CN111518809A - siRNA interfering expression of novel coronavirus COVID-19 gene and application thereof - Google Patents

siRNA interfering expression of novel coronavirus COVID-19 gene and application thereof Download PDF

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CN111518809A
CN111518809A CN202010398329.3A CN202010398329A CN111518809A CN 111518809 A CN111518809 A CN 111518809A CN 202010398329 A CN202010398329 A CN 202010398329A CN 111518809 A CN111518809 A CN 111518809A
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王喆明
谭昊
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Hangzhou Yongchengrui Biotechnology Co ltd
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Abstract

The invention discloses siRNA for interfering the expression of a novel coronavirus COVID-19 gene, which is one of the following double-stranded RNA sequences: the sense strand is a double-stranded RNA sequence of SEQ ID NO. 5 in the sequence table, and the antisense strand is a double-stranded RNA sequence of SEQ ID NO. 6 in the sequence table; the sense strand is a double-stranded RNA sequence of SEQ ID NO. 7 and the antisense strand is a double-stranded RNA sequence of SEQ ID NO. 8; has more than 95 percent of similarity with the double-stranded RNA sequence defined above and has the double-stranded RNA sequence with the function of silencing RDRP gene or S gene. The siRNA sequence provided by the invention is designed aiming at the mRNA sequence of the COVID-19 virus S protein and the RDRP enzyme, can effectively inhibit the expression of virus key protein, and provides important reference significance for the development of anti-COVID-19 virus medicaments.

