CN1904900A - Human autogenous siRNA sequence, its application and screening method - Google Patents

Abstract

The present invention integrates RNA interference technology, human gene bank search technology, biological computer information technology, computer algorithm language and genetic engineering technology, and develops a computer algorithm language and related application software, which can screen and predict endogenous siRNA genes and siRNA molecules. The present invention particularly relates to a method for discovering a complete endogenous siRNA gene and an endogenous siRNA, and obtains 255 brand-new siRNAs and their target mRNAs derived from coding gene introns. These siRNA molecules can be used to study the development, differentiation and growth of cells, tissues and organs, explore the function and expression regulation network of genes, and reveal the molecular mechanism of cell cycle control and apoptosis. These siRNA molecules can also be used to develop corresponding siRNA-based gene medicines to prevent and treat various diseases such as viral infection, tumor occurrence and growth, hereditary and metabolic diseases, and immune and nervous system diseases.

Description

Human autogenous siRNA sequence and application and screening technique

Technical field

The present invention relates to bioinformatics, more specifically, the present invention relates to the method for a kind of screening siRNA (smallinterfering RNA, small molecules interference RNA).The invention still further relates to autogenous siRNA sequence and application thereof by the mankind of this method screening.

Background technology

Micro RNAs (miRNAs) is the long noncoding single stranded RNA molecules of a big class～22 nucleotide.On evolving, they extensively are present in from plant, nematode to the mankind's cell ^[1]That find the earliest is lin-4 and its said target mrna, i.e. lin-14 ^[2]Lee etc. ^[2]In C.elegans (nematode), cloned the lin-4 gene with the method for the positional cloning of classics in 1993, and found not encoding proteins of lin-4, but produced a kind of small RNA molecular by rite-directed mutagenesis.This small RNA molecular can interact with the specific region of 3 ' untranslated end of the mode of incomplete complementation and its said target mrna and suppress the expression of lin-14, finally causes the minimizing of lin-14 protein synthesis, and this phenomenon is called translates inhibition.Found that lin-4 is controlling the C.elegans larva by the conversion of L1 phase to the L2 phase.Does why a RNA molecule that has only 22nt play important regulatory role like this? people can't explain at that time, can only think a kind of some phenomena of rareness.But, second miRNA let-7 in 2000 ^[3]And the discovery of homologue in human and the fruit bat, having changed people's view, miRNA may be that a class is evolved and gone up the molecule of guarding that plays the important regulating and controlling effect in life.They can suppress the synthetic of related protein effectively, can cause the degraded of said target mrna, and perhaps the regulation mechanism of other form suppresses target gene expression, produce gene silencing ^[4,5]Discovered in recent years miRNA may play vital role beyond imagination in the gene expression regulation field, the diversity of miRNA sequence, structure, abundance and expression way makes it may be as strong adjusting of protein coding mRNA.The understanding of people to protein synthesis control has been enriched in the discovery of miRNA, has replenished at rna level the said target mrna molecule is carried out rapider and effective adjusting, has represented the comprehensive multi-level network system of gene expression regulation in the cell ^[6]The discovery of miRNA also is the important supplement to the less important role of intermediary of RNA in the central dogma, and it will impel the biologist to think deeply the major issue of aspects such as cytogenetics regulation and control and growth thereof again.

The major function of miRNA is that cell is regulated and control gene expression at rna level, and its adjusting is similar to siRNA, can suppress the synthetic of related protein, also can cause the cracking of said target mrna, produces gene silencing.Recently find that miRNA may play vital role beyond imagination in the gene expression regulation field.The diversity of miRNA sequence, structure, abundance and expression way makes it may be as strong adjusting of protein coding mRNA.The understanding of people to protein synthesis control has been enriched in the discovery of miRNA, has replenished at rna level the said target mrna molecule is carried out rapider and effective adjusting, has represented the comprehensive multi-level network system of gene expression regulation in the cell.The discovery of miRNA also is the important supplement to the less important role of intermediary of RNA in the central dogma, and it will impel the biologist to think deeply the major issue of aspects such as cytogenetics regulation and control and growth thereof again.Along with going deep into of research, miRNA will play more far-reaching effect in origin of life and spore, complicacy, the disease generation of gene expression regulation and the aspects such as mechanism that develop.Simultaneously, the research of miRNA also will provide new foundation, thinking and space for the RNAi The Application of Technology.Now, developed countries such as America and Europe will extensively and in depth apply to the research of relevant miRNA the orientation growth of histoorgan, the space-time of cell Growth and Differentiation is regulated, the opening and closing of signal path, the monitoring of cell cycle and regulation and control, learning and Memory, the contrary differentiation of tumour, fat, old and feeble and dead is on the control of disease and the autotelic gene expression regulation.This should cause the close attention of China scientific circles.

Be easy to associate another small RNA molecular-siRNA from miRNA, they have the very strong function of closing gene, can disturb (RNA interference by mediate rna, RNAi) phenomenon, and instruct specific said target mrna to degrade, the gene regulatory system of siRNA mediation is an intracellular natural antagonism foreign heredity substance such as virus and the genetic immunization system that monitors the spatial and temporal expression of inherent gene.This system utmost point promises to be the identified gene function, and regulate gene expression and modifying gene show a kind of strategy approach of characteristic outward.People can copy the pharmacy pattern of the Nature to design a synthetic ectogenic 19-21 siRNA, carry out the drug therapy of related gene.The research and development of the biological strategic arms of this siRNAs are just causing showing great attention to of world research mechanism and biological medicine company, the silence that Britain and Holland jointly plan to finish at 2004-2005 with the RNAi perturbation technique whole 35000 human genes.The U.S. plans to come 1 with the RNAi perturbation technique) set up the siRNA library of molecules: intend finding out losing of those genes and will cause tumour, those genes lose that death, those genes that will cause tumour cell are relevant with the fat adjusting of human body, the aging of those genes and human body is about relevant with the stability of those genes and human genome; 2) the new gene target spot of screening treatment tumour; 3) research and development are the based gene medicine with RNAi; 3) specificity and the mechanism of action of appraisal new drug.The direct achievement of these researchs will be impelled human widespread use to gene expression product, drive farming, woods simultaneously, will herd, the development of related industries such as doctor, medicine, gardening, make biological industry become the leading industry of 21 century.

What astonish is that big pharmaceuticals falls over each other to add, as Novartis, and Aventis, GSK, Merck, Ffize ^r, Abbot Pharmacia, AstraZeneca, P ﹠amp; G, Wyeth, Roche, Bristol-Myers Squibb, Amgen, Schering-Plough, Procter﹠amp; Gamble, Glaxo-SmithKline, Agouron, Genentech Rigel Pharmaceuticals, power-power cooperation constantly occurs, as Merk-Alnylam, Affymatrix-Qiagen, Lily-ISIS etc.Be difficult to the medicine and the biotech company that find a family not use the RNAi technology in the U.S..Even to this day, risk investment has showed to such an extent that " drooled with envy " to the RNAi technology.At the beginning of setting up, the Alnylam drugmaker of RNAi drug development company that one family is positioned at Cambridge, Massachusetts just obtained 1,750 ten thousand dollars risk investment---know, risk investment is dripped only not to the biotech company of firm establishment and is not executed in 2 years in the past, and Alnylam can obtain so writing investment." undoubtedly, the RNA perturbation technique be in the past few years in institute's revolutionary breakthrough appearing in one's mind out, " the first executive officer John Maraganore of Alnylam company mentions.Main scientist of the former Millennium Pharmaceuticals before this of John Maraganore (Millennium Pharmaceuticals)." RNAi will become the main drug categories after recombinant protein and monoclonal antibody." he says.Ribozyme company has also obtained 4,800 ten thousand dollars of commercial developments that carry out RNAi.Ribozyme wishes can proceed to a clinical trial phase by 2005.The first executive officer Howard Robin of Ribozyme drugmaker says, RNAi is through chemistry and the pharmacological research of nearly several years, " going to be engaged in this for the great confidence of company can infusive field of medicaments commercialized development ".In August, 2004, U.S. Acuity company submits the clinical application of I phase of first RNAi medicine to FDA.In a word, the research and development of RNAi genomic medicine will bring a revolutionary change to field of biological pharmacy.

Fundamental research and medicinal industry exploitation having become the most important commanding elevation that countries in the world are seized in the biotechnology field about the RNA EVAC (Evacuation Network Computer Model).Nobel laureate Sharp and Boshor think the RNA The Study of Interference will be the most breathtaking in the biological study in following 10 years also be most possible one of the field of achievement of enriching that produces.But do not find human autogenous siRNA so far in the world as yet, so the present invention is very important to the announcement of human autogenous siRNA and its target gene, it will promote the research of little RNA greatly, mechanism of action to RNA provides the value research data, and research provides new approach to human diseases.

