CN117821463A - A nucleic acid specifically resisting viral proliferation and its application - Google Patents

Abstract

The invention discloses a specific antiviral proliferation nucleic acid and application thereof, wherein the nucleic acid has a free terminal or annular structure, and depends on nucleic acid aptamer and virus infected cells to mediate targeted delivery into cells, and a chimeric body is formed by the following 3 parts: (1) Targeting nucleic acid aptamers that bind to viral surface proteins as elements; (2) Antisense oligonucleotides targeted to silence viral RNAs as elements; (3) Nucleic acid sequence Linker connecting the elements in (1) and (2). For example, the nucleic acid is a nucleic acid that specifically resists SARS-CoV-2 inflammation and SARS-CoV-2 proliferation; the aptamer is targeted to bind SARS-CoV-2Spike protein; the antisense oligonucleotide is antisense oligonucleotide for targeted silencing of SARS-CoV-2 RNA; the chimera has the capacity of inhibiting virus, such as SARS-CoV-2 virus, to induce inflammation reaction and antiviral duplication, and has the advantages of high stability, low immunogenicity, etc. The invention can be applied in the fields of prevention and treatment, diagnosis, biological imaging and the like of viruses (such as SARS-CoV-2 virus).

Description

A nucleic acid specifically resisting viral proliferation and its application

Technical Field

The present invention relates to the fields of biotechnology and medicine, and to a nucleic acid that specifically resists viral proliferation and its application, and in particular to a nucleic acid aptamer-oligonucleotide chimera that binds to the spike protein of the new coronavirus (SARS-CoV-2) and its application.

Background technique

The novel coronavirus pneumonia is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is defined as a pandemic by the World Health Organization (WHO). Patients with this disease usually show typical symptoms of fever and dry cough, in addition to fatigue, headache and other phenomena. In severe cases, they may experience shortness of breath or difficulty, or even death. The new coronavirus is an RNA virus, and because it is highly conservative with the SARS-CoV virus, it is named SARS-CoV-2.

The protein structure of the new coronavirus mainly contains spike glycoprotein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). Among them, the S protein is the key protein for SARS-CoV-2 virus to enter cells. It is located on the surface of the virus and belongs to the surface protein of the virus. It can bind to the human angiotensin-converting enzyme 2 (ACE2) receptor and mediate its infection of host cells. In addition, the S protein can also bind to the pattern recognition receptor (TLR4) on the human cell membrane to activate the immune response. The N protein is the only protein present in the nucleocapsid. It forms a complex structure by binding to the genomic RNA, which plays an important role in the subsequent replication of the virus. Secondly, the N protein can also activate the intracellular NF-κB signaling pathway and cause an inflammatory response in the cell.

Aptamers are a type of single-stranded DNA or RNA molecules obtained by in vitro screening technology. They are ligands with high affinity and specificity. The targets that can bind include carbohydrates, cells, nucleic acids, peptides, protein small molecules and viruses. Compared with natural antibodies in organisms, aptamers are obtained by chemical synthesis and modification, without the need for animal immunization, and their affinity and specificity with the target can be controlled through the screening process. Therefore, aptamers are also called "chemical antibodies" and have the advantages of small molecular weight and low immunogenicity. Antisense oligonucleotides (ASOs) are single-stranded oligonucleotide molecules, usually containing 15-25 nucleotides. After entering the cell, they bind to their complementary target mRNA through the principle of base complementary pairing under the action of ribonuclease H1 (RNase H1), inhibiting the expression of the target gene. However, the current delivery of ASO drugs generally only relies on chemical and biotechnological construction, such as liposome nanoparticles, sugar-nucleic acid conjugates, lipid-nucleic acid conjugates or exosome membranes, etc., and the targeted delivery capability is limited. Efficient delivery systems are still a major demand in the field of nucleic acid drugs. In addition, the stability of nucleic acids in the body is also a problem.

In order to overcome these difficulties in the treatment of SARS-CoV-2 or other viral patients, a nucleic acid drug structure that can solve the targeted delivery and stability of ASO needs to be constructed. At present, there are no reports on the use of chimeras of nucleic acid aptamers and antisense oligonucleotides for antiviral proliferation, especially anti-SARS-CoV-2 inflammation and inhibition of its replication, nor are there any reports on the cyclization of chimeras of Aptamer and ASO.

Summary of the invention

The technical problem to be solved by the present invention is to provide a specific anti-viral proliferation nucleic acid and its application, so that the nucleic acid can achieve targeted delivery of ASO nucleic acid drugs, has the advantages of strong stability and low immunogenicity, and can also achieve the effect of anti-viral proliferation.

