CN1427008A - Method of designing and selecting natural siRNA as gene medicine and medicine formulation - Google Patents

Abstract

A practical method integrating RNA interference, human gene laboratory search, computer information, biochip and genetic engineering technologies into one whole for comparatively well designing and predicting high-specificity gene fragment to prepare efficient gene medicine for deactivating the pathogenic genom. A complete process for preparing gene medicine includes such steps as choosing the pathogenic target gene, discriminating endogenous siRNA sequence, predicting special SDSO moleculae, and preparing gene medicine from SDSO moleculae, carrier component, target pilot component and assistant component. A prescription of gene medicine is also disclosed.

Description

Design and method and the formula of medicine of selecting natural siRNA as genomic medicine

1 the invention under or direct applied technical field the field of the invention be a kind of micromolecular double-stranded oligonucleotide and a kind of method and process of making genomic medicine.

The appearance of 2 the most approaching prior art situation technical background computer chips has been carved human intelligence and wisdom into from refining guided missile, electric network to a series of high-tech products such as palm PC, and the birth of biochip has brought another pleasantly surprised to the mankind, makes people can see the gene structure of self, the expression imbalance of understanding oncogene and the variation of identifying gene structure in the astogeny process.Combination along with computer science and biology techniques, scientists has been finished great human genome plan, discharged the sequence of 32,000,000,000 bases in the human genome, identified tumor-necrosis factor glycoproteinss a large amount of in the genome, extrapolated and approximately contain 32000 genes in the human genome.And these genes only account for 5% (Pandey, A.et al.2001, Nature 405:837-846 of human body all DNA sequence; Shoemaker, DD et al., 2001, Nature 409:922-927).The drafting of the single-nucleotide polymorphism of human genome (SNP) collection of illustrative plates determine and located 1,420,000 SNP (The international SNP map working group, 2001, Nature409:928-933).Converging of biocomputer information technology and network high-speed highway, make the different local scientists in the world carry out the arrangement of gene order by some common gene databases such as BLAST and FASTA, the evaluation of the comparison of similarity, sequence pattern and high conservative fragments detect (Brown SA, 2000, Bioinformatics Eaton Publishing).In a word, a large amount of information material that comes from the human genome 26S Proteasome Structure and Function and fast computer processing mode constructing a broad bioinformation motorway.It will quicken to research and develop the genomic medicine of the genomic function mRNA molecule of direct guiding of novel wide spectrum.These chip technologies that change the world are given biomedical worker a kind of powerful ability, make them can analyze thousands of gene simultaneously, just as The Book of Life of fast reading.Gene sequencing and biochip technology are recognized scientists, and the gene of one group of unconventionality expression makes tumour cell be different from normal cell.Recently noticeable monogenic discovery from rare inherited disease will be made way for the pleasantly surprised of tomorrow, as easily suffering from a heart complaint or the patient of senile dementia is because the variation (Marcotte of certain the fragment gene sequence pattern in the gene, et al, 2001, Trends inPharmacological Science 22:426-437).More mirable is that these hi-techs are forcing them to research and develop the medicine of a new generation to the annual impact that will spend the pharmaceutical companies of 200,000,000 U.S. dollars on the earth.These new drugs will also be treated different diseases with varying with each individual efficiently, and can prevent those diseases all sidedly.The more important thing is that it also will cause a revolution on the pharmacology, change form, target and the composition of medicine widely.If biochip allows people can side by side identify multiple heterogeneic expression in the tissue and understands the overall picture of the generation of differing molecular incident in the lysis, so, the biocomputer information technology can be authorized the stronger ability of people and be removed to find apace high conservative sequence and specific sequence pattern in a certain gene, the high special dna fragmentation of search is as target, and is that template is made the active ingredient of genomic medicine efficiently with it.Along with finishing of human genome collection of illustrative plates, gene database can become a muca gene group information, compares the conserved sequence between different genera, the important tool of design efficient gene medicine.Increasing website begins to set up its distinctive gene database.These databases relate to the information material of the genes involved of various various disease, database (Marcotte as relevant tumour, diabetes, neuropathy, acquired immune deficiency syndrome (AIDS), heart trouble, obesity, hypertension etc., et al, 2001, Trends in Pharmacological Science 22:426-437).As seen, the interests of leaving people's maximum for of finishing of genome plan are from known genome sequence, directly removing to identify target gene that potential need treat and go to design those and they complementary genomic medicine sequences mutually easily, is that the bio-pharmaceutical that acts on protein level is researched and developed on the basis at leisure and do not need indirectly physicochemical property and crystalline structure with corresponding proteins matter.Obviously, biotech drug of future generation will be the outstanding achievement that proteinic genomic research and development are brought to bodily fuctions's property, rather than classical protein chemistry is given people's inspiration.Why cancer is difficult to be overcome by existing medicine and methods of treatment, and its answer is not remote from us.New biochip technology will allow medical personnels go to analyze from cancer cells up to 65000 expression of gene forms, and then compared with normal gene expression of cells.Millions of data can be carried out Computer Analysis, and the interaction of medicine and target also can be simulated by computer.Infusive is that many gene therapies likely are designed and develop.Scientists has found a double-stranded oligonucleotide (SDSO) that 19-25 Nucleotide is long, and they are the function of its homologous of deactivation efficiently RNA molecule veritably.Now main attention has focused on and how has selected and located special target area in the mRNA molecule, with its as template come synthesizing efficient genomic medicine (Lockhart, et al., 2000, Nature405:827-838).Since finding that RNA disturbed in so far several years of (RNAi) this phenomenon in 1998, it has become very clear: the natural functions of this process is a kind of system of defense of ancient biological gene group, and it is used to resist the infringement such as movable genetic material such as transposon, viruses.RNAi, ancient, the most polyenergic this antivirus system, the mechanism of expressing with posttranscriptional gene in plant and the fungi has confidential relation.In insect, batrachia, rat, mouse, monkey and human body, also be observed then and exist this RNA to disturb (RNAi) phenomenon.In nearest experiment, a kind of enzyme that can make the fluorescence worm send unusual radiance---luciferase gene is imported into one group of mammal cell, comprises human embryos nephridial tissue and Chinese hamster tissue.And the short chain RNA interfering (siRNA) that is imported into these cells in succession can reduce the function of luciferase gene effectively.Soon, flat put out such as prostate cancer cell in aspect the improper expression of several abiogenous genes such as bcl-2 gene, RNAi also is proved to be (Carthew, R.W.2001, Curr.Opin.Cell Biol.13, the 244-248 that Zhuo has special effect; Bernstein, E., et al., 2001, Nature, (London) 409,363-366; Tuschl, T., et al., 1999, Genes Dev.13,3191-3197; Oelgeschlager, M., et al., 2000, Nature, (London) 405,757-763).2.2 the power resources of the bio-pharmaceutical that background each big medicine manufacturer research and development in market are novel are in following aspects: in the past decade, viral and fungal infection presents the trend of growth in worldwide, adds that acquired immune deficiency syndrome (AIDS) is uncontrollable to be spread.The active drug of anti-human tumor is still very few, and especially the mortality ratio of cancers such as liver cancer, lung cancer and leukemia is high.Nature or obtain to the resistance of chemicals in continuous increase, and the continuous appearance of the malicious side effect of chemicals.No special and effective medicine can be used in the fact of those heredity illness of treatment.From the infection of ectogenic virus, bacterium and fungi, show all that to endogenic cancer, hyperlipidemia, senile dementia and some other heredity illness abnormal genetic expression is the major cause that causes numerous disease in the human body.The most important purpose of medical science and health care is exactly to put forth effort to find the also efficient ways of various science, stops these abnormal genetic expression, so that the development of control disease and propagation effectively, until finally curing them for a full due.Nature, large quantities of talented and scientists and each big pharmaceutical companies ability are all addressing the problems referred to above, attempt is found more to be hopeful and more profitable methods of treatment, and genomic medicine is becoming the jewel in the new drug of future generation that pharmaceutical companies endeavours to research and develop without doubt.Now clear, new biology techniques can provide the more deep understanding to the disease molecular mechanism, scientists is being used RNA to suppress and is being promoted technology such as son (Promoter) interferences, identifies those and viral, fungi and the growth of bacterium, the generation of tumour and the relevant gene of essence of inherited disease.The nature, when these genes as the treatment target the time, their homologous nucleotide sequences will be special and the most effective medicines.Based on this imagination, the research and development of novel drugs will depend on the evaluation of the particular target gene with unique effect pattern and the design and the making of their homologous nucleotide sequences to a great extent.The research and development method of this new drug also will reduce the cost of research and development and required time widely.Identify a medicine and the interactional structure of its target molecule, deep understanding to its mechanism of drug action usually can be provided, evaluate this most reliable interactional method and be by experiment program and understand the structure of medicine and target mixture, self-evident, this experiment needs expensive equipment, exacting terms, great amount of manpower and long time.Typical program is: people understand the physics and the chemical property of drug molecule earlier, and then seek and target complementary zone with them, for example, a drug molecule most probable that carries negative charge combines with a target with complementary region of positive charge characteristic.Obviously, genomic medicine itself just can ideally solve these puzzlement new drugs planners' a difficult problem, because the active ingredient SDSO molecular energy in the genomic medicine by the hydrogen bond of Watson-Crick base pairing and its target mRNA molecular specific combine, thereby suppress the function of mRNA efficiently.If to as a kind of interest of target of medicine being with RNA since some excellent of RNA in traditional protein target, perhaps, research and develop RNA is because RNA itself also has the characteristic more useful than other bio-pharmaceutical as a kind of medicine so, strategicly.In addition, because the information money section that finishes a large amount of relative dna sequence of being brought of human genome plan makes scientists than the biological function of whenever easier understanding RNA and the characteristic and the variation of molecular structure such as their elementary and three-dimensional structure in the past more.Moreover modern computer technology also gives that developers can search for and than the segmental ability of relatively large gene order.When all these fully and necessary conditions when being attached to technology, design and the genomic medicine of development of new just might come true, become the most compelling problem on the 21st century pharmacology.RNA is the fairly individual target of a class, because it is a kind of both sexes biomolecules, not only has the attribute (similar to DNA) of the genetic information of carrying, and shows the activity (as a kind of proteolytic enzyme) of catalysis in addition.Similar to protein, RNA can combine with some peptide molecules or other small molecules, adjusts its special three-dimensional structure, to obtain its unique biological function.Multi-form oligonucleotide has a kind of potential to can be used as the active medicine of treatment usefulness.Its principle is to combine with one section homologous sequence in the corresponding mRNA molecule by the hydrogen bond between the Watson-Crick base, thereby disturb the biological function of the mRNA molecule contain this section sequence, the genomic medicine that utilizes this feature of RNA molecule to be mixed with also will suppress the expression of the RNA molecule of those Disease-causing genes.Therefore, this medicine research that can be used to tackle tumour, viral infection and heredity illness and be used for other biology purpose.Three different strategic methods have been used to carry out the design of gene therapy, and the strategy of these three different gene therapies is paid close attention to by everybody.These gene therapies are employed three kinds of different RNA nucleases, that is: RNase L, RNase H and RNase III.These enzymes remove to cut off corresponding RNA molecule under the guiding of one section special nucleic acid, thereby make it lose due function.Because the activation of different RNA nuclease needs different nucleic acid as activator, studies show that 2-5A molecule, cDNA and dsRNA can activate RNase L respectively, RNase H and RNase III.In general, but the mRNA molecule of RNase L deactivation strand, and RNase H cuts off the mRNA molecule (cDNA-mRNA) in the two strands, and RNase III can disturb the mRNA molecule (dsRNA-mRNA) in three chains.Is a noticeable imagination with mRNA as the medicine target, because mRNA is easier to approaching than its corresponding gene.The method of Shu Xiing is the most, an anti-sense nucleic acid chain is imported a cell, this anti-sense nucleic acid chain can form the Watson-Crick base pairing with its said target mrna, and the mRNA of hybridization no longer can exercise its biological function, so that the mRNA molecule of hybridization is degraded by this RNase H enzyme.RNase H endonuclease capable cuts off the RNA chain in the RNA-DNA duplex specifically.Therefore, activated RNase H enzyme will cause the fracture of RNA target, can improve the benefit of antisense strand DNA inhibition of gene expression thus.Though many researchs and clinic trial are being carried out always, but, suffer the challenge of many practical problemss with antisense strand as the strategy of gene therapy means, such as, the stability of cDNA molecule, to the resistance of nuclease, with and the effect of treatment very not satisfactory.Second adopted gene therapy method is the nucleic acid molecule that produces a kind of heterozygosis artificially, it has a sequence formula, that is: (dN) m of sp5 ' A2 ' [p5 ' A2 '] 3O (CH2) 4OpO (CH2) 4Op5 ', its formula can be abbreviated as: 2-5A4-Bu2-(dN) m.5 ' end of 2-5A molecule contains a 5-monothiophosphoryl group, and this molecule links to each other with an anti-sense nucleic acid chain, anti-sense nucleic acid can with corresponding mRNA molecular hybridization, corresponding mRNA molecule thereby a RNase L enzyme that combines with 2-5A leads.Like this, this RNase L enzyme just can be that mRNA molecular degradation of hybridizing mutually with 2-5A heterozygosis chain.Discover that now RNase L is a nonspecific nuclease.After it is activated, can cut off all and its bonded mRNA molecule, 2-5A then be the prothetic group that activates this enzyme (Maitra RK: et al., 1995, J Biol Chem270:15071; Cirino NM, et al., 1997, Proc Natl Acad Sci USA 94:1937; Szczylik C, et al., 1991., Science 253:562; Lesiak K, et al. .1993, Bioconjugate Chem 4:467).Recently there is laboratory report to point out, the nucleic acid fragment of this heterozygosis, that is: 2-5A anti-sense nucleic acid chain, the infection of control breathing road syncytial virus effectively.The result shows that the drug effect of 2-5A anti-sense nucleic acid chain is the best medicine of present anti respiratory syncytial virus, that is: Ribavirin 50-90 doubly.But, the stability of 2-5A anti-sense nucleic acid chain and the performance of nuclease-resistant with and specificity still await illustrating further.The third also is the method that the present invention recommended, and is a kind of RNA perturbation technique.Have now found that the RNA interference phenomenon is present in from plant, lower animal until the cell of human body, these organisms comprise plant, protozoon, nematode, insect, fish, birds, mammals and the mankind etc.It is the natural defence system that a kind of primary cell is used for resisting virus, transposon and foreign heredity substance thereof that RNA disturbs.RNA disturbs the double stranded rna molecule by means of a kind of gene specific, and this molecule is called under the effect of RNase III enzyme at a kind of nuclease, is transformed into a series of short and small disturbance RNA molecule (siRNA).A siRNA molecule can combine with its homologous mRNA molecule specifically, causes corresponding mRNA molecule degraded specifically by RNase III enzyme (Fire, A.et al., 1999 Trends Genet.15,358-363; Cogoni, C.﹠amp; Macino, G.2000, Curr.Opin.Genet.Dev.10,638-643; Matzke, M.A., et al., 2001, Curr.Opin., Genet.Dev.11,221-227; Zamore, P.D., Tuschl, T., Sharp, P.A.﹠amp; Bartel, D.P.2000, Cell 101,25-33).The present invention adopts siRNA molecular method own in these natural cells of amplification in vitro, then the multiple different siRNAs transfered cell as active ingredient main in the genomic medicine of therapeutic dose is treated those because the caused illness of some gene overexpression.Therefore, genomic medicine of the present invention has following distinguished characteristics, they including, but not limited to:

1. brand-new theory: adopt natural to be present in siRNA molecule in the viable cell as the genomic medicine of treatment specified disease.

2. Gao Du resistance: adopt the long double-stranded oligonucleotide of 19-25 Nucleotide, avoided single strain oligonucleotide easily by nuclease degradation

Shortcoming.

3. special target is led: adopt human body gene library searching method, selected special siRNA molecule is an active substance.These siRNA molecules with

Specific fragment 100% in the mRNA molecule of same family gene identical.

4. deactivation efficiently: the siRNA molecule that it is the center that usefulness has three strong enzyme point of contacts, can guarantee to cut off efficiently corresponding mRNA branch

Son.

5. long lasting effect: because the siRNA molecule may have the ability of self-replacation and make the methylated ability of corresponding DNA fragments, its biology

Effect may continue the long period in viable cell.

6. Man Yi treatment: the long double-stranded oligonucleotide molecule of multiple 19-25 Nucleotide of different sorts and various dose can form unlimited group

Incompatible adaptation various disease and different patients' needs.

The purpose of 3 inventions and the problem that will solve

It is one that the present invention collects RNA perturbation technique, human body gene library searching technology, biocomputer information technology, biochip technology and genetic engineering technique, researches and develops and prepare a kind of natural genomic medicine.Two main purposes of the present invention are: ● provide practicality and the method for system.These methods relate to a series of process of developing genomic medicine, and they comprise how screening, predict, reflect

Fixed, synthesize, prepare and assemble a kind of natural genomic medicine, and multiple different viral infection, the tumour of how to treat the animal and human

Corresponding scheme with heredopathia.Wherein, a kind of prediction of special recommendation and selected efficiently, short and small interference two strands oligonucleotide

(SDSO) short-cut method.● the long double-stranded oligonucleotide of the preparation of the effective ingredient of a series of genomic medicine, a particularly 19-25 Nucleotide is described.The knot of SDSO

The structure feature comprises one with the VITAMIN B4 beginning, and with thymus pyrimidine or uridylic ending, the double-stranded oligomerization of three strong enzyme cleavage sites is contained in the centre

Nucleotide.Special SDSO molecular energy and its homologous nucleic acid interaction, thus the expression and the adjusting that suppress corresponding RNA molecule contain

The function that the segmental gene of homologous dna is arranged.

The present invention also at large sets forth the efficacy component of some genomic medicines, and the method for the treatment of and preventing some illness of some persistent ailment and human or animal's susceptible also is provided further.These illness can reach the purpose of treatment and prevention by said a kind of or the double-stranded oligonucleotide that several 19-25 Nucleotide are long of the present invention who gives various dose and different types.

The problem to be solved in the present invention mainly comprises the specificity of genomic medicine, high efficiency and stability.Genomic medicine of the present invention is the natural SDSO molecule that is present in the viable cell, and it is a kind of oligonucleotide of short and small two strands, has a powerful enzyme the hit heart and high specificity.When the SDSO of number of different types molecule and other auxiliary composition are assembled into genomic medicine; these genomic medicines are compared with other bio-pharmaceutical, can show following multiple advantage: design that (1) is brand-new and development are theoretical: exist a kind of natural RNAi securing system in the cell.Effect composition in this system can be at body

Amplified and strengthened by bionic method outward, then with in its defeated object of bringing back to life.It can be special and deactivation efficiently those with

The Disease-causing gene in source.Sequence pattern CGGAU (T), CGGGA in the effect composition of the present invention or their derived sequence are designs

With the important indicator of the selected molecule of SDSO efficiently as genomic medicine.(2) short new drug development cycle: by technology such as computer and gene chips, the most conservative part in the selected specific mRNA sequence

As the target of genomic medicine, and its homologous sequence is as the active ingredient of genomic medicine.This new research and development strategy can be widely

Reduce the time of the chemistry be used to study a kind of drug molecule and physicals and find out time of its action site on target molecule.(3) lower medicament research and development cost: study a new drug and and its target molecule between interaction usually need the reasearch funds of great number and big

The search time of amount, the free use of Computing method fast and common gene database, it is new to reduce research and development significantly

The cost of the genomic medicine of type.(4) high specificity: the present invention can filter out the target fragment of the tool potential in the specific mRNA sequence, does with its homologous SDSO

Be genomic medicine.They are discerned mutually and act on by the principle of the Watson-Crick base pairing of classics.Moreover, the fragment of selecting

Only common by homologous genes family members' member, and few similarity is only arranged with other gene family members' member, thus make the spy of medicine

The opposite sex is guaranteed fully.(5) extremely low malicious side effect: the main active ingredient in the genomic medicine of the present invention is a kind of natural nucleic acid fragment, and it is present in from low etc.

