CN101851618B - Modified small interfering RNA and preparation method thereof - Google Patents

Abstract

The invention provides a modified small interfering RNA, which comprises a first fragment and a second fragment, wherein the first fragment and the second fragment can form a double-chain area; the first fragment comprises at least one continuous UA sequence; the second fragment comprises at least one continuous UA sequence which is complementary to the UA sequence of the first fragment; the UA sequence of the first fragment and the UA sequence of the second fragment form a UA/UA locus; and at least one nucleotide at the UA/UA locus is modified, and the modification makes the stability of the modified small interfering RNA higher than that of an unmodified small interfering RNA. In the invention, the improvement in the serum stability of the modified small interfering RNA can be achieved with a small amount of modification introduced, so the potential cell toxicity of the modified small interfering RNA molecules and the influences of the modification on the biologic activity of the small interfering RNA are reduced.

Description

Small RNA of a kind of modification and preparation method thereof

Technical field

The present invention relates to small RNA of a kind of modification and preparation method thereof.

Background technology

RNA disturbs (RNA interference, RNAi) to be closed homogenic expression or make the phenomenon of this genetic expression silence in mRNA level by double-stranded RNA (double-strandedRNA, dsRNA) molecule.RNA perturbation technique is called again Knockdown (knock-down) or gene silencing (gene silencing) visually, it is a kind of typical post-transcriptional gene regulation method, claim again PTGS (post-transcriptional gene silencing, PTGS).The report that relevant RNA disturbs the earliest appears at nineteen ninety, report the RNA interference phenomenon in transgenic plant by two different research groups simultaneously, in nearly all eukaryotes such as nematode, fruit bat, zebra fish and mouse, observed RNA interference phenomenon again later.1999, Hamilton and Baulcombe detected that length is the RNA fragment of 21-25 Nucleotide in the plant that RNA interference occurs, and it is necessary that these RNA segments are proved to be RNA interference, is called as small molecule interference nucleic acid (siRNA).The silencing complex (RNA-inducedsilencing complex, RISC) of the relevant enzyme of double-stranded siRNA and cell source and Protein formation RNA induction.In RNA interfering process, the positive-sense strand in double-stranded siRNA is excluded from complex body, and antisense strand instructs RISC to be attached to the homologous site of said target mrna, then by the rnase iii degraded said target mrna in mixture, thereby closes the expression of target gene.

But, due to the less stable of small molecule interference nucleic acid (siRNA), in vivo easily by nuclease degradation, therefore people carry out chemically modified to synthetic siRNA, to increase the serum stability of siRNA, thereby effectively suppress the expression of goal gene.

At present, due to the people's degradation process in serum and enough understandings of mechanism shortage to siRNA, can only rely on experience separately to select randomly the multiple Nucleotide in siRNA molecule to carry out chemically modified.Modify the tactful serum stability that can improve well siRNA molecule although this, but owing to lacking theoretical guidance, conventionally in siRNA molecule, introduced excessive modification, increase the cytotoxicity of the siRNA after modifying, and reduced under many circumstances the biologic activity of siRNA, thereby restrict siRNA after modifying application in vivo.

In addition, in siRNA, in Nucleotide, introduce blindly a large amount of ways of modifying, also limited some and there is better stabilising effect, but the application of the relatively large modifying method of cytotoxicity in studying in vivo.

Therefore, design pointed modification protocols, realizing optimum stability object by minimum modification is current problem in the urgent need to address.

Summary of the invention

Thereby the object of the invention is to overcome and in the modification protocols of existing siRNA, have the large problem of small RNA cytotoxicity that a large amount of modifications causes the modification obtaining of introducing blindly, provide that a kind of serum is stable, small RNA that have good biological activity and the modification that cytotoxicity is lower, and the preparation method of this small molecule interference nucleic acid.

The invention provides a kind of small RNA of modification, it comprises the first fragment and the second fragment, and described the first fragment and the second fragment can form double-stranded region, described the first fragment comprises at least one continuous UA sequence, the continuous UA sequence of the UA sequence complementation that described the second fragment comprises at least one and described the first fragment, the UA sequence of described the first fragment and the UA sequence of described the second fragment form UA/UA site, wherein, at least one Nucleotide in described UA/UA site is through modifying, this modification makes the stability of the small RNA of modifying higher than the small RNA of unmodified.

The present invention is by carrying out specific modification to UA/UA site, thereby in the situation that only introducing a small amount of modification, can reach the object of the serum stability that increases the small RNA after modifying, thereby reduce the potential cytotoxicity of the small RNA molecule after modifying, and modified the impact of the biologic activity on small RNA.

Embodiment

The small RNA of modification provided by the invention, it comprises the first fragment and the second fragment, and described the first fragment and the second fragment can form double-stranded region, described the first fragment comprises at least one continuous UA sequence, the continuous UA sequence of the UA sequence complementation that described the second fragment comprises at least one and described the first fragment, the UA sequence of described the first fragment and the UA sequence of described the second fragment form UA/UA site, wherein, at least one Nucleotide in described UA/UA site is through modifying, this modification makes the stability of the small RNA of modifying higher than the small RNA of unmodified.

The present inventor has carried out careful research to small RNA molecule degradation process in vivo, the in the situation that of finding only to introduce a small amount of modification in UA/UA site, can improve the stability of the small RNA molecule of modification, greatly reduce owing to introducing randomly the cytotoxicity that a large amount of modifications causes highlyer, and modify the impact of the biologic activity on small RNA.

According to the present invention, described " the first fragment " refers to the nucleotide fragments having with the sequence of all or part of homology of sequence of genes encoding chain, and described " the second fragment " refers to the nucleotide fragments having with the sequence of the sequence complementation of genes encoding chain.Described " complementation " refers to that two Nucleotide can match under hybridization conditions, for example, between VITAMIN B4 (A) and thymus pyrimidine (T) or uridylic (U), can match, and pairing between cytosine(Cyt) (C) and guanine (G).

According to the present invention, the target of described small RNA molecule can be range gene, for example, can will there is gene to be analyzed as target in intracellular function, also can be using the gene that need to suppress its expression as target, for example, can using to disease or disorderly relevant gene as target, as the gene on oncogene, virogene, surface of cell membrane acceptor gene, nuclear receptor gene or cell signaling path etc.Those skilled in the art is according to its target gene, can design and obtain small RNA molecule (siRNA), for example, by the target sequence of goal gene or goal gene, the sequence number in NCBI Genbank is inputted various small RNAs and is designed program, as Insert Design Tool for the shRNAVectors (Ambion), shRNA Explorer (Gene Link), siDirect (Yuki Naito et al.University of Tokyo), SiRNA at Whitehead (Whitehead Institute for BiomedicalResearch), BLOCK-iT RNAi Designer (invitrogen), RNAi Design (IDT), RNAiExplorer (Gene Link), siRNA Target Finder (Ambion), or siSearch (StockholmBioinformatics Center) etc., this is designed program will be according to the principle of design of planner's requirement and siRNA, for provided gene or sequences Design siRNA.And some program can be done full genome or mRNA transcribes the homology analysis of group to the siRNA designing, thereby designs target gene or the special siRNA of target sequence.The above-mentioned siRNA mentioning designs program and the principle that relates to is conventionally known to one of skill in the art, and its full content is incorporated herein by reference in the lump at this.

According to the present invention, the small RNA of described modification can comprise multiple UA/UA site, and at least one Nucleotide in the plurality of UA/UA site is through modifying.

In addition,, in the small RNA of described modification, the Nucleotide in described UA/UA site, other Nucleotide is not through modifying.In this case, not only can improve the small RNA of described modification stability, maintain its biological activity, can also make the modification quantity of introducing in the small RNA molecule of modifying minimize, thereby reduce the cytotoxicity of modifying the small molecule interference nucleic acid causing further.

Under preferable case, in the UA/UA site of the small RNA of described modification, only has a uridylate through modifying.The present inventor is surprised to find that, as long as a uridylate in described UA/UA site is modified, just can improve the stability of the small RNA of modification, and maintain its biological activity, in this case, can make the modification quantity of introducing in the small RNA of modifying minimize, thereby further reduce the cytotoxicity of modifying the small molecule interference nucleic acid causing.

According to the present invention, the small RNA of described modification can be that single chain molecule can be also duplex molecule.

In the time that the small RNA of described modification is single chain molecule, the complementary region between described the first fragment and the second fragment forms double-stranded region.

In the time that the small RNA of described modification is duplex molecule, the small RNA of this modification comprises positive-sense strand and antisense strand, and described positive-sense strand can be continuous nucleotide chain, can be also discontinuous nucleotide chain; Described antisense strand is continuous nucleotide chain.

According to an aspect of the present invention, the small RNA of modification provided by the invention comprises positive-sense strand and antisense strand, and described positive-sense strand and antisense strand be continuous nucleotide chain, and described first section is positioned at positive-sense strand, and described the second fragment is positioned at antisense strand.

According to another aspect of the present invention, the small RNA of modification provided by the invention comprises positive-sense strand and antisense strand, and described positive-sense strand is the discontinuous nucleotide chain that comprises two or more positive-sense strand parts; Described antisense strand is continuous nucleotide chain; Described first section is positioned at one or more positive-sense strand parts, and described the second fragment is positioned at antisense strand.

In the present invention, term " positive-sense strand " refers in the time that the small RNA of described modification is duplex molecule, there is the nucleotide fragments with the sequence of all or part of homology of sequence of genes encoding chain, described " antisense strand " refer in the time that the small RNA of described modification is duplex molecule, has the nucleotide fragments with the sequence of the sequence complementation of genes encoding chain.And when the small RNA of described modification is duplex molecule and described positive-sense strand and antisense strand while being continuous nucleotide chain, term " positive-sense strand " and " the first fragment " can be exchanged use, term " antisense strand " and " the second fragment " can be exchanged use.

In the present invention, term " positive-sense strand part " refers in the time that described positive-sense strand is discontinuous Nucleotide, for forming a part of nucleotide chain of positive-sense strand; In described positive-sense strand, the total length of all positive-sense strand parts of this positive-sense strand and described positive-sense strand equal in length, and in the time comprising UA/UA site in described positive-sense strand part, term " positive-sense strand part " can exchange use with " the first fragment ".

