阅读说明：本技术 抗新型冠状病毒的核酸及其药物组合物与应用 (Anti-novel coronavirus nucleic acid, and pharmaceutical composition and application thereof ) 是由 徐可 蓝柯 于 2021-07-19 设计创作，主要内容包括：本发明提供了一种抗新型冠状病毒的核酸及其药物组合物与应用,所述抗新型冠状病毒的核酸包括：核酸分子SL：具有如SEQ ID NO:1所示的核苷酸序列,能够形成茎环结构SL,抑制病毒蛋白质的合成,进而抑制病毒的复制,所述茎环结构SL包括SL1、SL2、SL3、SL4、SL4.5和SL5共6个茎环分段；以及单独的SL1、SL2、SL3、SL4、SL4.5和SL5核酸分子以及将其进行不同长度的串联结构中的至少一种；所述核酸可以形成茎环结构,在转入细胞后可以靶向NSP1,抑制病毒蛋白质的合成,进而抑制病毒的复制,可为治疗SARS-CoV-2患者提供新的思路和方案。(The invention provides a nucleic acid for resisting a novel coronavirus, a pharmaceutical composition and an application thereof, wherein the nucleic acid for resisting the novel coronavirus comprises: nucleic acid molecule SL: 1, and can form a stem-loop structure SL, inhibit the synthesis of virus protein and further inhibit the replication of viruses, wherein the stem-loop structure SL comprises 6 stem-loop segments including SL1, SL2, SL3, SL4, SL4.5 and SL 5; and at least one of separate SL1, SL2, SL3, SL4, SL4.5, and SL5 nucleic acid molecules and tandem structures thereof of different lengths; the nucleic acid can form a stem-loop structure, can target NSP1 after being transferred into cells, inhibits the synthesis of virus protein, further inhibits the replication of virus, and can provide a new idea and scheme for treating SARS-CoV-2 patients.)

1. An anti-novel coronavirus nucleic acid capable of forming a stem-loop structure and resisting viruses by inhibiting protein synthesis of SARS-CoV-2; the anti-novel coronavirus nucleic acid comprises at least one of the following nucleic acid molecules:

nucleic acid molecule SL: has a nucleotide sequence shown as SEQ ID NO. 1; capable of forming a stem-loop structure SL comprising 6 stem-loop segments of SL1, SL2, SL3, SL4, SL4.5 and SL 5;

nucleic acid molecule SL 1: has a nucleotide sequence shown as SEQ ID NO. 2 and can form a stem-loop structure SL 1;

nucleic acid molecule SL 2: has a nucleotide sequence shown as SEQ ID NO. 3 and can form a stem-loop structure SL 2;

nucleic acid molecule SL 3: has a nucleotide sequence shown as SEQ ID NO. 4 and can form a stem-loop structure SL 3;

nucleic acid molecule SL 4: has a nucleotide sequence shown as SEQ ID NO. 5 and can form a stem-loop structure SL 4;

nucleic acid molecule SL 4.5: has a nucleotide sequence shown as SEQ ID NO. 6 and can form a stem-loop structure SL 4.5;

nucleic acid molecule SL 5: has a nucleotide sequence shown as SEQ ID NO. 7 and can form a stem-loop structure SL 5;

nucleic acid molecule SL 1-2: a series configuration of SL1+ SL 2;

nucleic acid molecule SL 1-3: a series structure of SL1+ SL2+ SL 3;

nucleic acid molecule SL 1-4: has a series structure of SL1+ SL2+ SL3+ SL 4;

nucleic acid molecule SL 1-4.5: has a series structure of SL1+ SL2+ SL3+ SL4+ SL 4.5;

nucleic acid molecule SL 1-5: has a series structure of SL1+ SL2+ SL3+ SL4+ SL4.5+ SL 5;

nucleic acid molecule SL 2-5: has a series structure of SL2+ SL3+ SL4+ SL4.5+ SL 5;

nucleic acid molecule SL 3-5: has a series structure of SL3+ SL4+ SL4.5+ SL 5;

nucleic acid molecule SL 4-5: a series configuration of SL4+ SL4.5+ SL 5;

nucleic acid molecule SL 4.5-5: with a series configuration of SL4.5+ SL 5.

