For suppressing the oligonucleotide molecule of HIF1A target genes mRNA expression and its into set groupCompoundThe application be the applying date be 2016-08-18, Application No. 201610687850.2, entitled " one kind is used forThe divisional application of the application for a patent for invention of the oligonucleotide molecule and its composition set of suppression target gene mRNA expression ".
Technical field
The present invention relates to biology field, more particularly to a kind of widow for being used to suppress HIF1A target genes mRNA expressionNucleic acid molecules and its composition set.
Background technology
From AndrewFire and Craig Mello et al. head in nematode (Caenorhabditis elegans) in 1998After secondary discovery RNAi phenomenons, Tuschl and Phil Sharp et al. 2001 are confirmed in mammal there is also RNAi, closeA series of progress is achieved in researchs such as RNAi mechanism principles, gene function and clinical practice.RNAi is not only in defence diseasePoison infection, the jump of anti-rotation stand etc. play in a variety of body protective mechanism key effect (Huntvagner et al, 2001；Tuschl,2001；Waterhouse et al,2001；Zamore 2001), its Related product is also very promising candidateMedicine.
Elbashir et al. had found that siRNA suppresses the silence of specific gene in mammalian cell in 2001, studied tableBright siRNA can be specifically the complementary said target mrna of same sequence combine, and make its degraded.The double-stranded RNA of long segment is by Dicer cleavagesInto 21-23 bases longs short-movie section RNA, the chain combined in two of which chain with said target mrna is referred to as antisense strand or guiding chain, separatelyOne chain is referred to as positive-sense strand or passerby chain.Research finds that the siRNA of iii vitro chemical synthesis similarly plays RNAi after entering cellEffect, and effectively reduce immune response caused by long-chain RNA.
But because siRNA validity is influenceed by many factors such as sequence-specific, target cell specificity, target spot, it is based onThe siRNA that existing design principle obtains not is each to be attained by effective silencing efficiency；The siRNA being typically designed there are aboutMore than 50% has the effect of silence said target mrna, and only 25% siRNA has more than 75% silencing efficiency, therefore follow-up pair setsThe siRNA of meter synthesis also needs to experimental verification, screening or optimization, time-consuming effort；It is a kind of general, efficient, quick based on thisRNAi technology is urgently developed with product.
The content of the invention
It is an object of the present invention to provide a kind of siRNA for suppressing or reducing HIF1A expression of target gene.
SiRNA provided by the invention, formed by positive-sense strand and with the antisense strand of its reverse complemental (being completely reversed complementation)；
The positive-sense strand is made up of 19-27 nucleotides, and the positive-sense strand from 5 ' ends 5-9 continuous nucleotide and5-9 continuous nucleotide carries out the modification of 2 ＇-O- ribose from 3 ' ends.
The antisense strand and the section reverse complemental on the target gene, the target gene is HIF1A；
The base composition sequence of the positive-sense strand is selected from SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQID NO.7,SEQ ID NO.9,SEQ ID NO.11,SEQ ID NO.13；
The base composition sequence of the antisense strand is selected from SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQID NO.8,SEQ ID NO.10,SEQ ID NO.12,SEQ ID NO.14。
SiRNA is combined by its antisense strand with the target sequence reverse complemental on target gene.
In some embodiments of invention, a non-natural nucleotides is comprised at least in siRNA molecule, is such as repaiied through chemistryThe nucleotides of decorations, the preferably chemical modification in positive-sense strand or just area.In certain embodiments, the 2 ＇-O- ribose modification of riboseSpecially 2 ＇-O- methyl are modified, the modification of 2 ＇-O- fluoro, 2 ＇-MOE modifications.The positive-sense strand modification of the present invention can be played：(1) strengthenSiRNA molecule stability；(2) missing the target property of siRNA molecule is reduced；(3) siRNA molecule specificity is improved；(4) immune activation is reducedThe effect such as reaction.
In above-mentioned siRNA, the positive-sense strand is made up of 24,25 or 26 nucleotides；
Or, the positive-sense strand 6 or 7 or 8 continuous nucleotides and 6 or 7 or 8 continuous kernels from 3 ' ends from 5 ' endsThuja acid carries out the modification of 2 ＇-O- ribose.
In above-mentioned siRNA, the 2 ＇-O- ribose is modified to 2 ＇-O- methyl (2 ＇-O-Me) modification, 2 ＇-O- fluoro are modified,Or 2 ＇-MOE modification.
