CN1648249A - Small Molecule Interfering RNA Inhibiting SARS Virus Gene Expression - Google Patents

Abstract

The invention discloses a group of target sequences and small molecule interfering ribonucleic acid (siRNA) capable of effectively inhibiting the expression of SARS virus RNA and protein, its carrier, mammalian cells and applications thereof, and the siRNA can be used for preparing treatment and/or prevention Drugs for atypical pneumonia and related diseases caused by SARS virus.

Description

Small Molecule Interfering RNA Inhibiting SARS Virus Gene Expression

technical field

The present invention relates to the field of antisense technology, and specifically the present invention relates to a small molecule interfering ribonucleic acid capable of inhibiting SARS virus replication and/or SARS virus gene expression and its application.

Background technique

The causative agent of atypical pneumonia, Severe Acute Respiratory Syndrome (Severe Acute Respiratory SyndromeSARS), has been identified as a coronavirus, which is a positive-strand RNA virus (GENEBANK accession number: NC_004718), with a genome of single-stranded RNA containing poly (A), the total length is nearly 30 kb. The surface of the virus has a rod-shaped structure of about 20nm, which is crown-shaped. The pathogen is highly contagious and contagious. After the virus undergoes an early replication process in the patient's body, it triggers a large-scale inflammatory response in the lungs, which eventually leads to lung tissue damage.

Infectious atypical pneumonia caused by SARS virus infection has seriously endangered the health of people in my country and many countries in the world, and is seriously interfering with the normal development of my country's economy. It has become a serious social, political and economic problem in my country. For the SARS virus, there is no effective drug to inhibit its growth. Small molecule ribonucleic acid (siRNA) is developed on the basis of RNAi (RNA interference) technology. The technology related to it was rated as the second of the top ten international scientific and technological breakthroughs in 2001 by the US "Science", the first of the top ten international scientific and technological breakthroughs in 2002, and the fourth in 2003. Its principle of action is that the siRNA is partially unzipped, and the fragment reversely complementary to the target mRNA binds to the target sequence, and the enzyme system in the body cuts the mRNA. In this way, the expression of the target gene can be suppressed. It is one of the most promising nucleic acid drug technologies.

RNA interference is a type of gene silencing induced by double-stranded RNA. During this process, messenger RNA (mRNA), which has a homologous sequence to the double-stranded RNA, is degraded, thereby inhibiting the expression of the gene. RNA interference technology has broad application prospects in probing gene functions and treating human diseases. The detailed mechanism of RNA interference is still unclear. The existing experimental data suggest that the process of RNA interference mainly involves two steps: (1) the long double-stranded RNA is cut into short double-stranded RNA of 21-23 base pairs by the cell-derived double-stranded RNA-specific nuclease Dicer, It is called small interfering RNA (small interfering RNA, siRNA); (2) small interfering RNA forms a complex with some cell-derived enzymes and proteins, called RNA-induced silencing complex (RNA-induced silencing complex) , RISC), this complex can recognize the mRNA with homologous sequence with the small molecule interfering RNA, and cut off the mRNA at a specific site. The discovery of small molecule interfering RNA not only deepens people's understanding of the mechanism of RNA interference, but also breaks through an obstacle often encountered when using long double-stranded RNA to inhibit gene expression in mammalian cells, that is, non-specific effects. Double-stranded RNA longer than 30 base pairs often activates protein kinases and induces nonspecific inhibition of protein synthesis. Small interfering RNAs generally do not induce such nonspecific repression in mammalian cells. Artificially synthesized small molecule interfering RNA can specifically inhibit the expression of exogenous or endogenous genes in mammalian cells. Experiments have shown that small interfering RNAs with a length of 21-23 bases and 2 bases protruding from the 3' end have higher activity. Small interfering RNA-induced gene suppression is highly sequence-specific. RNA interference can be seen as a defense mechanism similar to that of the immune system. Then, it is theoretically feasible to treat viral infectious diseases through RNA interference. At present, many studies have shown that RNA interference can inhibit the replication of viruses in cells, such as human immunodeficiency virus (HIV), poliovirus, human papillomavirus, hepatitis B virus and hepatitis C virus. However, these studies are only at the level of in vitro cultured cells in the laboratory and preliminary animal experiments, and there is no result of using small molecule interfering RNA in human trials. There is still a lot of research work to be done to apply RNA interference technology to clinical treatment of human diseases. How to safely and effectively introduce small molecule interfering RNA into target tissues or cells is a problem that must be solved. Nevertheless, a large number of in vitro experimental research data have shown the application prospect of this technology in the study of gene function and the great potential in the development of new drugs.

SARS virus RNA encodes multiple protein genes. Among them, the M protein, E protein, and N protein are all unique to the SARS virus, and there are no other proteins that replace their functions in human cells. If any one of these proteins is inhibited, the growth cycle of the SARS virus cannot be completed, and the growth of the virus is inhibited.

Contents of the invention

The purpose of the present invention is exactly to provide a small molecule interfering ribonucleic acid (siRNA) target sequence of a group of SARS virus and application thereof.

Another object of the present invention is to provide a set of small molecule interference ribonucleic acid, its carrier, host cell and its application.

