CN102174520B - Interference nucleic acid molecule and application thereof - Google Patents
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Abstract
The invention relates to a nucleic acid molecule and application thereof, in particular relates to an interference nucleic acid molecule and application thereof. The interference nucleic acid molecule is characterized by being formed by annealing a sense strand and an antisense strand, the length of each strand is at least of 30 nucleotides, at least two parts in the sense strand or the antisense strand respectively target at RNAs (Ribonucleic Acids) of different genes or different sites of the RNA of the same gene, and the nucleic acid molecule cannot arouse an interferon effect after entering cells. The nucleic acid molecule can interfere a translation process after transcription so as to inhibit or block gene expression, and the interference nucleic acid molecule cannot arouse the interferon effect. The nucleic acid molecule is an active component for preparing a medicament for regulating one ore more gene functions in cells.
Description
Technical field
The present invention relates to a kind of nucleic acid molecule and application, refer in particular to interfere RNA molecule and application thereof.
Background technology
RNA disturbs (RNA interference, RNAi) be by double-stranded RNA (double-strand RNA, dsRNA) (Nature. 1998,391:806-811) to cause a kind of PTGS form of its homology messenger RNA(mRNA) (messenger RNA, mRNA) enzymolysis.After long dsrna enters cell, by the combination of Dicer enzyme and cutting.The cleaved products length of Dicer enzyme is generally 20～25bp(base pair, base pair), and the siRNA (small interference RNA, siRNA) that has 2 Nucleotide to dangle at 3 ' end of every chain.The chain of siRNA is incorporated in the reticent mixture that RNA-induces (RNA-induced silencing complex, RISC), with the sequence pairing of complementary RNA.RISC at first mediates the siRNA two strands and untwists, and is combined with said target mrna in sequence-specific mode with the siRNA of the strand of RISC coupling, and the catalyst component by RISC cuts said target mrna afterwards.The cutting said target mrna can suppress its translation, finally suppresses the expression of this gene.Now being proved having great potentiality in the treatment of multiple disease of viral infection and tumour, is the treatment means that desirable blocking gene is expressed.
RNAi has broad application prospects at field of medicaments, such as antiviral, antitumor and anti-inflammatory etc.Double-stranded interfere RNA can be designed to the long double chain form that is called the Dicer substrate that can be sheared by the Dicer enzyme, or short, do not need the Dicer enzyme to shear direct form as the RISC substrate.Synthetic double-stranded RNA and goal gene sequence are complementary, behind transfered cell or the organism, by endogenous genetic material identification, activator RNA i approach.Utilize this mechanism, RNAi sharply descends the expression level of target gene.
The RNAi technology has been opened up a brand-new treatment field, and existing more than ten kind siRNA medicines enter clinical stage in the world at present.The diseases such as the siRNA of application treatment senile macular degeneration SMD, diabetic macular edema, respiratory syncytial virus disease, noumenal tumour, liver cancer and acute injury of kidney are wherein arranged.
The dsRNA of RNAi effect can be designed to the long double chain form that is called the Dicer substrate that can be sheared by Dicer, or do not need Dicer shear can be directly as the short siRNA form of RISC substrate.In mammalian cell, because long dsRNA easily causes nonspecific Interferon, rabbit effect.Therefore, generally believe when being applied to Mammals, the RNA interfering two strands must be shorter than 30 bp.But, designing long double-chain interference RNA still with practical value for RNAi, long RNA interfering two strands can be with a plurality for the treatment of targets, and the different loci of the mRNA of a plurality of different target genes of target or the mRNA of same target gene increases Gene silencing efficacy, the broadened application scope.The generation of disease, develop the common complex biological that participates in of several genes often and learn process, be subject to the impact of many factors, in the treatment of disease, the inhibition of term single gene can not suppress or reverse generation and the development of disease fully, and single target treatment has its limitation.So, research and develop many targets, noiseless plain long interfere RNA two strands of reacting, suppress simultaneously the expression of a plurality of disease genes, multi-level, multipath treatment disease, energy Effective Raise result for the treatment of can be used as the new way for the treatment of disease.