Summary of the invention

It is one that the present invention collects RNA perturbation technique, human body gene library searching technology, biocomputer infotech, algorithmic language and technique for gene engineering, researches and develops the new endogenic siRNA molecule of a class.Fundamental purpose of the present invention is:

1. create a kind of prediction and the existence of screening autogenous siRNA gene and the algorithmic language and the method for their target gene.

2. the subarea that includes of proof encoding gene exists the autogenous siRNA molecule, and they are different from the miRNA molecule of having found.

3. describe the sequential structure of 255 new autogenous siRNA molecules in detail.

4. the biology of biometric authentication autogenous siRNA molecule takes place.

5. predict the target gene of these autogenous siRNA molecules and may using of they.

Detailed Description Of The Invention

1. vocabulary of terms

In the context of the present invention, used term " 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 derivant are by wide coverage.Those nucleotide of being modified or substituting usually are 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.This autogenous siRNA molecule of the present invention is the RNA or the dna molecular of homology in cell of deactivation effectively.SiRNA molecule of the present invention is including, but not limited to those thiophosphate oligonucleotides and other the oligonucleotide through modifying.

Term as used herein " autogenous siRNA molecule " is meant that has the long specific nucleic acid binary of 19-25 nucleotide.The a certain dna fragmentation 100% of its just anticipate chain and its target gene identical, its antisense strand can with corresponding mRNA molecular hyridization, guiding RISC/RITC suppresses this target cdna molecule specifically, and function that can reticent other RNA molecule.

Term as used herein " homologous nucleic acid or homologous sequence ", the dna molecular that comprises the RNA of those energy encoding proteins and other function, the RNA molecule that produces from these DNA comprises immature mRNA, the identical segments in ripe mRNA and other RNA molecule and these dna moleculars.The interaction of autogenous siRNA 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 autogenous siRNA 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 (from producing the place of albumen to it in examining) RNA, 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 autogenous siRNA molecular energy specifically with the nucleic acid interaction of a plurality of homologies, has advantages of higher stability and validity, but aim of the present invention mainly is to disclose human autogenous siRNA and its target gene, promote the research of little RNA, mechanism of action to RNA provides the value material, and research provides new approach to human diseases.

2. the design of algorithmic language

We have developed a kind of method of computer forecast, it integrates multiple algorithmic language such as Smith-Whatman, HMM, neuronetwork etc. are in one, this algorithm has added the program of RNA secondary structure prediction, can also add systematic growth relatively from the stability of dynamics angle prediction siRNA gene and siRNA molecule, to determine the conservative property of sequence.We are called the dynamic system planning algorithm.This method has been considered following characteristics: 1. the oppositely complete complementary pairing of local location must be greater than 21nt; 2.RNA-RNA the prediction of double-stranded free energy; 3. the minimum free energy of target site; 4. hunting speed is fast and accurate.

3. write prediction and screening software

Autogenous siRNA is the loop-stem structure precursor by one～150nt, and guards on evolving, so the method for available computers is discerned.We develop a kind of new screening software according to three characteristics of siRNA, 1. must come from intension of encoding gene, produce the conservative property of siRNA, need to form the precursor of one～100nt; 2. in similar species, siRNA is very conservative; 3. between the species of apart from each other, siRNA has certain difference.It comprises following three steps: 1. the sequence of seeking the oppositely complete complementary pairing in the human encoding gene introne by software of the present invention; 2. recognize by software whether this sequence can form conservative loop-stem structure, and give this structure evaluation; 3. estimate the difference pattern of siRNA in different plant species.At last, by biochemical method, verified.

4. structure database

Making up database is the essential condition that autogenous siRNA is predicted and screened, so we have set up following relevant database:

Human body gene storehouse size 100,000MB;

Introne database scale 100MB;

MRNA sequence library 80MB;

And other relevant nucleic acid database as:

3’UTR；

5’UTR；

Promoter;

MiRNA；

Little RNA and;

The DNA database of other species etc.

5. predict and screening autogenous siRNA molecule

The present invention introduces one can search for autogenous siRNA gene computing method effectively.Identify that oppositely accurate complementation is the committed step that whether contains the autogenous siRNA gene in special genomic dna sequence of search.Because the autogenous siRNA gene should contain one greater than the long distinguished sequence of 21 nucleotide, it can suppress the function of corresponding target gene effectively.This sequence is highly keeping the composition and the order of original nucleotide all the time through the long river of very long Natural Selection and Evolution.Therefore, the autogenous siRNA gene is a kind of mechanism as the encoding gene regulation and control.Simultaneously, it also can save the time of the special genomic dna sequence of search as a kind of genomic medicine.

Though finishing of human genomic sequence provides a gene of studying most of encoding proteins, the biological information storehouse of tRNA and rRNA is identified the gene of those non-encoding proteins rapidly but still have any problem.Especially these novel autogenous siRNA sequences are out in the cold chronically.The present invention proposes the high conserved sequence of RNA, and particularly oppositely accurately complementary dried ring texture can be used as an important identification mark, and it can be used to screen the siRNA sequence.Computer software method such as dynamic programming language that the present invention uses us to create are searched for the siRNA molecule with other the special computers software that can predict the dried ring texture of RNA in the human genome information bank.In addition, other several different methods also are useful, seek structure with the common property special seized with terror of microRNA as computing machine, microarray assays between gene and they include in the subarea complementary series and from the protein that combines with RNA such as desaminase (siRNA is in conjunction with albumen) the isolation of RNA molecule.In a word, whether search exists the function steps necessary that corresponding autogenous siRNA molecule is the little RNA of research in the human genome information bank.

SiRNA is defined as a class RNA molecule, and their complete open reading frames of not encoding, these RNA molecules from different genera have high conservative sequence.According to the conservative degree of sequence randomly in the introne zone of non-encoding gene zone or gene screening siRNA sequence be a kind of feasible method.Therefore, the present invention proposes to screen siRNA sequence standard and is:

The dried ring texture of RNA,

High conservative cadre zone,

The length in cadre zone is 23-45 nucleotide,

Its precursor can produce a siRNA to several maturations,

This sequence is positioned at the subarea that includes of encoding gene.

All possible siRNA 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 in the gene intron district of all kinds and data.This has constituted the major obstacle of searching for corresponding siRNA sequence.Yet we have researched and developed special software now.The principle that these special softwares adopt is that everybody knows: that is exactly, and the first area of a nucleotide accurately is 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 23-45 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 thymine in adenine in the first area and the second area or uracil in antiparallel mode is connected.Similarly, a cytimidine in the first area can also match in antiparallel mode mutually with the guanine in the second area.Length according to conservative degree of sequence and conserved sequence, the sequence that includes the subarea to this encoding gene of coming from human body, nematode and fruit bat is measured, found that the highly conserved sequence of two class nucleotide, a class contains the long accurate complementary preface of 23 nucleotide.The another kind of long accurate complementary series of 45 nucleotide that contains.But this two class all contains a ring that differs in size, and is made up of 20 to 100 nucleotide.This shows that encoding gene includes a dried ring texture in subarea and can be used as the standard of a siRNA sequence of evaluation greater than the high conserved dna sequence of 21 length of nucleotides.

6. target gene

Cross thousand miRNA though found now, and propose them and in cell proliferation, differentiation, metabolism and death, bringing into play important regulatory role, but up to now, the miRNA of real affirmation function still is very little, found first miRNA till now from 1993, the miRNA of the affirmation function of finding in animal is no more than ten.Yet we have searched for human body mRNA database and have found that each autogenous siRNA molecule all has more than one target gene, and most target gene can reach more than 100, adopt the ICT-Blast program that their target genes have been carried out further checking.Now row cite an actual example as follows:

The target gene of table 1.SEQ ID No.1.