The first aspect of the present invention provides a group of small molecule interfering ribonucleic acid (siRNA) target sequences of SARS virus, it is characterized in that, described target sequence is the continuous 19-25 nucleic acid on M, N, E gene of SARS virus.

Preferably, the siRNA target sequence is the same sequence as the 18 consecutive nucleic acid sequences of any one of SEQ ID NO: 1-SEQ ID NO: 26. More preferably, the siRNA target sequence is any sequence shown in SEQ ID NO: 1-SEQ ID NO: 26.

The second aspect of the present invention provides the application of the above-mentioned siRNA target sequence in screening anti-SARS virus drugs. The anti-SARS virus drug is a drug for treating or preventing atypical pneumonia and other related diseases caused by the SARS virus.

The third aspect of the present invention provides a group of small molecule interfering ribonucleic acid (siRNA), characterized in that, said ribonucleic acid is specifically combined with the mRNA of the above-mentioned SARS virus site, and can inhibit the expression of SARS virus gene and/or replication of the SARS virus.

Preferably, the ribonucleic acid is a double-stranded RNA complex with 19-25 base pairs, and at least 18 base pairs in the double strand are complementary.

In a preferred example, the ribonucleic acid includes a sense strand and an antisense strand, the sequence of the antisense strand has at least 18 bases complementary to the above-mentioned siRNA target sequence, and the sequence of the sense strand is complementary to the sequence of the antisense strand There are at least 18 bases complementary. More preferably, the binding region of the sense strand and the antisense strand contains 19, 20 or 21 base pairs of complementarity.

In another preferred example, the antisense strand sequence has at least 18 bases complementary to any one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 29. Preferably, the antisense strand sequence is any sequence shown in SEQ ID NO: 30 to SEQ ID NO: 55 or a sequence having 18 consecutive bases identical to the aforementioned sequence.

In another preference, the double strand of the ribonucleic acid is selected from:

(1) The sequence of the antisense strand is SEQ ID NO: X, the sequence of the sense strand is SEQ ID NO: (X-29), and X is any natural number in 30-55;

(2) The sequence of the antisense strand is SEQ ID NO: X, and the sequence of the sense strand is SEQ ID NO: (X-29), but the bases at the corresponding positions on the sense strand paired with the 5' end of the antisense strand are replaced by different paired bases, X is any natural number in 30-55;

(3) The sequence of the antisense strand is SEQ ID NO: 41, and the sequence of the sense strand is SEQ ID NO: 27;

(4) The sequence of the antisense strand is SEQ ID NO: 54, and the sequence of the sense strand is SEQ ID NO: 28;

(5) The sequence of the antisense strand is SEQ ID NO: 53, and the sequence of the sense strand is SEQ ID NO: 29.

Preferably, the substitution of bases at corresponding positions on the sense strand paired with the 5' end of the antisense strand is selected from:

(1) C → U;

(2) U → C;

(3) A→G or I;

(4) G → A or I.

In another preferred example, the 3' ends of the sense strand and the antisense strand of the above-mentioned ribonucleic acid are connected with more than two ends of deoxyoligonucleotides. Preferably, the end is dTdT, dAdA, dCdC, dGdG or dIdI. More preferably, the end is dTdT.

The fourth aspect of the present invention provides a vector, characterized in that the vector comprises at least one ribonucleic acid. Preferably, the ribonucleic acid includes a sense strand region and an antisense strand region, the antisense strand region comprises a sequence complementary to the above-mentioned siRNA target sequence, and the sense strand region sequence is complementary to the antisense strand region sequence.

In a preferred example, the double strand of the ribonucleic acid is selected from:

(1) The sequence of the antisense strand is SEQ ID NO: X, the sequence of the sense strand is SEQ ID NO: (X-29), and X is any natural number in 30-55;

(2) The sequence of the antisense strand is SEQ ID NO: X, and the sequence of the sense strand is SEQ ID NO: (X-29), but the bases at the corresponding positions on the sense strand paired with the 5' end of the antisense strand are replaced by different paired bases, X is any natural number in 30-55;

(3) The sequence of the antisense strand is SEQ ID NO: 41, and the sequence of the sense strand is SEQ ID NO: 27;

(4) The sequence of the antisense strand is SEQ ID NO: 54, and the sequence of the sense strand is SEQ ID NO: 28;

(5) The sequence of the antisense strand is SEQ ID NO: 53, and the sequence of the sense strand is SEQ ID NO: 29.

The fifth aspect of the present invention provides a host cell, characterized in that the host cell comprises the above-mentioned vector. Preferably, the host cells are mammalian cells. More preferably, the mammalian cells are human cells.

The sixth aspect of the present invention provides the application of the above-mentioned ribonucleic acid, which is characterized in that the above-mentioned ribonucleic acid is used in the preparation of medicines for the treatment and/or prevention of atypical pneumonia and other related diseases caused by the SARS virus.

The seventh aspect of the present invention provides a composition, which is characterized in that it includes a safe and effective amount of the above ribonucleic acid and a pharmaceutically acceptable carrier. Preferably, the ribonucleic acid comprises a sense strand and an antisense strand, the antisense strand is selected from any nucleotide sequence shown in SEQ NO: 30 to SEQ NO: 55, and the region sequence of the sense strand is the same as The sequences of the antisense strand regions are complementary.