2. The anti-novel coronavirus nucleic acid of claim 1, further comprising: a series structure of one of SL5a, SL5b and SL5c and two-by-two combinations between said SL5a, SL5b and SL5 c; wherein, the SL5a, the SL5b and the SL5c are 3 stem-loop segments of SL5, and the nucleotide sequences are respectively shown as SEQ ID NO 8-SEQ ID NO 10.

3. The anti-novel coronavirus nucleic acid of claim 2, further comprising: SL5 or a series structure of at least one of SL5a, SL5b and SL5c with at least one of SL1, SL2, SL3, SL4, SL 4.5.

4. The nucleic acid of any of claims 1 to 3, wherein the tandem structures are linked using a linker comprising a sequence contained in the full length SL or a flexible protein linker comprising glycine G and serine S (GGGS)_nOr (GGGGS)_nOr (G)_nAnd n is an integer not less than 1.

5. The anti-novel coronavirus nucleic acid of claim 1, further comprising: the nucleic acid molecule is modified by at least one modification, including at least one of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotyping.

6. The anti-novel coronavirus nucleic acid of claim 5, further comprising: at least one of the nucleic acid molecules is linked with a substance for labeling or treatment; the substance for labeling or treatment includes: at least one of a fluorescent marker, a radioactive substance, a therapeutic substance, biotin, digoxin, a nano-luminescent material, a small peptide, and siRNA.

7. A method for the in vitro preparation of a nucleic acid against a novel coronavirus according to any one of claims 1 to 6, said method comprising:

adding a motif of T7 or SP6 to the 5' end of the nucleotide of the nucleic acid molecule, and amplifying by PCR to obtain cDNA of the nucleic acid molecule;

and (3) carrying out in vitro transcription on the cDNA of the nucleic acid molecule to obtain the nucleic acid for resisting the novel coronavirus.

8. A pharmaceutical composition against a novel coronavirus, said pharmaceutical composition comprising:

an effective amount of a pharmacologically acceptable nucleic acid against a novel coronavirus according to any one of claims 1-7;

and a pharmaceutically acceptable carrier.

9. Use of the anti-neocoronavirus nucleic acid according to any one of claims 1-7 and the anti-neocoronavirus pharmaceutical composition according to claim 6 for the preparation of a medicament for the treatment of SARS-CoV-2.

10. The use according to claim 9, wherein the therapeutic agent for SARS-CoV-2 is characterized by the mechanism that the anti-novel coronavirus nucleic acid and the anti-novel coronavirus pharmaceutical composition are used for inhibiting the protein synthesis of SARS-CoV-2.

Technical Field

The invention relates to the technical field of biomedicine, in particular to a novel coronavirus resistant nucleic acid, a pharmaceutical composition and application thereof.

Background

Vaccines are effective prophylactic against viral disease infection, and antiviral drugs provide effective therapeutic means for patients already infected with the virus. Because of the incomplete understanding of the interaction between virus and host, it is difficult to develop highly effective and safe antiviral chemical drugs. The broad spectrum antiviral drugs, Reidcisvir and dexamethasone, have been reported to exhibit activity against SARS-CoV-2. However, the global shortage of Reidesciclovir, the relatively high price and the lack of significant efficacy in patients with severe recurrent coronary pneumonia have not been widely used so far. Dexamethasone is used for the treatment of patients with severe new coronary pneumonia who require mechanical ventilation, however, has limited efficacy in patients with mild new coronary pneumonia who do not require respiratory support. New, non-traditional antiviral drug design and development ideas and approaches are therefore urgently needed.

The nucleic acid is also called as nucleotide medicine, is various oligoribonucleotide (RNA) or oligodeoxyribonucleotide (DNA) with different functions, the action target of the nucleic acid is the base sequence of the gene, and the design is relatively easy, so the nucleic acid medicine has wide application prospect. More than 10 nucleic acid drugs are currently approved worldwide. For example, ASONs can bind to target messenger RNA to form duplexes, thereby degrading mRNA; fomivirsen (trade name Vitravene), norcisane sodium (nusnersen, trade name Spinraza), etc. are approved for clinical treatment by the U.S. FDA and European Commission (EC). To date, no clinical reports have been found for the use of nucleic acid drugs in the treatment of SARS-CoV-2 patients.

