Use of fad synthetase flad1 in preparation of a drug for preventing or treating novel coronavirus infection

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses application of FAD synthetase FLAD1 in preparation of a medicine for preventing or treating novel coronavirus infection, the application is that a substance for targeting and inhibiting expression of FLAD1 is used to prepare the medicine for preventing or treating the novel coronavirus infection. Experiments prove that novel coronavirus replication in cells is significantly reduced after FLAD1 knockout treatment, which proves that the FAD synthetase FLAD1 can effectively inhibit proliferation of the novel coronavirus. The application provides a new treatment strategy for prevention and treatment of infectious diseases caused by the novel coronavirus infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of FAD synthase FLAD1 in the preparation of drugs for the prevention or treatment of novel coronavirus infection. Background Technology

[0002] COVID-19 is a severe acute respiratory syndrome caused by a novel coronavirus (SARS-CoV-2). The novel coronavirus is an enveloped, single-stranded, positive-sense RNA virus that can cause respiratory infections, cardiac dysfunction, and other symptoms in humans, with a mortality rate of approximately 3.8%.

[0003] The novel coronavirus genome has an m7GTP cap at the 5' end, a polyA tail at the 3' end, and six ORFs (open reading frames). ORF1 encodes 16 non-structural proteins involved in viral genome replication and transcription; the remaining ORFs encode seven accessory proteins (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, and ORF10) and four structural proteins (S, E, M, and N). The emergence of mutant strains such as Delta and Omeprone has challenged vaccine efficacy, making the screening of a conserved and effective vaccine and drug target crucial. The N protein gene is highly conserved and stable; the amino acid homology between SARS-CoV-2 and SARS-CoV N proteins is as high as 91%. The multifunctionality and conservation of the N protein make it a potentially promising target for coronavirus drug intervention. Summary of the Invention

[0004] To address the aforementioned problems, the primary objective of this invention is to provide a novel therapeutic target for patients infected with the novel coronavirus, namely, flavin adenine dinucleotide (FAD) synthase FLAD1, and the application of FAD synthase FLAD1 in the preparation of drugs for the prevention or treatment of novel coronavirus infection.

[0005] The specific technical solution of the present invention includes:

[0006] This invention provides the application of FAD synthase FLAD1 in the preparation of drugs for the prevention or treatment of novel coronavirus infection.

[0007] As a further optimization of the present invention, the application is to prepare a drug for the prevention or treatment of novel coronavirus infection using a substance that targets and inhibits FLAD1 expression.

[0008] As a further optimization of the present invention, the substance that targets and inhibits FLAD1 expression includes any one of FLAD1 inhibitors, FLAD1 siRNA, or gene editing tools that knock out FLAD1 expression.

[0009] As a further optimization of the present invention, the FLAD1 siRNA is any one of (1) to (4):

[0010] (1) siRNA formed by annealing two single strands as shown in SEQ ID No. 3 and SEQ ID No. 4;

[0011] (2) siRNA formed by annealing two single strands as shown in SEQ ID No. 5 and SEQ ID No. 6;

[0012] (3) siRNA formed by annealing two single strands as shown in SEQ ID No. 7 and SEQ ID No. 8;

[0013] (4) siRNA formed by annealing two single strands as shown in SEQ ID No. 9 and SEQ ID No. 10.

[0014] The present invention also provides a drug for the prevention or treatment of novel coronavirus infection, wherein the drug is a FLAD1 siRNA that targets and inhibits FLAD1 expression.

[0015] As a further optimization of the present invention, the FLAD1 siRNA is an siRNA formed by annealing two single strands as shown in SEQ ID No. 7 and SEQ ID No. 8.

[0016] In summary, the beneficial effects of the present invention are as follows:

[0017] This invention provides a novel therapeutic target for novel coronavirus infection, namely FAD synthase FLAD1. Studies have shown that knocking out the gene expressing FAD synthase FLAD1 significantly reduces novel coronavirus replication and effectively inhibits the proliferation of the novel coronavirus in cells. FLAD1 shows promise as a potential therapeutic target for novel coronavirus infection. This invention provides a new strategy for the prevention and control of infectious diseases caused by novel coronavirus infection and has significant application value in drug development and vaccine development. Attached Figure Description

[0018] Figure 1 To detect NP using immunoprecipitation and Western blotting SARS-CoV-2 Interaction with FAD synthase FLAD1;

[0019] Figure 2 To detect NP using immunoprecipitation and Western blotting SARS-CoV-2 Is the interaction with FAD synthase FLAD1 a direct interaction?