According to the present invention, the small RNA of described modification can be made up of ribonucleotide, can be also the hybrid molecule that comprises ribonucleotide and at least one deoxyribonucleotide.

According to the present invention, term " cytotoxicity " refers to the toxic action to cell causing due to nucleic acid molecule is modified.

In the present invention, the mode of described modification is conventionally known to one of skill in the art, and for example, the chemically modified that the present invention carries out described small molecule interference nucleic acid is following one or more:

(1) to connecting the modification of the phosphodiester bond of Nucleotide in the nucleotide sequence of described small molecule interference nucleic acid;

(2) to ribose in the nucleotide sequence of described small molecule interference nucleic acid modification;

(3) modification to base in the nucleotide sequence of described small molecule interference nucleic acid.

The modification of described phosphodiester bond refers to modifies the oxygen in phosphodiester bond, comprises thiophosphoric acid modification (Phosphorthioate) and borine phosphate modified (Boranophosphate).As shown in the figure respectively with the oxygen in sulphur and borine displacement phosphodiester bond.Two kinds of modifications can stable micro-molecular interfere RNA structure, keep high specific and the high-affinity of base pairing.And the hydrophobicity of the small RNA of borine phosphate modified is strong, be easy to form hydrated protein in blood plasma, to the toxic side effect of human body lower than thiophosphatephosphorothioate.

Thiophosphoric acid is modified borine phosphate modified

Described ribose is modified the modification referring to hydroxyl in Nucleotide pentose (2 '-OH).The hydroxy position of ribose introduce some substituting group as methoxyl group or fluorine after, make serum rnase small RNA not easy to identify, increased the stability of small RNA.Make small RNA there is the performance of stronger opposing nuclease hydrolysis.The modification of hydroxyl in Nucleotide pentose is comprised to 2 '-fluorine modification (2 '-fluro modification); 2 '-oxygen methyl is modified (2 '-OME); 2 '-methoxyethyl is modified (2 '-MOE); 2,4 '-dinitrophenol(DNP) is modified (2 '-DNP modification); Lock nucleic acid (LNA); 2 '-amido modified (Amina modification); (2 '-Deoxy modification) etc. modified in 2 '-deoxidation.

2 '-fluorine is modified 2 '-oxygen methyl and is modified

2 '-methoxyethyl is modified 2,4 '-dinitrophenol(DNP) and is modified

Lock nucleic acid 2 '-amido modified

2 '-deoxidation is modified

Described base modification refers to be modified the base of Nucleotide, if 5 '-bromouracil (5 '-bromo-uracil) and 5 '-iodouracil (5 '-iodo-uracil) modification of introducing bromine or iodine in 5 sites of uridylic are the normal base modification methods using, other also have N3-methyl uracil (N3-methyl-uracil) to modify, 2,6-diaminopurine (2,6-diaminopurine) modification etc.

5 '-bromouracil 5 '-iodouracil

N3-methyl uracil 2,6-diaminopurine

Under preferable case, described in be modified to the modification to 2 '-OH of ribose in the nucleotide sequence of described small molecule interference nucleic acid.More preferably, the 2 '-OH that is modified to ribose in the nucleotide sequence of described small molecule interference nucleic acid described in is replaced by methoxyl group or fluorine.Above-mentioned modification all can increase the serum stability of described small RNA, strengthens its resistivity to the hydrolysis of serum nuclease.

The present invention also provides a kind of preparation method of small RNA of modification, wherein, the method comprises: according to the nucleotide sequence of the small RNA of unmodified, and use is through the Nucleotide of corresponding position in the nucleotide sequence of the small RNA of the alternative described unmodified of modified nucleotide, carry out the small RNA of synthetic modification, make the small RNA of the modification obtaining comprise the first fragment and the second fragment, and described the first fragment and the second fragment can form double-stranded region, described the first fragment comprises at least one continuous UA sequence, the continuous UA sequence of the UA sequence complementation that described the second fragment comprises at least one and described the first fragment, the UA sequence of described the first fragment and the UA sequence of described the second fragment form UA/UA site, wherein, at least one Nucleotide in described UA/UA site is through modifying, this modification makes the stability of the small RNA of modifying higher than the small RNA of unmodified.

According to an aspect of the present invention, the preparation method of the small RNA of described modification provided by the invention can comprise: according to the nucleotide sequence of the small RNA of unmodified, and use is through the Nucleotide of corresponding position in the nucleotide sequence of the small RNA of the alternative described unmodified of modified nucleotide, carry out the small RNA of synthetic modification, make the small RNA of the modification obtaining comprise multiple UA/UA site, and at least one Nucleotide in the plurality of UA/UA site is through modifying.

According to another aspect of the present invention, the preparation method of the small RNA of described modification provided by the invention can comprise: according to the nucleotide sequence of the small RNA of unmodified, and use is through the Nucleotide of corresponding position in the nucleotide sequence of the small RNA of the alternative described unmodified of modified nucleotide, carry out the small RNA of synthetic modification, make in the small RNA of the modification obtaining, Nucleotide in described UA/UA site, other Nucleotide is not through modifying.And, under preferable case, in the UA/UA site of the small RNA of the modification obtaining, only have a uridylate through modifying.

In the present invention, the structure of the small RNA of modification provided by the invention is conventionally known to one of skill in the art, can be the structure that exists of various small RNAs.

For example, according to an aspect of the present invention, the small RNA of modification provided by the invention can be the single chain molecule of hairpin structure, and the complementary region between described the first fragment and the second fragment forms double-stranded region.

According to another aspect of the present invention, the small RNA of modification provided by the invention can comprise positive-sense strand and antisense strand, and described positive-sense strand and antisense strand are continuous nucleotide chain, and described first section is positioned at positive-sense strand, and described the second fragment is positioned at antisense strand.

According to another aspect of the present invention, the small RNA of modification provided by the invention can comprise positive-sense strand and antisense strand, and described positive-sense strand is the discontinuous nucleotide chain that comprises two or more positive-sense strand parts; Described antisense strand is continuous nucleotide chain; Described first section is positioned at one or more positive-sense strand parts, and described the second fragment is positioned at antisense strand.

According to another aspect of the present invention, the small RNA of modification provided by the invention can be the hybrid molecule for comprising ribonucleotide and at least one deoxyribonucleotide.

In the present invention, the method of synthetic small RNA can be the synthetic method of the small RNA of various routines, or it is synthetic that the synthetic biotech company of nucleic acid is specialized in trust, as entrusted Shanghai JiMa pharmacy Technology Co., Ltd (GenePharma), Rui Bo bio tech ltd, Guangzhou or handsome Bioisystech Co., Ltd (invitrogen) to synthesize.

In general, comprise following Four processes for the synthesis of the method for small RNA: (1) oligomerization ribonucleotide synthetic; (2) deprotection; (3) purifies and separates; (4) desalination.

For example, there are the concrete steps of the small RNA chemosynthesis of nucleotide sequence shown in SEQ ID NO:1 as follows:

(1) oligomerization ribonucleotide is synthetic: at automated DNA/RNA synthesizer (for example, AppliedBiosystems EXPEDITE8909) the upper RNA that sets synthetic 1 mmole, the coupling time of simultaneously setting each circulation is 10-15 minute, initiator be solid phase connect 5 '-O-to dimethoxy-thymidine upholder, first circulates in and on solid support, connects a base, then in the n time (19 >=n >=2) circulation, in the base connecting the n-1 time circulation, connect a base, repeat this circulation until complete the synthetic of whole nucleotide sequences.

(2) deprotection

The solid support that is connected with small RNA is joined in test tube, and in this test tube, add the ethanol/ethamine (volume ratio is 1: 3) of 1 milliliter, then sealing, be placed in 55-70 ℃ of incubator, hatch 2-30 hour, fech connection has the solid support of small RNA and with distilled water drip washing 2 times (each 1 milliliter), collects elutriant, and at room temperature dry 30 minutes.Then, add the tetrahydrofuran solution (1M) of 1 milliliter of tetrabutyl ammonium fluoride, room temperature is placed 4-12 hour, then adds 2 milliliters of ethanol, and collecting precipitation obtains the crude product of small RNA.

(3) purifies and separates

It is in the ammonium acetate solution of 1 mole/milliliter that the crude product of the small RNA obtaining is dissolved in to 2 ml concns, then separates by C18 high pressure liquid chromatography, obtains the small RNA product of purifying.

(4) desalination

Be small RNA product 2-4 time (each 2 milliliters) of the aqueous ethanolic solution washing purifying of 75 % by weight by concentration, remove salt, and dry under room temperature.Then by oligomerization Yeast Nucleic Acid mixed dissolution (10mM Tris in the damping fluid of 1-2 milliliter of positive-sense strand and antisense strand, pH=7.5-8.0,50mM NaCl), this solution is heated to 95 ℃, then slowly this solution is cooled to room temperature, and maintain room temperature 16-22 hour, obtain the solution that contains small RNA.

Above provide the object of numerous embodiments only for exemplarily the present invention will be described, rather than in order to limit the scope of the invention.

Further illustrate the present invention below in conjunction with embodiment, unless stated otherwise, the present invention's reagent used, substratum are commercial goods.

Embodiment 1

The gene of choosing in following table is target gene, and entrusts the siRNA in the synthetic table 1-127 of Shanghai JiMa pharmacy Technology Co., Ltd (GenePharma).

Embodiment 2

SiRNA shown in his-and-hers watches 1-127 carries out serum stability detection respectively by the following method, gene silencing efficiency detects and cytotoxicity detects:

1. serum stability detects

Be the modifying small RNA with different chemical and join and contain in 4 μ L foetal calf serums and 32 μ L 1 × PBS solution of unmodified of 20 μ M by 4 μ L concentration, the final concentration of serum is 10%; After reaction system being hatched to the regular hour under 37 ℃ of conditions, sample, be generally 0,3 and 6 hour; Get 10 μ L samples at every turn and carry out liquid nitrogen flash freezer, to end immediately the effect of serum nuclease to siRNA, then that Sample preservation is for subsequent use under-80 ℃ of conditions.