The eighth aspect of the present invention provides the application of the above carrier in the preparation of medicines for treating and/or preventing atypical pneumonia and other related diseases caused by SARS virus.

The ninth aspect of the present invention provides the application of the above-mentioned host cells in the preparation of medicines for treating and/or preventing SARS virus-induced atypical pneumonia and other related diseases. Preferably, the host cells are mammalian cells. More preferably, the mammalian cells are human cells.

As used in the present invention, the term "small molecule interfering ribonucleic acid" refers to siRNA and its analogues comprising sequences shown in SEQ ID NO: 1-SEQID NO: 55, and also refers to a series of chemical substances, which have a series of nuclear The nucleotide base sequence can recognize the SARS virus sequence or its partial sequence by interacting with the nucleotide base hydrogen bond of the SARS virus.

These RNA analogs include, but are not limited to, 2'-O-alkyl sugar modifications, methylphospholipids, phosphorothioates, diphosphates, formacetals, 3'-thioformals (3' -thioformacetal), sulfone, sulfamate, and nitro backbone modifications, amino compounds, and analogs in which the base moiety has been modified. Furthermore, analogs of molecules may be polymers in which the ribose moiety has been modified or replaced by other suitable moieties, resulting polymers include but are not limited to morphine analogs and peptide nucleic acid (PNA), locked nucleic acid ( LNA) and other analogues. These analogs include different combinations of the above modifications, including structural modifications of linking groups and/or sugars or bases to improve RNaseH-mediated destruction of SARS virus RNA, binding affinity, nuclease resistance and/or its specificity.

In the present invention, the terms "small molecule ribonucleic acid", "small molecule interfering ribonucleic acid" or "siRNA" and "ribonucleic acid of the present invention" can be used interchangeably, and they all refer to ribonucleic acid sequences that inhibit the expression of SARS virus, including sense The ribonucleic acid (RNA) of the strand region (any one of SEQ ID NO: 1 to SEQ ID NO: 29) and the antisense strand region (any one of SEQ ID NO: 30 to SEQ ID NO: 55).

At present, the RNA sequence encoding the present invention can be obtained completely through chemical synthesis. This RNA sequence can then be introduced into various existing RNA molecules (or eg, vectors) and cells known in the art.

The present invention also relates to a vector comprising the ribonucleic acid of the present invention and a host cell produced by genetic engineering using the vector of the present invention. The vectors can be in the form of plasmids, virus particles and other vectors. The ribonucleic acid sequence in the expression vector can be operably linked to the expression control sequence, and also contains ribosome binding sites for translation initiation and transcription termination, and preferably contains one or more selectable markers. The host cells may be bacteria such as Escherichia coli, fungi such as yeast, insect cells such as Sf9, animal cells (especially mammalian cells) such as CHO, COS or human cells, etc. Based on the description herein, the selection of appropriate vectors and host cells is within the knowledge of those skilled in the art.

In therapeutic applications, the ribonucleic acid of the present invention can be formulated into preparations according to different administration methods, including oral, topical or topical administration. Techniques and formulations are generally found in the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. The active ingredient is siRNA, which is usually combined with a carrier such as dilute excipients, including fillers, extenders, binders, wetting agents, disintegrants, surfactants, degradable polymers, or lubricants , depending on the nature of the mode of administration and the form of dosing. Typical dosage forms include tablets, powders, granules and capsules, and liquid preparations include suspensions, emulsions and solutions.

The ribonucleic acid of the present invention is particularly suitable for oral administration, which requires exposure of the drug to gastric acid for up to 4 hours under conventional drug delivery conditions, and can last up to 12 hours when presented in a sustained release form. It is very beneficial to formulate these siRNAs into preparations in the form of sustained release for the treatment of atypical pneumonia and diseases caused by SARS virus.

Systemic administration of siRNA, or oral complexes can also be achieved by transmucosal or transdermal methods. For transmucosal or transdermal administration, penetrants suitable to penetrate the barrier may be used in the formulation. Such penetrants are generally known in the art and include, for example, cholates and fusidic acid derivatives for transmucosal administration. Additionally, detergents can be used to assist penetration. Transmucosal administration can be, for example, by nasal spray, and formulations suitable for inhalation or suppository administration. The nasal cavity spray preparation can be used for treating and/or preventing atypical pneumonia and diseases caused by SARS virus.

The ribonucleic acid of the present invention can also be combined with a pharmaceutically acceptable carrier suitable for administration to a subject. Examples of suitable pharmaceutical carriers are various cationic liposomes including, but not limited to, N-(1-2,3-dioleyloxy)propyl-n,n,n-trimethylammonium chloride (N-( 1-2,3-dioleyloxy)ProPyl)-n,n,n-trimethylammonium chloride) (DOTMA) and dioleylphophotidyl ethanolamine (DOPE). Liposomes are also suitable carriers for the siRNAs of the invention. Other suitable carriers are slow release gels or polymers containing the desired siRNA.

The ribonucleic acid of the present invention can be administered to patients by any effective route, including intramuscular injection, intravenous injection, intrathoracic injection, intranasal injection, peritoneal injection, subcutaneous injection, and oral administration. Oral administration requires enteric coatings to protect the desired antisense molecules and their analogs from degradation in the gastrointestinal tract. siRNA can be mixed with a certain amount of physiologically acceptable carrier and diluent, such as saline solution or other suitable liquid.

