CN101182517A - Multi-target point cocktail double strand small interfere RNA used for curing disease and method of producing same - Google Patents

Abstract

The invention discloses the multi-target cocktail duplexes of small interfering RNA and the preparation method. According to three or more than three whole or part of open reading frames or the target sequences of mRNA, the multi-target cocktail duplexes of small interfering RNA is designed. The multi-target cocktail duplexes of small interfering RNA is mixed by three or more than three small interfering RNA sequences; the gene knock-out function of the multi-target cocktail duplexes of small interfering RNA is better than that of a single duplexes of small interfering RNA; the invention can be a new and strong drug and method for the treatment of disease.

Description

Be used for multi-target point cocktail double strand small interfere RNA of disease treatment and preparation method thereof

Technical field

The present invention relates to multi-target point cocktail double strand small interfere RNA and sequence signature thereof, also related to the preparation method of multi-target point cocktail double strand small interfere RNA; The siRNA of a plurality of chemical property unanimities (siRNA) molecule can be worked in coordination with the expression of striking low multiple Disease-causing gene, will be expected to become new medicine and method.

Background technology

Disease is owing to due to the undesired expression of autogene or foreign gene or due to the two all undesired expression, be a kind of pathological change process of complexity.The complicated diversity of disease progression process and resistance make the single medicine treatment face great challenge, and a kind of strategy that overcomes such difficult problem is the multiple pharmacological agent of combined utilization.

Unite in recent years that to use two kinds of medicines be general phenomenon comparatively, and to unite the medicine that uses more than 3 kinds or 3 kinds in a treatment plan be a kind of trend of modern medicine.In cancer therapy, unite and use chemotherapy to obtain tangible curative effect.For example, unite the multiple myelomatosis pars oralis pharyngis cancer of following of using paclitaxel analog compound Docetaxel, ifosfamide and the transfer of plus cisplatin in treatment prostate cancer, unite and use cortin, metronidazole and vancomycin treatment ulcerative colitis, unite and use rosiglitazone, Ge Liemei ketone and Mei Difu (diformazan allophanamide) treatment type ii diabetes.Though these combinational therapeutic methods have been obtained good curative effect, because differences such as the source of these medicines, chemical property, manufacture crafts, the security of the higher and long-time use of dosage is the subject matter that these methods face.(RNA disturbs as RNAi, RNAinterfering) siRNA of effector molecule (small interfering RNA, siRNA) can be in same treatment plan a plurality of Disease-causing genes of target, and the chemical property of each siRNA, source and manufacture craft are identical.Therefore unite and use the siRNA molecule not only to treat diseases such as cancer, communicable disease and inflammation clinically to obtain better therapeutic, and toxicity is littler and security is better.

Cancer

Cancer causes owing to multiple inherited genetic factors and environmental hazard.The heredity of oncogene and the sudden change of proto-oncogene are the principal elements of multiple cancer.The overexpression of the cancer suppressor genes such as Rb, p53 of somatomedins such as FGF, VEGF, PDGF, EGF and sudden change is the principal feature in the malignant tissue.The growth of cancer or early cancer is the result that changes of proliferous type cell pathology and relevant with malignant tumour.In near malignant tumour infiltration and the intrusion the tissue.Some malignant tumours can be diffused into whole body by blood and lymphsystem, and the unpredictable and uncontrolled growth of malignant tumour causes malignant cancer.Usually also can recur after the malignant tumour excision.What treat proliferative disease methods such as malignant tumour or malignant cancer is that target promotes the active factor of proliferative cell, and cuts off this process.Different steps in propagation has different specific cellular biochemical approach to be activated.

The anoxia condition that oxygen is under-supply is the early stage factor that causes the vasculogenesis key, by generating nitric oxide synthetase control blood vessel state and regulating vascular endothelial growth factor (VEGF), fibroblast growth factor, angiogenesis hormone and their acceptor.It is reported that the gene that participates in matrix metabolism comprises that matrix metalloproteinase, plasminogen activator receptor and inhibitor, collagen protein Protocollagen prolyl hydroxylase play a crucial role in angiogenesis.The vital role of tumor-blood-vessel growth in the g and D process of solid tumor generally acknowledged.The vital role of tumor-blood-vessel growth in tumor growth not only is proved, and from malignant tumour early stage to the infiltrative type cancer, to perceptible metastatic tumor clinical lesion process, have keying action from the small metastatic tumor of dormancy.

1.VEGF the factor in the path

VEGF family is made up of five kinds of factors of VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF (placenta growth factor), respectively combination and three kinds of acceptor molecules of activation.VEGF-A is in conjunction with VEGFR-1, VEGFR-2, and VEGF-B and PIGF (placenta growth factor) are only in conjunction with VEGFR-1, and VEGF-C and VEGF-D are in conjunction with VEGFR-2 and VEGFR-3.The process that some diseases is relevant also can raise the expression of VEGF.Specific siRNA suppress HIF-1 α, Shb, IQGAP1, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR-1, VEGFR-2 isogenic be expressed in the cell model and the different animals model in show the curative effect of angiogenesis inhibitor.

2. matrix metalloproteinase and adhesion molecule

SiRNA suppresses α v β 3 and integrates expression plain, that alpha 6 beta 4 is integrated the film correlation factor of element, Lyn albumen, PDGFr, ILK, CEACAM6, Smad2, FAK, Pacsin3, ADAM12, Ets-2, ADAM10, Mint-3, MMP-2 and MMP-9, reaches the vasculogenesis ability that reduces different cell types.

3. participate in other acceptors of vasculogenesis

Except VEGF and PDGF, striking low EGF-R ELISA (EGFR or erbB1) also is a kind of method of up-and-coming angiogenesis inhibitor.Use RNAi and strike endogenous erbB1 in low A341 people's epidermoid carcinoma cell, can suppress EGF inductive tyrosine phosphorylation enzyme, suppress the propagation of cell, inducing cell takes place to transfer dies.Studies show that can raise epidermic cell by the expression of siRNA inhibition TACE, amphiregulin, CXCR4, S100A10, receptor associated factors such as Her-2/neu, ErbB-1 transfers die activity and blocking-up cell proliferation.

4. the correlation factor of signal transduction pathway

VEGF stimulates migration, propagation, endothelial tissueization etc. to promote vasculogenesis by the activation signal transduction pathway.VEGF not only influences the endotheliocyte signal to the stimulation of Sphingosine kinase (SPK) also can promote the cell expressing vegf receptor.Studies show that by siRNA and suppress TRIP6, PDK1, DAB2, ILKAP, B-Raf, Mcl-1, the expression of unlike signal transduced elements such as Dgk α and SPK1 can blocking VEGF inductive vasculogenesis.

5. the siRNA of anti-VEGF treats cancer

The angiogenesis inhibitor treatment of cancer has two kinds of diverse ways: A. to activate endogenous anti-angiogenesis or imports the anti-angiogenesis of external source; B. the inhibition medicine that imports external source reduces the activity of endogenous anti-angiogenesis.SiRNA becomes a kind of up-and-coming inhibition medicine owing to its sequence-specific restraining effect, and has confirmed its angiogenesis inhibitor curative effect on cytology.But the siRNA transmission method that has only and come personally feasible is in conjunction with achieving success clinically.In transplantation tumor models such as MDA-435 and MCF-7 breast cancer cell, observe the effect of inhibition tumor growth of the siRNA of anti-VEGF.The nano particle parcel siRNA of cancer target can significantly suppress growth of tumor by the mouse of the method importing load neuroblastoma that is administered systemically behind repeat administration.Confirmed that topical only has effect to the part tumour, and these tumours and clinical and uncorrelated mutually.Therefore the clinical feasible system that the is administered systemically treatment of siRNA malignant tumour must solve.Peaceful thrombus albumen-1 (TSP1) and VEGF have an expression in tumour cell, after the siRNA downward modulation that VEGF is imported by system is expressed, and the effect that recovers TSP1 minimizing vascularization and tumor growth; But behind the topical, fail to find the variation of vegf expression.End collagen imports to PC-3 subcutaneous transplantation knurl by medicine-feeding technology in the knurl with the siRNA of target VEGF as the siRNA carrier, can extremely significantly suppress the vasculogenesis and the tumor growth of tumour.More than the methods of treatment of flourish malignant tumour angiogenesis inhibitor based on siRNA is being represented in research.

Approach such as somatomedin, cytokine, kinases and transcription factor also play important effect in growth of tumor.Many factors overexpression in tumour cell in these approach is the good action target spot of siRNA treatment tumor disease.In the present invention, unite the expression that the siRNA that uses more than three strikes multiple Disease-causing gene in low same approach or the different approaches, obtained stronger anticancer result of treatment.Also multi-joint the closing of clinical anticancer used multiple treatment means and medicine at present.

6. inflammatory disease

Rheumatic arthritis, scleroderma, psoriatic arthritis, lupus, dermatomyositis, fibromyalgia, ankylosing spondylitis, polymyositis, psoriatic arthritis, conjunctivo-urethro-synovial syndrome, juvenile rheumatoid arthritis etc. are the most common autoimmune disorders.These comprise that some make us the disease of serious weakness, and the people of country nearly 1% is influenced by this in prosperity, and millions of patients are arranged in the world approximately.Many because partial immune response causes, causes pain and normal function impaired, influences patient's daily life.

More existing medicines can suppress the immune response of these diseases, but curative effect is relatively poor, cause severe side effect, and the resistance of these medicines is also often had generation.New tool based on the siRNA medicine is the powerful mean of treatment rheumatism, and can avoid side effect; Can also help treatments such as cortin, antibody, gene therapy, soluble receptors, decoy receptor, receptor antagonist.Successful Application siRNA strikes TNF, IL-1 and their receptor expression in the low mammalian cell.Striking low cytokine and its receptor expression simultaneously can the blocking immunity reactive behavior, the development of the process that palliates a disease.

7. ophthalmic

Many ophthalmic diseasess are because due to eye new vessel or the undesired hyperplasia of blood vessel.In a lot of serious ophthalmic diseasess, the new vessel hyperplasia is early stage pathological change, and eyesight is produced infringement, and the new vessel hyperplasia is the major cause that the America and Europe causes permanent loss vision.After the kapillary of retina supply oxygen sustained damage, blood and other body fluid can flow into retina, brought out the new vessel growth, thereby caused urinating disease proliferative diabetic retinopathy (PDR).These new vesseles are implanted in the vitreum usually and are comparatively fragile, affect one's power of vision.If it is very high that serious PDR suffers from without the probability of treatment eyesight major injury.Here said new vessel is that the institute of disease causes, and new vessel can further increase the weight of the deterioration of the state of an illness.The main illness of these eye diseases is that ocular neovascular generates, and is the principal element of blinding among the patient.It is the result of vegf protein and acceptor overexpression thereof that new vessel generates.Therefore, the expression of striking low these short angiogenesis factors will be a kind of effective treatment means, and uniting the result of treatment of using many siRNA can be better.

8. infectious diseases

8.1 at sars coronavirus treatment SARS

Many infectious diseases are accompanied by human whole history.SARS epidemic situation in China and Canada's outburst has been killed hundreds of people.The novel coronavirus that causes the SARS disease is a kind of positive chain RNA virus of strand by World Health Organization's called after sars coronavirus.The hazardness of this novel sars coronavirus also is also not have effective treatment means and vaccine to prevent and treat SARS.The former albumen replicative enzyme 1 (pp1) of sars coronavirus be first also be unique be the gene expression product of template with the viral RNA genome.Pp1a and pp1b are further processed into about 12 Nonstructural Proteins.Nsp1 is to the ripe crucial a kind of proteolytic enzyme of viral protein.Nsp9 is the RNA synthetic enzyme that catalysis viral RNA synthetic RNA relies on.We predict that the siRNA at nsp1 and nsp9 may be the inhibitor that the most effectively suppresses virus transcription and duplicate.The spike protein that is positioned at the virion surface is responsible for inducing of virus tropism, acceptor identification, cell absorption and neutralizing antibody, and therefore, the coding region of spike protein is a siRNA effective function target spot, diffusion that can blocking virus.

8.2 siRNA treatment respiratory system infection at RSV

The caused diseases of respiratory system toxicity of respiratory syncytial virus disease is to use another respiratory system disease of siRNA treatment among the present invention.Rsv infection is the major cause that causes serious infantile respiratory tract infection.Have 2/3rds baby can infect RSV approximately in one-year-old, nearly all children can infect RSV at 2 years old with interior.Rsv infection tends to burst and can continue the several months.In fact, also there is not special and effective treatment means can reduce respiratory virus infection patient's virus load at present.For example, recently the monoclonal antibody drug Synagis of the anti-RSV of new listing (Medimmune Inc) can not alleviate the state of an illness of inpatient's RSV virus infection, this may be since antibody can not in and virus in the cell.Also there is dispute in ribavirin as the curative effect of antiviral.Concerning the treatment of RSV and other virus infections, can suppress the expression of virus replication and pathogenic host's factor safely and effectively, and then there is great demand in the medicine of virus in the cell killing.Therefore, strike the generation that low virus mRNA can stop viral protein, the infection that suppresses virus with duplicate.The present invention has showed that the siRNA at the design of the rna gene group of SARS has suppressed duplicating of virus really and infects in inhuman primate cell.Unite the curative effect of using many effective SARS-siRNA and be better than effective SARS-siRNA of independent use.

9.siRNA double-stranded inhibitor

The application of RNAi on cell model and fruit bat, nematode and zebra fish model obtained develop rapidly.RNAi is naturally occurring a kind of mechanism in the organism, and double-stranded RNA is cut into 19-23nt by RNA enzyme III, Dicer and has 2nt 3 ' terminal double stranded rna molecule, i.e. the siRNA molecule that dangle.SiRNA molecule and multi-component rnase constitute RNA inductive silencing complex (RISC), a strand in the double-stranded subsequently siRNA molecule still combines with RISC and and guides mixture to combine with complementary RNA, degradation of rna under the effect of rnase then, thus reach the effect of silencer.RNAi was chosen as annual scientific breakthrough in 2002 by " science " magazine, science and the further elaboration of RNAi mechanism and the deep expansion of application will be the key breakthrough of coming decade biological medicine.SiRNA also causes people's attention day by day as new treatment means.

The RNAi degradation of rna has sequence-specific, can suppress all expression of gene in theory, for downward modulation or the expression of silencer provides directly and means fast.The siRNA molecule that studies show that the 21-25nt of chemosynthesis has the RNAi effect in mammalian cell, the end of the double-stranded hybrid molecule of siRNA and the thermodynamic stability of central region have determined the function of siRNA molecule.There is research that RISC, siRNA molecule and RNAi characteristic are furtherd investigate.

There is research to report that chemosynthesis or shRNA expression vector produce the siRNA molecule and produce the RNAi effect in the laboratory or the application of potential clinical treatment in mammalian cell.The shRNA expression vector produces little hair fastener shape RNA (shRNA) at first in vivo, and shRNA is cut into siRNA by Dicer subsequently.Research thinks that siRNA treatment human diseases has huge prospect, especially aspect the treatment virus disease.

Summary of the invention

It importantly is not the expression that each potential siRNA molecule can both effectively suppress target gene, do not have method to guarantee that the siRNA that designs can suppress the expression of target gene, its inhibition effect to target gene must be tested and analyze to the curative effect of siRNA molecule.Application-dependent in the treatment is in the part of optimizing and the technology that is administered systemically.The advantage of siRNA medicine is the natural sex of its specificity, stability and mechanism.Therefore the siRNA of 21nt has unique sequence signature, just uses many siRNA to obtain synergistic functions with uniting.

Unite use at the different sequences of same gene or the multi-target-spot siRNA of heterogeneic sequence based on above understanding, reticent endogenous or expression of exogenous gene; The object of the present invention is to provide a kind of multi-target point cocktail double strand small interfere RNA that is used for disease treatment and preparation method thereof, multi-target point cocktail double strand small interfere RNA contains three kinds at least at three kinds of heterogeneic siRNA molecules among the present invention, unites and uses many siRNA to obtain synergistic function.

The technical measures that realize the object of the invention are as follows, a kind of multi-target point cocktail double strand small interfere RNA preparation method, design multi-target point cocktail double strand small interfere RNA according to target sequence complete or part open reading frame or mRNA more than three or three, form multi-target point cocktail double strand small interfere RNA and mix by the sequences of small interfering RNAs more than three or three.

Homology on the target sequence behaviour of siRNA of the present invention and the gene order of mouse or non-human primate, the identical or similar albumen of encoding; SiRNA is made up of natural Nucleotide or by the Nucleotide of synthetic or the duplex molecule structure of being made up of manually modified Nucleotide.

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation is selected for use by at the siRNA of encoding SARS virus Spike protein mRNA (5 '-AAGCTCCTAATTACACTCAAC-3 '), at the siRNA of encoding SARS virus nsp-9mRNA

(5 '-AAGGATGAGGAAGGCAATTTA-3 '), mix at the siRNA of encoding SARS virus nsp-10mRNA (5 '-AAGGATAAGTCAGCTCAATGC-3 ') with at least three or three above sequences of small interfering RNAs in the siRNA of encoding SARS virus nsp-13mRNA (5 '-ACTGGCACACTACTTGTCGA-3 ').

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation is selected for use by at the siRNA of coding mVEGFAmRNA (5 '-AAGCCGTCCTGTGTGCCGCTG-3 '), siRNA (5 '-AACGATGAAGCCCTGGAGTGC-3 ') at coding mVEGFAmRNA, siRNA (5 '-AAGTTAAAAGTGCCTGAACTG-3 ') at coding mVEGFR1mRNA, siRNA (5 '-AAGCAGGCCAGACTCTCTTTC-3 ') at coding mVEGFR1mRNA, mix at the siRNA (5 '-AAGCTCAGCACACAGAAAGAC-3 ') of coding mVEGFR2mRNA with at least three or three above sequences of small interfering RNAs in the siRNA of coding mVEGFR2mRNA (5 '-AATGCGGCGGTGGTGACAGTA-3 ').