Access?No	Gene?Name	Region
			NM_012468.3	T-cell?leukemia/lymphoma?6	5’UTR(861-878)
NM_018672.2	ATP-binding?cassette，sub-family?A	5’UTR(85-101)
			AF083390	WHSC1?protein(WHSC1)mRNA，alternative?splice	5’UTR(39-57)
NM_014919.1	Wolf-Hirsch?horm?syndrome?candidate?1	5’UTR(38-55)
			NM_198506.1	FLJ44691?protein(FLJ44691)，	5’UTR(38-55)
NM_014925.2	KIAA1002?protein	5’UTR(205-222)
			XM_498589.1	Hypothetical?gene?supported?by?AK128797	5’UTR(2029-2046)
XM_371108.3	Similar?to?KIAA1314?protein(LOC388462)，	5’UTR(1498-1615)
			NM_032378	Eukaryotic?translation?elongation?factor?1?delta(EEF1D)	5’UTR(136-153)
AF452411.1	AKT2	5’UTR(72-89)
			AF148213	Aggrecanase-1	3’UTR(4085-4102)
NM_130786.2	Alpha-1-B?glycoprotein(A1BG)	3’UTR(3136-3153)
			NM_017873.2	Ankyrin?repeat?and?SOCS?box-containing?6	3’UTR(3063-3080)
NM_181869	APAF1	3’UTR(2405-2512)
			NM_003661	APOL1	3’UTR(1666-1683)
NM_030882	APOL2	3’UTR(1674-1690)
			NM_053286.1	Aquaporin?6，kidney?specific(AQP6)，	3’UTR(4428-4445)
AF084555	ARPP-19	3’UTR(4147-4164)
			AF082283	BCL10	3’UTR(2390-2407)

NM_001745	CAMLG	3’UTR(1981-1998)
			AF196175	Capsaicin?receptor	3’UTR(3292-3308)
NM_014959	CARD8	3’UTR(4260-4276)
			NM_033357.1	Caspase?8，apoptosis-related?cysteine?protease	3’UTR(2143-2160)
NM_033355.1	Caspase?8，apoptosis-related?cysteine?protease	3’UTR(2018-2035)
			NM_033356.1	Caspase?8，apoptosis-related?cysteine?protease	3’UTR(1794-1810)
NM_033332.1	CDC14?cell?division?cycle?14?homolog?B	3’UTR(2682-2699)
			NM_138413.2	Chromosome?10?open?reading?frame?65	3’UTR(1922-1939)
NM_014356.2	Chromosome?6?open?reading?frame?123(C6orf123)	3’UTR(785-802)
			NM_017437.1	Cleavage?and?polyadenylation?specific?factor?2	3’UTR(3988-4015)
AF439325	CRF2/12	3’UTR(4026-4043)
			NM_000761.2	Cytochrome?D450，family?1	3’UTR(2588-2605)
AF313469	Dendritic?cell-associated?C-type?lectin-1?beta	3’UTR(1148-1165)
			NM_024423.1	Desmocollin?3	3’UTR(5038-5055)
NM_001941.2	Desmocollin?3(DSC3)，transcript?variant?Dsc3a	3’UTR(4994-5010)
			AB037161	DSCR6d	3’UTR(1159-1176)
NM_022549.2	Fasciculation?and?elongation?protein?zeta?1	3’UTR(1484-1501)
			NM_012174.1	F-box?and?WD-40?domain?protein?8(FBXW8)，	3’UTR(3372-3389)
NM_133279.1	FC?fragment?of?IgA	3’UTR(1310-1327)

7. Bioexperiment proves

See inventive embodiment and optimum implementation.

Compare all advantages with prior art

Comparing the advantage that the present invention has with prior art comprises:

Brand-new design and development are theoretical: the autogenous siRNA molecule that exists some in the introne of encoding gene, this molecule has the scapus structure greater than 21 base-pairs of complete reverse complemental, the loop sample structure that 20-100 base constitutes, and have complete complementation greater than the long said target mrna of 21 bases.

Brand-new sequential structure: comprise that with existing ncRNA and other little RNA the sequence of the RNA of miRNA database compares, all little RNA all are newfound siRNA molecules.

New and binding mode target gene: different with endogenous miRNA molecule, each autogenous siRNA molecule all has more than one said target mrna, and the different parts target spot is arranged as handling the subarea, 3 ' UTR, 5 ' UTR includes the subarea, with different binding mode as handling the subarea dna methylation, inhibition etc. is translated in the degraded cutting.

Description of drawings

Fig. 1. the gene pairs telephone network of little RNA (siRNA/miRNA) mediation.

Fig. 2. deposit the source book table of newfound siRNA molecule.

Fig. 3. the precursor of autogenous siRNA and ripe siRNA

Fig. 4. autogenous siRNA is to the effect of genetically manipulated subarea dna methylation.

Fig. 5. autogenous siRNA acts on 5 ' the UTR target area of a mRNA.

Fig. 6. autogenous siRNA acts on 3 ' the UTR target area of a mRNA.

Fig. 7. autogenous siRNA acts on the introne target area of a pre-mRNA

Specific embodiments

Be described in further detail understanding the present invention below in conjunction with accompanying drawing, but be not that the present invention is limited.

1. materials and methods

1.1 bioinformatics

The software that uses dynamic programming algorithm and our autonomous Design thereof and write on the dawn 4000H uniprocessor of Chinese Academy of Sciences's computer research institute, screening and predict the sequential structure of autogenous siRNA from human body introne database.In addition, from human body mRNA sequence library the search have given characteristic sequence sequence, verified with other biological information software again.About the research and analysis of sequence, we use have full-text database ( Http:// www.ensembl.org), the NIBC online resource ( Http:// www.nibc.nlm.nih.gov), the genome association network address that DOE connects ( Http:// www.igi.doe.gov), UCSC genome bioinformatics network address ( Http:// gonome.ucsc.edu), the network address of European biological information association ( Http:// www.ebi.ac.uk/clustalw/) and the folded net server ( Http:// www.bioinfo.rpi.edu/applications/mfold/old/rna).

1.2 the extraction and purification of sample gene group and siRNA plasmid

Genomic DNA utilizes conventional phenol/chloroform extracting preparation in this laboratory, the siRNA plasmid extracts with the alkaline lysis method.RNAi-Ready pSIREN-DNR-DsRed-Express (BDBiosciences Clontech) carrier is that this little RNA is from the encoding gene introne with the little RNA of reorganization pRNA vector encoded.Advance carrier with chemical method synthetic oligonucleotide and insertion, in addition, we have also made up the complex carrier that has luciferase or contain fluorescent protein report gene.

1.3 the cultivation of cell and transfection

The human breast cancer cell is that MCF-7 and human body cervix neoplasms clone Hela are that (Rockville MD) obtains from ATCC.Cell is grown in DMEM, is supplemented with the penicillin of 100U/ml and the streptomysin of 100 μ g/ml in the nutrient culture media, contains 5% CO2 in the humidifying air of 10% heat-labile FBS under 37 ℃.Oncocyte is cultivated in each hole of six porose discs, and the carrier impact damper after the processing is independently controlled each group.Transfection in Prna carrier (1 μ g) or the empty carrier again, with lipofectamine2000 according to the description of product go to carry out (Life Technologies, NewEngland).

1.4 design of primers and probe mark

According to a pair of primer of 5 ' end design of the sequence of relating to persons gene, the PCR product is used for probe mark after cloning and sequencing is identified.Primer is given birth to worker's bioengineering company limited by Shanghai and is synthesized.Primer sequence is determined pF and pR according to the gene of being studied.The general procedure that is used for the Southem hybridization of ripe siRNA is to extract total little RNA, and pulse electrophoresis is transferred on the nylon membrane, with PCR-DIG, or the hybridization of isotope-labeled probe hybridization liquid.

Total RNA separates with TRI REAGENT (Molecular Research Center).CDNA is transported out with SUPERSCRIPT II reverse transcriptase effect 30min down at 50 ℃ by reverse transcription by total RNA of 2 μ g.Its result make cDNA mainly by pcr amplification and specific primer (1 μ M) in 20 μ l potpourris.Usually, carry out amplification cycles 35 times with the TaqDNA polymerase of Promage company, the agar that the PCR product adds 1-2% carries out gel electrophoresis.Circulation law determines to realize optimization in the PCR of each experiment response line sexual stage.

1.5 luciferase analysis

Two luciferase analyses are the instructions according to manufacturer, and 24 hours carry out after transfection with an OptoompI illuminometer (MGM Instruments).Insert Rr-luc (Promega) destination carrier of autogenous siRNA homologous fragment and the carrier cotransfection cell of only expressing autogenous siRNA, Pp-luc destination carrier cotransfection obtains the transfection product after 24 hours, and the activity of the plain enzyme of analysis of fluorescence continuously, identify the inhibiting effect of autogenous siRNA to target gene.

1.6Westerm?blotting

Protein extracted with former method.Total protein (40 μ g) decomposes under the 10%SDS-polyacrylamide gel electrophoresis and the band that has protein transport to move is gone to polychloroethylene film (Amersham).The sealing of film is spent the night on distant bed with 5% skimmed milk TBS, hatches primer antibody 2 hours.The Actin gene of expressing is used as and adds loaded regulate and control, is that the antibody of usefulness comprises CDC6, Actin and EEL etc.Film produced the sheep anti mouse or the anti-sheep second generation of the rabbit antibody of HRP coupling after 1 hour.Immune complex manifests by autoradiographic technique.