The present invention takes the SARS-CoV-2 virus as the research basis, and uses the nucleic acid aptamer targeting the S protein of SARS-CoV-2 and the antisense oligonucleotide targeting the N gene of SARS-CoV-2 as examples, and combines the nucleic acid aptamer (SApt1) of the S protein with the ASO (NASO2) targeting the N gene to construct linear SApt1-NASO2 and circular SApt1-NASO2 (circ SApt1-NASO2). The chimera is delivered into cells by relying on SApt1 and SARS-CoV-2 virus infection, and exerts the ability to specifically inhibit inflammation and resist viral proliferation.

Based on the above research, the present invention has developed nucleic acids that specifically resist viral proliferation and their applications, as follows:

The present invention provides a specific antiviral proliferation nucleic acid having a free end or a ring structure, which relies on nucleic acid aptamers and virus-infected cells to mediate targeted delivery into cells, and is composed of the following three parts:

(1) nucleic acid aptamers that target and bind to viral surface proteins as components;

(2) antisense oligonucleotides targeting silencing viral RNA as elements;

(3) Linker, a nucleic acid sequence that connects the elements in (1) and (2).

One of the main design concepts of the present invention is to utilize nucleic acid aptamers to bind to viral surface proteins, thereby simultaneously delivering antisense oligonucleotides when the virus infects cells, and then achieving an antiviral proliferation effect through the action of antisense oligonucleotides.

As a further improvement of the present invention, the virus is the novel coronavirus SARS-CoV-2, the nucleic acid is a nucleic acid that specifically resists SARS-CoV-2 inflammation and SARS-CoV-2 proliferation; the nucleic acid aptamer is a nucleic acid aptamer that targets and binds to the SARS-CoV-2 Spike protein; the antisense oligonucleotide is an antisense oligonucleotide that targets and silences SARS-CoV-2 RNA; the elements in (1) and (2) and the linker in (3) are DNA or RNA.

Among them, the Spike protein is the surface protein of the SARS-CoV-2 virus. Binding the nucleic acid aptamer to it can achieve the delivery of antisense oligonucleotides that target silencing SARS-CoV-2 RNA. It also has the ability to inhibit the inflammatory response induced by the SARS-CoV-2 virus and resist the replication of the SARS-CoV-2 virus.

As a further improvement, the ring structure is to connect the 5' end and the 3' end of the nucleotide sequence into a ring; the connection method is any one of the following: T4 DNA ligase connection, biosynthetic connection, chemical connection; the nucleic acid sequence Linker used for connection is any one of the following: DNA, RNA with or without a marker; the marker is a fluorescent marker, biotin, Protac, digoxin, a small peptide, a nanoluminescent material, a biological small molecule, a therapeutic substance, a radioactive substance, siRNA or an enzyme marker; the chimera of the ring structure contains a stem-loop structure.

Although one of the T4 DNA ligase connection methods is used in the embodiments of the present invention, it is understandable that those skilled in the art can also adopt other conventional connection methods in the prior art as described above based on the design concept of the present invention, and can also achieve similar technical effects as the present invention.

As a further improvement, the nucleic acid aptamer includes a derivative, which is a modified thiophosphate derivative that can specifically bind to a viral surface protein, or a peptide nucleic acid that specifically binds to a viral surface protein; when the virus is the new coronavirus SARS-CoV-2, the derivative is a modified thiophosphate derivative that can specifically bind to the SARS-CoV-2 Spike protein, or a peptide nucleic acid that specifically binds to the Spike-Tri protein of SARS-CoV-2.

That is, in the present invention, in addition to conventional nucleic acid aptamers, those skilled in the art may also adopt derivatives thereof without affecting their functions, which can also achieve similar or enhanced effects.

Furthermore, the antisense oligonucleotide is a targeted knockdown of viral RNA, such as an antisense oligonucleotide targeted to knockdown SARS-CoV-2 RNA, including targeted knockdown of genes expressing non-structural proteins, genes expressing structural proteins, genes expressing auxiliary proteins, and regulatory genes. By knocking down the RNA of the above-mentioned virus, the proliferation of the gene can be inhibited.

Furthermore, the antisense oligonucleotide is an antisense oligonucleotide that targets and knocks down the N gene of SARS-CoV-2.

Furthermore, the specific antiviral proliferation nucleic acid is a modified nucleotide sequence, and the modification method adopts any one or more of the following: phosphorylation, amination, methylation, sulfhydrylation, isotopization, replacement of oxygen with sulfur, and replacement of oxygen with selenium.