Therefore, there is a need to develop a nucleic acid that provides new ideas and protocols for the treatment of SARS-CoV-2 patients.

Disclosure of Invention

The invention aims to provide a novel coronavirus resistant nucleic acid, a pharmaceutical composition and an application thereof, wherein the novel coronavirus resistant nucleic acid comprises a plurality of nucleic acid molecules, the sequences of the nucleic acid molecules can form Stem-loop Structures (SL), after the nucleic acid molecules are transferred into cells, the SL can target NSP1 to inhibit the synthesis of viral proteins, so that the replication of the virus is inhibited, and a novel thought and scheme can be provided for treating SARS-CoV-2 patients.

In a first aspect of the present invention, there is provided a nucleic acid against a novel coronavirus capable of forming a stem-loop structure, which is antiviral by inhibiting protein synthesis of SARS-CoV-2; the nucleic acid resisting the novel coronavirus comprises at least one of the following nucleic acid molecules:

nucleic acid molecule SL: has a nucleotide sequence shown as SEQ ID NO. 1; capable of forming a stem-loop structure SL comprising 6 stem-loop segments of SL1, SL2, SL3, SL4, SL4.5 and SL 5;

nucleic acid molecule SL 1: has a nucleotide sequence shown as SEQ ID NO. 2 and can form a stem-loop structure SL 1;

nucleic acid molecule SL 2: has a nucleotide sequence shown as SEQ ID NO. 3 and can form a stem-loop structure SL 2;

nucleic acid molecule SL 3: has a nucleotide sequence shown as SEQ ID NO. 4 and can form a stem-loop structure SL 3;

nucleic acid molecule SL 4: has a nucleotide sequence shown as SEQ ID NO. 5 and can form a stem-loop structure SL 4;

nucleic acid molecule SL 4.5: has a nucleotide sequence shown as SEQ ID NO. 6 and can form a stem-loop structure SL 4.5;

nucleic acid molecule SL 5: has a nucleotide sequence shown as SEQ ID NO. 7 and can form a stem-loop structure SL 5;

nucleic acid molecule SL 1-2: a series configuration of SL1+ SL 2;

nucleic acid molecule SL 1-3: a series structure of SL1+ SL2+ SL 3;

nucleic acid molecule SL 1-4: has a series structure of SL1+ SL2+ SL3+ SL 4;

nucleic acid molecule SL 1-4.5: has a series structure of SL1+ SL2+ SL3+ SL4+ SL 4.5;

nucleic acid molecule SL 1-5: has a series structure of SL1+ SL2+ SL3+ SL4+ SL4.5+ SL 5;

nucleic acid molecule SL 2-5: has a series structure of SL2+ SL3+ SL4+ SL4.5+ SL 5;

nucleic acid molecule SL 3-5: series structure 5 with SL3+ SL4+ SL4.5+ SL;

nucleic acid molecule SL 4-5: a series configuration of SL4+ SL4.5+ SL 5;

nucleic acid molecule SL 4.5-5: with a series configuration of SL4.5+ SL 5.

Further, the nucleic acid against a novel coronavirus further comprises: a series structure of one of SL5a, SL5b and SL5c and two-by-two combinations among the SL5a, SL5b and SL5 c; wherein, the SL5a, the SL5b and the SL5c are 3 stem-loop segments of SL5, and the nucleotide sequences are respectively shown as SEQ ID NO 8-SEQ ID NO 10.

Further, the nucleic acid against a novel coronavirus further comprises: a series structure of at least one of SL5a, SL5b, and SL5c and at least one of SL1, SL2, SL3, SL4, SL 4.5.

Furthermore, the tandem structures are all connected by a linker, the linker comprises a sequence contained in the SL full length or a flexible protein linker, and the flexible protein linker is formed by glycine G and serine S (GGGS)_nOr (GGGGS)_nOr (G)_nAnd n is an integer not less than 1.

