CN115887683A - DNA tetrahedron and application thereof in resisting swine fever virus - Google Patents

Abstract

The invention provides a DNA tetrahedron, which consists of DNA single strands, wherein the DNA single strands comprise DNA tetrahedron structure sequences, and at least one DNA single strand also comprises an siRNA binding sequence. Preferably, two different siRNAs are simultaneously modified in the TDN, so that the anti-virus effect of the molecule is obviously enhanced, the virus escape phenomenon caused by mutation is reduced, the advantages of the molecule in antiviral research are fully shown, and a new thought is provided for the prevention and control of viruses. The TDN is a siRNA delivery carrier, so that the stability of siRNA is remarkably enhanced, and the action time of siRNA is prolonged. In addition, the TDN has low cost, is easy to synthesize and reform, is convenient for industrial production, and has good application prospect.

Description

DNA tetrahedron and application thereof in resisting swine fever virus

Technical Field

The invention belongs to the field of biological materials and antiviral drugs, and particularly relates to a DNA tetrahedron and application thereof in resisting swine fever viruses.

Background

Classical Swine Fever (CSF) is an acute, febrile, highly contagious disease caused by Classical Swine Fever Virus (CSFV). CSF can be spread horizontally or vertically, both domestic and wild boars are extremely infected, and the mortality rate of piglets can reach as high as 90% within 4 weeks after CSFV infection, so CSFV seriously threatens the economic development of the pig industry (Blome, S., et al., classic Swine river-An Updated review. Viruses,2017.9 (4)). Congenital CSFV infection can lead to persistent infection in an animal, resulting in little or no production of antibodies specific for the virus in the animal, and can present diagnostic difficulties. The persistently infected animals will continue to shed virus and become a potential source of new outbreaks of swine fever. In addition, infected pork and pork products are also potential sources of CSFV.

Strategies for controlling CSFV mainly include systemic vaccination and total culling of non-vaccinated vaccines. In 2016, a forced vaccination campaign (OIE WAHIS) was officially reported in 22 countries. Despite efforts in many countries to destroy CSF, CSFV is still widely spread in several countries in south and central america, parts of eastern europe and neighboring countries, as well as parts of asia (including india) and africa. Therefore, there is an urgent need to develop new methods for efficiently removing CSFV. With the continuous development of biotechnology, the anti-virus strategy of the transgenic pig (TG) comes into play, and the method can cultivate the transgenic pig (TG) which is resistant to CSFV infection on the gene. In 2018, ziconv Xie et al screened two antiviral shRNAs, siRNA-C3 and siRNA-C6, respectively. Subsequently, insertion of these two shrnas into the porcine Rosa26 (drosa 26) locus by CRISPR/Cas 9-mediated knock-in strategy resulted in TG pigs resistant to CSFV, which were finally found to have significantly enhanced resistance to viral infection (Xie, z., et al, genetic modified pigs with protected from viral pig farm virus, plos pathway, 2018.14 (12): p.e 1007193). This result suggests that RNAi technology is an effective strategy for inhibiting viral replication, but that the biological safety of transgenic pigs is to be further evaluated (Guzman-Villanueva, D., et al., formulation approaches to short interfering RNA and MicroRNA: transformations and injections. J Pharm Sci,2012.101 (11): p.4046-66.). Naked siRNA is generally digested by a series of ribonucleases (ribonucleases) in biological systems and fluids or rapidly eliminated by renal filtration due to its small size, and siRNA has hydrophilic and polyanionic properties, and thus is less efficiently taken up by cells (Xue, h., et al, DNA tetrahedron-based nanogels for siRNA delivery and gene cloning. Chem comm (Camb), 2019.55 (29): p.4222-4225), which greatly limit the application of siRNA. Therefore, there is an urgent need to find a suitable delivery system to protect siRNA from nuclease and improve its delivery efficiency. Studies have found that a variety of materials for delivering siRNA, such as viral vectors, liposomes and polymer systems, have been found, but these vectors still have some problems in terms of safety or size.