Animal is in the cell of human body.Because its high specific and high efficiency make its consumption be lower than other medicine significantly.Obviously, its poison

Side effect also is lower than other medicine out and away.(6) advantages of higher stability: SDSO molecule of the present invention is a kind of double-stranded oligonucleotide, can with some protein and other small molecules mutually

Mutual effect has nucleic acid such as cDNA than other strand that much better stability is arranged.It can resist the degraded of nuclease effectively, easily with

Some protein form stabilized complex.Its some base also is easy to modify, and is used to strengthen the performance of nuclease-resistant.(7) multiple use: genomic medicine of the present invention is the mixture of a kind of dissimilar, various dose and multiple SDSO molecule.They can

Adjusted according to patient's the particular case and the severity of disease, and be can be made into the preparation of different types, be used for different by way of,

Will start the New Times of diagnosing a disease and writing a prescription for each patient.(8) high efficiency: can interrupt corresponding mRNA molecule efficiently can several different Disease-causing genes of while deactivation be genomic medicines of the present invention also

Characteristics, the breakthrough on this methodology is particularly useful for treatment for cancer and prevention, virus and the persistent ailment that causes of fungi and that

The heredity illness of a little refractories.(9) high anti-mutation: according to the principle of mathematics theory of probability, but the energy rate that the sudden change of a Nucleotide takes place in a short and small sequence will be big

The earth is less than the probability in one section very long sequence.In addition, also can contain in the genomic medicine of the present invention and suppress drug resistance gene

The SDSO molecule.The concrete technical scheme of 4 inventions

Perhaps, genomic medicine will become the leading medicine for the treatment of various disease in the world soon, and in the U.S., gene therapy is in different development, from studying, develop the on probation of clinical one, two, three phases.Have now found that antisense strand treatment strategy exists some tangible weakness, as unstable and inefficiencies.Many knowledgeable people are just beginning to attempt to find out more reasonable method and are preparing a kind of genomic medicine with high efficiency and high stability.In order to reach this two big purpose, a brand-new relevant strategic thinking is suggested.That is exactly a kind of natural efficient and stable genomic medicine of preparation.This strategy imagination has demonstrated fully us has had the genomic medicine of the tool potential of understanding better, selecting to be direct medicine target with mRNA and other RNA molecule interest and confidence and to natural selection and computer can make more fully appraisal on molecular level to the whole bag of tricks of gene therapy.Along with the mankind grasp and use to the progressively raising of the understanding of the gene order of disease and human genome with to computer science, biochip and siRNA perturbation technique comprehensively, a kind of brand-new methods of treatment will be used to treat those puzzlement people with acquired immune deficiency syndrome (AIDS) of a specified duration, tumour, senile dementia and other multiple heredity illness.Of the present invention will guarantee the selected of genomic medicine and the method identified the genomic medicine prepared safe, special and effective, reach and finally can cure diseases associated.The present invention also illustrates the prescription and the corresponding compound method of the crucial composition of genomic medicine, also at large descriptive system ground and easily selected genomic medicine complete procedure, treat those related example of incurable diseases and application still at present and come preventing disease, promotion health and beautify the possibility of appearance with genomic medicine of the present invention with the active ingredient in the genomic medicine of the present invention.

5.1 vocabulary of terms

In the context of the present invention, term " genomic medicine " is meant double-stranded nucleic acid oligomers (SDSO) that have an enzyme cutting center of one or more different amounts and these SDSO molecules can be imported an animal especially people's special cell and the mixture of a kind of biofilm carrier that can accept accordingly on drug effect.Term " genomic medicine " further also comprises the SDSO molecule that those are exposed and/or the mixture of other auxiliary composition.They can be directly used in clinical treatment, the prevention of disease and fundamental research.

Term as used herein " double-stranded oligonucleotide " is a kind of polymer of Nucleotide or binary of oligomer of containing.As the RNA molecule (dsRNA) of a two strands, the dna molecular of a two strands (dsDNA), the sRNA-cDNA hybrid molecule of a two strands.This term further comprises by the connection between the Nucleotide of natural Nucleotide, sugar and covalency, and the oligonucleotide through modifying or non-natural Nucleotide is formed.Each type in these oligomer and their countless derivative are by wide coverage.Those are modified or alternate Nucleotide usually is superior to the Nucleotide of nature, as are used to synthetic corresponding oligonucleotide, and its product has stronger resistance to enzymolysis performance, are better taken in performance and Geng Gao and affine performance its target nucleic acid by cell.

Term as used herein " double-stranded RNA molecule (dsRNA), double-stranded dna molecular (dsDNA), double-stranded sRNA-cDNA hybrid molecule " is meant a kind of nucleic acid binary.Their each bar chain is made up of 19-25 Nucleotide.They can be write a Chinese character in simplified form into the SDSO molecule.This SDSO molecule of the present invention is homologous RNA molecule in cell of deactivation effectively.SDSO molecule of the present invention is including, but not limited to those phosphorothioate oligonucleotides and other the oligonucleotide through modifying.

Term as used herein " special SDSO molecule " is meant that has the long specific nucleic acid binary of 19-25 Nucleotide.Most of or all members' of its just anticipate chain and its homologous genes family a certain dna fragmentation 100% identical, and do not have with the gene order of other gene family or only less than 80% similarity.Its antisense strand can with corresponding mRNA molecular hybridization, guiding RNase III enzyme this mRNA molecule of degrading specifically, and function that can other RNA molecule of deactivation.The research report of several lines has pointed out that this siRNA molecule just can not suppress the activity of that said target mrna molecule so if the siRNA molecule has more than one Nucleotide different with its said target mrna is intermolecular.

Term as used herein " SDSO molecule efficiently " is meant a kind of short chain oligonucleotide binary.It comprises enzyme cutting center, and this enzyme hits the sequence of the heart including, but not limited to CGGAU (T), CGGAA, CGGAC, CGGAG, CGGGC, CGGGA and CGGGU (T).These sequences contain two to three high-intensity restriction enzyme sites, and they are GG, GA and AU.Therefore, contain the mRNA molecule that the SDSO molecule of two to three high-intensity restriction enzyme sites can guiding RNase III enzyme efficiently and be specifically degraded and contained homologous sequence.

Term as used herein " homologous nucleic acid or homologous sequence ", the dna molecular that comprises the RNA of those energy proteins encoded and other function, the RNA molecule that produces from these DNA comprises immature mRNA, the identical segments in sophisticated mRNA and other RNA molecule and these dna moleculars.The interaction of SDSO molecule and target nucleic acid can influence the corresponding function of this nucleic acid.This inhibition by the numerator mediated target nucleic acid function of SDSO is generally defined as " RNA or DNA disturb ".The disturbed function of RNA comprises transcribing of those mRNA, the splicing (producing one or more mRNA molecules) of the transposition of RNA (in examining, producing proteic place) RNA to tool, and RNA translates and other specific function by the RNA mediation.The disturbed function of DNA comprises that DNA duplicates, transcribes, repairs and recombinates.The disturbed net result of these target nucleic acids is, the degraded of the mRNA of synthetic proteins or polypeptide, the deactivation of the specific function of other RNA molecule, and the methylating of homologous DNA sequence.Though the SDSO molecular energy specifically with one or more homologous nucleic acid interactions, has advantages of higher stability and validity, but aim of the present invention mainly is to suppress the function of those genomic RNA molecules, thereby can take precautions against and treat cancer, viral infection and heredity illness.Nucleic acid molecule involved in the present invention is including, but not limited to the mRNA molecule, and they are: ● the mRNA of coding oncoprotein, as H-ras, K-ras, N-ras, C-myc, rhoB, BRCA1﹠amp; 2, mdm-2, p-53, p-21, ● the mRNA of coding somatomedin, as EGF, HGF, IGF-1﹠amp; 2, NGF, PDGF, TNF, VEGF, alpha-FGF, beta-FGF, TGF-

Alpha, TGF-beta, GGF, ● the mRNA of coding growth factor receptors, as EGF-R, HGF-R, IGF-1﹠amp; 2-R, NGF-R, PDGF-R, TNF-R, VEGF-R,

Alpha-FGF-R, beta-FGF-R, TGF-alpha-R, TGF-beta-R, GGF-R, HuH-7, ● coded signal transmits the mRNA of molecule, as PKC-alpha, and Stat3﹠amp; 5, CDK-2﹠amp; 4, Ras, Raf, FAK, Src, MEK, CDKN2A, ● the mRNA of coding hormone receptor, as estrogen (SERMs), progesterone, testosterone, aldosterone, corticosterone, ● coding survives the mRNA of molecule, as BCL-2, BCL-6, BCL-xl, Telomerase, ● the mRNA relevant that encode with cellular localization, as Integrins, E-cadherin, ● coding and relevant mRNA, as Cytokines, Interleukins, interferons, ● coding and relevant mRNA, as CD401/CD40, ICAM-1/LFA-1, Hyalurin/CD44, ● the mRNA relevant with hereditary illness encodes, as LDL-R, Amyloid protein, WNKs, ● the mRNA of the viral proteins associated matter of encoding, as RSV, HPV-16﹠amp; 18, EBV, Protease (PROT), polymerase (POL),

integrase(INT)，gp120?and?gp41，transactivating?protein(TAT)，regulator?of?expression?of?virion?protein

(REV)，and?viral?infectivity?factor(VIF)。

5.2 identify the mRNA molecule relevant with disease

Normally with the use of abnormal human genomic sequence database and the development of strong biochip technology, the permission brainstrust can promptly be identified the transcription product of those gene molecules in any sufferer tissue and cell and those undesired expression and design special genomic medicine targetedly.These new and high technologies also make people can understand RNA molecule and proteinic all respects better.The active ingredient of the genomic medicine among the present invention can be determined and selected by other method of reporting for work in biochip technology and the scientific literature.

Biochip technology is being given the method that people understand the cancer true nature in depth, makes people recognize that cancer is not a kind of product of bad cdna, but the disastrous effect that the gene of one group of undesired expression is created.In recent years, scientists has been identified the change of many gene expression patterns in the cell of many cancers.These cancers comprise leukemia, lymphoma, prostate cancer, mammary cancer, squamous cell carcinoma, melanoma, brain tumor etc.Some brainstrusts can not be replied with which can determine which cancer cells may reply present treatment.In addition, investigators recognize that also cancer cells their development by one group of gene rather than by single Gene Handling, keep diffusion and shift.These discoveries will be the design of genomic medicine and the preparation data of submitting necessary information.Obviously, choosing the most effective target sequence further in these oncogene, design corresponding SDSO active ingredient, then they are assembled into a kind of specific genomic medicine, is aim of the present invention place.

Very clear now, constantly perfect along with biochip technology, the variation that detects all genetic expression forms exactly will become possibility.Which gene is the biochip technology that increasing biotech firm just is being devoted to develop a new generation come in the recognizing cells in specified time, and which gene is being closed opening, the change of finding out which genetic expression relates to the generation of tumour, development and transfer, with to identify which gene relevant with hereditary illness.Moreover, some company is is researching and developing biochip is being diagnosed general illness as the method for routine, for example people's body fluid and blood are through after certain processing, can be used for special biochip, so that a patient's saliva, with regard to diagnosable patient get sick toxinfection or bacterial infection.Equally, coming from one with one of biochip test has cancer family history patients'blood, just can know whether the patient attacked by cancer.In clinical application, whether the patient that biochip technology has been used to relatively to suffer from melanoma is easy to suffer the threat of metastases or is easy to obtain the result that sb.'s illness took a turn for the worse.

The present invention uses the main purpose of biochip to be to identify which gene overexpression in an abnormal cell, and wherein which is the key gene that directly causes disease.Because the gene that is not all high expression level all can produce excess protein or polypeptide, perhaps proteic change synthetic and its amount is one and reflects the whether index of overexpression of its gene reliably.It can be used to evaluate more accurately the danger of the changes of function of genes involved.The combination of gene chip and protein chip among the present invention will provide more comprehensively and special detection information.The relatively more normal variation of genetic expression and protein synthesis with abnormal cells and/or in organizing, relatively they will provide valuable information and foundation with the corresponding SDSO molecule of design as genomic medicine for our selected target RNA molecule in the variation of patient's different times and the difference between a kind of genetic expression and its protein synthesis.

5.3 identify autogenous siRNA s

After the information that obtains relevant target gene and their rna transcription, the present invention introduces one can search for endogenous RNAi gene effectively ... the method of the template of a SDSO molecule.Identify that endogenous RNFA interference base is because of being special genomic dna sequence conduct of search committed step of the active ingredient of genomic medicine efficiently.Because endogenous RNA interference base is because of containing one the 21 long distinguished sequence of Nucleotide, it can suppress the function of corresponding mRNA effectively.This sequence is keeping the composition and the order of original Nucleotide all the time through the long river of very long Natural Selection and Evolution.Therefore, endogenous RNAi gene is the best choice as genomic medicine.Simultaneously, it also can save the special genomic dna sequence of search as time of the active ingredient of genomic medicine efficiently.

Though finishing of human genomic sequence provides a gene of studying most of proteins encoded, the bioinformation storehouse of tRNA and rRNA is identified the gene of those non-proteins encoded rapidly but still have any problem.Especially these novel endogenous RNA interference sequences are out in the cold chronically, and do not have unique identifier.The present invention proposes the high conserved sequence of RNA, particularly does ring texture and can be used as an important identification mark, and it can be used to screen the RNA interference sequence, because the RNA interference sequence must contain one section reverse complementary sequence.The present invention uses existing computer software method to search for rnai molecule as folding ranking method (FOLDALIGN) and other the special computers software that can predict the dried ring texture of RNA in the human genome information storage.In addition, other several different methods also are useful, seek structure with the common property special seized with terror of microRNA as computer, microarray assays between gene and they include in the subarea complementary sequence and from RNA bonded protein such as desaminase (siRNA is conjugated protein) the isolation of RNA molecule.In a word, whether search exists the critical step that corresponding endogenous rnai molecule is the template of selected special SDSO molecule in the human genome information storage, because natural rnai molecule is the best original shape type as the corresponding genomic medicine of design.

RNA disturbs and to be defined as a class RNA molecule, their complete open reading frames of not encoding, and these RNA molecules from different genera have high conservative sequence.For example, the RNA interference base of people and nematode has the high conserved sequence greater than 95% because of almost completely the same, and typical case is from the gene of the coded protein of different genera, usually only less than 70% similarity.According to the conservative degree of sequence randomly in the intron zone of non-encoding gene zone or gene screening RNA interference sequence be a kind of feasible method.Therefore, the present invention proposes to screen RNA interference sequence standard: the dried ring texture of (1) RNA, (2) high conservative cadre zone, the length in (3) cadre zone is 19-25 Nucleotide, (4) this sequence be positioned at distinguish between encoding gene or encoding gene include the subarea.

All possible rnai molecule all derives from the subarea that includes of intergenic region or gene, and is so far, still imperfect or lack fully about the dna sequence dna information of the intergenic region of all kinds and data.This has constituted the major obstacle of searching for corresponding RNA sequence.Yet, now had some human body gene storehouses of developing and special software.The principle that these special softwares adopt is that everybody knows: that is exactly, and the first area of a Nucleotide may be complementary to the second area of same Nucleotide, and arrange in antiparallel mode in these two zones.A Nucleotide in the first area can be right with the oligonucleotide ligand in the second area.When arrange in antiparallel mode in first and second zones, have at least the oligonucleotide ligand in 95% Nucleotide and the second area right in the first area.The length in these two zones is approximately crossed over 19-25 length of nucleotides.It would be desirable all Nucleotide in the first area and all the Nucleotide complementations in the second area.Forming hydrogen bond as the thymus pyrimidine in VITAMIN B4 in the first area and the second area or uridylic in antiparallel mode is connected.Similarly, a cytosine(Cyt) in the first area can also match in antiparallel mode mutually with the guanine in the second area.For example, let-7, a RNA interference base is because of being positioned at an intergenic region.Length according to conservative degree of sequence and conserved sequence, sequence to this intergenic region of coming from human body, nematode and fruit bat compares mensuration, found that let-7 contains the highly conserved sequence of 21 Nucleotide, can obtain the highest score, BLAST:42 (Fig. 1).According to documents and materials, promote subregion can not obtain such high score and have all these constructional features from the great majority of different genera.Sequence 1 has shown the human body let-7 interference sequence that a cover is come out by blast search, and it is positioned at different intergenic regions, and its 21 Nucleotide are almost identical with the sequence from this gene of nematode and fruit bat.This shows that the dried ring texture of intergenic region and the high conserved dna sequence of 21 length of nucleotides can be used as the standard of identifying a RNA interference sequence.

5.4 search for high conservative sequence with the analysis of structure homology

If in existing human body gene database, can not detect desired RNA interference fragment, be necessary to carry out the analysis of the homology sequence of a gene family, with all members of search corresponding gene family.In this step of search, main task is most of members or whole structure homologous sequence of being shared of member of a certain gene family of search different genera.Structural homology is the attribute of a macromole three-dimensional structure.Its closely related Nucleotide in primary structure is formed and is put in order.High conserved sequence (Motif) contains most important genetic information, under these information are preserved in the process of evolving constantly and be presented among the conserved sequence.It is made up of specific sequence and certain structure restriction usually, and some part of its primary structure is changed, and its entire structure also can obtain keeping.The segmental important precaution of search specific genes are, in the same gene family of different kinds, find out high conservative sequence and in the different mutation of same gene family, identify special sequence pattern (Pattern), and these conserved sequences and sequence pattern seldom are that other gene family is common.For example for all members in a kind of oncogene of deactivation family, it is common for all members be able to identify which section sequence.Like this when this section sequence of selecting during as the active fragments of genomic medicine, it is each member in this family of deactivation effectively.These characteristics are very beneficial for treating the bigger illness of those genovariations.As the different variants that different patients is carrying same gene, genomic medicine of the present invention just can be cured to same degree these patients' illness, otherwise, can only be used for a certain patient, and invalid to other patients.

Multisequencing is arranged formula can detect total sequence in the same gene of different genera, for plural series arrangement, conclusion type formula is only selection, and multisequencing is arranged at first and finished with progressive type arrangement formula usually.These computer formula computings are very fast, do not need the bigger gene chip of capacity, just can move on microcomputer like this.Use in the software of this formula at all, first-elected CLUSTAL W and MUSCA are the most handy.CLUSTAL W also can be used for arranging a certain specific region of many sequences, and does not need to change the arrangement of other parts.If in the arrangement that requires for the first time, all sequences demonstrate each other similarity all, so, this must be best result, needn't be determined with other any softwares again.The gene order that the present invention arranged is to obtain (Fig. 2) with BLAST algorithm formula from different kind gene databases, for example, sequence from all members of the IGF-2 gene of different genera is arranged in CLUSTAL W software, resulting rank results can be carried out artificial and accurately be adjusted, with reflection common conservative region, at last, the series arrangement of IGF-2 can be used as the basis of further analysis and application (Fig. 3 and Fig. 4 is a).

Yet, if run into the sequence of some differences in height, as contain big disappearance, or bad conservative region, be necessary to use different modes or different softwares to come more resulting result, Fig. 5 shows two shared homology segments by one section non-conservative section separately, and this situation usually occurs in the genomic dna sequence, carry out the arrangement formula of formula as use, especially easily produce mistake.Mainly due to the method for successive comparison long disappearance tendency is given and bigger penalty index.For fear of this shortcoming, the two series arrangement formulas of BLAST are best choice.The sensitivity of sequence search similarity can also be improved by the length according to the size balance institute favored area of conserved sequence.Like this, once several homologous sequence is identified that the general picture searching method of use BLAST can be arranged out those sequences at a distance of the different members of bigger same gene family.

5.5 come selected desired sequence with the analysis of the genomic sequence pattern of human body

In this section, it is not only common by the different members of same gene family to be necessary to make clear which high conserved region territory, and is shared by other gene family.The method of an analytical sequence is earlier this sequence to be divided into different sections, and these different sections belong to different gene families, and they on the structure, or are correlated with on the function on evolving, and are keeping their total feature or patterns.The dna sequence dna of existing known road high conservative necessarily relates to a certain important function, and the pattern of this sequence can be used to distinguish the member of family and the difference between non-member.The discovery operational method of binding sequence pattern and strict multisequencing are arranged formula, and the zone that has in sequence that a kind of effective means identifies reflection family differences and the family can be provided.At last, this is present in the basis that a kind of intragenic constant sequence pattern will be used as the active ingredient of selection genomic medicine among the present invention.

In order to detect the homologous dna sequence dna, available BLAST and FASTA search engine are retrieved and they matching databases, as Genebank, and Swiss-port EMBL.These databases all pass through special arrangement and favorable tissue, have all nucleotide sequences of delivering at present, retrieve very convenient.But still can not only rely on a note in the database to find all homologous sequences of same gene so far.Recently effective means is, takes out a certain member of same gene family, as the enquirement sequence, open the corresponding calculated formula then, compared with each sequence in the database, finally obtain all homologous sequences.In a series of independently test of the present invention, special dna sequence dna such as IGF-2 are used to seek corresponding transcripton, and the homologous sequence (Fig. 2) of one section siRNA is arranged in this transcripton.This sequence can be used as the individual active ingredient in the genomic medicine, is used to suppress the function of IGF-2 mRNA, promptly stops its synthetic corresponding IGF-2 protein sequence.Therefore, the present invention recommends to seek corresponding homologous sequence with the blast search engine in NCBI human body gene storehouse.In addition, in order to guarantee a search up hill and dale more comprehensively, another method also is feasible, that be exactly be used in that the first step identifies repeatedly carry out this searching process from the several homologous sequences that differ greatly of different genera, up to finding all homologous sequences.After carrying out blast search, it can point out that how many sequences were compared, and has how many sequences with puing question to sequence in various degree similarity to be arranged.In the result of blast search, there is the sequence of certain similarity to be arranged from top to down by the size of their similarity degrees.According to the difference of score, having the sequence set of high similarity can see at a glance, can totally calculate from the member's of homologous genes family and different genes family number.More different Search Results, can choose best sequence, most of members of it and its gene family or whole common sequence patterns of having of member, and have only less similarity with the member of other gene families, and the similar sum of all sequences also is minimum.