The polyacrylamide gel of configuration 20%, mixes the sample-loading buffer of 3 μ L (30mM EDTA, 36% glycerine, 0.06% tetrabromophenol sulfonphthalein) then loading, electrophoresis under the constant current conditions of 80mA with the degraded sample of siRNA.After electrophoresis finishes, carry out taking a picture after the dyeing of 10 minutes with 1 × Sybr Gold dyestuff (Invitrogen, Cat.11494), result is as shown in table 1-127.

2. gene silencing efficiency detects

The human embryonic kidney cell (HEK293) that will cultivate in the DMEM substratum (10%FBS, 2mM L-glutaminate, the Streptomycin sulphate of the penicillin of 100 units per ml and 100 μ g/ml) is inoculated in 24 orifice plates (1 × 10 ⁵cell/0.5ml substratum/hole).After 24 hours, when the degrees of fusion of cell is 50% left and right, substratum is changed to Opti-MEM substratum (Gibco company) until Growth of Cells.Then control plasmid (the Promega company of restructuring Photinus pyralis LUC reporter plasmid and pRL-TK (coding Renilla luciferase) of target site will be separated with siRNA, Madison WI, USA) pass through Lipofectamine 2000 (Invitrogen company with the siRNA of chemosynthesis, the U.S.) carry out cell transfecting, every hole is containing 0.17g recombinant plasmid and 0.017g pRL-TK control plasmid, and the final concentration of siRNA is 13nM.Three multiple holes of every kind of parallel transfection of siRNA, with two kinds of reporter plasmids of the same amount of a transfection, three multiple holes of not transfection siRNA in contrast.After 4 hours, again transfection medium is changed into 1ml DMEM substratum (10%FBS, 2mM L-glutaminate, the Streptomycin sulphate of the penicillin of 100 units per ml and 100 μ g/ml).Harvested cell after 24 hours, with 10 μ l cell pyrolysis liquid cells, utilize Relative luciferase activity assay system (Dual-Luciferase Assay System, Promega company) and microplate reader (Novostar, BMG Labtechnologies GmbH, Germany) activity of two kinds of luciferases of mensuration, using the expression amount of the reporter gene in the hole of untransfected siRNA as standard control, calculate the silence efficiency of siRNA to target site by following formula, result is as shown in table 2.Every kind of siRNA tests parallel 3 the multiple holes of doing at every turn, and each experiment repeats 2 times, and result is as shown in table 1-127.

Silence efficiency=(expression amount/experimental group Renilla luciferase expression amount of experimental group Firefly luciferase)/(expression amount/control group Renilla luciferase expression amount of control group Firefly luciferase)

The cytotoxicity of 3, modifying siRNA detects

The human embryonic kidney cell (HEK293) that will cultivate in the DMEM substratum (10%FBS, 2mM L-glutaminate, the Streptomycin sulphate of the penicillin of 100 units per ml and 100 μ g/ml) is inoculated in 24 orifice plates (1 × 10 ⁵cells/well); After 24 hours, when the degrees of fusion of cell is 50% left and right, substratum is changed to Opti-MEM substratum (Gibco company) until Growth of Cells; Then the siRNA of chemosynthesis is passed through to Lipofectamine 2000 (Invitrogen company, the U.S.) carry out cell transfecting, the final concentration of siRNA is 13nM, and holes are answered in every kind of three of parallel transfection of siRNA, answer hole in contrast without three transfections of siRNA; After 4 hours, again transfection medium is changed into 1ml DMEM substratum (10%FBS, 2mML-glutamine, the Streptomycin sulphate of the penicillin of 100 units per ml and 100 μ g/ml); After 24 hours, suck nutrient solution, wash once with PBS, every hole adds 1ml PBS and 10 μ l MTT (tetrazolium bromide) dye liquors, at 37 ℃ of 5%CO ₂cO2gas incubator in cultivate 4～6 hours; Every hole adds 0.1ml acidifying Virahol, on vibrator, vibrates and mixes, and allows reduzate fully dissolve; Enzyme-linked immunosorbent assay instrument is measured each hole, 570nm place A value, and calculates inhibitory rate of cell growth according to following formula, and result is as shown in table 1-127.

Inhibitory rate of cell growth (%)=(control group average A value-experimental group average A value)/control group average A value × 100%.

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Table 127

In upper table 1-127:

" A ", " U " that bold-faced letter represents, " G " or " C " Nucleotide of letter representation through modifying;

"+" represents after serum is hatched, the completely dissolve of siRNA master tape, visible significantly degraded band;

" ++ " represents after serum is hatched, though siRNA master tape has degraded but still be high-visible, visible significantly degraded band;

" +++ " represents after serum is hatched, and siRNA master tape is clear has no degraded, has no obvious degraded band.

In addition, from table 1-127, can find out, with compared with the random small RNA of modifying, small RNA provided by the invention is in having improved stability, also reduce significantly the cytotoxicity of small RNA, and the expression inhibiting efficiency on small RNA does not affect substantially, as can be seen here, the present invention is in the situation that only introducing a small amount of modification, can reach the object of the serum stability that increases the small RNA after modifying, thereby reduce the potential cytotoxicity of the small RNA molecule after modifying, and the impact of the biologic activity of modification on small RNA.

SEQUENCE LISTING

<110> Peking University

Small RNA of a <120> modification and preparation method thereof

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gcccgggccc gggagccgcg gccagcgcct cggcggcggg ggactcggcg gcgacgaaca 480