A DNA tetrahedron (TDN, or tFNAs for short) is a three-dimensional DNA nanostructure composed of four single-stranded DNAs (ssdnas) assembled by complementary base pairing, each ssDNA constituting one face of the TDN. In the dry state, the height of TDN is typically 2-3nm (Kim, K.R., et al, drug delivery by a self-assembled DNA tetrahedron for over-compounded Drug resistance in break reactor cells, camb, 2013.49 (20): p.2010-2.) and is sized for applications in which small molecules are delivered in animal systems. Previous studies have reported numerous properties of DNA nanostructures in biomedical applications: a) TDN has excellent biocompatibility and biosafety (Walsh, a.s., et al., DNA cage delivery to mechanical cells. Acs Nano,2011.5 (7): p.5427-32); b) TDN can independently permeate the cell membrane by endocytosis; c) TDN is relatively stable, resistant to degradation in the biological system, and remains intact within the cell for at least 48 hours (Li, J., et al, self-assisted multiple DNA nomenclature for systemic intracellular delivery of immunological CpG oligonucleotides. ACS Nano,2011.5 (11): p.8783-9.); d) TDN can be modified by different small molecules to play multiple functions (charoenphl, P.and H.Bermudz, aptamer-targeted DNA nanostructres for therapeutic delivery. Mol Pharm,2014.11 (5): p.1721-5; jiang, D.D., et al, multiple-arm genomic DNA nanostructres for Tumor-Targeting, dual-modification in Vivo imaging. ACS Appl Mater Interfaces,2016.8 (7): p.4378-84.). Therefore, compared with naked siRNA, TDN can deliver siRNA more efficiently and stably, and the combination of TDN and RNAi technology has the potential to become a candidate of a novel antiviral strategy.

Disclosure of Invention

In order to improve the stability, transfection efficiency and safety of siRNA and enhance the action effect, the invention provides a novel delivery system, namely DNA tetrahedron (TDN, also called tFNAs).

The invention provides a DNA tetrahedron, which consists of DNA single strands, wherein the DNA single strands comprise DNA tetrahedron structure sequences, and at least one DNA single strand also comprises a SiRNA binding sequence.

Preferably, the DNA tetrahedron comprises 4 DNA single strands. Each DNA single-chain structure respectively comprises a tetrahedral structure sequence, and each tetrahedral structure sequence is used for forming one face of the TDN. At least 1 of the 4 DNA single strands further comprises an siRNA binding sequence. The siRNA binding sequence is connected to the 5 'or 3' end of the tetrahedron structural sequence, and the siRNA binding sequence is used for binding siRNA to the DNA tetrahedron.

Preferably, the single-stranded DNA comprises 4 strands of Seq ID NO: 1. seq ID NO: 2. seq ID NO: 3. seq ID NO:4 in sequence table.

Preferably, any of the above, the at least two single strands of DNA further comprise an siRNA binding sequence.

Preferably of any of the above, the siRNA binding sequence nucleotide sequence is as defined in Seq ID NO:5 and Seq ID NO: and 6, respectively.

Preferably, in any of the above, the DNA tetrahedron is composed of 4 DNA single strands having nucleotide sequences such as Seq ID NO: 1. seq ID NO: 2. seq ID NO:7 and Seq ID NO: shown in fig. 8.

When 4 DNA single strands are shown as Seq ID NO: 1. seq ID NO: 2. seq ID NO: 3. seq ID NO:4, 4 DNA single strands form a DNA tetrahedron, and each DNA single strand forms one face of the DNA tetrahedron. When 4 DNA single strands are as shown in Seq ID NO: 1. seq ID NO: 2. seq ID NO:7 and Seq ID NO:8, seq ID NO: 1. seq ID NO:2 and Seq ID NO:7 and Seq ID NO:8 for forming a DNA tetrahedral structure, seq ID NO:7 and Seq ID NO:8 for binding to an siRNA.

The invention also provides application of the DNA tetrahedron in resisting swine fever viruses.

The invention also provides a combination of agents for combating classical swine fever virus comprising a DNA tetrahedron according to any one of claims 1-5 and an siRNA.

Preferably, the siRNA comprises siRNA-C3 and/or siRNA-C6.

Preferably, any of the above, the siRNA-C3 sense strand sequence is as set forth in Seq ID NO:9, the siRNA-C3 antisense chain sequence is shown as Seq ID NO:11, respectively.

Preferably of any of the above, the siRNA-C6 sense strand sequence is as set forth in Seq ID NO:10, the siRNA-C3 antisense chain sequence is shown as Seq ID NO: shown at 12.

The invention constructs a safe, stable, efficient and specific swine fever virus (CSFV) small-molecule inhibitor, and provides an effective candidate scheme for preparing a CSFV prevention and control preparation. The invention firstly synthesizes a DNA Tetrahedron (TDN) with stable structure, modifies one or two different siRNAs specifically targeting CSFV respectively, and judges the inhibition effect of the molecule on CSFV by detecting the copy number or titer of virus in cells.

In a preferred embodiment of the invention, the DNA tetrahedron employed is represented by the nucleic acid sequences shown in Table 1 in Tris-HCl (10 mM) and MgCl ₂ ·6H ₂ O (50 mM), pH adjusted to 8.0, was annealed at 95 ℃ for 10 minutes, 4 ℃ for 20 minutes.