5.6 select the SDSO sequence by special cut mode

Another problem of the relevant distinguished sequence of the present invention is the putting in order and special sequence pattern of number of the Nucleotide in the sequence.The double-stranded oligonucleotide that purine is abundant, particularly those contain the double-stranded oligonucleotide of four guanine bases, under physiological condition, have the tendency that forms stable tetramer structure.Guanine in the single strain oligonucleotide does not have the space constraint in the double-stranded oligonucleotide, therefore, can form various hydrogen bonds in some non-Watson-Crick base pairings, so can give birth to a kind of tetramer that everybody knows, i.e. guanine limbs.The dissociation yield of this limbs is low-down, and will influence and its said target mrna molecular hybridization cause the active ingredient in the genomic medicine to lose efficacy.As if another what is interesting is that RNase III nuclease prefers the oligonucleotide of a plurality of urine purines.Like this, if a 19-25 oligonucleotide contains more uridylic, perhaps the affinity between them strengthens to some extent.Special combination and cutting efficiently are the most important indexs of a design and a selected effective SDSO molecule.The present invention organically integrates one group of powerful enzyme cleavage site and high special sequence, as the basic structure of genomic medicine active ingredient.A kind of method of simplification also is provided simultaneously, just can correctly predicts a SDSO molecule efficiently.The system of selection of this simplification is a seed with an enzyme cutting center mainly, extends about 7 length of nucleotides to both sides respectively on its both sides then.The sequence that this enzyme cutting pericardium is drawn together a cover cleavage site, they are made up of CGGAU (T), CGGGA and their derived sequence.Several groups of researchs show that RNA nuclease (RNase III) can be at GG, cuts off the covalent linkage between them between GA or the AU apace, make one complete and long mRNA chain becomes two corresponding short and small nucleotide chains, translate proteinic function thereby lose it.Perhaps, the hit CGG sequence of the heart of this enzyme is the reaction center of a methylase, and it will wave certain function in the function of regulating DNA.As seen, the enzyme that the present invention the proposes heart that hits not only helps predicting effective SDSO molecule and saves and select and the time of searching for relevant special order, and is that exploration regulatory gene group function has been opened up another approach.

Carefully analyzing an enzyme hits and can find that there are core sequence, i.e. a CGG in each center behind the heart.CGG is that a high conservative order is closely related with the high specific of forecasting sequence, if change CGG, even only change the C in the CGG sequence, will cause the sequence-specific fundamental change of SDSO, generally speaking, the complementary sequence of nonspecific pairing or part will increase (table 1 and table 2).Enzyme hits due to the derived sequence of core structure replaces mainly due to the 4th and the 5th Nucleotide in this sequence.Even the 4th can be A, C, G or U, but best choice is A and G, and this is because these two Nucleotide not only can form a strong enzyme cleavage site with they previous Nucleotide G, and they also are relevant to the specificity of SDSO molecule nearly.Though the 4th is that C or T also can select more special SDSO molecule sometimes, has and bigger may select a large amount of nonspecific sequences (as table 3 and table 4).Equally, the 5th Nucleotide is any one in four Nucleotide also, but T and A can form strong restriction enzyme site with the 4th A and G respectively.The specificity influence less (as table 3 and 4) of different here nucleotide pair SDSO molecules.In a word, the present invention is used for predicting the sequence member that the enzyme of an effective SDSO molecule hits the heart, including, but not limited to CGGAU (T), CGGAA, CGGAC, CGGAG, CGGGA, CGGGC and CGGGU.In other words, contain the hit SDSO molecule of the heart of above-mentioned arbitrary enzyme, can be elected to be the candidate of active ingredient in the genomic medicine, they are the corresponding mRNA molecule of deactivation effectively.

Specific enzymes among the present invention heart that hits is CGGAU (T), CGGGA or their derived sequence, these are centered close on the just meaning chain of SDSO molecule, and their complementary sequence, AU (T) CCG, U (T) CCCG and their derived sequence must be positioned on the antisense strand, enzyme hit second G of the heart should be positioned at the SDSO molecule the beginning downstream the tenth or the 11 on because RNA nuclease (RNase III) usually the tenth or the 11 on interrupt mRNA molecule or other RNA molecule with the SDSO complementary element.As seen, the restriction enzyme site of bunch change helps guaranteeing the efficient and the success ratio of enzyme cutting.

5.7 select the special and easy method of SDSO molecule efficiently

Except that the method for the complexity of a said system ground search and a selected effective SDSO, the present invention also provide one of an easy prediction effectively inhibitory phase answer the method for the SDSO molecule of genetic expression, the characteristics of this method mainly are whether the antisense strand of decision SDSO molecule can be complementary fully with a certain sequence on the homogenic RNA molecule.This sequence also contains an enzyme the hit heart such as CGGAT, CGGGA or their derived sequence.

The first step of this method is which section sequence contains the enzyme heart such as CGGAT or CGGGA or other the corresponding enzyme heart that hits that hits among specific gene group DNA of search, one section complementary sequence of hitting the heart with enzyme should be arranged, as AU (T) CCG or U (T) CCCG on the antisense strand of SDSO molecule.This contains the hit nucleotide fragments of heart complementary sequence of enzyme, can with the RNA molecular hybridization of corresponding genomic dna sequence.

Second step was to arrange the enzyme position of the heart on the SDSO molecule of hitting.Second G of the heart and place on the SDSO molecule the tenth with its complementary C normally hits enzyme on the SDSO molecule.

The 3rd step was to be that 7 Nucleotide are extended in its both sides of middle mind-set with this enzyme heart that hits, thereby obtained the long SDSO molecule of 19 Nucleotide.

The 4th step was to serve as to put question to sequence with this sequence, went other similar sequence of search in the human body gene storehouse, thereby obtained a series of identical or sequences that part is identical.

The 5th step, compare the resulting result of different enquirement sequences, can find out the member of great majority or whole same gene families, seldom maybe can not find out any sequence similar for being elected to sequence to the member of other gene family and can only find out.

The 6th step in all elected sequences, be VITAMIN B4 with the beginning, or its previous Nucleotide was a VITAMIN B4, ended up into the thymus pyrimidine person is optimal selection, as the active ingredient of genomic medicine.

Have now found that having the hit sequence of the heart of enzyme can show the specificity of its height, it does not contain or only contains few sequence similar to other gene family member.The present invention has proved the complementary fragment that the enzyme on the antisense strand of SDSO molecule hits the heart, is a reliable enzyme trimscript will as AUCCG, and it can guarantee the corresponding mRNA molecule of selected SDSO molecular energy deactivation efficiently.Like this, have reason to recommend to contain the fragment that enzyme hits the heart on the genomic dna sequence, can be used as the theoretical basis of design SDSO molecule.Recognize the hit biological significance of the heart of enzyme on the effective SDSO molecule fully, embodied and be superior to method of design in the past on the inventive method, the enzyme existence of the heart of hitting on the SDSO molecule also reflects high efficiency and the high specific of this molecule in the expression that suppresses corresponding RNA molecule.The present invention will describe the practical application (table 1,2,3 and 4) of present method for example.

Following table shows that with the enzyme heart that hits be the example that sign comes a selected high special SDSO molecule.Have hit some oligonucleotides of the heart of enzyme and be used to seek other identical wholly or in part sequence in the human body gene storehouse, the specificity of a SDSO molecule of selecting can be evaluated with following method.Method is as puing question to sequence with a SDSO molecule, it is compared with about 960000 different sequences in the human body gene storehouse, the similar wholly or in part nucleotide sequence that all seek out by it can be divided into three groups in various degree according to their similaritys, first group is 100% pairing, second group is the pairing of 80-95%, and the 3rd group is the pairing less than 80%.Each specific gene in following every table all has a special code name near the human genome sequence database, and each SDSO molecule all has corresponding sequence code name, by base composition, enzyme in the sum of the sequence found out and not homotactic Origin And Destination, the sequence the hit heart and paired number in various degree.Under the matcher hurdle, m represents the member of same family, and n represents the member of non-same family, and what of sequence are the size of numeral represent.According to the different similar gene order of being found out what, the SDSO molecule of selecting is compared, just may estimate the height of the specificity and the susceptibility of selected SDSO molecule.

Table 1 and table 2 have shown that by three letters be hit in the heart core of enzyme that CGG forms, if the C in this core sequence is replaced by other A, G or T, the sequence sum of finding out so will increase.Table 1. people amyloid beta (A4) precursor protein (gi|14780094)

Seq. ID#1	?Total ?Hits	?100％ ?Match	?80-95％ ?Match	＜80％ Match	?Cleav. Pattern	?Start????Sequence???????????End ?Point????(19?Bases)???????Point
							?1	?120	?10m	?????2n	????108n	?aggtc	?1????atgtcccagg?tcatgagag????19
?2	?56	?17m??3n	?????1n	?????35n	?cggag	?756??atcaagacggaggagatct???774
							?3	?205	?16m??3n	?????8n	????178n	?atgca	?1079?tgagcagatgcagaactag?1097
?4	?248	?15m??4n	?????8n	????221n	?aggat	?454??gagattcaggatgaagttg?472
							?5	?205	?19m??4n	????11n	????161n	?tggat	?789?gtgaagatgga?tgcagaat?807
?6	?505	?14m??4n	7m??39n	????441n	?gggaa	?16???agaga?atgggaagag?gcag?34
							?7	?18	?13m??4n		??????1n	?cggaa	?542??tcagttacg?gaaacgatgc?460

Table 2 has shown that by three letters be hit in the heart core of enzyme that CGG forms, if this core sequence is replaced by other sequence, the sequence sum of finding out so is just big, otherwise just little.Relatively can find that by CGGAT or by the enzyme that other sequence the is formed heart that hits with the enzyme of being recommended among the present invention heart that hits serve as that sign comes selected VEGF siRNA to have much higher specificity.Table 2. human vascular endothelial growth factor (gi|15422108)

Seq. ID#2	?Total ?Hits	?100％ Match	?80-95％ ?Match	＜80％ Match	?Pattern	?Start????Sequence?????????End ?Point???????????????????Point
							?1	?201	?22m?4n	?5n	?170n	?ttggg	?21?tgctgtcttg?ggtgcattg?39
?2	?81	?16m	?5n?4m	?56n	?tgaca	?551?gcagatgtga?caagccgag?569
							?3	?59	?18m	?1n	?40n	?gaggg	?261?caatgacgag?ggcctggag?279
?4	?23	?21m		?2n	?cggat	?315?gattat?gcggatcaaa?cct?333
							?5	?157	?21m	?20n	?116n	?tcatg	?121??gtgaagttca?tggatgtct?139
?6	?520	?22m	?11n	?487n	?gttcc	?481?tgtaaatgtt?cctgcaaaa?499
							?7	?102	?21m	?4n	?77n	?gccat	?148??agctactgccatccaatcg??166

Table 3 and table 4 are that example illustrates and constitutes the hit CGGAT of the heart of enzyme with people BCL2 and human protein kinase, and CGGGA or their derived sequence are to the specific influence of the siRNA molecule of a selected PRKWNK4.Examine and to find to hit enzyme the 4th and the 5th Nucleotide can change in the heart sequence.Even the 4th can be A, C, G or U, but best choice is A and G.Though the 4th is that C or T also can select more special SDSO molecule sometimes, has and bigger may select a large amount of nonspecific sequences.Table 3. people BCL2, B-cell CLL/lymphoma 2 (gi|13646672)

Seq. ID#3	?Total ?Hits	?100％ Match	?80-95％ Match	＜80％ Match	?Pattern	?Start????Sequence???????????End ?Point?????????????????????Point
							??2	??18	??8m	?????3m	??7n	??cggtc	??187?cggg?acccggtcgc?cagga?205
??3	??152	??11m	??5n	??136n	??cggct	??217?caga?ccccggctgc?ccccg?235
							??4	??81	??11m		??70n	??cggtg	??256?ctcag?cccggtgcca?cctgtg?276
??5	??89	??11m		??78n	??cggtg	??388?ttt?gccacggtgg?tggagg?406
							??6	??25	??6m		??19n	??cggcc	??599?aa?ctgtacggcc?ccagcat?617
??7	??41	??10m		??30n?1m	??cgggg	??372?caccgcgcg?gggacgcttt?390
							??8	??35	??8m	??2n	??22n?3m	??cgggc	??120?cccgcaccggg?catcttct?138

Table 4. human protein kinase, lysine deficient 4 (PRKWNK4), mRNA (NM 032387.1 GI:15277311)

Seq.ID# 4

?Total ?Hit

?100％ ?Match

?80-95％ ?Match

＜80％ Match

?Pattern

?Start????Sequence????End ?Point??????????????Point

??1	??13	??4m	??1n	??8n	??cggaa	??1029?gggaccccggaattcatgg?1047
							??2	??12	??3		??9	??cggaa	??366?aaggctgcggaagactccg?384
??3	??21	??3	??7	??11	??cggaa	??632?gcagactcggaaactgtct?650
							??4	??24	??3	??3	??18	??cggac	??270?gatcctccggactccgctg?288
??5	??66	??3	??1	??62	??cggac	??393?gagctcccggactctgcag?411
							??6	??44	??3	??5	??36	??cggag	??30??ccggccacggagaccaccg?48
??7	??12	??3		??9	??cggag	??2193?ctgccttcggagcgagatg?2211
							??8	??5	??4		??1	??cggat	??1254?atccgcacggataagaacg?1272
??9	??7	??3		??4	??cggat	??1752?accacttcggattgcgaga?1770
							??10	??4	??3		??1	??cggat	??2216?tctcagacggattcgggag?2234
??11	??56	??4		??52	??cggca	??653?agctgagcggcagcgcttc?671
							??12	??6	??4		??2	??cggca	??1093?acgcgttcggcatgtgcat?1111
??13	??53	??2	??1	??50	??cggcc	??24???caatccccggccacggaga?42
							??14	??136	??3	??5	??128	??cggcc	??2990?tcctgctcggcccctccca?3008
??15	??128	??3	??2	??123	??cggcg	??458?cctagagcggcggcgggag?476
							??16	??171	??3	??1	??167	??cggcg	??1397?ggacgcgcggcgcgggggg?1415
??17	??34	??3		??31	??cggct	??1872?ctgccctcggcttttgccc?1890
							??18	??66	??3	??2	??61	??cggga	??151?gcttctccgggaaggctga?169
??19	??48	??4	??3	??41	??cggga	??911?cctgcaccgggatctcaag?929
							??20	??15	??4		??11	??cggga	??942?tttatcacgggacctactg?960
??21	??72	??3	??1	??68	??cgggc	??102?ggcaccgcggggcagcccc?120
							??22	??25	??4		??19	??cgggc	??786?atgacctcgggcacgctca?804
??23	??26	??4	??5	??17	??cgggg	??866?aatcctgcggggacttcat?884
							??24	??9	??4		??5	??cgggt	??833?gaagccgcgggtccttcag?851
??25	??8	??3		??5	??cgggt	??1547?acgtgaacgggttgctgcc?1565
							??26	??52	??3	??1	??48	??cggtc	??1654?tggcccccggtccccccag?1672
??27	??7	??3		??4	??cggtg	??570?ttcaagacggtgtatcgag?588
							??28	??33	??4		??29	??cggtg	??735?tggaagtcggtgctgaggg?753
??29	??23	??3		??20	??cggtg	??1318?aggagcgcggtgtgcacgt?1336
??30	??292	??3	??10	??279	??gagga	??481??aagaaaaggaggacatgga?499
							??31	??153	??3	??15	??135	??attct	??2183?cgagttcattctgccttcg??2201

5.8 the sensitivity of SDSO molecule and specificity

Though about the specificity and the existing many reports of sensitivity valuation methods of an anti-meaning oligonucleotide, along with the foundation in human genome dna sequence data storehouse and the development of biocomputer information technology, some new concept requirements are illustrated.In order to evaluate sensitivity or the specificity of a SDSO molecule to the human body gene storehouse, the Matthews relation conefficient, a measure that is generally used for the biological computation machine information, it can be used to evaluate predicting the outcome and quantitative analysis and the SDSO molecule of prediction and human body gene library searching result's consistence of an effective SDSO molecule.The sensitivity of a mentioned SDSO molecule means that but it and specific gene family member share the energy rate of all or part of sequence among the present invention, its specificity then refer to it with other gene family member between the probability of shared all or part of sequence.Other relevant term and definition is as follows:

True positives (TP) is the positive findings that a SDSO molecule homology is measured, and it shows that the member of a specific gene family has the similar probability size of all or part of sequence to other.

True negative (TN) is the negative findings that a SDSO molecule homology is measured, and it shows that the member of other gene family does not have and all or part of similar possibility of its sequence.

False positive (FP) is the positive findings that a SDSO molecule homology is measured, and it shows that the member of other gene family has and all or part of similar possibility of its sequence.

False negative (FN) is the negative findings that a SDSO molecule homology is measured, and it shows that the member of a specific gene family does not have and all or part of similar possibility of its sequence.

In the context of the present invention, the sensitivity of a specific SDSO molecule and specificity are relevant to the length of a sequence, enzyme is cut the variation of sequence pattern in the attribute of a conservative region and the corresponding RNA molecule, as everyone knows, when the length of an oligonucleotide sequence shortens, the possibility of other homologous sequence similarity will rise in this sequence and the human genome database, sequence of forming by 20 Nucleotide for example, when other homologous sequence in the human genome database and Nucleotide paired number wherein when 20 drop to 10, it will search out increasing homologous sequence from the human genome database.In other words, the false positive (FP) that this oligonucleotide molecule searches out homologous sequence will increase, and its specificity will reduce.When one section high conserved region territory common by a specific gene family member, and when also being shared by other gene family member, a SDSO molecule identical with certain section sequence in this section high conserved region territory just not only can with the member's of this specific gene family transcripton hybridization, and can act on mutually with the coded mRNA molecule of other gene family, so this SDSO molecule just has higher sensitivity and lower specificity.Hit aspect the heart sequence pattern difference at enzyme, as long as a SDSO molecule contains the sequence that arbitrary enzyme hits the heart, when being CGG AU (T), CGGGA or their derived sequence, it just may obtain higher specificity, otherwise, as to adopt other sequence be the enzyme heart that hits, in most of the cases, that SDSO molecule will obtain may be higher sensitivity and lower specificity.In a word, if want a SDSO molecule to obtain the highest specificity, the present invention recommends optimal scheme to be but is not limited to: the member of SDSO molecule and its special genes family has 100% sequence similarity, high conserved region section in the homologous gene sequence is only enjoyed by specific gene family and enzyme hits, and the heart must be CGGAU, CGGGA or their derived sequence.The sensitivity and the specificity of a SDSO molecule of balance are if desired used method among the present invention to remove to regulate these correlation parameters and just can be realized.The active benefit of its cognate rna molecule of the inhibition of a SDSO molecule is the major issue that design gene therapy scheme must think better of, and it is the sensitivity and the specificity of substantial connection to a SDSO molecule also.Yet, how to evaluate the benefit of a SDSO molecule, in many patents of asking in the past and all seldom mention on the research document, mainly be owing to exist some technical difficulties.Along with constantly perfect, the evaluation of most of gene structures of the finishing of human body gene storehouse plan, human genome sequence database and the application of biocomputer information technology, these technical difficulties now progressively are resolved.Self-evident, after the oligonucleotide of a small segment is introduced in the cell, with the many RNA not of the same race of its homologous will be competitively and its hybridization.If these homologous RNA molecule is many more, so a certain amount of SDSO molecule also will be low more to the deactivation effect of specific mRNA molecule.Its two, the benefit of SDSO molecule also is relevant to the content in a cell of the mRNA molecule that will suppress with it.If content is high more, required SDSO molecule is also many more, just can reach the effect of complete inactivation.Its three, the benefit of SDSO molecule and the enzyme heart that hits is relevant, if SDSO molecule contains enzyme that a plurality of strong restriction enzyme sites the form heart that hits, so, the ability to express of the inhibition corresponding mRNA molecule of this SDSO molecule is also just strong more, otherwise then.Last factor is the pairing degree of relevant target RNA and SDSO molecule.Available data shows that fully complementary when a certain section of SDSO molecule and its target RNA, its bonded Rnase III nuclease just can be brought into play the cutting effect so, otherwise Rnase III nuclease just can not cut off corresponding mRNA molecule.The result is that the SDSO molecule combines the activity that the physics mode that is caused disturbs the corresponding mRNA developed by molecule with a kind of with the mRNA molecular moiety.Therefore, improve the specificity and the sensitivity of a SDSO molecule, the method that the present invention introduced is one and reaches the easy of this purpose and reliable approach.Simultaneously, these methods also syntheticly provide new selection for what accurately adjust a particular proteins.