Description

siRNA interfering expression of novel coronavirus COVID-19 gene and application thereof
Technical Field
The invention relates to the field of RNA interference and virus resistance, in particular to siRNA for interfering the expression of a novel coronavirus COVID-19 gene and application thereof.
Background
The new coronavirus COVID-19 belongs to a new strain of severe acute respiratory syndrome related coronavirus, and is a sister strain which is similar to SARS-CoV found in 2003 and belongs to the genus of B-coronavirus in the family of coronavirus. The human transmissibility of the COVID-19 virus is very strong, and the infectivity and the absolute fatality rate caused by the infection are far more than those of SARS-CoV and MERS-CoV.
The invasion of the COVID-19 virus into host cells mainly depends on the combination of virus surface spike protein (SProtein) encoded and expressed by an S gene and angiotensin converting enzyme 2(ACE2) on the surface of the host cells, so that the virus genome enters into human cells to start infection. In two functional units of the S protein, the S1 subunit contains an important C-terminal receptor binding domain to promote the binding of the virus to a host cell receptor, so that the coding gene of the S protein is silenced, the generation of the S protein is inhibited, and the attachment and the binding of the virus and human cells can be effectively prevented.
After the COVID-19 virus invades the host cell, it relies on the RDRP enzyme to perform mass replication. The covi-19 virus is a positive single-stranded RNA virus of the family coronaviridae, and can direct the synthesis of proteins by mRNA strands, or can use viral genomic RNA as a template, translate RDRP enzyme by the protein synthesis system of host cells, and then use the enzyme to perform mRNA synthesis of other structural proteins to generate negative strands to complete replication of viral genomic RNA, thereby starting the life cycle of virions. Therefore, the RDRP enzyme is an essential enzyme in the process of COVID-19 virus replication and plays an important role in the replication and transcription of the viral genome.
RNAi (RNAInterference) technology, RNA interference technology, is a process of inducing degradation of target mRNA having a homologous sequence to a small interfering RNA of 21-23bp in a eukaryote, thereby inhibiting the expression of the gene. RNAi technology is taken as a Nobel medical prize technology, and has made great progress in recent research, and the Patisiran which is successfully marketed in 2018 becomes the first RNAi medicine which is approved to be marketed for 20 years since the RNAi phenomenon is found; subsequently, givosiran for the treatment of acute hepatic porphyrins in 11 months in 2019 became approved for the market for the second RNAi drug. RNAi technology can stimulate high-efficiency antiviral effect in animals, so the RNAi technology is widely applied to the research of viral diseases, pharmaceutical companies such as Alnylam, Arrowhead, Dicerna and the like adopt RNAi technology to make major breakthrough in antiviral research, and various antiviral RNAi drugs such as developed HIV, HBV, HCV and the like enter clinical research stages.
The COVID-19 virus is an mRNA positive-sense single-chain virus, and is more suitable for inducing the degradation of virus mRNA by adopting an RNAi technology. As the gene sequence of the COVID-19 virus is already clear and the genome length is about 29903 nucleotides, the research on the special medicine for resisting the COVID-19 virus by using the RNAi technology is completely possible.
Disclosure of Invention
The invention aims to provide siRNA for interfering with expression of a novel coronavirus COVID-19 gene and application thereof, so as to solve the defects of the prior art.
The invention adopts the following technical scheme:
the first aspect of the invention provides siRNA for interfering the expression of the COVID-19 gene of the new coronavirus, wherein the corresponding target sequence of the siRNA is 21-25 continuous nucleotides on the gene sequence of the RDRP enzyme or S protein on the COVID-19 gene of the new coronavirus.
Further, the target sequence is a sequence shown as SEQ ID NO. 3 or SEQ ID NO. 4 in the sequence table.
Further, the siRNA is one of the following double-stranded RNA sequences:
1) the sense strand is a double-stranded RNA sequence of SEQ ID NO. 5 in the sequence table, and the antisense strand is a double-stranded RNA sequence of SEQ ID NO. 6 in the sequence table;
2) the sense strand is a double-stranded RNA sequence of SEQ ID NO. 7 and the antisense strand is a double-stranded RNA sequence of SEQ ID NO. 8;
3) the double-stranded RNA sequence has more than 95 percent of similarity with the double-stranded RNA sequence defined in 1) and has the function of silencing RDRP gene;
4) the double-stranded RNA sequence has more than 95 percent of similarity with the double-stranded RNA sequence defined in the step 2) and has the function of silencing the S gene.
Furthermore, the 3' end of the double-stranded RNA also contains a dTdT structure.
According to the siRNA design principle, the content of cDNA sequence GC of selected siRNA (RDRP enzyme) is 33%, the content of cDNA sequence GC of siRNA (S protein) is 33%, the cDNA sequence GC of the selected siRNA corresponds to 84-104 th nucleotide of RDRP gene mRNA sequence and 215 th 235 th nucleotide of S gene mRNA sequence, and the dTdT structure at the 3' end increases the stability of the siRNA.
The second aspect of the invention provides the application of the siRNA interfering the expression of the new coronavirus COVID-19 gene in the preparation of a medicine for resisting the new coronavirus COVID-19.
The invention has the beneficial effects that:
the invention discloses a target sequence and an siRNA sequence for effectively interfering the expression of a novel coronavirus COVID-19 gene. A group of siRNA molecules are respectively designed aiming at the gene sequences of the RNA polymerase (RDRP) and the surface S protein which are dependent on the CoVID-19RNA of the new coronavirus, and the siRNA molecules which can effectively resist the COVID-19 virus in theory are screened according to the siRNA design rule. The COVID-19 virus target related in the invention can be used as an anti-COVID-19 virus target for drug development; the siRNA molecule designed by the invention can effectively inhibit the expression of virus key protein, can be used for preparing a novel coronavirus pneumonia drug for treating or preventing COVID-19 virus, and provides effective technology and means for developing an RNAi drug for resisting the COVID-19 virus.
Drawings
FIG. 1 is a diagram of the structure of pcDNA3.1(+) plasmid.
FIG. 2 shows PCR amplification of VeroE6 cells transfected with RDRP and S genes.
FIG. 3 shows the inhibition ratios of RDRP-siRNA and S-siRNA to genes.
FIG. 4 shows the relative expression amounts of RDRP-siRNA and S-siRNA to proteins.
Detailed Description
The invention is explained in more detail below with reference to exemplary embodiments and the accompanying drawings. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