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation is selected for use by mixing at least three or three above sequences in the siRNA of the mRNA sequence of VEGF, VEGFR1 and VEGFR2, and sequence is seen sequence table or specification sheets table SS1; The above-mentioned multi-target point cocktail siRNA that mixes as clinical angiogenesis inhibitor, treat moist AMD, diabetes type retinopathy disease, interstitial keratitis, glaucoma and the peripheral retinal region new vessel generates ophthalmic diseases, treats various types of cancers, the medicine of treatment rheumatic arthritis and lung vasculogenesis disease.

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation, select for use by mixing at least three or three above sequences in the siRNA of EGF acceptor, FGF and VEGF mRNA sequence, sequence is seen sequence table or specification sheets table SS1, table SS2 and table SS3, and the above-mentioned multi-target point cocktail siRNA that mixes comprises the medicine of vasculogenesis diseases such as cancer as treatment.

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation; select for use by at Ursodeoxycholic Acid (UDCA) acceptor, VEGF and Alpha-Methyl acyl-CoA racemase (α-methylacyl-CoA racemase; AMACR) at least three or three above sequences mix in the siRNA of mRNA sequence; sequence is seen sequence table or specification sheets table SS2 and table SS53, and the above-mentioned multi-target point cocktail siRNA that mixes is as the medicine of treatment prostate cancer.

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation, select for use by mixing at least three or three above sequences in the siRNA of VEGF, c-Met and PCDP10mRNA sequence, sequence is seen sequence table or specification sheets table SS2, table SS4 and table SS5, and the above-mentioned multi-target point cocktail siRNA that mixes is as the medicine of treatment liver cancer, lung cancer and colorectal carcinoma.

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation, select for use by mixing at least three or three above sequences in the siRNA of VEGF, c-Met and HGF mRNA sequence, sequence is seen sequence table or specification sheets table SS2, table SS3 and table SS4, and the above-mentioned multi-target point cocktail siRNA that mixes is as the medicine of treatment liver cancer.

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation, select for use by mixing at least three or three above sequences in the siRNA of EGF acceptor, VEGF and p53 mutant mRNA sequence, sequence is seen sequence table or specification sheets table SS1, SS2, SS3 and SS5, and the above-mentioned multi-target point cocktail siRNA that mixes is as the treatment lung cancer drugs.

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation, select for use by at the E6 gene of HPV16 and HPV 18 bacterial strains, mix at least three or three above sequences in the siRNA of the EE7 gene of HPV16 and HPV 18 bacterial strains and human P 53 mutant mRNA sequence, sequence is seen sequence table or specification sheets table SS5, and the above-mentioned multi-target point cocktail siRNA that mixes is as the medicine of treatment cervical cancer.

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation, select for use by at least three or three above sequences in the siRNA of integrating plain mRNA sequence at matrix metalloproteinase-2, at placenta growth factor acceptor and α v β 3 and mix, sequence is seen sequence table or specification sheets table SS8, SS9, SS10, and the above-mentioned multi-target point cocktail siRNA that mixes comprises the medicine of the vasculogenesis disease of cancer as treatment.

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation, select for use by mixing at least three or three above sequences in the siRNA of TNF α, IL-1 and IL-1 receptor mrna sequence, sequence is seen sequence table or specification sheets table SS5, and the above-mentioned multi-target point cocktail siRNA that mixes comprises the medicine of the vasculogenesis disease of rheumatic arthritis, uveitis, psoriatic and Crohn's disease (crohn ' s disease) as treatment.

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation, select for use by mixing at least three or three above sequences in the siRNA of RSV virus nucleocapsid albumen (N), non-glycosylated stromatin (M) and transmembrane glycoprotein (F) mRNA sequence, sequence is seen sequence table or specification sheets table SS6, and the above-mentioned multi-target point cocktail siRNA that mixes is as suppressing the RSV medicine for treating viral infections.

The multi-target point cocktail double strand small interfere RNA of the inventive method preparation, select for use by mixing at least three or three above sequences in the siRNA of sars cov spike protein, sars coronavirus RNA polymerase and sars coronavirus rna replicon enzyme mRNA sequence, sequence is seen sequence table or specification sheets table SS7, and the above-mentioned multi-target point cocktail siRNA that mixes is as suppressing the medicine that SARS virus infects.

The purposes of the multi-target point cocktail double strand small interfere RNA of the inventive method preparation; medicine as treatment cancer, transmissible disease and inflammation; described multi-target point cocktail siRNA is by combination and degrade messenger RNA(mRNA) or geneome RNA undesired or that the overexpression gene is transcribed, thus the inhibition advancing of disease.

The purposes of the multi-target point cocktail double strand small interfere RNA of the inventive method preparation, multi-target point cocktail siRNA and other drug are united use as the treatment disease medicament.

Figure of description

Fig. 1 multi-target point cocktail siRNA strikes low VEGF pathway gene and expresses.(CRL-2278 is ATCC) with SVR cell (CRL-2280, ATCC) cell transfecting multi-target point cocktail siRNA for RAW264.7gamma NO (-), dosage is 0,0.1,0.5,1.0 or 2.0ug/2ml/ hole is extracted RNA and is carried out RT-PCR reaction detection expression conditions after 24 hours.(CRL-1573, ATCC) cell detection siVEGFR2 strikes the effect of low external source VEGFR2 genetic expression to the plasmid transfection 293 of expression mVEGFR2.PCI-VEGFR2 plasmid (0.2ug/2ml/ hole) and siVEGFR1 or siLuc (0,0.1,0.5,1.0, or 2.0ug/2ml/well) cotransfection cell extracts RNA after 48 hours, RS-PCR reaction detection expression conditions.

Fig. 2. the multi-target point cocktail siRNA at the VEGF pathway gene suppresses CpG inductive vasculogenesis through topical.Respectively at implant the CpG particle after 24 hours subconjunctival injection siRNA (10ug/10ul pereye) carry out topical, or under deep anaesthesia infective virus after 1 day and 3 days behind corneal limbus the 2mm place inject siRNA.After implanting 4 days and 7 days, CpG ODN checks new vessel and corneal limbus.A: between different treatment vasculogenesis there were significant differences ( ^*, P_0.05; ^*, P_0.01); B: implant CpG and take pictures after 7 days.Amplify 40 times.

Fig. 3. the multi-target point cocktail siRNA at the VEGF pathway gene suppresses CpG inductive vasculogenesis through being administered systemically.Respectively at implant the CpG particle after 24 hours tail vein injection siRNA be administered systemically.After implanting 4 days and 7 days, CpG ODN checks new vessel.A: between different treatment vasculogenesis there were significant differences ( ^*, P_0.05; ^*, P_0.01); B: implant CpG and take pictures after 7 days.Amplify 40 times.

Fig. 4. by the effect of drug administration carrier and the injection siRNA of dosage experiments raising system.Respectively at implant CpG particle 6 and after 24 hours tail vein injection siRNA be administered systemically.After implanting 4 days and 7 days, CpG ODN checks new vessel.A: between different treatment vasculogenesis there were significant differences (*, P_0.05), dosage relies on experiment (10,20,40 and 80g siRNA); B: calculate every group of laboratory animal new vessel area, relatively the effect of various dose angiogenesis inhibitor.

Fig. 5. multi-target point cocktail siRNA suppresses the result of treatment of VEGF pathway gene in the HSK model.Cut gently on cornea, and to splash into titre be 1 * 10 ⁵The HSV-1RE virus of plaque-forming units (PFUs) is set up the HSK model.After 1 day and 3 days, 10ug multi-target point cocktail siRNA carries out topical by subconjunctival injection at virus infection, and 40ug is administered systemically by tail vein injection.Virus infection calculates HSK damage average (A) and new vessel score (B) after 10 days.Each stain is represented the clinical score of each, the average score that the digitized representation on the sea line should be organized.C: multi-target point cocktail siRNA handles back edge of cornea new vessel and reduces.

Fig. 6. multi-target point cocktail siRNA reduces the expression of VEGF mRNA in the HSV-1 virus infection cornea.With 1 * 10 ⁵The HSV-1 RE virus infection cornea of plaque-forming units (PFUs), the siRNA treatment group imports siRNA by subconjunctival injection and tail vein injection at virus infection after 1 day and 3 days, behind virus infection 4 days. and collect the cornea of each treatment group 7 days the time, extract RNA and carry out RT-PCR and analyze (A) or real-time quantitative PCR analysis (B).

Fig. 7. multi-target point cocktail siRNA reduces vegf protein level in the HSV-1 virus infection cornea.After infecting 7 days, detect the variation of vegf protein level.

Fig. 8 .A: reduce virus genomic copy number.The real-time quantitative PCR result shows that uniting the special siRNA of many SARS of use has stronger inhibition effect than using one separately.B: unite the research that the prevention of using many special siRNA of SARS continues actual effect.Preventive effect can continue until transfection after 72 hours.Unite the combination and the dosage that use many siRNA: SC2+SC5+SC14; SC14+SC15; SC14+SC5; SC14+SC2; SC5+SC14+SC15; 3xSC2 (0.9 μ g SC2 siRNA/well; 3XSC5 (0.9 μ gSC5 siRNA/well); Contrast: untransfected siRNA.

Fig. 9 unites and uses many special siRNA of SARS can significantly improve inhibition effect (P＜0.05) to virus.1:SC2+SC5; 3:SC14+SC15; 4:SC14+SC5; 6:SC14+SC2; 7:SC5+SC15; 8:SC2+SC5+SC14+SC15; 9:SC2+SC5+SC14; 2XSC2:0.6 μ g SC2/ hole; 2XSC5:0.6 μ g SC5 siRNA/ hole; 3XSC5:0.9 μ g SC5 siRNA/ hole; 4XSC5:1.2 μ g siRNA/ hole; Contrast: untransfected siRNA.

Figure 10. the angiogenesis inhibitor effect of multi-target point cocktail siRNA

(A-B) .LacZ-siRNA (A) or multi-target point cocktail siRNA (Cock tail siRNA) (B) tumor tissue section of administration through H﹠amp; The E processing of dyeing: the coloration result in LacZ-siRNA (A) group shows chromosome condensation in the nuclear, shows to have the proliferation activity that enlivens.(C-D) .VEGF monoclonal antibody coloration result shows: LacZ-siRNA (C) group is compared with hVEGF-siRNA administration group and is had a large amount of capillary vesseies, and the VEGF high expression level.(E-F) .TUNEL dyeing more the depth represent the natural death of cerebral cells vigor.The coloration result of multi-target point cocktail siRNA administration group shows the natural death of cerebral cells that existence is active.

Figure 11. multi-target point cocktail siRNA suppresses xenotransplantation growth of tumor (I).Multi-target point cocktail siRNA per week is administered once, and measures gross tumor volume before each administration.

Figure 12. multi-target point cocktail siRNA suppresses xenotransplantation growth of tumor (II).Multi-target point cocktail siRNA per week is administered once, and measures gross tumor volume before each administration.

Embodiment

The design of embodiment 1. multi-target point cocktail double strand small interfere RNAs

Be used to strike the siRNA duplex molecule of low genetic expression, no matter its at be what gene, all adopt same chemical process synthetic; And the pathways metabolism unanimity of each siRNA component in the multi-target point cocktail double strand small interfere RNA.Therefore in the potion medicine, unite and use many side effects that siRNA can not cause expecting.And unite effect that use shows at heterogeneic many siRNA of disease-related and be better than single siRNA at term single gene, show and unite the synergistic function that uses many siRNA.

1. the selection of template sequence (gene of multi-target point cocktail double strand small interfere RNA target or mRNA).

Although the sequence of the same a kind of gene in the different biologies has very high homology sometimes, still can there be difference; And medicament research and development need be tested its drug effect and security in multiple biologies such as mouse and monkey, if can design the time and the cost that will shorten simultaneously medicament research and development greatly at the gene of humans and animals model, therefore simultaneously at the siRNA inhibitor of humans and animals model sequence be a kind of be suitable for simultaneously clinical before and the splendid way of clinical study.If the while is at the siRNA of effect not as only designing at the human sequence of the siRNA of humans and animals model sequences Design, we can use alternative method, the siRNA that promptly can be used for clinical study, and the siRNA that filters out on data backer's cell model of siRNA in animal model of utilization at gene order of the same race in the animal model by the screening of people's cell model.SiRNA at sequence be the zone of the covering 21-27m in the goal gene mRNA sequence, sequence is seen sequence table or table SS1, SS2, SS3, SS4, SS5, SS6, SS7, SS8, SS9 and SS10.

2. the selection of each component sequence in the multi-target point cocktail double strand small interfere RNA.

Multi-target point cocktail double strand small interfere RNA refers at least 3 siRNA molecules of associating use, at least 3 different zones of at least 3 genes or same gene, and the expression of downward modulation Disease-causing gene or disease controlling gene (disaese control genes).The siRNA molecule is united to use and is had fine or treat the effect of disease preferably.SiRNA at sequence be the zone of the covering 21-27nt in the goal gene mRNA sequence.Select the sequence of institute's target according to different algorithms.The associating use also needs to consider at the design of heterogeneic not homotactic three above siRNA more than three kinds: except the effect of considering to avoid to miss the target, only at particular sequence, be easy to the requirement of design siRNA such as combination of RISC, also needing to verify between the siRNA of associating use does not have sequence homology.Can finish by information biology instrument and direct aligned sequences.

3. the composition of multi-target point cocktail double strand small interfere RNA.

The siRNA at same specific gene that is screened can unite use, also can with unite use at other gene siRNAs.Each contains the siRNA molecule that is no less than three at three gene multi-target point cocktail double strand small interfere RNAs.At each component ratio in the same specific gene siRNA cocktail agent identical (calculating) according to molecular weight; When a plurality of expression of gene levels of target not simultaneously, the ratio of each siRNA can be decided according to specific requirement.

4. the evaluation of multi-target point cocktail double strand small interfere RNA function.

Filter out siRNA with silencer expressive function ( embodiment 2 and 3 etc.) by different biological assessments, as RT-PCR, Western Blot, ELASIA, protein function analysis and physiological function analysis etc.The best multi-target point cocktail double strand small interfere RNA of effect will be used to further clinical preceding and clinical study.

5. according to treatment cancer, inflammation and the transmissible disease multi-target point cocktail double strand small interfere RNA of aforesaid method screening and design.The independent siRNA that forms multi-target point cocktail double strand small interfere RNA can select from table SS1, SS2, SS3, SS4, SS5, SS6, SS7, SS8, SS9 and SS10 according to following requirement.

(1) can both bring into play its effect at the different combination of siRNA formation of the different VEGF path factors on same the path, make up as following multi-target point cocktail double strand small interfere RNA:

VEGF-A, VEGF-B and VEGF-R2

VEGF-A, VEGF-B and PIGF

VEGF-B, VEGF-R1 and VEGF-R2

VEGF-A, PIGF and VEGF-R2

Or at other combinations of above-mentioned factor siRNA.

(2) can both bring into play its effect at the different combination of different siRNA formation of matrix metalloproteinase and adhesion molecule correlation factor mRNA sequence, make up as following multi-target point cocktail double strand small interfere RNA:

MMP-2, MMP-9 and PDGF-R

α v β 3 integrin, α v β 5 integrin and alpha 6 beta 4 integrin

Lyn, PDGFr and ILK

CEACAM6, Smad2 and FAK

Pacsin 3, ADAM12 and Ets-2

Mint-3, MMP-2 and MMP-9

Or at other combinations of above-mentioned factor siRNA.

(3) different combinations also can effectively be treated cancer, neovascular diseases of the eye, inflammatory disease at the different siRNA formation of the different receptor pathway factor mRNA sequences relevant with the vasculogenesis process, makes up as following multi-target point cocktail double strand small interfere RNA:

EGF, FGF and VEGF

EGF, VEGF and HGF

ErbB-1, Her-2 and VEGF-R2

ErbB-1, VEGF-R2 and FGF-R

TACE, Amphiregulin and CXCR4

S100A10, Her-2/neu and FGF-R

Or at other combinations of above-mentioned factor siRNA.

(4) different combinations also can effectively be treated cancer at the different siRNA formation of the signal transduction pathway factor mRNA sequence relevant with the vasculogenesis process, makes up as following multi-target point cocktail double strand small interfere RNA:

TRIP6, Grb-2 and PDK1

DAB2, ILKAP and B-Raf

C-Raf, Mcl-1 and Dgk α

A-Raf, Grb-2 and SPK1

Or at other combinations of above-mentioned factor siRNA.

(5) transferring the different siRNA of the plastosome associated protein factor mRNA sequence of the process of dying to constitute different combinations at participation also can effectively treat cancer, make up as following multi-target point cocktail double strand small interfere RNA:

Bcl-2, BCL2L1 and Trail

Trail, Bcl-2 and Fas

Or at other combinations of above-mentioned factor siRNA.

(6) at short angiogenesis factor, short multiplicaiton factor with anti-ly transfer the combination of the siRNA inhibitor of dying can very effective treatment cancer.The associating use will be the medicine of strong treatment lung cancer, kidney and colorectal carcinoma at the multi-target point cocktail double strand small interfere RNA of VEGF, EGF and FGF and their acceptor; The associating use will be the medicine of strong treatment mammary cancer at the multi-target point cocktail double strand small interfere RNA of VEGF, Her-2 and p53 mutant.

(7) expression that can reduce the oncogene specified mutant very selectively at the combination of the siRNA inhibitor of proto-oncogene mutant sequence.The associating use will be the medicine of strong treatment mammary cancer at the siRNA cocktail agent of K-ras mutant, ERCC1 mutant and BRCA1 mutant.

(8) Human papilloma virus HPV (HPV), endogenous oncogene and proto-oncogene are the major causes that causes cervical cancer.Therefore, use siRNA simultaneously: unite the siRNA cocktail agent of use at E6, E7 and p53 sequence at HPV (as E6 and E7 sequence) and p53 gene.E6 and E7 gene source are in HPV16 or HPV18 bacterial strain, and p53 is the sequence of human mutant.