5.9 synthetic, purifying and identify selected siRNA molecule

The method of a synthetic double-stranded oligonucleotide molecule is existing many introductions (Needham-VanDevanter et al.1984 on document, Nucleic Acids Res., 12:6159-6168, Beaucage and Caruthers, 1981, Tetrahedron Letts, 22:1859-1862).Oligonucleotide molecule needs through further purifying after synthetic, method commonly used be anionite-exchange resin or polyacrylamide electrophoresis (Pearson andRegnier, 1983, J.Chrom.255:137-149.).Oligonucleotide molecule behind the purifying can be identified (Maxam and Gilbert with chemical degradation method and nucleic acid sequence analysis instrument, 1980, in Grossman and Moldave (eds.) Academic Press, New York, Methods inEnzymology 65:499-560).Here just enumerate the synthetic of a kind of double-stranded oligonucleotide molecule and be illustrated relevant process with the method for purifying, nucleic acid synthetics commonly used is a kind of RNA/DNA synthesizer of automatization.The present invention includes but be not limited to and synthesize a SDSO molecule with following method.5.9.1 the G-base post (iPr-Pac-G-RNA 500) of synthetic (1) 1 mmole of RNA and contain 2 '-O-TBDMS protective material (t-Butyl-dimethylsilyl)

The oligomerization ribonucleotide (Bz-A-CE Phosphoramidite, U-CE Phosphoramidite, dmf-G-CE Phosphoramidite,

Ac-C-CE Phosphoramidite) and the synthetic activator (0.25 M 5-Ethylthio-1H-Tetrazole in acetonitrile) of RNA

Be used for the synthetic of RNA.(2) synthesize the just meaning chain (+) and the antisense strand (-) of double-stranded oligonucleotide with DNA/RNA synthesizer (model is 392),

As

(+) RNA:5 '-ACUUAGUCGGAUCAAGGUATT-3 ' or DNA

(-) RNA:5 '-UACCUUGAUCCGACUAAGUTT-3 ' or DNA (3) set synthesis cycle on synthesizer be the RNA of 1 mmole, and adjusting coupling time simultaneously is 10-15 minute.(4) the synthetic of whole oligomer approximately needs about 4 hours.5.9.2 product is separated with synthetic post, and removes base and phosphate blocking group (1) is opened synthetic post, pour support into one in vitro, do not need aseptic technique this moment.(2) in this test tube, add ethanol/amine (volume ratio is 1: 3) of 1 milliliter,, place 55 ℃ incubator, hatch 28 then with its deadend

Hour.(3) this test tube is placed on the ice cube cool off, test tube is carefully opened in centrifugation then.From then on begin, all operations needs to advance under aseptic condition

OK.Remove supernatant,, collect all elutriants simultaneously, will collect then with 2 distilled water drip washing solid upholders of 1 milliliter

Elutriant is evaporated to dried.In having the oligomerization ribonucleotide of blocking group, add 0 5.9.3 remove 2 '-O-silyl blocking group (TBDMS) (1).1 mole of HTF's (tetrabutylammonium floride) of 4 milliliters is molten

Liquid rocks test tube lightly, is placed on room temperature then 6 hours.(2) in pipe, add 1 mole of TEAA solution (aqueous triethylammonium acetate) of 0.4ml again, add the two of 1ml then again

Steam water.5.9.4 the sanitas in the desalting column is poured out in RNA oligomer desalination (1), clean pillar with 15 milliliters distilled waters, RNA solution is added on the post, with 1 milliliter pair

Steam the water wash pillar, collect elutriant, note should not containing in these elutriants any RNA molecule.(2) further from post, the RNA molecule is eluted, collect this elutriant of 4 milliliters, wherein contain desired with 4 milliliters of distilled waters

The RNA molecule is washed once with distilled water more again, can obtain remaining on a small quantity by the RNA molecule of salt pollution.(3) these RNA molecules of lyophilize.5.9.5 come purifying RNA molecule (1) preparation a kind of urea polyacrylamide gel (7.3 moles urea, 20% polyacrylamide) with urea and polyacrylamide electrophoresis.The blob of viscose size is 16 * 30cm.

● urea 70.4 grams

● 16 milliliters in 10 times TBE mother liquor

● 38: 2 mother liquor 80ml

●10％APS?1.6ml

●TEMED?60ml

● total amount=160ml (2) preparation RNA sample liquid

● the RNA sample is dissolved in the sample buffer of 600 microlitres (distilled water of 400ul+100ul RNA dyestuff damping fluid+100ul's

100% glycerine)

● be heated to 100 degree, kept 2 minutes, then immediately in cooled on ice.(3) sample liquid is added to the top of glue, and under 500V voltage, ran glue 2 hours.(4) the RNA band is cut down from glue.

● glue is placed on the TLC plate, under the UV lamp, checks the position of RNA band.

● the adhesive tape that will contain the RNA molecule downcuts, and adhesive tape is cut into little sheet piece.(5) from glue, extract the RNA molecule.

● little blocky RNA glue is immersed in the TBE solution of 20ml, and places on the automatic rocker, spend night in 4.

● collect solution and film is placed the TBE solution of 20ml once more, spend night in 4.

● merge these liquid.(6) concentrate the liquid that contains RNA.

● add the sodium-acetate (ultimate density is 0.3 mole) of 3 moles of 9ml and the isopropanol (ultimate density is 50%) of 45ml

● solution is placed-20 the degree spend the night or-80 the degree 30 minutes,

● RNA is placed whizzer, under 4 degree centrifugal 30 minutes with 15000rpm,

● outwell supernatant liquor, wash the RNA throw out with 80% cold ethanol, and then under 4 degree with 10000rpm centrifugal 30 minutes.

● throw out is dry in vacuum vessel.

● RNA is dissolved in the distilled water of 0.5ml.(7) with the purified RNA desalination, method such as IV go on foot, lyophilize and be stored in-20 degree down.Ultimate capacity is that every 1mmol post is 1mg.5.9.6 dsRNA's is synthetic Just anticipate chain and the antisense strand of the 50uM volumetric molar concentration of 25ul placed the HEPES-KOH damping fluid of 20 mmoles of 150ul, wherein contain the Potassium ethanoate of 50mM, magnesium acetate and the pH 7.2 of 3mM.

Reaction mixture is heated to 95 ℃ and continues 3 minutes, then it is cooled to room temperature gradually, keeps 16 to 20 hours.As not being all, also will change double-stranded oligomer into for most strand oligomer. Be stored in-20 degree down, can freezing repeatedly dissolving 5 times.This is a specific examples among the present invention, select and the double-stranded oligonucleotide of synthetic have with RNA molecule accordingly in the similarity of certain specific fragment.In the tissue of a tumour cell or a pathology, the function of corresponding RNA molecule may be by the complete or most of inhibition of the SDSO molecule among the present invention, by suppressing the expression of Disease-causing gene, the infection or the heredity illness of the growth of cancer cells, virus can be controlled effectively.

5.10 select suitable carriers

Because oligonucleotide exposed in PBS solution is difficult to enter cell, can effectively transmit genomic medicine be treat a successful important step.The transfer system of oligonucleotide is divided into two big classes, promptly biological and pipeline machinery.Biological transmission can be divided into two kinds, and a kind of is viral, and a kind of is non-viral, and mechanical transfer can be divided into manual injection method and particle gun injection.The present invention mainly adopts but is not limited to a kind of mixture of being made up of liposome and polymer.

The non-virus type mixture of ideal comprises lipid acid and lipid, cationic-liposome, and positively charged ion is pounced on quinoline, melts gene polypeptide and artificial virosome.These mixtures can form a kind of mixture with oligonucleotide, and its principle is to be combined into one by the electrostatic interaction with the phosphate group of secondary electric charge and positively charged carrier granule on the oligonucleotide.In addition, these carriers also can provide the protection to oligonucleotide, in case the degraded of nuclease (De Smedt et al., 2000, Pharmaceutical Research 17:113-126).

Some lipid acid, fatty acid ester, huge legendary turtle compound and surface-active material, perhaps all can be used as carrier, assist oligonucleotide to enter cell, lipid acid that often is used and various fat, including, but not limited to 1-dodecylazacycloheptan-2-one, arachidonic acid, caprylic acid (caprylic acid), goat resin acid (capric acid), glycerine two lauric acid fat (dilaurin), triglyceride (diglyceride), dicaprate, eicosanoic acid (eicosanoic acid), glyceryl 1-monocaprate, lauric acid, linoleic acid, linolenic acid, monoglyceride (monoglyceride) glycerine monooleate (monoolein) Semen Myristicae acid (myrtstic acid), oleic acid (oleic acid), palm fibre acid (palmiticacid), stearic acid (stearic acid) and tricaprate. etc.

Cationic-liposome is one of optimum carrier that is used for the human body gene treatment, because their right and wrong are infective, does not almost have immunogenicity and toxicity.Aspect morphology, cationic-liposome can be divided into three main types, that is: small-sized monolayer vesicle, large-scale monolayer vesicle and multiwalled vesicle.Commonly used lipid and liposome comprise neutral fat, promptly DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DiPPE (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine) and DOPE.These lipids and liposome can be assisted the breaking of endosome behind the born of the same parents.Cation lipid such as DOTAP (dioleoyltetramethylaminopropyl), DOTMA (the cytofectinN-[1-(2,3-dioleoyl) phosphatidyl]-N, N, N trimethyl ammonium chloride) and TMAG (N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride).The ideal lipid carrier is a kind of cation lipid of equal proportion and the mixture of neutral lipid normally.

Another kind of cation lipid is that positively charged ion is pounced on quinoline.Tetra (4-methylpyridyl) porphyrin (TMP) and tetraanilinium porphyrin (TAP) can more effectively assist oligonucleotide to enter cell than exposed oligonucleotide itself.In addition, positively charged ion is pounced on quinoline and can not only be helped oligonucleotide to enter cell, also it can be delivered in the nucleus, so that oligonucleotide can act on mutually with corresponding mRNA molecule and RNase III nuclease there.The synthetical virosome is another kind of transmission media, the sort of natural ability that it can utilize a virus to enter cell is helped oligonucleotide and is entered cell, the influenza virus after birth of reorganization is a kind of famous virosome, it can enter cell by receptor mediated endocytosis, and is integrated with the interior body film of biting.Recently research has been fused cation lipid and virosome for one and has been used as a kind of novel launch vehicle.

The polycation body is another kind of useful carrier, can be used to strengthen cationic-liposome-mediated endocytosis, available positively charged ion polymer comprises: poly L type Methionin (poly-L-lysine), protamine sulfate (protamine sulfate), recombinant human histone h1 (recombinanthumanHl histone protein), arginine (spermidine) and PEI (polyethylenimine).Polyaziridine (PEI) has been proved to be a kind of effective non-viral carrier, gene delivery can be advanced dissimilar cells, with promote oligonucleotide to enter the karyon of cells of mamma animals, other feature of PEI comprises: can combine with nucleic acid, with nucleic acid compression, has the activity of biting body in the surge capability and intrinsic dissolving efficiently.In addition, it can also protect the degraded of nucleic acid to nuclease-resistant.Behind the PEI and oligonucleotide mixture of 22KDa size of external application or injection wire, can see that in cell the marker gene of its transmission can be expressed efficiently.Discover that further the PEI of the 25KDa of branch-like is except having the similar effect in the in-vitro transfection experiment to the PEI of wire, it also is proved to be the PEI that its intravital gene transmission effect is better than wire far away.When together the time, can more effectively gene being imported various tumour cell with part bonded PEI in conjunction with the characteristics of all these PEI and receptor-mediated gene pass through mechanism.

In addition.Other method that also need mention comprises binding peptide and particle gun.Binding peptide can form a polypeptide circle around oligonucleotide, help oligonucleotide and taken in by cell.Many binding peptides that contain poly-lysine can cause the stability of going of film.In general, these binding peptides have lower cytotoxicity than lipid, and have similar transmission effect.Except old-fashioned manual injection method, a kind of novel particle gun has also been declared to be born, its principle is that the highly compressed helium can produce a kind of ultransonic speed, thereby by high acceleration the dna molecular of gold grain parcel is squeezed in the cell alive, this method can be used for transmitting oligonucleotide effectively and directly and enter cell.

5.1 1 selects special cellular targets to lead molecule

An important topic of living of related gene medicine is how to have the genomic medicine of result of treatment to be delivered in target cell or the target tissue a kind of, and does not damage normal cell and tissue.Tissue target is led can be by directly being injected into corresponding tissue with genomic medicine, or finish the existing in the literature a large amount of reports of the method for many this respects by means of a kind of navigation molecule such as antibody, part or virion.The present invention lays particular stress on special target guiding systems including, but not limited to following several aspects: 5.11.1 target adpedance body (1) is efficient, and affine monoclonal antibody--AF-20 can discern the glycoprotein of the cell surface of a kind of 180KDa that can be bitten in the speed.This anti-

The carrier of the bootable SDSO of the containing molecule of body acts on mutually with liver cancer cell specifically, and corresponding genomic medicine is imported liver cancer cell.(2) the CD3 antibody of high specific.It can link to each other with poly-lysine, the latter again can with the SDSO interaction of molecules, like this this antibody can with

CD3 acceptor interaction on the lymphocyte, thus specifically the SDSO molecule is imported the leukemia cell.(3) with a kind of single-chain fragment and liposome coupling of antibody of high-molecular weight melanocytoma related antigen, be used for relevant SDSO branch

Son imports in the cell of melanocytoma of those transfers specifically.5.11.2 target lead polysaccharide or a kind of glycoprotein of protein ligands (4) can with a kind of special acceptor interaction on the CD4 male T cell, this glycoprotein can be used to SDSO molecule spy

The strange land imports those T cells, is used for the treatment of acquired immune deficiency syndrome (AIDS) or T chronic myeloid leukemia.(5) cholesterol and arginic combination can import those with the SDSO molecule by a kind of glycoprotein of discerning the liver cancer cell surface specifically

In the AF-20 male liver cancer cell.(6) adenovirus or retrovirus or Coxsackie virus all can be sent the SDSO molecule into the thin of this acceptor by relevant acceptor such as CAR acceptor

In the born of the same parents, in lung carcinoma cell.(7) be that one can combine with ldl receptor specifically in conjunction with poly-lysine, low-density lipoprotein and SDSO molecule, the SDSO molecule is imported

The adipocyte of obesity.5.11.3 other target is led media

The target of other kind is led media also by different biotech firm's research and development, and Pentratin is exactly wherein a kind of, and it is a peptide species, and 16 amino are arranged

Acid is formed, and it can combine with DNA, and its transposition is entered in the endochylema and karyon of viable cell.

5.12 other composition

Perhaps also have other multiple auxiliary composition in the genomic medicine of the present invention except active double-stranded oligonucleotide, as traditional medicine, these compositions are perhaps including, but not limited to following all kinds of:

Antiphlogistic active ingredient such as non-steroid antiphlogiston and cortisone etc., antioxidant, local anesthetic, lubricant, sanitas, stablizer, intensifier, wetting agent, dyestuff, spices, glue.

Yet so auxiliary composition is fashionable when adding, and is prerequisite with the biological activity that does not influence the SDSO molecule, otherwise can not uses.

5.13 the assembling of genomic medicine

The assembling of a genomic medicine relates to the many factors of every aspect; comprise SDSO molecule, ratio, their concentration, pH value, ionic strength and the stable material of its increase of damping fluid with lipid; the item that mainly notes is to avoid or reduce the precipitation of mixture, and protection SDSO molecule is in order to avoid be degraded and improve the benefit of conversion.It is considered herein that following all conditions are the assurances that constitutes the optimal drug assembling.

(1) contain the PBS damping fluid of 5% glucose, it is used to dilute the concentration of SDSO molecule

(2) pH value of PBS damping fluid is 5.5-6

(3) low ionic strength

(4) 1: 6 SDSO and lipids

(5) concentration of SDSO is no more than 4ug/ul (not comprising intravenously, intra-arterial and encephalic administration)

(6) carrier size

In addition, ideal is 30-60nm (nanometer) by the size of the required conversion mixture of administrations such as mucocutaneous epithelium, the size that is used for the conversion mixture of inhalation route is 50-200nm, and the size that is used for the conversion mixture of intravenous administration is preferably 200-600nm.

Active ingredient is a kind of or a different set of special SDSO molecule, they can suppress the target RNA molecule of being correlated with effectively, degree according to one group of gene overexpression in the disease cell, regulate the mode of kind, dosage and the combination of SDSO molecule, so that obtain maximum result of treatment and minimum malicious side effect.

Although genomic medicine can be mixed with many not isotypes, the preparation of only describing the watery suspension of genomic medicine here is as an example.(1) the SDSO molecule of 10ug is dissolved in (no ion distilled water preparation) in the Glucose Liquid of 10ul.(2) the dendritic PIE (PBS with 0.01M contains 100mM sodium-chlor, pH value 5.5) of the branch of the 25kDa of the 20mM of 10ul.(3) solution (1) is splashed into solution (2), mix being placed on room temperature following 10 minutes gently.(4) with the cationic-liposome of 30ul, the PBS that is dissolved in the 0.01M of 50ul as FuGene6 contains 150mM sodium-chlor, pH value 7.4.(5) solution (3) is slowly splashed into solution (4) after, jiggle and make their thorough mixing, put then in the room temperature 40 minutes.(6) final mixing solutions can be used for transfection or other purposes of cell.

The shape of the suspension that genomic medicine is watery as shown in Figure 7.

5.14 genomic medicine

Because medicine is defined as a kind of chemical substance that can adjust the particular procedure of organism, so genomic medicine can be regarded as the oligonucleotide of the function of a kind of energy affect live cells in a kind of chemical substance.5.14.1 the characterizing gene medicine of genomic medicine has following feature: (1) does not change the structure sequence of gene and entrained genetic information.(2) can with a certain specific region complementation in genomic dna, mRNA or other RNA molecule.(3) can disturb specifically, the function or the expression of inhibition or complete inactivation corresponding gene.5.14.2 the structure of genomic medicine active ingredient

One of specific examples of the present invention is the structure of relevant SDSO molecule.An ideal SDSO molecule is the long double-stranded oligonucleotide of 19 Nucleotide, it should be before VITAMIN B4 or first base to be the base beginning of VITAMIN B4, a phosphoric acid residue is arranged on this base, and be ending, a free hydroxyl is arranged on thymus pyrimidine with the thymus pyrimidine.The enzyme heart that hits is arranged in molecule, and there are 3 strong restriction enzyme sites, i.e. G in this center ^*G, G ^*A or A ^*U.3 ' end of every chain of this molecule is gone up by a projection of being made up of 2 free thymus pyrimidines.Therefore, every chain of an ideal SDSO molecule should be that 21 Nucleotide are long, and its total length is 23 Nucleotide.The SDSO molecule comprises three types the long double-stranded oligonucleotide of 21-23 Nucleotide, i.e. 21-23nt siRNA (table 5), 21-23nt sRNA-cDNA (table 6) and 21-23ntsiDNA (table 7).The hit sequence pattern of the heart of enzyme comprises CGGAU, and CGGGA and their derived sequence, these derived sequences be including, but not limited to CGGAA, CGGAC, CGGAG, CGGGC, CGGGU (T) and CGGGA.

Short and small RNA interfering s (siRNAs) is the long double-stranded RNA of a 21-23 Nucleotide.21-23nt sRNA-cDNA is the double-stranded oligonucleotide of a heterozygosis, and wherein sRNA is an antisense strand for the chain cDNA that just anticipates.The long double-stranded DNA of 21-23 Nucleotide of siDNA.The main level structure of table 5. 21-23nt siRNA.

			??1	??2	??3	??4	??5	??6	??7	??8	??9	??10	??11	??12	??13	??14	??15	??16	??17	??18	??19	??20	??21	??3’
																									??Sense	??5’	??P	??A	??U	??A	??C	??A	??U	??C	??C	??G	??G	??A	??U	??U	??A	??A	??G	??C	??U	??U	??T	??T	??OH
			??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|
																									??OH	??T	??T	??U	??A	??U	??G	??U	??A	??G	??G	??C	??C	??U	??A	??A	??U	??U	??C	??G	??A	??A	??p	??5’
	??21	??20	??19	??18	??17	??16	??15	??14	??13	??12	??11	??10	??9	??8	??7	??6	??5	??4	??3	??2	??1

The main level structure of table 6. 21-23nt sRNA-cDNA.

??1

??2

??3

??4

??5

??6

7

??8

??9

??10

??11

??12

??13

??14

??15

??16

??17

??18

??19

??20

??21

??3’

Sense	?5’	?P	?A	?U	?A	?C	?A	?U	?C	?C	?G	?G	?A	?U	?U	?A	?A	?G	?C	?U	?U	?T	?T	?OH
																												?|	?|	?|	?|	?|	?|	?|	?|	?|	?|	?|	?|	?|	?|	?|	?|	?|	?|	?|
?3’OH	?T	?T	?T	?A	?T	?G	?T	?A	?G	?G	?C	?C	?T	?A	?A	?T	?T	?C	?G	?A	A	P	?5’
																										?21	?20	?19	?18	?17	?16	?15	?14	?13	?12	?11	?10	?9	?8	?7	?6	?5	?4	?3	?2	?1

The main level structure of table 7. 21-23nt siDNA.