That is, in the present invention, without affecting its function, those skilled in the art can also perform the above-mentioned conventional modifications on the nucleic acid to achieve similar or enhanced effects.

Furthermore, the delivery also includes other enhanced delivery carriers coordinated with the specific anti-viral proliferation nucleic acid, including delivery relying on chemical and biotechnology construction; the delivery relying on chemical and biotechnology construction adopts any one of liposome nanoparticles, sugar-nucleic acid conjugates, lipid-nucleic acid conjugates or exosome membranes.

The present invention utilizes nucleic acid aptamers to bind to viral surface proteins, thereby achieving the delivery of antisense oligonucleotides simultaneously when the virus infects cells. It is understandable that under this design concept, on the basis of affecting normal functions, in order to further enhance its delivery effect, technical personnel in this field can also combine the above-mentioned conventional delivery methods.

Furthermore, the SARS-CoV-2 is a wild-type SARS-CoV-2 or a mutant thereof, such as a Delta, Lambda, Omicron mutant, etc.

Based on the above research, the present invention further provides the use of the above-mentioned specific antiviral proliferation nucleic acid, wherein the nucleic acid is used for any one or more of the following groups:

(1) Isolating and purifying virus surface proteins, such as isolating and purifying the Spike protein of SARS-CoV-2;

(2) Marking the surface protein/gene of the virus, such as marking the Spike protein/gene of SARS-CoV-2;

(3) Imaging of viral surface proteins/genes, such as the Spike protein/gene of SARS-CoV-2;

(4) Enrichment of viral surface proteins/genes, such as enrichment of SARS-CoV-2 Spike protein/gene;

(5) Quantitative or qualitative detection of viral surface proteins/genes, such as quantitative or qualitative detection of the Spike protein/gene of SARS-CoV-2;

(6) Preparation of nucleic acid inhibitors of viral surface proteins/genes, such as preparation of nucleic acid inhibitors of SARS-CoV-2 Spike protein/gene;

(7) Preparing reagents or drugs targeting viral surface proteins/genes, such as preparing reagents or drugs targeting the Spike protein/gene of SARS-CoV-2;

(8) preparing inhibitors that block the binding between viral surface proteins and TLR4 proteins, such as preparing inhibitors that block the binding between SARS-CoV-2 Spike proteins and TLR4 proteins;

(9) Preparation of reagents or drugs for the diagnosis and treatment of viral infections or inflammation-related diseases; such as the preparation of reagents or drugs for the diagnosis and treatment of pneumonia infections or inflammation-related diseases caused by SARS-CoV-2;

(10) Preparing kits for detecting, imaging, diagnosing and treating viruses, such as preparing kits for detecting, imaging, diagnosing and treating SARS-CoV-2.

By adopting the above technical solution, the present invention has at least the following advantages:

1. A specific antiviral proliferation nucleic acid of the present invention utilizes a nucleic acid aptamer to bind to a viral surface protein, thereby being able to simultaneously deliver an antisense oligonucleotide when the virus infects cells, and has the advantages of strong stability and low immunogenicity. In addition, in combination with an antisense oligonucleotide that targets silencing viral RNA, antiviral proliferation can be achieved, especially when the nucleic acid aptamer and the antisense oligonucleotide chimera form a ring, the stability is significantly enhanced, and the antiviral proliferation effect is greatly improved.

2. The present invention combines a nucleic acid aptamer (Aptamer) that targets and binds to the Spike protein of the new coronavirus (SARS-CoV-2) and an antisense oligonucleotide (ASO) that targets and silences SARS-CoV-2 RNA to construct a nucleic acid aptamer-antisense oligonucleotide chimera and a cyclized circular chimera. The chimera and its circular chimera achieve targeted delivery of nucleic acid drugs by relying on nucleic acid aptamers and viral infection-mediated methods. The chimera and its cyclic structure have the ability to significantly inhibit SARS-CoV-2 virus-induced inflammatory response and resist SARS-CoV-2 virus replication, and at the same time have the advantages of strong stability and low immunogenicity, and can be used in the fields of prevention, diagnosis, and biological imaging of the new coronavirus.