The actual amount of any particular siRNA administered depends on many factors, such as the type and stage of the disease or infection, the toxicity of the siRNA to other cells in the body, its rate of uptake by cells, the age and weight of the patient to whom the antisense molecule is being administered. An effective amount for a patient can be determined by routine methods such as gradually increasing the dose of siRNA from ineffective to effective in inhibiting cell proliferation. Concentrations ranging from 5 nM to approximately 5 μM presented to diseased cells were effective in inhibiting gene expression and exhibiting a detectable phenotype. Preferably, the concentration ranges from 10 nM to about 100 nM, for example, the preferred concentration of siRNA No. 15 is 13 nM. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

Description of drawings

Figure 1. Determination of the cycle number of RT-PCR.

Figure 2. The relationship curve between RT-PCR cycle number and DNA yield.

Figure 3. Screening of siRNA against the E gene.

Figure 4. Screening of siRNAs against the M gene.

Figure 5. Screening of siRNAs against the N gene.

Figure 6. No.90 siRNA's inhibitory effect on SARS virus E gene expression and its dose relationship.

Figure 7. No. No. 15 siRNA's inhibitory effect on SARS virus M gene expression and its dose relationship.

Figure 8. No.58 siRNA's inhibitory effect on SARS virus N gene expression and its dose relationship.

Figure 9. Inhibition of GFP-E fusion protein expression by No. 90 siRNA.

Figure 10. Inhibition of GFP-M fusion protein expression by No. 15 siRNA.

Figure 11. Inhibition of GFP-N fusion protein expression by No. 58 siRNA.

Figure 12. Inhibition of SARS gene expression in Vero E6 cells by unpaired siRNA at the 5' end of the antisense strand.

Figure 13. Secondary structure simulation of siRNA target site mRNA for E gene

Figure 14. Secondary structure simulation diagram of siRNA target site mRNA for M gene

Figure 15. Secondary structure simulation of siRNA target site mRNA for N gene

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions.

The design of embodiment one siRNA target site

Computer simulation of the secondary structure of SARS virus RNA. Select the following sites in the M, N, E protein coding regions and in the negative strand RNA: target gene serial number target site sequence serial number Small envelope protein E (smallenvelopepritein E) 19 26167 5'uuu cgu ggu auu cuu gcu agu 3'26187 8 51 26263 5'aac ggu uua cgu cua cuc gcg 3'26283 25 88 26211 5'uuc gau ugu gug cgu acu gcu 3'26231 9 89 26135 5'uua aua guu aau agc gua cuu 3'26155 10 90 26305 5'uga agg agu ucc uga ucu ucu 3'26325 26 M protein (proteinM) 7 26768 5'cca gac cgc uca ugg aaa gug 3'26788 11 8 26927 5'cgu cgc agc gug uag gca cug 3'26947 12 15 26414 5'agc uua aac aac ucc ugg aac 3'26434 13 16 26985 5'aaa cua uaa auu aaa uac aga 3'27005 1 52 26580 5'ugc ugc ugu cua cag aau uaa 3'26600 2 53 26658 5'uag cua cuu cgu ugc uuc cuu 3'26678 14 54 26877 5'aga gau cac ugu ggc uac auc 3'26897 15 55 26637 5'uau ugu agg cuu gau gug gcu 3'26657 16 56 26824 5'uug cga aug gcc gga cac ucc 3'26844 3 57 26957 5'uug cug cauu aca acc gcu acc 3'26977 17 N protein (Nucleocapsidprotein) 10 28525 5'aau aca ccc aaa gac cac auu 3'28545 18 17 28555 5'aau ccu aau aac aau gcu gcc 3'28575 19 58 28111 5'gau aau gga ccc caa uca aac 3'28131 20 59 28288 5'aag gag gaa cuu aga uuc ccu 3'28308 twenty one 60 28483 5'aac aaa gaa ggc auc gua ugg 3'28503 4 61 28863 5'uga ggc auc uaa aaa gcc ucg 3'28883 5 62 28246 5'aau aau acu gcg ucu ugg uuc 3'28266 twenty two 63 28371 5'aag agc uac ccg acg agu ucg 3'28391 26 64 28593 5'uca agg aac aac auu gcc aaa 3'28613 twenty three 65 28889 5'aac gua cug cca caa aac agu 3'28909 twenty four 91 28212 5'aag gcc aaa aca gcg ccg acc 3'28232 7

      Table 1 (see attached drawings 13, 14, and 15 for the secondary structure simulation diagram)

The target site sequence shown in Table 1 is the same as the positive-sense strand sequence of the siRNA with the same number, and the sequence number in the sequence table is shown in the case on the far right. The sequences shown in this table are only used to illustrate the present invention and not to limit the scope of the present invention.

Example 2. Synthesis of siRNA

1. Use Bechman Oligo 1000M synthesizer (Bechman) to synthesize the sense strand and antisense strand of siRNA (see Table 2 and Table 3 for the sequence).

2. Annealing to form siRNA:

1) Dissolve the synthetic siRNA sense and antisense strands in diethyl pyrophosphate (DEPC)-treated water.