(9) (α-methylacyl-CoA racemase AMACR) is the method for effectively treating prostate cancer at Ursodeoxycholic Acid (UDCA) acceptor, VEGFR2 and Alpha-Methyl acyl-CoA racemase to unite use.

(10) also can effectively treat inflammatory disease at the different combination of different siRNA formation of the factor mRNA sequence that participates in inflammatory disease, make up as following siRNA cocktail agent:

TNF-α, TNF-β and IL-1

TNF-α, IL-1 and IL-1r

IL-1 β, GG2-1 and TNF-α

CIAS1, Ark and TNF-α

TNF-β, IL-1 α and IL-1 β

NF-κ B, TNF-α and IL-1

Or at other combinations of above-mentioned factor siRNA.

(11) the eye stromal keratitis is the inflammatory disease of human herpes simplex vicus (HSV) infection-induced.The associating use is effective treatment means at the multi-target point cocktail double strand small interfere RNA of HSV virus gene sequence, pro-inflammatory cytokine (as IL-17, IL-12) and angiogenesis factor (VEGF or VEGFR).

(12) in suppressing African green time nephrocyte SARS virus infect and the research of duplicating in find at nsp-1 albumen, at nsp-9 albumen, at nsp-10 albumen with show the effect (siRNA at first transfection enters cell, uses virus infected cell again) of very strong prophylaxis of viral infections respectively at four siRNA molecules of spike protein; But the effect of treatment virus infection is weak (at first use virus infected cell, siRNA transfection again enters cell).Use these to have the active siRNA of inhibition but ought mix in varing proportions, its effect is significantly increased.

(13) by the respiratory tract lead-in mode, will be the method that effectively suppresses the RSV virus infection at the siRNA cocktail agent of RSV virus nucleocapsid albumen (N), non-glycosylated stromatin (M) and transmembrane glycoprotein (F) gene.

(14) the eye new vessel is the typical genius morbis of many eye illnesses.We have designed the siRNA at mouse VEGF-A, VEGFR-1 and VEGFR-2 gene, strike the expression of low corresponding gene, blocking-up vasculogenesis process.These siRNA are imported eye by local and the approach that is administered systemically respectively separately, the vasculogenesis that can significantly suppress virus infection and brought out.Unite the curative effect of using these siRNA and be much better than independent siRNA.

6. multi-target point cocktail double strand small interfere RNA and other medicines are united to use and are had better effect.

In the treatment disease, uniting use at the cocktail siRNA (at 3 or 3 above sequences) and the other drug of many target spots is up-and-coming treatment plan.For example, unite the siRNA cocktail agent, monoclonal antibody drug, antibiotic medicine (chemotherapy) and other small-molecule drugs that use many target spots (as Gleevec etc.) treatment cancer.

Table SS1.VEGF approach

Table SS1.1.VEGF-A

The VEGF gene: people VEGF, Accession:XM_052681, GeneID:14781453, mouse VEGF, Accession:M95200, Gene ID:202350. is according to this sequences Design and screen 10 siRNA:

Numbered positions target sequence

VEGF-A-1 64-84 AAGTGGTCCCAGGCTGCACCC

VEGF-A-2 467-487 AAGATCCGCAGACGTGTAAAT

VEGF-A-3 498-518 AAACACAGACTCGCGTTGCAA

VEGF-A-4 499-519 AACACAGACTCGCGTTGCAAG

VEGF-A-5 517-537 AAGGCGAGGCAGCTTGAGTTA

VEGF-A-6 537-557 AAACGAACGTACTTGCAGATG

VEGF-A-7 538-558 AACGAACGTACTTGCAGATGT

VEGF-A-8 542-564 AACGTACTTGCAGATGTGACA

VEGF-A-9 162-182 AATCGAGACCCTGGTGGACAT

VEGF-A-10 338-358 AAGGCCAGCACATAGGAGAGA

Table SS1.2.VEGF-B

VEGF-B gene: people VEGF-B, Accession:NM_003377.3, Gene ID:39725673

According to this sequences Design and screen 10 siRNA:

Numbered positions target sequence

VEGF-B-1 140-160 AAAGTGGTGTCATGGATAGAT

VEGF-B-2 141-163 AAGTGGTGTCATGGATAGATG

VEGF-B-3 236-258 AAACAGCTGGTGCCCAGCTGC

VEGF-B-4 327-349 AAGTCCGGATGCAGATCCTCA

VEGF-B-5 390-412 AAGAACACAGCCAGTGTGAAT

VEGF-B-6 393-415 AACACAGCCAGTGTGAATGCA

VEGF-B-7 424-446 AAAGGACAGTGCTGTGAAGCC

VEGF-B-8 425-447 AAGGACAGTGCTGTGAAGCCA

VEGF-B-9 440-462 AAGCCAGACAGGGCTGCCACT

VEGF-B-10 670-692 AACCCAGACACCTGCAGGTGC

Table SS1.3.

VEGF R-1 gene: human VEGF-R1, (hFLT-1), and Accession:AF063657, Gene ID:3132830, mouse VEGF-R1, (mFLT-1), Accession:D88689, Gene ID:2809068),

According to this sequences Design and screen 8 siRNA:

The numbered positions sequence

VEGFR1-1 1706-1728 AAGGAGAGGACCTGAAACTGT

VEGFR1-2 2698-2720 AAGCAAGGAGGGCCTCTGATG

VEGFR1-3 2702-2724 AAGGAGGGCCTCTGATGGTGA

VEGFR1-4 2755-2777 AACTACCTCAAGAGCAAACGT

VEGFR1-5 3014-3036 AAGTGGCCAGAGGCATGGAGT

VEGFR1-6 3048-3070 AAAGTGCATTCATCGGGACCT

VEGFR1-7 3049-3071 AAGTGCATTCATCGGGACCTG

VEGFR1-8 2140-2160 AGCACGCTGTTTATTGAAAGA

Table SS1.4.

VEGF R-2 gene: people VEGF-R2, (hKDR), and Accession:AF063658, Gene ID:3132832, mouse VEGF-R2, (mFLK-1), Accession:X70842, Gene ID:57923),

According to this sequences Design and screen 11 siRNA:

The numbered positions sequence

VEGFR2-1 523-545 AACAGAATTTCCTGGGACAGC

VEGFR2-2 2387-2409 AACTGAAGACAGGCTACTTGT

VEGFR2-3 2989-3011 AAGGACTTCCTGACCTTGGAG

VEGFR2-4 3032-3054 AAGTGGCTAAGGGCATGGAGT

VEGFR2-5 3040-3062 AAGGGCATGGAGTTCTTGGCA

VEGFR2-6 3401-3423 AAATGTACCAGACCATGCTGG

VEGFR2-7 3632-3654 AATTCCATTATGACAACACAG

VEGFR2-8 3676-3698 AACAGTAAGCGAAAGAGCCGG

VEGFR2-9 3641-3661 ATGACAACACAGCAGGAATCA

VEGFR2-10 357-377 AAGCTCAGCACACAGAAAGAC

VEGFR2-11 493-513 AATGCGGCGGTGGTGACAGTA

Table SS2.EGF approach

Table SS2.1.

EGF gene: people EGF, Accession:NM_001963, Gene ID:6031163.

According to this sequences Design and screen 20 siRNA:

The numbered positions sequence

EGF-1 2042-2062 AAGTGGATAGAGAGAGCTAAT

EGF-2 3873-3893 AAGGCTGCTGGATTCCAGTAT

EGF-3 2426-2446 AAGCAGTCTGTGATTGAAATG

EGF-4 2621-2641 AAGCCCTCATCACTGGTTGTG

EGF-5 1273-1293 AAAGGACATGGTTAGAATTAA

EGF-6 2328-2348 AAGGCCTTGGCCGTCTGGTTA

EGF-7 174-194 AAGGGTGTCAGGTATTTCTTA

EGF-8 3922-3942 AATGGAGCGAAGCTTTCATAT

EGF-9 1496-1516 AAGTACTGTGAAGATGTTAAT

EGF-10 1274-1294 AAGGACATGGTTAGAATTAAC

EGF-11 531-551 AAGGTACTCTCGCAGGAAATG

EGF-12 2686-2706 AAACGGAGGCTGTGAACATAT

EGF-13 2263-2283 AATGGCCAAGAGATTATTCTG

EGF-14 1292-1312 AACCTCCATTCATCATTTGTA

EGF-15 261-281 AAGGTCTCTCAGTTGAAGAAA

EGF-16 3218-3238 AATGCCAGCTGCACAAATACA

EGF-17 1019-1039 AAGGCTCTGTTGGAGACATCA

EGF-18 2576-2596 AAGAGGACTGGCAAAGATAGA

EGF-19 760-780 AAGGCAAGAGAGAGTATGTAA

EGF-20 765-785 AAGAGAGAGTATGTAATATAG

Table SS2.2.

EGF R gene: Human EGF-R, Accession:NM_005228, Gene ID:41327737), mouse EGF-R, Accession:NM_207655, Gene ID:46560581,

According to this sequences Design and screen 5 siRNA:

The numbered positions sequence

EGFR-1 483-505 AAAGACCATCCAGGAGGTGGC

EGFR-2 2869-2889 AAAGTGCCTATCAAGTGGATG

EGFR-3 2870-2890 AAGTGCCTATCAAGTGGATGG

EGFR-4 3751-3771 AACCCTGACTACCAGCAGGAC

EGFR-5 3755-3775 CTGACTACCAGCAGGACTTCT

Table SS2.3.

The HER-2 gene: people HER-2, Accession:M11730, Gene ID:183986, mouse HER-2, Accession:BC053078, Gene ID:31419374,

According to this sequences Design and screen 5 siRNA:

The numbered positions sequence

HER2-1 1255-1275 AAGATCTTTGGGAGCCTGGCA

HER2-2 1253-1273 AAGAAGATCTTTGGGAGCCTG

HER2-3 2797-2817 AAGGTGCCCATCAAGTGGATG

HER2-4 3019-3039 AAATGTTGGATGATTGACTCT

HER2-5 3805-3825 AACCTCTATTACTGGGACCAG

Table SS2.4.

The HER-3 gene: people HER-3, Accession:M34309, Gene ID:183990, mouse HER-3, Accession:XM 125954, Gene ID:38091004,

According to this sequences Design and screen 13 siRNA:

The numbered positions sequence

HER3-1 678-698 AATTGACTGGAGGGACATCGT

HER3-2 1264-1284 AAGATCCTGGGCAACCTGGAC

HER3-3 1537-1557 AAGGAAATTAGTGCTGGGCGT

HER3-4 2404-2424 AAGATTCCAGTCTGCATTAAA

HER3-5 2857-2877 AAATACACACACCAGAGTGAT

HER3-6 2858-2878 AATACACACACCAGAGTGATG

HER3-7 3770-3790 AAGATGAAGATGAGGAGTATG

HER3-8 3776-3796 AACCTCTATTACTGGGACCAG

HER3-9 1118-1138 CTGACAAGATGGAAGTAGATA

HER3-10 1119-1139 TGACAAGATGGAAGTAGATAA

HER3-11 2402-2422 TCAAGATTCCAGTCTGCATTA

HER3-12 2403-2423 CAAGATTCCAGTCTGCATTAA

HER3-13 2805-2825 TGAGGCCAAGACTCCAATTAA

Table SS2.5.

HER-4 gene: people HER-4, Accession:NM_005235, Gene ID:4885214, mouse HER-4, Accession:XM_136682, Gene ID:38049556.

According to this sequences Design and screen 7 siRNA:

The numbered positions sequence

HER4-1 462-482 AAATGGTGGAGTCTATGTAGA

HER4-2 463-483 AATGGTGGAGTCTATGTAGAC

HER4-3 731-751 AATGTGCTGGAGGCTGCTCAG

HER4-4 838-860 AATCCAACCACCTTTCAACTG

HER4-5 1227-1247 AACAGGTTTCCTGAACATACA

HER4-6 1450-1470 AACTGGACAACACTCTTCAGC

HER4-7 1909-1929 AACGGTCCCACTAGTCATGAC

Table SS3.FGF approach

Table SS3.1.

FGF-2 gene: people FGF-2 (basic FGF), Accession:NM_002006, Gene ID:41352694.

According to this sequences Design and screen 20 siRNA:

The numbered positions sequence

FGF-2-1 630-650 AAGAGCGACCCTCACATCAAG

FGF-2-2 661-681 AAGCAGAAGAGAGAGGAGTTG

FGF-2-3 849-869 AAACGAACTGGGCAGTATAAA

FGF-2-4 880-900 AAACAGGACCTGGGCAGAAAG

FGF-2-5 854-874 AACTGGGCAGTATAAACTTGG

FGF-2-6 648-668 AAGCTACAACTTCAAGCAGAA

FGF-2-7 850-870 AACGAACTGGGCAGTATAAAC

FGF-2-8 881-901 AACAGGACCTGGGCAGAAAGC

FGF-2-9 667-687 AAGAGAGAGGAGTTGTGTCTA

FGF-2-10 723-743 AAGGAAGATGGAAGATTACTG

FGF-2-11 734-754 AAGATTACTGGCTTCTAAATG

FGF-2-12 781-801 AACGATTGGAATCTAATAACT

FGF-2-13 690-710 AAAGGAGTGTGTGCTAACCGT

FGF-2-14 818-838 AAGGAAATACACCAGTTGGTA

FGF-2-15 804-824 AATACTTACCGGTCAAGGAAA

FGF-2-16 750-770 AAATGTGTTACGGATGAGTGT

FGF-2-17 822-842 AAATACACCAGTTGGTATGTG

FGF-2-18 655-675 AACTTCAAGCAGAAGAGAGAG

FGF-2-19 823-843 AATACACCAGTTGGTATGTGG

FGF-2-20 798-818 AACTACAATACTTACCGGTCA

Table SS3.2.

FGF-1 gene: people FGF-1 (acidic FGF),

transcript?variant?1，Accession：NM_000800，Gene?ID：15055546；

transcript?variant?2，Accession：NM_033136，Gene?ID：15055540；

transcript?variant?3，Accession：NM_033137，Gene?ID：15055544.

According to this sequences Design and screen 20 siRNA:

The numbered positions sequence

FGF-1-1 447-467 AAGGCTGGAGGAGAACCATTA

FGF-1-2 214-234 AAGCCCAAACTCCTCTACTGT

FGF-1-3 190-210 AATCTGCCTCCAGGGAATTAC

FGF-1-4 114-134 AAGCGCCACAAGCAGCAGCTG

FGF-1-5 484-504 AAGAAGCATGCAGAGAAGAAT

FGF-1-6 539-559 AACGCGGTCCTCGGACTCACT

FGF-1-7 460-480 AACCATTACAACACCTATATA

FGF-1-8 97-117 AAGCTCTTTAGTCTTGAAAGC

FGF-1-9 469-489 AACACCTATATATCCAAGAAG

FGF-1-10 221-241 AACTCCTCTACTGTAGCAACG

FGF-1-11 288-308 AAGGGACAGGAGCGACCAGCA

FGF-1-12 487-507 AAGCATGCAGAGAAGAATTGG

FGF-1-13 113-133 AAAGCGCCACAAGCAGCAGCT

FGF-1-14 502-522 AATTGGTTTGTTGGCCTCAAG

FGF-1-15 520-540 AAGAAGAATGGGAGCTGCAAA

FGF-1-16 211-231 AAGAAGCCCAAACTCCTCTAC

FGF-1-17 538-558 AAACGCGGTCCTCGGACTCAC

FGF-1-18 526-546 AATGGGAGCTGCAAACGCGGT

FGF-1-19 220-240 AAACTCCTCTACTGTAGCAAC

FGF-1-20 424-444 AATGAGGAATGTTTGTTCCTG

Table SS3.3.

FGFR2 gene: people FGFR2

transcript?variant?1，Accession：NM_000141，Gene?ID：13186239；

transcript?variant?2，Accession：NM_022969，Gene?ID：13186252；

transcript?variant?3，Accession：NM_022970，Gene?ID：13186254.

transcript?variant?4，Accession：NM_022971，Gene?ID：13186256；

transcript?variant?5，Accession：NM_022972，Gene?ID：13186258；

transcript?variant?6，Accession：NM_022973，Gene?ID：13186260.

transcript?variant?7，Accession：NM_022974，Gene?ID：13186262；

transcript?variant?8，Accession；NM_022975，Gene?ID：27754768；

transcript?variant?9，Accession：NM_022976，Gene?ID：13186266.

transcript?variant?10，Accession：NM_023028，Gene?ID：13186268；

transcript?variant?11，Accession：NM_023029，Gene?ID：13186242；

transcript?variant?12，Accession：NM_023030，Gene?ID：13186270.

transcript?variant?13，Accession：NM_023031，Gene?ID：13186272；

According to this sequences Design and screen 20 siRNA:

The numbered positions sequence

FGFR2-1 1368-1388 AAGCCGGACTGCCGGCAAATG

FGFR2-2 2610-2630 AAGCCCTGTTTGATAGAGTAT

FGFR2-3 2088-2108 AAGCAGTGGGAATTGACAAAG

FGFR2-4 2297-2317 AAAGGCAACCTCCGAGAATAC

FGFR2-5 1753-1773 AATCGCCTGTATGGTGGTAAC

FGFR2-6 2010-2030 AATGGGAGTTTCCAAGAGATA

FGFR2-7 699-719 AAGAGCCACCAACCAAATACC

FGFR2-8 2843-2863 AAGCAGTTGGTAGAAGACTTG

FGFR2-9 1187-1207 AAGCAGGAGCATCGCATTGGA

FGFR2-10 1082-1102 AAGCGGCTCCATGCTGTGCCT

FGFR2-11 1557-1577 AAGAGATTGAGGTTCTCTATA

FGFR2-12 1771-1791 AACAGTCATCCTGTGCCGAAT

FGFR2-13 2762-2782 AAGCCAGCCAACTGCACCAAC

FGFR2-14 1178-1198 AAGGAGTTTAAGCAGGAGCAT

FGFR2-15 2151-2171 AAGATGATGCCACAGAGAAAG

FGFR2-16 2745-2765 AAGGACACAGAATGGATAAGC

FGFR2-17 1171-1191 AAACGGGAAGGAGTTTAAGCA

FGFR2-18 1222-1242 AAACCAGCACTGGAGCCTCAT

FGFR2-19 2732-2752 AAGCTGCTGAAGGAAGGACAC

FGFR2-20 1556-1576 AAAGAGATTGAGGTTCTCTAT

Table SS3.4.