BRIEF DESCRIPTION OF THE DRAWINGS

The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

FIG1 is a schematic diagram showing the structural comparison of SAPT-NASO and its cyclized form cirSApt-NASO;

FIG2 is a fluorescence image verifying the expression of N-GFP gene knocked down by 4 NASO in HEK-293T cells;

Figure 3 shows the effect of chimera structure on cyclization efficiency; wherein (a) is a schematic diagram and a proof diagram of SAPT2-NASO2 cyclization; (b) is a schematic diagram and a proof diagram of SAPT1-NASO2 cyclization;

FIG4 is a diagram evaluating the effect of linkers of different lengths on chimera circularization;

Figure 5 is a diagram for evaluating the stability of circularized chimeras and linear chimeras; wherein (a) is the electrophoresis analysis of SAPT1-NASO2 and its circularized form and random sequence and its quantitative data; (b) is the electrophoresis analysis of Linker-13 and Linker-18 and their circularized form and its quantitative data;

Figure 6 is an analysis diagram of the ability of the chimera SAPt1-NASO2 and its cyclized form to bind to the Spike protein of the SARS-CoV-2 virus and to inhibit the binding of S-TLR4; wherein, (a) is an analysis diagram of the ability of the chimera SAPt1-NASO2 and its cyclized form to inhibit the binding of S-TLR4; (b) is a diagram of the ability of the chimera SAPt1-NASO2 and its cyclized form to bind to the Spike protein of the SARS-CoV-2 virus;

Figure 7 is a graph showing the KD values of the chimera SAPt1-NASO2 and its cyclized form and the SARS-CoV-2 virus Spike protein; wherein (a) is the KD value of SAPt1; (b) is the KD value of the linear SAPt1-NASO2; (c) is the KD value of the cyclized SAPt1-NASO2;

FIG8 is a fluorescence graph showing that different concentrations of chimera SAPt1-NASO2 and its cyclized form inhibit N-GFP gene expression in HEK293T cells;

Figure 9 is a diagram showing the expression of inflammatory factors induced by Spike protein in THP-1 cells by the chimera SAPt1-NASO2 and its cyclized form, wherein (a) is the expression of IL-1β mRNA; (b) is the expression of IL-6 mRNA; (c) is the expression of TNF-α mRNA;

FIG10 is a graph showing the ability of the chimera SAPt1-NASO2 and its cyclized form to inhibit inflammation-induced expression in Calu-3 cells; wherein (a) is the expression of IL-6 mRNA; (b) is the expression of IL-8 mRNA; (c) is the expression of TNF-α mRNA;

FIG11 evaluates the ability of targeted delivery of nucleic acids mediated by nucleic acid aptamers and viral infection; wherein (a) fluorescence confocal and flow cytometry analysis verify that the delivery of the chimera depends on nucleic acid aptamers; (b) is a comparison of the delivery efficiency of pseudoviruses and transfection reagents at different concentrations and a flow cytometry analysis diagram;

Figure 12 is a graph showing the ability of cyclized SAPT1-NASO2 to inhibit SARS-CoV-2-induced inflammation at the viral level; wherein (a) shows the inhibition of IL-1β mRNA expression in THP-1 cells induced by WT SARS-CoV-2 virus by cyclized SAPT1-NASO2; (b) shows the inhibition of IL-1β mRNA expression in THP-1 cells induced by Omicron SARS-CoV-2 virus by cyclized SAPT1-NASO2; (c) shows the inhibition of IL-6 mRNA expression in Calu-3 cells induced by WT SARS-CoV-2 virus by cyclized SAPT1-NASO2; (d) shows the inhibition of IL-6 mRNA expression in Calu-3 cells induced by WT SARS-CoV-2 virus by cyclized SAPT1-NASO2;

Figure 13 is a graph showing the ability of cyclized SAPt1-NASO2 to inhibit the proliferation of SARS-CoV-2 at the viral level, wherein (a) is a fluorescence graph showing that different concentrations of cyclized SAPt1-NASO2 inhibit the proliferation of SARS-CoV-2 virus in Vero E6 cells; (b) is a quantitative graph showing that different concentrations of cyclized SAPt1-NASO2 inhibit the proliferation of SARS-CoV-2 virus in Vero E6 cells.

Detailed ways

The exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

This example uses the SARS-CoV-2 virus as the research basis, and uses the nucleic acid aptamer targeting the S protein of SARS-CoV-2 and the antisense oligonucleotide targeting the N gene of SARS-CoV-2 as examples. The nucleic acid aptamer (SApt) of the S protein is chimerized with the ASO (NASO) targeting the N gene to construct linear SAPT-NASO and circular SAPT-NASO (circSApt-NASO) (as shown in Figure 1). The chimera is delivered into cells by relying on SAPT and SARS-CoV-2 virus infection, and exerts the ability to specifically inhibit inflammation and antiviral proliferation.