1.7 methylation analysis

The methylation status analysis on the CpG island on the target gene promoters be by be methylated special PCR (MSP) analytical approach as previously mentioned.DNA genome in all samples has all been isolated DNAzoL reagent (Life Technologies).The MSP primer according to the genome sequence around the target gene transcription initiation site by Methprimer ( Http:// www.urogene.org/methprimer/indexl.html) check in.The sulphite modifying DNA is distinguished with the pcr amplification primer and to be methylated and non-methylate DNA.The archaeal dna polymerase of Promage company is used for full amplification.45 PCR circulations can be carried out farther analysis.

2 results and discussion

Our hypothesis exists the gene pairs telephone network of a little RNA (siRNA/miRNA) mediation.Little RNA can be encoded by endogenous gene, is positioned between structural gene the introne or the extron district of district or structural gene, is transcribed by RNA polymerase, and its precursor contains and emits shape structure and polyadenylic acid.The siRNA/miRNA precursor of bunch change can be cut from pri-miRNA by spliosome and Drosha enzyme, forms ripe miRNA molecule under the effect of Dicer enzyme.Single miRNA can in conjunction with and a plurality of different mRNA molecules of adjusting.Equally, a plurality of different siRNA/miRNA also can combine with same mRNA molecule and regulate and control its biologically active.Along with the expression of encoding gene, the siRNA/miRNA that shears out from introne and extron can combine with the different structure zone of the target mRNA of its upstream and downstream, suppresses or cutting degraded (Fig. 1) thereby instruct to translate.

Regulate molecule in order to exist siRNA in the introne that proves the human body encoding gene, we use dynamic programming algorithm and our autonomous Design thereof and the software write Chinese Academy of Sciences's computer research dawn 4000H uniprocessor on the dna sequence dna in the human body introne database has been carried out the extensive search of system.255 new endogenous small RNA moleculars have been found for the first time.The result as shown in Figure 2.

In order to prove that 255 new endogenous small RNA moleculars in the human body encoding gene introne that we find exist really, we have studied their generation with biological method.Fig. 3 has enumerated an example.Northern Blotting can detect the existing of siRNA (SEQ ID No.1) of a maturation that derives from the BRCA1 introne.

In order to prove that this siRNA that derives from the maturation of BRCA1 introne has corresponding said target mrna molecule, we have searched for human body mRNA database, found that about 130 target mRNA (table 1).Analyze the seat of target sequence place mRNA, find that this siRNA can act on heterogeneic different loci, comprise and handle the subarea, 5 ' UTR, 3 ' UTR and include the subarea.

In order to prove that this siRNA acts on heterogeneic different loci and can produce different gene silencing effects, we have detected the situation of telomerase gene expression with methods such as MSP.Compare in control group, the cell of transfection siRNA, telomerase gene expression is subjected to obvious inhibition (Fig. 4).Simultaneously, detect ELL and the also obviously decline (Fig. 5) of gene expression that has target site at 5 ' UTR section with RT-PCR.

In addition, the gene expression that has target site at introne and 3 ' UTR section has been carried out detecting (Fig. 6 and Fig. 7), compared, do not had the difference that can detect with control group with RT-PCR.Then, we change experiment with the active height of Lucifease and show that this siRNA can suppress the target mRNA molecule of the 3 ' UTR that contains its homologous sequence, and difference is up to about 50%.

List of references

1David?P?B.MicroRNAs：Genomics，Biogenesis，Mechanism，and?Function.Cell，2004，116：281～297

2Lee，R?C，Feinbaum?R?L，Ambros，V.The?C.elegans?heterochronic?gene?lin-4?encodessmall?RNAs?with?antisense?complementarity?to?lin-14.Cell，1993，75：843～854

3Reinhart?B?J，Slack?F?J，Basson?M，et?al.The?21?nucleotide?let-7?RNA?regulatesdevelopmental?timing?in?Caenorhabditis?elegans.Nature，2000，403：901～906

4Yin?J?Q，Wang?Y.siRNA-mediated?gene?regulation?system：now?and?the?future.Int?J?MolMed，2002，10：355～65.

5Tan?F?L，Yin?J?Q.Application?of?RNAi?to?cancer?research?and?therapy.Front?Biosci，2005，01，10：1946～1960.

6Yin?J?Q，Jiang?L.MiRNA-directed?gene?intertalk.Chin?Acad?Forum，2004，11(20)：1-4

7Lee?Y，Kim?M，Han?J，et?al.MicroRNA?genes?are?transcribed?by?RNA?polymerase?II.EMBO?J，2004，23(20)：4051～60.

8Rodriguez?A，Griffiths-Jones?S，Asburst?JL，et?al.Identification?of?mammalianmicroRNA?host?genes?and?transcription?units.Genome?Res，2004，14(10A)：1902～10.

9Lee?Y，Ahn?C，Han?J，et?al.The?nuclear?RNase?III?Drosha?initiates?microRNA?processing.Nature，2003，425：415-419

10Ahmet?M.Denli，Bastiaan?B.J.Tops，Ronald?H.A.Plasterk，et.al.Processing?of?primarymicroRNAs?by?the?Microprocessor?complex.Nature，2004，432：231～5

11Gregory?R.I.，Yan?K，Amuthan?G，et.al.The?microprocessor?complex?mediates?thegenesis?of?microRNAs.Nature，2004，432：235～40

12HEN?Fang?&?YIN?Q.James(2005)Gene?expression?regulator，MicroRNAs.ChineseScience?Bulletin?50(15)1-12.

13Tan?FL?&?Yin?JQ.RNAi，a?new?therapeutic?strategy?against?viral?infection.Cell?Res.2004Dec，14(6)：460-6.

The gene silencing of 14 solicitous big (2004) siRNA mediation.Acta Biophysica Sinica 20,85-90

15Liu?TG，Yin?JQ，Shang?BY，Min?Z，He?HW，Jiang?JM，Chen?F，Zhen?YS，Shao?RG.Silencing?of?hdm2?oncogene?by?siRNA?inhibits?p53-dependent?human?breast?cancer.Cancer?Gene?The..2004?Nov?11(11)：748-56.

16Ma?JP，Zhan?WH，Wang?JP，Peng?JS，Gao?JS，Yin?Q.Specific?inhibition?of?hTERT?geneexpression?by?short?interfering?RNAs?in?gastric?cancer?SGC7901?cell.Zhonghua?Wai?KeZa?Zhi.2004?Nov?22；42(22)：1372-6.

17 Liu Tie are firm, and Shang Baiyang is solicitous big, Zhang Min, He Hongwei, Zhen Yongsu, Shao Rongguang. the antitumor action research of (2004) multiple siRNA. and Chinese Journal of New Drugs 13; 6-10

18 Tan Yu are good, and are solicitous big. (2005) SiRNA: a kind of medicine of novel treatment tumour.Acta Pharmaceutica Sinica.40(3)；193-198

19 Tan Yu are good, and are solicitous big. the application of (2005) RNAi perturbation technique in the gene defect model.Acta Genetica Sinica.32(4)；5-9

Sequence table

＜110〉Chinese Academy of Sciences's biophysics research institute

Solicitous big

＜120〉Ren Lei autogenous siRNA sequence and application thereof and screening technique

<130>siRNA

<160>255

<170>PatentIn?version?3.3

<210>1

<211>22

<212>DNA

＜213〉people

<220>

＜221〉nucleotide

<222>(1)..(22)