Summary of the invention
The present invention aims to provide a kind of nucleic acid molecule and application thereof, especially interfere RNA molecule and application thereof.The RNA of the different target genes of the antisense strand target of this interfere RNA molecule or the different loci of same target gene RNA, this RNA can be mRNA, Microrna (microRNA, miRNA) or the subsequence of mRNA or miRNA, and this interfere RNA molecule do not cause ifn response in the body.
The invention discloses a kind of interfere RNA molecule, it is characterized in that, formed by positive-sense strand and antisense strand annealing, the length of every chain is at least 30 Nucleotide, at least two parts in its positive-sense strand or the antisense strand are distinguished the different loci of the heterogeneic RNA of target or same gene RNA, and this nucleic acid molecule can not cause the Interferon, rabbit effect after entering cell.
The invention also discloses a kind of interfere RNA molecule, formed by positive-sense strand and antisense strand annealing, the length of every chain is at least 30 Nucleotide, at least two parts in its antisense strand are distinguished the different loci of the heterogeneic RNA of target or same gene RNA, wherein, exist at least one length to be at least the Cyclic Nucleotides structure of self complementation of 7 Nucleotide in the described positive-sense strand, this nucleic acid molecule can not cause the Interferon, rabbit effect after entering cell.
Preferably, described interfere RNA molecule is comprised of a positive-sense strand and two antisense strands, and every positive-sense strand and antisense strand all contain 5 ' end and 3 ' end, wherein: 3 ' end complementation of 5 ' end of positive-sense strand and article one antisense strand; 3 ' end of positive-sense strand is complementary with 5 ' end of second antisense strand; Article one, 3 ' end of 5 ' of antisense strand end and second antisense strand is complementary, after annealing, forms the nucleotide double with secondary structure.Use this ifn response that long interfere RNA secondary structure, that be made of positive-sense strand or antisense strand does not excite mammalian cell that has.
Preferably, the ribonucleotide that contains at least one sugar ring modification or backbone modification in the described two strands.
The present invention also provides the application of described interfere RNA molecule in preparing the medicine of regulating at least a gene function in the cell.Described gene is disease related gene.Described disease related gene comprises pathogenic agent genes involved such as virogene, and Non Communicable Diseases (NCD) genes involved such as tumor-related gene.Described tumour is at least a in liver cancer, lung cancer, cancer of the stomach, cervical cancer, multiple myeloma, Skin Squamous Cell Carcinoma, colorectal carcinoma, melanoma, bladder cancer, osteosarcoma, nasopharyngeal carcinoma and the oral carcinoma.The present invention is expected to suppressing, alleviating or alleviate the state of an illness or prevent from obtaining more significant result for the treatment of aspect the recurrence of some disease symptoms.
The present invention compared with prior art, this nucleic acid molecule can suppress the expression of a plurality of target genes simultaneously, perhaps a plurality of sites of a target gene of target.In addition, do not cause the Interferon, rabbit effect, can be applicable to mammalian cell.Nucleic acid molecule disclosed by the invention is the application form of a kind of new siRNA, is having wide practical use aspect the gene therapy of disease.
Description of drawings
Fig. 1 is nucleic acid molecule By-Pass structural representation.The hair fastener Cyclic Nucleotides structure that has self complementation on this nucleic acid molecule positive-sense strand, and 5 ' end of positive-sense strand is complementary with 3 ' end of antisense strand 1; 3 ' end of positive-sense strand is complementary with 5 ' end of antisense strand 2; 5 ' end of antisense strand 1 and 3 ' end of antisense strand 2 are complementary, after annealing, form the nucleotide double with secondary structure.Among the figure, 1 is positive-sense strand, and 2 is that antisense strand 1,3 is that antisense strand 2,4 is the hair fastener Cyclic Nucleotides structure of self complementation.