FGFR1 gene: people FGFR1

transcript?variant?1，Accession：NM_000604，Gene?ID：13186232；

transcript?variant?2，Accession：NM_015850，Gene?ID：13186250；

transcript?variant?3，Accession：NM_023105，Gene?ID：13186233.

transcript?variant?4，Accession：NM_023106，Gene?ID：13186235；

transcript?variant?5，Accession：NM_023107，Gene?ID：13186237；

transcript?variant?6，Accession：NM_023108，Gene?ID：13186240.

transcript?variant?7，Accession：NM_023109，Gene?ID：13186244；

transcript?variant?8，Accession：NM_023110，Gene?ID：13186246；

transcript?variant?9，Accession：NM_023111，Gene?ID：13186248.

According to this sequences Design and screen 20 siRNA:

The numbered positions sequence

FGFR1-1 2701-2721 AACGGCCGACTGCCTGTGAAG

FGFR1-2 2275-2295 AAGTCGGACGCAACAGAGAAA

FGFR1-3 2422-2442 AAGGGCAACCTGCGGGAGTAC

FGFR1-4 2255-2275 AAGTGGCTGTGAAGATGTTGA

FGFR1-5 2319-2339 AATGGAGATGATGAAGATGAT

FGFR1-6 2237-2257 AACCCAACCGTGTGACCAAAG

FGFR1-7 2887-2907 AAGCCCAGTAACTGCACCAAC

FGFR1-8 1540-1560 AACGTGGAGTTCATGTGTAAG

FGFR1-9 2236-2256 AAACCCAACCGTGTGACCAAA

FGFR1-10 2332-2352 AAGATGATCGGGAAGCATAAG

FGFR1-11 1153-1173 AACACCAAACCAAACCGTATG

FGFR1-12 1303-1323 AATGGCAAAGAATTCAAACCT

FGFR1-13 2905-2925 AACGAGCTGTACATGATGATG

FGFR1-14 1636-1656 AACCTGCCTTATGTCCAGATC

FGFR1-15 2857-2877 AAGCTGCTGAAGGAGGGTCAC

FGFR1-16 1596-1616 AAAGCACATCGAGGTGAATGG

FGFR1-17 2230-2250 AAGGACAAACCCAACCGTGTG

FGFR1-18 2968-2988 AAGCAGCTGGTGGAAGACCTG

FGFR1-19 2254-2274 AAAGTGGCTGTGAAGATGTTG

FGFR1-20 1444-1464 AACCACACATACCAGCTGGAT

Table SS3.5.

FGFR3 gene: people FGFR3, Accession:M58051, Gene ID:182568

transcript?variant?1，Accession：NM_000142，Gene?ID：13112046；

transcript?variant?2，Accession：NM_022965，Gene?ID：13112047；

According to this sequences Design and screen 20 siRNA:

The numbered positions sequence

FGFR3-1 1969-1989 AACCTCGACTACTACAAGAAG

FGFR3-2 1627-1647 AAGATGATCGGGAAACACAAA

FGFR3-3 1588-1608 AAGGACCTGTCGGACCTGGTG

FGFR3-4 865-885 AAGGTGTACAGTGACGCACAG

FGFR3-5 2263-2283 AAGCAGCTGGTGGAGGACCTG

FGFR3-6 652-672 AAGCTGCGGCATCAGCAGTGG

FGFR3-7 1540-1560 AAGCCTGTCACCGTAGCCGTG

FGFR3-8 1571-1591 AAGACGATGCCACTGACAAGG

FGFR3-9 1321-1341 AACGCGTCCATGAGCTCCAAC

FGFR3-10 1297-1317 AAGCGACAGGTGTCCCTGGAG

FGFR3-11 2191-2211 AACTGCACACACGACCTGTAC

FGFR3-12 994-1014 AAGGAGCTAGAGGTTCTCTCC

FGFR3-13 1570-1590 AAAGACGATGCCACTGACAAG

FGFR3-14 982-1002 AACACCACCGACAAGGAGCTA

FGFR3-15 1873-1893 AAGTGCATCCACAGGGACCTG

FGFR3-16 331-351 AATGCCTCCCACGAGGACTCC

FGFR3-17 1813-1833 AAGGACCTGGTGTCCTGTGCC

FGFR3-18 2152-2172 AAGCTGCTGAAGGAGGGCCAC

FGFR3-19 1723-1743 AACCTGCGGGAGTTTCTGCGG

FGFR3-20 265-285 AAGGATGGCACAGGGCTGGTG

Table SS3.6.

FGFR4 gene: people FGFR4, Accession:L03840, Gene ID:182570

transcript?variant?1，Accession：NM_002011，Gene?ID：47524172；

transcript?variant?2，Accession：NM_022963，Gene?ID：47524176；

transcript?variant?3，Accession：NM_213647，Gene?ID：47524174；

According to this sequences Design and screen 20 siRNA:

The numbered positions sequence

FGFR4-1 726-746 AAGGATGGACAGGCCTTTCAT

FGFR4-2 2403-2423 AAGGTCCTGCTGGCCGTCTCT

FGFR4-3 1743-1763 AAGCTGATCGGCCGACACAAG

FGFR4-4 1085-1105 AAAGACTGCAGACATCAATAG

FGFR4-5 292-312 AAGAGCAGGAGCTGACAGTAG

FGFR4-6 1657-1677 AAGCCAGCACTGTGGCCGTCA

FGFR4-7 753-773 AACCGCATTGGAGGCATTCGG

FGFR4-8 1833-1853 AAGGGAAACCTGCGGGAGTTC

FGFR4-9 1392-1412 AAGCTCTCCCGCTTCCCTCTG

FGFR4-10 1078-1098 AAGTCCTAAAGACTGCAGACA

FGFR4-11 1692-1712 AACGCCTCTGACAAGGACCTG

FGFR4-12 604-624 AAGCACCCTACTGGACACACC

FGFR4-13 1086-1106 AAGACTGCAGACATCAATAGC

FGFR4-14 1686-1706 AAAGACAACGCCTCTGACAAG

FGFR4-15 666-686 AACACCGTCAAGTTCCGCTGT

FGFR4-16 1454-1474 AAGCTCATCCCTGGTACGAGG

FGFR4-17 984-1004 AAGGTGTACAGCGATGCCCAG

FGFR4-18 1687-1707 AAGACAACGCCTCTGACAAGG

FGFR4-19 1764-1784 AACATCATCAACCTGCTTGGT

FGFR4-20 504-524 AATCTCACCTTGATTACAGGT

Table SS4.HGF approach

SS4.1.

HGF acceptor gene: people HGF receptor (MET), Accession:NM_000245, Gene ID:42741654:

According to this sequences Design and screen 20 siRNA:

The numbered positions sequence

MET-1 341-361 AACACCCATCCAGAATGTCAT

MET-2 505-525 AAGCCAATTTATCAGGAGGTG

MET-3 1494-1514 AAGTCCTCTTAACATCTATAT

MET-4 1021-1041 AATCAGGTTCTGTTCCATAAA

MET-5 2723-2743 AAGCCAGTGATGATCTCAATG

MET-6 3929-3949 AAGTGGATGGCTTTGGAAAGT

MET-7 3747-3767 AAGTAGCCAAAGGCATGAAAT

MET-8 1066-1086 AATGCCTCTGGAGTGTATTCT

MET-9 281-301 AAGTCCGAGATGAATGTGAAT

MET-10 2111-2131 AATGGCCACGGGACAACACAA

MET-11 1682-1702 AATGGCTACACACTGGTTATC

MET-12 2722-2742 AAAGCCAGTGATGATCTCAAT

MET-13 838-858 AAGGCTAAAGGAAACGAAAGA

MET-14 3154-3174 AAGCCCAACTACAGAAATGGT

MET-15 1681-1701 AAATGGCTACACACTGGTTAT

MET-16 1382-1402 AATAGGACACTTCTGAGAAAT

MET-17 734-754 AAAGTCCTTTCATCTGTAAAG

MET-18 1364-1384 AATCATGAGCACTGCTTTAAT

MET-19 2529-2549 AAGCAGGAAGGAACTTTACAG

MET-20 334-354 AACACCCATCCAGAATGTCAT

Table SS5.

Table SS5.1.

Gene title sequence

PAK4-1 AACTTCGAGCACCGCGTGCAC

PAK4-2 AAGACCATCGTGCGGGGCAGC

Hepsin-A AAGGTGGCAGCTCTCACTGCG

Hepsin-B AACAGCGAGGAGAACAGCAAC

Antrogen?R-A AAGACCTACCGAGGAGCTTTC

Antrogen?R-B AAGAGACTAGCCCCAGGCAGC

Table SS5.2.

Gene title sequence

mouse?IL-1b AGGCTCCGAGATGAACAACAA

mouse?IL-1b TACCTGTCCTGTGTAATGAAA

mouse?IL-1r ACCATCGAGGTTACTAATGAA

mouse?IL-1r TCGGAATATCTCCCATCATAA

mouse?IL-1a TCGGGAGGAGACGACTCTAAA

mouse?IL-1a CCAGAGTGATTTGAGATACAA

mouse?IL-1r2 CACGTTTATCTCGGCTGCTTA

mouse?IL-1r2 AAGACTGATAGTCCCGTGCAA

mouse?TNF?receptor?a AAGGAAAGTATGTCCATTCTA

mouse?TNF?receptor?a CCGCAACGTCCTGACAATGCA

mouse?TNF?receptor?b CCAGGTTGTCTTGACACCCTA

mouse?TNF?receptor?b CTGGCTATTCCCGGAAATGCA

mouse?TNF CACGTCGTAGCAAACCACCAA

mouse?TNF CAGCCGATTTGCTATCTCATA

Table SS5.3.

Gene title sequence location

HP?BRCA2-A AAGTCAACCACAGAGTCGTAT 247-268

HP?BRCA2-B AAGTAACGAGTGAGCCACGCT 215-235

NOXA-A AAGTCGAGTGTGCTACTCAAC 238-258

NOX AACTGAACTTCCGGCAGAAAC 277-297

Novel?ZF?Protein AATGCGGAGAACACTAATTAT 345-365

Novel?ZF?Protein AACTTCCATAAATGTGAAATC 381-401

NFAT4 AAGTGATACTCCCGCCTCAGC 726-746

NFAT4 AAGTAGCTGGCACTACGGGCA 752-772

Co-factor?of?SP1 AATCAGGTTCCAATGTGATGA 200-221

Co-factor?of?SP1 AAGGCTTAGCTCCCAAGCCTC 145-165

Ets2?Repressor AAGGCAGATCCAGCTGTGGCA 194-214

Ets2?Repressor AAGCCAGAGTCGTCCCCTGGC 171-191

PKC?related AAGTCTTCCGTTTTCTGAGAA 69-89

PKC?related AATGGTGCAGCAGAAATTGGA 126-136

PKC?eta AAGAAGGGCCACCAGCTGCTG 269-289

PKC?eta AACGTCACCGACGGCGGCCAC 389-409

Mitochondrial?F0 AACCTCGGGCAGAAGAGGAGA 164-184

Mitochondrial?F0 AACTGAAACGGATTGCCAGAG 211-231

Bcl-2?TF AAGAAGCGATACAGGTCTCGT 91-111

Bcl-2?TF AAGGTCTCGTAGTAGAGATCG 126-146

Bcl-2?A1 AACCTGGATCAGGTCCAAGCA 257-277

Bcl-2?A1 AATCTGAAGTCATGCTTGGAC 334-354

P53-1 TGTGAGGCACTGCCCCCAC

P53-2 TGAACTGGAGGCCCATCCT

P53-3 TGAACCAGAGGCCCATCCT

P53-4 TTTGAGGTGCATGTTTGTG

RAP1 AACAGAGGAGGACTACATTCC 267-287

RAP1 AACCACGAAATCACCAGCATC 379-399

Table SS5.4.

Hpv-16E6 AAGTTACCAGATTTATGCACA

Hpv-16E6 AACAGTTACTGCGACGTGAGG

Hpv-16E7 AATATATGTTAGATTTGCAAC

Hpv-16E7 AATAGATGGTCCAGCTGGACA

Hpv-18E6 AACACGGCGACCCTACAAGCT

Hpv-18E6 AACTTACAGAGGTATTTGAAT

Hpv-18E7 AAGGCAACATTGCAAGACATT

Hpv-18E7 AATAGATGGAGTTAATCATCA

Table SS5.5.

DICER-A AB028449 AATGGGTCCTTTCTTTGGACT

DICER-B AACTGCTTGAAGCAGCTCTGG

MD2?PROTEIN-A NM_015364 AAGCTCAGAAGCAGTATTGGG

MD2?PROTEIN-B AATGCAATACCCAATTTCAAT

GAGE-2-A U19143 AATGATTGGGCCTATGCGGCC

GAGE-2-B AAGTGGAACCAGCAACACCTG

BREAST?TA?84-A NM_015966 AAGACTTTGGAGGACTTCCGG

BREAST?TA?84-B AAGTCGCGGGGAGATAAACTG

EGFR-RP-A AK026010 AAGCTGGACATTCCCTCTGCG

EGFR-RP-B AAGAGCCCAGCTTCCTGCAGC

ENDOPLASMIN?94-A AK025862 AACTGTTGAGGAGCCCATGGA

ENDOPLASMIN?94-B AATCTGATGATGAAGCTGCAG

FOLATE?BP-A AF000381 AACCGCGGTCCTATTCCATTA

FOLATE?BP-B AACACTCCAATTTTTCAAAGT

RALA?BP-A NM_006788 AACACCGCAGGGTGGAGCATG

RALA?BP-B AAGAGATCAGCCCTACTAAGT

GRB2?BP-A BC000631 AAGGGGGGACATCCTCAAGGT

GRB2?BP-B AATCCCCAGAGCCAAGGCAGA

CDR-62-A L02867 AAGCGCCAGGCCCCGCGTGGG

CDR-62-B AAGAGGAGTCCTGGTACGACC

A-RAF-A U33821 AAGAGTTACCTTCCTAATGCA

A-RAF-B AAGATTGGGTTGGTATATTCA

NOVEL-1-A NM_017873 AATCCTTGTTCTCACTGAGCT

NOVEL-1-B AAGATGGCTGAGCTGGGGCTG

MAC30-A L19183 AACCGACAGACTATGGGGGCT

MAC30-B AACCTGCTGAAGTGGTATGCT

GRANULIN-A NM_002087 AACGCGGTGCCCAGATGGTCA

GRANULIN-B AATGGCCCACAACACTGAGCA

HCA?ANTI.58-A NM_016436 AAGTGGGAGCCCAGTTGGAAG

HCA?ANTI.58-B AAGACATTGACTACGAGGAAG

MI2-BETA-A NM_001273 AATGAAGAGGACCCAGAAGAG

MI2-BETA-B AAGCCTAAGAAACCTCGGGAC

EGF?FACTOR?8-A NM_005928 AACCCCTGCCACAACGGTGGT

EGF?FACTOR?8-B AACCACTGTGAGACGAAATGT

APRIL-A AK090698 AACTGCCCCAGCGATCTCTGC

APRIL-B AACCTAATTCTCCTGAGGCTG

PGF?PRECURSOR-A AK023843 AAGAGTGACACTGTGGCTTCC

PGF?PRECURSOR-B AATGGGCTGAGCTGCTGCTCC

MELA.ANTIGEN-A AB014518 AATCAGCTCAACACTGTCCTC

MELA.ANTIGEN-B AAGGAGACAGTACTGAGTGCC

RALA?B.PROTEIN-A NM_006788 AACACCGCAGGGTGGAGCATG

RALA?B.PROTEIN-B AAGAGATCAGCCCTACTAAGT

Table SS6. is at the siRNA of respiratory syncytial virus (RSV)

Table SS6.1.

The RSV virus strains A2﹠amp of siRNA target; F/P, B1 and 9230 genome sequences

The target gene sequence in A2 strain gene group in B1 strain gene group in 9230 strain gene groups

The position (AY353550) of the position of the position (M734568) of (5 ' to 3 ')

(NC-001781)

Leader/NS1 AATGGGGCAAATAAGAATTTG 42-62 42-62 42-62

(-)strand

Leader/NS1 AATGGGGCAAATAAGAATTTg 42-62 42-62 42-62

N AAGATGGCTCTTAGCAAAGTc 1137-1157 1137-1157 1135-1155

P AATTCCTAGAATCAATAAAGg 2401-2421 2403-2423 2401-2421

M AAGCTTCACGAAGGCTCCACA 3279-3299 3281-3301 3279-3299

SH NA

G NA

F AATGATATGCCTATAACAAAt 6444-6464 6449-6469 6447-6467

M2 AAGATAAGAGTGTACAATACT 7975-7995 7987-8007 7986-8006

M2/L NA

L AACATCCTCCATCATGGTTAA 9090-9110 9101-9121 9100-9120

L AAGTACTAATTTAGCTGGACA 12973-12993 12984-13004 12983-13003

L AAGATTGCAATGATCATAGTT 14133-14153 14144-14164 14143-14163

L AACATTCATTGGTCTTATTTA 14243-14263 14254-14274 14253-14273

Table SS6.2.