<400>1

ggcctcccaa?agtgctggga?tt 22

<210>2

<211>22

<212>DNA

＜213〉people

<400>2

aatcccagca?ctttgggagg?cc 22

<210>3

<211>52

<212>DNA

＜213〉people

<400>3

cccgcctcgg?cctcccaaag?tgctgggatt?acaggcgtga?gccaccgcgc?cc 52

<210>4

<211>52

<212>DNA

＜213〉people

<400>4

gggcgcggtg?gctcacgcct?gtaatcccag?cactttggga?ggccgaggcg?gg 52

<210>5

<211>26

<212>DNA

＜213〉people

<400>5

gcctcccaaa?gtgctgggat?tacagg 26

<210>6

<211>26

<212>DNA

＜213〉people

<400>6

cctgtaatcc?cagcactttg?ggaggc 26

<210>7

<211>40

<212>DNA

＜213〉people

<400>7

gtacacttgg?attgtacatc?acttttagtg?tacaaatata 40

<210>8

<211>40

<212>DNA

＜213〉people

<400>8

tatatttgta?cactaaaagt?gatgtacaat?ccaagtgtac 40

<210>9

<211>22

<212>DNA

＜213〉people

<400>9

tacaattgta?tcataaaatat?at 22

<210>10

<211>22

<212>DNA

＜213〉people

<400>10

atatatttat?gatacaattg?ta 22

<210>11

<211>26

<212>DNA

＜213〉people

<400>11

gtttttatgg?ttttaggtct?aacgtt 26

<210>12

<211>26

<212>DNA

＜213〉people

<400>12

aacgttagac?ctaaaaccat?aaaaac 26

<210>13

<211>24

<212>DNA

＜213〉people

<400>13

ctcccaaagt?gctgggatta?cagg 24

<210>14

<211>24

<212>DNA

＜213〉people

<400>14

cctgtaatcc?cagcactttg?ggag 24

<210>15

<211>24

<212>DNA

＜213〉people

<400>15

ctcccaaagt?gctgggatta?cagg 24

<210>16

<211>24

<212>DNA

＜213〉people

<400>16

cctgtaatcc?cagcactttg?ggag 24

<210>17

<211>23

<212>DNA

＜213〉people

<400>17

ccaggctggt?cttgaactcc?tgg 23

<210>18

<211>23

<212>DNA

＜213〉people

<400>18

ccaggagttc?aagaccagcc?tgg 23

<210>19

<211>32

<212>DNA

＜213〉people

<400>19

cccgcctcgg?cctcccaaag?tgctgggatt?ac 32

<210>20

<211>32

<212>DNA

＜213〉people

<400>20

gtaatcccag?cactttggga?ggccgaggcg?gg 32

<210>21

<211>31

<212>DNA

＜213〉people

<400>21

ccttggcctc?ccaaagtgct?gggattacag?g 31

<210>22

<211>31

<212>DNA

＜213〉people

<400>22

cctgtaatcc?cagcactttg?ggaggccaag?g 31

<210>23

<211>22

<212>DNA

＜213〉people

<400>23

gcctcccaaa?gtgctgggat?ta 22

<210>24

<211>22

<212>DNA

＜213〉people

<400>24

taatcccagc?actttgggag?gc 22

<210>25

<211>24

<212>DNA

＜213〉people

<400>25

tcccaaagtg?ctgggattac?aggc 24

<210>26

<211>24

<212>DNA

＜213〉people

<400>26

gcctgtaatc?ccagcacttt?ggga 24

<210>27

<211>22

<212>DNA

＜213〉people

<400>27

gtgctgggat?tacaggcatg?ag 22

<210>28

<211>22

<212>DNA

＜213〉people

<400>28

ctcatgcctg?taatcccagc?ac 22

<210>29

<211>21

<212>DNA

＜213〉people

<400>29

tatatatgta?tacatatata?t 21

<210>30

<211>21

<212>DNA

＜213〉people

<400>30

atatatatgt?atacatatat?a 21

<210>31

<211>21

<212>DNA

＜213〉people

<400>31

atatatatgt?atacatatat?a 21

<210>32

<211>21

<212>DNA

＜213〉people

<400>32

tatatatgta?tacatatata?t 21

<210>33

<211>21

<212>DNA

＜213〉people

<400>33

tatatatgta?tacatatata?t 21

<210>34

<211>21

<212>DNA

＜213〉people

<400>34

atatatatgt?atacatatat?a 21

<210>35

<211>21

<212>DNA

＜213〉people

<400>35

tatatatgta?tacatatata?t 21

<210>36

<211>21

<212>DNA

＜213〉people

<400>36

atatatatgt?atacatatat?a 21

<210>37

<211>21

<212>DNA

＜213〉people

<400>37

tatatatgta?tacatatata?t 21

<210>38

<211>21

<212>DNA

＜213〉people

<400>38

atatatatgt?atacatatat?a 21

<210>39

<211>22

<212>DNA

＜213〉people

<400>39

ctttcactta?gcataatgtt?tt 22

<210>40

<211>22

<212>DNA

＜213〉people

<400>40

aaaacattat?gctaagtgaa?ag 22

<210>41

<211>48

<212>DNA

＜213〉people

<400>41

gcggtgagcc?gagatcgcgc?cactgcactc?cagcctgggc?gacagagc 48

<210>42

<211>48

<212>DNA

＜213〉people

<400>42

gctctgtcgc?ccaggctgga?gtgcagtggc?gcgatctcgg?ctcaccgc 48

<210>43

<211>21

<212>DNA

＜213〉people

<400>43

cctcccaaag?tgctgggatt?a 21

<210>44

<211>21

<212>DNA

＜213〉people

<400>44

taatcccagc?actttgggag?g 21

<210>45

<211>28

<212>DNA

＜213〉people

<400>45

ggcctcccaa?agtgctggga?ttacaggc 28

<210>46

<211>28

<212>DNA

＜213〉people

<400>46

gcctgtaatc?ccagcacttt?gggaggcc 28

<210>47

<211>39

<212>DNA

＜213〉people

<400>47

gcctcccaaa?gtgctgggat?tacaggcgtg?agccaccgc 39

<210>48

<211>39

<212>DNA

＜213〉people

<400>48

gcggtggctc?acgcctgtaa?tcccagcact?ttgggaggc 39

<210>49

<211>24

<212>DNA

＜213〉people

<400>49

agcctcccaa?agtgctggga?ttac 24

<400>50

gtaatcccag?cactttggga?ggct 24

<210>51

<211>22

<212>DNA

＜213〉people

<400>51

gggattacag?gcgtgagcca?cc 22

<210>52

<211>22

<212>DNA

＜213〉people

<400>52

ggtggctcac?gcctgtaatc?cc 22

<210>53

<211>23

<212>DNA

＜213〉people

<400>53

acagagcgag?actccgtctc?aaa 23

<210>54

<211>23

<212>DNA

＜213〉people

<400>54

tttgagacgg?agtctcgctc?tgt 23

<210>55

<211>46

<212>DNA

＜213〉people

<400>55

gcctggccag?gttttatgct?ttttaagaca?attttgggct?gggcac 46

<210>56

<211>46

<212>DNA

＜213〉people

<400>56

gtgcccagcc?caaaattgtc?ttaaaaagca?taaaacctgg?ccaggc 46

<210>57

<211>23

<212>DNA

＜213〉people

<400>57

cctcggcctc?ccaaagtgct?ggg 23

<210>58

<211>23

<212>DNA

＜213〉people

<400>58

cccagcactt?tgggaggccg?agg 23

<210>59

<211>23

<212>DNA

＜213〉people

<400>59

cctcggcctc?ccaaagtgct?ggg 23

<210>60

<211>23

<212>DNA

＜213〉people

<400>60

cccagcactt?tgggaggccg?agg 23

<210>61

<211>24

<212>DNA

＜213〉people

<400>61

taatccgccc?gcctcggcct?ccca 24

<210>62

<211>24

<212>DNA

＜213〉people

<400>62

tgggaggccg?aggcgggcgg?atta 24

<210>63

<211>28

<212>DNA

＜213〉people

<400>63

gcctcccaaa?gtgctgggat?tacaggcg 28

<210>64

<211>28

<212>DNA

＜213〉people

<400>64

cgcctgtaat?cccagcactt?tgggaggc 28

<210>65

<211>25

<212>DNA

＜213〉people

<400>65

ctccactaaa?aatacaaaaaa?ttagc 25

<210>66

<211>25

<212>DNA

＜213〉people

<400>66

gctaattttt?gtatttttag?tggag 25

<210>67

<211>30

<212>DNA

＜213〉people

<400>67

ctcggcctcc?caaagtgctg?ggattacagg 30

<210>68

<211>30

<212>DNA

＜213〉people

<400>68

cctgtaatcc?cagcactttg?ggaggccgag 30

<210>69

<211>26

<212>DNA

＜213〉people

<400>69

gcctcccaaa?gtgctgggat?tacagg 26

<210>70

<211>26

<212>DNA

＜213〉people

<400>70

cctgtaatcc?cagcactttg?ggaggc 26

<210>71

<211>21

<212>DNA

＜213〉people

<400>71

gcctcccaaa?gtgctgggat?t 21

<210>72

<211>21

<212>DNA

＜213〉people

<400>72

aatcccagca?ctttgggagg?c 21

<210>73

<211>25

<212>DNA

＜213〉people

<400>73

ctccactaaa?aatacaaaaa?ttagc 25

<210>74

<211>25

<212>DNA

＜213〉people

<400>74

gctaattttt?gtatttttag?tggag 25

<210>75

<211>25

<212>DNA

＜213〉people

<400>75

atatatataa?atatatttat?ataaa 25

<210>76

<211>25

<212>DNA

＜213〉people

<400>76

tttatataaa?tatatttata?tatat 25

<210>77

<211>24

<212>DNA

＜213〉people

<400>77

ctcccaaagt?gctgggatta?cagg 24

<210>78

<211>24

<212>DNA