			??1	??2	??3	??4	??5	??6	??7	??8	??9	??10	??11	??12	??13	??14	??15	??16	??17	??18	??19	??20	??21	??3’
																									??Sense	??5’	??P	??A	??T	??A	??C	??A	??T	??C	??C	??G	??G	??A	??T	??T	??A	??A	??G	??C	??G	??T	??T	??T	??OH
			??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|	??|
																									??3’OH	??T	??T	??T	??A	??T	??G	??T	??A	??G	??G	??C	??C	??T	??A	??A	??T	??T	??C	??G	??C	??A	??P	??5’
	??21	??20	??19	??18	??17	??16	??15	??14	??13	??12	??11	??10	??9	??8	??7	??6	??5	??4	??3	??2	??1

5.14.3 the double-stranded oligonucleotide that the prescription of genomic medicine is dissimilar

Another example of the present invention is to adopt dissimilar double-stranded oligonucleotides as a kind of SDSO molecule, and these dissimilar SDSO molecules are formed a kind of mixture with identical or different dosage.Perhaps, the different compatibilities of these three kinds dissimilar SDSO molecules can be used to design the treatment plan that produces different length curative effects, as the compatibility of traditional medicine.For example, the antisense strand of every type SDSO molecule can produce effect immediately.Since they all can with corresponding mRNA molecular hybridization, thereby block its biological function.Thereby perhaps the chain of just anticipating among type siRNA and the sRNA-cDNA can produce the usefulness that more antisense strand enlarges the SDSO molecule as template.Perhaps they may combine with methyltransgerase, to the modification that methylates of homologous dna fragmentation, cause the active decline of corresponding gene or close fully.On the contrary, siDNA does not but have these functions.But the antisense strand DNA among siDNA and the sRNA-cDNA but can activate the activity of another nuclease.Just can make enzyme cut benefit as these three kinds different double-stranded oligonucleotides of hybrid combination like this increases greatly, because they can activate the activity of two kinds of different nucleases.The double-stranded oligonucleotide that table 8 is dissimilar, involved enzyme and their function

Name	siRNA	sRNA-cDNA	siDNA
				Short curative effect	Antisense?RNA	cDNA	Antisense?DNA
Long curative effect	Sense?RNA	Sense?RNA	None
				The type of activating enzyme	RNase?III，Helixase，	RNase?H.Helixase？	RNase?H，Helixase？
Duplicate	RNA?polymerase?II？	RNA?polymerase?II？
				Suppress the DNA activity	Methyltransferase	?Methyltransferase？

The compatibility of the double-stranded oligonucleotide that one or more are dissimilar

The compatibility of the double-stranded oligonucleotide that one or more are dissimilar is another example of relevant genomic medicine characteristics of the present invention.The genomic medicine active ingredient comprises the double-stranded oligonucleotide that one or more are dissimilar, especially the different SDSO molecule in the genomic medicine can be respectively interacts with different said target mrna, suppress a mRNA molecule as first kind of SDSO molecular energy, second, third ... n SDSO molecular energy suppresses second, third ... n different mRNA molecule.This multiple special active ingredient is combined, tackle an animal particularly among the human inner cell one group of corresponding prescription of crossing the mRNA molecule of expressing be another important example of the present invention.Because this method is specially adapted to the prevention and the treatment of tumour, two or more bonded SDSO molecule can be simultaneously or transfered cell one after the other, to bring into play their functions separately.At example of the present invention with in using, also the details of this method will be continued to illustrate.The consumption of double-stranded oligonucleotide

Making up a prescription for each patient's diagnosis and treatment, is a new trend of 21st century the world of medicine.Suffer from different patients with a kind of disease, perhaps their expression of gene pattern is also different, detected result according to gene chip, can obtain pattern and abundance that those Disease-causing genes are expressed, calculate the various dose of each corresponding SDSO molecule, then they being assembled into corresponding genomic medicine, is symptomatic treatment patient's optimal path.Therefore, the SDSO molecule a kind of, that two or more are dissimilar is mixed with the special genes medicine with identical or different dosage, the disease that this genomic medicine could really fundamentally be cured different patients.The form of genomic medicine

Genomic medicine can be mixed with different forms, as: aqua, pulvis, finish, ointment, tablet, pill, liquid medicine, jelly, propellant, subcutaneous embedded block.Perhaps, traditional drug effect carrier, hydrate, powder or oily matter or other shape are essential or wish.The composition of oral medicine and preparation comprise meal, granular substance, finely particulate, the emboliform mixture of sodium, watery or non-watery suspension, capsule, tablet or small pill.In addition, intensifier, spices, thinner, emulsifying agent, dispersion agent or wedding agent perhaps be need or require.Route of administration

Effective ingredient of the present invention or compatibility comprise the double-stranded oligonucleotide that 21-23 Nucleotide is long, i.e. 21-23nt siRNA (table 5), 21-23nt sRNA-cDNA (table 6) and 21-23nt siDNA (table 7).Except double-stranded oligonucleotide, other relevant composition comprises the carrier of accepting on the drug effect, or other is commonly used to strengthen and promote the auxiliary composition of drug absorption.Route of administration is including, but not limited to following all kinds of: (1) external application: comprise eye, ear, nose, rectum, vagina etc.(2) suck: comprise in the tracheae, nasal cavity, cross skin or outer pulvis or the propellant of skin.(3) oral (4) injection or drop: comprise intravenously, intra-arterial, subcutaneous, intraperitoneal or intramuscular etc.(5) encephalic administration: sheath is interior, Intraventricular.The usage of genomic medicine

About the existing a large amount of report of the research of treatment compatibility and relevant administration number of times, the size of dosage depends on the severity of disease, conformability and patient's state of health.Therapeutic process can be cured or obviously be alleviated from several days to several weeks, several months, several years or up to disease, and ideal dosage yardstick can decide in the situation of intravital distribution of people and accumulation by the tolerance medicine.Brainstrust is easy to determine ideal dosage usually.

Perhaps, ideal dosage can change along with the variation of the relative efficiency of concrete different double-stranded oligonucleotide, evaluates according to EC50S usually.Because the EC50S zootype inside and outside why is a kind of reliable index in studying, in general, the dosage range of SDSO molecule is 5ng～200mg/kg body weight, this dosage can be once a day for several times or weekly or for several times, every month once or for several times, annual or for several times.Brainstrust usually can be according to measuring institute's medicine of giving in people intravital residence time, medicine at body fluid or the concentration in organizing decide the number of times and the frequency of administration.After the treatment along with success, patient is kept treatment also usually to be needed with the recurrence that wards off disease.At this moment, the double-stranded oligonucleotide of a maintenance dose is necessary, and usually dosage range is 5ng～200mg/kg body weight, this dosage can be once a day for several times or weekly or for several times, every month once or for several times, once a year or give patient for several times.The optimal dose scope is 5mg～50mg/kg body weight/day.The metabolic mechanism of genomic medicine

Suppress those expression of gene pair cells of not wanting and can manage it that to execute its normal function be very important.Make the principle of inactivation of gene comprise a series of process of supervising after transcribing and transcribing.Many researchs point out that the flat mechanism of putting out gene activity of endogenic or ectogenic double-stranded oligonucleotide relates to four processes of supervising after transcribing at least.First is mediated by the protein kinase (PKR) that dsRNA relies on the nonspecific main path of replying of double-stranded oligonucleotide.This endonuclease capable phosphorylation, translation factor eIF2a, the eIF2a after the phosphorylation just loses activity, thereby can not cause proteinic synthetic, cause the non-specific inhibition of all proteins synthetic in the cell, so that open pathologic or non-pathologic necrocytosis passage.The mode activated protein kinase PKR that DsRNA relies on a kind of length.Have now found that when dsRNA length surpassed 80 Nucleotide, it just can fully activate PKR, and, just can not cause that PKR's transfers activated form to from the inhibition type when dsRNA length during less than 30 Nucleotide.Therefore, for fear of activating PKR, opens a nonspecific necrocytosis passage and the malicious side effect that causes, the present invention emphasizes the double-stranded oligonucleotide molecule that only curative 19-25 Nucleotide of importing is grown in cell.

Second mechanism is relevant to one by an approach that depends on the nuclease medium of 2-5A.This dsRNA response pathway relates to the synthetic of dsRNA inductive 2-5A (polyadenylic acid).2-5A can activate a nonspecific nuclease, and promptly RNase L can non-ly cut off all mRNA molecules after this enzyme activation specifically, thereby suppresses any proteinic synthetic in the cell, finally causes the death of cell.Yet, the long double-stranded oligonucleotide molecule of 19-25 Nucleotide but can inductive 2-5A (polyadenylic acid) synthetic, thereby can not activate that nonspecific nuclease RNase L (Maitra RK: et al., 1995, J Biol Chem 270:15071; Cirino NM, et al., 1997, ProcNatl Acad Sci USA 94:1937; Szczylik C, et al., 1991., Science 253:562; Lesiak K, et al. .1993, Bioconjugate Chem 4:467).

The 3rd mechanism is disturbed relevant with RNAi.Article one, long dsRNA can be cut into a series of short and small double-stranded oligonucleotide molecules by a nuclease that is called RNaseIII, and it is long that they are generally 19-25 Nucleotide.These short and small double-stranded oligonucleotide molecules (siRNA) can act on mutually with they homologous mRNA molecules, thereby guide the nuclease of the RNaseIII that combines with them to remove to cut off specifically those mRNA molecules.Have now found that; the organism inner cell can adopt this mechanism to come those interior unusual RNA molecules of as killed cells; to some proteinic needs, the protection cell is removed external genetic material to regulating cell in the different growth perioies, the infringement of pathogenic agent such as antagonism virus.As seen the RNAi interference is the natural defense mechanism of an intracellular cover, just as the human immune system.Although their protection level is different, purpose is roughly the same arranged.

Article four, supervising approach relates to dna molecular.Behind dsDNA or sRNA-cDNA transfered cell, their intermediary cDNA chains can cause one typically by the nuclease-mediated pathways metabolism of RNase H.When the cDNA chain with after its said target mrna combines, RNase H nuclease just can cut off that mRNA molecular chain, thereby stops its biological function.What should arouse attention here is, the anti-DNA of meaning exists with duplex molecule a kind of heterozygosis or that isozygoty, and perhaps they have the performance of stronger opposing nuclease, otherwise they need modifiedly could prolong its transformation period in vivo.

Recently, the evidence of several lines shows that 19-25 the long siRNA of Nucleotide obviously is better than the inhibition of the mRNA expression of strand cDNA mediation to the interference effect of mRNA.Usually the latter has only several minutes in the intracellular transformation period.And by sustainable several days of the interference mediated effect of RNAi, and even the longer time.Equally, as if many researchs prove that also siRNA is highly stable, and it does not need further chemically modified like this, has reduced the synthetic cost yet.The most important thing is that siRNA can also suppress target gene expression specifically.Contrasting to antisense strand treatment technology and RNA perturbation technique in several laboratories, found that ssRNA can only partly suppress an expression of gene, and siRNA can cause the inhibition to several times of mRNA expression and even hundred times.Obviously, these observations also provide strong support for the high efficiency and the specificity of genomic medicine of the present invention.Simultaneously, this is also firm, and people remove further to develop RNAi or DNA perturbation technique, with its health of bringing benefit to the mankind and happiness (Aravin AA., etal, 2001, Curr Biol Jul 10; 11 (13): 1017-27).

In addition, the long siRNA of 19-25 Nucleotide is found also relevant with methylating of DNA, and the expression that dna methylation can not only suppressor gene also may increase the possibility that influenced gene is undergone mutation.Though dna methylation plays an important role in normal biological procedures, abnormal dna methylation also can cause the sudden change of cell and the generation of cancer.Especially some evidences have proved that methylating of DNA in the promotion subarea of tumor suppressor gene such as p53 and Rb can cause the decline of these cancer suppressor gene functions or completely lose.In addition, the dna methylation in proteins encoded district also can cause the unusual of amino acid whose sudden change and protein function.All these molecular events can both promote the generation and the development of cancer, and constitute the significant obstacle to the modern treatment cancer.A new imagination is to be used as a kind of medicine for the treatment of cancer with siRNA.Because siRNA can combine with a kind of methyltransgerase.When the specific region on it and its homologous dna fragmentation interacts, it just can be this methyltransgerase this specific zone of leading, thereby make the dna sequence dna in this specific region carry out nonspecific methylating, the function of gene thereby inhibition is methylated.Moreover, also can design a SDSO molecule, this SDSO molecule is the function of the mRNA molecule of the interior coding of anticancer methyltransgerase specifically, thereby removes the inhibition to tumor suppressor gene.Therefore, if certain Disease-causing gene in SDSO molecule and the cell interacts, impel it to be methylated, just might suppress that expression of gene function (Matzke, M.A., et al. for a long time, (2001) Curr.Opin., Genet.Dev.11,221-227; Carthew, R.W. (2001) Curr.Opin.Cell Biol.13,244-248; Elbashir, S.M., Lendeckel, W.﹠amp; Tuschl, T. (2001) Genes Dev.15,188-200).5 compare all advantages with prior art compares the advantage that the present invention has with prior art and comprise: design that (1) is brand-new and development are theoretical: exist a kind of natural RNAi securing system in the cell.Effect composition in this system can be at body

Amplified and strengthened by bionic method outward, then with in its defeated object of bringing back to life.It can be special and deactivation efficiently those with

The Disease-causing gene in source.Sequence pattern CGGAU (T), CGGGA in the effect composition of the present invention or their derived sequence are designs

With the important indicator of the selected molecule of SDSO efficiently as genomic medicine.(2) short new drug development cycle: by technology such as computer and gene chips, the most conservative part in the selected specific mRNA sequence

As the target of genomic medicine, and its homologous sequence is as the active ingredient of genomic medicine.This new research and development strategy can be widely

Reduce the time of the chemistry be used to study a kind of drug molecule and physicals and find out time of its action site on target molecule.(3) lower medicament research and development cost: study a new drug and and its target molecule between interaction usually need the reasearch funds of great number and big

The search time of amount, the free use of Computing method fast and common gene database, it is new to reduce research and development significantly

The cost of the genomic medicine of type.(4) high specificity: the present invention can filter out the target fragment of the tool potential in the specific mRNA sequence, does with its homologous SDSO

Be genomic medicine.They are discerned mutually and act on by the principle of the Watson-Crick base pairing of classics.Moreover, the fragment of selecting

Only common by homologous genes family members' member, and few similarity is only arranged with other gene family members' member, thus make the spy of medicine

The opposite sex is guaranteed fully.(5) extremely low malicious side effect: the main active ingredient in the genomic medicine of the present invention is a kind of natural nucleic acid fragment, and it is present in from low etc.

Animal is in the cell of human body.Because its high specific and high efficiency make its consumption be lower than other medicine significantly.Obviously, its poison

Side effect also is lower than other medicine out and away.(6) advantages of higher stability: SDSO molecule of the present invention is a kind of double-stranded oligonucleotide, can with some protein and other small molecules mutually

Mutual effect has nucleic acid such as cDNA than other strand that much better stability is arranged.It can resist the degraded of nuclease effectively, easily with

Some protein form stabilized complex.Its some base also is easy to modify, and is used to strengthen the performance of nuclease-resistant.(7) multiple use: genomic medicine of the present invention is the mixture of a kind of dissimilar, various dose and multiple SDSO molecule.They can

Adjusted according to patient's the particular case and the severity of disease, and be can be made into the preparation of different types, be used for different by way of,

Will start the New Times of diagnosing a disease and writing a prescription for each patient.(8) high efficiency: can interrupt corresponding mRNA molecule efficiently can several different Disease-causing genes of while deactivation be genomic medicines of the present invention also

Characteristics, the breakthrough on this methodology is particularly useful for treatment for cancer and prevention, virus and the persistent ailment that causes of fungi and that

The heredity illness of a little refractories.(9) high anti-mutation: according to the principle of mathematics theory of probability, but the energy rate that the sudden change of a Nucleotide takes place in a short and small sequence will be big

The earth is less than the probability in one section very long sequence.In addition, also can contain in the genomic medicine of the present invention and suppress drug resistance gene

The SDSO molecule.(10) long lasting effect: because the siRNA molecule may have the ability of self-replacation and make the methylated ability of corresponding DNA fragments, it is given birth to

The thing effect may continue the long period in viable cell.

6 about explanation and accompanying drawing totally 10 width of cloth accompanying drawings and 10 description of drawings.Fig. 1. from an endogenic RNAi interference base of not of the same race bunch because of homologous sequence.Fig. 2. the homologous sequence of the gene of the human vascular endothelial growth factor that a cover is typically found out by BLAST ordering formula.Fig. 3. the homologous sequence that a cover is typically discharged by CLUSTAL W multisequencing ordering formula from the gene of not of the same race bunch insulinoid growth factor-2.The blast search of the growth factor gene sequence of Fig. 4 a. human insulin-like.The similar sequences of the insulinoid somatomedin sequence of Fig. 4 b. different people.Fig. 5. the result of the blast search of two series arrangement.The specificity of the SDSO molecule that the sequence pattern predicted is cut in Fig. 6 appraisal with enzyme.The mode chart of Fig. 7 genomic medicine.Fig. 8 A is the SDSO molecule (redness) that contains of a kind of large-scale monolayer vesicle liposome (blueness) and it, divides dendritic PIE (grey), navigation molecule (purple).Fig. 8 B has described the mutual relationship between small-sized monolayer vesicle liposome and SDSO molecule and other composition.Fig. 8 .Dermogene can suppress propagation and the survival of cell A375 on nude mice of human body melanoma effectively.Fig. 9 .Leukogene is to the propagation of CML cell and the effect of survival inhibition.Figure 10. relatively Leukogene and empty liposome are to the propagation of CML cell and the inhibition of survival.

7 working of an invention examples and optimum implementation example 1--analyze the specificity of the selected SDSO molecule of easy back-and-forth method with BLAST series arrangement formula

Table 9 shows that easy back-and-forth method can obtain to have the sequence that high specific and enzyme are cut benefit reliably.In people c-myc proto-oncogene, depositing and containing five different fragments that enzyme is cut.Yet when being searched in the human body gene storehouse by the enquirement sequence with these five different fragments, they all can find the homologous sequence of high specific, as 2 among the Seq.ID#5,3,4,5 and 6.On the contrary, random choice method but obtains many low specific similar sequences, as 1 among the Seq.ID#5 and 7.The similar sequences (gi|11493193 :) of the different fragments among the table 9 people c-myc proto-oncogene mRNA

Seq. ID#5	?Total ?Hits	?100％ ?Match	?80-95％ ?Match	?＜80％ ?Match	?Pattern	?Start????Sequence???????End?Point ?Point
							?1	?118	?19m??3n	?1n	?94n?1m	?aggaa	?21?caccaacagg?aactatgacc?39
?2	?29	?17m??2n	?1n	?9n	?cggaa	?1296?acagc?tacggaactc?ttgt?1314
							?3	?34	?15m??3n		?16n	?cggaa	?1254?cttgttg?cggaaacgac?ga?1272
?4	?41	?16m??3n		?22n	?cggaa	?939?ct?ccactcggaa?ggactat?957
							?5	?39	?15m??3n		?21n	?cggag	?1107?gcta?aaacggagct?ttttt?1125
?6	?24	?17m??3n		?4n	?cggac	?349?tg?cgacccggacgacgaga?367
							?7	?217	?18m??3n		?196n	?ccgcc	?541?ctgagcgccg?ccgcctcag?559

It is the homologous sequence of puing question to sequence to be searched in the human body gene storehouse that table 10 has been enumerated with the long sequences of 19 Nucleotide different in the mdm2 gene.Wherein 4 sequences of selecting at random can be found out up to 347 similarity sequences, have only 1 sequence of selecting at random to have higher specificity.On the contrary, a sequence with enzyme is cut the design of sequence pattern and selected just can obtain higher specificity (1 among the Seq.ID#6).The result shows that the reliability of Xuan Zeing is less at random, and need trial repeatedly that the more important thing is that they more lack strong restriction enzyme site, this will influence the benefit of genomic medicine.(XM 052466, GI:14762555) for the similar sequences of the different fragments among the mRNA of table 10 people mdm2 sample

Seq. ID#6	?Total ?Hits	?100％ ?Match	?80-95％ ?Match	＜80％ ?Match	?Pattern	?Start????Sequence?????????End ?Point???????????????????Point
							?1	?52	?31m		?21n	?cggaa	?58?ccagcttcggaac?aagaga?76
?2	?135	?35m	?3n	?97n	?aactt	?371?ttgtgctaac?ttatttccc?389
							?3	?302	?34m	?11n	?257n	?gtgca	?301?tttacatgtg?caaagaagc?319
?4	?111	?32m	?1m	?78n	?gtctg	?11??ccaacatgtc?tgtacctac?29
							?5	?39	?31m		?8n	?gacct	?241?caaggtcgac?ctaaaaatg?259
?6	?347	?33m	?17n	?307n	?agaaa	?161?aaagggaaga?aacccaaga?179