Fig. 2 is the horizontal column diagram of mRNA relative expression of Survivin gene in the SMMC-7721 liver cancer cell.Among the figure, ordinate zou represents the mRNA relative expression level of Survivin gene, and X-coordinate represents the different experiments group.
Fig. 3 is the horizontal column diagram of mRNA relative expression of Bcl-2 gene in the SMMC-7721 liver cancer cell.Among the figure, ordinate zou represents the mRNA relative expression level of Bcl-2 gene, and X-coordinate represents the different experiments group.
Fig. 4 is the horizontal column diagram of mRNA relative expression of Interferon, rabbit genes involved OAS1 gene in the SMMC-7721 liver cancer cell.Among the figure, ordinate zou represents the mRNA relative expression level of OAS1 gene, and X-coordinate represents the different experiments group.
Fig. 5 is the growth curve chart of SMMC-7721 liver cancer cell.Among the figure, ordinate zou represents the ultraviolet light absorption value, i.e. OD value, and X-coordinate represents the different time of different IPs acid molecule function cells.
Fig. 6 is that the different IPs acid molecule is to the growth inhibition ratio column diagram of liver cancer cell SMMC-7721.Among the figure, ordinate zou represents inhibitory rate of cell growth, and X-coordinate represents the different experiments group.
Fig. 7 is the growth curve chart of HepG2 liver cancer cell.Among the figure, ordinate zou represents the ultraviolet light absorption value, i.e. OD value, and X-coordinate represents the different time of different IPs acid molecule function cells.
Fig. 8 is that the different IPs acid molecule is to the growth inhibition ratio column diagram of liver cancer cell HepG2.Among the figure, ordinate zou represents inhibitory rate of cell growth, and X-coordinate represents the different experiments group.
Embodiment
The definition of term is not to be to limit the invention among the present invention.
For simplicity, hereinafter, term " RNA molecule ", " nucleic acid molecule " can exchange, and the meaning of their expressions is identical with scope.
The antisense strand of interfere RNA molecule of the present invention is targeted to the mRNA of two or more target genes or is targeted to two or more Micrornas (microRNA, miRNA) or is targeted to the subsequence of two or more mRNA or miRNA.
MiRNA is the single stranded RNA molecule of 21～23 length of nucleotides of regulatory gene expression.MiRNA is by the genes encoding of transcribing from DNA, but do not translate into protein (non-coding RNA); The miRNA forming process is by the short loop-stem structure that is called the pri-miRNA primary transcript and is processed into pre-miRNA, finally forms functional miRNA.Ripe miRNA molecular moiety is complementary to one or more mRNA molecules, and their major function is the expression of down-regulated gene.
The antisense strand of nucleic acid molecule of the present invention, positive-sense strand Nucleotide can be the nucleotide analogs of sugar ring or backbone modification.
There are in the art a lot of embodiment explanations in Nucleotide, to introduce sugar, base and phosphoric acid modification and can significantly increase nuclease stability and validity.As, the nuclease tolerance group of modified oligonucleotide comes enhanced stability and/or strengthens biologic activity, nucleotide base such as 2 '-amino, 2 '-C-allyl group, 2 '-fluorine, 2 '-methoxyl group, 2 '-oxygen-allyl group, 2 '-hydrogen is modified (Usman and Cedergren, TIBS 17:34,1992; Usman etc., Nucleic Acids Symp. Ser. 31:163,1994; Burgin etc., Biochemistry 35:14090,1996).Nucleic acid molecule sugar-modified (Perrault etc., Nature 344:565-568,1990; Pieken etc., Science 253:314-317,1991; Usman and Cedergren, Trends in Biochem. Sci. 17:334-339,1992; Burlina etc., Bioorg. Med. Chem. 5:1999-2010,1997).
In some specific embodiments, the interfere RNA molecule comprises independently justice or antisense strand, this justice or antisense strand are covalently bound by Nucleotide or non-nucleotide joint, perhaps carry out non-covalent combination by ionic reaction, hydrogen bonded, Van der Waals force, hydrophobic reactant or stacking reaction.