Furthermore, the 5' ends of the nucleic acid molecules are added with motifs of a T7 promoter, and the nucleotide sequence of the T7 promoter is shown as SEQ ID NO. 11.

Further, the nucleic acid against a novel coronavirus further comprises: the nucleic acid molecule is modified by at least one modification, including at least one of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotyping.

Further, the nucleic acid against a novel coronavirus further comprises: at least one of said nucleic acid molecules having attached thereto a substance for labeling or treatment; the substance for labeling or treatment includes: at least one of a fluorescent marker, a radioactive substance, a therapeutic substance, biotin, digoxin, a nano-luminescent material, a small peptide, and siRNA.

In a second aspect of the invention, there is provided a method for the in vitro preparation of said nucleic acid against a novel coronavirus, said method comprising:

adding a motif of T7 or SP6 to the 5' end of the nucleotide of the nucleic acid molecule, and amplifying by PCR to obtain cDNA of the nucleic acid molecule;

and (3) carrying out in vitro transcription on the cDNA of the nucleic acid molecule to obtain the nucleic acid for resisting the novel coronavirus.

In a specific embodiment, the T7 or SP6 motif can be added to the 5' end of the SL nucleotide by a kit, and the T7 or SP6 promoter can be used for in vitro transcription to obtain the nucleic acid against the novel coronavirus according to the method of the kit. The nucleotide sequence of the T7 promoter is shown as SEQ ID NO. 11.

In a third aspect of the present invention, there is provided a pharmaceutical composition against a novel coronavirus, the pharmaceutical composition comprising:

an effective amount of a pharmacologically acceptable nucleic acid against said novel coronavirus;

and a pharmaceutically acceptable carrier.

In the fourth aspect of the invention, the invention provides the application of the nucleic acid for resisting the novel coronavirus and the pharmaceutical composition for resisting the novel coronavirus in the preparation of the therapeutic drugs for SARS-CoV-2.

Furthermore, the mechanism of the therapeutic drug for SARS-CoV-2 is that the anti-novel coronavirus nucleic acid and the anti-novel coronavirus pharmaceutical composition are used for inhibiting the protein synthesis of SARS-CoV-2.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the invention provides a novel coronavirus resistant nucleic acid, a pharmaceutical composition and an application thereof, wherein the novel coronavirus resistant nucleic acid comprises a plurality of nucleic acid molecules, the sequences of the nucleic acid molecules can form a Stem-loop Structure (SL), after the nucleic acid molecules are transferred into cells, the SL can target NSP1 to inhibit the synthesis of viral proteins and further inhibit the replication of viruses, and a novel thought and scheme can be provided for treating SARS-CoV-2 patients.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a schematic diagram of tandem design of nucleic acids SL;

FIG. 2 is a schematic diagram of the viral genome escape NSP1 translational repression function;

FIG. 3 is a graph demonstrating the translational repression of the viral genome escape NSP 1; FIG. 3A shows the result of inhibition of host gene expression by NSP1 (reported as CMV-renilla activity), and FIG. 3B shows the translation inhibition function of viral genome escape NSP1 (reported as 5' UTR-luciferase activity)

FIG. 4 shows the effect of different tandem forms of SL on the expression activity of the viral 5 'UTR promoter (reported as the 5' UTR-luciferase activity);

FIG. 5 shows SL inhibition of SARS-CoV-2 virus N protein expression (reported as activity of 5' UTR-N);

FIG. 6 shows the result of suppression of viral 5' UTR promoter N protein expression by a truncated small stem-loop nucleotide of SL 5;

FIG. 7 shows that SL5 inhibits protein expression of SARS-CoV-2 live virus;

FIG. 8 is a pattern diagram of each fragment of the stem loop of the full-length nucleic acid SL.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are provided to illustrate the invention, and not to limit the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, 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 belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.