The RSV virus strains A2﹠amp of siRNA target; The F/P genome sequence

The target gene sequence is in A2 strain gene group

The position (M734568) of (5 ' to 3 ')

Leader AAATGCGTACAACAAACTTGC 9-29

(-)strand

Leader AACAAACTTGCATAAACCAAA 19-39

NS1 AAGAATTTGATAAGTACCACT 54-74

NS1 AACTAACGCTTTGGCTAAGGC 209-229

NS2 AATAAATCAATTCAGCCAACC 602-622

NS2 AACTATTACACAAAGTAGGAA 830-850

N AACAAAGATCAACTTCTGTCA 1176-1196

N AAGAAATGGGAGAGGTAGCTC 1558-1578

P AATTCAACTATTATCAACCCA 2520-2530

P AACAATGAAGAAGAATCCAGC 2676-2696

M AAATAAAGATCTGAACACACT 3770-3790

M AAATATCCACACCCAAGGGAC 3442-3462

M AAATAAAGATCTGAACACACT 3770-3790

SH AACATAGACAAGTCCACACAC 4266-4286

SH AACAATAGAATTCTCAAGCAA 4320-4340

G AAACAAGGACCAACGCACCGC 4696-4716

G AACTTCACTTATAATTGCAGC 4840-4860

F AAATAAGTGTAATGGAACAGA 5858-5878

F AAACAATCGAGCCAGAAGAGA 5969-5989

M2 AAATAAGTGGAGCTGCAGAGT 7781-7801

M2 AACAATCAGCATGTGTTGCCA 7880-7900

M2/L NA

L AAGTTACATATTCAATGGTCC 8593-8613

L AACTAAATATAACACAGTCCT 8685-8905

Trail NA

SS6.3.

The RSV virus strains B1 of siRNA target and 9320 genome sequences

Target gene sequence (5 ' to 3 ') in B1 strain gene group in 9032 strain gene groups

The position (AY353550) of position (NC-001781)

Leader AATGCGTACTACAAACTTGCA 10-30 10-30

(-)strand

Leader AAATGCGTACTACAAACTTGC 9-29 9-29

NS1 AATTAATTCTTCTGACCAATG 196-216 196-216

NS1 AACAAGCAGTGAAGTGTGCCC 278-298 278-298

NS2 AATAATAACATCTCTCACCAA 700-720 700-720

NS2 AATGTATTGGCATTAAGCCTA 936-956 936-956

N AAATAAGGATCAGCTGCTGTC 1175-1195 1173-1193

N AACAAACTATGTGGTATGCTA 1272-1292 1270-1290

P AATAAAGGGCAAGTTCGCATC 2416-2436 2414-2434

P AACAAATGACAACATTACAGC 2725-2745 2723-2743

M AATATGGGTGCCTATGTTCCA 3361-3381 3359-3379

M AACATACTAGTGAAGCAGATC 3428-3448 3426-3446

SH AAATACATCCATCACAATAGA 4308-4328 4306-4326

SH AAACATTCTGTAACAATACTC 4445-4465 4443-4463

G AATCTATAGCACAAATAGCAC 4796-4816 4794-4814

G AATATTCATCATCTCTGCCAA 4866-4886 4864-4884

F AAAGAAACCAAATGCAATGGA 5858-5878 5856-5876

F AAACAAAGCTGTAGTCAGTCT 6187-6207 6185-6205

M2 AAATAAGTGGAGCTGCTGAAC 7793-7813 7792-7812

M2 AACAATCAGCATGTGTTGCTA 7892-7912 7892-7911

M2/L NA

L AAATAACATCACAGATGCAGC 9591-9611 9590-9610

L AATACCTACAACAGATGGCCC 9931-9951 9930-9950

Trail NA

SS7

SS7.1.siRNA target sars coronavirus genome sequence

Numbering position, coding region (nt) sequence (5 '-3 ')

SC07 5’UTR 146-166 aacgagtaactcgtccctctt

SC08 ORF1a，nsp-1 594-614 aattgcataccgcaatgttct

SC06 ORF1a，nsp-3 2721-2741 aacctttggagaagatactgt

SC03 ORF1a，nsp-3 2772-2792 aatcacatttgagcttgatga

SC09 ORF1a，nsp-3 3236-3256 aacctacacctgaagaaccag

SC10 ORF1a，nsp-3 4172-4192 aaggatgtgctggttatacac

SC11 ORF1a，nsp-3 5758-5778 aaaggaccagtgactgatgtt

SC12 ORF1a，nsp-3 8096-8116 aaggtgttgttgataccgatg

SC13 ORF1a，nsp-6 11074-11094 aagcacgcattcttgtgcttg

SC05 ORF1b，nsp-12 13530-13550 aaggatgaggaaggcaattta

SC01 ORF1b，nsp-12 13603-13623 aagagactatttataacttgg

SC16 ORF1b，nsp-12 14758-14778 aactcctattcgtagttgaag

SC17 ORF1b，nsp-13 16756-16776 aaggtgactatggtgatgctg

SC14 ORF1b，nsp-13 17544-17564 aaggataagtcagctcaatgc

SC18 ORF1b，nsp-14 18264-18284 aacctacctctccagctagga

SC15 ORF1b，nsp-16 20843-20863 aactggcacactacttgtcga

SC02 ORF2，Spike 21553-21573 aagctcctaattacactcaac

SC04 ORF2，Spike 21669-21689 aatgttacagggtttcatact

SC19 ORF2，Spike 22068-22088 aagggctatcaacctatagat

SC20 ORF2，Spike 22289-22309 aatcacagatgctgttgattg

SC21 ORF2，Spike 22951-22971 aaccttacagagttgtagtac

SC22 ORF2，Spike 23272-23292 aagatgttaactgcactgatg

SC23 ORF2，Spike 24871-24891 aagagctggacaagtacttca

SC37 ORF3a 25330-25350 aagtactgttcatgctacagc

SC38 ORF3a 25599-25619 aatgcatcaacgcatgtagaa

SC39 ORF3a 25618-25638 aattattatgagatgttggct

SC40 ORF3a 25764-25784 aaggtgacggcatttcaacac

SC41 ORF3a 25805-25825 aaattactacagacactggta

SC42 ORF3a 25929-25949 aaaatgctacattcttcatct

SC43 ORF3a 25984-26004 aatacacacaatcgacggctc

SC24 ORF4，E-protein 26121-26141 aagaaacaggtacgttaatag

SC25 ORF4，E-protein 26137-26157 aatagttaatagcgtacttct

SC34 ORF4，E-protein 26170-26190 aagcacattgacgcatacaaa

SC26 ORF4，E-protein 26219-26139 tgtgcgtactgctgcaatatt

SC36 ORF4，E-protein 26230-26250 aagactgatgaagctcagcct

SC27 ORF4，E-protein 26307-26327 aaggagttcctgatcttctgg

SC28 ORF5，M-protein 26440-26460 aacctagtaataggtttccta

SC29 ORF5，M-protein 26628-26648 aatggcttgtattgtaggctt

SC30 ORF5，M-protein 26760-26780 aattgtgaccagaccgctcat

SC33 ORF5，M-protein 26789-26809 aaccagcttgagagcaaagtt

SC31 ORF5，M-protein 26876-26896 aagagatcactgtggctacat

SC32 ORF5，M-protein 26968-26988 aaccgctaccgtattggaaac

SC44 ORF7 27355-27375 aaccttgcccatcaggaacat

SC45 ORF7 27425-27445 aacttgcactagcacacactt

SC46 ORF7 27541-27561 aagagctctactcgccacttt

SC47 ORF9a，N-protein 28176-28196 aactgacaataaccagaatgg

SC48 ORF9a，N-protein 28355-28375 aaattggctactaccgaagag

SC35 ORF9a，N-protein 28904-28924 aacagtacaacgtcactcaag

SS8: at the target sequence of placenta growth factor sequences Design siRNA.

Numbering target sequence

1 gtttgttaggaccaaacctca

2 taggaccaaacctcaaagcat

3 accaaacctcaaagcatggct

4 cctattccactggattgggaa

5 ctattccactggattgggaaa

SS9: at the target sequence of metal matrix albumen-2 sequences Design siRNA

Numbering target sequence

1 cccttgtttccgctgcatcca

2 catcatcaagttccccggcga

3 gacaaagagttggcagtgcaa

4 gcaacccagatgtggccaact

5 caagcccaagtgggacaagaa

SS10. at the target sequence of integrin alpha V β 3 sequences Design siRNA

Numbering target sequence

1 cagggattttgtcaaggagga

2 gggattttgtcaaggaggatt

3 ggattcagcattgattttact

4 ggtggtcctggtagcttttat

5 ggtagcttttattggcaaggt

Embodiment 2 unites the gene therapy neovascular diseases of the eye that uses on many target spots double-chain small disturbance RNA inhibition VEGF path

The CpG sequence that is rich in biologically active in the dna sequence dna of HSV virus can generate factor VEGF (vascular endothelial growth factor) by induction of vascular, and the antibody of anti-VEGF can reduce HSV institute inductive vasculogenesis; Inducing VEGF to cause new vessel to generate by the oligonucleotide that contains the CpG sequence of biologically active sets up animal model, can be used to estimate situation and the effect that siRNA suppresses vegf expression.

1. multi-target point cocktail siRNA

From sequence table, select siRNA preparation multi-target point cocktail siRNA, as at three gene mVEGF A, mVEGFR1 in the VEGF approach and mVEGFR2 design siRNA, according to rules such as Tuschl two siRNA of different sites design at the mRNA of each genetic transcription, by Qiagen (Valencia, CA) siRNA of synthetic design, all siRNA are has 3 ' the dTdT long double stranded rna molecule of terminal 21nt that dangles.Selecting siRNA preparation multi-target point cocktail siRNA from sequence table, be (a) AAGCCGTCCTGTGTGCCGCTG and (b) AACGATGAAGCCCTGGAGTGC at the siRNA of mVEGFA gene for example; At the siRNA of mVEGF R1 gene is (a) AAGTTAAAAGTGCCTGAACTG and (b) AAGCAGGCCAGACTCTCTTTC; SiRNA at mVEGF R2 gene is AAGCTCAGCACACAGAAAGAC and (b) ATGCGGCGGTGGTGACAGTA.Negative control siRNA is the siRNA at LacZ and firefly luciferase (Luc) gene, at the siRNA sequence of LacZ is LacZ (a) AACAGTTGCGCAGCCTGAATG and (b) AACTTAATCGCCTTGCAGCAC, is Luc (a) AAGCTATGAAACGATATGGGC and (b) AACCGCTGGAGAGCAACTGCA at the siRNA sequence of Luc.Mole such as a molecule and b molecule mixes use in the present embodiment.

2. laboratory animal and animal model

Use the female BALB/c rat in 5-6 week.Use phosphorylation ODNs1466 (TCAACGTTGA) and 1555 (GCTAGACGTTAGCGT) making animal model in the present embodiment.

3. viral

Present embodiment use HSV-l strain RE (provide by Robert doctor Lausch, University ofAlabama, Mobile AL), uses individual layer Vero cell culture and virus (catalog no.CCL81; American Type Culture Collection, Manassas VA), after the mensuration titre, is stored in-80 ℃.

4.siRNA in-vitro transfection

(there is VEGFA genetic expression in this cell for CRL-2278, ATCC) the external curative effect of clone checking siRNA, and culturing cell in 6 orifice plates, substratum are RPMI and 10% foetal calf serum, and culture condition is 37 ℃ and 5%CO to use RAW264.7gamma NO (-) ₂, incubated overnight.Used in second day Lipofectamine 2000 (Invitrogen, Carlsbad is CA) with siVEGFA or siLuc (0,0.1,0.5,1.0, or 2.0ug/2ml/ hole) transfectional cell extracted RNA after 24 hours, and carried out RT-PCR reaction detection expression conditions.

(checking siVEGFR1 strikes the effect of low VEGFR1 genetic expression to the VEGFR1 gene in this cell for CRL-2280, ATCC) middle constitutive expression at the SVR cell.Culturing cell in 6 orifice plates, substratum are Dulbecco ' s modified Eagle ' s medium and 5% foetal calf serum, and culture condition is 37 ℃ and 5%CO ₂, incubated overnight.Used in second day Lipofectamine 2000 (Invitrogen, Carlsbad is CA) with siVEGFR1 or siLuc (0,0.1,0.5,1.0, or 2.0ug/2ml/ hole) transfectional cell extracted RNA after 48 hours, and carried out RS-PCR reaction detection expression conditions.

(CRL-1573, ATCC) cell detection siVEGFR2 strikes the effect of low external source VEGFR2 genetic expression to use the plasmid transfection 293 of expressing mVEGFR2 in the present embodiment.Culturing cell in 6 orifice plates, substratum are Dulbecco ' s modified Eagle ' s medium and 5% foetal calf serum, and culture condition is 37 ℃ and 5%CO ₂, incubated overnight.Used Lipofectamine 2000 (Invitrogen in second day, Carlsbad, CA) with pCI-VEGFR2 plasmid (0.2ug/2ml/ hole) and siVEGFR1 or siLuc (0,0.1,0.5,1.0, or 2.0ug/2ml/well) the cotransfection cell, extract RNA after 48 hours, and carry out RS-PCR reaction detection expression conditions.

5. cornea crystallite lattice experimental analysis

(Vaerlose is Denmark) with hydron polymer (120mg/1ml ethanol for 10mg, Bulch Meditec with CpG ODNs, sucralfate; Interferon Sciences, New Brunswick NJ) mixes, and has the 15mm in aperture ²Synthetic grid (the Sefar America of size, Inc., KansasCity, MO) middle preparation contains the particle of 1ug of CpG ODNs, (LeicaMicrosytems under stereoscopic microscope, Wetzlar, Germany) make the crystallite lattice (every group of four eyes) of 1mm size at corneal limbus, implant the particle that contains CpG ODNs, use calipers (BiomedicalResearch Instruments after 4 days and 7 days, Rockville MD) estimates the vasculogenesis situation.Evaluation originates from corneal limbus blood vessel annular and is developed to the per hour width of clock sector region (colock hours) of the length of new vessel of cornea central authorities and ring-type new vessel, and per hour the clock sector region is 30 °.Calculate the blood vessel hyperplasia area according to following formula: X0.4 (vessel length in mm) Xpi A=[(colock hours)]/2.

6.siRNA vivo medicine-feeding

Use 32-gauge Hamilton syringe (Hamilton Co. after 6 hours and 24 hours respectively at implanting the CpG particle, Reno, NV) siRNA (10ug/10ul pereye) of subconjunctival injection phosphoric acid buffer dilution carries out topical, or under deep anaesthesia infective virus after 1 day and 3 days behind corneal limbus the 2mm place inject siRNA.

Be administered systemically by intravenous injection after siRNA (40ug/100ul per mice) and the mixed with polymers, with 32-gauge syringe the CpG particle after 6 hours and 24 hours or infective virus to pass through after 1 day and 3 days be intravenous administration.

7. cornea HSV-1 virus infection

Anaesthetize mouse with Tribromoethyl alcohol, with 30-gauge needle cut gently on cornea, and to splash into titre be 1 * 10 ⁵The HSV-1 RE virus of plaque-forming units (PFUs) is massaged eye face, every group of 6 mouse gently after handling.

8. clinicing symptom observation (HSK symptom and blood vessel hyperplasia scoring)

Carry out the living tissue micrography by slit lamp and infect the progress of clinical lesion after the different number of days and the occurring degree of keratitis, points-scoring system is: 0 expression normal cornea; + 1 expression moderate is dim; + 2 expression cornea moderate muddiness or scars; + 3 expression severe corneal muddinesses but irises as seen; Opaque and the keratohelcosis of+4 expression corneas; + 5 expression keratorhexises and downright bad bag.By following means vasculogenesis situation: the new vessel 1.5mm that entad grows is represented grade in each quadrant, be divided into four grades, the score addition of four quadrants is derived put each ocular neovascular preset time and generate index.

9. result

9.1 in external expression of striking low VEGF pathway gene

In sequence table or specification sheets subordinate list, select three siRNA, be respectively siVEGFA, siVEGFR1 and siVEGFR2, the efficient of its silencer of checking in different clone respectively.RAWNO (-) scavenger cell can produce endogenous VEGFA, be used to verify the effect of siVEGFA, difference transfection siVEGFA and control group siRNA (siLuc), transfection is carried out RT-PCR and is analyzed after 24 hours, siVEGFA has reduced the expression of VEGF120 and VEGFA164 as shown in Figure 1, and is dose-dependently.SVR can produce endogenous VEGFR1, and the RS-PCR analysis revealed after transfection siVEGFR148 hour has reduced VEGFR1 expression of gene (as shown in Figure 1), and siLuc siRNA is then to the not influence of VEGFR1 expression of gene.Express the plasmid transfection 293 (CRL-1573 of mVEGFR2, ATCC) cell detection siVEGFR2 strikes the effect of low external source VEGFR2 genetic expression, pCI-VEGFR2 plasmid (0.2ug/2ml/ hole) and siVEGFR1 or siLuc (0,0.1,0.5,1.0, or 2.0ug/2ml/well) cotransfection cell, carry out the expression that RS-PCR analysis revealed siVEGFR2 has reduced external source mVEGFR2 after 48 hours, control group siRNA does not then influence the mVEGFR2 expression of gene, as shown in Figure 1.These show that these siRNA can be in the vitro inhibition expression of gene.

9.2 the siRNA at the VEGF pathway gene suppresses CpG inductive vasculogenesis through topical

There are some researches show that the ODN that will contain CpG is embedded in the hydrophilic polymer, and implant the vasculogenesis that to induce VEGF to mediate in the crystallite lattice of cornea.Present embodiment is estimated the effect of local implanting needle to VEGFA, VEGFR1 and VEGFR2 gene siRNA with this method.The consumption of every eye siRNA is 10ug in every group, implants CpG and import siRNA in subconjunctival injection after ODN24 hour in the crystallite lattice.Article three, siRNA (siVEGFA, siVEGFR1 and siVEGFR2) imports in the eye respectively, and three siRNA are prepared in the multi-target point cocktail siRNA importing eye with 1: 1: 1 mixed.After implanting 4 days and 7 days, CpGODN checks new vessel and corneal limbus (Fig. 2), with contrast siRNA (siLacZ), siVEGFA, siVEGFR1, siVEGFR2 and multi-target-spot siRNA all significantly suppress the generation (p＜0.05) of new vessel, wherein the inhibition of multi-target-spot siRNA is most effective, and 60% inhibition efficient (p＜0.01) is arranged approximately.