2) Prepare annealing buffer (2x): 200mM potassium acetate, 4mM magnesium acetate, 60mM Hepes-KOH (pH7.4).

3) Prepare 20uM siRNA stock solution:

            2x Annealing Buffer 100ul

    siRNA sense strand Xul (making the final concentration 20uM)

    siRNA antisense strand Yul (making the final concentration 20uM)

                                                                                                                            

The above ingredients were mixed well, placed at 90°C for 1 minute, and then placed at 37°C for 1 hour. Store at -20°C.

Example 3. Identification of siRNA inhibiting the expression of M, N, and E proteins in vero-E6 cells

1. Method:

(1) Cell transfection

Cell transfection experiments were performed using 24-well cell culture plates.

a. One day before transfection, inoculate 0.5×10 ⁵ Vero-E6 cells (Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) per well on a 24-well cell culture plate, and culture overnight at 37°C in a 5% CO ₂ incubator. When the cells are transfected, the cells cover 30-50% of the well area.

b. Cationic liposome Lipofectamine 2000 (Invitrogen) transfection reagent was used for transfection of M, N, E protein expression vectors, and the transfection experiment was carried out according to its operating instructions. The specific dosage is 50ng of target gene carrier per well of cells, 0.75ug of siRNA, and 2ul of Lipofectamine 2000.

c. After 6 hours, add fetal bovine serum to a final concentration of 10%. Continue culturing at 5% CO ₂ at 37°C until 48 hours before detection.

(2) RT-PCR detection

a. 48 hours after transfection, remove the medium and wash the cells three times with sterile PBS buffer.

b. Extract total cellular RNA with Trizol Reagent (GIBCO) and dissolve in 30ul of water (RNase free). Digest with RNase free DNase (DNase) at 37°C for 30 minutes, and then treat at 60°C for 10 minutes to heat inactivate the DNase.

c. Use the One step RNA PCR kit (TaKaRa) to extract 5ul of the total RNA as a template for RT-PCR detection to determine the inhibitory effect of siRNA and perform screening. RT-PCR conditions are as follows:

50℃, 30min;

94°C, 2min;

72℃, 10min

The total reaction volume was 50ul.

d. Determination of RT-PCR cycle number:

Taking the M gene as a sample, transfer 50ng of the expression vector of the M gene into a 24-well plate. After 24 hours, the total RNA was extracted and dissolved in 30ul water (RNase free). Take 5ul as a template, and do RT-PCR under the above conditions. 20ul samples were taken at 20, 25, 26, 27, 28, 29, 32, and 34 cycles respectively for 1% agarose gel electrophoresis and EB staining. The experimental results are shown in Figure 1 and Figure 2. It can be seen from Figure 2 that taking 28 cycles to do RT-PCR can achieve a more realistic reaction template amount.

(3) Calculation of the inhibition rate of gene expression by siRNA

Among them: A is the gray scale of the target mRNA RT-PCR band in the drug-dosing group; A' is the gray level of the β-actine mRNA RT-PCR band in the drug-dosing group as the RT-PCR control; B is the target mRNA RT-PCR band in the control group -The gray scale of the PCR band; B' is the gray scale of the control group as the β-actine mRNA RT-PCR band of the RT-PCR control.

(4) Detection of siRNA inhibitory effect on SARS virus gene expression at the protein level

Since there are no antibodies against SARS virus structure and functional proteins, the mammalian cell expression vectors of SARS virus E, M and N protein genes and reporter gene-green fluorescent protein (GFP) fusion are constructed: pEGFP/E, pEGFP/ M, and pEGFP/N (the GFP expression vector pEGFP-N3 was purchased from Clontech). The siRNA and this GFP fusion protein expression vector were co-transfected into Vero E6 cells. After 48 hours, the expression level of the protein was determined by detecting the intensity of fluorescence with a fluorescence microscope (Olympus BX-50).

(5) A method for screening siRNAs against E, M, and N genes.

Co-transfect siRNA with E, M or N gene expression vectors (pCDNA3.1/E, pCDNA3.1/M or pCDNA3.1/N) into Vero E6 cells, extract total RNA 48 hours later for RT-PCR detection, The RT-PCR product was run on 2% Agarose gel electrophoresis. A and B are the electrophoresis results of two independent experiments. C. The band density data was measured by scanning the gel image of the two results, and calculated with the result of only transfecting the pCDNA3.1/X (X is E, M or N) vector as a reference (the inhibitory effect is set as 0%) The inhibitory strength of various siRNAs was plotted by taking the average value of the two results. The experimental results are shown in Figures 3, 4, and 5, respectively. See Table 2 and Table 3 for specific results.

