CN103275978A - Modified small interfering RNA and preparation method thereof - Google Patents
Modified small interfering RNA and preparation method thereof Download PDFInfo
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- CN103275978A CN103275978A CN2013100534714A CN201310053471A CN103275978A CN 103275978 A CN103275978 A CN 103275978A CN 2013100534714 A CN2013100534714 A CN 2013100534714A CN 201310053471 A CN201310053471 A CN 201310053471A CN 103275978 A CN103275978 A CN 103275978A
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Abstract
The invention provides a modified small interfering RNA and preparation method thereof, which comprises a first fragment and a second fragment, and the first fragment and the second fragment form a double-chain area, and the first fragment comprises at least a continuous CA sequence or UG sequence, and the second fragment comprises at least one continuous UG sequence or CA sequence which is complementary to the CA sequence or UG sequence of the first fragment, and the CA sequence or UG sequence of the first fragment and the UG sequence or CA sequence of the second fragment form CA/UG locus, wherein, at least one nucleotide of the CA/UG locus is modified, thereby the stability of the modified small interfering RNA is higher than the unmodified small interfering RNA, and the first fragment is 5'-UGAACGUCACCGAGGAGAATT-3', and the second fragment is 3'-TTACUUGCAGUGGCUCCUCUU-5'. The invention increases the serum stability of the modified small interfering RNA, and reduces the latent cytotoxicity of the modified small interfering RNA and influence of modification to the biological activity of the small interfering RNA.
Description
The application be that April 3, application number in 2009 are 200910081145.8 the applying date, denomination of invention divides an application for the application for a patent for invention of " 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 that (RNA interference is that (double-stranded RNA, dsRNA) molecule is closed homogenic expression or made the phenomenon of this genetic expression silence in the mRNA level by double-stranded RNA RNAi).The RNA perturbation technique is called clpp gene low (knock-down) or gene silencing (gene silencing) again visually, it is a kind of typical posttranscriptional gene regulate and control method, claim again PTGS (post-transcriptional gene silencing, PTGS).The report of relevant RNA interference appears at nineteen ninety the earliest, report the RNA interference phenomenon in the transgenic plant simultaneously by two different research groups, in nearly all eukaryotes such as nematode, fruit bat, zebra fish and mouse, observed the RNA interference phenomenon again later on.1999, Hamilton and Baulcombe have detected length in the plant that the RNA interference takes place be the RNA fragment of 21-25 Nucleotide, and it is necessary that these RNA segments are proved to be the RNA interference, is called as small RNA (siRNA).The silencing complex that the relevant enzyme of double-stranded siRNA and cell source and protein formation RNA induce (RNA-induced silencing complex, RISC).In the RNA interfering process, the positive-sense strand among the double-stranded siRNA is excluded out complex body, and antisense strand instructs RISC to be attached to the homologous site of said target mrna, then by the degraded of the rnase iii in mixture said target mrna, thereby closes target gene expression.
But because the less stable of small RNA (siRNA), in vivo easily by nuclease degradation, so people carry out chemically modified to synthetic siRNA, increasing the serum stability of siRNA, thereby suppress the expression of goal gene effectively.
At present, because people lack enough understandings to the degradation process of siRNA in serum and mechanism, can only rely on experience separately to select a plurality of Nucleotide in the siRNA molecule to carry out chemically modified randomly.Though this modification strategy can improve the serum stability of siRNA molecule well, but owing to lack guide of theory, usually in the siRNA molecule, introduced excessive modification, increased the potential cytotoxicity of the siRNA after modifying, and reduced the biologic activity of siRNA under many circumstances, thereby restricted the siRNA application in vivo after modifying.
In addition, in siRNA, in Nucleotide, introduce a large amount of ways of modifying blindly, also limited some and had better stabilising effect, but the application of the relatively large modifying method of cytotoxicity in studying in vivo.
Therefore, design pointed modification protocols, realize that by minimum modification optimum stable purpose is the problem that presses for solution at present.
Summary of the invention
Introduce the big problem of small RNA cytotoxicity that a large amount of modifications causes the modification that obtains blindly thereby the objective of the invention is to overcome exist in the modification protocols of existing siRNA, provide that a kind of serum is stable, small RNA that have the good biological activity and the modification that cytotoxicity is lower.