Table 11 has been enumerated other example and has been illustrated that enzyme hits heart sequence in one of the prediction importance in the SDSO molecule efficiently.Compare easy back-and-forth method and random choice method that the present invention recommends, can find that the former can guarantee selected sequence fully, making the transition as the people, somatomedin sequence 3 can have high specificity and strong enzyme is cut effect, and the latter's success ratio is very little, specificity is very low also, as other 6 sequences (1 among the Seq.ID#7,2,4,5,6 and 7) in the table 5.The similar sequences (gi|31959) of the different fragments among the mRNA of table 11. people transforming growth factor-beta2

Seq. ID#7	?Total ?Hits	?100％ ?Match	?80-95％ ?Match	＜80％ Match	?Pattern	?Start????Sequence??????????End ?Point????????????????????Point
							?1	?193	?6m	?25n	?162n	?ctgat	?31?cgcttttctg?atcctgcat49
?2	?196	?5m	?7n	?184n	?tttct	?1201?gaacagcttt?ctaatatgat?1219

??3	??12	??5m	??1n	??6n	??cggat	??486?tgaac?aacggattga?gcta504
							??4	??106	??5m	??2n	??99n	??gggat	??976?ttcaa?gagggatcta?gggt?994
??5	??112	??6m??1n	??13n	??92n	??agatc	??121?cgcgggcaga?tcctgagca139
							??6	??211	??7m	??85n	??109n	??ccctt	??321?catgccgccc?ttcttcccct?339
??7	??241	??5m	??14n	??222n	??gggaa	??819?aa?acagtgggaa?gacccca837

With the different fragments among the human telomerase mRNA in the table 12 serves as that the sequence of puing question to sequence, Search Results to show that further easy back-and-forth method is selected has high specific and high enzyme is cut benefit, as 2 and 4 among the Seq.ID#8.The similar sequences (AF221907) of the different fragments among the table 12 human telomerase mRNA

??Seq. ??ID#8	??Total ??Hits	??100％ ??Match	??80-95％ ??Match	??＜80％ ??Match	??Pattern	??Start????Sequence??????End?Point ??Point
							??1	??54	??2m	??1????1m	??48n??2	??gactc	??1????agagagtgac?tctcacgag????19
??2	??20	??4m		??16n	??cggaa	??223?cagcgggc?ggaaaagcctc?241
							??3	??67	??4m	??4n	??59n	??cagga	??521?gtgcacccag?gactcggct?539
??4	??12	??4m	??1n	??8n	??cggag	??469?ag?aggaacggag?cgagtcc487
							??5	??528	??4m??1n	??25n	??499n	??gggag	??111?tgggcctggg?aggggtggt??129
??6	??66	??3m??1n	??3n	??59n	??ccgaa	??327?ccag?cccccgaacc?ccgcc?345

Table 13 further shows the uncertainty of random choice method.It can select more special sequence such as sequence 2 and 5 sometimes, and to cut the possibility of benefit be very little but obtain high specific and high enzyme simultaneously, contain restriction enzyme site efficiently as sequence 7, but its specificity is very low.The make the transition similar sequences (gi|10863872 :) of the different fragments among somatomedin beta 1 (TGFB1) mRNA of table 13. people

Seq. ID#9	?Total ?Hits	?100％ ?Match	?80-95％ ?Match	＜80％ Match	?Pattern	?Start????Sequence?????End ?Point???????????????Point
							?1	?72	?6m??1n	?2n	?63n	?cctcc	?1?atgccgccct?ccgggctgc9
?2	?22	?7m??1n		?14n	?tgatc	?1141tccaacatga?tcgtgcgctc1159
							?3	?18	?8m??1n		?9n	?cggag	?599?at?gtcaccggag?ttgtgcg?617
?4	?50	?7m??1n	?8n	?34n	?cggag	?767?gcag?aaccggagcc?cgagc?785
							?5	?46	?8m??1n	?1n	?36n	?tccgc	?901?attgacttcc?gcaaggacct?929
?6	?319	?8m??1n	?14n	?296n	?tgttc	?391?atatatatgt?tcttcaaca?409
							?7	?244	?7m??1n	?28n	?208n	?gggga	?189?ga?gccagggggaggtgccg207

Though table 14 shows easy back-and-forth method and can obtain to have the sequence that high specific and enzyme are cut benefit reliably, but in selected those sequences, be to exist difference (1,3 and 4 among the Seq ID#10), through further comparing, can therefrom choose best sequence, as Seq.ID#4.The circulate similar sequences (gi|14759971 :) of the different fragments among plain kinases 2 (CDK2) mRNA that relies on of table 14. people

Seq. ID#10	?Total ?Hits	?100％ ?Match	?80-95％ ?Match	＜80％ Match	?Pattern	?Start????Sequence?????????End ?Point ?Point
							??1	??51	??10m	?3m??5n	??33n	??cggag	??23??aaaagatc?ggagagggca?c?41
??2	??53	??10m		??43n	??caagc	??761?atgtgaccaa?gccagtacc?779
							??3	??27	??10m	????1n	??16n	??cggac	??540?catctttcgga?ctctgggg?558
??4	??20	??9m		??10n?1m	??cgggc	??489?ga?ctcgccgggc?cctattc?507
							??5	??503	??10m	????90n	??403n	??cagct	??321?tctgttccag?ctgctccag?339
??6	??150	??10m	????3n	??137n	??tgcac	??241?gaatttctgc?accaagatc?259
							??7	??77	??10m	????1n	??66n	??ggagc	??161?tgcttaagga?gcttaacca?179

Another high specific of selecting with easy back-and-forth method of table 15 is the sequence of SDSO molecule (4 among the Seq.ID#11) efficiently.It can be used for the function of the mRNA of deactivation human hepatocyte growth factor.The similar sequences (gi|4750937) of the different fragments among the table 15. human hepatocyte growth factor HGF mRNA

??Seq. ??ID#11	??Total ??Hits	??100％ ??Match	??80-95％ ??Match	??＜80％ ??Match	??Pattern	??Start????Sequence??????End ??Point????????????????Point
							??1	??359	??17m?2n	??17n	??326n	??cctgc	??11?ccaaactcctgccagccct?19
??2	??87	??16m?2n		??69n	??gggat	??697?cagc?gctgggatca?tcaga?716
							??3	??139	??13m?2n	??1n	??126n	??cttgc	??1381?tgggattatt?gccctattt?1399
??4	??43	??12m?2n	??1n	??28n	??cggaa	??1655?atgtcc?acggaagaggaga?1673
							??5	??81	??12m?2n	??1n	??66n	??taagg	??2161?ttaacatata?aggtaccac?2179
??6	??90	??17m?2n	??2n	??69n	??gggaa	??403?gctacaa?gggaacagta?tc?422

The selected SDSO molecule of the easy back-and-forth method of example 2--can suppress the function of corresponding mRNA efficiently

The cell A375 of human body melanoma and SK-Mel-28 cultivate collection center (ATTCC) from U.S.'s tissue-type and obtain, all cells maintain (DMEM in improved Dulbecco ' the s nutrient solution, the glucose of 5g/l, 10% foetal calf serum, the penicillin of 100 units/ml, 100ug/ml Streptomycin sulphate and 0.25ug/ml amphoterin B), nude mice (6-8 week) is raised under bioclean condition.

With a wide scalp acupuncture 3mm size tumor piece is transplanted to the dorsal part of nude mice, tumour to be transplanted is long to be divided into five groups randomly with the nude mice that has inoculated tumour time the 1cm size, be used for the treatment of experiment.The PBS of each day injection 15ul in first group tumour; The empty liposome of the 150 sodium moles of each day injection 15ul in second group tumour; The empty liposome of the 150 sodium moles of each day injection 15ul and the double-stranded oligonucleotide of 5ug in the 3rd group tumour; Each day injection 15ul contains the empty liposome of 150 sodium moles of the endoxan of 50mg in the 4th group tumour; In the 5th group tumour, inject the empty liposome of the 150 sodium moles that contain 15ul and the double-stranded oligonucleotide of 5ug every day.In the 5th group experiment, liposome needs injection in per two days once.In addition, the per five days sizes with miniature kind of calliper tumour are so that estimate the growth-inhibiting of medicine to transplantation tumor.Tumor size can be calculated by following formula: ab ²/ 2.Here a is the width of tumour, and b is the length of tumour.Tumour size (per-cent) can obtain by another formula relatively, that is: T _n/ To X100.The To here is the weight of the tumour in when injection in tumour, T _nBe the tumor weight after the medication.

Fig. 8 shows one group of cell A375 propagation and survival on nude mice that can be suppressed the human body melanoma by 7 different SDSO molecules.Inoculating in 35 days observation period of tumour, in first group tumour each day injection PBS and in second group tumour each day injection empty liposome to the propagation of human body melanoma and survival all without any effect.The liposome that each day injection contains the liposome of 7 kinds of different SDSO molecules and each day injection contains the endoxan of 50mg in the 4th group tumour in the 3rd group tumour in preceding 15 days, can enough suppress to some extent the human body melanoma propagation, but in back 20 days, still lose their inhibition ability gradually.Yet the liposome that every day injection contains 7 kinds of different SDSO molecules in the 5th group tumour can suppress propagation and the survival of human body melanoma on nude mice effectively.The result showed the tumour of inoculation under the effect of 7 kinds of different SDSO molecules, and tumour from large to small and fade away can not find the cell of human body melanoma in the obtained nude mice tissue at 35 days.

Another group experiment is about the long siRNA of 21 Nucleotide the propagation and the survival of CML cell to be suppressed observation on effect.In order to determine the influence of the long siRNA of 21 Nucleotide to the chronic myeloid leukemia cell, the cell of three different patients' CML marrow is used to this experiment.These cells are carried out chromosome analysis and RT-PCR detection, found to contain in these cells the bcr/abl of b3/a2 type.Cell from this three patients' marrow is cultivated, handled with the SDSO molecule of specific homologous sequence among the mRNA of the mRNA that contains bcr and abl and bcl-6 and N-ras then, just can suppress the propagation of cell with the SDSO of 50ng/ml respectively effectively.On the contrary, empty liposome without any effect (Fig. 9), from normal people's medullary cell, but can be resisted the retarding effect (Figure 10) of SDSO cell growth to the chronic myeloid leukemia cell.Example 3--analyzes those are had the active anti-meaning nucleotide chain of efficient inhibition by report specificity with BLAST series arrangement formula.

In order to identify the specificity of the effective ASO that report in other laboratory, this research pertinent literature to nearly ten years in the PubMed database has carried out a comprehensive search, select those have higher inhibition to corresponding mRNA molecule ASO molecule, then, with these molecules as puing question to sequence, whether 960,000 different sequences in the human genome database, observing these sequences has real higher specificity if being compared.

In literature search, following standard is used to select those ASO molecules: (1) to the benefit of the ASO molecule more than 10 contrast research experiment in the ASO molecule.(2) the ASO molecule during the benefit of the antisense strand of sections different on the same mRNA molecule has been done to observe.(3) the ASO molecule that can be used for clinical experiment of some FDA approvals.(4) some are used for the ASO molecule of patent application

Table 16 has been enumerated a series ofly has the ASO molecule of different retarding effects to the ubiquitin ligase mRNA of human body Nedd-4 sample, and compared the selected segmental ASO molecule of WWP2 dna homology in other laboratory simultaneously and the SDSO molecule selected with the short-cut method among the present invention between specificity and sensitivity.The WWP2-ASO molecule that other laboratory is selected can be divided into three groups, and first group is high efficiency, as the inhibiting rate of High1,2,3,4 and 5 couples of WWP2 from 80-100%; Second group is middle effect, as the inhibiting rate of Med1,2 and 3 couples of WWP2 from 40-60%; The 3rd group is inefficiencies, as the inhibiting rate of Low 1,2,3 and 4 couples of WWP2 from 0-20%.The ubiquitin ligase mRNA of table 16 human body Nedd-4 sample (XM 028151.2 GI:15318611)

Seq. ID#12

?Total ?Hit

?100％ ?Match

?80-95％ ?Match

＜80％ Match

?Cleav. Pattern

?Start????Sequence????End ?Point??????????????Point

??High1	?16	??6????1n		?9n	?cggt	??54?cttcacggtgatgatatgg?72
							??High2	?39	??6????1n		?32	?cggt	??52?agcttcacggtgatgatat?70
??High3	?24	??5????1n	????1n	??17	??cggt	??50?cagcttcacggtgatgatat?69
							??High4	??14	??6????1n		??7		??142?gtgtccgcaa?agcccaaggt160
??High5	??7	??7				??173?acctcgaa?ttaactccta?c?191
							??Low1	??93	??5	??12	??76		??2800?tggtcccacacagggccaca?2781
??Low2	??123	??2	??26	??97		??1360?cattgtcctgtcttttctcc?1341
							??Low3	??59	??3	??18	??38	??ggga	??1961?tgtagaaagggagggtgaag?1942
??Low4	??84	??3	??25	??56		??530?aggaaaattgtcagttttcc?511
							??Med1	??59	??6????1n	??14	??38		??917?ttcctctccttcagccggtg?898
??Med2	??25	??4????1n	??10	??10		??1035?tattgtggtcaacataatag?1016
							??Med3	??28	??2	??8n??1m	??17		??1239?aggaatctttggctgaag?1222
??CGG1	??15	??6????1n		??7	??cggac	??635?aagatcccggacgcacaga?653
							??CGG2	??47	??6????1n	??1	??39	??cggag	??435?ctgcagacggagaacaaag?453
??CGG3	??56	??3????1n	??1	??51	??cggag	??463?tctcaggcggagagctgac?481
							??CGG4	??22	??6????1n		??15	??cggag	??704?cggtgctcggagccggcac?722
??CGG5	??10	??6????1n		??3	??cgggt	??921?agcacttcgggtacacagc?939
							??CGG6	??6	??4????1n		??2	??cggac	??1000?tgcccaacggacgtgtcta?1018
??CGG7	??31	??3		??28	??cgggc	??1931?atcgacacgggcttcaccc?1949
							??CGG8	??16	??3		??13	??cggat	??1957?ctacaagcggatgctcaat?1975
??CGG9	??51	??1	??1	??47??2m	??cgggt	??2143?gagcatccgggtcacagag?2161
							??CGG10	??12	??3		??9	??cggac	??2508?gtagcaacggaccacagaa?2526

Can find after examining that in efficient group, each ASO molecule can be found out less homologous sequence, 20 of average out to.Each molecule can be sought out the member of average 6 100% same gene family.Almost do not have the similar sequences of 80-95%, and contain that 7-32 do not wait be less than 80% similar non-familial homologous sequence.In middle effect group, each ASO molecule on average can be sought out 30 homologous sequences, wherein, average 4 is 100% identical same family member's sequence, the identical non-familial sequence of 12 80-95% with 10-38 do not wait less than 80% similar non-homology sequence.In the poor efficiency group, each ASO molecule on average can be found out 89 homologous sequences, wherein average 3 is the member of 100% identical homogenic family, and 20 is the similar non-familial member's of 80-95% sequence, with 38-97 the sequence less than 80% similar non-familial member that does not wait.In a word, the ASO molecular energy detects the member of most same family efficiently, only with few non-familial dna sequence dna lower similarity is arranged.On the contrary, the ASO molecule of poor efficiency can only detect the member of same family of part, and with more non-familial member's dna sequence dna the similarity of higher degree is arranged.

The specificity of the dna sequence dna of selecting with the short-cut method that the present invention recommended can compare favourably with the specificity of the selected segmental ASO molecule of WWP2 dna homology in other laboratory.Any one dna sequence dna of being selected by short-cut method all has than high specific, and on average each dna sequence dna can be found out 17 homologous sequences.The sequence found out sum even be lower than above-mentioned efficient group of number that is detected.It should be noted that most in efficient group a kind of in the core sequence that has three sequences also to contain the present invention to be recommended, i.e. CGGTG.This shows that the standard of selecting efficient double-stranded oligonucleotide that the present invention recommended is effective.Not only the method for ASO molecule is more convenient efficiently than any selection for it, and better result is arranged.Simultaneously, it not only is applicable to selects the SDSO molecule, also can be used for the selection of other ASO molecule.

Table 17 has been enumerated 9 anti-sense nucleic acid chains the most profitable reporting on the document.Wherein each antisense strand all has their used title in report.The specificity of the starting point of chain and terminal point, chain is represented by the number size under the similarity hurdle.Validity is meant that each antisense strand can inhibitory phase answers the degree of genetic expression, represents with high, medium and low degree inhibiting rate.The ASO molecule that has only those to have high inhibition degree is selected.BCL means B cell, chronic myeloid leukemia/lymphoma.2 molecule VCAM are meant the blood vessel adhesion molecule.PKC is meant protein kinase C.P53 is an oncogene arrestin.The specificity of 9 anti-sense nucleic acid chains the most profitable reporting on table 17. document.

?Seq.ID	?Total ?Hit	?100％ ?Match	?80-95％ ?Match	＜80％ Match	?Pattern	?Start????Sequence???????End ?Point?????????????????Point
							?BCL-2	?34	?9????1n	?1n??1m	?12		?33?tggcgcacgctgggagaac?51
?Cotter?et?al.，1994，Oncogene?9：3049-3055
							?TNF	?22	?12???3n		?10		?582?agcatgatccgggacgtgg?600
?d′Hellencourt?et?al.，996，Biochim.Biophys.Acta?1317：168-174
							?VCAM	?40	?6	?8	?22		?2866?aacccagtgctccctttgct?2847

Lee?et?al.，1995，Shock?4：1-10
							?P53	?91	?30????2	?1n	?59		?1224?cctgctcccccctggctcc?1206
?Bishop?et?al.，1996，J.Clin.Oncol.14：1320-1326
							?PGY1	?8	?3m	?1m	?5n		?428?ccatcccgacctcgcgct?411
?Alahari?et?al.，1996，Mol.Pharmacol.50：808-819
							?RAF	?27	?5????2n	?7	?13		?2503?tcccgcctgtgacatgcatt?2484
?Monia?et?al..1996，Nature?Med.2：668-675
							?PKC-a	?18	?4	?2	?12		?41?aaaacgtcagccatggtccc?22
?Dean?et?al.，1994，J.Biol.Chem.269：16416-6424
							?CD54	?336	?8????1n	?7	?320		?1952?tgagaggggaagtggtggg?1970
?Lee?et?al.，1995，Shock?4：1-10
							?bcr	?21	?18m	?1n	?2n		?3203?gtctccggggctctatgggt?3222
?Maran?et?al，1998，Blood；92(11)：4336-43

What can find after examining similar sequences in every example, if the member of an ASO molecular energy and same gene family has 100% homology, and extremely low similarity arranging with the member of less other gene family, the corresponding genetic expression of this ASO molecule just has higher inhibition benefit so.Go out as referred to here, inhibitory phase answers the ASO molecule of genetic expression that one section dna sequence dna that specificity is higher is all arranged effectively.In the middle of them, there are two sequences to contain the core sequence that the present invention recommends, i.e. CGGGA and CGGGG.Similarly, the specificity of the dna sequence dna of selecting with the short-cut method that the present invention recommended is all very high, and it can have 100% homology with the member of same gene family all or part, and with the member of other gene family less similarity is arranged.In addition, the dna sequence dna that easy back-and-forth method is selected also has the strong enzyme heart that hits, so the dna sequence dna of being selected by easy back-and-forth method is the also expression that suppresses corresponding gene in the cell surely effectively of the SDSO molecule made of template.The selected SDSO molecule of the easy back-and-forth method of example 4--will be used for the treatment of diseases associated and dysfunction

A good genomic medicine should have the stability of height, easily is imported into target cell, can obtain sufficiently high IC, can combine specifically with target RNA height, cuts off said target mrna efficiently, and low and even nontoxic side effect.Genomic medicine of the present invention is the natural SDSO molecule that is present in the viable cell, when these molecules combine with a kind of effective carrier, just can be imported specifically in the target cell, suppressed the gene of those unconventionality expressions, thereby reached treatment disease and parafunctional purpose.Simultaneously, it also can be used for preventing or postponing the generation of inflammation, infection and tumour.The treatment tumour

There are many gene therapy strategies to be used for tackling tumour, but their common feature is to suppress a special genes in the cell with a kind of anti-meaning nucleotide chain, and the result of study of recent gene chip is pointed out, tumour is not a kind of gene, but the comprehensive result of one group of gene unconventionality expression.Therefore, the present invention adopts a kind of strategy for the treatment of cancer to be, set about from the mRNA molecular level, cure the expression of one group of oncogene in the tumour cell simultaneously with one group of SDSO molecule, releasing is to the inhibition of suppressor oncogene, rational growth of blocking-up cytopathy and splitted approach, the dead passage in the reconstituted cell.Obviously, this just requires the biological characteristics of curative medicine is proposed higher requirement.In order to reach these purposes, it is active ingredient that the present invention adopts one group of special SDSO molecule.These SDSO molecules can be divided into two groups.One group is at the common feature of cancer cells, as the activity decline of cancer suppressor gene, and non-controlling growth and division, vasostimulant growth is to obtain nutrition etc.Another group then is the feature of being had by oneself at the different carcinoma cell, specific oncogene active, the high expression level of certain acceptor or the proteic unusual lofty tone of certain specific function etc.The active ingredient of first group of genomic medicine among the present invention (their corresponding sequence is shown in table 18) is including, but not limited to CDK-2 and 4, Stat-3 and 5, Mdm-2, PKC-alpha, Telomerase, VEGF, b-FGF.The active ingredient of second group of genomic medicine is including, but not limited to Dermogene:HPV (E6), CDKN2A, HDC, N-Ras, Spermidine or PEI.Lungene:IGF, K-RAS, Neu, HGF, BCL-2 and-xl, liposome, Spermidine, or virosome.Hepatogene:HuH-7 (Hepatoma-derived Growth Factor), rhoB, c-myc, TR3 orphan receptor, TGF-alpha, N-RAS, and HGF, liposome, or Fugene.Leukogene:BCL-6, Bcr-Abl, N-Ras, liposome, or Fugene.Breastogene：BRCA1?and?2，erbB-2，Estrogen?receptor，。Braintumogene:N-RAS, liposome, or Fugene.