Example 1: Evaluation of the ability of antisense oligonucleotides (ASOs) to knock down the N gene of SARS-CoV-2

In this example, 4 antisense oligonucleotides targeting the N gene (named NASO1, NASO2, NASO3, and NASO4) and pCMV-N-GFP plasmid were simultaneously transfected into 293T cells, and the knockdown effect on N gene expression was detected 48 hours later. As shown in Figure 2, the expression of N-GFP protein was observed by fluorescence imaging, and the results showed that the fluorescence intensity of cells transfected with NASO2 was the weakest, indicating that NASO2 had the best knockdown ability. Therefore, NASO2 was selected for the next experiment.

The sequence information is as follows:

NASO1:CCAATGTGATCTTTTGGTGT

NASO2:ATTGTTAGCAGGATTGCGGG

NASO3:ATTTCCTTGGGTTTGTTCTG

NASO4:GGCCAATGTTTGTAATCAGT

Example 2: Effect of chimera structure on cyclization efficiency

In this embodiment, the nucleic acid aptamer SAPt2 and the antisense oligonucleotide NASO2 are chimerized together using three consecutive bases TTT (i.e., linker) to obtain a linear nucleic acid aptamer-antisense oligonucleotide chimera-SApt2-NASO2. This embodiment intends to improve the stability of the chimera after cyclization to enhance its knockdown ability. It was found through experiments that a stem-loop structure could not be formed in the SAPT2-NASO2 chimera. After treatment with T4 DNA ligase, it was found that the SAPT2-NASO2 chimera could not be successfully cyclized, that is, it was easily degraded by exonuclease I (Exo I) (as shown in Figure 3 (a)). Next, consider using SAPT1 to chimerize with NASO2. The chimera has a clear stem-loop structure. After phosphorylation at the 5 end, T4 DNA ligase is used for connection. It is found that the cyclized SAPT1-NASO2 is hardly degraded by exonuclease I (Exo I), proving that it has successfully formed a cyclization result, indicating that designing a stem structure in the chimera is conducive to the cyclization of the chimera.

The sequence information is as follows:

ST-6-2 (SApt2): GGGGAGGGCGGGTGGATTGGATGCCGA

SApt2-NASO2:GGGGAGGGCGGGTGGATTGGATGCCGATTTATTGTTAGCAGGATTGCGGG

ST-6-1(SApt1):AGGGCATCAAAGGGGGGAGGGCGGGTGGATTGGATGCCGA

SApt1-NASO2:

AGGGCATCAAAGGGGGGAGGGCGGGTGGATTGGATGCCGATTTATTGTTAGCAGGATTGCGGG

circSApt1-NASO2:

AGGGCATCAAAGGGGGGAGGGCGGGTGGATTGGATGCCGATTTATTGTTAGCAGGATTGCGGGTTT

Example 3: Evaluation of the effect of linker lengths on chimera circularization

In this example, we want to observe the effect of linker lengths on the circularization of the chimera. We increase the linker length to 13 and 18, and design two chimeras (Linker-13 and Linker-18). Also when the 5-end is phosphorylated, we use T4 DNA ligase to connect them. We find that Linker-13 or Linker-18 can be successfully circularized, that is, they are hardly degraded by exonuclease I (Exo I) (as shown in Figure 4), indicating that the linker length has little effect on the circularization efficiency.

The sequence information is as follows:

Linker-13:

AGATGAGGGCATCAAAGGGGGGAGGGCGGGTGGATTGGATGCCGATTTATTGTTAGCAGGATTGCGGG

circLinker-13:

AGATGAGGGCATCAAAGGGGGGAGGGCGGGTGGATTGGATGCCGATTTATTGTTAGCAGGATTGCGGGTTT

Linker-18:

ATCGAAGGGCATCAAAGGGGGGAGGGCGGGTGGATTGGATGCCGAGGTATTTTATTGTTAGCAGGATTGCGGG

circLinker-18:

ATCGAAGGGCATCAAAGGGGGGAGGGCGGGTGGATTGGATGCCGAGGTATTTTATTGTTAGCAGGATTGCGGGTTT

Example 4: Evaluation of the stability of circularized chimeras and linear chimeras

In this example, the successfully cyclized SAPT1-NASO2, Linker-13 and Linker-18 were incubated in 10% fetal bovine serum (FBS) to observe their stability in serum. As shown in Figure 5, compared with the linear SAPT1-NASO2, Linker-13 and Linker-18, the stability of the cyclized SAPT1-NASO2, Linker-13 and Linker-18 in serum was significantly improved.