tggagaaccc ccagcttgga gcgcaagtgc tcctgcggga agaagtgtcg cggctccagg 540

aggaagttca ccttctccgg cagatgaagg agatgttggc gaaggacctg gaggagtcgc 600

agggcggcaa gtcctctgag gtcctctcgg ccaccgagct cagggtccag ctggcccaga 660

aggagcagga gctagccaga gccaaagaag ccttgcaggc catgaaagct gatcggaagc 720

gcttaaaggg cgagaagaca gacctggtga gccagatgca gcagctgtat gccacactgg 780

agagccgcga ggagcagctc cgagacttca tccgcaacta tgagcagcac cgcaaggaga 840

gcgaggatgc ggtcaaagcg ctggccaagg agaaggacct gctggagcgt gagaagtggg 900

agctgcggcg ccaagccaag gaggccacag accacgccac ggcactgcgc tcccagctgg 960

acctcaagga caaccggatg aaggagctgg aggccgagct ggccatggcc aaacagtcct 1020

tagctacgct gaccaaggac gtccccaagc ggcattccct cgccatgccg ggcgagacgg 1080

tgctcaatgg caaccaggag tgggtggtgc aggcggacct cccgctgacc gcagccatcc 1140

ggcagagtca acagactctc taccactcac acccccctca ccctgcggac cggcaagcgg 1200

tcagggtgag cccctgccac tcccggcagc cctctgtcat ctccgacgca tctgccgccg 1260

aaggcgaccg gtcgtccaca ccgagcgaca tcaactcccc tcgacaccgg acacactccc 1320

tctgcaacgg cgacagtccc ggcccagttc agaagaacct gcacaaccct attgtacagt 1380

cactagagga tcttgaagac caaaaacgga aaaagaagaa agagaagatg ggattcggct 1440

ccatctcccg cgtcttcgcc agagggaagc agcggaagtc cctcgacccc ggcctctttg 1500

atgactcgga cagccagtgc agccccacgc ggcagagcct cagcctgtcg gaaggcgagg 1560

agcagatgga ccggctgcag caggtggagc tggtgaggac cacccctatg tcccactgga 1620

aggcgggcac cgtccaggcc tggctggagg tggtgatggc catgcctatg tacgtcaagg 1680

cctgcacgga gaacgtgaag agcgggaagg tgctgctgag cctgagtgac gaggacctgc 1740

agctgggcct tggggtgtgc agctccctgc accggcgcaa gctgcgcctg gccatcgagg 1800

actaccgtga tgccgaggca ggccgcagcc tgtccaaagc tgccgagctg gaccatcact 1860

gggtggccaa ggcctggctg aatgacattg gcctgtccca gtactcccag gcctttcaga 1920

accacctggt tgatgggcgg atgctgaatt ccctgatgaa gcgagacctg gagaagcacc 1980

tgaacgtgtc caagaagttc caccaggtca gcatcctgct ggggatcgag ctgctgtacc 2040

aagtgaactt cagcagggag gccctccagg agcgccgggc ccgctgcgag acgcagaaca 2100

ttgaccccgt ggtgtggacc aaccagcggg tgctcaagtg ggttcgagac atcgacctga 2160

aggagtacgc agacaacctg accaacagcg gcgtccatgg tgctgtgctg gtgctggagc 2220

ccacattcaa tgccgaggcc atggccactg ccctgggcat ccccagtggg aagcacatcc 2280

tccggagaca cctggcagag gagatgagcg ccgtcttcca cccagccaac tccacaggca 2340

tccgggaggc tgagcgtttt ggaacgcccc ctggcagggc ctccagcgtc acgcgggcag 2400

gaaaggagga gaacagcagc ggtctcaagt acaaggctgg ccggctgccc ctggggaaga 2460

taggaagggg cttcagcagc aaagatcccg atttccatga tgactatggc tctcttcaaa 2520

acgaagattg cggagacgat gacccccaga gcaggctgga acagtgccgt ctggaaggct 2580

acaacagcct ggaggtcacc aacgtgtaag gaactggtgg ctccaccaga cccaacgtga 2640

gagacccagg aaggaagaga agccagatgg ccccaggtgt cgttctcact gtacatagcg 2700

gccgcaggct gaggatgtcc cttgctcctg ggcaaaatcc cgatggactc tgcggtttca 2760

gctccacagc gcccaggaga gagaagacac cagcccacct gtcttgggtg ggccatggac 2820

tttcctgttc agctggagat gggcccagag gacctgtcac agtgtccggc cctgcctcca 2880

tccaggatac acaggctcca cctcagagtg accgtcactg tggagcagcc aagcagtccc 2940

tggagcctta aacggagctg ccaaggtggg aggaggccca cagttcccta aaacaccctt 3000

ccggcgggag caggggggac cccaacccca caccccagcg cccagtgcat tggcagagcc 3060

gggtgcagga agtgctgcct cttgccgaga cgtcggacag ggcgggggtt ggggaactct 3120

cggctacagc atcttaccct tgactgagaa cttgggtcct gacttggctc actgaatctc 3180

tcttgggaga atgcaaaatc cttccacctg aaaagctctg tgacacatgg gggtggacgt 3240

attgaagagc tgtttgccga tccacccagg agtggctacg ctgagtgggg agccggtgaa 3300

tgatccgtgc aggagtgggg cttagcagcc acatttctag gagatgcaga tatcctatca 3360

ccagaatgaa agctattggg acaacaggat cggggatgac cgacggcccc atatggtgaa 3420

tctctggcct gtggtttggc tttactgaga ttccaaaccc cactatctgc actccgtgac 3480

agtggtatgg agtgtggcaa tgagtttggg gtctggggca gggaaatgct tgacattgtt 3540

aacccaacaa acctttgttg tgatgtccct gtcacctgaa acataggtga catagctcac 3600

caatgtccta accgagacac aaactccaca gagcaaaatc atttggtatt ggtggggaga 3660

accccagccc ttttcttgac ctgccactgt tatgctgtgt ggcttcttcc cagtggcctc 3720

acctctctgt gcctcgatgt cttcatctac gatacttctg gttccctccc agggacatcg 3780

tgaggattaa cacttgctaa tatctgtaac acaatttgta acctctcagg agacaatggg 3840

aagttatggg gtagctaatt tcccatttac aacacagaaa tgatatagag ctagttcgct 3900

ccaactcttt aggttgaagc agtgtgcaaa aggaagaaaa gaaatgttta atgttcagac 3960

ctgccaagag cctccaacag ggctcaagaa acatataaat cccatgagca cagccttgaa 4020

aaccagtttg actcaagcct tcgggcctca gttcattgac cagatgacag ccacgtgatg 4080

attagggaag gacggatgca ttgcgattct gcttacacat cgggttatca aagcgagtca 4140

cttgttggaa ccatgatgct cgacctcctt caaggccgtt tgcactgggg cttgagtttc 4200

caagattcac aacaggtgtc agcctctgag aaccctcaaa gcgtgtgttc ttcaacctgg 4260

caaattgttt cctctcatgg gggaagccga gctctgatga acttgagaat tacacctctc 4320

tcatgccgaa gaccgtggtg ttccccctaa tgacataaac gcagcctttc ttgctgtctg 4380

agaccaaatg tctagttggt agacaggtgg atgtttggcc tcctaagggc acacttctga 4440

tcctgggccc caggtggtga atctctggca tgtggcttgg ctttgttgag actccaaatt 4500

ccattatctt catgacattc ggcctcatcc atagggtcct gaagctgcag tccacagctc 4560

agaaaggaga ggtgagacct ccctccaacc tggtgccaca ggtctctccc aagccacatc 4620

cagcctggat gacctgggac cccagaaact gccgtttggg aggcagcaac agcaacgtgc 4680

ccaggcaggc agttattccc acagagtgag ccagaattgt agcagggcac ttgaatgcag 4740

agctgatgat ttgaaaccaa cgttcaccca acttgtcaga aatggcactt acatggttcg 4800

atcttgctgg agacaagtgg acaattgggg gtcactggca gagacagtat tgcccaaaat 4860

gttcacagca ggaggccagc aggcctgagg caacacgggc aaccgcgaat gcctcttttg 4920

gtttaaatta tgccatcaca accctctttc acccatgagg ctccccatcc ctgacagcca 4980

ggtgagcatt tggagctggt ttctcaacat gaggatgggt tggttgttaa attaacaacc 5040

tccacagtat cagattgagt gagctttgtc tgctggaaaa acctgaaacg tcaactctgc 5100

ttcaaggtcg gcaagaagaa cagaaggcgg agacttggca gagagactca agctgattgt 5160

cacaggctac agaggggcca gctccagaac agtgaccagc tacatcctgt ccaagcagcc 5220

cgagtgtggt cttggtccct gcagggcaat gtgggcatct ggacctgggg acgatgtgga 5280

tgcacttctt ggaaagctgt tgtagcttgt gcctgtgggt ggagaaggca cctgcctggt 5340

agactctcag ctttctgacc cccaggagcc tctgcgaggc ccctttgtcc ttggctgagc 5400

cggacctttc ttttggaaat ctgtctgtct gttggcatcg ctgttttcag accccaggct 5460

gcagaggagg ggagaagcca cacaacaatc tggacccaat aaagtggaga gaagggcgtc 5520

tctacacagc ccggccagcg tggagggccc caggacaggg acccaaaagc ttgacgtcac 5580

tgaacagggc tgggtactgg cagaacagga agatttggcc agaggtgacc tcagtgttcc 5640

ctccaggggc atccaggccc ctctgacctg gggagaagaa ggcccatgct caggcccacc 5700

tccctcttcc catcagagcc catgcgtcct gggcaccacc acttccactc tgcttttcga 5760

ggctccggag ggctcttcct gctgtgaaag gaaaggagaa gaaagcctgt gggcaatggc 5820

aacctctgag tctggcattc ttgccaatgg ctggccagcg aggagaatct cccgagccct 5880

gacacacaaa ggcattttgt ggctgcagag gaaatgggtt ggctctgaac aaagatgcag 5940

tttctagggc cgtggcccca aatcgcttcc ccgagagtga attttaacac tgtaacaata 6000

aatactactg cacagcactt taaaaaaaaa aaaaaaaaaa aaaaaaa 6047

<210>3

<211>2413

<212>DNA

The <213> mankind (Homo sapiens CDKN1B)

<400>3

cttcttcgtc agcctccctt ccaccgccat attgggccac taaaaaaagg gggctcgtct 60

tttcggggtg tttttctccc cctcccctgt ccccgcttgc tcacggctct gcgactccga 120

cgccggcaag gtttggagag cggctgggtt cgcgggaccc gcgggcttgc acccgcccag 180

actcggacgg gctttgccac cctctccgct tgcctggtcc cctctcctct ccgccctccc 240

gctcgccagt ccatttgatc agcggagact cggcggccgg gccggggctt ccccgcagcc 300

cctgcgcgct cctagagctc gggccgtggc tcgtcggggt ctgtgtcttt tggctccgag 360

ggcagtcgct gggcttccga gaggggttcg ggctgcgtag gggcgctttg ttttgttcgg 420

ttttgttttt ttgagagtgc gagagaggcg gtcgtgcaga cccgggagaa agatgtcaaa 480

cgtgcgagtg tctaacggga gccctagcct ggagcggatg gacgccaggc aggcggagca 540

ccccaagccc tcggcctgca ggaacctctt cggcccggtg gaccacgaag agttaacccg 600

ggacttggag aagcactgca gagacatgga agaggcgagc cagcgcaagt ggaatttcga 660

ttttcagaat cacaaacccc tagagggcaa gtacgagtgg caagaggtgg agaagggcag 720

cttgcccgag ttctactaca gacccccgcg gccccccaaa ggtgcctgca aggtgccggc 780

gcaggagagc caggatgtca gcgggagccg cccggcggcg cctttaattg gggctccggc 840

taactctgag gacacgcatt tggtggaccc aaagactgat ccgtcggaca gccagacggg 900

gttagcggag caatgcgcag gaataaggaa gcgacctgca accgacgatt cttctactca 960

aaacaaaaga gccaacagaa cagaagaaaa tgtttcagac ggttccccaa atgccggttc 1020

tgtggagcag acgcccaaga agcctggcct cagaagacgt caaacgtaaa cagctcgaat 1080

taagaatatg tttccttgtt tatcagatac atcactgctt gatgaagcaa ggaagatata 1140

catgaaaatt ttaaaaatac atatcgctga cttcatggaa tggacatcct gtataagcac 1200

tgaaaaacaa caacacaata acactaaaat tttaggcact cttaaatgat ctgcctctaa 1260

aagcgttgga tgtagcatta tgcaattagg tttttcctta tttgcttcat tgtactacct 1320

gtgtatatag tttttacctt ttatgtagca cataaacttt ggggaaggga gggcagggtg 1380

gggctgagga actgacgtgg agcggggtat gaagagcttg ctttgattta cagcaagtag 1440

ataaatattt gacttgcatg aagagaagca attttgggga agggtttgaa ttgttttctt 1500

taaagatgta atgtcccttt cagagacagc tgatacttca tttaaaaaaa tcacaaaaat 1560

ttgaacactg gctaaagata attgctattt atttttacaa gaagtttatt ctcatttggg 1620

agatctggtg atctcccaag ctatctaaag tttgttagat agctgcatgt ggctttttta 1680

aaaaagcaac agaaacctat cctcactgcc ctccccagtc tctcttaaag ttggaattta 1740

ccagttaatt actcagcaga atggtgatca ctccaggtag tttggggcaa aaatccgagg 1800

tgcttgggag ttttgaatgt taagaattga ccatctgctt ttattaaatt tgttgacaaa 1860

attttctcat tttcttttca cttcgggctg tgtaaacaca gtcaaaataa ttctaaatcc 1920

ctcgatattt ttaaagatct gtaagtaact tcacattaaa aaatgaaata ttttttaatt 1980

taaagcttac tctgtccatt tatccacagg aaagtgttat ttttcaagga aggttcatgt 2040

agagaaaagc acacttgtag gataagtgaa atggatacta catctttaaa cagtatttca 2100

ttgcctgtgt atggaaaaac catttgaagt gtacctgtgt acataactct gtaaaaacac 2160

tgaaaaatta tactaactta tttatgttaa aagatttttt ttaatctaga caatatacaa 2220

gccaaagtgg catgttttgt gcatttgtaa atgctgtgtt gggtagaata ggttttcccc 2280

tcttttgtta aataatatgg ctatgcttaa aaggttgcat actgagccaa gtataatttt 2340

ttgtaatgtg tgaaaaagat gccaattatt gttacacatt aagtaatcaa taaagaaaac 2400

ttccatagct att 2413

<210>4

<211>600

<212>DNA

<213> chimpanzee (Pan troglodytes SOD2)