9.3 the siRNA at the VEGF pathway gene suppresses the generation of CpG inductive new vessel through being administered systemically

With siVEGFA, siVEGFR1, the multi-target point cocktail siRNA that siVEGFR2 reaches by three kinds of siRNA preparations imports in the rat of implanting CpG ODN crystallite lattice with the sosimetric system of 40ug respectively, detects the effect of siRNA angiogenesis inhibitor and the efficient of the importing siRNA of the interior system of body.The carrier that is used for the importing siRNA of system in the present embodiment is TOP Polymer.After CpG ODN implants 4 days and 7 days, compare siVEGFA with control group, siVEGFR1, siVEGFR2 all remarkable (p＜0.05) suppresses vasculogenesis (Fig. 3), as the result in the topical experiment, the inhibition of multi-target point cocktail siRNA is most effective, on average reaches 40% (p＜0.01).Multi-target-spot siRNA is combined with TOP polymer and phosphoric acid buffer (PBS) respectively, import in the body by system's injection, the result shows that the siRNA that is dissolved among the PBS has obvious suppression effect (p＜0.05) in early days, and HK polymer significantly is better than PBS (Fig. 4) as the effect that carrier imports the angiogenesis inhibitor of siRNA.The result shows the expression that can suppress the VEGF pathway gene by intravenous injection siRNA, and uses the purpose that TOP polymer reaches the raising curative effect.

Present embodiment has also further been verified the be administered systemically dose effect of angiogenesis inhibitor of siRNA, after implanting CpG ODN, the crystallite lattice imported respectively by siVEGFA in 6 hours and 24 hours, multi-target point cocktail siRNA and siLuc that siVEGFR1 and siVEGFR2 are mixed with, importing carrier is HK polymer, and dosage is respectively 10,20,40,80 υ g, by the tail vein injection administration, the result as shown in Figure 4.SiRNA has dosage effect to CpG inductive vasculogenesis.

9.5 multi-target point cocktail siRNA suppresses the result of treatment of VEGF pathway gene in the HSK model

Studies show that VEGF is the key factor in the HSV institute inductive vasculogenesis in the HSK model.Cut gently on cornea, and to splash into titre be 1 * 10 ⁵The HSV-1 RE virus of plaque-forming units (PFUs) is set up the HSK model and is used to estimate the effect that suppresses the HSK progress at the siRNA of VEGF pathway gene.With multi-target point cocktail siRNA (siVEGFA, the mixture of mol ratio such as siVEGFR1 and siVEGFR2) combines with polymer support and be used for local and be administered systemically, at virus infection after 1 day and 3 days, 10ug multi-target point cocktail siRNA carries out topical by subconjunctival injection, and 40ug is administered systemically by tail vein injection.Compare with control group, no matter topical still is administered systemically, further develop (p＜0.05) that significantly suppresses vasculogenesis and HSK at the multi-target point cocktail siRNA of VEGF pathway gene, control group siLuc treatment group has 80% to develop into tangible clinical lesion (2 grades or more serious), and topical has 42% similar symptom to occur, and the similar symptom of 50% appearance has been administered systemically.After 10 days, in treatment group, 9 grade scorings are arranged greater than 6 in 12 eyes, and at VEGF pathway gene multi-target point cocktail siRNA treatment group, 5 grade scorings are arranged greater than 6 (Fig. 5) in 12 eyes.The result shows that the siRNA molecule at the VEGF pathway gene can alleviate the HSK symptom by suppressing vasculogenesis.

9.6 reduce the expression of VEGFmRNA in the HSV-1 virus infection cornea at the target point cocktail siRNA of VEGF pathway gene

With 1 * 10 ⁵The HSV-1 RE virus infection cornea of plaque-forming units (PFUs), the siRNA treatment group imports siRNA at virus infection after 1 day and 3 days, behind virus infection 4 days. and collect the cornea of each treatment group 7 days the time, extract RNA and carry out RT-PCR analysis or real-time quantitative PCR analysis, the result as shown in Figure 6, compare with control group, can significantly reduce the level of VEGF mRNA at the siRNA molecule of VEGF pathway gene.

9.7 the target point cocktail siRNA at the VEGF pathway gene reduces vegf protein level in the HSV-1 virus infection cornea

By detecting the variation that HSV infects vegf protein level in the virus infection cornea that the cornea neutralization crosses at the siRNA molecular therapy of VEGF pathway gene, checking is at the influence to vegf protein of the siRNA molecule of VEGF pathway gene, the result shows, compare with control group (siLuc+ imports carrier TOP polymer), be mixed with multi-target point cocktail siRNA after carrier TOP polymer combines and effectively reduce vegf protein level (p＜0.05) with importing at the siRNA molecule of VEGF pathway gene, as shown in Figure 7.

The multi-target point cocktail siRNA molecule control SARS that embodiment 3 sars coronavirus genome sequences are special

1.SARS the preventive effect of special multi-target point cocktail siRNA.Adopt the special multi-target point cocktail siRNA strategy of SARS than using a siRNA to suppress the better effects if of virus replication separately.The special multi-target point cocktail siRNA of SARS from sequence table, select or specification sheets table SS7 in select.

SARS CoV (3PFU/ hole) infects the FRhK-4 cell, and cells infected is transfection siRNA after 1 hour.Behind the virus infection 36 hours, collecting cell and substratum are analyzed the mRNA copy number and are measured virus titer (TCID ₅₀), Fig. 8 A shows the virus genomic copy number of reduction.The real-time quantitative PCR result shows that uniting the special siRNA of many SARS of use has stronger inhibition effect than using one separately.Fig. 8 B shows the research of uniting the lasting actual effect of prevention of using many special siRNA of SARS.Preventive effect can continue until transfection after 72 hours.

2. uniting use duplicates in cell at the multi-target point cocktail siRNA treatment SARS virus infected monkey embryonic kidney cells (FRhK-4) and the SARS virus of sars coronavirus genome sequence.The special multi-target point cocktail siRNA of SARS from sequence table, select or specification sheets table SS7 in select.

SARS CoV (3PFU/ hole) infects the FRhK-4 cell, and cells infected is transfection siRNA after 1 hour.Behind the virus infection 36 hours, collecting cell and substratum are analyzed the mRNA copy number and are measured virus titer (TCID ₅₀).Fig. 9 shows and unites the result of treatment of using many special siRNA of SARS.Verified the potential treatment application of multi-target point cocktail siRNA.

Embodiment 4 multi-target point cocktail siRNA treat cancer

1. multi-target point cocktail siRNA

At three gene EGF acceptors in the VEGF approach, FGF and VEGF design siRNA, according to rules such as Tuschl two siRNA of different sites design at the mRNA of each genetic transcription, by Qiagen (Valencia, CA) siRNA of synthetic design, all siRNA are has 3 ' the dTdT long double stranded rna molecule of terminal 21nt that dangles.Select siRNA preparation multi-target point cocktail siRNA from sequence table or specification sheets table SS1, SS2 and SS3, for example the siRNA at the EGF acceptor gene is CTGACTACCAGCAGGACTTCT; SiRNA at the FGF gene is AAGGCTGGAGGAGAACCATTA; SiRNA at the VEGF gene is AAGTGGTCCCAGGCTGCACCC.Negative control siRNA is the siRNA at LacZ and fireflyluciferase (Luc) gene, is LacZAACAGTTGCGCAGCCTGAATG at the siRNA sequence of LacZ, is LucAAGCTATGAAACGATATGGGC at the siRNA sequence of Luc.

2. cell and animal model

MCF-7/VEGF165 and MDA-MB-435 are for treating transfectional cell; Use MCF7/VEGF ₁₆₅Cell tumour model evaluation siRNA curative effect in vivo

3. the evaluation of external curative effect

Select MCF-7/VEGF165 and MDA-MB-435 for treating transfectional cell.During transfection, get up to use with three kinds of various dose and Oligofectamine reagent mix, the transfection program can be according to agent delivery person (Invitrogen, CA) described carrying out.With LacZ-siRNA transfectional cell under the same terms is control group, also has a untreated fish group in addition.After 48 hours, supernatant liquor is collected and carries out elisa assay and collecting cell and carry out RT-PCR and analyze.Select two siRNA molecules the most effective by above-mentioned experiment, and use bioinformatics method that multi-target point cocktail siRNA is further analyzed, determine its validity and specificity.

4. the evaluation of interior curative effect

Use MCF7/VEGF ₁₆₅Cell tumour model evaluation multi-target point cocktail siRNA curative effect in vivo.10 to 15 micrograms mark and unlabelled multi-target point cocktail siRNA import in the tumour.24h, 48h after multi-target point cocktail siRNA administration, 72h, 96h and all equi-time points are estimated the influence of administration then.

5. result

5.1 immunohistochemical method is analyzed the specificity downward modulation effect of multi-target point cocktail siRNA to tumour

Multi-target point cocktail siRNA after 24 hours, carries out the administration second time in administration, and our the learn from else's experience tumor tissues of multi-target point cocktail siRNA or twice processing of LacZ-siRNA carries out H/E dyeing, monoclonal antibody (mAb) dyeing and the TUNEL group analysis that dyes then.Wherein the chromosomal situation in the neoplastic cell nuclei is investigated in H/E dyeing, if the karyomit(e) in the neoplastic cell nuclei obviously concentrates, just mean to have active mitotic division in the tumor tissues, in the control group tumor tissues of LacZ-siRNA effect, have the active mitotic division stage.And monoclonal antibody (mAb) special in conjunction with hVEGF, the painted result of mAb illustrates further, VEGF is distributed in the inner capillary blood vessel of control group tumor tissues in a large number, yet rarely found in the tumor tissues of VEGF-siRNA effect.TUNEL dyeing is used for studying the apoptosis vigor, and TUNEL analysis revealed tumor tissues after multi-target point cocktail siRNA effect has strong apoptosis activity, and strong apoptosis activity appears in there be no evidence to suggest in control group.In addition, in two experimental group and control group, all do not observe multi-target point cocktail siRNA cause obvious toxic-side effects (Figure 10).

Therefore, these discoveries have effectively illustrated following importance: 1) multi-target point cocktail siRNA can reduce or close the expression of VEGF, thereby suppresses the quick growth of MCF-7/VEGF transplanted tumor; 2) in tumor model, multi-target point cocktail siRNA can suppress overexpression activated tumor-blood-vessel growth; 3) these downward modulation effects finally cause the reduction of proliferative effect and the rising of natural death of cerebral cells; 4) suppress tumor cell proliferation and acceleration death of neoplastic cells, break the balance in the tumour cell, and tumor growth is suppressed.

5.2 the transplanted tumor that multi-target point cocktail siRNA causes is suppressed

1438 Transplanted cells knurls in nude mice experimentize.When the tumour size reaches average 200mm ³When (behind the cell inoculation 7 days), import 30 μ l and contain 10 μ g multi-target point cocktail siRNA solution.Three cohorts are respectively experimental group, injecting normal saline control group and multi-target point cocktail siRNA administration control group, and every group has 5 mouse processed.Tumour is handled through 5 dosage altogether, every 7 days once.To measure the tumour size before each the processing.Compare with control group, the retarding effect of experimental group tumour clearly, thereby cause tumour dwindle (Figure 11, Figure 12).

The result shows that multi-target point cocktail siRNA administration really can stop the growth of transplanted tumor, and is effective and have no side effect.

Sequence table

＜110〉Guangzhou Top Genomics Ltd.

＜120〉be used for the multi-target point cocktail double strand small interfere RNA and preparation method thereof of disease treatment

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uuucuucaac?ugagagacct?t 21

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uguauuugug?cagcuggcat?t 21

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ugaugucucc?aacagagcct?t 21

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ucuaucuuug?ccaguccuct?t 21

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<222>(1)..(19)

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<223>

<400>58

uuacauacuc?ucucuugcct?t 21

<210>59

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>59

cuauauuaca?uacucucuct?t 21

<210>60

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>60

gccaccuccu?ggauggucut?t 21

<210>61

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>61

cauccacuug?auaggcacut?t 21

<210>62

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>62

ccauccacuu?gauaggcact?t 21

<210>63

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>63

guccugcugg?uagucagggt?t 21

<210>64

<211>21

<212>RNA

<213>Homo?sapiens/Mus?musculus

<400>64

agaaguccug?cugguaguca?g 21

<210>65

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>65

ugccaggcuc?ccaaagauct?t 21

<210>66

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>66

caggcuccca?aagaucuuct?t 21

<210>67

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>67

cauccacuug?augggcacct?t 21

<210>68

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>68

agagucaauc?auccaacaut?t 21

<210>69

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>69

cuggucccag?uaauagaggt?t 21

<210>70

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>70

acgauguccc?uccagucaat?t 21

<210>71

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>71

guccagguug?cccaggauct?t 21

<210>72

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>72

acgcccagca?cuaauuucct?t 21

<210>73

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<400>73

uuuaaugcag?acuggaauct?t 21

<210>74

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>74

aucacucugg?uguguguaut?t 21

<210>75

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>75

caucacucug?guguguguat?t 21

<210>76

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>76

cauacuccuc?aucuucauct?t 21

<210>77

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>77

cuggucccag?uaauagaggt?t 21

<210>78

<211>21

<212>RNA

<213>Homo?sapiens/Mus?musculus

<400>78

uaucuacuuc?caucuuguca?g 21

<210>79

<211>21

<212>RNA

<213>Homo?sapiens/Mus?musculus

<400>79

uaaugcagac?uggaaucuug?a 21

<210>80

<211>21

<212>RNA

<213>Homo?sapiens/Mus?musculus

<400>80

uuaaugcaga?cuggaaucuu?g 21

<210>81

<211>21

<212>RNA

<213>Homo?sapiens/Mus?musculus

<400>81

uuaauuggag?ucuuggccuc?a 21

<210>82

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>82

ucuacauaga?cuccaccautt 21

<210>83

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>83

gucuacauag?acuccaccat?t 21

<210>84

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>84

cugagcagcc?uccagcacat?t 21

<210>85

<211>21

<212>RNA

<213>Homo?sapiens/Mus?musculus

<400>85

uuaucuacuu?ccaucuuguc?a 21

<210>86

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>86

caguugaaag?gugguuggat?t 21

<210>87

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>87

uguauguuca?ggaaaccugt?t 21

<210>88

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<400>88

uguauguuca?ggaaaccugt?t 21

<210>89

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>89

gucaugacua?gugggaccgt?t 21

<210>90

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>90

cuugauguga?gggucgcuct?t 21

<210>91

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>91

caacuccucu?cucuucugct?t 21

<210>92

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>92

uuuauacugc?ccaguucgut?t 21

<210>93

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>93

cuuucugccc?agguccugut?t 21

<210>94

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>94

ccaaguuuau?acugcccagt?t 21

<210>95

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>95

uucugcuuga?aguuguagct?t 21

<210>96

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>96

guuuauacug?cccaguucgt?t 21

<210>97

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>97

gcuuucugcc?cagguccugt?t 21

<210>98

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>98

uagacacaac?uccucucuct?t 21

<210>99

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>99

caguaaucuu?ccaucuucct?t 21

<210>100

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>100

cauuuagaag?ccaguaauct?t 21

<210>101

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>101

aguuauuaga?uuccaaucgt?t 21

<210>102

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>102

acgguuagca?cacacuccut?t 21

<210>103

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>103

uaccaacugg?uguauuucct?t 21

<210>104

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>104

uuuccuugac?cgguaaguat?t 21

<210>105

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>105

acacucaucc?guaacacaut?t 21

<210>106

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>106

cacauaccaa?cugguguaut?t 21

<210>107

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>107

cucucucuuc?ugcuugaagt?t 21

<210>108

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>108

ccacauacca?acugguguat?t 21

<210>109

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>109

ugaccgguaa?guauuguagt?t 21

<210>110

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>110

uaaugguucu?ccuccagcct?t 21

<210>111

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>111

acaguagagg?aguuugggct?t 21

<210>112

<211>21

<212>DNA

<213>Homo?sapiens

<400>112

guaauucccu?ggaggcagat?t 21

<210>113

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>113

cagcugcugc?uuguggcgct?t 21

<210>114

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>DNA

<222>(20)..(21)

<223>

<220>

<221>RNA

<222>(1)..(19)