[0020] Figure 3To detect NP using DuoLink immunofluorescence assay SARS-CoV-2 Co-location with FLAD1;

[0021] Figure 4 To confirm whether FLAD1 has been knocked out using immunoblotting.

[0022] Figure 5 To detect the mRNA level of FLAD1 after knocking down FLAD1 in cells using FLAD1 siRNA, quantitative real-time PCR was used.

[0023] Figure 6 To detect the viral replication level after knocking out FLAD1 in cells using CRISPER-Cas9, quantitative real-time PCR was employed. Detailed Implementation

[0024] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0025] I. Materials

[0026] 1. Plasmid

[0027] Flag-NP SARS-CoV-2 HA-FLAD1 was constructed in our laboratory. The target fragment was directionally cloned into the pCDNA3.1 vector using double restriction enzyme sites of BamH1 and EcoR1. The Flag (HA) tag is located at the N-terminus of the NP (FLAD1) gene and is fused with the target protein for expression. The NP gene was obtained from our research group, and the FLAD1 gene was cloned from the A549 cell genome. The nucleotide sequence of the FLAD1 gene is shown in SEQ ID NO.1, and the FLAD1 protein sequence is shown in SEQ ID NO.2.

[0028] pGEX-4T-1-FLAD1 was purchased from GENERALCBIOL. For structural details, please refer to the article published in JBC by Hartenina et al. in 2020. (Hartenian E, Nandakumar D, Lari A, Ly M, Tucker JM, Glaunsinger BA. The molecular virology of coronaviruses. J Biol Chem. 2020 Sep11; 295(37):12910-12934. doi:10.1074 / jbc.REV120.013930. Epub 2020 Jul 13. PMID:32661197;PMCID:PMC7489918.).

[0029] 2. Reagents

[0030] The quantitative PCR kit was purchased from Promega (catalog number A6020); the DuoLink immunofluorescence kit was purchased from Sigma (catalog number DUO92004-100RXN); the Lipofectamine 3000 transfection reagent was purchased from Thermo Fisher Scientific; F-12K medium (catalog number 2958713), fetal bovine serum (catalog number 16000-044), trypsin (catalog number 25200-056), and PBS (catalog number C10010500BT) were all purchased from GIBCO; the TransIT X2 transfection reagent was purchased from Mirus Bio (catalog number MIR6003); the FLAD1 antibody was purchased from Proteintech (catalog number 14118-1-AP); and the β-Actin antibody was purchased from Proteintech (catalog number 66009-1-Ig).

[0031] 3. Cells

[0032] Human embryonic kidney 293 cells and human lung adenocarcinoma A549 cells were purchased from the National Experimental Cell Resource Sharing Platform.

[0033] Human lung adenocarcinoma A549 (FLAD1 KO) was constructed as follows: based on the CRISPR-Cas9 system, gRNA targeting the FLAD1 gene was designed, and the gRNA and Cas9 were co-expressed in the target cells. Positive clones were obtained by resistance selection.

[0034] Human lung adenocarcinoma A549 (ACE2 OE) is used. ACE2 is the cell surface receptor for SARS-CoV-2. Since the expression level of ACE2 in A549 cells is relatively low, stable expression of the ACE2 protein in A549 cells is necessary to ensure normal SARS-CoV-2 infection. The specific construction method is as follows: first, an ACE2 plasmid is constructed, then it is transfected into cells, and clones that stably express ACE2 are obtained through antibiotic selection.

[0035] Unless otherwise specified, all reagents and materials used in this invention are commercially available products.

[0036] II. Methods

[0037] Unless otherwise specified, the methods used below are conventional methods known to those skilled in the art.