<400>4

atgaagcaca gcctccccga cctgccctac gactacggcg ccctggaacc tcacatcaac 60

gcgcagatca tgcagctgca ccacagcaag caccacgcgg cctacgtgaa taacctgaac 120

gtcaccgagg agaagtacca ggaggcgttg gccaagggag atgttacagc ccagatagct 180

cttcagcctg cactgaagtt caatggtggt ggtcatatca atcatagcat tttctggaca 240

aacctcagcc ctaacggtgg tggagaaccc aaaggggagt tgctggaagc catcaaacgt 300

gactttggtt cctttgacaa gtttaaggag aagctgacgg ctgcatctgt tggtgtccaa 360

ggctcaggtt ggggttggct tggtttcaat aaggaacggg gacacttaca aattgctgct 420

tgtccaaatc aggatccact gcaaggaaca acaggcctta ttccactgct ggggattgat 480

gtgtgggagc acgcttacta ccttcagtat aaaaatgtca ggcctgatta tctaaaagct 540

atttggaatg taatcaactg ggagaatgta actgaaagat acatggcttg caaaaagtaa 600

<210>5

<211>3233

<212>DNA

<213> rhesus monkey (Macaca mulatta calcium-sensing receptor)

<220>

<221>misc_feature

<222>(2570)..(2654)

<223>n is a，c，g，or t

<400>5

aaaacttctg ggagcctcca aactcctagc tgtcttgtcc ctcgccctgg agagaaggca 60

gaaccatggc attttattgc tgcttctggg tcctcttggc actcacctgg cacacctctg 120

cctatgggcc cgaccagcga gcccaaaaga aaggggacat tatacttggc gggctctttc 180

ctattcattt tggagtagca gctaaagatc aagatctcaa atcaaggccg gagtctgtgg 240

aatgcatcag gtataatttc cgtgggtttc gctggttaca ggctatgata tttgccatag 300

aggagataaa cagcagccca gcccttcttc ccaacttgac gctgggatac aggatatttg 360

acacttgcaa caccgtttct aaggccttgg aagccaccct gagttttgtt gctcaaaaca 420

aaattgattc tttgaacctt gatgagttct gcaactgctc agagcacatt ccctctacga 480

ttgctgtggt gggagcaact ggctcaggcg tctccacggc agtggcaaat ctgctggggc 540

tcttctacat tccccaggtc agttacgcct cctccagcag actccttagc aacaagaatc 600

aattcaagtc cttcctccga accatcccca atgatgagca ccaggccact gccatggcag 660

acatcattga gtatttccgc tggaactggg tgggcacaat tgcagctgat gatgactatg 720

ggcggccagg gattgagaag ttccgagagg aagctgagga aagggacatc tgcattgact 780

tcagtgaact catctcccag tactctgatg aggaagagat ccagcatgtg gtggaggtga 840

ttcaaaattc cacggccaaa gtaatcgtgg ttttctccag tggcccagac cttgagcccc 900

tcatcaagga gattgtccgg cgcaatatca caggcaagat ctggctggcc agtgaggcct 960

gggccagctc ctccctgatc gccatgcccg agtacttcca cgtggttgga ggcaccattg 1020

gatttgctct gaaggctggg cagatcccag gctttcggga attcctgaag aaggtccatc 1080

ccaggaagtc tgtccacaat ggttttgcca aggagttttg ggaagaaaca tttaactgcc 1140

acctccaaga aggtgccaaa ggacctttac ctgtggacac ctttctgaga ggtcacgaag 1200

aaagtggagg caggtttagc aacagttcga cagccttccg acccctctgt acaggggatg 1260

agaacatcag cagtgttgag accccttaca tagattacac acatttacgg atatcctaca 1320

atgtgtactt agcagtctac tccattgccc atgccttgca agatatatat acctgcttac 1380

ctgggagagg gctcttcacc aacggctcct gtgcagacat caagaaagtt gaggcgtggc 1440

aggtcctgaa gcacctacgg cacctaaact ttaccaacaa tatgggggag caggtgacct 1500

ttgatgagtg tggtgacctg gtggggaact attccatcat caactggcac ctctccccag 1560

aggatggctc catcgtgttt aaggaagtcg ggtattacaa cgtctatgcc aagaagggag 1620

aaagactctt catcaacgag gagaaaatcc tgtggagtgg gttctccagg gaggtgccct 1680

tctccaactg cagccgagac tgtctggcag ggaccaggaa agggatcatt gagggggagc 1740

ccacctgctg ctttgagtgt gtggagtgtc ctgatgggga gtacagcgat gagacagatg 1800

ccagtgcctg taacaagtgc ccagatgact tctggtccaa tgagaaccac acctcctgca 1860

ttgccaagga gatcgagttt ctgtcgtgga cagagccctt tgggattgca ctcaccctct 1920

ttgccgtgct gggcattttc ctgacagcct ttgtgctggg tgtgtttatc aagttccgca 1980

acacgcccat cgtcaaggcc acgaaccgag agctctccta ccttctcctc ttctccctgc 2040

tctgctgctt ctccagctcc ctgttcttca ttggggagcc ccaggactgg acttgccgcc 2100

tgcgccaacc ggcctttggc atcagcttcg tgctctgcat ctcgtgcatc ctggtgaaaa 2160

ccaaccgtgt cctcctggtg tttgaggcca agatccccac cagcttccac cgcaagtggt 2220

gggggctcaa cctgcagttt ctgctggttt tcctctgcac cttcatgcag attgtcatct 2280

gtgtgatctg gctctacacc gcgcccccct caagctaccg caaccacgag ctggaggatg 2340

agatcatctt catcacgtgc cacgagggct cactcatggc cctgggcttc ctgatcggct 2400

acacctgcct gctggctgcc atctgcttct tctttgcctt caagtcccgg aagctgccag 2460

agaacttcaa tgaagccaag ttcatcacct tcagcatgct catcttcttc attgtctgga 2520

tctccttcat tccagcctat gccagcacct acggcaagtt tgtctctgcn nnnnnnnnnn 2580

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2640

nnnnnnnnnn nnnnccatcc cgcaacacca tcgaggaggt gcgttgcagc accgcagctc 2700

acgctttcaa ggtggctgcc cgggccacgc tgcgccgcag caacgtctcc cgcaagcggt 2760

ccagcagcct tgggggctcc actggatcca ccccctcctc ctccatcagc agcaagagca 2820

acagcgaaga cccattccca cagcccgaga ggcagaagca gcagcagccg ctgccaagat 2880

gcaagcagaa ggtcatcttc ggcagtggca cggtcacctt ctcactgagc ttcgatgagc 2940

ctcagaagaa cgccatggcc cacaggaatt ctacacacca gaactccctg gaggcccaga 3000

aaagcagcga tacgctggcc cgacaccagg cattactccc gctgcagtgc ggggaagcgg 3060

actcagatct gagcgtccaa gaaacaggtc tgcaaggacc agtgggtgga gaccaccggc 3120

cagaggtgga ggtccctgaa gagttgtccc cagcacttgt agtgtccagt tcacagagct 3180

ttgtcatcag cggtggagga agcactgtta cagaaaatgt actgcattca taa 3233

<210>6

<211>2694

<212>DNA

<213> dog (Canis BCL2-related)