＜213〉people

<400>78

cctgtaatcc?cagcactttg?ggag 24

<210>79

<211>23

<212>DNA

＜213〉people

<400>79

cccaaagtgc?tgggattaca?ggc 23

<210>80

<211>23

<212>DNA

＜213〉people

<400>80

gcctgtaatc?ccagcacttt?ggg 23

<210>81

<211>24

<212>DNA

＜213〉people

<400>81

gcctcccaaa?gtgctgggat?taca 24

<210>82

<211>24

<212>DNA

＜213〉people

<400>82

tgtaatccca?gcactttggg?aggc 24

<210>83

<211>24

<212>DNA

＜213〉people

<400>83

gcctcccaaa?gtgctgggat?taca 24

<210>84

<211>24

<212>DNA

＜213〉people

<400>84

tgtaatccca?gcactttggg?aggc 24

<210>85

<211>23

<212>DNA

＜213〉people

<400>85

tcccaaagtg?ttgggattac?agg 23

<210>86

<211>23

<212>DNA

＜213〉people

<400>86

cctgtaatcc?caacactttg?gga 23

<210>87

<211>134

<212>DNA

＜213〉people

<400>87

catttcgcat?tatttcatgt?aacttgaacc?tattgcttca?aaaatagcac?agaacgaatg 60

ttgattctgt?ttctttcatt?aaaaaatgtg?ctactgtgca?ttttttccca?cttctacgac 120

tttaaaggcg?cgcg 134

<210>88

<211>27

<212>DNA

＜213〉people

<400>88

gctgggatta?caggcgtgag?ccaccac 27

<210>89

<211>27

<212>DNA

＜213〉people

<400>89

gtggtggctc?acgcctgtaa?tcccagc 27

<210>90

<211>21

<212>DNA

＜213〉people

<400>90

tatatatagt?agttatatat?a 21

<210>91

<211>21

<212>DNA

＜213〉people

<400>91

tatatataac?tactatatat?a 21

<210>92

<211>29

<212>DNA

＜213〉people

<400>92

tatatttata?tataaatata?tttatatat 29

<210>93

<211>29

<212>DNA

＜213〉people

<400>93

atatataaat?atatttatat?ataaatata 29

<210>94

<211>28

<212>DNA

＜213〉people

<400>94

tcccaaagtg?ctgggattac?aggcgtga 28

<210>95

<211>28

<212>DNA

＜213〉people

<400>95

tcacgcctgt?aatcccagca?ctttggga 28

<210>96

<211>26

<212>DNA

＜213〉people

<400>96

tgctgggatt?acaggcgtga?gccact 26

<210>97

<211>26

<212>DNA

＜213〉people

<400>97

agtggctcac?gcctgtaatc?ccagca 26

<210>98

<211>41

<212>DNA

＜213〉people

<400>98

ctcggcctcc?caaagtgctg?ggattacagg?catgagccac?t 41

<210>99

<211>41

<212>DNA

＜213〉people

<400>99

agtggctcat?gcctgtaatc?ccagcacttt?gggaggccga?g 41

<210>100

<211>25

<212>DNA

＜213〉people

<400>100

ctcccaaagt?gctgggatta?caggc 25

<210>101

<211>25

<212>DNA

＜213〉people

<400>101

gcctgtaatc?ccagcacttt?gggag 25

<210>102

<211>26

<212>DNA

＜213〉people

<400>102

atatattata?tgatatatta?tattat 26

<210>103

<211>26

<212>DNA

＜213〉people

<400>103

ataatataat?atatcatata?atatat 26

<210>104

<211>45

<212>DNA

＜213〉people

<400>104

atatattata?ttatatgata?tattatatga?tatattatat?tatat 45

<210>105

<211>45

<212>DNA

＜213〉people

<400>105

atataatata?atatatcata?taatatatca?tataatataa?tatat 45

<210>106

<211>40

<212>DNA

＜213〉people

<400>106

atatattata?tgatatatta?tattatatga?tatattatat 40

<210>107

<211>40

<212>DNA

＜213〉people

<400>107

atataatata?tcatataata?taatatatca?tataatatat 40

<210>108

<211>27

<212>DNA

＜213〉people

<400>108

tatattatat?gatatattat?attatat 27

<210>109

<211>27

<212>DNA

＜213〉people

<400>109

atataatata?atatatcata?taatata 27

<210>110

<211>25

<212>DNA

＜213〉people

<400>110

atatattata?tgatatatta?tatta 25

<210>111

<211>25

<212>DNA

＜213〉people

<400>111

taatataata?tatcatataa?tatat 25

<210>112

<211>25

<212>DNA

＜213〉people

<400>112

atatattata?tgatatatta?tatta 25

<210>113

<211>25

<212>DNA

＜213〉people

<400>113

taatataata?tatcatataa?tatat 25

<210>114

<211>29

<212>DNA

＜213〉people

<400>114

aagtgctggg?attacaggcg?tgagccacc 29

<210>115

<211>29

<212>DNA

＜213〉people

<400>115

ggtggctcac?gcctgtaatc?ccagcactt 29

<210>116

<211>29

<212>DNA

＜213〉people

<400>116

aagtgctggg?attacaggcg?tgagccacc 29

<210>117

<211>29

<212>DNA

＜213〉people

<400>117

ggtggctcac?gcctgtaatc?ccagcactt 29

<210>118

<211>29

<212>DNA

＜213〉people

<400>118

aagtgctggg?attacaggcg?tgagccacc 29

<210>119

<211>29

<212>DNA

＜213〉people

<400>119

ggtggctcac?gcctgtaatc?ccagcactt 29

<210>120

<211>29

<212>DNA

＜213〉people

<400>120

aagtgctggg?attacaggcg?tgagccacc 29

<210>121

<211>29

<212>DNA

＜213〉people

<400>121

ggtggctcac?gcctgtaatc?ccagcactt 29

<210>122

<211>24

<212>DNA

＜213〉people

<400>122

gggtttcacc?atgttggcca?ggct 24

<210>123

<211>24

<212>DNA

＜213〉people

<400>123

agcctggcca?acatggtgaa?accc 24

<210>124

<211>23

<212>DNA

＜213〉people

<400>124

agaatatttt?ggattcttaa?taa 23

<210>125

<211>23

<212>DNA

＜213〉people

<400>125

ttattaagaa?tccaaaatat?tct 23

<210>126

<211>27

<212>DNA

＜213〉people

<400>126

tggcctccca?aagtgctggg?attacag 27

<210>127

<211>27

<212>DNA

＜213〉people

<400>127

ctgtaatccc?agcactttgg?gaggcca 27

<210>128

<211>21

<212>DNA

＜213〉people

<400>128

ctttaggagg?ccgaggcagg?a 21

<210>129

<211>21

<212>DNA

＜213〉people

<400>129

tcctgcctcg?gcctcctaaa?g 21

<210>130

<211>24

<212>DNA

＜213〉people

<400>130

gcctcggcct?cccaaagtgc?tggg 24

<210>131

<211>24

<212>DNA

＜213〉people

<400>131

cccagcactt?tgggaggccg?aggc 24

<210>132

<211>29

<212>DNA

＜213〉people

<400>132

ctccagcctg?ggcaacaaga?gcaagactc 29

<210>133

<211>29

<212>DNA

＜213〉people

<400>133

gagtcttgct?cttgttgccc?aggctggag 29

<210>134

<211>29

<212>DNA

＜213〉people

<400>134

ctccagcctg?ggcaacaaga?gcaagactc 29

<210>135

<211>29

<212>DNA

＜213〉people

<400>135

gagtcttgct?cttgttgccc?aggctggag 29

<210>136

<211>41

<212>DNA

＜213〉people

<400>136

ctcttataaa?tagtggacca?tcaccctata?cccactaaat?t 41

<210>137

<211>41

<212>DNA

＜213〉people

<400>137

aatttagtgg?gtatagggtg?atggtccact?atttataaga?g 41

<210>138

<211>25

<212>DNA

＜213〉people

<400>138

gattacaggt?gtgagccacc?gcacc 25

<210>139

<211>25

<212>DNA

＜213〉people

<400>139

ggtgcggtgg?ctcacacctg?taatc 25

<210>140

<211>22

<212>DNA

＜213〉people

<400>140

ggcctcccaa?agtgctggga?tt 22

<210>141

<211>22

<212>DNA

＜213〉people

<400>141

aatcccagca?ctttgggagg?cc 22

<210>142

<211>25

<212>DNA

＜213〉people

<400>142

gcctcccaaa?gtgctgggat?tacag 25

<210>143

<211>25

<212>DNA

＜213〉people

<400>143

ctgtaatccc?agcactttgg?gaggc 25

<210>144

<211>35

<212>DNA

＜213〉people

<400>144

ggaccacctc?cttgtgcccc?ctcctctgcc?atggc 35

<210>145

<211>35

<212>DNA

＜213〉people

<400>145

gccatggcag?aggagggggc?acaaggaggt?ggtcc 35

<210>146

<211>22

<212>DNA

＜213〉people

<400>146

ggatgagcaa?agaaagtggt?tt 22

<210>147

<211>22

<212>DNA

＜213〉people

<400>147

aaaccacttt?ctttgctcat?cc 22

<210>148

<211>29

<212>DNA

＜213〉people

<400>148

cagcctgggc?aacagagtga?gactctgtc 29

<210>149

<211>29

<212>DNA

＜213〉people

<400>149

gacagagtct?cactctgttg?cccaggctg 29

<210>150

<211>54

<212>DNA

＜213〉people

<400>150