<223>

<400>114

auucuucucu?gcaugcuuct?t 21

<210>115

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>115

agugaguccg?aggaccgcgt?t 21

<210>116

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>116

uauauaggug?uuguaauggt?t 21

<210>117

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>117

gcuuucaaga?cuaaagagct?t 21

<210>118

<211>21

<212>DNA

<213>Homo?sapiens

<400>118

cuucuuggau?auauaggugt?t 21

<210>119

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>119

cguugcuaca?guagaggagt?t 21

<210>120

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>120

ugcuggucgc?uccuguccct?t 21

<210>121

<211>21

<212>DNA

<213>Homo?sapiens

<400>121

ccaauucuuc?ucugcaugctt 21

<210>122

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>122

agcugcugcu?uguggcgcut?t 21

<210>123

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>123

cuugaggcca?acaaaccaat?t 21

<210>124

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>124

uuugcagcuc?ccauucuuct?t 21

<210>125

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>125

guagaggagu?uugggcuuct?t 21

<210>126

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>126

gugaguccga?ggaccgcgtt?t 21

<210>127

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>127

accgcguuug?cagcucccat?t 21

<210>128

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>128

guugcuacag?uagaggagut?t 21

<210>129

<211>21

<212>DNA

<213>Homo?sapiens

<400>129

caggaacaaa?cauuccucat?t 21

<210>130

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>130

cauuugccgg?caguccggct?t 21

<210>131

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>131

auacucuauc?aaacagggct?t 21

<210>132

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>132

cuuugucaau?ucccacugct?t 21

<210>133

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>133

guauucucgg?agguugccut?t 21

<210>134

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>134

guuaccacca?uacaggcgat?t 21

<210>135

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>135

uaucucuugg?aaacucccat?t 21

<210>136

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>136

gguauuuggu?ugguggcuct?t 21

<210>137

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>137

caagucuucu?accaacugct?t 21

<210>138

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>138

uccaaugcga?ugcuccugct?t 21

<210>139

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>139

aggcacagca?uggagccgct?t 21

<210>140

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>140

uauagagaac?cucaaucuct?t 21

<210>141

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>141

auucggcaca?ggaugacugt?t 21

<210>142

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>142

guuggugcag?uuggcuggct?t 21

<210>143

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>143

augcuccugc?uuaaacucct?t 21

<210>144

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>144

cuuucucugu?ggcaucauct?t 21

<210>145

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>145

gcuuauccau?ucugugucct?t 21

<210>146

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>146

ugcuuaaacu?ccuucccgut?t 21

<210>147

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>147

augaggcucc?agugcuggut?t 21

<210>148

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>148

guguccuucc?uucagcagct?t 21

<210>149

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>149

auagagaacc?ucaaucucut?t 21

<210>150

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>150

cuucacaggc?agucggccgt?t 21

<210>151

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>151

uuucucuguu?gcguccgact?t 21

<210>152

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>152

guacucccgc?agguugccct?t 21

<210>153

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>153

ucaacaucuu?cacagccact?t 21

<210>154

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>154

aucaucuuca?ucaucuccat?t 21

<210>155

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>155

cuuuggucac?acgguugggt?t 21

<210>156

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>156

guuggugcag?uuacugggct?t 21

<210>157

<211>21

<212>DNA

<213>Homo?sapiens

<400>157

cuuacacaug?aacuccacgt?t 21

<210>158

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>158

uuuggucaca?cgguugggut?t 21

<210>159

<211>21

<212>DNA

<213>Homo?sapiens

<400>159

cuuaugcuuc?ccgaucauct?t 21

<210>160

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>160

cauacgguuu?gguuuggugtt 21

<210>161

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>161

agguuugaau?ucuuugccat?t 21

<210>162

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>162

caucaucaug?uacagcucgt?t 21

<210>163

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>163

gaucuggaca?uaaggcaggtt 21

<210>164

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>164

gugacccucc?uucagcagct?t 21

<210>165

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>165

ccauucaccu?cgaugugcut?t 21

<210>166

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>166

cacacgguug?gguuugucct?t 21

<210>167

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>167

caggucuucc?accagcugct?t 21

<210>168

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>168

caacaucuuc?acagccacut?t 21

<210>169

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>169

auccagcugg?uauguguggt?t 21

<210>170

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>170

cuucuuguag?uagucgaggt?t 21

<210>171

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>171

uuuguguuuc?ccgaucauct?t 21

<210>172

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>172

caccaggucc?gacaggucct?t 21

<210>173

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>173

cugugcguca?cuguacacct?t 21

<210>174

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>174

cagguccucc?accagcugct?t 21

<210>175

<211>21

<212>DNA

<213>Homo?sapiens

<400>175

ccacugcuga?ugccgcagct?t 21

<210>176

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>176

cacggcuacg?gugacaggct?t 21

<210>177

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>177

ccuugucagu?ggcaucguct?t 21

<210>178

<211>21

<212>DNA

<213>Homo?sapiens

<400>178

guuggagcuc?auggacgcgt?t 21

<210>179

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>179

cuccagggac?accugucgct?t 21

<210>180

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>180

guacaggucg?ugugugcagt?t 21

<210>181

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>181

ggagagaacc?ucuagcucct?t 21

<210>182

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>182

cuugucagug?gcaucgucut?t 21

<210>183

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>183

uagcuccuug?ucgguggugt?t 21

<210>184

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>184

caggucccug?uggaugcact?t 21

<210>185

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>185

ggaguccucg?ugggaggcat?t 21

<210>186

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>186

ggcacaggac?accaggucct?t 21

<210>187

<211>21

<212>DNA

<213>Homo?sapiens

<400>187

guggcccucc?uucagcagct?t 21

<210>188

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>188

ccgcagaaac?ucccgcaggt?t 21

<210>189

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>189

caccagcccu?gugccaucct?t 21

<210>190

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>190

augaaaggcc?uguccaucct?t 21

<210>191

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>191

agagacggcc?agcaggacct?t 21

<210>192

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>192

cuugugucgg?ccgaucagct?t 21

<210>193

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>193

cuauugaugu?cugcagucut?t 21

<210>194

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>194

cuacugucag?cuccugcuct?t 21

<210>195

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>195

ugacggccac?agugcuggct?t 21

<210>196

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>196

ccgaaugccu?ccaaugcggt?t 21

<210>197

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>197

gaacucccgc?agguuuccct?t 21

<210>198

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>198

cagagggaag?cgggagagct?t 21

<210>199

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>199

ugucugcagu?cuuuaggactt 21

<210>200

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>200

cagguccuug?ucagaggcgt?t 21

<210>201

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>201

ggugugucca?guagggugct?t 21

<210>202

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>202

gcuauugaug?ucugcaguct?t 21

<210>203

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>203

cuugucagag?gcguugucut?t 21

<210>204

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>204

acagcggaac?uugacggugt?t 21

<210>205

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>205

ccucguacca?gggaugagct?t 21

<210>206

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>206

cugggcaucg?cuguacacct?t 21

<210>207

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>207

ccuugucaga?ggcguuguct?t 21

<210>208

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>208

accaagcagg?uugaugaugt?t 21

<210>209

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>209

accuguaauc?aaggugagat?t 21

<210>210

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>210

augacauucu?ggaugggugt?t 21

<210>211

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>211

caccuccuga?uaaauuggct?t 21

<210>212

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>212

auauagaugu?uaagaggact?t 21

<210>213

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>213

uuuauggaac?agaaccugatt 21

<210>214

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>214

cauugagauc?aucacuggct?t 21

<210>215

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>215

acuuuccaaa?gccauccact?t 21

<210>216

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>DNA

<222>(20)..(21)

<223>

<220>

<221>RNA

<222>(1)..(19)

<223>

<400>216

auuucaugcc?uuuggcuact?t 21

<210>217

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>217

agaauacacu?ccagaggcat?t 21

<210>218

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>218

auucacauuc?aucucggact?t 21

<210>219

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>219

uuguguuguc?ccguggccat?t 21

<210>220

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>220

gauaaccagu?guguagccat?t 21

<210>221

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>221

auugagauca?ucacuggcut?t 21

<210>222

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>222

ucuuucguuu?ccuuuagcct?t 21

<210>223

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>223

accauuucug?uaguugggct?t 21

<210>224

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>224

auaaccagug?uguagccaut?t 21

<210>225

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>225

auuucucaga?aguguccuat?t 21

<210>226

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>226

cuuuacagau?gaaaggacut?t 21

<210>227

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>227

auuaaagcag?ugcucaugat?t 21

<210>228

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>228

uguaaaguu?ccuuccugct?t 21

<210>229

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>229

augacauucu?ggaugggugt?t 21

<210>230

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>230

gugcacgcgg?ugcucgaagt?t 21

<210>231

<211>21

<212>DNA

<213>Homo?sapiens/Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>231

gcugccccgc?acgaugguct?t 21

<210>232

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>232

cgcagugaga?gcugccacct?t 21

<210>233

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>233

guugcuguuc?uccucgcugt?t 21

<210>234

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>234

gaaagcuccu?cgguagguct?t 21

<210>235

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>235

gcugccuggg?gcuagucuct?t 21

<210>236

<211>21

<212>RNA

<213>Mus?musculus

<400>236

uuguuguuca?ucucggagcc?u 21

<210>237

<211>21

<212>RNA

<213>Mus?musculus

<400>237

uuucauuaca?caggacaggu?a 21

<210>238

<211>21

<212>RNA

<213>Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>238

uucauuagua?accucgaugg?u 21

<210>239

<211>21

<212>RNA

<213>Mus?musculus

<400>239

uuaugauggg?agauauuccg?a 21

<210>240

<211>21

<212>RNA

<213>Mus?musculus

<400>240

uuuagagucg?ucuccucccg?a 21

<210>241

<211>21

<212>RNA

<213>Mus?musculus

<400>241

uuguaucuca?aaucacucug?g 21

<210>242

<211>21

<212>RNA

<213>Mus?musculus

<400>242

uaagcagccg?agauaaacgu?g 21

<210>243

<211>21

<212>DNA

<213>Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>243

uugcacggga?cuaucaguct?t 21

<210>244

<211>21

<212>DNA

<213>Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>244

uagaauggac?auacuuucct?t 21

<210>245

<211>21

<212>RNA

<213>Mus?musculus

<400>245

ugcauuguca?ggacguugcg?g 21

<210>246

<211>21

<212>RNA

<213>Mus?musculus

<400>246

uaggguguca?agacaaccug?g 21

<210>247

<211>21

<212>RNA

<213>Mus?musculus

<400>247

ugcauuuccg?ggaauagcca?g 21

<210>248

<211>21

<212>RNA

<213>Mus?musculus

<400>248

uuggugguuu?gcuacgacgu?g 21

<210>249

<211>21

<212>RNA

<213>Mus?musculus

<400>249

uaugagauag?caaaucggcu?g 21

<210>250

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>250

auacgacucu?gugguugact?t 21

<210>251

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>251

agcguggcuc?acucguuact?t 21

<210>252

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>252

guugaguagc?acacucgact?t 21

<210>253

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>253

guuucugccg?gaaguucagt?t 21

<210>254

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>254

auaauuagug?uucuccgcat?t 21

<210>255

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>255

gauuucacau?uuauggaagtt 21

<210>256

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>256

gcugaggcgg?gaguaucact?t 21

<210>257

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>257

ugcccguagu?gccagcuact?t 21

<210>258

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>258

ucaucacauu?ggaaccugat?t 21

<210>259

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>259

gaggcuuggg?agcuaagcct?t 21

<210>260

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>260

ugccacagcu?ggaucugcct?t 21

<210>261

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>261

gccaggggac?gacucuggct?t 21

<210>262

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>262

uucucagaaa?acggaagact?t 21

<210>263

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>263

uccaauuucu?gcugcaccat?t 21

<210>264

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>264

cagcagcugg?uggcccuuct?t 21

<210>265

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>265

guggccgccg?ucggugacgt?t 21

<210>266

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>266

ucuccucuuc?ugcccgaggt?t 21

<210>267

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>267

cucuggcaau?ccguuucagt?t 21

<210>268

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>268

acgagaccug?uaucgcuuct?t 21

<210>269

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>269

cgaucucuac?uacgagacct?t 21

<210>270

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>270

ugcuuggacc?ugauccaggt?t 21

<210>271

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>271

guccaagcau?gacuucagat?t 21

<210>272

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>272

ggaauguagu?ccuccucugt?t 21

<210>273

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>273

gaugcuggug?auuucguggt?t 21

<210>274

<211>21

<212>DNA

<213>Human?herpesvirus?type?16

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>274

ugugcauaaa?ucugguaact?t 21

<210>275

<211>21

<212>DNA

<213>Human?herpesvirus?type?16

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>275

ccucacgucg?caguaacugt?t 21

<210>276

<211>21

<212>DNA

<213>Human?herpesvirus?type?16

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>276

guugcaaauc?uaacauauat?t 21

<210>277

<211>21

<212>DNA

<213>Human?herpesvirus?type?16

<400>277

uguccagcug?gaccaucuat?t 21

<210>278

<211>21

<212>DNA

<213>Human?herpesvirus?type?18

<400>278

agcuuguagg?gucgccgugt?t 21

<210>279

<211>21

<212>DNA

<213>Human?herpesvirus?type?18

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>279

auucaaauac?cucuguaagt?t 21

<210>280

<211>21

<212>DNA

<213>Human?herpesvirus?type?18

<400>280

aaugucuugc?aauguugcct?t 21

<210>281

<211>21

<212>DNA

<213>Human?herpesvirus?type?18

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>281

ugaugauuaa?cuccaucuat?t 21

<210>282

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>282

aguccaaaga?aaggacccat?t 21

<210>283

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>283

ccagagcugc?uucaagcagt?t 21

<210>284

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>284

cccaauacug?cuucugagct?t 21

<210>285

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>285

auugaaauug?gguauugcat?t 21

<210>286

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>286

ggccgcauag?gcccaaucatt 21

<210>287

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>287

cagguguugc?ugguuccact?t 21

<210>288

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>288

ccggaagucc?uccaaaguct?t 21

<210>289

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>289

caguuuaucu?ccccgcgact?t 21

<210>290

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>290

cgcagaggga?auguccagct?t 21

<210>291

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>291

gcugcaggaa?gcugggcuct?t 21

<210>292

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>292

uccaugggcu?ccucaacagt?t 21

<210>293

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>RNA

<222>(20)..(21)

<223>

<400>293

cugcagcuuc?aucaucagat?t 21

<210>294

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>294

uaauggaaua?ggaccgcggt?t 21

<210>295

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>295

acuuugaaaa?auuggagugt?t 21

<210>296

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>296

caugcuccac?ccugcggugt?t 21

<210>297

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>297

acuuaguagg?gcugaucuct?t 21

<210>298

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>298

accuugagga?ugucccccct?t 21

<210>299

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>299

ucugccuugg?cucuggggat?t 21

<210>300

<211>21

<212>DNA

<213>Homo?sapiens

<400>300

cccacgcggg?gccuggcgct?t 21

<210>301

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>301

ggucguacca?ggacuccuctt 21

<210>302

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>302

ugcauuagga?agguaacuct?t 21

<210>303

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>303

ugaauauacc?aacccaauct?t 21

<210>304

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>304

agcucaguga?gaacaaggat?t 21

<210>305

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>RNA

<222>(20)..(21)

<223>

<400>305

cagccccagc?ucagccauct?t 21

<210>306

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>306

agcccccaua?gucugucggt?t 21

<210>307

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>307

agcauaccac?uucagcaggt?t 21

<210>308

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>308

ugaccaucug?ggcaccgcgt?t 21

<210>309

<211>21

<212>DNA

<213>Homo?sapiens

<400>309

ugcucagugu?ugugggccat?t 21

<210>310

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>310

cuuccaacug?ggcucccact?t 21

<210>311

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>311

cuuccucgua?gucaauguct?t 21

<210>312

<211>21

<212>DNA

<213>Homo?sapiens

<220>

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<223>

<220>

<221>DNA

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cucuucuggg?uccucuucat?t 21

<210>313

<211>21

<212>DNA

<213>Homo?sapiens

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<221>RNA

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<220>

<221>DNA

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gucccgaggu?uucuuaggct?t 21

<210>314

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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accaccguug?uggcaggggt?t 21

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acauuucguc?ucacaguggt?t 21

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<221>RNA

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<220>

<221>DNA

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gcagagaucg?cuggggcagt?t 21

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<221>DNA

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cagccucagg?agaauuaggt?t 21

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<221>RNA

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ggaagccaca?gugucacuct?t 21

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ggagcagcag?cucagcccat?t 21

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<221>RNA

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<221>DNA

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gaggacagug?uugagcugat?t 21

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<212>DNA

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<221>DNA

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ggcacucagu?acugucucct?t 21

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<221>RNA

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caugcuccac?ccugcggugt?t 21

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<221>RNA

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<220>

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acuuaguagg?gcugaucuct?t 21

<210>324

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<221>RNA

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<220>

<221>DNA

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caaauucuua?uuugccccat?t 21

<210>325

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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gacuuugcua?agagccauct?t 21

<210>326

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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ccuuuauuga?uucuaggaat?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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uguggagccu?ucgugaagct?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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auuuguuaua?ggcauaucat?t 21

<210>329

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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aguauuguac?acucuuauct?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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uuaaccauga?uggaggaugtt 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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uguccagcua?aauuaguact?t 21

<210>332

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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aacuaugauc?auugcaauct?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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uaaauaagac?caaugaaugt?t 21

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<212>DNA

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<221>RNA

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<221>DNA

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gcaaguuugu?uguacgcaut?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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uuugguuuau?gcaaguuugt?t 21

<210>336

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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agugguacuu?aucaaauuct?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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gccuuagcca?aagcguuagt?t 21

<210>338

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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gguuggcuga?auugauuuat?t 21

<210>339

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<212>DNA

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uuccuacuuu?guguaauagt?t 21

<210>340

<211>21

<212>DNA

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<221>RNA

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<220>

<221>DNA

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ugacagaagu?ugaucuuugt?t 21

<210>341

<211>21

<212>DNA

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<221>RNA

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<223>

<220>

<221>DNA

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gagcuaccuc?ucccauuuct?t 21

<210>342

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<212>DNA

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<221>RNA

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<223>

<220>

<221>DNA

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uggguugaua?auaguugaat?t 21

<210>343

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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gcuggauucu?ucuucauugt?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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aguguguuca?gaucuuuautt 21

<210>345

<211>21

<212>DNA

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<221>RNA

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<220>

<221>DNA

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gucccuuggg?uguggauaut?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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guguguggac?uugucuaugt?t 21

<210>347

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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uugcuugaga?auucuauugt?t 21

<210>348

<211>21

<212>DNA

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<221>RNA

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<220>

<221>DNA

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gcggugcguu?gguccuugut?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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gcugcaauua?uaagugaagt?t 21

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<211>21

<212>DNA

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<221>RNA

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<220>

<221>DNA

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ucuguuccau?uacacuuaut?t 21

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<212>DNA

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<221>RNA

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<223>

<220>

<221>DNA

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ucucuucugg?cucgauugut?t 21

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<211>21

<212>DNA

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<221>RNA

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<220>

<221>DNA

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acucugcagc?uccacuuaut?t 21

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<212>DNA

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uggcaacaca?ugcugauugt?t 21

<210>354

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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ggaccauuga?auauguaact?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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aggacugugu?uauauuuagt?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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ugcaaguuug?uaguacgcat?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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gcaaguuugu?aguacgcaut?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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cauuggucag?aagaauuaat?t 21