<400>6

gggggggggg ggccggactc aactctactg tggagtcggg gccatccctc atttttctca 60

ctcaggagcg gctggctggc ggcggctggc tggcggcagg agctggcaat gtttggcctc 120

aagagaaacg cagtaatccg gactcaactc tactgtgggg gggccgggct gggggccggc 180

agcggcggcg cctcctcttc gggagggcgg cttttggctt cggggaggga ggccacgacc 240

agacgggagg gagggggagg ggaagccggt gcggtgattg gcggaagcgc cggcgcaagt 300

cccccgacca ctctggcgcc ggacgcccgg agggtcgcgc ggccctcacc cattggcgct 360

gagggcccca acgtcagcgc gacccccccg aggctgctgc tgctcgcgcc cccctgccgc 420

gcgtcgccgc ctgaagagat ggaaggcccg gccgccgacg ccatcatgtc gcccgaagag 480

gagctagacg ggtacgagcc ggaacctttg gggaagcggc cggcggtcct gcctctgctg 540

gagctggtgg gggaggccag cagtggcccc ggcatggacg gctcgctacc ctcgacgcca 600

cccccggcgg aggaggagga agatgagttg taccggcagt ccctggagat tatctctcgg 660

taccttcggg aacaggccac aggcgccaag gacgcgaaac cactgggcgg gtctcgggcg 720

gccagccgga aggcgttaga gaccctccag cgagtcgggg acggggtaca gcgcaaccac 780

gagacagcct tccaaggcat gcttcggaaa ctggacatca aaaacgaaga cgatgtcaaa 840

tcgttgtctc gagtgattgt ccatgttttc agtgacggag taacaaactg gggcaggatt 900

gtgactctta tttcctttgg tgcctttgtg gccaaacact tgaagagtat aaaccaagaa 960

agctgcatcg aaccattagc agaaagcatc acagatgttc tcgtaaggac gaaacgagac 1020

tggctagtca aacaaagagg ctgggatggg tttgtggagt tcttccatgt agaggaccta 1080

gaaggcggca tcagaaatgt gctgctggct tttgcaggtg ttgctggagt aggagctggt 1140

ttggcatatc taataagata gccttttaag tgcaataatt aacttttaaa caaccccagg 1200

caccaaaacc acatatgact gctgtgaaat caaatgtatt tatgaagttg gactttgagc 1260

tgtccaggct gtaacctcgg agagttctac tctagcaacg tagaaaagca agtggcaaga 1320

ggattatggc taacaggaat aaatacatgg gagaagtagt cccccttgaa gagtcactgt 1380

ctaaaagaag cagagctcag tttcagcaac aggcaaactt ggggaggcca tggaggagga 1440

cttttagatt tagtaaagtt ggtagggttg aagagactta attttcttgt ctagaacagg 1500

agagtggcca gtagccaggc tagtcataga gtccattaaa tatgtccact gaattcatta 1560

acttcccata tagtgttaaa agagaagcac taacaatggc attgatctgt atgaaatgga 1620

tttaagctac aggtgataga actatgacta gaagccgcag tactgtacaa cagtgtgaag 1680

gaaagctttt tctctgtaat tagctttccc aagtatactt cttgaaagtc caagtgctca 1740

ggacgtttta cctgttctac tttggcttgg tttgagtggt ttagtttatt agcctagtaa 1800

tggccaacaa tacttgactt agggtcaata attacaattg caaatgtggg aatagcctca 1860

atttttaagg caaaaacaaa ttataaatgt atttgtctgt aaaaattgtg tatattttta 1920

cagaaagtct atttctttga aatgtaaagg gatgaagagt ctcaaaatat attagttttt 1980

ttttcatgcc cgtttgaaat ttacaacttc tgtagttagg aatctatttc ttacagcttt 2040

tctaaatttt gtcctggtca gttctagatt gtatacagaa ccaattgatg taattgtatg 2100

caacttggtt gtagtggaac aattccaatt cataactatg cagacttttt aattttccta 2160

tctgattggt aagtattctt tagtggtttt gggtgttttg ttttgtttta aacctgggat 2220

tgagaagttg atgaatggaa attcatcctt taacttcatt acatgtgggt ttacaataat 2280

tgagtcaagg tgaagtttga agtttggggg cagggggcgt gtgtgggttg ataacttaaa 2340

aaaataatgg gctctgattg ggcagctact catttgagtt ccttccattt gacctaattt 2400

aactggtgaa atttaaattg agtttatgag ctcatcttca aaacttttgc tagaagattt 2460

tcagctgttc caaatgggac ttactaacag tatgtatata aaaagatcac atcagtggat 2520

gagagacatt tgatcccttg tttgcttaat aaattgtaaa atgatggctt ggaaaagcag 2580

gcttatagtc taaccatggt gctattatta ggcttgcttg ttaaacacag gtctaagcct 2640

agtatatcaa taaaacaaat acttatttca tttgaaaaaa aaaaaaaaaa aaaa 2694

<210>7

<211>3863

<212>DNA

<213> rat (Rattus norvegicus kazrin)

<400>7

ctgcgccgga ggagccggcg cttgccggtg cctgggggtc ggggcgcggg gaaacccggg 60

ccgctggggt gacccaacag gtagagccgg ggccacggcc ggccacgcgc cccccgcgtg 120

catcatgcag ctctttgtca cctctctcgc ccccaggcca aaatcctgag catgatggaa 180

gacaataagc agctcgcgct ccgcatcgat ggggcggtcc agtcggccag ccaggaggtg 240

accaacctgc gagccgaact cacggccacc aaccggagac tggcggaatt gagcggcgga 300

ggcggcggcc ccggctcggg cccgggagct gcgaccagcg cctcggcagc agcggtgacg 360

gtggctgact cggcggtggc gaccatggag aaccatcagc acggagctca agtgctcctg 420

cgggaggaag tggtccagct tcaggaggaa gtccaccttc tccgacagat gaaggaaatg 480

ctggcaaagg acctagagga gtcacagggc ggcaaatgct ccgaggtgct gtcggccact 540

gaactccggg tgcagctggt acagaaggag caggagctgg ccagagccag ggaagccttg 600

caagccatga aagctgaccg gaagcgctta aagggcgaga agaccgacct ggtgagccag 660

atgcagcagt tgtacgccac gctggagagc cgcgaggagc agttgcgaga cttcatccgc 720

aactatgaac aacaccgcaa ggagagtgag gatgctgtca aagcgctggc caaggagaaa 780

gacctgctgg agcgagagaa gtgggagctc cgacggcagg ccaaggaggc cacggaccat 840

gctgcggccc tgcgctccca gctggacctc aaggacaacc gcatgaagga gctggaggcc 900

gagctagcca tggccaagca gtccttagcc acactgacca aggatgttcc caagaggcat 960

tcactcgcca tgcccggcga gacggtgctc aatggcaacc aggaatgggt ggtgcaggct 1020

gacctcccac tgactgccgc catccggcag agccaacaga ctctctacca ctcccaccct 1080

ccccaccctg cagaccgaca agcggtcagg gtgagcccct gccactcaag acagccttcc 1140

gtcatctctg atgcttccgc tgctgaaggt gaccggtcat ctacaccgag cgacatcaac 1200

tccccaagac accggacaca ctccctctgc aacggcgaca gtcccggccc agttcagaag 1260

agcctacaca accctattgt acagtcacta gaggatcttg aagaccaaaa acggaaaaag 1320

aagaaagaga agatgggatt tggctccatc tctcgagtct tcgccagagg gaaacaacgg 1380

aagtccctcg acccgggcct ctttgatggt accgcccctg attattacat agaggaagac 1440

gcggactggt gataacccac cgccctccgc ccgttgtcca cgggcgtgtc tgtgtgtgga 1500

tgagccttgg agagtgtgcg agagtgtgcg tgcgcgtggg gatgcggagc ctgtgtgtgg 1560

ggtgtgtgtg cacggaggca ctcgccctcg tgggtgcgtg tagccaagcg cctggaacag 1620

acagaaacac aaatgtgacc cccaaccccc ctccgcgtcg tcacctctgt aatcgctgta 1680

cataccacaa accgtgtgtg actctgtcaa ccccgttgtc tctgagcgat acacttgtgt 1740

ttgtttctct cgtttgtttt gtttttcgat gttttttggg ggggggggtt gtttgtttgt 1800

ttctgtttaa atccgtcgtg tttttgtttt taatttgccc ctttctcctc cctcctccct 1860

ttttatgaaa cttgaacact tgaaggactg ctgtgtattt gtaaataaca aaagtagtgt 1920

gcactttgtg cttgtaaagt ccctggtcca gccgctactc ctacagccca cgtcgccaag 1980

gacgcgcggt ctgtgtttca tgtcccctcc ttcttcggtg tgaacgtgtg ggaccagacc 2040

ttgttctcgg ggtgtgcccg agtcacttta accacaaaaa cacccccgcc atcgtcgtca 2100

gggtaaggtc actctgcaga gacccgtgag cccatccaag actttttggc tgggccacag 2160

tgtttataag ggggtcatat gggctctttt taatggcctg cctgttttta aattgtgttg 2220

ctgtttcttt ctttatggag caaaaagaga gaaagaaaac ttgcatttaa tttatttgca 2280

caaatgcaga aagcgtattc tatctaaatt attacgtaaa tattggaatg tatatttttc 2340

cacggggtgg gcgggaggcg ggcgtttctg ctgacttgtc tattctgtgt tcatgccgct 2400

gcagacactg tgcaaggtag aattcaggtg gctagagctg gggacctcag acgaccagct 2460

tgcttcttct ttttttctct tttctgtccc cccccccccg gctgctgtcc ctgcatgaga 2520

ggactcccac cttctgctct ggcctttctc tctagatatt ctctctcttt atatatatat 2580

ctatatattt atttatctga catacagatc atgacttcgg tgagcagaga acactaacag 2640

tcaaattccc cttctcttgg acctgtcttt ggaaccatgt tgattgagac aagggaaggt 2700

tagcctgggg cacatctgtg gggtctaacc agacatctgg gcttgagtca gtgggtaaga 2760

ggctcctggt ggcacataca caatgcctct ctatccttag cttattccaa acatagggca 2820

agccagtgtc aacaaagatg atgcccacct tctgggaggg gctgttgggg gcagtatcta 2880

gcaggcaagc cctttgccct ctttcgtcca gttgacttct ttgcccacac atggactttc 2940

tcataatccc tggatccctg tcctctgatt atatcagcga ctcaagcaga ctaagcccaa 3000

aagggctggc aggtgcctgt gcctgtttgt gggggtagca ctgtccctat ggggcctcac 3060

ctggggcatg gagaacccac aagtttccag ccagtaccac cccacccccg aactcagttt 3120

cctctttctc tcctccccat ccccttttaa ctgatcctaa accactaata actcagccca 3180

caccccttat ggccactgct gtgctccctc taacagaaac attctcaact cttgaaagat 3240

ttttcagtac cggatggaaa atctggaatg tataggatct tgtgtccaaa gtctctaaaa 3300

aaacaagcca aaaagcccct caccaggtca cctcttccct cccgacctgg cccagaggcc 3360

ggagagagag caggcaggga caccagatgg gccttcctgg gtacacaaca accctttcgg 3420

ctcagctgat taacccagcc tggtggtgag ggagagaggc cagagctggc atcagaggga 3480

ggtcttagaa aacactaccc ccaccccatg aattttcggg ttttcttttc ctttcttttt 3540

tttatggttc ccccacacac acccttcctt ttaacttaca agaaaataag gttgcaacca 3600

ctcccggtta atggcttagt cactcctctt cgtttcagtt ttttggaaat taggagatta 3660

tcctgtgagt tactaaggat ggtttatttt cgagaaccac atcagatagg gaatgagtta 3720

tgggtaactc tagatgaaaa tgacccttcc cccccccccg ggagaggttt ctcgaaggca 3780

tggatgcaaa atataaaata ttaaaaaaaa gtctaaataa agcttatttt aaaataaaaa 3840

aaaaaaaaaa aaaaaaaaaa agg 3863

<210>8

<211>903

<212>DNA

<213> rat (Rattus Birc5)

<400>8

gatcatgggt gctacggcgc tgcccccgat ctggcagatg taccttaagg accaccggat 60

ctacaccttc aagaactggc ccttcctgga ggattgctcc tgcaccccag agcggatggc 120

ggaggctggc ttcatccact gccctaccga gaatgagcct gatttggccc agtgtttttt 180

ctgctttaag gaactggaag gctgggaacc ggatgacaac cctatagagg agcataggaa 240

gcactcccct ggctgcgcct tccttacagt caagaagcag gtggaagaac tgaccgtcag 300

cgagtttttg aaactggaca aacaaagagc caagaacaaa attgcaaagg agaccaacaa 360

caaacagaaa gagttcgagg agacccggag gaccgtccgg cagtccattg agcagctggc 420

tgccttacgc tgagcctttg ctgggcaacc tggacctgag tgacatgcca cggctaagcc 480

acttgtccca gctttccagg cctgcctggc cgccttggtg tcttacagga gaccgtgaca 540

tttcgaaact ggacgtcaga tgatttgggg ttttgcttta aaggggctca gcctgcgtgg 600

ccacctctct ttggttttgt ggctttgctc tattgtgacc tggacttaag cactgaggaa 660

gggagtggat gagggacagg attctctgac aggatctatg ggtggggagg ggggtgaagg 720

gagggttgtg caaggccttt ctggtcttga tgtttccatg cctggcagct gtcgcagccc 780

atgtgtaggt gttggtttat atatgtttgt gctgataatt cttcttctgt ccttctgatg 840

agtcctccta ccatggggta atggaataaa ataacttaac aaaaaaaaaa aaaaaaaaaa 900

aaa 903

<210>9

<211>8229

<212>DNA

<213> mouse (Mus musculus Pten)