TABLE 1

The invention has the beneficial technical effects that:

1) According to the research, two different siRNAs are simultaneously modified in the TDN, so that the anti-virus effect of the molecule is obviously enhanced, the virus escape phenomenon caused by mutation is reduced, the advantages of the molecule in antiviral research are fully shown, and a new thought is provided for the prevention and control of viruses.

2) Naked siRNA has low transfection efficiency and extremely short half-life in cells, thereby greatly limiting the research and application of the siRNA in vivo. In the research, the TDN is used as an siRNA delivery carrier, so that the stability of the siRNA is obviously enhanced, and the action time of the siRNA is prolonged. In addition, the TDN has low cost, is easy to synthesize and reform, is convenient for industrial production, and has good application prospect.

Drawings

FIG. 1 is a schematic diagram of a tetrahedron constructed in accordance with preferred embodiment 1 of the present invention.

FIG. 2 shows the tetrahedral Native-PAGE results constructed in the preferred embodiment 1 of the present invention.

FIG. 3 is a tetrahedron dispersion constructed in accordance with preferred embodiment 1 of the present invention.

FIG. 4 is a graph showing the transcript level of t-siRNA transferred into cells detected by qRT-PCR in the preferred embodiment 2 of the present invention.

FIG. 5 is a graph showing the antiviral ability of t-C3 and t-C6 measured by immunofluorescence in accordance with a preferred embodiment 2 of the present invention.

FIG. 6 is a diagram of the copy number of CSFV in PK-15 cells measured by qRT-PCR in the preferred embodiment 2 of the present invention.

FIG. 7 is a graph showing the antiviral ability of t-C3-C6 measured by immunofluorescence in accordance with a preferred embodiment 2 of the present invention.

FIG. 8 is a diagram of the copy number of CSFV in PK-15 cells measured by qRT-PCR in the preferred embodiment 2 of the present invention.

FIG. 9 shows the CSFV virus titer in PK-15 cells in the preferred embodiment 2 of the present invention.

Detailed Description

The present invention will be more clearly and completely described in the following embodiments, but the described embodiments are only a part of the embodiments of the present invention, and not all of them. The examples are provided to aid understanding of the present invention and should not be construed to limit the scope of the present invention.

Example 1

Construction of siRNA modified DNA tetrahedron:

ssDNA and siRNA as shown in Table 1 were synthesized.

Synthetic ssDNA and siRNA equimolar solutionsDissolving in TM buffer (10 mmol/L Tris-HCl, 50mmol/L MgCl ₂ pH 8.0) was heated to 95 ℃ for 10 minutes, 4 ℃ for 20 minutes using a PCR instrument. The sizes and dispersions of DNA tetrahedrons were examined by Native-PAGE and Atomic Force Microscopy (AFM), and the results are shown in FIGS. 1 to 3.

FIG. 1 is a schematic diagram of construction of DNA tetrahedrons (t-C3, t-C6) linked with siRNA-C3 and siRNA-C6. As shown in fig. 1, the tetrahedral structure in example 1 is represented by Seq ID NO: 1. seq ID NO:2 and Seq ID NO:7 and Seq ID NO:8, siRNA-C3 and siRNA-C6 are linked to the DNA tetrahedron. Wherein, seq ID NO: 1. seq ID NO:2 and Seq ID NO:7 and Seq ID NO:8 for forming a DNA tetrahedral structure, seq ID NO:7 and Seq ID NO:8 for the tetrahedral ligation of siRNA-C3 and siRNA-C6 to said DNA.

FIG. 2 is a diagram of detecting TDN size by Native-PAGE (wherein in each lane: 1.

In fig. 3, the dispersibility of TDN was examined by atomic force microscopy.

Example 2

And (3) antiviral effect detection:

PK-15 cells are inoculated in a 96-well plate (PK-15, pig kidney cells, which are cell lines reported in the prior art and can be purchased as commercial products, the culture method is the same as the method in the prior art), 250nmol/L of DNA tetrahedrons respectively connected with siRNA-C3, siRNA-C6, siRNA-C3 and siRNA-C6 are added when the cell fusion rate is about 70 percent, and 200TCID is added after incubation for 24 hours ₅₀ CSFV (phylum shimen strain, the strain used in the validation experiment of the present invention was a strain reported in the prior art), 72 hours after infection, cells were fixed overnight with pre-cooled 80% acetone, followed by addition of 1:100 diluted anti-CSFV positive serum (from CSFV vaccine)Preparing CSFV antibody from peripheral blood of immunized pig, obtaining serum from peripheral blood of immunized animal is a conventional method in the prior art, and is not described herein), adding FITC labeled anti-pig IgG, observing under a fluorescence microscope, and taking a picture. The results are shown in FIGS. 4-6 and FIGS. 7-9.