Another object of the present invention is to provide a kind of complete siRNA for suppressing or reducing expression of target gene.
Complete siRNA provided by the invention, including the siRNA that at least five is above-mentioned.
Each siRNA corresponds to the different target sequences of same target gene.
In above-mentioned complete siRNA, the complete siRNA is made up of 5,6,7,8,9 or 10 siRNA.
In above-mentioned complete siRNA, the amount of the material of single siRNA molecule can be random in the complete siRNA, appointThe amount ratio of 2 siRNA material is 1:1-1:5；It is preferred that the amount of the material of single siRNA molecule equal 1:1.
1) or 2) another object of the present invention is to provide another following material.
Or 2) 1) material provided by the invention：
1) suppress or reduce the reagent of expression of target gene, it is following A or B：
A includes claim 1-3 any described siRNA and transfection reagent；
B includes the complete siRNA and transfection reagent described in claim 4 or 5；
2) suppress or reduce the kit of expression of target gene, it includes above-mentioned siRNA or above-mentioned complete siRNA or describedReagent.
In above-mentioned substance, total concentration of all siRNA molecules in the reagent is 2-100nM in the complete siRNA；Each concentration of the siRNA molecule in the reagent is 10-20nM；
The application of above-mentioned siRNA or above-mentioned complete siRNA or above-mentioned material in suppressing or reducing expression of target geneAnd the scope of protection of the invention；
Or above-mentioned siRNA or above-mentioned complete siRNA or above-mentioned material are suppressing or are reducing the expression of cell target geneIn application be also the scope of protection of the invention；
Or above-mentioned siRNA or above-mentioned complete siRNA or above-mentioned material are preparing suppression or are reducing expression of target gene productionApplication in product is also the scope of protection of the invention；
Or above-mentioned siRNA or above-mentioned complete siRNA or above-mentioned material are preparing suppression or are reducing cell target geneThe application of product is also the scope of protection of the invention in expression；
Or above-mentioned siRNA or above-mentioned complete siRNA or above-mentioned material are preparing prevention or alleviation or treated by target baseBecause the application in the product in disease caused by expression is also the scope of protection of the invention.
Method provided by the invention, comprises the following steps：Will suppress or reduce expression of target gene siRNA positive-sense strand from5 ' ends play 5-9 continuous nucleotide and 5-9 continuous nucleotide carries out the modification of 2 ＇-O- ribose from 3 ' ends；
The siRNA forms by positive-sense strand and with the antisense strand of its reverse complemental；
The positive-sense strand is made up of 19-27 nucleotides；
The antisense strand and the section reverse complemental on the target gene.
Or the 2 ＇-O- ribose modification is specially the modification of 2 ＇-O- methyl, the modification of 2 ＇-O- fluoro or 2 ＇-MOE modifications.
4th purpose of the invention is to provide a kind of product.
Product provided by the invention, including above-mentioned siRNA or above-mentioned complete siRNA or above-mentioned material；
And/or the product has following 1) -3) at least one of function：
1) suppress or reduce expression of target gene；
2) suppress or reduce the expression of cell target gene；
3) prevention or alleviation or treatment disease as caused by expression of target gene.
In above-mentioned, the target gene is tumour, cancer, angiocardiopathy, inflammation, infectious disease or rare sick related gene；
Or, the tumour, cancer, angiocardiopathy, inflammation, infectious disease or rare sick related gene are specially HIF1A；
The cell is specially vertebrate cells, mammalian cell, primates zooblast, human cell；
Or, target gene unconventionality expression (expression is higher than normal subjects) or have gene defect (such as chromosome is differentOften or gene mutation) cancer cell, tumour cell, inflammatory cell, blood cell, leucocyte, brain cell, liver cell, pneumonocyte,Nephrocyte, mammary glandular cell, cervical cell, endothelial cell, nerve cell, Deiter's cells；
Or, the cell is specially HeLa, 293T, A549 or HUVEC cell；
Or, the product is specially medicine.
Above-mentioned target gene can be tumour or cancer related gene, preferably HIF1A genes.
SiRNA corresponding to above-mentioned target gene is as follows.