atgtattgta?tattacattt?gaaatatata?ttgaaatata?tattgttgag?tata 54

<210>151

<211>54

<212>DNA

＜213〉people

<400>151

tatactcaac?aatatatatt?tcaatatata?tttcaaatgt?aatatacaat?acat 54

<210>152

<211>22

<212>DNA

＜213〉people

<400>152

atatttttta?tatatataaa?aa 22

<210>153

<211>22

<212>DNA

＜213〉people

<400>153

tttttatata?tataaaaaaat?at 22

<210>154

<211>30

<212>DNA

＜213〉people

<400>154

agtgatattt?gtgtttgtta?aattatataa 30

<210>155

<211>30

<212>DNA

＜213〉people

<400>155

ttatataatt?taacaaacac?aaatatcact 30

<210>156

<211>26

<212>DNA

＜213〉people

<400>156

actgcactcc?agcctgggtg?acagag 26

<210>157

<211>26

<212>DNA

＜213〉people

<400>157

ctctgtcacc?caggctggag?tgcagt 26

<210>158

<211>22

<212>DNA

＜213〉people

<400>158

atatgtatat?gcatatacat?at 22

<210>159

<211>22

<212>DNA

＜213〉people

<400>159

atatgtatat?gcatatacat?at 22

<210>160

<211>25

<212>DNA

＜213〉people

<400>160

tgggattaca?ggcatgagcc?actgt 25

<210>161

<211>25

<212>DNA

＜213〉people

<400>161

acagtggctc?atgcctgtaa?tccca 25

<210>162

<211>26

<212>DNA

＜213〉people

<400>162

gcctcccaaa?gtgctgggat?tacagg 26

<210>163

<211>26

<212>DNA

＜213〉people

<400>163

cctgtaatcc?cagcactttg?ggaggc 26

<210>164

<211>23

<212>DNA

＜213〉people

<400>164

agcctcctga?gtagctggga?tta 23

<210>165

<211>23

<212>DNA

＜213〉people

<400>165

taatcccagc?tactcaggag?gct 23

<210>166

<211>24

<212>DNA

＜213〉people

<400>166

atatgtatac?atatgcatat?acag 24

<210>167

<211>23

<212>DNA

＜213〉people

<400>167

tgtatatgca?tatgtataca?tat 23

<210>168

<211>28

<212>DNA

＜213〉people

<400>168

aagtgctggg?attacaggca?tgagccac 28

<210>169

<211>28

<212>DNA

＜213〉people

<400>169

gtggctcatg?cctgtaatcc?cagcactt 28

<210>170

<211>30

<212>DNA

＜213〉people

<400>170

agtgcagtgg?cgcgatctcg?gctcactgca 30

<210>171

<211>30

<212>DNA

＜213〉people

<400>171

tgcagtgagc?cgagatcgcg?ccactgcact 30

<210>172

<211>24

<212>DNA

＜213〉people

<400>172

gcctcccaaa?gtgctgggat?taca 24

<210>173

<211>24

<212>DNA

＜213〉people

<400>173

tgtaatccca?gcactttggg?aggc 24

<210>174

<211>29

<212>DNA

＜213〉people

<400>174

ccaaagtgct?gggattacag?gcatgagcc 29

<210>175

<211>29

<212>DNA

＜213〉people

<400>175

ggctcatgcc?tgtaatccca?gcactttgg 29

<210>176

<211>22

<212>DNA

＜213〉people

<400>176

tgggattaca?ggcgtgagcc?ac 22

<210>177

<211>22

<212>DNA

＜213〉people

<400>177

gtggctcacg?cctgtaatcc?ca 22

<210>178

<211>31

<212>DNA

＜213〉people

<400>178

cctcggcctc?ccaaagtgct?gggattacag?g 31

<210>179

<211>31

<212>DNA

＜213〉people

<400>179

cctgtaatcc?cagcactttg?ggaggccgag?g 31

<210>180

<211>36

<212>DNA

＜213〉people

<400>180

gcccacctcg?gcctcccaaa?gtgctgggat?tacagg 36

<210>181

<211>36

<212>DNA

＜213〉people

<400>181

cctgtaatcc?cagcactttg?ggaggccgag?gtgggc 36

<210>182

<211>24

<212>DNA

＜213〉people

<400>182

tcccaaagtg?ctgggattac?aggc 24

<210>183

<211>24

<212>DNA

＜213〉people

<400>183

gcctgtaatc?ccagcacttt?ggga 24

<210>184

<211>26

<212>DNA

＜213〉people

<400>184

ctccagcctg?ggcaacagag?cgagac 26

<210>185

<211>26

<212>DNA

＜213〉people

<400>185

gtctcgctct?gttgcccagg?ctggag 26

<210>186

<211>63

<212>DNA

＜213〉people

<400>186

gaggcggagc?ttgcagtgag?ccgagatcgc?tgggctgcac?tccagcctgg?gcgacagagc 60

gag 63

<210>187

<211>63

<212>DNA

＜213〉people

<400>187

ctcgctctgt?cgcccaggct?ggagtgcagc?ccagcgatct?cggctcactg?caagctccgc 60

ctc 63

<210>188

<211>30

<212>DNA

＜213〉people

<400>188

aaagtgctgg?gattacaggc?atgagccacc 30

<210>189

<211>30

<212>DNA

＜213〉people

<400>189

ggtggctcat?gcctgtaatc?ccagcacttt 30

<210>190

<211>26

<212>DNA

＜213〉people

<400>190

gcctcccaaa?gtgctgggat?tacagg 26

<210>191

<211>26

<212>DNA

＜213〉people

<400>191

cctgtaatcc?cagcactttg?ggaggc 26

<210>192

<211>34

<212>DNA

＜213〉people

<400>192

tatataatta?tatattatat?ataatatata?atta 34

<210>193

<211>34

<212>DNA

＜213〉people

<400>193

taattatata?ttatatataa?tatataaatta?tata 34

<210>194

<211>28

<212>DNA

＜213〉people

<400>194

atatataata?tataattata?tattatat 28

<210>195

<211>28

<212>DNA

＜213〉people

<400>195

atataatata?taattatata?ttatatat 28

<210>196

<211>31

<212>DNA

＜213〉people

<400>196

atatataata?tataattata?tattatatat?t 31

<210>197

<211>31

<212>DNA

＜213〉people

<400>197

aatatataat?atataattat?atattatata?t 31

<210>198

<211>28

<212>DNA

＜213〉people

<400>198

atatataata?tataattata?tattatat 28

<210>199

<211>28

<212>DNA

＜213〉people

<400>199

atataatata?taattatata?ttatatat 28

<210>200

<211>24

<212>DNA

＜213〉people

<400>200

gcctcccaaa?gtgctgggat?taca 24

<210>201

<211>24

<212>DNA

＜213〉people

<400>201

tgtaatccca?gcactttggg?aggc 24

<210>202

<211>22

<212>DNA

＜213〉people

<400>202

tgggattaca?ggcatgagcc?ac 22

<210>203

<211>22

<212>DNA

＜213〉people

<400>203

gtggctcatg?cctgtaatcc?ca 22

<210>204

<211>24

<212>DNA

＜213〉people

<400>204

tttttgtatt?tttagtagag?acgg 24

<210>205

<211>24

<212>DNA

＜213〉people

<400>205

ccgtctctac?taaaaataca?aaaa 24

<210>206

<211>37

<212>DNA

＜213〉people

<400>206

cctcagcctc?ccaaagtgct?gggattacag?gcgtgag 37

<210>207

<211>37

<212>DNA

＜213〉people

<400>207

ctcacgcctg?taatcccagc?actttgggag?gctgagg 37

<210>208

<211>21

<212>DNA

＜213〉people

<400>208

gcccacctcg?gcctcccaaa?g 21

<210>209

<211>21

<212>DNA

＜213〉people

<400>209

ctttgggagg?ccgaggtggg?c 21

<210>210

<211>37

<212>DNA

＜213〉people

<400>210

cctcagcctc?ccaaagtgct?gggattacag?gcgtgag 37

<210>211

<211>37

<212>DNA

＜213〉people

<400>211

ctcacgcctg?taatcccagc?actttgggag?gctgagg 37

<210>212

<211>21

<212>DNA

＜213〉people

<400>212

gcccacctcg?gcctcccaaa?g 21

<210>213

<211>21

<212>DNA

＜213〉people

<400>213

ctttgggagg?ccgaggtggg?c 21

<210>214

<211>21

<212>DNA

＜213〉people

<400>214

gggattacag?gtgtgagcca?c 21

<210>215

<211>21

<212>DNA

＜213〉people

<400>215

gtggctcaca?cctgtaatcc?c 21

<210>216

<211>24

<212>DNA

＜213〉people

<400>216

tttttgtatt?tttagtagag?acgg 24

<210>217

<211>24

<212>DNA

＜213〉people

<400>217

ccgtctctac?taaaaataca?aaaa 24

<210>218

<211>36

<212>DNA

＜213〉people

<400>218

cctcagcctc?ccaaagtgct?ggg?attacag?gcgtga 36

<210>219

<211>36

<212>DNA

＜213〉people

<400>219

tcacgcctgt?aatcccagca?ctttgggagg?ctgagg 36

<210>220

<211>36

<212>DNA

＜213〉people

<400>220

cctcagcctc?ccaaagtgct?gggattacag?gcgtga 36

<210>221

<211>36

<212>DNA

＜213〉people

<400>221

tcacgcctgt?aatcccagca?ctttgggagg?ctgagg 36

<210>222

<211>32

<212>DNA

＜213〉people

<400>222

ggcctcccaa?agtgctggga?ttacaggcgt?ga 32

<210>223