[0038] 1. The interaction between FAD synthase FLAD1 and the novel coronavirus NP was confirmed by immunoprecipitation and Western blotting. The specific methods are as follows:

[0039] (1) Combine HA-FLAD1 and Flag-NP SARS-CoV-2 The plasmid was transfected using Lipofectamine 3000 reagent according to... Figure 1 The combination shown was used to transfect human embryonic kidney 293 cells (purchased from the National Experimental Cell Resource Sharing Platform). 3 μg of plasmid was transfected, and after 48 h, the culture medium was aspirated. The cells were collected and resuspended using pre-cooled 1×PBS, washed 3 times, and the supernatant was completely discarded. After adding cell lysis buffer (Tris-HCl 50mM pH 8.0, NaCl 150mM, NP40 1%, protease inhibitor 1 tablet / 50mL), place on ice for 30min, centrifuge at 12000rpm for 10min at 4℃, and aspirate the supernatant into a clean 1.5mL EP tube. Add 15μL of agarose beads conjugated with Flag antibody, and incubate at 4℃ for 2h for immunoprecipitation. Centrifuge at 8000rpm for 1min at 4℃, discard the supernatant, and wash 3 times with cell lysis buffer without protease inhibitor. Add 75μL of 1×SDS-PAGE loading buffer to the immunoprecipitate, boil in water for 10min, centrifuge at 8000rpm for 3min at 4℃, and take 15μL of supernatant for polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis at 80V for 30min, adjust the voltage to 120V until bromophenol blue migrates to the bottom of the gel and stop electrophoresis.

[0040] (2) Activate the PVDF membrane with 10 mL of methanol for 10 s, wash with deionized water, and place it in 1× semi-dry transfer buffer (Tris-HCl 24 mM, glycine 5 mM, 20% methanol). Then, place the SDS-PAGE gel that has undergone electrophoresis on the PVDF membrane. Place the layers sequentially on the transfer apparatus in the order of filter paper-gel-membrane-filter paper from top to bottom, using a pressing rod to remove air bubbles between layers. Transfer at 18V for 2 h. After transfer, block with TBST containing 5% skim milk powder at room temperature for 1 h, and wash three times with 1×TBST for 5 min each time. Incubate with antibodies (anti-HA and anti-Flag) diluted 1:1000 at room temperature for 1 h, and wash three times with 1×TBST for 5 min each time. Finally, develop using enhanced chemiluminescence (ECL) using a developing apparatus.

[0041] Figure 1 The results showed that FLAD1 was present in the immunoprecipitation product of Flag-NP, proving that FLAD1 and NP... SARS -CoV-2 There are interactions between them. And it is known that NP... SARS-CoV-2 FLAD1 plays a crucial role in the replication process of the novel coronavirus. As a FAD synthase, it also plays a vital role in cellular metabolism and oxidative phosphorylation. Based on the above experimental results and comprehensive functional analysis of FLAD1, it was determined that FLAD1 is involved in the replication process of the novel coronavirus. Therefore, altering the activity of FLAD1 can further affect the expression of related genes.

[0042] 2. The interaction between FAD synthase FLAD1 and the novel coronavirus NP was confirmed by Far-Wester and immunoblotting methods. The specific methods are as follows:

[0043] (1) Flag-NP SARS-CoV-2 The plasmid was transfected using Lipofectamine 3000 reagent according to... Figure 2The cells were transfected into human embryonic kidney 293 cells using the combination shown. 3 μg of plasmid was transfected, and after 48 h, the culture medium was aspirated. The cells were collected and resuspended using pre-cooled 1×PBS, washed 3 times, and the supernatant was completely discarded. After adding cell lysis buffer (Tris-HCl 50mM pH 8.0, NaCl 150mM, NP40 1%, protease inhibitor 1 tablet / 50mL), place on ice for 30min, centrifuge at 12000rpm for 10min at 4℃, and aspirate the supernatant into a clean 1.5mL EP tube. Add 15μL of agarose beads conjugated with Flag antibody, and incubate at 4℃ for 2h for immunoprecipitation. Centrifuge at 8000rpm for 1min at 4℃, discard the supernatant, and wash 3 times with cell lysis buffer without protease inhibitor. Add 75μL of 1×SDS-PAGE loading buffer to the immunoprecipitate, boil in water for 10min, centrifuge at 8000rpm for 3min at 4℃, and take 15μL of supernatant for polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis at 80V for 30min, adjust the voltage to 120V until bromophenol blue migrates to the bottom of the gel and stop electrophoresis.