<400>9

gcggcaggat acgcgcttgg gcgtcgggac gcggctgcgc tcagctctct cctctcggaa 60

gctgcagcca tgatggaagt ttgagagttg agccgctgtg aggccaggcc cggcgcaggc 120

gagggagatg agagacggcg gcggccacgg cccagagccc ctctcagcgc ctgtgagcag 180

ccgcgggggc agcgccctcg gggagccggc cgggcggcgg cggcggcagc ggcggcgggc 240

ctcgcctcct cgtcgtctgt tctaaccggg cagcttctga gcagcttcgg agagagacgg 300

tggaagaagc cgtgggctcg agcgggagcc ggcgcaggct cggcggctgc acctcccgct 360

cctggagcgg gggggagaag cggcggcggc ggccgcggct ccggggaggg ggtcggagtc 420

gcctgtcacc attgccaggg ctgggaacgc cggagagttg ctctctcccc ttctcctgcc 480

tccaacacgg cggcggcggc ggcggcacgt ccagggaccc gggccggtgt taagcctccc 540

gtccgccgcc gccgcacccc ccctggcccg ggctccggag gccgccggag gaggcagccg 600

ctgcgaggat tatccgtctt ctccccattc cgctgcctcg gctgccaggc ctctggctgc 660

tgaggagaag caggcccagt ctctgcaacc atccagcagc cgccgcagca gccattaccc 720

ggctgcggtc cagggccaag cggcagcaga gcgaggggca tcagcgaccg ccaagtccag 780

agccatttcc atcctgcaga agaagcctcg ccaccagcag cttctgccat ctctctcctc 840

ctttttcttc agccacaggc tcccagacat gacagccatc atcaaagaga tcgttagcag 900

aaacaaaagg agatatcaag aggatggatt cgacttagac ttgacctata tttatccaaa 960

tattattgct atgggatttc ctgcagaaag acttgaaggt gtatacagga acaatattga 1020

tgatgtagta aggtttttgg attcaaagca taaaaaccat tacaagatat acaatctatg 1080

tgctgagaga cattatgaca ccgccaaatt taactgcaga gttgcacagt atccttttga 1140

agaccataac ccaccacagc tagaacttat caaacccttc tgtgaagatc ttgaccaatg 1200

gctaagtgaa gatgacaatc atgttgcagc aattcactgt aaagctggaa agggacggac 1260

tggtgtaatg atttgtgcat atttattgca tcggggcaaa tttttaaagg cacaagaggc 1320

cctagatttt tatggggaag taaggaccag agacaaaaag ggagtcacaa ttcccagtca 1380

gaggcgctat gtatattatt atagctacct gctaaaaaat cacctggatt acagacccgt 1440

ggcactgctg tttcacaaga tgatgtttga aactattcca atgttcagtg gcggaacttg 1500

caatcctcag tttgtggtct gccagctaaa ggtgaagata tattcctcca attcaggacc 1560

cacgcggcgg gaggacaagt tcatgtactt tgagttccct cagccattgc ctgtgtgtgg 1620

tgatatcaaa gtagagttct tccacaaaca gaacaagatg ctcaaaaagg acaaaatgtt 1680

tcacttttgg gtaaatacgt tcttcatacc aggaccagag gaaacctcag aaaaagtgga 1740

aaatggaagt ctttgtgatc aggaaatcga tagcatttgc agtatagagc gtgcagataa 1800

tgacaaggag tatcttgtac tcaccctaac aaaaaacgat cttgacaaag caaacaaaga 1860

caaggccaac cgatacttct ctccaaattt taaggtgaaa ctatacttta caaaaacagt 1920

agaggagcca tcaaatccag aggctagcag ttcaacttct gtgactccag atgttagtga 1980

caatgaacct gatcattata gatattctga caccactgac tctgatccag agaatgaacc 2040

ttttgatgaa gatcagcatt cacaaattac aaaagtctga tttttttttt cttatcaaga 2100

gggataaaat accatgaaaa aaaaaaaact tgaataaact gaaatggacc tttttttttt 2160

tttttttttt ttaaatggca ataggacatt gtgtcagatt gcagttatag gaacaattct 2220

cttctcctga ccaatcttgt tttaccctat acatccacag ggttttgaca cttgttgtcc 2280

agttaaaaaa aggttgtgta gctgtgtcat gtatatacct ttttgtgtca aaaggacatt 2340

taaaattcaa ttaggataaa taaaagatgg cactttccca ttttattcca gttttataaa 2400

aagtggagac aggctgatgt gtatacgcag gagtttttcc tttattttct gtcaccagct 2460

gaagtggctg aagagctctg attcccgggt tcacgtccta cccctttgca cttgtggcaa 2520

cagataagtt tgcagttggc taaggaagtt tctgcagggt tttgttagat tctaatgcat 2580

gcacttgggt tgggaatgga gggaatgctc agaaaggaat gtttctacct gggctctgga 2640

ccatacacca tctccagctc cttagatgca cctttcttta gcatgctcca cttactaatc 2700

tggacatccg agagattggc tgctgtcctg ctgtttgttt gtgcatttta aagagcatat 2760

tggtgctaga caaggcagct agagtgagta tatttgtagt ggggtacagg aatgaaccat 2820

ctacagcatc ttaagaatcc acaaaggaag ggatataaaa aaagtggtca tagatagata 2880

aaagacacag cagcaatgac ttaaccatac aaatgtggag gctttcaaca aaggatgggc 2940

tggaaacaga aaatttgaca atgatttatt cagtatgctt tctcagttgt aatgactgct 3000

ccatctccta tgtaatcaag gccagtgcta agagtcagat gctattagtc cctacatcag 3060

tcaacacctt acctttattt ttattaattt tcaatcatat acctactgtg gatgcttcat 3120

gtgctggctg ccagtttgtt tttctcctta aatattttat aattcttcac aggaaatttc 3180

aacttgagat tcaacagtaa gcaggttttg tttttttttt ttcctagaga ttgatgatgc 3240

gcgtcctcag tccagtggct gtcagacgtt cagccccttt gaccttacac attctattac 3300

aatgagtttt gcagttttgc acattttttt taaatgtcat taactgttag ggaattttac 3360

ttgaatactg aatacatata atgtgtatat taaaaaagtc attgtttgtg ttaaaaaaga 3420

aattagagtt gcagtaaatt tacagcactg cacgaataat aaggcattga agtttttcag 3480

tagaaattgt cctacagatg ctttatcgac ttgctattgg aagaatagat cttcttaaat 3540

gtgcagtgtt gagtcacttc gttatagtgg tagagttggg attagggctt caattttact 3600

tcttaaatat cattctatgt ttgatatgcc cagactgcat acaatttaaa gcaagagtac 3660

aactactatc gtaatggtaa tgtgaagatg ctattacaaa ggatctcctc ccaacccctc 3720

gggaatttgg tgtctttcaa attatatctt gaccttgaca tttgaatatc cagccattat 3780

tagatttctt aatggtgtga agtcccattt tcaataactt attggtgctg aaattgttca 3840

ctagctgtgg tctgacctag ttaatttaca agtacagatt gcataggacc cactagagaa 3900

gcatttatag tttgatggta agtagattag gcagaacgcc atctaaaata ttcttagaaa 3960

ataatgttga tgtattttcc atacctcatc agtttcactc aaccaataaa gtttttaaaa 4020

ttgtaacaaa gctcttagga tttacacatt tatatttaaa cattgataca tgaatattga 4080

ctgactgttg ataaagtcag agacaacttt tcctgagatc tcaccatgga aatctgtaca 4140

cccccttgtc tttcctaaaa gctgaaagtg gctgactaaa atgcaaagca gctgttgatg 4200

ttttgaagat agtgataaac actgttcttt gttagttttg ggcacagcat gctaaactat 4260

aacttgtatt gttccaatat gtaacacaga gggccaggtc atgaataatg acattacaat 4320

gggctgttgc actgttaata tttttccttt ggaatgtgaa ggtctgaatg agggttttga 4380

ttttgaatgt ttcagtgttt ttgagaagcc ttgcttacat tttatggtgt agtcattgga 4440

aatggaaaaa tggcattata tatatattat atatatataa atatatatat tatacatact 4500

ctccttactt tatttcagtt accatcccca tagaatttga caagaattgc tatgactgaa 4560

agggttttga gtcctaattc aaactttctt tatgacagta ttcacgatta gcctgaagtg 4620

cattctgtag gtgatctctc ccgtgtttct ggaatgcttt cttagactct tggatgtgca 4680

gcagcttatg tgtctgaaat gacttgaagg catcaccttt aagaaggctt acagttgggc 4740

cccgtacatc ccaagtcctc tgtaattcct cttggacatt tttgccataa ttgtaaaagg 4800

gtagttgaat taaatagcgt caccattctt tgctgtggca caggttataa acttaagtgg 4860

agtttaccgg cagcatcaaa tgtttcagct ttaaaaataa aagtaggtta caagttacat 4920

gtttagtttt agaaaatttg tgcaatatgt tcataacgat ggctgtggtt gccacaaagt 4980

gcctcgttta cctttaaata ctgttaatgt gtcgtgcatg cagacggaag gggtggatct 5040

gtgcactaaa cggggggctt ttactctagt attcggcaga gttgccttct acctgccagc 5100

tcaaaagttc gatctgtttt catatagaat atatatacta aaaccatcca gtctgtaaaa 5160

cagccttacc ccgattcagc ctcttcagat actcttgtgc tgtgcagcag tggctctgtg 5220

tgtaaatgct atgcactgag gatacacaaa tatgacgtgt acaggataat gcctcatacc 5280

aatcagatgt ccatttgtta ctgtgtttgt taacaaccct ttatctctta gtgttataaa 5340

ctccacttaa aactgattaa agtctcattc ttgtcattgt gtgggtgttt tattaaatga 5400

gagtatttat aattcaaatt gcttaaatcc attaaaatgt tcagtaatgg gcagccacat 5460

atgattacaa agttcctgtg catttttcta tttttccccc tccttgctat ccttccaagc 5520

aaagcatctt tctgtcatct tggtagacac atacctgtct actcatggtt aagaagagca 5580

ctttaagcct tagtcatcac ttaataagtt attccaggca cagtaaaaag ttcaaggttc 5640

ttggaaaacg gtgcttattt ctcttcttat aagccagatg tctgaagata gccctaaccc 5700

caagaacggg cttgatgtct caggtctgtt ctgtggcttt ctgttttttt taacactgca 5760

gttggccatc agcacatggg aggtttcatc gggacttgtc cagagtagta ggctcaaata 5820

tactatctcc tttctaatat tcttaaaggc taaggagtcc tttcaatata acagtaagat 5880

aacttgtgat gttttagaag taagcagacc attaatgtca atgtggagtc ttaatgttac 5940

atgaagttga tagtttctct gtgacccatt taaaaataca aaccgagtag catgcaatta 6000