In order to solve the technical problems, the general idea of the embodiment of the application is as follows:

the prior art discloses that SARS-CoV-2 is a single strand positive strand RNA virus with enveloped genome size varying from 29.8kb to 29.9kb, belonging to the genus beta of the coronavirus family. SARS-CoV-2 genome gene 1 encodes two large overlapping open reading frames (ORF1a and ORF1b) and various structural and non-structural accessory proteins. After infection, SARS-CoV-2 hijacks the translation machinery of the host cell, synthesizing ORF1a and ORF1b polyproteins, which are subsequently cleaved by proteolytic cleavage into 16 mature non-structural proteins, namely NSP1 to NSP 16. Among them, NSP1 is an important virulence factor of coronavirus, and through direct binding with 40S small ribosome subunit, effectively inhibits translation of host mRNA, and plays an important role in inhibiting host gene expression, promoting virus replication and immune evasion. On the other hand, NSP1 can facilitate translation of SARS-CoV-2 by binding to its 5' UTR. Thus NSP1 is a potential therapeutic target for limiting the replication of SARS-CoV-2.

The invention takes NSP1 as a target point, discovers a plurality of nucleic acids, and the nucleic acid sequence can form a Stem-loop Structure (SL), after the SL is transferred into cells, the SL can target NSP1, inhibit the synthesis of virus protein, further inhibit the replication of virus, and provide a new thought and scheme for treating SARS-CoV-2 patients. Specifically, the method comprises the following steps:

the Applicant has first found that the nucleic acid molecule 1: has a nucleotide sequence shown as SEQ ID NO. 1; can form a stem-loop structure SL, inhibit the synthesis of virus protein and further inhibit the replication of virus, wherein the stem-loop structure SL comprises 6 stem-loop segments of SL1, SL2, SL3, SL4, SL4.5 and SL 5;

subsequently, the applicant found through experiments that the individual SL1, SL2, SL3, SL4, SL4.5 and SL5 nucleic acid molecules and the tandem connection thereof with different lengths are SL1+ SL2, SL1+ SL2+ SL3, SL1+ SL2+ SL3+ SL4, SL1+ SL2+ SL3+ SL4+ SL4.5, SL1+ SL2+ SL3+ SL4+ SL4.5+ SL5, SL2+ SL3+ SL4+ SL4.5+ SL5, SL3+ SL4+ SL4.5+ SL5, SL4+ SL4.5+ SL5, and SL4.5+ SL5 can also form stem-loop structures, thereby inhibiting the synthesis of viral proteins and further inhibiting the replication of viruses.

Therefore, one or any combination of more than one of the above nucleic acid molecules can be used as a nucleic acid drug against the novel coronavirus.

The details of a novel coronavirus resistant nucleic acid, its pharmaceutical composition and its application are described in the following examples and experimental data.

Example 1 tandem design and in vitro Synthesis of Stem-Loop structures

1. Stem-loop SL nucleotide tandem design

As shown in FIG. 1, six RNA sequences named SL1, SL2, SL3, SL4, SL4.5 and SL5 are constructed and connected in series with different lengths, namely SL1+ SL2, SL1+ SL2+ SL3, SL1+ SL2+ SL3+ SL4, SL1+ SL2+ SL3+ SL4+ SL4.5, SL4+ SL4+ SL4+ SL4+ SL4.5+ SL4, SL4+ SL4+ 4.5+ SL4, SL4+ SL4+ 4.5+ SL4, SL4.5+ 4, SL 4-2, SL 4-3, SL 4-4-5-SL 4, SL4-SL 4, SL 365-SL 4 and SL 365-SL 4 are constructed in series.

2. In vitro synthesis of stem-loop SL nucleotide tandem

(1) Obtaining cDNA of each stem-loop SL nucleotide

As shown in the following table, the motif TAATACGACTCACTATAGGG (SEQ ID NO:11) obtained by adding the T7 promoter to the 5' end of the SL nucleotides of different tandem lengths described above was assigned to the Oncology Biotechnology Ltd. Motif ATTTAGGTGACACTATAGAAGNG of sp6 (SEQ ID NO:12) may also be employed in other embodiments; and carrying out PCR amplification by taking the synthesized SL sequence as a template to obtain the cDNA of SL1, SL2, SL3, SL4, SL4.5, SL5, SL1-SL2, SL1-SL3, SL1-SL4, SL1-SL4.5, SL1-SL5, SL2-SL5, SL3-SL5, SL4-SL5 and SL4.5-SL 5.