[0044] (2) Activate the PVDF membrane with 10 mL of methanol for 10 s, wash with deionized water, and place it in 1× semi-dry transfer buffer (Tris-HCl 24 mM, glycine 5 mM, 20% methanol). Then place the SDS-PAGE gel that has undergone electrophoresis on the PVDF membrane. Place the layers sequentially on the transfer apparatus in the order of filter paper-gel-membrane-filter paper from top to bottom, using a pressing rod to remove air bubbles between layers. Transfer at 18V for 2 h. After transfer, block with TBST containing 5% skim milk powder at room temperature for 1 h, and wash three times with 1×TBST for 5 min each time. Incubate with GST-FLAD1 purified protein diluted 1:100 at room temperature for 1 h, then incubate with antibody diluted 1:1000 (anti-GST, anti-Flag) at room temperature for 1 h, and wash three times with 1×TBST for 5 min each time. Finally, develop using enhanced chemiluminescence (ECL) using a developing apparatus.

[0045] like Figure 2 The results show that, using Far-Western Blot analysis, FLAD1 is still present in Flag-NP. SARS-CoV-2 The immunoprecipitate products showed that this interaction occurred directly between the two.

[0046] 3. The co-localization of FAD synthase FLAD1 and novel coronavirus NP in cells was confirmed using DuoLink immunofluorescence. The specific method is as follows:

[0047] (1) Place a coverslip in a six-well plate to prepare a human lung adenocarcinoma A549 cell crawling slide and infect the cells with SARS-CoV-2.

[0048] (2) After 48 hours, aspirate the culture medium, wash three times with 1×PBS, and discard the supernatant completely. Add 1 mL of 4% paraformaldehyde, aspirate after 15 minutes, wash three times with 1×PBS, and discard the 4% paraformaldehyde completely. Add 1 mL of 0.3% Triton X-100, aspirate after 15 minutes, wash three times with 1×PBS, and discard the 0.3% Triton X-100 completely. Add 100 μL of blocking agent, aspirate after 1 hour, wash three times with 1×PBS, and discard the blocking agent completely. Add 100 μL of primary antibody (100 μL antibody dilute contains 2 μL each of FLAD1 and NP antibodies), aspirate after 1 hour, wash three times with 1×PBS, and discard the primary antibody completely. Add 100 μL of probe system (100 μL deionized water contains 20 μL each of minus and plus), aspirate after 1 hour, wash three times with 1×PBS, and discard the probe system completely. Add 40 μL of ligation system (40 μL of deionized water containing 1 μL of ligase and 8 μL of ligation), aspirate after 30 min, wash three times with 1×PBS, and discard the ligation system completely. Add 40 μL of amplification system (40 μL of deionized water containing 0.5 μL of polymerase and 8.5 μL of amplification), aspirate after 100 min, wash three times with 1×PBS, and discard the amplification system completely. Place a mounting medium containing DAPI onto a glass slide, invert a coverslip onto the slide, and after approximately 30 min, image using a ZISSE (LSM-800) laser confocal microscope.

[0049] like Figure 3 As shown, the red fluorescence indicates that FLAD1 and NP are co-localized in the cytoplasm, further confirming their interaction at the cellular level.

[0050] 4. The knockout efficiency of FAD synthase FLAD1 was confirmed by immunoblotting. The specific method is as follows:

[0051] (1) Human lung adenocarcinoma A549 (WT), human lung adenocarcinoma A549 (ACE2 OE), and human lung adenocarcinoma A549 (ACE2 OE / FLAD1 KO) cells were passaged simultaneously. After 48 h, the culture medium was aspirated, and the cells were collected and resuspended with pre-cooled 1×PBS. The cells were washed three times, and the supernatant was completely discarded. 600 μL of cell lysis buffer (Tris-HCl 50 mM pH 8.0, NaCl 150 mM, NP40 1%, protease inhibitor 1 tablet / 50 mL) was added, and the cells were placed on ice for 30 min. After centrifugation at 12000 rpm for 10 min at 4 °C, the supernatant was transferred to a clean 1.5 mL EP tube. Add 200 μL of 1×SDS-PAGE loading buffer to the cell lysate, boil in water for 10 min, centrifuge at 4℃ and 8000 rpm for 3 min, take 15 μL of sample for polyacrylamide gel electrophoresis (SDS-PAGE), electrophoresis at 80V for 30 min, then adjust the voltage to 120V until bromophenol blue migrates to the bottom of the gel and stop electrophoresis.