FIGS. 4 to 6 show the results of the examination of anti-CSFV effects of t-C3 and t-C6. In FIG. 4, qRT-PCR detects the transcription level of t-siRNA after it is transferred into cells; in FIG. 5, immunofluorescence is used to detect the antiviral ability of t-C3 and t-C6, the cells with green fluorescence are CSFV-infected cells, and the number of the cells with green fluorescence in the t-C3 and t-C6 groups is significantly reduced compared with the control group, i.e. the number of the cells infected with CSFV is significantly reduced; in FIG. 6, the copy number of CSFV in PK-15 cells was determined by qRT-PCR.

FIGS. 7 to 9 show the results of examination of t-C3-C6 for anti-CSFV effect. In FIG. 7, immunofluorescence measures t-C3-C6 antiviral ability, and cells with green fluorescence infect CSFV, the number of cells with green fluorescence is significantly reduced in t-C3-C6 group compared to control group, i.e., CSFV-infected cells are significantly reduced; in FIG. 8, copy number of CSFV in PK-15 cells was determined by qRT-PCR; in FIG. 9, CSFV virus titer in PK-15 cells.

In the figure, t-siRNA is a DNA tetrahedron connected with siRNA, siRNA is siRNA not connected with the DNA tetrahedron, and the specific expression is as follows:

t-C3: a DNA tetrahedron with siRNA-C3 formed by the sequence S1+ S2+ S3L + S4L + siRNA-C3.

t-C6: a DNA tetrahedron with siRNA-C6 formed by the sequence S1+ S2+ S3L + S4L + siRNA-C6.

t-C3-C6: is a DNA tetrahedron formed by a sequence S1+ S2+ S3L + S4L + siRNA-C3+ siRNA-C6 and simultaneously carrying siRNA-C3 and siRNA-C6.

the t-siNC is a DNA tetrahedron which is formed by random sequences of sequences S1+ S2+ S3L + S4L + siRNA and has random sequence siRNA, and the random sequence is non-swine fever virus specificity. Random sequences were purchased from the general siRNA oligo/NC of Jima Gen., suzhou.

The result shows that DNA Tetrahedrons (TDN) respectively connected with siRNA-C3 and siRNA-C6 and simultaneously connected with siRNA-C3 and siRNA-C6 can reduce the PK-15 cell infection caused by CSFV, but the DNA tetrahedrons simultaneously connected with siRNA-C3 and siRNA-C6 have better effect of resisting virus infection.

Claims (10)

1. A DNA tetrahedron comprising a single DNA strand comprising a DNA tetrahedron structural sequence, wherein at least one DNA strand further comprises an siRNA binding sequence.

2. The DNA tetrahedron of claim 1 wherein the DNA single strands are 4 strands each comprising Seq ID NO: 1. seq ID NO: 2. seq ID NO: 3. seq ID NO:4 in sequence of DNA tetrahedral structure.

3. The DNA tetrahedron of claim 2 wherein the at least two single strands of DNA further comprise an siRNA binding sequence.

4. The DNA tetrahedron of claim 3, wherein the siRNA binding sequence nucleotide sequence is selected from the group consisting of Seq ID NO:5 and Seq ID NO: and 6, respectively.

5. The DNA tetrahedron of claim 4, wherein the DNA tetrahedron is composed of 4 DNA single strands, the nucleotide sequence of the DNA single strands being as defined in Seq ID NO: 1. seq ID NO: 2. seq ID NO:7 and Seq ID NO: shown in fig. 8.

6. Use of a DNA tetrahedron according to any one of claims 1-5 for combating classical swine fever virus.

7. A combination of agents for combating classical swine fever virus, comprising a DNA tetrahedron of any one of claims 1-5 and an siRNA.

8. The combination of agents of claim 7, wherein the siRNA comprises siRNA-C3 and/or siRNA-C6.

9. The combination of agents of claim 8, wherein the siRNA-C3 sense strand sequence is as set forth in Seq ID NO:9, the siRNA-C3 antisense chain sequence is shown as Seq ID NO:11, respectively.

10. The combination of agents of claim 8, wherein the siRNA-C6 sense strand sequence is as set forth in Seq ID NO:10, the siRNA-C3 antisense chain sequence is shown as Seq ID NO: shown at 12.

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