Wherein Dermogene, Lungene, Hepatogene, Leukogene, Breastogene and Braintumogene are the titles of genomic medicine among the present invention, and other term is the title of different SDSO molecule, and their corresponding sequence is shown in table 18.These SDSO molecules can suppress the function of corresponding mRNA molecule effectively, and form the genomic medicine for the treatment of different tumours together with other auxiliary composition.Treatment virus and fungi infestation to virus and treatment of fungal infections strategy is, the defense mechanism of the double-stranded oligonucleotide that is present in biology intravital antiviral and fungi by strengthening nature reaches therapeutic purpose.Short and small DsRNA is a kind of fabulous antiviral active ingredient, and it after external amplification, by suitable approach, sucks anti-ly to cure cold with the infection of syncytial virus etc. as atomizing, by being used for suppressing the caused skin diseases of born of the same parents' exanthema virus outward.Inject by system and to tackle acquired immune deficiency syndrome (AIDS) etc.For example, here screen and designed SDSO molecule to the gene of anti-AIDS, this genomic medicine is named as AIDSogene, and active ingredient wherein is including, but not limited to proteolytic enzyme (Protease), polymerase (polymerase), intergrase (integrase), transactivation albumen (transactivating protein), viral protein expression regulon (regulator of expression of virion protein), the virus infection factor (viral infectivity factor), gp120 and gp41 molecule.

Similar, other many methods of being recommended in according to the present invention can design and choose other multiple special SDSO molecule.Treat other virus not of the same race or fungi infestation with them.Prevent and treat these by virus or fungus-caused disease by different route of administration.Treatment heredopathia inherited disease is another kind of refractory disease.It relates to the change of gene structure and degree of gene expression.So, how to close those abnormal genes and cross the gene of expressing, also be one of main object of genomic medicine of the present invention.The same with the principle of diseases such as treatment tumour and viral infection, design and choose those special SDSO molecules, make they have with corresponding Disease-causing gene in certain specific region 100% homology is arranged, under the special guiding of these molecules, remove to cut off the mRNA molecule that those are not wanted with relevant nuclease, thereby go to cure relevant heredity illness, as senile dementia and essential hypertension etc.The present invention has disclosed and has enumerated the application of some concrete examples and method.Obviously, those people with this respect knowledge and technology will can make similar or further improved technology and method at an easy rate according to prompting of the present invention and imagination, and obtain direct or indirect result.So the additional application of the present invention is including, but not limited to all relevant specific exampless and modification or improvement of equal value.

1 one endogenic RNAl interference bases of 8 nucleotide sequence sequences are because of, the relevant sequence (black) of people Iet-7 rna gene.Its Smalt is represented just the anticipating sequence of chain of Iet-7RNA gene, and red represent one section with the Iet-7RNA gene with antiparallel mode complementary sequence mutually. Sequence 2. by MUSCA sequence type formula the relevant CGGAG that from people PRKWNK4 gene; searches, CGGGA and their derived sequence pattern. ( PRKWNK4 ) NM_032387.1 GI：15277311 1 gccctgctct ttcctcatgt tggcaatccc cggccacgga gaccaccgtc ctcatgtccc 61 agactgaggc cgacctggcc ctgcggcccc cgcctcctct tggcaccgcg gggcagcccc 121 gcctcgggcc ccctcctcgc cgagcgcgcc gcttctccgg gaaggctgag ccccggccgc 181 gctcttctcg tctcagccgc cgtagctcag tcgacttggg gctgctgagc tcttggtccc 241 tgccagcctc acccgctccg gacccccccg atcctccgga ctccgctggt cctggccccg 301 cgaggagccc accgcctagc tccaaagaac cccccgaggg cacgtggacc gagggagccc 361 ctgtgaaggc tgcggaagac tccgcgcgtc ccgagctccc ggactctgca gtgggcccgg 421 ggtccaggga gccgctaagg gtccctgaag ctgtggccct agagcggcgg cgggagcagg 481 aagaaaagga ggacatggag acccaggctg tggcaacgtc ccccgatggc cgatacctca 541 agtttgacat cgagattgga cgtggctcct tcaagacggt gtatcgaggg ctagacaccg 601 acaccacagt ggaggtggcc tggtgtgagc tgcagactcg gaaactgtct agagctgagc 661 ggcagcgctt ctcagaggag gtggagatgc tcaaggggct gcagcacccc aacatcgtcc 721 gcttctatga ttcgtggaag tcggtgctga ggggccaggt ttgcatcgtg ctggtcaccg 781 aactcatgac ctcgggcacg ctcaagacgt acctgaggcg gttccgggag atgaagccgc 841 gggtccttca gcgctggagc cgccaaatcc tgcgg act tcatttccta cactcccggg 901 ttcctcccat cctgcaccgg gatctcaagt gcgacaatgt ctttatcacg ggacctactg 961 gctctgtcaa aatcgg ac ctgggcctgg ccacgctcaa gcgcgcctcc tttgccaaga1021 gtgtcatcgg gaccccggaa ttcatggccc ccgagatgta cgaggaaaag tacgatgagg1081 ccgtggacgt gtacgcgttc ggcatgtgca tgctggagat ggccacctct gagtacccgt1141 actccgagtg ccagaatgcc gcgcaaatct accgcaaggt cacttcgggc agaaagccga1201 acagcttcca caaggtgaag atacccgagg tgaaggagat cattgaaggc tgcatccgca1261 cggataagaa cgagaggttc accatccagg acctcctggc ccacgccttc ttccgcgagg1321 agcgcggtgt gcacgtggaa ctagcggagg aggacgacgg cgagaagccg ggcctcaagc1381 tctggctgcg catggaggac gcgcggcgcg g ggcgccc acgggacaac caggccatcg1441 agttcctgtt ccagctgggc cgggacgcgg ccgaggaggt ggcacaggag atggtggctc1501 tgggcttggt ctgtgaagcc gattaccagc cagtggcccg tgcagtacgt gaacgggttg1561 ctgccatcca gcgaaagcgt gagaagctgc gtaaagcaag ggaattggag gcactcccac1621 cagagccagg acctccacca gcaactgtgc ccatggcccc cggtcccccc agtgtcttcc1681 cccctgagcc tgaggagcca gaggcagacc agcaccagcc cttccttttc cgccacgcca1741 gctactcatc taccacttcg gattgcgaga ctgatggcta cctcagctcc tccggcttcc1801 gctgagtccc atctctgcct gccctcggct tttgccctat ccattccacg ttctggccct1861 ggaagtgact tttcccccgg ggacagctat gcctcagatg cagcttcagg ccttagcgat1921 gtgggagaag ggatgggaca aatgaggaga cccccaggga ggaatctccg gcgcagaccc1981 cgatcccggc tgcgggtcac tagtgtctca gaccagaatg acagagtggt tgagtgccag2041 ctacagaccc ataacagcaa gatggtgacc ttccgatttg atctggatgg ggacagcccg2101 gaagagattg cagctgccat ggtatataac gagttcattc tgccttcgga gcgagatgga2161 tttctcagac ggattcggga gattatccag cgagtggaga ccctgttgaa gagagacact2221 ggccccatgg aggctgctga agacacccta agcccccagg aggagccagc accattacct2281 gccctgcccg tccccctccc agacccatcc aatgaagagc tccagagcag cacctccctg2341 gagcacagga gctggacagc cttctccacc tcctcatctt ctcctggaac tcctttgtct2401 cctggaaacc cattttcccc tggaaccccc atttccccag gtcccatctt ccccatcact2461 tctcccccat gtcatcccag cccctcccca ttctccccca tttcttccca ggtctcctca2521 aatccctctc cacaccccac cagctctcca cttccattct cctccagcac acccgagttt2581 ccggtcccac tctctcagtg tccctggagt tctctcccca cgacttctcc acctacgttc2641 tctcccactt gttctcaggt cactcttagt tcccctttct ttcctccgtg cccctccact2701 tcttccttcc cctccaccac agcagcccct ctcctttctc tggctagtgc cttctcactg2761 gctgtgatga ctgtggccca gtccctgctg tccccctcac ctgggctcct ttcccagtct2821 cctccagccc ctcctagtcc cctccctagc ctgccccttc cccctcccgt tgctcctggt2881 ggccaggaaa gcccttcacc ccacacagct gaggtggaga gtgaggcctc accacctcct2941 gctcggcccc tcccagggga agccaggctg gcgcccatct ctgaagaggg aaagccgcag3001 cttgttgggc gtttccaagt gacttcatcc aaggaaccgg ctgagcctct tcccttgcag3061 ccaacatccc ccactctctc tggttctcca aaaccttcaa cccctcagct cacttcagag3121 agctcagata cagaggacag tgctggaggc gggccagaga ccagggaagc tctggctgag3181 agcgaccgtg cagctgaggg tctgggggct ggagttgagg aggaaggaga tgatgggaag3241 gaaccccaag ttgggggcag cccccaaccc ctgagccatc ccagcccagt gtggatgaac3301 tactcctaca gcagcctgtg tttgagcagc gaggagtcag aaagcagtgg ggaagatgag3361 gagttctggg ctgagctgca gagtcttcgg cagaagcact tgtcagaggt ggaaacacta3421 cagacactac agaaaaaaga aattgaagat ttgtacagcc ggctggggaa gcagccccca3481 ccgggtattg tggccccagc tgctatgctg tccagccgcc agcgccgcct ctccaagggc3541 agcttcccca cctcccgccg caacagccta cagcgctctg agcccccagg ccctggcatc3601 atgcgaagga actctctgag tggcagcagc accggctccc aggagcagcg ggcaagcaag3661 ggggtgacat tcgccgggga tgttggcagg atgtga

The sequence that table 18 is chosen by systematic selection among the present invention and easy back-and-forth method.

Sequence number	Sequence forms (19 bases), starting point and terminal point	The type of sequence	The source of sequence	Sequence	The gene pool sequence number
						1	542 tcagttacg gaaacgatgc 460	RNA/DNA	Artificial sequence	2	gi|14780094
2	315 gattat gcggatcaaa cct 333	RNA/DNA	Artificial sequence	4	gi|15422108
						3	187 cggg acccggtcgc cagga 205	RNA/DNA	Artificial sequence	2	gi|13646672
4	1254 atccgcacggataagaacg 1272	RNA/DNA	Artificial sequence	9	GI：15277311
						5	349 tgcgacccggacgacgaga 367	RNA/DNA	Artificial sequence	6	gi|11493193
6	58  ccagcttcggaac aagaga 76	RNA/DNA	Artificial sequence	1	GI：14762555
						7	486 tgaac aacggattga gcta504	RNA/DNA	Artificial sequence	3	gi|31959
8	469 ag aggaacggag cgagtcc487	RNA/DNA	Artificial sequence	4	AF221907
						9	599 at gtcaccggag ttgtgcg 617	RNA/DNA	Artificial sequence	3	gi|10863872
10	489 ga ctcgccgggc cctattc 507	RNA/DNA	Artificial sequence	4	gi|14759971
						11	1655 atgtcc acggaagaggaga 1673	RNA/DNA	Artificial sequence	4	gi|14750937
12	635 aagatcccggacgcacaga 653	RNA/DNA	Artificial sequence	1	GI：15318611
						13	114 ccttcag cggccagtag ca 132	RNA/DNA	Artificial sequence	2	GI：180638
	289 aaa gctccgggtcttaggc 307	RNA/DNA	Artificial sequence	3	GI：180638
							40 g agtctccggg gctctatg 58	RNA/DNA	Artificial sequence	1	GI：180638
14	197 tgccccccggagccgcgag 215	RNA/DNA	Artificial sequence	1	GI：183986
							441 gaggctgcggattgtgcga 459	RNA/DNA	Artificial sequence	2	GI：183986
	1060 ctttctacggacgtgggat 1078	RNA/DNA	Artificial sequence	3	GI：183986
							1276 tttctgccggagagctttg 1294	RNA/DNA	Artificial sequence	4	GI：183986
	3051 aagattccgggagttggtg 3069	RNA/DNA	Artificial sequence	5	GI：183986
						15	78 gcc ggcccggatt gacgag 96	RNA/DNA	Artificial sequence	1	gi|4758515
16	405 aagggg tcggtggaccggt 423	RNA/DNA	Artificial sequence	1	gi|333031
							413 ggtggacc ggtcgatgta t431	RNA/DNA	Artificial sequence	2	gi|333031
18	49  ct gtgcacggaa ctgaaca  67	RNA/DNA	Artificial sequence	1	gi|60876
							312 ggtgcctgcg gtgccagaaa 330	RNA/DNA	Artificial sequence	2	gi|60876
19	813 gcaagttc ggcagcagct t 831	RNA/DNA	Artificial sequence	1	gi|14737359

	793 atagttgc ggagagtctg c 821	RNA/DNA	Artificial sequence	2	gi|14737359
							1206 tgaat ttcggcacct gcaa 1224	RNA/DNA	Artificial sequence	3	gi|14737359
	1858tcc cagaacggag gcgaac 1876	RNA/DNA	Artificial sequence	4	gi|14737359
						20	602 tacattccg gaaagattgt 620	RNA/DNA	Artificial sequence	1	gi|15296805
	301 gttattttgg ttcgagaga  319	RNA/DNA	Artificial sequence	2	gi|15296805
							501 taatgggggc gagctgttt 519	RNA/DNA	Artificial sequence	3	gi|15296805
21	1056 tggaccccggattgctgct 1074	RNA/DNA	Artificial sequence	1	GI：340193
							1160 ctctgagcgggaaggtgag 1178	RNA/DNA	Artificial sequence	2	GI：340193
	2008 aaaaaagcggagacaggag 2026	RNA/DNA	Artificial sequence	3	GI：340193
						22	428 ccatcccgacctcgcgct 411	RNA/DNA	Artificial sequence	1	GI：187468
	1816 gtttctacgggaaatcatt 1834	RNA/DNA	Artificial sequence	2	GI：187468
							2041 cgccattgcacgtgccctg 2059	RNA/DNA	Artificial sequence	3	GI：187468
23	1709 tccagtcggatgtctactc 1727	RNA/DNA	Artificial sequence	1	GI：35841
							243 tcagcgccgggcatcagat 261	RNA/DNA	Artificial sequence	2	GI：35841
	549 ctttgctcggaagacgttc 567	RNA/DNA	Artificial sequence	3	GI：35841
							1074 aagagagcgggcaccagta 1092	RNA/DNA	Artificial sequence	4	GI：35841
	2503 tcccgcctgtgacatgcatt 2484	RNA/DNA	Artificial sequence	5	GI：35841
						24	959 cttcgagcggatccgcaag 977	RNA/DNA	Artificial sequence	1	gi|29420
	1071 gaggtgtcggaccgcatca 1089	RNA/DNA	Artificial sequence	2	gi|29420
							1571 catgttccgggacaaaagc 1589	RNA/DNA	Artificial sequence	3	gi|29420
	2275 acaactacggagttgccat 2293	RNA/DNA	Artificial sequence	4	gi|29420

9 reference The international SNP map working group, 2001, Nature 409:928-933Aravin AA., etal, 2001, Curr Biol Jul 10; 11 (13): 1017-27Bernstein, E., et al., (2001) Nature, (London) 409,363-366.Brown, 2000, Bioinformatics Eaton PublishingCaplen, N.J., Fleenor, J., Fire, A.﹠amp; Morgan, R.A. (2000) Gene 252,95-105.Carthew, R.W. (2001) Curr.Opin.Cell Biol. 13,244-248.Cirino NM, et al., 1997, Proc Natl Acad Sci USA 94:1937.Cogoni, C.﹠amp; Macino, G. (2000) Curr.Opin.Genet.Dev.10,638-643.Durbin et al, 1998, Biological sequence analysis.Cambridge University PressElbashir, S.M., Lendeckel, W.﹠amp; Tuschl, T. (2001) Genes Dev.15,188-200.Fire, A.﹠amp; Mello, C.C. (1999) Cell 99,123-132.Fire, A. (1999) Trends Genet.15,358-363.Frischmeyer, P.A.﹠amp; Dietz, H.C. (1999) Hum.Mol.Genet.8,1893-1900.Hamilton, A.J.﹠amp; Baulcombe, D.C. (1999) Science 286,950-952.Hammond, S.M., etal., 2000, Nature (London) 404,293-296.Higgns et al, 2000 Bioinformatics.Oxford University PressKasschau, K.D.﹠amp; Carrington, J.C. (1998) Cell 95,461-470.Ketting, R.F., et al., (1999) Cell 99,133-141.Lesiak K, et al. .1993, Bioconjugate Chem 4:467.Llave, C., et al., (2000) Proc.Natl.Acad.Sci.US4 97,13401-13406.Lockhart, et al., 2000, Nature 405:827-838Maitra RK.:et al., 1995, J Biol Chem 2701:5071.Marcotte, et al, 2001, Trends in Pharmacological Science 22:426-437Matzke, M.A., et al., (2001) Curr.Opin, Genet.Dev.11,221-227.Mitchell, P.﹠amp; Tollervey, D. (2000) Curr.Opin.Genet.Dev.10,193-198.Nakano, H., et al., (2000), Biochem.Biophys.Res.Commun.274,434-439.Oelgeschlager, M., et al., (2000), Nature, (London) 405,757-763.Parrish, S., etal., (2000) Mol.Cell.6,1077-1087.Svoboda, P., et al., (2000), Development, (Cambridge, U.K.) 127,4147-4156.Szczylik C, et al., 1991., Science 253:562.Tabara, H., Sarkissian, M., Kelly, W.G., Fleenor, J., Grishok, A., Timmons, L., Tuschl, T., et al., (1999) Genes Dev.13,3191-3197.Ui-Tei, K., Zenno, S., Miyata, Y.﹠amp; Saigo, K. (2000) FEBS Lett.479,79-82.Wargelius, A., et al., (1999), Biochem.Biophys.Res.Commun.263,156-161.Wianny, F.﹠amp; Zernicka-Goetz, M. (2000) Nat.Cell Biol.2,70-75.Wolffe, A.P.﹠amp; Matzke, M.A. (1999) Science 286,481-486.Zamore, P.D., Tuschl, T., Sharp, P.A.﹠amp; Bartel, D.P. (2000) Cell 101,25-33.10 SEQUENCE LISTING＜110〉Yin, Dongsheng Yin, James Q.＜120〉Method for design and selection of short double-stranded