[0052] (2) Activate the PVDF membrane with 10 mL of methanol for 10 s, wash with deionized water, and place it in 1× semi-dry transfer buffer (Tris-HCl 24 mM, glycine 5 mM, 20% methanol). Then place the SDS-PAGE gel, which has undergone electrophoresis, on the PVDF membrane. Place the layers sequentially on the transfer apparatus in the order of filter paper-gel-membrane-filter paper from top to bottom, using a pressing rod to remove air bubbles between layers. Transfer at 18V for 2 h. After transfer, block with TBST containing 5% skim milk powder at room temperature for 1 h, and wash three times with 1×TBST for 5 min each time. Incubate with primary antibodies (anti-β-actin, anti-FLAD1) diluted 1:1000 at room temperature for 1 h, and wash three times with 1×TBST for 5 min each time. Incubate with secondary antibodies diluted 1:1000 at room temperature for 1 h, and wash three times with 1×TBST for 5 min each time. Finally, develop using the ECL method with a developing apparatus.

[0053] like Figure 4 The results showed that after knocking out FLAD1 in A549 cells using CRISPER-Cas9 technology, no FLAD1 band was detected by Western blotting. This demonstrates that the knockout was effective.

[0054] 5. Use quantitative real-time PCR to screen for siRNAs that most effectively knock down FLAD1. The specific method is as follows:

[0055] (1) 8 μL of NC siRNA and 8 μL of 4 kinds of FLAD1 siRNA were transfected into human embryonic kidney 293 cells using TransIT X2 transfection reagent. After 48 h, the culture medium was aspirated, and the cells were collected and resuspended with pre-cooled 1×PBS. The cells were washed 3 times and the supernatant was completely discarded.

[0056] Information on the four FLAD1 siRNAs is shown in Table 1.

[0057] Table 1. Summary of information on four FLAD1 siRNAs

[0058]

[0059] (2) Refer to Qiagen Company The Mini Kit instructions describe extracting total RNA from cells, adding 350 μL of Buffer RLT to lyse cells, mixing well, then adding 350 μL of 70% ethanol, mixing thoroughly, and then transferring the mixture to an RNeasy MiniSpin Column. Centrifuge at 8000 rpm for 1 min and discard the waste liquid. Add 700 μL of Buffer RW1 to the column, centrifuge at 8000 rpm for 1 min, and discard the waste liquid. Add 500 μL of Buffer RPE to the column, centrifuge at 8000 rpm for 1 min, and discard the waste liquid. Repeat this step. Transfer the column to a new 2 mL Collection Tube, centrifuge at 13000 rpm for 5 min, discard the waste liquid, transfer the column to a new 1.5 mL EP tube, add 60 μL of RNase-free water, centrifuge at 13000 rpm for 1 min, and finally obtain the total RNA from the cells for RNA concentration determination.

[0060] (3) Refer to Promega Perform RT-qPCR according to the 1-Step RT-qPCR System kit instructions. Primer information is as follows: SEQ ID No. 11: FLAD1-F: 5'-TGACCCCTACTCCTGTAGCC-3'; SEQ ID No. 12: FLAD1-R: 5'-TCTGTAGGTCCAGTCCAGCA-3'.

[0061] (4) This experiment was performed using a Bio-Rad iQ5 Real Time PCR instrument. 1 μL of RNA was used to prepare a 20 μL reaction mixture. The reaction conditions were: 40℃ for 15 min; 95℃ for 10 min; 95℃ for 10 s; 60℃ for 30 s (collecting fluorescence signal); 72℃ for 30 s; 40 cycles.

[0062] like Figure 5The results showed that knocking down FLAD1 in human embryonic kidney 293 cells with four siRNAs significantly reduced FLAD1 expression, demonstrating that the knockdown was effective, with siRNA-3 showing the best efficiency.