The invention provides a kind of small RNA of modification, it comprises first fragment and second fragment, and described first fragment and second fragment can form double-stranded region, described first fragment comprises CA sequence or the UG sequence that at least one is continuous, described second fragment comprises at least one and the CA sequence of described first fragment or continuous UG sequence or the CA sequence of UG sequence complementation, the UG sequence of the CA sequence of described first fragment or UG sequence and described second fragment or CA sequence form the CA/UG site, wherein, at least one Nucleotide in the described CA/UG site is through modifying, this modification makes the stability of the small RNA of modification be higher than the small RNA of unmodified, described first fragment is 5 '-UGAACGUCACCGAGGAGAATT-3, and second fragment is 3 '-TTACUUGCAGUGGCUCCUCUU-5 '.
Preferably, in the described CA/UG site, has only cytidylic acid(CMP) through modifying.
Preferably, the Nucleotide in described CA/UG site, other Nucleotide is not through modifying.
The small RNA of modification provided by the invention wherein, describedly is modified at least a in the following modification:
(1) to connecting the modification of the phosphodiester bond of Nucleotide in the nucleotide sequence of the small RNA of described modification;
(2) to the modification of ribose in the nucleotide sequence of the small RNA of described modification;
(3) to the modification of base in the nucleotide sequence of the small RNA of described modification.
Preferably, the described modification that is modified to 2 '-OH of ribose in the nucleotide sequence of the small RNA of described modification.
Further preferably, 2 '-OH of ribose is replaced by methoxyl group or fluorine in the nucleotide sequence of the described small RNA that is modified to described modification.
The present invention is by carrying out specific modification to the CA/UG site, thereby only introducing under a small amount of situation of modifying, can reach the purpose of the serum stability that increases the small RNA after modifying, thereby reduced the potential cytotoxicity of the small RNA molecule after modifying, and modified the influence to the biologic activity of small RNA.
Fig. 1 modifies and borine phosphoric acid salt modification structure figure for thiophosphoric acid;
Fig. 2 is 2 '-fluorine modifies, 2 '-the oxygen methyl modify (2 '-OME), 2 '-methoxyethyl modify (2 '-MOE) and 2,4 '-dinitrophenol(DNP) modification structure figure;
Fig. 3 for lock nucleic acid, 2 '-amido modified and 2 '-deoxidation modification structure figure;
Fig. 4 is 5 '-bromouracil, 5 '-iodouracil, N3-methyl uracil (N3-methyl-uracil) modify and the 2,6-diaminopurine structure iron.
Embodiment
The present inventor has carried out careful research to small RNA molecule degradation process in vivo, discovery is only introduced under a small amount of situation of modifying in the CA/UG site, can improve the stability of the small RNA molecule of modification, greatly reduce owing to introducing the potential cytotoxicity that a large amount of modifications causes randomly higherly, and modify influence to the biologic activity of small RNA.
According to the present invention, the target of described small RNA molecule can be range gene, for example, can will gene to be analyzed be arranged as target in intracellular function, also needs can be suppressed the gene of its expression as target, for example, can with disease or disorderly relevant gene as target, as the gene on oncogene, virogene, surface of cell membrane acceptor gene, nuclear receptor gene or the 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, the target sequence of goal gene or the sequence number of goal gene in NCBI Genbank are imported various small RNAs designs program, as Insert Design Tool for the shRNA Vectors (Ambion), shRNA Explorer (Gene Link), siDirect (Yuki Naito et al. University of Tokyo), SiRNA at Whitehead (Whitehead Institute for Biomedical Research), BLOCK-iT RNAi Designer (invitrogen), RNAi Design (IDT), RNAi Explorer (Gene Link), siRNA Target Finder (Ambion), or siSearch (Stockholm Bioinformatics Center) etc., this is designed program will be according to planner's requirement and the principle of design of siRNA, at the gene that provides or sequences Design siRNA.And some program can do full genome to the siRNA that designs or mRNA transcribes the homology analysis of group, thereby designs target gene or the special siRNA of target sequence.The above-mentioned siRNA that mentions 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 a plurality of CA/UG site, and at least one Nucleotide in this a plurality of CA/UG site is through modifying.
According to an aspect of the present invention, in the small RNA of described modification, the Nucleotide in described CA/UG site, other Nucleotide is not through modifying.In this case, not only can improve the small RNA of described modification stability, keep its biological activity, the modification quantity of introducing in the small RNA molecule of modifying is minimized, thereby reduce the potential cytotoxicity of modifying the small RNA that causes further.
Among the present invention, term " positive-sense strand " refers to when the small RNA of described modification is duplex molecule, has the nucleotide fragments with the sequence of all or part of homology of sequence of genes encoding chain, described " second fragment " refer to refer to when 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 when being continuous nucleotide chain, term " positive-sense strand " and " first fragment " can be exchanged use, and term " antisense strand " and " second fragment " can be exchanged use.