Target gene is HIF1A, and complete siRNA corresponding to it is RB-HIF-D1, RB-HIF-D2, RB-HIF-D3, RB-At least six in HIF-D4, RB-HIF-D5, RB-HIF-D6, RB-HIF-D7.Present invention additionally comprises one kind to reduce target base in cellBecause of the method for expression, including the use of above-mentioned mixture, method includes a) obtaining siRNA molecule or its mixture methods described,The siRNA molecule mixture comprises at least 5,6,7,8,9,10 siRNA；B) siRNA molecule mixture is delivered into cell.
The present invention is by introducing siRNA molecule or its mixing to cell in siRNA molecule mixture (complete siRNA)Thing, suppress the expression of target gene；Introducing can be to be introduced directly into or introduce indirectly, other known in addition to using transfection reagentVarious all to use the siRNA molecule delivering such as mode of cell, such as injection, (carrier can be plasmid or disease for carrier transfectionPoison), electroporation, liposome transfection etc..
" complementation " refers to the ability matched between core base.
Also the length of nucleotides or RNA molecule chain can be represented in the present invention with base or bases longs.
The tolerance mispairing of the siRNA molecule of the present invention is at least 1-5 nucleotides, and its preferred tolerant position is single-stranded or doubleChain end, the base number of tolerance are influenceed by complementary section length." mispairing " refers to that core base can not match.
The preferred solid-phase synthesis of siRNA molecule mixture obtains, and can also be synthesized by the method for transcribing or other method.
The kit of the present invention may also include buffer solution, label in addition to siRNA molecule and/or siRNA molecule mixture(label can be dyestuff, radioactive marker substance or fluorescence labeling material, and the position of mark can be in antisense strand or positive-sense strandEnd), transfection reagent, container, test tube, reagent of annealing, control siRNA (including NC controls, N controls) etc.；In kitSiRNA molecule mixture is placed in a vessel after can also can be combined by single packing；Kit components can be frozenDry powder or solution.
The experiment proves that single siRNA molecule stability is good, external or internal suppression examination is on the one hand improvedTo the resistance of nuclease in testing；On the other hand be advantageous to store and transport；The effect of missing the target of siRNA molecule is reduced simultaneously.IntoIt is even more a kind of general, effectively and rapidly RNAi instruments to cover siRNA, it is advantageous that：(1) more than 60% inhibiting rate is can ensure that,More than 75% siRNA molecule mixture can reach more than 75% inhibiting rate, and more than 50% siRNA molecule mixture can reachTo more than 85% inhibiting rate, relative to single siRNA molecule, the probability of silence and the efficiency of silence are integrally improved；ExistingHave in technology, the probability that the siRNA being typically designed only has 50% can suppress expression of target gene, and only 25% siRNA can reachMore than 75% inhibiting rate.(2) particular design is not required to, is subsequently not required to screen siRNA, optimizes, or its effect is carried outExperimental verification, it is time saving and energy saving.(3) solve in different cell lines, different transfection reagents processing when, siRNA effect it is inconsistentThe problem of, can in multiple cell lines effective reticence target gene, and do not influenceed by transfection reagent.(4) single siRNA is enhancedThe Gene silencing efficacy of molecule, serve the effect of Synergistic.(5) effect of missing the target of siRNA molecule is reduced.
SiRNA molecule mixture can influence at least 50%, 55%, 60%, 65%70%, 75%, 80% in the present invention,85%, the expression of 90% cell target gene, inhibiting rate is at least 45%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%.Can be true when the siRNA molecule mixture of the present invention is in different cell lines, different transfection reagents are handledProtect at least 60% target gene inhibition." suppression " of the present invention or similar expression can refer to RNA or protein level or related rawChange the reduction of the expressing of index, activity or index with respect to negative control.