The following test methods and test materials used were all conventional methods unless otherwise specified, and were obtained from commercial companies.
EXAMPLE 1 design and Synthesis of siRNA of the CoVID-19RDRP Gene and S Gene of the novel coronavirus
Referring to the siRNA design principle, an ideal siRNA sequence is searched from 50-100nt downstream of an initiation codon AUG of target mRNA, a 21nt sequence adjacent to the 3' end of the siRNA sequence is used as a candidate target point, siRNA sequences with the GC content of 30-60% are selected, a group of siRNA is respectively designed and synthesized according to corresponding target points, and the siRNA sequences are compared with human genome sequences through Blast function to ensure that the siRNA sequences have no homology with human genes. The RDRP enzyme gene sequence finally selected is CCCUACUAUAACUCAAAUGAA, GC% content, corresponding to 84-104 th nucleotide of mRNA; the S protein gene sequence is 33% in GACAUAUGUGACUCAACAAUU, GC content and corresponds to nucleotide 215-235 of mRNA. The sequence is shown in the following table:
Figure BDA0002488504060000031
Figure BDA0002488504060000041
example 2 in vitro construction of RDRP Gene and S Gene of novel coronavirus COVID-19
The method comprises the steps of using a CoVID-19RDRP gene (sequence number: 1449584) and an S gene (sequence number: 43740568) of the new coronavirus as target sequences, entrusting Nanjing Kingsler Biotechnology Limited to synthesize oligonucleotide fragments in a virus-free system, enabling the tail ends of the oligonucleotide fragments to be complementary in pairs, mutually extending through a PCR process, and finally respectively assembling the complete RDRP gene and the S gene. The assembled RDRP gene segment and S gene segment are cloned to a plasmid pcDNA3.1(+) (as shown in figure 1) respectively to obtain two eukaryotic expression plasmids pcDNA3.1-RDRP and pcDNA3.1-S, and the obtained RDRP gene and S gene sequences are consistent with the RDRP gene and S gene sequences of the new coronavirus COVID-19 through sequencing analysis and completely accord with the expected design.
Example 3 transfection of RDRP Gene and S Gene constructed in vitro and cell culture
VeroE6 cells were cultured in a flask containing 10% (v/v) fetal bovine serum DMEM (high-sugar) medium, and the flask was placed at 37 ℃ in sterile CO2In the cell culture box, VeroE6 cells with uniform size and good state are added to about 1.2 × 106Divided into 6-well plates and distributed uniformly. After 24 hours, the constructed pcDNA3.1-RDRP and pcDNA3.1-S plasmids were separately prepared according to the SuperFect Transfection protocol. After 6 hours of transfection, cell exchange was performed 1-2 times, and after 48 hours, cell supernatant was harvested and centrifuged at 45000 rpm for 2 hours at 4 deg.CAt this time, cells centrifuged to the bottom of the tube were resuspended in a small amount of DMEM high-sugar medium. The total RNA of the transfected cells is extracted and subjected to reverse transcription and then PCR analysis, and the result shows that the RDRP and S gene segments are successfully amplified (as shown in figure 2), which indicates that the RDRP gene and S gene constructed in vitro can correctly transcribe the target gene in the host cells.
Example 4 transfection of siRNA
VeroE6 cells infected with pseudo virus containing RDRP gene and S gene are about 1.2 × 104The cells were divided into 24-well plates and transfected 24 hours later. Transfection was performed according to lipofectamine RNAiMAX transfection instructions. The final siRNA concentration in each well of the macropore was 50nM and 1Ul of lipofectamine RNAiMAX was added. After 6 hours of transfection, cell supernatants were removed, and cultured in fresh DMEM high-sugar medium for 24 hours, after which the cells were divided into 96-well plates and transfected for 48 hours.
Example 5 detection of Gene inhibitory Effect of siRNA by real-time fluorescent quantitative PCR
The cells of example 4 were collected, total RNA was extracted with trizol reagent, and after synthesizing cDNA by reverse transcription using TaKa reverse transcription kit, the effect of siRNA on inhibition of the gene was observed by real-time fluorescent quantitative PCR using fluorescent quantitative PCR kit (QIAGEN) and GAPDH for the internal reference gene. The results showed (as shown in FIG. 3) that the inhibition ratio of RDRP-siRNA to RDRP gene was 79.5% compared with the negative control group; the inhibition rate of S gene by S-siRNA was 83.5%.
Example 6 flow cytometry detection of RDRP, S protein expression in VeroE6 cells
72 hours after siRNA transfection, respectively collecting VeroE6 cells in each well, digesting the cells by pancreatin, adding 5ml of DMEM high-sugar medium, repeatedly blowing and beating, centrifuging for 5 minutes at 1000 rpm, washing for 2 times by PBS buffer solution (0.01M, pH 7.4), then suspending in PBS, detecting the average fluorescence intensity and the ratio of fluorescence positive cells in each well by a flow cytometer at the wavelength of 488nm laser, and calculating the total fluorescence intensity of each well of cells. The results showed (as shown in FIG. 4) that the inhibition rate of RDRP protein expression by RDRP-siRNA was 68.3% compared with that of the negative control group; the inhibition rate of S protein expression by S-siRNA was 72.3%.
Sequence listing
<110> Yongcheng Rui Biotech Co., Ltd
<120> siRNA interfering expression of novel coronavirus COVID-19 gene and application thereof
<160>8
<170>SIPOSequenceListing 1.0
<210>1
<211>338
<212>DNA
<213> RDRP Gene cDNA (Artificial sequence)
<400>1
tggggtaagg ctagacttta ttatgattca atgagttatg aggatcaaga tgcacttttc 60
gcatatacaa aacgtaatgt catccctact ataactcaaa tgaatcttaa gtatgccatt 120
agtgcaaaga atagagctcg caccgtagct ggtgtctcta tctgtagtac tatgaccaat 180
agacagtttc atcaaaaatt attgaaatca atagccgcca ctagaggagc tactgtagta 240
attggaacaa gcaaattcta tggtggttgg cacaacatgt taaaaactgt ttatagtgat 300
gtagaaaacc ctcaccttat gggttgggat tatcctaa 338
<210>2
<211>493
<212>DNA
<213> S Gene cDNA (Artificial sequence)
<400>2
ttcaagactc actttcttcc acagcaagtg cacttggaaa acttcaagat gtggtcaacc 60
aaaatgcaca agctttaaac acgcttgtta aacaacttag ctccaatttt ggtgcaattt 120
caagtgtttt aaatgatatc ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg 180
ataggttgat cacaggcaga cttcaaagtt tgcagacata tgtgactcaa caattaatta 240
gagctgcaga aatcagagct tctgctaatc ttgctgctac taaaatgtca gagtgtgtac 300
ttggacaatc aaaaagagtt gatttttgtg gaaagggcta tcatcttatg tccttccctc 360
agtcagcacc tcatggtgta gtcttcttgc atgtgactta tgtccctgca caagaaaaga 420
acttcacaac tgctcctgcc atttgtcatg atggaaaagc acactttcct cgtgaaggtg 480
tctttgtttc aaa 493
<210>3
<211>21
<212>RNA
<213> RDRP-RNA (Artificial sequence)
<400>3
cccuacuaua acucaaauga a 21
<210>4
<211>21
<212>RNA
<213> S-RNA (Artificial sequence)
<400>4
gacauaugug acucaacaau u 21
<210>5
<211>21
<212>RNA
<213> RDRP-siRNA sense strand (Artificial sequence)
<400>5
cccuacuaua acucaaauga a 21
<210>6
<211>21
<212>RNA
<213> RDRP-siRNA antisense strand (Artificial sequence)
<400>6
gggaugauau ugaguuuacu u 21
<210>7
<211>21
<212>RNA
<213> S-siRNA sense strand (Artificial sequence)
<400>7
gacauaugug acucaacaau u 21
<210>8
<211>21
<212>RNA
<213> S-siRNA antisense strand (Artificial sequence)
<400>8
cuguauacac ugaguuguua a 21