According to the present invention, the small RNA of described modification can be made up of ribonucleotide, also can be for comprising the hybrid molecule of ribonucleotide and at least one deoxyribonucleotide.
According to the present invention, term " potential cytotoxicity " refers to the toxic action to cell that causes owing to nucleic acid molecule is modified.
Among 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) to the modification of base in the nucleotide sequence of described small molecule interference nucleic acid.
The modification of described phosphodiester bond refers to the oxygen in the phosphodiester bond is modified, and comprises that thiophosphoric acid is modified (Phosphorthioate) and borine phosphoric acid salt is modified (Boranophosphate).As shown in Figure 1 respectively with the oxygen in sulphur and the borine displacement phosphodiester bond.Two kinds of modifications can both the stable micro-molecular interfere RNA structure, keep high specific and the high-affinity of base pairing.And the hydrophobicity of the small RNA that borine phosphoric acid salt is modified is strong, is easy to form in blood plasma hydrated protein, and the toxic side effect of human body is lower than thiophosphatephosphorothioate.
Described ribose modify refer to hydroxyl in the Nucleotide pentose (2 '-OH) modification.After the hydroxy position of ribose is introduced some substituting group such as methoxyl group or fluorine, make serum rnase small RNA not easy to identify, increased the stability of small RNA.Make small RNA have the performance of stronger opposing nuclease hydrolysis.To the modification of hydroxyl in the Nucleotide pentose comprise 2 '-fluorine modifies (2 '-fluro modification); 2 '-modification of oxygen methyl (2 '-OME); 2 '-the methoxyethyl modification (2 '-MOE); 2,4 '-the dinitrophenol(DNP) modification (2 '-DNP modification); Lock nucleic acid (LNA); 2 '-amido modified (Amina modification); 2 '-the deoxidation modification (2 '-Deoxy modification) etc., as shown in Figures 2 and 3.
Described base modification refers to the base of Nucleotide is modified, as introduce in 5 sites of uridylic 5 of bromine or iodine '-bromouracil (5 '-bromo-uracil) and 5 '-iodouracil (5 '-iodo-uracil) to modify be the normal base modification method of using, other also have N3-methyl uracil (N3-methyl-uracil) to modify, 2,6-diaminopurine (2,6-diaminopurine) modify etc., as shown in Figure 4.
Under the preferable case, the described modification that is modified to 2 '-OH of ribose in the nucleotide sequence of described small molecule interference nucleic acid.More preferably, 2 '-OH of ribose is replaced by methoxyl group or fluorine in the described nucleotide sequence that is modified to described small molecule interference nucleic acid.Above-mentioned modification all can increase the serum stability of described small RNA, strengthens it to the resistivity of serum nuclease hydrolysis.
The present invention also provides a kind of preparation method of small RNA of modification, wherein, this 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, come the small RNA of synthetic modification, make the small RNA of the modification that obtains comprise first fragment and second fragment, and described first fragment and second fragment can form double-stranded region, described first fragment comprises CA sequence or the UG sequence that at least one is continuous, described second fragment comprises at least one and the CA sequence of described first fragment or continuous UG sequence or the CA sequence of UG sequence complementation, the UG sequence of the CA sequence of described first fragment or UG sequence and described second fragment or CA sequence form the CA/UG site, wherein, at least one Nucleotide in the described CA/UG site is through modifying, and this modification makes the stability of the small RNA of modification be higher than the small RNA of unmodified.Under the preferable case, make in the CA/UG site of small RNA of the modification that obtains, have only cytidylic acid(CMP) in the antisense strand through modifying.
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, come the small RNA of synthetic modification, make the small RNA of the modification that obtains comprise a CA/UG site, and at least one Nucleotide in this CA/UG site is through modifying.Under the preferable case, make in the CA/UG site of small RNA of the modification that obtains, have only cytidylic acid(CMP) 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, come the small RNA of synthetic modification, make the small RNA of the modification that obtains comprise a plurality of CA/UG site, and at least one Nucleotide in this a plurality of CA/UG site is through modifying.Under the preferable case, make in the CA/UG site of small RNA of the modification that obtains, have only cytidylic acid(CMP) through modifying.
Under a kind of preferred situation, the preparation method of the small RNA of described modification provided by the invention makes in the small RNA of the modification that obtains, and the Nucleotide in described CA/UG site, other Nucleotide is not through modifying.
Among the present invention, it is conventionally known to one of skill in the art that the preparation method of the small RNA of described modification provided by the invention makes the structure of the small RNA of the modification that obtains, and can be the structure that exists of various small RNAs.
According to another aspect of the present invention, the preparation method of the small RNA of described modification provided by the invention makes the small RNA of the modification that obtains can be for being the hybrid molecule that comprises ribonucleotide and at least one deoxyribonucleotide.