Brief description of the drawings
Fig. 1 is siRNA vitro stability measurement results.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The preparation of embodiment 1, siRNA
All single siRNA of design can target all transcripts of target gene, and design method is with reference to Elbashir etal.2002；Paddison et al.2002；Reynoldset al.2004；Ui-Tei et al.2004 et al. method(Elbashir,S.M.,Harborth,J.,Weber,K.,and Tuschl,T.2002.Analysis of genefunction in somatic mammalian cells using small interfering RNAs.Methods 26:199–213；Paddison,P.J.,Caudy,A.A.,Bernstein,E.,Hannon,G.J.,and Conklin,D.S.2002.Short hairpin RNAs(shRNAs)induce sequence-specific silencing inmammalian cells.Genes&Dev.16:948–958；Reynolds,A.,Leake,D.,Boese,Q.,Scaringe,S.,Marshall,W.S.,and Khvorova,A.2004.Rational siRNA design for RNAinterference.Nat.Biotechnol.22:326–330；Ui-Tei,K.,Naito,Y.,Takahashi,F.,Haraguchi,T.,Ohki-Hamazaki,H.,Juni,A.,Ueda,R.,and Saigo,K.2004.Guidelines forthe selection ofhighly effective siRNA sequences for mammalian and chick RNAinterference.Nucleic Acids Res.32:936–948)；To ensure siRNA specificity, BLAST is used(Basic Local Alignment Search Too,http://www.ncbi.nlm.gov)) analytical sequence homogeneity(identity), the selection sequence minimum with other sequences homogeneity.
The SS positive-sense strands and formed with the antisense strand AS of its reverse complemental that siRNA is made up of 25 nucleotides, and be flat endEnd；
Each siRNA is combined by its antisense strand with the target sequence reverse complemental on the target gene；
AS chains and SS chains are completely reversed complementation, and AS chains are completely reversed complementation with the target sequence on target gene, and SS's is last from 5 '7 continuous nucleotides are held and 7 continuous nucleotides are modified by 2 ＇-O-Me from 3 ' ends.
Target gene is as shown in table 1, and siRNA particular sequences are as shown in table 2 corresponding to target gene.
Table 1 is target gene
Table 2 is siRNA molecule sequence table
In above-mentioned table, mA, mU, mC and mG are respectively that U, 2 ＇-O-Me are modified after A, 2 ＇-O-Me modification after 2 ＇-O-Me are modifiedG after C and 2 ＇-O-Me modifications afterwards.
The preparation of embodiment 2, complete siRNA
1st, design principle
Complete siRNA is made up of 5-10 siRNA molecule；
The SS positive-sense strands and formed with the antisense strand AS of its reverse complemental that each siRNA is made up of 25 nucleotides, and beFlat end；Each siRNA combines by the way that the target sequence on its antisense strand and target gene is complementary；
AS chains and SS chains are completely reversed complementation, and AS chains are completely reversed complementation with the target sequence on target gene, and SS's is last from 5 '7 continuous nucleotides are held and 7 continuous nucleotides are modified by 2 ＇-O-Me from 3 ' ends.
Target gene is as shown in table 1, and siRNA is as shown in table 2 corresponding to target gene.
The complete siRNA of target gene can include 5,6,7 or 10 siRNA, and packet situation is as shown in table 3.
The complete siRNA compositions of the target gene of table 3
Respectively will be each in above-mentioned complete siRNA groups RM-2 (7 siRNA are mixed), RM-3 (6 siRNA are mixed)SiRNA requires mixing according to the packet shown in table 5, and each siRNA is equimolar ratio mixing.
Embodiment 3, complete siRNA suppress the research of expression of target gene
The following examples exemplified by the complete siRNA groups RM-2 (7 siRNA mixing) in table 3 with being tested：
First, complete siRNA groups RM-2 is compared with single siRNA molecule is to HeLa cell target gene inhibiting rates
By TP53, BIRC5, CTNNB1, COPS5, CLU, STAT3, VEGFA, KRAS in embodiment 2 shown in table 3 this 7Complete siRNA groups RM-2 difference transfection HeLa cell corresponding to target gene, method is same as Example 2, wherein, RM-2 mixturesIn the total concentrations of all siRNA molecules be 100nM, and the amount of material of each siRNA molecule to wait mixes.
To transfect single siRNA molecule as control, wherein, the concentration of single siRNA molecule is 100nM.
Complete siRNA groups RM-2 inhibiting rates and single siRNA inhibiting rates result are as shown in table 4, it can be seen that for 7Target gene, complete siRNA groups RM-2 inhibitions are all higher than 90%；And 7 lists of the same concentration of complete siRNA groups RM-2Individual siRNA molecule is compared, and serves the effect of Synergistic.