The primer pairs used are shown in table 1:

TABLE 1 amplification of cDNA corresponding primers

cDNA sequence of interest	Upstream primer	Downstream primer
			SL1	SL1 F	SL1 R
SL2	SL2 F	SL2 R
			SL3	SL3 F	SL3 R
SL4	SL4 F	SL4 R
			SL4.5	SL4.5 F	SL4.5 R
SL5	SL5 F	SL5 R
			SL1-SL2	SL1 F	SL2 R
SL1-SL3	SL1 F	SL3 R
			SL1-SL4	SL1 F	SL4 R
SL1-SL4.5	SL1 F	SL4.5 R
			SL1-SL5	SL1 F	SL5 R
SL2-SL5	SL2 F	SL5 R
			SL3-SL5	SL3 F	SL5 R
SL4-SL5	SL4 F	SL5 R
			SL4.5-SL5	SL4.5 F	SL5 R

TABLE 2 primers

The PCR reaction system is shown in Table 3:

TABLE 3

Composition (I)
		ddH2O	32μL
Upstream primer	1μL
		Downstream primer	1μL
10xPCR buffer	5μL
		dNTP	5μL
MgSO4	3μL
		SARS-CoV-2 genome	1μL
KOD-plus-neo	1μL

PCR reaction procedure: at 94 ℃, cycle entry after 2 min: 10s at 98 ℃; 30s at 55 ℃; 68 ℃ for 30 s. For a total of 35 cycles, and then stored at 4 ℃. The cDNA for each SL nucleotide was subsequently recovered by DNA agarose electrophoresis. Then through OMEGA e.z.n.a.Gel Extraction Kit, purification of the cDNA obtained above, the procedure was as follows:

adding 100 mu L binding buffer and 100 mu L PCR product into an EP tube, uniformly mixing, adding the mixture into an adsorption column, centrifuging at the maximum revolution for 1min, and discarding waste liquid.

② adding 300 mu L binding buffer into the adsorption column, centrifuging for 1min at the maximum revolution, and discarding the waste liquid.

③ adding 700 mu L of SPW buffer into the adsorption column, centrifuging for 1min at the maximum revolution, and discarding the waste liquid.

Fourthly, repeat

Fifthly, the adsorption column is put back to the collecting pipe again, and the centrifugation is carried out for 1min at the maximum revolution.

Sixthly, putting the adsorption column into a new centrifugal tube with 1.5mL, dripping 20 mu L of Elution buffer on the centrifugal column, standing for 2min at room temperature, and centrifuging for 1min at the maximum revolution.

(2) The RNA sequence corresponding to each cDNA can be obtained by in vitro Transcription of the purified cDNA by using a full-scale gold T7 High Efficiency Transcription Kit, and the reaction system and the steps are as follows:

TABLE 4

Composition (I)
		Form panel	1μg
5xT7 buffer	4μL
		NTP Mix	8μL
T7 Transcripton Reation Buffer	2μL
		ddH2O	5μL

Mixing, incubating at 37 deg.C for 2h, adding 1 μ L DNase I, reacting at 37 deg.C for 15min, adding 1 μ L500 mM EDTA (pH 8.0) to terminate the reaction to obtain RNA;

the RNA obtained in the above step needs further purification, and the operation steps are as follows:

adding phenol: chloroform: shaking the mixed solution with isoamyl alcohol volume ratio of 25:24:1 to form emulsion, centrifuging at 12000rpm and 4 ℃ for 1min, taking supernatant, and adding into a new 1.5mL EP tube

② 1/10 volume of 5M ammonium acetate and 2 volume of absolute ethyl alcohol are added, and the mixture is stood for 15min at-20 ℃.

③ centrifuging at 13000rpm for 15min at 4 ℃, discarding the supernatant, drying in the air, adding 100 mu L DEPC water, and preserving at-80 ℃.