tgtaaagaaa tatgaagatt atatgtagtc acacattttc tttagaattc ttagtttggt 6060

gaaaacttga atataaaggt attttgattt atatgacatt ttgatgatat ttgaaaaaaa 6120

ggaatttcct gacattttgc ttttagatca tgtcccccat tgtgctgtaa tttaagccaa 6180

cttggttcag tgaatgccat caccatttcc attgagaatt taaaactcac cagtgtttaa 6240

catgcaggct tctgagggct cccggagaat cagaccttaa gcccagttga tttacttcta 6300

acgtgaaact tcgagttcct gtatactttg ctagataatt tgtggtacat ctaaagctta 6360

gtcttaagtg gcttgtgtgt ggattttatt caacattctt gttgctaggg tagagagaaa 6420

tgttgctgag tagaaacaag agtacccagt tcaatgtggt acagagagca gtccctaaaa 6480

tctgtacaca gtgtaatgga ccactttagg agtcaagagg ctgatttttc ctatgaaatt 6540

acattgcaac aggaagcctt ctagtatagt tccttttact gttagaatat gtttttatgc 6600

atacgctata gctgctttcc catcttccaa caacaggtat caggatgtaa gcaagcttta 6660

aacagtgtga agatggcagg atagtgtcat cggtaacagt cctctgactc taaatgtagt 6720

tgctctgtaa cactttgtga atataacatc acaattctca tgtccttggg gggggggggc 6780

atacccagta ttagtatgtt ttagtgacta agcaatcatt tttctgttta ctcatgtaca 6840

ttttctcttt aaaactaaaa cctgtactgt gtatgtctcc aaagcctttt agcttagttt 6900

ttaggaaatg aacactgaat ggatcacttt ttagtgtagc aggtatggga tatgtgcatt 6960

atagagagac cttgtcagct ctctgggcct atttgaatgt ttattgttgg tgtgaggatg 7020

gtaggggaat cagtaaatac aagttacgtt ggtttagcag agcaagctca gtgtgggtat 7080

ttctctttga agcgtggtgc gtgacgcact gtgagtagag aatttggtca ccctttgagt 7140

cctcttgcat tttgcaaact tgctcagcaa atgcgtacct accttgcccc ctaggtaaaa 7200

gcaggaacta ctactgattt atctgtcact cagctgtctt tatatgtgtg cttctgtgac 7260

ttgtatcaca caagaatctt aaagatttca caaattgtta ccttttagct ctgaatgttg 7320

agtattctgg tgggctaaca acaagacaaa ctcttgacag tcatttgaga attttcatga 7380

aacatttagc tgaaaacatt ttataattta tgaaaaaaat gtgttacctt aaacttttac 7440

atatgtggga gacattaact gccatatttg agcatactga attttaaatt taaaataaag 7500

ctgcatattt ttaaatgaaa tgtttaacaa ggattcatat tttttgtttt ttaagattaa 7560

aaataattta tgtcttctca tgtggaacct catctgtcac aatggttaga ttatacagaa 7620

tggagcaagg cttgtagtgg tttagcttac agtaaaattc ttaatgttta gatgtgttta 7680

cttactggct gttatgtata cttttgagat tttccacctg ttctgtgtag ttttctaaat 7740

gatactccta cttaaaaaca gcattttagt atctattttc tgtctccatt aaatggtcct 7800

cattttctat tgagtttgga agtgtgcaca ttgtgtgtgt gtgtgtgtgt gtgtgtgtgc 7860

acacgtgtgc gcgcccgtgc gtgtgtctat ttgtggagtt tgtatgggag aattagtttt 7920

gaaagtgcta gaatagagat gaaatttggt tcaagtaaaa ttttcccact gggattttac 7980

agtttattgt aataaaatgt taattttgga tgaccttgaa tattaatgaa tttgttagcc 8040

tcttgatgtg tgcattaatg agatatatca aagttgtata ttaaaccaaa gttggagttg 8100

tggaagtgtt tttatgaagt tccgtttggc taccaatgga cataagacta gaaatacctt 8160

cctgtggaga atatttttcc tttaaacaat taaaaaggtt cattattttt gaaaaaaaaa 8220

aaaaaaaaa 8229

<210>10

<211>1329

<212>DNA

<213> aspergillus tubigensis (Aspergillus fumigatus Af293 asparaginase family protein)

<400>10

atgagtcaac cacagagagg tggaggtatc tctgccatct tcgtccatgt aggggcggga 60

tatcacggcc atggaaacga gagagcccac cttcaggtat gcgaaaacgc ttgtaaagcc 120

gcaatgggca ttttaaaaag cggtggatca gctctggatg ctgttgagat ggccatcatt 180

gtaatggagg atgacgagat tacaaatgct ggctatggaa gcaacctgac gattgaagga 240

gctgtcgaat gtgatgctac tattgtcgac caccatggca ggagcggcgc agctggagcc 300

gtctcgccgc gaatgctact tgaaacctct gcaaagcctt taacttgcca cagagtgcct 360

ccaagcttcc tcgttggtga gggtgcaacg gactttgctt atgagcaagg attggtcata 420

ctgcctcctg acgggctcat atcgcgcttt tcgaaagaga gatggcaccg ctggctgcag 480

gatctcgagg ctgctgaact agtcgaaaga aaacgggatc cttcacgttt ccgcatggaa 540

gaagatagag catccttcct tcgtcggcca atgctgaatc gtaatccagc ccgcctcatt 600

gacaatacgc gtttacgccc acatctaccc agctctccct tcaccggagt cgacttgctg 660

aacccccttc ctcgtctcca ggctcttgga agcagacaaa ccatagctcc tgcaccacca 720

gctatgcctt cgaaccaagg acacggcatg ggtcctgcaa ttggacaacc tatggctgtg 780

gcaggtggac aaagtatggc ttctgcacct gagcaaggta tggctgcctc ggccaacacc 840

catgcagatg gtgcgacttt ccgcgcgtgc cctacaaaag caccttgtgc acctgacact 900

tcgacgactg ctcacccggg cgctgcagac gaggatgaag atatgatcag cgatacagtc 960

ggggccatag ctgtagactg ttatggcaat attgcggcag gttcttcgtc tggcgggatt 1020

ggcgcgaagc atcgcggtcg aattgggcct gctgcgctgg tgggcatcgg cacctatgtg 1080

attcctgtgg atcccagcga tcccgaacaa gtatcagtcg cttcagttac atcgggcaca 1140

ggcgagcaca ttgctacaac tatggctgcg cagacgtcag ctgcaagact ctattactgt 1200

caaaagaagt gtaaggacgg tactttcgaa agtgtatccg aagacgaagc tatgaatgca 1260

atcatagcca ctgagttcat gggtgagtta ttgtattcca tattccttac gaccattgat 1320

agagaataa 1329

Claims (7)

1. the small RNA of a modification, it comprises the first fragment and the second fragment, and described the first fragment and the second fragment can form double-stranded region, described the first fragment comprises at least one continuous UA sequence, the continuous UA sequence of the UA sequence complementation that described the second fragment comprises at least one and described the first fragment, the UA sequence of described the first fragment and the UA sequence of described the second fragment form UA/UA site, it is characterized in that, at least one Nucleotide in described UA/UA site is through modifying, this modification makes the stability of the small RNA of modifying higher than the small RNA of unmodified, in the described nucleotide sequence that is modified to small RNA, 2 '-OH of ribose is replaced by methoxyl group or fluorine, Nucleotide in described UA/UA site, other Nucleotide is not through modifying.

2. the small RNA of modification according to claim 1, wherein, the small RNA of this modification comprises multiple UA/UA site, and at least one Nucleotide in the plurality of UA/UA site is through modifying.

3. according to the small RNA of the modification described in any one in claim 1 or 2, wherein, in described UA/UA site, only has a uridylate through modifying.

4. according to the small RNA of the modification described in any one in claim 1 or 2, wherein, the single chain molecule that the small RNA of this modification is hairpin structure, the complementary region between described the first fragment and the second fragment forms double-stranded region.

5. according to the small RNA of the modification described in any one in claim 1 or 2, wherein, the small RNA of this modification comprises positive-sense strand and antisense strand, and described positive-sense strand and antisense strand are continuous nucleotide chain, described first section is positioned at positive-sense strand, and described the second fragment is positioned at antisense strand.

6. according to the small RNA of the modification described in any one in claim 1 or 2, wherein, the small RNA of this modification comprises positive-sense strand and antisense strand, and described positive-sense strand is the discontinuous nucleotide chain that comprises two or more positive-sense strand parts; Described antisense strand is continuous nucleotide chain; Described first section is positioned at one or more positive-sense strand parts, and described the second fragment is positioned at antisense strand.

7. according to the small RNA of the modification described in any one in claim 1 or 2, wherein, the small RNA of this modification is the hybrid molecule that comprises ribonucleotide and at least one deoxyribonucleotide.