<210>359

<211>21

<212>DNA

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<221>RNA

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<220>

<221>DNA

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gggcacacuu?cacugcuugt?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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uuggugagag?auguuauuat?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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uaggcuuaau?gccaauacat?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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gacagcagcu?gauccuuaut?t 21

<210>363

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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uagcauacca?cauaguuugt?t 21

<210>364

<211>21

<212>DNA

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<221>RNA

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<220>

<221>DNA

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gaugcgaacu?ugcccuuuat?t 21

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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gcuguaaugu?ugucauuugt?t 21

<210>366

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<212>DNA

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<221>RNA

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<220>

<221>DNA

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uggaacauag?gcacccauat?t 21

<210>367

<211>21

<212>DNA

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<221>RNA

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<220>

<221>DNA

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gaucugcuuc?acuaguaugt?t 21

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<211>21

<212>DNA

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<221>RNA

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<220>

<221>DNA

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ucuauuguga?uggauguautt 21

<210>369

<211>21

<212>DNA

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<220>

<221>RNA

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<223>

<220>

<221>DNA

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gaguauuguu?acagaaugut?t 21

<210>370

<211>21

<212>DNA

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<220>

<221>RNA

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<223>

<220>

<221>DNA

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<223>

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gugcuauuug?ugcuauagat?t 21

<210>371

<211>21

<212>DNA

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<220>

<221>RNA

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<223>

<220>

<221>DNA

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<223>

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uuggcagaga?ugaugaauat?t 21

<210>372

<211>21

<212>DNA

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<220>

<221>RNA

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<223>

<220>

<221>DNA

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<223>

<400>372

uccauugcau?uugguuucut?t 21

<210>373

<211>21

<212>DNA

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<220>

<221>RNA

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<223>

<220>

<221>DNA

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<223>

<400>373

agacugacua?cagcuuugut?t 21

<210>374

<211>21

<212>DNA

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<221>RNA

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<223>

<220>

<221>DNA

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<223>

<400>374

guucagcagc?uccacuuaut?t 21

<210>375

<211>21

<212>DNA

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<220>

<221>RNA

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<223>

<220>

<221>DNA

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<223>

<400>375

uagcaacaca?ugcugauugt?t 21

<210>376

<211>21

<212>DNA

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<220>

<221>RNA

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<223>

<220>

<221>DNA

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<223>

<400>376

gcugcaucug?ugauguuaut?t 21

<210>377

<211>21

<212>DNA

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<220>

<221>RNA

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<223>

<220>

<221>DNA

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<223>

<400>377

gggccaucug?uuguagguat?t 21

<210>378

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

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<223>

<400>378

aagagggacg?aguuacucgt?t 21

<210>379

<211>21

<212>DNA

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<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>379

agaacauugc?gguaugcaat?t 21

<210>380

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>380

acaguaucuu?cuccaaaggt?t 21

<210>381

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>381

ucaucaagcu?caaaugugat?t 21

<210>382

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>382

cugguucuuc?agguguaggtt 21

<210>383

<211>21

<212>DNA

<213>SARS?coronavirus

<400>383

guguauaacc?agcacaucct?t 21

<210>384

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>384

aacaucaguc?acugguccutt 21

<210>385

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>385

caucgguauc?aacaacacct?t 21

<210>386

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>386

caagcacaag?aaugcgugct?t 21

<210>387

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>387

uaaauugccu?uccucaucct?t 21

<210>388

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>388

ccaaguuaua?aauagucuct?t 21

<210>389

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>389

cuucaacuac?gaauaggagt?t 21

<210>390

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>390

cagcaucacc?auagucacct?t 21

<210>391

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>391

gcauugagcu?gacuuaucct?t 21

<210>392

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>392

uccuagcugg?agagguaggt?t 21

<210>393

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

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ucgacaagua?gugugccagt?t 21

<210>394

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>394

guugagugua?auuaggagct?t 21

<210>395

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>395

aguaugaaac?ccuguaacat?t 21

<210>396

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>396

aucuauaggu?ugauagccct?t 21

<210>397

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>397

caaucaacag?caucugugat?t 21

<210>398

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>398

guacuacaac?ucuguaaggt?t 21

<210>399

<211>21

<212>DNA

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<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

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caucagugca?guuaacauct?t 21

<210>400

<211>21

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<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

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ugaaguacuu?guccagcuct?t 21

<210>401

<211>21

<212>DNA

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<220>

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<223>

<220>

<221>DNA

<222>(20)..(21)

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gcuguagcau?gaacaguact?t 21

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<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>402

uucuacaugc?guugaugcat?t 21

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<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

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agccaacauc?ucauaauaat?t 21

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<211>21

<212>DNA

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<220>

<221>RNA

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<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

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guguugaaau?gccgucacct?t 21

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<212>DNA

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<221>RNA

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<223>

<220>

<221>DNA

<222>(20)..(21)

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uaccaguguc?uguaguaaut?t 21

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<211>21

<212>DNA

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<220>

<221>RNA

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<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

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agaugaagaa?uguagcauut?t 21

<210>407

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>407

gagccgucga?uuguguguat?t 21

<210>408

<211>21

<212>DNA

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<220>

<221>DNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

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cuauuaacgu?accuguuuct?t 21

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<211>21

<212>DNA

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<220>

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<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

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agaaguacgc?uauuaacuat?t 21

<210>410

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>410

uuuguaugcg?ucaaugugct?t 21

<210>411

<211>21

<212>RNA

<213>SARS?coronavirus

<400>411

aauauugcag?caguacgcac?a 21

<210>412

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>412

aggcugagcu?ucaucaguct?t 21

<210>413

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>413

ccagaagauc?aggaacucct?t 21

<210>414

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>414

uaggaaaccu?auuacuaggt?t 21

<210>415

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

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aagccuacaa?uacaagccat?t 21

<210>416

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>416

augagcgguc?uggucacaat?t 21

<210>417

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>417

aacuuugcuc?ucaagcuggt?t 21

<210>418

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>418

auguagccac?agugaucuct?t 21

<210>419

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>419

guuuccaaua?cgguagcggt?t 21

<210>420

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>420

auguuccuga?ugggcaaggt?t 21

<210>421

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>421

aagugugugc?uagugcaagtt 21

<210>422

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>422

aaaguggcga?guagagcuct?t 21

<210>423

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>423

ccauucuggu?uauugucagt?t 21

<210>424

<211>21

<212>DNA

<213>SARS?coronavirus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>424

cucuucggua?guagccaaut?t 21

<210>425

<211>21

<212>DNA

<213>SARS?coronavirus

<400>425

cuugagugac?guuguacugt?t 21

<210>426

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>426

ugugaggcac?ugcccccact?t 21

<210>427

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>427

ugaacuggag?gcccauccut?t 21

<210>428

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>428

ugaaccagag?gcccauccut?t 21

<210>429

<211>21

<212>DNA

<213>Homo?sapiens

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>429

uuugaggugc?auguuugugt?t 21

<210>430

<211>21

<212>RNA

<213>Homo?sapiens

<400>430

guuuguuagg?accaaaccuc?a 21

<210>431

<211>21

<212>RNA

<213>Homo?sapiens

<400>431

uaggaccaaa?ccucaaagca?u 21

<210>432

<211>21

<212>RNA

<213>Homo?sapiens

<400>432

accaaaccuc?aaagcauggc?u 21

<210>433

<211>21

<212>RNA

<213>Homo?sapiens

<400>433

ccuauuccac?uggauuggga?a 21

<210>434

<211>21

<212>RNA

<213>Homo?sapiens

<400>434

cuauuccacu?ggauugggaa?a 21

<210>435

<211>21

<212>RNA

<213>Homo?sapiens

<400>435

cccuuguuuc?cgcugcaucc?a 21

<210>436

<211>21

<212>RNA

<213>Homo?sapiens

<400>436

caucaucaag?uuccccggcg?a 21

<210>437

<211>21

<212>RNA

<213>Homo?sapiens

<400>437

gacaaagagu?uggcagugca?a 21

<210>438

<211>21

<212>RNA

<213>Homo?sapiens

<400>438

gcaacccaga?uguggccaac?u 21

<210>439

<211>21

<212>RNA

<213>Homo?sapiens

<400>439

caagcccaag?ugggacaaga?a 21

<210>440

<211>21

<212>RNA

<213>Homo?sapiens

<400>440

cagggauuuu?gucaaggagg?a 21

<210>441

<211>21

<212>RNA

<213>Homo?sapiens

<400>441

gggauuuugu?caaggaggau?u 21

<210>442

<211>21

<212>RNA

<213>Homo?sapiens

<400>442

ggauucagca?uugauuuuac?u 21

<210>443

<211>21

<212>RNA

<213>Homo?sapiens

<400>443

ggugguccug?guagcuuuua?u 21

<210>444

<211>21

<212>RNA

<213>Homo?sapiens

<400>444

gguagcuuuu?auuggcaagg?u 21

<210>445

<211>21

<212>DNA

<213>Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>445

cagcggcaca?caggacggct?t 21

<210>446

<211>21

<212>DNA

<213>Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>446

gcacuccagg?gcuucaucgt?t 21

<210>447

<211>21

<212>DNA

<213>Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>447

caguucaggc?acuuuuaact?t 21

<210>448

<211>21

<212>DNA

<213>Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>448

gaaagagagu?cuggccugct?t 21

<210>449

<211>21

<212>DNA

<213>Mus?musculus

<220>

<221>RNA

<222>(1)..(19)

<223>

<220>

<221>DNA

<222>(20)..(21)

<223>

<400>449

gucuuucugu?gugcugagct?t 21

<210>450

<211>21

<212>DNA

<213>Mus?musculus

<400>450

uacugucacc?accgccgcat?t 21

Claims (17)

1. multi-target point cocktail double strand small interfere RNA preparation method, it is characterized in that designing multi-target point cocktail double strand small interfere RNA, form multi-target point cocktail double strand small interfere RNA and mix by the sequences of small interfering RNAs more than three or three according to target sequence complete or part open reading frame or mRNA more than three or three.

2. multi-target point cocktail double strand small interfere RNA preparation method according to claim 1 is characterized in that homology on the gene order of the target sequence behaviour of described siRNA and mouse or non-human primate, the identical or similar albumen of encoding; SiRNA is made up of natural Nucleotide or by the Nucleotide of synthetic or the duplex molecule structure of being made up of manually modified Nucleotide.

3. the multi-target point cocktail double strand small interfere RNA of the described method preparation of claim 1 is characterized in that selecting for use the siRNA by at encoding SARS virus Spike protein mRNA

(5 '-AAGCTCCTAATTACACTCAAC-3 '), at the siRNA of encoding SARS virus nsp-9mRNA (5 '-AAGGATGAGGAAGGCAATTTA-3 '), at the siRNA of encoding SARS virus nsp-10mRNA (5 '-AAGGATAAGTCAGCTCAATGC-3 ') with at the siRNA of encoding SARS virus nsp-13mRNA

At least three or three above sequences of small interfering RNAs mix in (5 '-ACTGGCACACTACTTGTCGA-3 ').

4. the multi-target point cocktail double strand small interfere RNA of the described method preparation of claim 1 is characterized in that selecting for use the siRNA by at coding mVEGFA mRNA

(5 '-AAGCCGTCCTGTGTGCCGCTG-3 '), at the siRNA of coding mVEGFA mRNA (5 '-AACGATGAAGCCCTGGAGTGC-3 '), siRNA (5 '-AAGTTAAAAGTGCCTGAACTG-3 ') at coding mVEGFR1mRNA, siRNA (5 '-AAGCAGGCCAGACTCTCTTTC-3 ') at coding mVEGFR1mRNA, mix at the siRNA (5 '-AAGCTCAGCACACAGAAAGAC-3 ') of coding mVEGFR2mRNA with at least three or three above sequences of small interfering RNAs in the siRNA of coding mVEGFR2mRNA (5 '-AATGCGGCGGTGGTGACAGTA-3 ').

5. the multi-target point cocktail double strand small interfere RNA of the described method of claim 1 preparation, it is characterized in that selecting for use by mixing at least three or three above sequences in the siRNA of the mRNA sequence of VEGF, VEGFR1 and VEGFR2, sequence is seen sequence table or specification sheets table SS1; The above-mentioned multi-target point cocktail siRNA that mixes as clinical angiogenesis inhibitor, treat moist AMD, diabetes type retinopathy disease, interstitial keratitis, glaucoma and the peripheral retinal region new vessel generates ophthalmic diseases, treats various types of cancers, the medicine of treatment rheumatic arthritis and lung vasculogenesis disease.

6. the multi-target point cocktail double strand small interfere RNA of the described method of claim 1 preparation, it is characterized in that selecting for use by mixing at least three or three above sequences in the siRNA of EGF acceptor, FGF and VEGF mRNA sequence, sequence is seen sequence table or specification sheets table SS1, table SS2 and table SS3, and the above-mentioned multi-target point cocktail siRNA that mixes comprises the medicine of vasculogenesis diseases such as cancer as treatment.

7. the multi-target point cocktail double strand small interfere RNA of the described method of claim 1 preparation is characterized in that selecting for use by at Ursodeoxycholic Acid (UDCA) acceptor, VEGF and Alpha-Methyl acyl-CoA racemase

(α-methylacyl-CoA racemase, AMACR) at least three or three above sequences mix in the siRNA of mRNA sequence, sequence is seen sequence table or specification sheets table SS2 and table SS53, and the above-mentioned multi-target point cocktail siRNA that mixes is as the medicine of treatment prostate cancer.

8. the multi-target point cocktail double strand small interfere RNA of the described method of claim 1 preparation, it is characterized in that selecting for use by mixing at least three or three above sequences in the siRNA of VEGF, c-Met and PCDP10mRNA sequence, sequence is seen sequence table or specification sheets table SS2, table SS4 and table SS5, and the above-mentioned multi-target point cocktail siRNA that mixes is as the medicine of treatment liver cancer, lung cancer and colorectal carcinoma.

9. the multi-target point cocktail double strand small interfere RNA of the described method of claim 1 preparation, it is characterized in that selecting for use by mixing at least three or three above sequences in the siRNA of VEGF, c-Met and HGF mRNA sequence, sequence is seen sequence table or specification sheets table SS2, table SS3 and table SS4, and the above-mentioned multi-target point cocktail siRNA that mixes is as the medicine of treatment liver cancer.

10. the multi-target point cocktail double strand small interfere RNA of the described method of claim 1 preparation, it is characterized in that selecting for use by mixing at least three or three above sequences in the siRNA of EGF acceptor, VEGF and p53 mutant mRNA sequence, sequence is seen sequence table or specification sheets table SS1, SS2, SS3 and SS5, and the above-mentioned multi-target point cocktail siRNA that mixes is as the treatment lung cancer drugs.

11. the multi-target point cocktail double strand small interfere RNA of the described method preparation of claim 1, it is characterized in that selecting for use by at the E6 gene of HPV16 and HPV 18 bacterial strains, mix at least three or three above sequences in the siRNA of the EE7 gene of HPV16 and HPV 18 bacterial strains and human P 53 mutant mRNA sequence, sequence is seen sequence table or specification sheets table SS5, and the above-mentioned multi-target point cocktail siRNA that mixes is as the medicine of treatment cervical cancer.

12. the multi-target point cocktail double strand small interfere RNA of the described method preparation of claim 1, it is characterized in that selecting for use by at least three or three above sequences in the siRNA of integrating plain mRNA sequence at matrix metalloproteinase-2, at placenta growth factor acceptor and α v β 3 and mix, sequence is seen sequence table or specification sheets table SS8, SS9, SS10, and the above-mentioned multi-target point cocktail siRNA that mixes comprises the medicine of the vasculogenesis disease of cancer as treatment.

13. the multi-target point cocktail double strand small interfere RNA of the described method preparation of claim 1, it is characterized in that selecting for use by mixing at least three or three above sequences in the siRNA of TNF α, IL-1 and IL-1 receptor mrna sequence, sequence is seen sequence table or specification sheets table SS5, and the above-mentioned multi-target point cocktail siRNA that mixes comprises the medicine of the vasculogenesis disease of rheumatic arthritis, uveitis, psoriatic and Crohn's disease (crohn ' s disease) as treatment.

14. the multi-target point cocktail double strand small interfere RNA of the described method preparation of claim 1, it is characterized in that selecting for use by mixing at least three or three above sequences in the siRNA of RSV virus nucleocapsid albumen (N), non-glycosylated stromatin (M) and transmembrane glycoprotein (F) mRNA sequence, sequence is seen sequence table or specification sheets table SS6, and the above-mentioned multi-target point cocktail siRNA that mixes is as suppressing the RSV medicine for treating viral infections.

15. the multi-target point cocktail double strand small interfere RNA of the described method preparation of claim 1, it is characterized in that selecting for use by mixing at least three or three above sequences in the siRNA of sars cov spike protein, sars coronavirus RNA polymerase and sars coronavirus rna replicon enzyme mRNA sequence, sequence is seen sequence table or specification sheets table SS7, and the above-mentioned multi-target point cocktail siRNA that mixes is as suppressing the medicine that SARS virus infects.

16. the purposes of the multi-target point cocktail double strand small interfere RNA of the described method preparation of claim 1; it is characterized in that medicine as treatment cancer, transmissible disease and inflammation; described multi-target point cocktail siRNA is by combination and degrade messenger RNA(mRNA) or geneome RNA undesired or that the overexpression gene is transcribed, thus the inhibition advancing of disease.

17. the purposes of the multi-target point cocktail double strand small interfere RNA of the described method of claim 1 preparation is characterized in that multi-target point cocktail siRNA and other drug are united to use as the treatment disease medicament.

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