RM-2 and single siRNA comparison in table 4HeLa cells
| Gene | RM-2 | D1 | D2 | D3 | D4 | D5 | D6 | D7 |
| TP53 | 91 | 83 | 82 | 87 | 87 | 84 | 92 | 75 |
| BIRC5 | 93 | 90 | 92 | 91 | 73 | 19 | 17 | 50 |
| CTNNB1 | 93 | 88 | 90 | 90 | 89 | 87 | 91 | 62 |
| COPS5 | 94 | 85 | 88 | 87 | 93 | 89 | 92 | 77 |
| STAT3 | 90 | 89 | 86 | 83 | 85 | 89 | 90 | 75 |
| VEGFA | 90 | 64 | 46 | 92 | 70 | 57 | 37 | 88 |
| KRAS | 94 | 94 | 90 | 92 | 90 | 68 | 26 | 72 |
D1-D7 in above-mentioned table is respectively 7 siRNA corresponding to each target gene, and RM-2 is 7 corresponding to each target geneIndividual siRNA biased sample.
2nd, complete siRNA RM-2 inhibition compares in different cell lines
By complete siRNA groups RM-2 corresponding to the HIF1A target genes in embodiment 2 shown in table 3 transfect respectively 4 kinds it is differentCell line (human embryonic kidney cells 293T, cervical cancer cell HeLa, non-small cell lung cancer cell A549 and Human umbilical vein endothelial cellsCell is observed after HUVEC (deriving from ATCC) inoculated and cultureds 24h, it is in good condition to start to transfect.
1st, transfect
(1) 50 μ L riboFECT rotaring redyeing systems, 5 μ L riboFECTTMCP Reagent (the sharp rich biotechnologies in GuangzhouCo., Ltd, C10511-05), complete siRNA groups RM-2 prepared by 5 μ L embodiments 2 (all siRNA total concentration is 100nM)With 40 μ L riboFECTTMCP Buffer (Guangzhou Ribo Bio Co., Ltd., C10511-05).
(2) 100 μ L LF2K rotaring redyeing systems：1 μ L LF2K (Invitrogen, 11668019), prepared by 5 μ L embodiments 3Complete siRNA groups RM-2 (all siRNA total final concentration of 100nM) and 94 μ L Opti-MEM cell culture mediums (ThermoFisher Scientific, 31985070).
To be separately added into each hole of 4 kinds of different cell line culture plates above-mentioned 50 μ L riboFECT rotaring redyeing systems or100 μ L LF2K rotaring redyeing systems, cell, the extraction of Trizol methods RNA, Reverse Transcription are collected after transfecting 48hMix reverse transcription reagent box obtains cDNA for reverse transcription (Guangzhou Ribo Bio Co., Ltd., C10170).
2nd, RT-PCR detects inhibiting rate
SiRNA cellular levels suppress experiment
By individually transfection HeLa cell (derives from siRNA corresponding to the target gene shown in table 2 prepared by embodiment 1ATCC), it is specific as follows：
1st, LF2K is transfected
100 μ L LF2K rotaring redyeing systems：1 μ L μ L LF2K (Invitrogen, 11668019), 5 μ L siRNA are (final concentration of100nM) and 94 μ L Opti-MEM cell culture mediums (Thermo Fisher Scientific, 31985070).
Above-mentioned siRNA is respectively siRNA corresponding to the target gene of the preparation of embodiment 1.
HeLa cells are cultivated on Tissue Culture Plate, then above-mentioned 100 μ L rotaring redyeing systems are added into each hole, transfect 48h,Obtain transfecting the cell that different target genes correspond to siRNA.
2nd, RT-PCR detects inhibiting rate
After transfecting 48h, collect and transfect the cell that different target genes correspond to siRNA, the extraction of Trizol methods RNA, ReverseTranscriptionmix reverse transcription reagent box is used for reverse transcription (Guangzhou Ribo Bio Co., Ltd., C10170), obtainsThe cDNA of siRNA cell is corresponded to the different target genes of transfection.Using cDNA as template, the target base corresponding to the siRNA shown in table 3The primer pair of cause carries out RT-PCR amplifications, and reference gene (Forward is used as using the house-keeping gene actin of people:5-TCAAGATCATTGCTCCTCCTGAG-3(SEQ ID NO.15)；Reverse:5-ACATCTGC
TGGAAGGTGGACA-3) (SEQ ID NO.16), Real-time PCR kit SYBR Premix (2 are utilized×) (BIO-RAD 750000131) progress real-time fluorescence quantitative PCR reaction.9 repetitions (each single sample of one sampleProduct have 3 repetitions in transfection, each repeat to do 3 multiple holes in qPCR) Ct errors ± 0.5, then entered with CFX 2.1Row relative quantitative assay.SPSS19.0 Software of Data Statistics data analyses, data are its average value in table, and P values are equal<0.05.