2. Results

1).SiRNA screening

I. Screening Results

siRNA with inhibition efficiency <30%: target gene siRNA ID serial number siRNA sequence(*) Inhibition efficiency Protein M No.16 1/30 (+)5'aaa cua uaa auu aaa uac aga tt 3'(-)5'ucu gua uuu aau uua uag uuu tt 3' <30% No.52 2/31 (+)5'ugc ugc ugu cua cag aau uaa tt 3'(-)5'uua auu cug uag aca gca gca tt 3' No.56 3/32 (+)5'uug cga aug gcc gga cac ucc tt 3'(-)5'gga gug ucc ggc cau ucg caa tt 3' Protein N No.60 4/33 (+)5'aac aaa gaa ggc auc gua ugg tt 3'(-)5'cca uac gau gcc uuc uuu guu tt 3' No.61 5/34 (+)5'uga ggc auc uaa aaa gcc ucg tt 3'(-)5'cga ggc uuu uua gau gcc uca tt 3' No.63 6/35 (+)5'aag agc uac ccg acg agu ucg tt 3'(-)5'cga acu cgu cgg gua gcu cuu tt 3' No.91 7/36 (+)5'aag gcc aaa aca gcg ccg acc tt 3'(-)5'ggu cgg cgc ugu uuu ggc cuu tt 3' Protein E No.19 8/37 (+)5'uuu cgu ggu auu cuu gcu agu tt 3'(-)5'acu agc aag aau acc acg aaa tt 3' No.88 9/38 (+)5'uuc gau ugu gug cgu acu gcu tt 3'(-)5'agc agu acg cac aca auc gaa tt 3' No.89 10/39 (+)5'uua aua guu aau agc gua cuu tt 3'(-)5'aag uac gcu auu aac uau uaa tt 3'

Table 2

siRNAs with >30% inhibition efficiency: target gene siRNA ID serial number .siRNA sequence(*) Inhibition efficiency Protein M No.7 11/40 (+)5'cca gac cgc uca ugg aaa gug tt 3'(-)5'cac uuu cca uga gcg guc ugg tt 3' 39.8% No.8 12/41 (+)5'cgu cgc agc gug uag gca cug tt 3'(-)5'cag ugc cua cac gcu gcg acg tt 3' 40.9% No.15 13/42 (+)5'agc uua aac aac ucc ugg aac tt 3'(-)5'guu cca gga guu guu uaa gcu tt 3' 75.8% No.53 14/43 (+)5'uag cua cuu cgu ugc uuc cuu tt 3'(-)5'aag gaa gca acg aag uag cua tt 3' 54.9% No.54 15/44 (+)5'aga gau cac ugu ggc uac auc tt 3'(-)5'gau gua gcc aca gug auc ucu tt 3' 47.2% No.55 16/45 (+)5'uau ugu agg cuu gau gug gcu tt 3'(-)5'agc cac auc aag ccu aca aua tt 3' 46.2% No.57 17/46 (+)5'uug cug cau aca acc gcu acc tt 3'(-)5'ggu agc ggu ugu aug cag caa tt 3' 62.9% Protein N No.10 18/47 (+)5'aau aca ccc aaa gac cac auu tt 3'(-)5'aau gug guc uuu ggg ugu auu tt 3' 35.8% No.17 19/48 (+)5'aau ccu aau aac aau gcu gcc tt 3'(-)5'ggc agc auu guu auu agg auu tt 3' 56.1% No.58 20/49 (+)5'gau aau gga ccc caa uca aac tt 3'(-)5'guu uga uug ggg ucc auu auc tt 3' 74.7% No.59 21/50 (+)5'aag gag gaa cuu aga uuc ccu tt 3'(-)5'agg gaa ucu aag uuc cuc cuu tt 3' 48.1% No.62 22/51 (+)5'aau aau acu gcg ucu ugg uuc tt 3'(-)5'gaa cca aga cgc agu auu auu tt 3' 47.5% No.64 23/52 (+)5'uca agg aac aac auu gcc aaa tt 3'(-)5'uuu ggc aau guu guu ccu uga tt 3' 51.0% No.65 24/53 (+)5'aac gua cug cca caa aac agu tt 3'(-)5'acu guu uug ugg cag uac guu tt 3' 37.3% Protein E No.51 25/54 (+)5'aac ggu uua cgu cua cuc gcg tt 3'(-)5'cgc gag uag acg uaa acc guu tt 3' 52.3% No.90 26/55 (+)5'uga agg agu ucc uga ucu ucu tt 3'(-)5'aga aga uca gga acu ccu uca tt 3' 70.1%

table 3

In Table 2 and Table 3, (+) indicates the sense strand; (-) indicates the antisense strand. Wherein, the sequence number of the sense strand is the same as the sequence number of the corresponding numbered target site sequence in Table 1, and the sequence number (Y) of the sense strand is the sequence number of the antisense strand (X, X is any natural number in 30-55) minus 29, that is, Y=X-29. The left side of the third column "/" in Table 2 and Table 3 is the sequence number of the sense strand, and the right side is the sequence number of the antisense strand. For example, the sense strand of siRNA numbered 65 is SEQ ID NO: 24, and the antisense strand is SEQ ID NO: 53.

Embodiment four No.90 No. siRNA is to the relation of the inhibitory effect of SARS virus E gene expression and its dosage.

Different concentrations of No.90 siRNA and E gene expression vector (pCDNA3.1/E) were co-transfected into VeroE6 cells, and total RNA was extracted for RT-PCR detection after 48 hours.

A. Run the RT-PCR product on 2% Agarose gel electrophoresis.

B. Gel image scanning of the results of two independent experiments measured the band density data, and used the results of co-transformation of the Chinese name (control siRNA) and pCDNA3.1/E vector as a reference (inhibition effect was set as 0%) to calculate The inhibitory strength of various siRNAs was obtained, and the average value of the two results was used to make a dose-effect relationship curve for the concentration of siRNA. Figure 6 shows that 15nM No.90 siRNA can achieve 50% inhibition efficiency.