Example 5: Evaluation of the binding ability of the chimera SAPt1-NASO2 and its cyclized form to the SARS-CoV-2 virus Spike protein and the ability to inhibit S-TLR4 binding

This example verifies the ability of the chimera SAPT1-NASO2 and its cyclized form to bind to the Spike protein by ELISA experiments. The Spike protein was coated on a 96-well plate, and the biotin-labeled chimera was bound to these proteins for 30 minutes and then washed 3 times. After adding HRP-streptavidin and incubating for 30 minutes, the mixture was washed 3 times, and then TMB reaction was added. After termination, the absorbance was measured at OD 450nm. As shown in Figure 6 (b), the chimera SAPT1-NASO2 and its cyclized form have a high binding force with the Spike protein. At the same time, flow analysis also found that it has a high binding ability with the S protein. Next, as shown in Figure 6 (a), the competitive ELISA experiment also shows that the chimera SAPT1-NASO2 and its cyclized form are consistent with SAPT1, and have a significant inhibitory effect on the S-TLR4 interaction.

Example 6: Evaluation of the ability of the chimera SAPt1-NASO2 and its cyclized form to bind to the SARS-CoV-2 virus Spike protein

After determining that the chimera SAPT1-NASO2 and its cyclized form bind to the Spike protein, the present embodiment uses the molecular interaction system Pall forteBio Octet K2 to measure the KD value of the chimera SAPT1-NASO2 and its cyclized form. The lower the KD value, the stronger the affinity between the molecules. Biotin-labeled SAPT1, the chimera SAPT1-NASO2 and its cyclized form are fixed on the biosensor, and the Spike protein is used as the analyte. After the steps of solidification, binding, dissociation, etc., the light absorption value received by the sensor changes and the corresponding rate is converted. The affinity constant KD value between the nucleic acid aptamer and the analyte Spike protein is calculated, as shown in Figure 7. Finally, the KD value of SAPT1 is 76.86nM, the KD value of SAPT1-NASO2 is 82.35nM, and the KD value of cyclized SAPT1-NASO2 is 87.55nM. The Ka of SAPT1 was 1.44×10 ⁴ (1/Ms), Kd was 1.11×10-4 (1/s), and KD was 76.86±1.21 (nM); the Ka of SAPT1-NASO2 was 1.34×105 (1/Ms), Kd was 1.10×10-3 (1/s), and KD was 82.35±1.19 (nM); the Ka of cyclized SAPT1-NASO2 was 7.64×104 (1/Ms), Kd was 6.89×10-4 (1/s), and KD was 87.55±2.46 (nM); this indicated that the chimera SAPT1-NASO2 and its cyclized form also had a high affinity with the Spike protein.

Example 7: Evaluation of the ability of the chimera SAPt1-NASO2 and its cyclized form to inhibit N gene expression in HEK293T cells

In this example, different concentrations (25nM, 50nM, 75nM, 100nM) of chimera SAPT1-NASO2 and its thio- and cyclized forms and pCMV-N-GFP plasmid were transfected into 293T cells at the same time, and the knockdown effect on N gene expression was detected after 48 hours. As shown in Figure 8, the expression of N-GFP protein was observed by fluorescence imaging, and it was found that the knockdown effect of chimera SAPT1-NASO2 and its thio-modified form was about 40%-50%, among which the fluorescence of cyclized SAPT1-NASO2 was the lowest, and the knockdown effect reached about 80%-85%, indicating that the chimera can significantly knock down the N gene.

Example 8: Evaluation of the ability of the chimera SAPt1-NASO2 and its cyclized form to inhibit inflammation induced by Spike protein in THP-1 cells

This example uses human monocytic leukemia cells (THP-1 cells) to verify the ability of the chimera SAPT1-NASO2 and its cyclized form to inhibit the inflammation caused by Spike protein at the cellular level. According to literature reports, the Spike-Tri protein of SARS-CoV-2 can trigger an intracellular inflammatory factor storm by interacting with the TLR4 receptor on the cell membrane, that is, the expression levels of inflammatory factors such as inflammatory factors IL-1β, IL-6, IL-8, TNF-α, etc. increase. In this example, RS, SAPT1, chimera SAPT1-NASO2 and its cyclized form were incubated with Spike protein in vitro for 0.5h, and then added to THP-1 cells together. After stimulation for about 2h, THP-1 cells were collected for total RNA extraction and the level of inflammatory factor mRNA was detected. As shown in the RT-qPCR quantitative results in Figure 9, compared with the random sequence RS, SAPt1, chimera SAPt1-NASO2 and its cyclized form have an inhibitory effect of about 60%-70% on the changes in inflammatory factor mRNA, indicating that the chimera SAPt1-NASO2 and its cyclized form can inhibit the ability of Spike protein to induce increased expression of inflammatory factors at the cellular level to a certain extent.