NC negative control groups：The siRNA of transfection is unrelated non-specific siRNA,
5'UUCUCCGAACGUGUCACGU dTdT 3'(SEQ ID NO.17)
5'ACGUGACACGUUCGGAGAA dTdT 3'(SEQ ID NO.18).
The primer of HIF1A target genes：
GCCCTAACGTGTTATCTGTC(SEQ ID NO.19)
CGCTTTCTCTGAGCATTCTG(SEQ ID NO.20)
N blank control groups：Normal cell, no siRNA transfections.
Real-Time PCR inhibiting rate calculations：
The mRNA relative expression levels of inhibiting rate=NC negative control group mRNA relative expression levels-siRNA groups
NC negative control group mRNA relative expression levels
NC negative control groups mRNA relative expression levels are 1.As a result as shown in table 5, compareed with respect to NC, for differentTarget gene, in different cell line, using different transfection reagents, RM-2 mixtures can reach more than 60% suppression effectFruit.
RM-2 inhibiting rate compares (%) in 5 different cell lines of table
3rd, in HeLa cells RM-2 mixtures compared with the mixture histamine result of the siRNA molecule of usual structure
Two target genes of SOD1, EIF4E are chosen, compare the siRNA molecule of its corresponding RM-2 mixture and usual structureMixture in HeLa cells inhibition.
The usual structure siRNA of above-mentioned each gene nucleotide sequence RM-2 corresponding with each gene prepared by embodiment 2The difference is that each nucleotides is modified without 2 '-O-Me in generally structure siRNA, length (removes positive-sense strand respectively for 19bp6 nucleotides that 6 nucleotides and antisense strand 3 ' at 5 ' ends are held), there are 2 dTdT pendencys, remaining all same in end.
Method is identical with above-mentioned one, the difference is that complete siRNA groups corresponding to SOD1, EIF4E prepared by embodiment 2RM-2 transfection HeLa cells (M).
To transfect the mixture of the siRNA molecule of usual structure corresponding to SOD1, EIF4E as control (S).Transfection reagent isLF2K。
In above-mentioned transfection, siRNA total concentration is 100nM.
As a result as shown in table 6, for 2 target genes, the inhibition of the mixture of RM-2 structures is better than usual structureThe mixture of siRNA molecule.
RM-2 modifies results contrast in the HeLa cells of table 6
4th, the inhibition of the complete siRNA transfection HeLa cells of different groups compares
This 2 target genes of selection SOD1, MYC, more different complete siRNA groups inhibitions in HeLa cells.
Method is identical with above-mentioned one, the difference is that complete siRNA groups RM- corresponding to SOD1, MYC prepared by embodiment 22nd, RM-3 and RM-6 difference transfection HeLa cell.Transfection reagent is LF2K.
As a result as shown in table 7,
The siRNAs mixtures that the different siRNA bars numbers of table 7 mix
| Gene | RM-1 (5) | RM-3 (6) | RM-2 (7) | RM-6 (10) |
| SOD1 | 80 | 82 | 85 | 91 |
| MYC | 63 | 66 | 74 | 80 |
As a result show, mixture RM-1, RM-3, RM-2, RM-6 can play a part of efficiently suppressing several genes.
Embodiment 4, vitro stability measure
Each siRNA RB-KRA-D1, RB-TP5-D6, RB-EIF-D3 such as are added at the body after being diluted to 5 μM without RNase waterLong-pending fresh rat serum (for Shanghai Yuan Mu bio tech ltd product), then 37 DEG C be incubated 6 hours after sample intoRow electrophoresis observation difference siRNA integrality.
It is as a result as shown in Figure 1, it will be seen that, siRNA is stable in serum, it is contemplated that it has more preferable effect in vivo.It is otherSiRNA Stability Determination experimental result is identical, and specific figure omits.
Each technical characteristic of the embodiment can be combined arbitrarily, to make description succinct, not to above-described embodimentIn each technical characteristic it is all possible combination be all described, as long as however, lance is not present in the combination of these technical characteristicsShield, all it is considered to be the scope of this specification record.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneouslyCan not therefore it be construed as limiting the scope of the patent.It should be pointed out that come for one of ordinary skill in the artSay, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present inventionScope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
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