<211>32

<212>DNA

＜213〉people

<400>223

tcacgcctgt?aatcccagca?ctttgggagg?cc 32

<210>224

<211>21

<212>DNA

＜213〉people

<400>224

ctcccaaagt?gctgggatta?c 21

<210>225

<211>21

<212>DNA

＜213〉people

<400>225

gtaatcccag?cactttggga?g 21

<210>226

<211>52

<212>DNA

＜213〉people

<400>226

atatatatac?gtatatatat?atacacatat?atatatacgt?atatatatat?at 52

<210>227

<211>52

<212>DNA

＜213〉people

<400>227

atatatatat?atacgtatat?atatatgtgt?atatatatat?acgtatatat?at 52

<210>228

<211>40

<212>DNA

＜213〉people

<400>228

tatatatata?tacacatata?tatatacgta?tatatatata 40

<210>229

<211>40

<212>DNA

＜213〉people

<400>229

tatatatata?tacgtatata?tatatgtgta?tatatatata 40

<210>230

<211>52

<212>DNA

＜213〉people

<400>230

atatatatac?gtatatatat?atacacatat?atatatacgt?atatatatat?at 52

<210>231

<211>52

<212>DNA

＜213〉people

<400>231

atatatatat?atacgtatat?atatatgtgt?atatatatat?acgtatatat?at 52

<210>232

<211>40

<212>DNA

＜213〉people

<400>232

tatatatata?tacacatata?tatatacgta?tatatatata 40

<210>233

<211>40

<212>DNA

＜213〉people

<400>233

tatatatata?tacgtatata?tatatgtgta?tatatatata 40

<210>234

<211>21

<212>DNA

＜213〉people

<400>234

cttggcctcc?caaagtgctg?g 21

<210>235

<211>21

<212>DNA

＜213〉people

<400>235

ccagcacttt?gggaggccaa?g 21

<210>236

<211>23

<212>DNA

＜213〉people

<400>236

gctctgttgc?ccaggctgca?gtg 23

<210>237

<211>23

<212>DNA

＜213〉people

<400>237

cactgcagcc?tgggcaacag?agc 23

<210>238

<211>23

<212>DNA

＜213〉people

<400>238

gctctgttgc?ccaggctgca?gtg 23

<210>239

<211>23

<212>DNA

＜213〉people

<400>239

cactgcagcc?tgggcaacag?agc 23

<210>240

<211>28

<212>DNA

＜213〉people

<400>240

aagtgctggg?attacaggca?tgagccac 28

<210>241

<211>28

<212>DNA

＜213〉people

<400>241

gtggctcatg?cctgtaatcc?cagcactt 28

<210>242

<211>25

<212>DNA

＜213〉people

<400>242

ccatgtccat?gtgcaactca?acctc 25

<210>243

<211>25

<212>DNA

＜213〉people

<400>243

gaggttgagt?tgcacatgga?catgg 25

<210>244

<211>22

<212>DNA

＜213〉people

<400>244

ccttggcctc?ccaaagtgct?gg 22

<210>245

<211>22

<212>DNA

＜213〉people

<400>245

ccagcacttt?gggaggccaa?gg 22

<210>246

<211>24

<212>DNA

＜213〉people

<400>246

gatcacacca?ctgcactcca?gcct 24

<210>247

<211>24

<212>DNA

＜213〉people

<400>247

aggctggagt?gcagtggtgt?gatc 24

<210>248

<211>23

<212>DNA

＜213〉people

<400>248

tcccaaagtg?ctggggttac?agg 23

<210>249

<211>23

<212>DNA

＜213〉people

<400>249

cctgtaaccc?cagcactttg?gga 23

<210>250

<211>21

<212>DNA

＜213〉people

<400>250

gagaccagcc?tgaccaacat?g 21

<210>251

<211>21

<212>DNA

＜213〉people

<400>251

catgttggtc?aggctggtct?c 21

<210>252

<211>29

<212>DNA

＜213〉people

<400>252

gctctgtcgc?ccaggctgga?gtgcagtgg 29

<210>253

<211>29

<212>DNA

＜213〉people

<400>253

ccactgcact?ccagcctggg?cgacagagc 29

<210>254

<211>21

<212>DNA

＜213〉people

<400>254

aggttggtgc?aaaagtgatt?g 21

<210>255

<211>21

<212>DNA

＜213〉people

<400>255

caatcacttt?tgcaccaacc?t 21

Claims (12)

1. method of screening siRNA, it comprises the algorithmic language design, computer software is write, the human body gene library screening, autogenous siRNA prediction and biological experiment are identified:

1) writes the software that screens and predict the autogenous siRNA gene with a kind of new algorithmic language;

2) screen and predict the dna sequence dna of the siRNA that comes from the encoding gene introne with biocomputer infotech and human body gene library searching technology;

3) with various biological experiments the siRNA that selects is identified.

2. the method for the siRNA sequence in the introne that screens the human body encoding gene, it comprises:

1) adopts dynamic programming algorithm from people's intron sequences storehouse, to seek and have oppositely accurately complementary dna structure;

2) use multisequencing to arrange the high conserved sequence that formula and specific sequence pattern determination techniques and human body gene database search technology are sought those homologous geneses that are positioned at different genera;

3) choose one section and have the dna sequence dna that the siRNA typical case does ring texture;

4) evaluate the siRNA sequence that this chooses, standard is to adopt the ICT-Blast program to check that this sequence must have accordingly, at least more than one said target mrna.

3. claim 1 or 2 method, wherein said siRNA gene segment be one section and contain oppositely accurately complementary dna structure, and can be transcribed into the siRNA precursor, and complementary district can produce one to several ripe siRNA molecules through cutting processing.

4. claim 1 or 2 method, wherein said siRNA typical case does ring texture should contain 19-25 scapus sequence and 20 ring texturees that base to 120 base does not wait that nucleotide is grown at least.

5. claim 1 or 2 method, the dna sequence dna of wherein said siRNA is one section dna fragmentation in the encoding gene introne, it can be used as the template of a synthetic autogenous siRNA.

6. claim 1 or 2 method, wherein said siRNA is long strand of 19-26 nucleotide or double-stranded RNA molecule, their 3 ' section can contain 1-3 nucleotide base that suddenlys change.

7. claim 1 or 2 method, the arbitrary or a plurality of nucleotide base in the wherein said siRNA molecule can be modified by different chemical groups, thereby obtains advantages of higher stability.

8. claim 1 or 2 method, wherein said siRNA molecule can be hybridized specifically with a coded protein or RNA or dna molecular with other biological function, thereby disturbs its activity or its function of deactivation.

9. claim 1 or 2 method, the antisense strand of wherein said siRNA molecule is the long oligonucleotide of 19-25 nucleotide of the corresponding mRNA molecule of a series of deactivation effectively, comprises the oligonucleotide molecule of following sequence number: 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 to 255.

10. claim 1,2 or 9 method, the dna sequence dna of wherein said siRNA can be integrated in plasmid or the viral vectors, is bred and transcribe to be processed into ripe siRNA molecule.

11. nucleotide sequence, it has any one nucleotide sequence among the SEQ ID No.1-255.

12. the nucleotides sequence of claim 11 is listed in the application in the medicine of preparation and exploitation treatment tumour, heredity and metabolic disease etc.

Images