Example 9: Evaluation of the ability of the chimera SAPt1-NASO2 and its cyclized form to inhibit inflammatory expression in Calu-3 cells

This example uses human lung adenocarcinoma cells (pleural effusion) Calu-3 to verify the ability of the chimera SAPt1-NASO2 and its cyclized form to inhibit the expression of the N gene and induce inflammation. In Calu-3 cells, the N protein expressed by the N gene will activate the NF-κB pathway to induce an increase in intracellular inflammatory responses (such as IL-6, IL-8, TNF-α). The chimera SAPt1-NASO2 and its cyclized form and the pCMV-N gene were co-transfected into Calu-3 cells, and the cells were collected after 48 hours. As shown in the RT-qPCR quantitative results of Figure 10, linear SAPt1-NASO2 can inhibit the production of about 40-50% of related inflammatory factors, while cyclized SAPt1-NASO2 can inhibit the production of about 80% of related inflammatory factors.

Example 10: Evaluation of the ability of targeted delivery of nucleic acids mediated by nucleic acid aptamers and viral infection

The present embodiment uses HEK293T-ACE2 cells and S pseudovirus (S-Pseudovirus) to evaluate the delivery mediated by nucleic acid aptamers and SARS-CoV-2 infection. Human angiotensinase 2 (ACE2) is overexpressed in the cell, which can be infected by SARS-CoV-2S pseudovirus, and GFP green fluorescent protein can be expressed after infection, so that its infection can be observed. RS, SAPT1, cyclized SAPT1-NASO2 and S-Pseudovirus labeled with Cy3 are incubated in vitro for 0.5h, and after being added to HEK293T-ACE2 cells and infected for 48h, fluorescence is observed under a confocal fluorescence microscope. In order to quantify the delivery efficiency, the cyclized SAPT1-NASO2 labeled with Cy3 is transfected to cells using a liposome transfection reagent. Meanwhile, cells are subjected to flow analysis and quantified. As shown in the results of confocal imaging and flow analysis in Figure 10, the Cy3-labeled cyclized SAPT1-NASO2 is consistent with SAPT1 (Figure 11a), indicating that the delivery of the chimera is dependent on nucleic acid aptamers. In addition, the chimera co-localizes with S-Pseudovirus, and the red fluorescence is proportional to the virus concentration (Figure 11b). In addition, compared with transfection, the delivery capacity is slightly lower than that of the transfection reagent, indicating that the ability of targeted delivery of SAPT1-NASO2 mediated by nucleic acid aptamers and SARS-CoV-2 infection is very efficient.

Example 11: Evaluation of the ability of cyclized SAPt1-NASO2 to inhibit SARS-CoV-2-induced inflammation at the viral level

This example selects the ability to inhibit inflammatory responses in THP-1 and Calu-3 cells. Cyclized SAPT1-mutNASO2 or cyclized SAPT1-NASO2 and WT or Omicron strain SARS-CoV-2 virus were incubated in vitro for 1 hour and added to THP-1 and Calu-3 cells. First, THP-1 cells were collected after 2 hours, total RNA was extracted for reverse transcription, and RT-qPCR experiments were performed to detect the expression of related inflammatory factors. Then, Calu-3 cells were collected 48 hours later and total RNA was also extracted, and RT-qPRC was used to detect the expression of related inflammatory factors. As shown in the RT-qPCR quantitative results of Figure 12, cyclized SAPT1-NASO2 can significantly reduce the production of inflammatory factors in THP-1 cells or Calu-3 cells.

Example 12: Evaluation of the ability of cyclized SAPt1-NASO2 to inhibit the proliferation of SARS-CoV-2 at the viral level

This embodiment selects African green monkey kidney cells (Vero cells) to evaluate the ability of cyclized SAPT1-NASO2 to inhibit the replication of SARS-CoV-2. Different concentrations of RS, SAPT-1, cyclized SAPT1-mutNASO2 or cyclized SAPT1-NASO2 and WT SARS-CoV-2 viruses were incubated in vitro for 1 hour and added to Vero cells. After 48 hours, a fluorescent immunoassay (Immunofluorescence assay, IFA) was performed, and after the cells were fixed, DAPI was used to stain the nuclei, and the N protein fluorescent antibody was used to observe the replication of SARS-CoV-2 viruses in the cells. As shown in Figure 13, according to the IFA experimental results, cyclized SAPT1-NASO2 can significantly inhibit the replication of SARS-CoV-2 in Vero cells.

The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art may make some simple modifications, equivalent changes or modifications using the technical contents disclosed above, which are all within the protection scope of the present invention.