[0063] 6. The effect of FLAD1 knockout on SARS-CoV-2 replication was detected using quantitative real-time PCR. The experimental group consisted of A549(ACE2 OE) cells with FLAD1 knockout infected with SARS-CoV-2 (i.e., A549(ACE2 OE / FLAD1 KO)), and the control group consisted of A549(ACE2 OE) cells infected with SARS-CoV-2. Viral replication was evaluated by measuring viral mRNA levels. The specific methods are as follows:

[0064] On day 1, A549 cells were seeded in 6-well plates and cultured for 24 hours until they adhered to the plates.

[0065] On day 2, cells were infected using SARS-CoV-2 viral supernatant.

[0066] On day 3, the supernatant was replaced with 5% FBS medium. On days 6 and 7, 350 μL of supernatant was collected each time, and 350 μL of Trizol was added. The cells were then stored at -80°C for later use. On day 8, 350 μL of supernatant and cells were collected, and 350 μL of Trizol was added. The cells were then stored at -80°C for later use.

[0067] Referencing Qiagen The Mini Kit instructions describe extracting total RNA from cells. Take the frozen sample, thaw it on ice, add 70% ethanol in equal proportions, mix thoroughly, and then add it to the RNeasy Mini Spin Column. Centrifuge at 8000 rpm for 1 min and discard the waste liquid. Add 700 μL of Buffer RW1 to the column, centrifuge at 8000 rpm for 1 min, and discard the waste liquid. Add 500 μL of Buffer RPE to the column, centrifuge at 8000 rpm for 1 min, and discard the waste liquid. Repeat this step. Transfer the column to a new 2 mL Collection Tube, centrifuge at 13000 rpm for 5 min, discard the waste liquid, transfer the column to a new 1.5 mL EP tube, add 60 μL of RNase-free water, and centrifuge at 13000 rpm for 1 min to obtain the final total RNA. RNA concentration was then measured.

[0068] Referencing Promega Perform RT-qPCR according to the 1-Step RT-qPCR System kit instructions.

[0069] The primer sequence information is as follows: SEQ ID No. 13: NSP10-F: 5'-CCCTGTGGGTTTTACACTTAA-3'; SEQ ID No. 14: NSP10-R: 5'-ACGATTGTGCATCAGCTGA-3'.

[0070] This experiment was performed using a Bio-Rad iQ5 Real-Time PCR instrument. 1 μL of RNA was used to prepare a 20 μL reaction mixture. The reaction conditions were: 40℃ for 15 min; 95℃ for 10 min; 95℃ for 10 s; 60℃ for 30 s (fluorescence signal collection); 72℃ for 30 s; 40 cycles.

[0071] The results are as follows Figure 6 As shown, compared with the control group A549 (ACE2 OE), the replication of the novel coronavirus was significantly inhibited after FLAD1 was knocked out.

[0072] III. Conclusion

[0073] This invention identifies a therapeutic target for flavin adenine dinucleotide (FAD) synthase FLAD1. Experiments have shown that knocking out FLAD1 using CRISPER-Cas9 technology significantly reduces SARS-CoV-2 replication in cells, demonstrating that FLAD1 can effectively inhibit SARS-CoV-2 proliferation. This invention provides a novel therapeutic strategy for the prevention and treatment of severe acute respiratory syndrome caused by SARS-CoV-2 infection.

[0074] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various improvements without departing from the concept of the present invention, and these improvements all fall within the scope of protection of the present invention.

Claims

1. A substance for targeting and inhibiting the expression of FLAD1 in the preparation of a drug for preventing or treating novel coronavirus infection, characterized in that, The substance for targeting and inhibiting the expression of FLAD1 is FLAD1 siRNA or knockout by CRISPR-Cas9 technology FLAD1 Gene editing system of gene The FLAD1 siRNA is any one of (1) to (4): (1) siRNA formed by annealing two single strands as shown in SEQ ID No. 3 and SEQ ID No. 4; (2) siRNA formed by annealing two single strands as shown in SEQ ID No. 5 and SEQ ID No. 6; (3) siRNA formed by annealing two single strands as shown in SEQ ID No. 7 and SEQ ID No. 8; (4) siRNA formed by annealing two single strands as shown in SEQ ID No. 9 and SEQ ID No 10.

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