Embodiment five.No.15 No. siRNA is to the relation of the inhibitory effect of SARS virus M gene expression and its dosage. Method is the same as embodiment four. Figure 7 shows that 13nM No.15 siRNA can achieve 50% inhibition efficiency.

The relationship between the inhibitory effect of No.58 siRNA on the expression of SARS virus N gene and its dose in embodiment six.

Method is the same as embodiment four. Figure 8 shows that 13nM No.58 siRNA can achieve 50% inhibition efficiency.

Example 7 Inhibition of No. 90 siRNA on the expression of GFP-E fusion protein.

30nM No.90 siRNA and the expression vector (pEGFP/E) of the fusion protein of SARS virus E protein and green fluorescent protein were co-transfected into Vero E6 cells, and the intensity of green fluorescence was observed with a fluorescence microscope after 48 hours.

A. Transfection of pEGFP/E vector only (none); B. Co-transfection of pEGFP/E with non-specific control siRNA (control);

C, co-transfection of pEGFP/E and No.90 siRNA. A, B, C are the results of fluorescence detection; D, E, F are the conditions of cells observed in bright field under the same field of view. G. Count the number of green fluorescent cells and the total number of cells in the four fields of view of each experiment, convert them into the number of fluorescent cells per 500 total cells, and take the average value. When the inhibition efficiency of control siRNA is set as 0%, the inhibition efficiency of No. 90 siRNA at 30 nM can be calculated as 88.0% (see Figure 9).

Example 8 Inhibition of No. 15 siRNA on the expression of GFP-M fusion protein.

30nM No.15 siRNA and the expression vector (pEGFP/M) of the fusion protein of SARS virus M protein and green fluorescent protein were co-transfected into Vero E6 cells, and the intensity of green fluorescence was observed with a fluorescent microscope after 48 hours.

A, pEGFP/M vector only (none); B, pEGFP/M co-transfected with non-specific control siRNA (control); C, pEGFP/M co-transfected with No.15 siRNA. A, B, C are the results of fluorescence detection; D, E, F are the conditions of cells observed in bright field under the same field of view. G. Count the number of green fluorescent cells and the total number of cells in the four fields of view of each experiment, convert them into the number of fluorescent cells per 500 total cells, and take the average value. When the inhibition efficiency of control siRNA is set as 0%, the inhibition efficiency of No. 15 siRNA of 30 nM can be calculated as 89.4% (see Figure 10).

Example 9. Inhibition of No. 58 siRNA on the expression of GFP-N fusion protein.

Vero E6 cells were co-transfected with 30nM No.58 siRNA and the fusion protein expression vector (pEGFP/N) of SARS virus N protein and green fluorescent protein, and the intensity of green fluorescence was observed with a fluorescent microscope after 48 hours.

A, pEGFP/N vector only (none); B, co-transfection of pEGFP/N and non-specific control siRNA (control); C, co-transfection of pEGFP/M and No.58 siRNA. A, B, C are the results of fluorescence detection; D, E, F are the conditions of cells observed in bright field under the same field of view. G. Count the number of green fluorescent cells and the total number of cells in the four fields of view of each experiment, convert them into the number of fluorescent cells per 500 total cells, and take the average value. When the inhibition efficiency of control siRNA is set as 0%, the inhibition efficiency of 30 nM siRNA No. 58 can be calculated as 88.8% (see Figure 11).

Example 10 Antisense strand 5' end unpaired siRNA inhibits SARS gene expression in Vero E6 cells

1. Basis:

According to the latest research by Dianne S. Schwarz et al., the stability of the pairing at both ends of the siRNA double strand determines which end is easier to unwind first, so that the strand that is the 5' end enters the RISC complex more (a A complex of siRNA and protein can specifically degrade target gene mRNA). (Cell, Vol 115, 199-208, 17 October 2003)

Select three siRNAs: No.8, No.51, and No.65, and mutate the last nucleotide (position 21) at the 3' end of the sense strand as follows

No.8*: (sense chain sequence number is SEQ ID NO: 27)

5’CGU CGC AGC GUG UAG GCA CUA dTdT 3’

||| ||| ||| ||| ||| ||| || о

3’dTdT GCA GCG UCG CAC AUC CGU GAC 5’

No.51*: (sense chain sequence number is SEQ ID NO: 28)

5’AAC GGU UUA CGU CUA CUC GCA dTdT 3’

||| ||| ||| ||| ||| ||| || о

3’dTdT UUG CCA AAU GCA GAU GAG CGC 5’

No.65*: (sense strand sequence number is SEQ ID NO: 29)

5’AAC GUA CUG CCA CAA AAC AGC dTdT 3’

||| ||| ||| ||| ||| ||| || о

3’dTdT UUG CAU GAC GGU GUU UUG UCA 5’

(Note: The mutation site is marked in bold italics with underline, * indicates the siRNA with mutation, the same below.)