Claims (10)

1. A nucleic acid with specific antiviral proliferation, characterized by having a free end or loop structure, dependent on nucleic acid aptamer and virus-infected cell-mediated targeted delivery into cells, a chimera consisting of the following 3 parts:

(1) Targeting nucleic acid aptamers that bind to viral surface proteins as elements;

(2) Antisense oligonucleotides targeted to silence viral RNAs as elements;

(3) Nucleic acid sequence Linker connecting the elements in (1) and (2).

2. The specific antiviral proliferative nucleic acid according to claim 1, wherein the virus is novel coronavirus SARS-CoV-2, and the nucleic acid is specific anti-SARS-CoV-2 inflammatory and anti-SARS-CoV-2 proliferative nucleic acid;

the nucleic acid aptamer is a nucleic acid aptamer which is targeted to bind SARS-CoV-2Spike protein; the antisense oligonucleotide is antisense oligonucleotide for targeted silencing of SARS-CoV-2 RNA;

the elements in (1) and (2) and the Linker in (3) are DNA or RNA.

3. The specific antiviral proliferative nucleic acid according to claim 1 or 2, wherein the loop structure is formed by ligating the 5 'end and the 3' end of the nucleotide sequence into a loop;

the connection mode adopts any one of the following modes: t4 DNA ligation, biosynthetic ligation, and chemical ligation;

the nucleic acid sequence Linker used for ligation employs any of the following: DNA, RNA with or without a marker; the marker is a fluorescent marker, biotin, protac, digoxin, small peptide, nano luminescent material, biological small molecule, therapeutic substance, radioactive substance, siRNA or enzyme marker;

the chimera of the ring structure comprises a stem-loop structure.

4. The specific antiviral proliferative nucleic acid according to claim 1 or 2, wherein the nucleic acid aptamer comprises a derivative which is an engineered phosphorothioate derivative capable of specifically binding to a viral surface protein, or a peptide nucleic acid specifically binding to a viral surface protein;

when the virus is a novel coronavirus SARS-CoV-2, the derivative is an engineered phosphorothioate derivative capable of specifically binding SARS-CoV-2Spike protein, or a peptide nucleic acid specifically binding SARS-CoV-2 Spike-Tri protein.

5. The specific antiviral proliferative nucleic acid according to claim 1 or 2, wherein the antisense oligonucleotide is an antisense oligonucleotide for targeted knockdown of viral RNA, comprising a gene for targeted knockdown of an expressed nonstructural protein, a gene for expression of a structural protein, and a gene for expression of an auxiliary protein, and a regulatory gene.

6. The specific antiviral proliferative nucleic acid according to claim 5, wherein said antisense oligonucleotide is an antisense oligonucleotide targeting the N gene of knockdown SARS-CoV-2.

7. The specific antiviral proliferative nucleic acid according to claim 1 or 2, wherein the specific antiviral proliferative nucleic acid is a modified nucleotide sequence, the modification being performed by any one or more of the following: phosphorylation, amination, methylation, sulfhydrylation, isotopicization, substitution of oxygen for sulfur, substitution of oxygen for selenium.

8. The specific antiviral proliferative nucleic acid according to claim 1 or 2, wherein said delivery further comprises an additional enhanced delivery vehicle that cooperates with said specific antiviral proliferative nucleic acid, including chemical and biotechnological construction dependent delivery; the delivery of the chemical and biotechnology dependent constructs employs any of liposome nanoparticles, sugar-nucleic acid conjugates, lipid-nucleic acid conjugates, or exosome membranes.

9. The specific antiviral proliferative nucleic acid according to claim 2, wherein the SARS-CoV-2 is wild-type SARS-CoV-2 or a mutant thereof.

10. Use of a specific antiviral proliferative nucleic acid according to any one of claims 1 to 9, wherein said nucleic acid is used in any one or more of the group consisting of:

(1) Separating and purifying surface proteins of viruses;

(2) Labeling the surface proteins/genes of the virus;

(3) Imaging of viral surface proteins/genes;

(4) Enriching surface proteins/genes of viruses;

(5) Quantitatively or qualitatively detecting surface proteins/genes of the virus;

(6) Preparing a nucleic acid inhibitor of a surface protein/gene of a virus;

(7) Preparing reagents or drugs targeting viral surface proteins/genes;

(8) Preparing an inhibitor for blocking the binding between the surface protein of the virus and the TLR4 protein;

(9) Preparing reagents or medicaments for diagnosing and treating viral-induced infection or inflammation-related diseases;

(10) Kits for detection, imaging, diagnosis and treatment of viruses are prepared.