Example 2 SL5 leads to transcriptional repression of viral RNA by competitive binding to NSP1

(1) Luciferase reporter System detecting inhibition of 5' UTR on translation of proteins by NSP1 protein

The document reports that SARS-CoV-2 non-structural protein NSP1 binds to host cell ribosome, prevents the entry of host cell mRNA into ribosome, and inhibits the translation of host cell mRNA, as shown in FIG. 2A. However, the 5' UTR of the viral genome forms a stem-loop structure that binds to NSP1 to detach it from the ribosome, and thus the viral RNA genome can enter the ribosome and be translated into viral proteins, as shown in fig. 2B. In order to verify the functions, a luciferase reporter plasmid with a virus 5 'UTR as a promoter is designed to simulate the transcription of virus RNA (5' UTR-luciferase), meanwhile, the reporter plasmid with a CMV promoter is adopted to simulate the transcription of cell mRNA (CMV-renilla), and the co-transfer NSP1 protein is used for detecting the promotion effect of the co-transfer NSP1 protein on the translation of the virus RNA and the inhibition effect of the translation of host RNA. The specific operation is as follows:

293A cells were plated in 24-well plates and when the cells grew to 30% -50%, group 1 was cotransformed with 500ng CMV-renilla and 100ng pCAG-unloaded or pCAG-NSP1 and group 2 was cotransformed with 500ng 5' UTR-luciferase and 100ng pCAG-unloaded or pCAG-NSP 1. Fluorescence was measured after incubation at 37 ℃ for 24 h. The experimental result is shown in fig. 3, NSP1 can significantly inhibit the expression of CMV-renilla (fig. 3A), which indicates that NSP1 has significant inhibitory effect on the translation of cellular mRNA; while NSP1 did not inhibit transcription of the 5 'UTR-luciferase reporter plasmid (fig. 3B), indicating that the 5' UTR of viral RNA can escape the translational repression of NSP 1.

(2) Detection of interference function of SL nucleotide on translation inhibition of NSP1

293A cells were plated in 24-well plates, and when the cells grew to 30% -50%, group 1 and group 2 were transfected with 20pmol ncRNA, and groups 3-18 were transfected with 20pmol stem-loop RNA SL1, SL2, SL3, SL4, SL4.5, SL5, SL1-2, SL1-3, SL1-4, SL1-4.5, SL1-5, SL2-5, SL3-5, SL4-5, SL4.5-5, and cultured at 37 ℃ for 8 h.

Group 1 co-transfected 500ng 5 'UTR-luc and 100ng pCAG, and groups 2-18 co-transfected 500ng 5' UTR-luciferase and 100ng pCAG-NSP 1. Fluorescence was measured after incubation at 37 ℃ for 24 h.

The experimental result shows that under the condition of expressing NSP1, the luciferase gene with SARS-CoV-2 virus 5' UTR can escape the protein translation inhibition effect of NSP1, and normal protein expression is carried out. At this time, the fluorescence values of 5 ' UTR-luciferase were decreased to different extents after transfection of different stem-loop nucleotides SL, wherein SL5 and the different truncated SLs containing SL5 inhibited the expression of 5 ' UTR-luciferase most significantly (the result is shown in FIG. 4), which indicates that the stem-loop nucleotides, especially SL5, could counteract the translational inhibition function of 5 ' UTR escape NSP1, i.e. SL5 led the translation of viral RNA to be inhibited by competitive binding to NSP1, thus SL5 has the potential function of inhibiting the expression of SARS-CoV-2 protein.

Example 3 detection of inhibitory Effect of SL on SARS-CoV-2 protein expression

(1) Detecting the function of different tandem SL nucleotides on the translation inhibition of 5' UTR-N protein

To verify whether the SL nucleotide designed by the present invention inhibits SARS-CoV-2 protein expression, this example uses SARS-CoV-2 structural protein N from the 5' UTR promoter to perform an experiment.

293A cells were plated in 24-well plates, and when the cells grew to 30% -50%, group 1 and group 2 were transfected with 20pmol ncRNA, and experimental group 3-group 13 were transfected with 20pmol stem-loop RNA SL1, SL1-2, SL1-3, SL1-4, SL1-4.5, SL1-5, SL2-5, SL3-5, SL4-5, SL4.5-5, SL5, and cultured at 37 ℃ for 8 h.