Among the present invention, the method of synthetic small RNA can be the synthetic method of the small RNA of various routines, it is synthetic that perhaps the synthetic biotech company of nucleic acid is specialized in trust, as entrusting Shanghai JiMa pharmacy Technology Co., Ltd (GenePharma), the sharp rich bio tech ltd in Guangzhou or handsome Bioisystech Co., Ltd (invitrogen) to synthesize.
In general, the method for the synthesis of small RNA comprises following four processes: (1) oligomerization ribonucleotide synthetic; (2) deprotection; (3) purifies and separates; (4) desalination.
For example, it is as follows to have concrete steps of small RNA chemosynthesis of nucleotide sequence shown in the table 1:
(1) the oligomerization ribonucleotide is synthetic: in that automated DNA/the RNA synthesizer (for example, Applied Biosystems EXPEDITE8909) goes up the RNA that sets synthetic 1 mmole, the coupling time of setting each circulation simultaneously is 10-15 minute, initiator is the dimethoxy of 5 '-O--thymidine upholder that solid phase connects, first circulates in and connects a base on the solid support, then in the n time (19 〉=n 〉=2) circulation, the base that connects the n-1 time circulation connects a base, repeats this circulation until finishing the synthetic of whole nucleotide sequences.
(2) deprotection
The solid support that is connected with small RNA is joined in the test tube, and in this test tube, add ethanol/ethamine (volume ratio is 1:3) of 1 milliliter, sealing then, place 55-70 ℃ of incubator, hatched 2-30 hour, taking-up is connected with 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 was placed 4-12 hour, added 2 milliliters of ethanol again, and collecting precipitation namely obtains the crude product of small RNA.
(3) purifies and separates
It is in 1 mole/milliliter the ammonium acetate solution that the crude product of the small RNA that obtains is dissolved in 2 ml concns, separates by the C18 high pressure liquid chromatography then, 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 weight % with concentration, remove salt, and drying under the room temperature.Then with 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 ℃, slowly this solution is cooled to room temperature then, and kept room temperature 16-22 hour, obtain containing the solution of small RNA.
Above provide the purpose of numerous embodiments only for exemplarily the present invention will be described, rather than in order to limit the scope of the invention.
Further specify the present invention below in conjunction with embodiment, unless stated otherwise, the used reagent of the present invention, substratum are the commercial goods.
Embodiment 1
The gene of choosing in the following table 1 is target gene, and entrusts the siRNA in the synthetic table 1 of Shanghai JiMa pharmacy Technology Co., Ltd (GenePharma).
Embodiment 2
SiRNA shown in the his-and-hers watches 1 carries out the serum stability detection respectively by the following method, gene silencing efficient 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, the 1 * PBS solution of unmodified of 20 μ M with 4 μ L concentration, the final concentration of serum is 10%; After under 37 ℃ of conditions reaction system being hatched the regular hour, take a 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 the serum nuclease immediately to the effect of siRNA, then sample is stored under-80 ℃ of conditions standby.
Configuration 20% polyacrylamide gel, with 3 μ L 5 * sample-loading buffer (30mM EDTA, 36% glycerine, 0.06% bromine atmosphere indigo plant) mix with the degraded sample of siRNA, go up sample then, electrophoresis under the constant current conditions of 80mA.After electrophoresis finishes, carry out with 1 * Sybr Gold dyestuff (Invitrogen, Cat. 11494) taking a picture after 10 minutes the dyeing, the result is as shown in table 1.
2. gene silencing efficient detects
Will be 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) cultured human embryo tire nephrocyte (HEK293) be inoculated in 24 orifice plates (1 * 10
5Cell/0.5 ml substratum/hole).Treat that the cell growth after 24 hours, when the degrees of fusion of cell is 50% left and right sides, is changed to Opti-MEM substratum (Gibco company) with substratum.To have control plasmid (the Promega company that siRNA separates reorganization Photinus pyralis LUC reporter plasmid and the pRL-TK (coding sea pansy luciferase) of target site then, Madison WI, USA) siRNA with chemosynthesis passes through Lipofectamine 2000 (Invitrogen company, the U.S.) carry out cell transfecting, every hole contains 0.17g recombinant plasmid and 0.017g pRL-TK control plasmid, and the final concentration of siRNA is 13 nM.Three multiple holes of every kind of parallel transfection of siRNA, with two kinds of reporter gene plasmids of the same amount of a transfection, three multiple holes of not transfection siRNA in contrast.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) after 4 hours.Harvested cell after 24 hours, with 10 μ l cell pyrolysis liquid cells, utilize two luciferase reporter gene analytical system (Dual-Luciferase Assay System, Promega company) and microplate reader (Novostar, BMG Labtechnologies GmbH, Germany) activity of two kinds of luciferases of mensuration, with the expression amount of the reporter gene in the hole of untransfected siRNA as standard control, calculate the reticent efficient of the target site of siRNA by following formula, the result is as shown in table 1.Every kind of siRNA tests parallel 3 the multiple holes of doing at every turn, and each experiment repeats 2 times, and the result is as shown in table 1.