Claims (5)

1. An siRNA for interfering the expression of a new coronavirus COVID-19 gene, wherein the corresponding target sequence of the siRNA is 21-25 continuous nucleotides in the gene sequence of RDRP enzyme or S protein on the new coronavirus COVID-19.
2. The siRNA interfering with expression of the CoVID-19 gene of the neocoronavirus of claim 1, wherein the target sequence is shown as SEQ ID NO. 3 or SEQ ID NO. 4 in the sequence table.
3. The siRNA interfering with expression of the CoVID-19 gene of neocoronavirus of claim 2, wherein the siRNA is one of the following double-stranded RNA sequences:
1) the sense strand is a double-stranded RNA sequence of SEQ ID NO. 5 in the sequence table, and the antisense strand is a double-stranded RNA sequence of SEQ ID NO. 6 in the sequence table;
2) the sense strand is a double-stranded RNA sequence of SEQ ID NO. 7 and the antisense strand is a double-stranded RNA sequence of SEQ ID NO. 8;
3) the double-stranded RNA sequence has more than 95 percent of similarity with the double-stranded RNA sequence defined in 1) and has the function of silencing RDRP gene;
4) the double-stranded RNA sequence has more than 95 percent of similarity with the double-stranded RNA sequence defined in the step 2) and has the function of silencing the S gene.
4. The siRNA of claim 3, wherein the 3' end of the double-stranded RNA further comprises a dTdT structure.
5. Use of the siRNA of any one of claims 1-4 interfering with expression of the CoVID-19 gene of neocoronavirus in the preparation of a medicament against CoVID-19 gene of neocoronavirus.
CN202010398329.3A 2020-05-12 2020-05-12 siRNA interfering expression of novel coronavirus COVID-19 gene and application thereof Withdrawn CN111518809A (en)

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WO2022049541A1 (en) * 2020-09-04 2022-03-10 Solstar Pharma Inc. Antiviral silencing rna molecules, chemically modified antiviral silencing rna molecules with enhanced cell penetrating abilities, pharmaceutical compositions comprising same and uses thereof for treatment of viral infections
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CN113817730B (en) * 2021-02-04 2023-02-07 南京吉迈生物技术有限公司 siRNA for inhibiting novel coronavirus (CoV 19) and composition and application thereof
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CN114250229B (en) * 2021-07-19 2023-12-26 深圳大学 SiRNA for inhibiting novel coronavirus 2019-nCoV and application thereof

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Application publication date: 20200811