oligonucleotides?as?gene?drugs?and?compounds?of?gene?drugs<130>??01-2793<140>??01144177.1<141>??2001-12-14<160>??53<170>??PatentIn?version?3.1<210>??1<211>??720<212>??DNA<213>??Homo?sapiens<400>??1tcacacagga?aaccaggatt?accgaggagg?aaaaaaagcc?ttcctgtggt?gctcaactgt??60gattcctttt?caccattcac?cctggatgtt?ctcttcactg?tgggatgagg?tagtaggttg?120tatagtttta?gggtcacacc?caccactggg?agataactat?acaatctact?gtctttccta?180acgtgataga?aaagtctgca?tccaggcggt?ctgatagaaa?gtcagttaac?taattgtaca?240gataatttta?tgttgaaatt?ttctttcgaa?agagattgta?ctttccattc?cagaagaaaa?300cattgctcta?tcagagtgag?gtagtagatt?gtatagttgt?ggggtagtga?ttttaccctg?360ttcaggagat?aactatacaa?tctattgcct?tccctgagga?gtagacttgc?tgcattattt?420tctttttatt?tagatgatat?taaaactcag?aagaattaat?tttgacattt?tgtatttaca?480aattagaaac?aaaactcaaa?gaacatgacc?taatttaaca?ggttaatttg?aagtgcatct?540gccaagtaga?agaccagcaa?gaaaaaaaaa?atgggttcct?aggaagaggt?agtaggttgc?600atagttttag?ggcagggatt?ttgcccacaa?ggaggtaact?atacgacctg?ctgcctttct?660tagggcctta?ttattcaccg?ataacctgtt?tccttgctac?tttgctttgg?tgtaagcaga?720<210>??2<211>??3696<212>??DNA<213>??Homo?sapiens<400>??2gccctgctct?ttcctcatgt?tggcaatccc?cggccacgga?gaccaccgtc?ctcatgtccc??60agactgaggc?cgacctggcc?ctgcggcccc?cgcctcctct?tggcaccgcg?gggcagcccc??120gcctcgggcc?ccctcctcgc?cgagcgcgcc?gcttctccgg?gaaggctgag?ccccggccgc??180gctcttctcg?tctcagccgc?cgtagctcag?tcgacttggg?gctgctgagc?tcttggtccc??240tgccagcctc?acccgctccg?gacccccccg?atcctccgga?ctccgctggt?cctggccccg??300cgaggagccc?accgcctagc?tccaaagaac?cccccgaggg?cacgtggacc?gagggagccc??360ctgtgaaggc?tgcggaagac?tccgcgcgtc?ccgagctccc?ggactctgca?gtgggcccgg??420ggtccaggga?gccgctaagg?gtccctgaag?ctgtggccct?agagcggcgg?cgggagcagg??480aagaaaagga?ggacatggag?acccaggctg?tggcaacgtc?ccccgatggc?cgatacctca??540agtttgacat?cgagattgga?cgtggctcct?tcaagacggt?gtatcgaggg?ctagacaccg??600acaccacagt?ggaggtggcc?tggtgtgagc?tgcagactcg?gaaactgtct?agagctgagc??660ggcagcgctt?ctcagaggag?gtggagatgc?tcaaggggct?gcagcacccc?aacatcgtcc??720gcttctatga?ttcgtggaag?tcggtgctga?ggggccaggt?ttgcatcgtg?ctggtcaccg??780aactcatgac?ctcgggcacg?ctcaagacgt?acctgaggcg?gttccgggag?atgaagccgc??840gggtccttca?gcgctggagc?cgccaaatcc?tgcggggact?tcatttccta?cactcccggg??900ttcctcccat?cctgcaccgg?gatctcaagt?gcgacaatgt?ctttatcacg?ggacctactg??960gctctgtcaa?aatcggggac?ctgggcctgg?ccacgctcaa?gcgcgcctcc?tttgccaaga?1020gtgtcatcgg?gaccccggaa?ttcatggccc?ccgagatgta?cgaggaaaag?tacgatgagg?1080ccgtggacgt?gtacgcgttc?ggcatgtgca?tgctggagat?ggccacctct?gagtacccgt?1140actccgagtg?ccagaatgcc?gcgcaaatct?accgcaaggt?cacttcgggc?agaaagccga?1200acagcttcca?caaggtgaag?atacccgagg?tgaaggagat?cattgaaggc?tgcatccgca?1260cggataagaa?cgagaggttc?accatccagg?acctcctggc?ccacgccttc?ttccgcgagg?1320agcgcggtgt?gcacgtggaa?ctagcggagg?aggacgacgg?cgagaagccg?ggcctcaagc?1380tctggctgcg?catggaggac?gcgcggcgcg?gggggcgccc?acgggacaac?caggccatcg?1440agttcctgtt?ccagctgggc?cgggacgcgg?ccgaggaggt?ggcacaggag?atggtggctc?1500tgggcttggt?ctgtgaagcc?gattaccagc?cagtggcccg?tgcagtacgt?gaacgggttg?1560ctgccatcca?gcgaaagcgt?gagaagctgc?gtaaagcaag?ggaattggag?gcactcccac?1620cagagccagg?acctccacca?gcaactgtgc?ccatggcccc?cggtcccccc?agtgtcttcc?1680cccctgagcc?tgaggagcca?gaggcagacc?agcaccagcc?cttccttttc?cgccacgcca?1740gctactcatc?taccacttcg?gattgcgaga?ctgatggcta?cctcagctcc?tccggcttcc?1800gctgagtccc?atctctgcct?gccctcggct?tttgccctat?ccattccacg?ttctggccct?1860ggaagtgact?tttcccccgg?ggacagctat?gcctcagatg?cagcttcagg?ccttagcgat?1920gtgggagaag?ggatgggaca?aatgaggaga?cccccaggga?ggaatctccg?gcgcagaccc?1980cgatcccggc?tgcgggtcac?tagtgtctca?gaccagaatg?acagagtggt?tgagtgccag?2040ctacagaccc?ataacagcaa?gatggtgacc?ttccgatttg?atctggatgg?ggacagcccg?2100gaagagattg?cagctgccat?ggtatataac?gagttcattc?tgccttcgga?gcgagatgga?2160tttctcagac?ggattcggga?gattatccag?cgagtggaga?ccctgttgaa?gagagacact?2220ggccccatgg?aggctgctga?agacacccta?agcccccagg?aggagccagc?accattacct?2280gccctgcccg?tccccctccc?agacccatcc?aatgaagagc?tccagagcag?cacctccctg?2340gagcacagga?gctggacagc?cttctccacc?tcctcatctt?ctcctggaac?tcctttgtct?2400cctggaaacc?cattttcccc?tggaaccccc?atttccccag?gtcccatctt?ccccatcact?2460tctcccccat?gtcatcccag?cccctcccca?ttctccccca?tttcttccca?ggtctcctca?2520aatccctctc?cacaccccac?cagctctcca?cttccattct?cctccagcac?acccgagttt?2580ccggtcccac?tctctcagtg?tccctggagt?tctctcccca?cgacttctcc?acctacgttc?2640tctcccactt?gttctcaggt?cactcttagt?tcccctttct?ttcctccgtg?cccctccact?2700tcttccttcc?cctccaccac?agcagcccct?ctcctttctc?tggctagtgc?cttctcactg?2760gctgtgatga?ctgtggccca?gtccctgctg?tccccctcac?ctgggctcct?ttcccagtct?2820cctccagccc?ctcctagtcc?cctccctagc?ctgccccttc?cccctcccgt?tgctcctggt?2880ggccaggaaa?gcccttcacc?ccacacagct?gaggtggaga?gtgaggcctc?accacctcct?2940gctcggcccc?tcccagggga?agccaggctg?gcgcccatct?ctgaagaggg?aaagccgcag?3000cttgttgggc?gtttccaagt?gacttcatcc?aaggaaccgg?ctgagcctct?tcccttgcag?3060ccaacatccc?ccactctctc?tggttctcca?aaaccttcaa?cccctcagct?cacttcagag?3120agctcagata?cagaggacag?tgctggaggc?gggccagaga?ccagggaagc?tctggctgag?3180agcgaccgtg?cagctgaggg?tctgggggct?ggagttgagg?aggaaggaga?tgatgggaag?3240gaaccccaag?ttgggggcag?cccccaaccc?ctgagccatc?ccagcccagt?gtggatgaac?3300tactcctaca?gcagcctgtg?tttgagcagc?gaggagtcag?aaagcagtgg?ggaagatgag?3360gagttctggg?ctgagctgca?gagtcttcgg?cagaagcact?tgtcagaggt?ggaaacacta?3420cagacactac?agaaaaaaga?aattgaagat?ttgtacagcc?ggctggggaa?gcagccccca?3480ccgggtattg?tggccccagc?tgctatgctg?tccagccgcc?agcgccgcct?ctccaagggc?3540agcttcccca?cctcccgccg?caacagccta?cagcgctctg?agcccccagg?ccctggcatc?3600atgcgaagga?actctctgag?tggcagcagc?accggctccc?aggagcagcg?ggcaagcaag?3660ggggtgacat?tcgccgggga?tgttggcagg?atgtga???????????????????????????3696<210>??3<211>??19<212>??DNA<213>??Artificial<400>??3tcagttacgg?aaacgatgc??????????????????????????????????????????19<210>??4<211>??19<212>??DNA<213>??Artificial<400>??4gattatgcgg?atcaaacct??????????????????????????????????????????19<210>??5<211>??19<212>??DNA<213>??Artificial<400>??5cgggacccgg?tcgccagga?????????????????????????????????????????19<210>??6<211>??19<212>??DNA<213>??Artificial<400>??6atccgcacgg?ataagaacg?????????????????????????????????????????19<210>??7<211>??19<212>??DNA<213>??Artificial<400>??7tgcgacccgg?acgacgaga?????????????????????????????????????????19<210>??8<211>??19<212>??DNA<213>??Artificial<400>??8ccagcttcgg?aacaagaga?????????????????????????????????????????19<210>??9<211>??19<212>??DNA<213>??Artificial<400>??9tgaacaacgg?attgagcta?????????????????????????????????????????19<210>??10<211>??19<212>??DNA<213>??Artificial<400>??10agaggaacgg?agcgagtcc??????????????????????????????????????????19<210>??11<211>??19<212>??DNA<213>??Artificial<400>??11atgtcaccgg?agttgtgcg?????????????????????????????????????????19<210>??12<211>??19<212>??DNA<213>??Artificial<400>??12gactcgccgg?gccctattc?????????????????????????????????????????19<210>??13<211>??19<212>??DNA<213>??Artificial<400>??13atgtccacgg?aagaggaga???????????????????????????????????????????19<210>??14<211>??19<212>??DNA<213>??Artificial<400>??14aagatcccgg?acgcacaga???????????????????????????????????????????19<210>??15<211>??19<212>??DNA<213>??Artificial<400>??15ccttcagcgg?ccagtagca???????????????????????????????????????????19<210>??16<211>??19<212>??DNA<213>??Artificial<400>??16aaagctccgg?gtcttaggc???????????????????????????????????????????19<210>??17<211>??19<212>??DNA<213>??Artificial<400>??17gagtctccgg?ggctctatg???????????????????????????????????????????19<210>??18<211>??19<212>??DNA<213>??Artificial<400>??18tgccccccgg?agccgcgag???????????????????????????????????????????19<210>??19<211>??19<212>??DNA<213>??Artificial<400>??19gaggctgcgg?attgtgcga???????????????????????????????????????????19<210>??20<211>??19<212>??DNA<213>??Artificial<400>??20ctttctacgg?acgtgggat???????????????????????????????????????????19<210>??21<211>??19<212>??DNA<213>??Artificial<400>??21tttctgccgg?agagctttg???????????????????????????????????????????19<210>??22<211>??19<212>??DNA<213>??Artificial<400>??22aagattccgg?gagttggtg???????????????????????????????????????????19<210>??23<211>??19<212>??DNA<213>??Artificial<400>??23gccggcccgg?attgacgag???????????????????????????????????????????19<210>??24<211>??19<212>??DNA<213>??Artificial<400>??24aaggggtcgg?tggaccggt???????????????????????????????????????????19<210>??25<211>??19<212>??DNA<213>??Artificial<400>??25ggtggaccgg?tcgatgtat???????????????????????????????????????????19<210>??26<211>??19<212>??DNA<213>??Artificial<400>??26ctgtgcacgg?aactgaaca???????????????????????????????????????????19<210>??27<211>??19<212>??DNA<213>??Artificial<400>??27gtgcctgcgg?tgccagaaa???????????????????????????????????????????19<210>??28<211>??19<212>??DNA<213>??Artificial<400>??28gcaagttcgg?cagcagctt?????????????????????????????????????????????19<210>??29<211>??19<212>??DNA<213>??Artificial<400>??29atagttgcgg?agagtctgc?????????????????????????????????????????????19<210>??30<211>??19<212>??DNA<213>??Artificial<400>??30tgaatttcgg?cacctgcaa?????????????????????????????????????????????19<210>??31<211>??19<212>??DNA<213>??Artificial<400>??31tcccagaacg?gaggcgaac?????????????????????????????????????????????19<210>??32<211>??19<212>??DNA<213>??Artificial<400>??32tacattccgg?aaagattgt?????????????????????????????????????????????19<210>??33<211>??19<212>??DNA<213>??Artificial<400>??33gttattttgg?ttcgagaga?????????????????????????????????????????????19<210>??34<211>??19<212>??DNA<213>??Artificial<400>??34taatgggggc?gagctgttt?????????????????????????????????????????????19<210>??35<211>??19<212>??DNA<213>??Artificial<400>??35tggaccccgg?attgctgct?????????????????????????????????????????????19<210>??36<211>??19<212>??DNA<213>??Artificial<400>??36ctctgagcgg?gaaggtgag?????????????????????????????????????????????19<210>??37<211>??19<212>??DNA<213>??Artificial<400>??37aaaaaagcgg?agacaggag?????????????????????????????????????????????19<210>??38<211>??19<212>??DNA<213>??Artificial<400>??38ccatcccgac?ctcgcgcta?????????????????????????????????????????????19<210>??39<211>??19<212>??DNA<213>??Artificial<400>??39gtttctacgg?gaaatcatt?????????????????????????????????????????????19<210>??40<211>??19<212>??DNA<213>??Artificial<400>??40cgccattgca?cgtgccctg?????????????????????????????????????????????19<210>??41<211>??19<212>??DNA<213>??Artificial<400>??41tccagtcgga?tgtctactc?????????????????????????????????????????????19<210>??42<211>??19<212>??DNA<213>??Artificial<400>??42tcagcgccgg?gcatcagat?????????????????????????????????????????????19<210>??43<211>??19<212>??DNA<213>??Artificial<400>??43ctttgctcgg?aagacgttc?????????????????????????????????????????????19<210>??44<211>??19<212>??DNA<213>??Artificial<400>??44aagagagcgg?gcaccagta?????????????????????????????????????????????19<210>??45<211>??20<212>??DNA<213>??Artificial<400>??45tcccgcctgt?gacatgcatt????????????????????????????????????????????20<210>??46<211>??19<212>??DNA<213>??Artificial<400>??46cttcgagcgg?atccgcaag?????????????????????????????????????????????19<210>??47<211>??19<212>??DNA<213>??Artificial<400>??47gaggtgtcgg?accgcatca?????????????????????????????????????????????19<210>??48<211>??19<212>??DNA<213>??Artificial<400>??48catgttccgg?gacaaaagc?????????????????????????????????????????????19<210>??49<211>??19<212>??DNA<213>??Artificial<400>??49acaactacgg?agttgccat?????????????????????????????????????????????19<210>??50<211>??19<212>??DNA<213>??Artificial<400>??50tcaaagtcgg?acagcctca?????????????????????????????????????????????19<210>??51<211>??19<212>??DNA<213>??Artificial<400>??51gtttctgcgg?atgcttctg?????????????????????????????????????????????19<210>??52<211>??19<212>??DNA<213>??Artificial<400>??52ctcttagcgg?ttatccacg?????????????????????????????????????????????19<210>??53<211>??19<212>??DNA<213>??Artificial<400>??53atgaccggga?gtcgtggcc?????????????????????????????????????????????19

Claims (20)

1. The process of 1 one complete preparation genomic medicines, it comprises double-stranded oligonucleotide molecule that screening, prediction, evaluation and synthetic 19-25 Nucleotide is long and is assembled into special them and genomic medicine efficiently, is used for the treatment of multiple different viral infection, tumour and heredopathia.Concrete step is as follows: ● with the Disease-causing gene of gene and selected those overexpressions of protein biochip technology; ● identify that with biocomputer information technology and human body gene library searching technology one special is positioned at the base that causes a disease

   High conserved dna sequence (Motif) in the cause; ● predict according to special genes sequence pattern (Sequence Pattern) which fragment can be used to join in the gene

   Make short and small double-stranded oligonucleotide (SDSO) of its homologous; ● synthesize short and small double-stranded oligonucleotide molecule with the DNA/RNA automatic DNA synthesizer DNA; ● assembling one group of relevant SDSO molecule becomes special and genomic medicine efficiently.
2. 2 one processes of identifying specific DNA sequence in the human genome (system, it comprises: ● identify endogenic short and small interference relevant in the human genome by computer and special software

   RNA (siRNA) sequence; ● use multisequencing to arrange formula and specific sequence pattern determination techniques and human body gene database search skill

   Art is sought the high conserved sequence of those homologous geneses that are positioned at different genera; ● choose one and have the long particular sequence of 19-25 Nucleotide, it with the major part of same gene family or

   All members' relevant dna fragmentation 100% identical; ● evaluate the long sequence of this 19-25 that chooses Nucleotide, standard be this sequence must with the becoming of its family

   A certain fragment 100% identical of member's gene, and do not have with the dna sequence dna of other gene family or only

   Similarity less than 80% is arranged.
3. 3 in the clause of statement (1) or (2), and the long sequence of 19-25 Nucleotide is the interior dna fragmentation of a fragment gene, and it can be used as the template of the long double-stranded oligonucleotide (SDSO) of a synthetic 19-25 Nucleotide.
4. 4 in the clause of statement (1), special genes sequence pattern is that an enzyme hits heart sequence as CGGA (U) T, CGGGA and their derived sequence, this enzyme heart that hits is positioned on the just meaning chain of SDSO molecule, and their complementary sequence, AU (T) CCG, U (T) CCCG and their derived sequence are positioned on the antisense strand of SDSO molecule, enzyme hit second G of the heart should be positioned at the SDSO molecule the beginning downstream the tenth or the 11 on.
5. 5 in statement (1) or (3) clause, and short and small double-stranded oligonucleotide (SDSO) is long dsRNA or sRNA-cDNA or a dsDNA molecule of a 19-25 Nucleotide.
6. 6 in statement (5) clause, and the cDNA among the said sRNA-cDNA is an antisense strand in the oligonucleotide two strands, and sRNA is the chain of just anticipating in the oligonucleotide two strands.
7. 7 in bright (1), (2), (3), (4) and (5) clause, 19-25 the long double-stranded oligonucleotide of Nucleotide is a SDSO molecule, it can or have the RNA molecular specific ground hybridization of other biological function with a coded protein, thereby disturbs its activity or its function of deactivation.In addition, it also may regulate methylating of specific fragment on the corresponding genomic dna, and causes closing of corresponding gene.
8. 8 in statement (1) clause, endogenic short and small RNA interfering (siRNA) sequence of indication is sequence that includes the subarea that is arranged in intergenic region or gene on the genomic dna, and this section sequence can form the antiparallel complementary double chain nucleotide molecule of a long 19-25 Nucleotide.
9. 9 in the clause of statement (1) and (8), and the source of endogenic siRNA comprises the subarea that includes of the intergenic region of coded protein not and proteins encoded plasmagene.
10. The prescription of 10 genomic medicines, it is including, but not limited to following all or part composition: receptible carrier, one or more special target are led protein or polypeptide, other active ingredient, auxiliary composition or additive on efficacy component, one or more nucleic acid compression composition, the one or more drug effect.
11. 11 at the efficacy component of statement in (10), it is the long double-stranded oligonucleotide (dsRNA, sRNA-cDNA and/or dsDNA) of a 19-25 Nucleotide, it has the enzyme heart that hits, and can suppress an animal, the particularly expression of the intravital a kind of mRNA of people effectively.
12. 12 at the efficacy component of statement in (10), and it is one group is made up of one or more double-stranded oligonucleotides, can with the molecule of specific target sequence hybridization in the corresponding RNA molecule.
13. 13 at the prescription of statement in (10), and it is the double-stranded oligonucleotide that 19-25 Nucleotide of the corresponding mRNA molecule of a series of deactivation is effectively grown.They comprise the double-stranded oligonucleotide molecule of following sequence number.They are: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37.
14. 14 in statement (1), (2), (3), (4) and (5) clause, 19-25 long double-stranded oligonucleotide of Nucleotide also can be used to as preventing disease, promotion health and beautify appearance a kind of by way of or a kind of product in composition.
15. 15 at the prescription of statement in (10), is a kind of mixture, by one or more multi-form double-stranded oligonucleotide molecules, in the mode of equivalent or inequality, or formed with any other compatibility form, is specially adapted to treat different tumours.
16. 16 in prescription in bright (10), it is a kind of short and small double-stranded oligonucleotide molecule, comprises the enzyme heart sequence of hitting in its just meaning chain, this enzyme hits the sequence of the heart including, but not limited to CGGAU (T), CGGAA, CGGAC, CGGAG, CGGGC, CGGGA, CGGGU (T), CGGGG, CGGU (T) A, CGGU (T) C, CGGU (T) G, and CGGU (T) U (T).The enzyme heart sequence of hitting is a key component in the long sequence of 19-25 Nucleotide, and this part can be used as the basis whether certain fragment in gene of prediction can be used as a kind of effective pharmaceutical compositions of that corresponding gene of deactivation.
17. 17 at the prescription of statement in (10), and it is a kind of double-stranded oligonucleotide molecule, and perhaps it is a kind of oligonucleotide molecule of heterozygosis formula.
18. 18 1 kinds of mixtures of forming by acceptable carrier or a kind of thinner on the mixture in the statement (14) and a kind of drug effect.
19. 19 in prescription in bright (10), it can further form a kind of dispersion system of colloidal substance.
20. 20 1 kinds of easy systems of selection, it comprises an enzyme hit determining of heart sequence and six concrete steps, this easy system of selection can be used for selected special and SDSO molecule and anti-meaning oligonucleotide molecule (ASO) efficiently.