Reticent efficient=(expression amount of experimental group Firefly luciferase/experimental group Renilla luciferase expression amount)/(expression amount of control group Firefly luciferase/control group Renilla luciferase expression amount)
3, the cytotoxicity of modifying siRNA detects
Will be 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) cultured human embryo tire nephrocyte (HEK293) be inoculated in 24 orifice plates (1 * 10
5Cells/well); Treat that the cell growth after 24 hours, when the degrees of fusion of cell is 50% left and right sides, is changed to Opti-MEM substratum (Gibco company) with substratum; SiRNA with chemosynthesis carries out cell transfecting by Lipofectamine 2000 (Invitrogen company, the U.S.) then, and the final concentration of siRNA is 13 nM, three multiple holes of every kind of parallel transfection of siRNA, and the multiple hole of three transfections of no siRNA is in contrast; 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) after 4 hours; Inhale after 24 hours and remove nutrient solution, with the PBS washing once, every hole adds 1ml PBS and 10 μ l MTT (tetrazolium bromide) dye liquors, at 37 ℃ of 5% CO
2CO2gas incubator in cultivated 4-6 hour; Every hole adds 0.1ml acidifying Virahol, at the vibrator mixing that vibrates, allows reduzate fully dissolve; Enzyme-linked immunosorbent assay instrument is measured each hole A value of 570nm place, and calculates inhibitory rate of cell growth according to following formula, and the result is as shown in table 1.
Inhibitory rate of cell growth (%)=(control group average A value-experimental group average A value)/control group average A value * 100%.
Table 1
In last table 1:
Bold-faced letter represent "
A", "
U", "
G" or "
C" Nucleotide of letter representation through modifying;
"
+" represent after serum is hatched the completely dissolve of siRNA master tape, visible significantly degraded band;
"
++" expression is after serum is hatched, though the siRNA master tape has degraded but still is high-visible, visible significantly degraded band;
"
+++" expression is after serum is hatched, the siRNA master tape is clear does not see degraded, does not see tangible degraded band.
In addition, from last table 1 as can be seen, with compare through the small RNA of modifying at random, small RNA provided by the invention improved stable in, also reduced the cytotoxicity of small RNA significantly, and the expression inhibiting efficient to small RNA does not have influence substantially, this shows, the present invention is only introducing under a small amount of situation of modifying, can reach the purpose of the serum stability that increases the small RNA after modifying, thereby reduced the potential cytotoxicity of the small RNA molecule after modifying, and modified the influence to the biologic activity of small RNA.
Claims (5)
1. the small RNA of a modification, it comprises first fragment and second fragment, and described first fragment and second fragment can form double-stranded region, described first fragment comprises CA sequence or the UG sequence that at least one is continuous, described second fragment comprises at least one and the CA sequence of described first fragment or continuous UG sequence or the CA sequence of UG sequence complementation, the UG sequence of the CA sequence of described first fragment or UG sequence and described second fragment or CA sequence form the CA/UG site, wherein, at least one Nucleotide in the described CA/UG site is through modifying, this modification makes the stability of the small RNA of modification be higher than the small RNA of unmodified, described first fragment is 5 '-UGAACGUCACCGAGGAGAATT-3, and second fragment is 3 '-TTACUUGCAGUGGCUCCUCUU-5 '.
2. the small RNA of modification according to claim 1 is characterized in that, in the described CA/UG site, has only cytidylic acid(CMP) through modifying.
3. the small RNA of modification according to claim 1 is characterized in that, the Nucleotide in described CA/UG site, other Nucleotide is not through modifying.
4. according to the small RNA of each described modification of claim 1-3, it is characterized in that the described modification that is modified to 2 '-OH of ribose in the nucleotide sequence of the small RNA of described modification.
5. the small RNA of modification according to claim 4 is characterized in that, 2 '-OH of ribose is replaced by methoxyl group in the nucleotide sequence of the described small RNA that is modified to described modification.
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| CN101370818A (en) * | 2005-11-01 | 2009-02-18 | 阿尔尼拉姆医药品有限公司 | RNAi inhibition of influenza virus replication |
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