Group 1 co-transferred 500ng 5 'UTR-luciferase and 100ng pCAG, and groups 2-13 co-transferred 500ng 5' UTR-N and 100ng pCAG-NSP 1. And culturing at 37 ℃ for 24h, and detecting the expression quantity of the N protein.

As shown in FIG. 5, in the case of NSP1 expression, the N protein expression level of the 5 'UTR promoter was not affected, but after co-transfection of SL nucleotides in different tandem forms, the N protein expression level was decreased to different extents, and especially the suppression of 5' UTR-N expression by SL5 or SL nucleotides in tandem with SL5 was most significant. The SL5 can obviously inhibit the N protein expression of the 5' UTR promoter; the nucleotide SL (SL1-5, SL2-5, SL3-5, SL4-5 and SL4.5-5) connected with SL5 in series has the function of inhibiting the N protein expression of a 5' UTR promoter.

(2) Detecting the function of the SL5 truncated stem-loop RNA on the translation inhibition of 5' UTR-N protein

In order to verify whether the SL5 nucleotide truncated stem-loop RNA (i.e., SL5a, SL5b, SL5c) designed by the present invention inhibits the expression of SARS-CoV-2 protein, an experiment was performed using SARS-CoV-2 structural protein N of the 5' UTR promoter.

293A cells are paved on a 24-well plate, when the cells grow to 30% -50%, each group is transfected with 100ng of pCAG-NSP1, meanwhile, group 1 is transfected with 20pmol of ncRNA, experimental group 2-group 5 are transfected with 20pmol of SL5, SL5a, SL5b and SL5c respectively, and the N protein expression amount is detected after the cells are cultured at 37 ℃ for 24 h.

The results are shown in fig. 6, under the condition of NSP1 expression, the expression level of N protein of 5' UTR of the group of transfected ncrnas is not affected, and after co-transfection of SL5, the expression level of N protein is obviously reduced; similarly, the 5' UTR-N expression level is also obviously reduced after the truncated nucleotides SL5a, SL5b and SL5c of the co-transfected SL 5. The truncated nucleotides of SL5 and SL5 are proved to have the function of inhibiting the N protein expression of a 5' UTR promoter.

(3) Detection of the inhibitory Effect of SL5 on protein expression of SARS-CoV-2 live Virus

In order to further verify whether the SL5 designed by the present invention inhibits the protein expression of the SARS-CoV-2 live virus, in this example, different doses of SL5 were used to transfect Vero-E6-ACE2 cells, then the cells were infected with the SARS-CoV-2 live virus, and the expression of the viral N protein and S protein in the cell lysate was detected, as follows:

293A cells were plated in 24-well plates, and when the cells grew to 30% -50%, group 1 was left untreated, group 2 was transfected with 200pmol ncRNA, and groups 3-5 were transfected with 50pmol, 100pmol, and 200pmol stem-loop RNA SL5, respectively, and cultured at 37 ℃ for 8 h.

Subsequently, the cell culture plate is moved into an ABSL-3 laboratory and is infected with SARS-CoV-2 with the MOI of 0.01, after the culture is continued for 24 hours, the cells are collected, 2x SDS loading buffer is added for sample preparation, 100-degree metal bath is carried out for 15min, and then the expression conditions of SARS-CoV-2N protein and S protein are analyzed through western blot.

The experimental results are shown in FIG. 6, after SARS-CoV-2 infection of the cell transfected with the control ncRNA, the expression of the virus N protein and S protein is not affected, while after SARS-CoV-2 infection of the cell transfected with different dosage of SL5, the expression of the virus N protein and S protein is dose-dependently inhibited by SL5, which shows that SL5 has the function of inhibiting the expression of SARS-CoV-2 live virus protein and is a potential nucleic acid drug for inhibiting the replication of SARS-CoV-2.

Combining the experimental results of FIG. 5 and FIG. 6, it can be concluded without any doubt that other stem-loop structures such as SL1-5, SL2-5, SL3-5, SL4-5 and SL4.5-5 have the effect of inhibiting the expression of SARS-CoV-2 protein. This shows that other SL nucleotides in tandem with SL5 all have the effect of inhibiting the expression of SARS-CoV-2 protein, and are potential nucleic acid drugs for inhibiting the replication of SARS-CoV-2.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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