Use pCDNA3.1/E and No.51 siRNA or No.51*siRNA, pCDNA3.1/M and No.8 siRNA or No.8*siRNA, pCDNA3.1/N and No.65 siRNA or No.65* siRNA co-transfected VeroE6 cells, and a single transfected SARS virus gene expression vector (none) or a non-specific group siRNA (control) co-transfected Vero E6 cells, and RT-PCR detected the level of SARS virus gene expression after 48 hours. 2% agarose electrophoresis, EB staining (A). The intensity of the strips was scanned in gray scale, and the inhibition rate of the control group was set as 0%, and the inhibition percentage of siRNA in each group was calculated (B).

The siRNA that does not pair with the first base at the 5' end of the antisense strand can inhibit the gene expression of SARS virus by 1.5 times that of the normal group siRNA, and the siRNA with poor inhibitory effect obtained during the screening process can be redesigned according to this method , should be able to make it more effectively suppress the expression of SARS virus gene (see Figure 12).

Preferably, the bases at the 3' end of the sense strand of the siRNA that do not pair with the first base at the 5' end of the antisense strand can be replaced according to the following principles: pyrimidine (including C, U) is replaced with pyrimidine, and purine (including G, U) is replaced with pyrimidine. A) Replacement with purines (including G, A, I (hypoxanthine)). For example, C and U are interchanged, and G is replaced by A or 工.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Sequence Listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

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Claims (18)

1. the small molecule interfering ribonucleic acid (siRNA) target sequence of a group of SARS virus, it is characterized in that, described target sequence is the continuous 19-25 nucleic acid on SARS virus M, N, E gene. 2. The siRNA target sequence as claimed in claim 1, wherein the target sequence is any sequence in SEQ ID NO: 1-SEQ ID NO: 26. 3. the application of the siRNA target sequence described in claim 1 or 2 in screening anti-SARS virus medicine. 4. a group of small molecule interfering ribonucleic acid (siRNA), it is characterized in that, described ribonucleic acid is specifically combined with the mRNA of SARS virus site according to claim 1, and can suppress the expression of SARS virus gene and/or Or the replication of the SARS virus. 5. The ribonucleic acid according to claim 4, wherein said ribonucleic acid is a double-stranded RNA complex with 19-25 base pairs, and at least 18 bases are complementary to each other in said double strand. 6. ribonucleic acid as claimed in claim 4 or 5, is characterized in that, described ribonucleic acid comprises a sense strand and an antisense strand, and described antisense strand sequence and the siRNA target sequence described in claim 2 have at least 18 18 bases are complementary, and the sense strand sequence has at least 18 bases complementary to the antisense strand sequence. 7. The ribonucleic acid according to claim 6, wherein said sense strand and antisense strand binding region contains 19, 20 or 21 pairs of complementary bases. 8. The ribonucleic acid according to claim 6, wherein the antisense strand sequence has at least 18 bases complementary to any one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 29. 9. ribonucleic acid as claimed in claim 4 or 5, is characterized in that, the double strand of described ribonucleic acid is selected from: (1) The sequence of the antisense strand is SEQ ID NO: X, the sequence of the sense strand is SEQ ID NO: (X-29), and X is any natural number in 30-55; (2) The sequence of the antisense strand is SEQ ID NO: X, and the sequence of the sense strand is SEQ ID NO: (X-29), but the bases at the corresponding positions on the sense strand paired with the 5' end of the antisense strand are replaced by different paired bases, X is any natural number in 30-55; (3) The sequence of the antisense strand is SEQ ID No: 41, and the sequence of the sense strand is SEQ ID NO: 27; (4) The sequence of the antisense strand is SEQ ID NO: 54, and the sequence of the sense strand is SEQ ID NO: 28; (5) The sequence of the antisense strand is SEQ ID NO: 53, and the sequence of the sense strand is SEQ ID NO: 29. 10. ribonucleic acid as claimed in claim 9, is characterized in that, the replacement of the base of corresponding position on the positive sense strand paired with antisense strand 5 ' end is selected from: (1) C → U; (2) U → C; (3) A→G or I; (4) G → A or I. 11. ribonucleic acid as claimed in claim 5 is characterized in that, the 3 ' end of described sense strand and antisense strand is connected with the end of more than two deoxyoligonucleotides. 12. ribonucleic acid as claimed in claim 6 is characterized in that, the 3 ' end of described sense strand and antisense strand is connected with the end of more than two deoxy oligonucleotides. 13. A vector, characterized in that the vector comprises at least one ribonucleic acid according to claim 4. 14. A mammalian cell, characterized in that the mammalian cell comprises the vector of claim 10. 15. The mammalian cell of claim 14, wherein the mammalian cell is a human cell. 16. The application of ribonucleic acid as claimed in claim 4, characterized in that said ribonucleic acid is used in the preparation of medicines for the treatment and/or prevention of SARS virus-induced atypical pneumonia and other related diseases. 17. A composition, characterized in that it comprises a safe and effective amount of the ribonucleic acid according to claim 4 or 5 and a pharmaceutically acceptable carrier. 18. compositions as claimed in claim 17 is characterized in that described ribonucleic acid comprises a sense strand and an antisense strand, and described antisense strand is selected from SEQ NO:30 shown in SEQ NO:55 For any nucleotide sequence, the sense strand region sequence is complementary to the antisense strand region sequence.

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