[0044]

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

[0046] Table 2 Sequences of antisense oligonucleotide O

[0047] Antisense Oligonucleotide O Sequence (5'-3') Serial 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 indicates 2'-OMe modification, * indicates PS 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 of the antisense oligonucleotide after binding to the frameshift element RNA. The results are shown in Table 3. The energy of O1 and O2 after binding to the frameshift element RNA is relatively high, which indicates that the double strands they form are relatively unstable, while O3, O4, O5, and O6 have lower energy after binding to the frameshift element RNA, indicating that the binding is more stable.

[0051] Table 3 Binding energy of antisense oligonucleotides to frameshift element RNA

[0052]

[0053]

[0054] Next, the subsequent binding experiment was carried out. The reaction mixture (5 μL) of the complementary pairing of the antisense oligonucleotide and polypeptide conjugates POC1, POC2, POC3, POC4, POC5 and POC6 with the target RNA contained 1 μM frameshift element RNA (as shown in Table 4), the concentration of the antisense oligonucleotide and polypeptide conjugate was 1-20 μM, and the whole system was complementary paired in buffer 1 (50 mMTris-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 antisense oligonucleotide and polypeptide conjugate 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. The loading solution of polyacrylamide is now prepared. The loading solution is added to the loading well of a 15% polyacrylamide gel with TBE (45 mM Tris-boric acid, 10 mM EDTA, pH = 8) as the running buffer. The results are shown in Figure 4 As shown, the binding effect of the conjugates of antisense oligonucleotides and polypeptides; POC1 was unable to bind to the frameshift element RNA at all concentrations. As the position of POC targeting *FSE35 changed, it was found that the conjugates of antisense oligonucleotides and polypeptides POC2, POC3, POC4, POC5 and POC6 could 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] 3. Test of the cleavage performance of POC1, POC2, POC3, POC4, POC5 and POC6 on frameshift element RNA

[0059] The antisense oligonucleotide and polypeptide conjugates POC1, POC2, POC3, POC4, POC5 and POC6 were respectively mixed with the reaction mixture (5 μL) for cutting the target RNA, containing 1 μM *FSE35 or *FSE52, 1-20 μM antisense oligonucleotide conjugates and polypeptide conjugates, and the entire system was used in buffer 1 (50 mM Tris-HCl, pH=7.0, 0.2 M KCl, 1 mM EDTA) for RNA cleavage experiments.

[0060] For experiments with RNase H, buffer 2 (20 mM Tris-HCl, pH = 7.8, 40 mM KCl, 8 mM MgCl2 and 1 mM DTT) was used as the buffer solution. The mixture was incubated at 37°C and transferred to -80°C to quench the reaction. 2 μL of the incubated solution was taken, and then 5 μL of 2× RNA loading buffer (95% formamide, 10 mM NaOH, 10 mM EDTA) was added, and the solution was diluted to 10 μL with buffer 1. The polyacrylamide loading solution was thus prepared.

[0061] The loading solution was added to the sample wells of a 15% polyacrylamide gel with TBE (45 mM Tris-boric acid, 10 mM EDTA, pH = 8) as the running buffer at a voltage of 150 V. To determine the cleavage site, FSE-RNA was cleaved with 2 M imidazole to generate markers for all cleavage sites. The results are shown in Figure 5As shown, the cleavage effects of the antisense oligonucleotide and polypeptide conjugates POC1, POC2, POC3, POC4, POC5 and POC6 on frameshift element RNA. Among them, POC1 has no cleavage effect on frameshift element RNA, but POC2, POC3, POC4, POC5 and POC6 all have a relatively obvious cleavage and cleavage effect on frameshift element RNA. Among them, the cleavage efficiency of POC4 and POC5 is relatively high compared with others. The 72h cleavage rates of POC4 and POC5 on *FSE52 are 87% and 67% respectively, which are higher than other POCs, such as Figure 8 Shown are the sites where POC4 and POC5 cleave the SARS-CoV-2 frameshift element RNA.

[0062] 4. Serum stability test of m(POC4-S)

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

[0064] 5. Test of intracellular frameshift element RNA degradation performance of m(POC4-S)

[0065] AD293 cells were grown in DMEM medium at 37°C and 5% CO2, supplemented with 10% fetal bovine serum, 100 unit / mL penicillin and 100 μg / mL streptomycin. In the previous experiment, POC4 was screened as the sequence with the best cutting effect, so m(POC4-S) was selected to test the degradation performance of frameshift element RNA in cells. Lipo3000 was used to transfect the frameshift element RNA plasmid and m(POC4-S) at different concentrations (0nM, 100nM, 250nM, 500nM). After culturing for 6 hours, the cells were replaced with DMEM antibiotic-free medium supplemented with 10% fetal bovine serum and cultured for another 24 hours at 37°C and 5% CO2. The results are shown in Figure 2. Figure 7 As shown, the red fluorescence of mcherry as a negative control did not decrease significantly, and the green fluorescence of the frameshift element RNA reporter gene decreased significantly, indicating that m(POC4-S) can effectively catalyze the degradation of frameshift element RNA in cells at a concentration of 500 nM.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all 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, wherein the 1st, 2nd, 14th and 15th positions of the nucleotide sequence shown in SEQ ID NO.1 are modified with phosphorothioate backbone, and the ribose of all nucleotides in the nucleotide sequence shown in SEQ ID NO.1 are modified with 2'-O-methyl to obtain the nucleotide sequence or the nucleotide sequence shown in SEQ ID NO.2; 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 drugs for treating coronavirus infection and related diseases.

6. Use of the conjugate of the antisense oligonucleotide and polypeptide according to any one of claims 1 to 3 in the preparation of drugs for treating SARS-CoV-2 infection and related diseases.

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

8. The use according to claim 6, 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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