Nucleic acid molecule of the present invention is comprised of independently positive-sense strand and antisense strand, and positive-sense strand and antisense strand nucleotide sequence complementary form double-stranded.Antisense strand can comprise nucleotide sequence complementary with target nucleotide or that part is complementary, and positive-sense strand can comprise nucleotide sequence consistent with target sequence or that part is consistent.
Term among the present invention " Nucleotide " refers to that deoxyribonucleotide or ribonucleotide reach by its strand that forms or double-stranded polymkeric substance.Nucleotide comprises the nucleotide analog known or rear key residue or the connection of modification in this term, can be synthetic, natural in non-natural, contain and binding property like the ucleotides and metabolic character.
Term among the present invention " RNA " refers to a kind of molecule that comprises at least a ribonucleotide residue." ribonucleotide " refers to 2 ' the Nucleotide that an oh group is arranged at β-D ribofuranose group.This term comprises the RNA of double-stranded RNA, single stranded RNA, separation, RNA, synthetic RNA, the RNA that recombinant chou produces such as partially purified RNA, Economical Purification, can change RNA by adding, delete, replace or change one or more Nucleotide, make it be different from natural RNA.These changes can comprise adds the non-nucleotide raw material, as is added on the terminal or middle of RNA interfering, such as one or more Nucleotide of RNA.The Nucleotide of RNA molecule of the present invention also can comprise off-gauge Nucleotide, and such as the Nucleotide of non-natural existence or Nucleotide or the deoxyribonucleotide of chemosynthesis, the RNA of these changes can be the analogue of RNA.
The complementary nucleotide base refers to by the interconnection a pair of nucleotide base of hydrogen bond among the present invention.VITAMIN B4 (A) is matched with thymus pyrimidine (T) pairing or with uridylic (U), guanine (G) and cytosine(Cyt) (C) pairing.Complementary fragment or the nucleotide chain of hybridizing mutually (passing through hydrogen bonded)." complementation " refers to that a nucleotide chain and another nucleotide chain can form hydrogen bond by Watson-Crick rule or other unconventional combination.
Among the present invention the hair fastener Cyclic Nucleotides structure of complementation " self " refer to exist on the positive-sense strand of nucleic acid molecule the nucleotide sequence of one section self complementation, its length is at least 7 Nucleotide, and this sequence forms the secondary structure of a hair fastener ring-type after positive-sense strand and reaction chain annealing.
Amplifying nucleic acid molecule of the present invention can come together to treat disease with nucleic acid transport agent and other known treatment composition.
The formulation of medicine of the present invention can be various ways, as long as be suitable for the administration of corresponding disease and keep rightly the activity of interfere RNA molecule.Such as, for the injection drug delivery system, formulation can be lyophilized powder.
Randomly, can comprise the acceptable auxiliary of any pharmacy in the said medicine formulation, as long as its activity that is suitable for corresponding drug delivery system and keeps rightly the interfere RNA molecule.
In any form administration of interfere RNA molecule among the present invention is as through skin or local injection.
The formulation of interfere RNA molecule can be tablet, capsule or the elixir of oral administration among the present invention, can be the suppository of rectal administration, can be sterile solution, the suspension of drug administration by injection, or other formulation of knowing in the art.
One preferred embodiment in, this interfere RNA molecule is comprised of antisense strand 1 and 2 two antisense strands of antisense strand and a positive-sense strand, the total length of molecule is at least 30 Nucleotide, and has the hair fastener Cyclic Nucleotides structure of self complementation on the positive-sense strand of interfere RNA molecule.5 ' end of positive-sense strand is complementary with 3 ' end of antisense strand 1; 3 ' end of positive-sense strand is complementary with 5 ' end of antisense strand 2; 5 ' end of antisense strand 1 and 3 ' end of antisense strand 2 are complementary.After annealing, form the nucleotide double with secondary structure, use this ifn response that long interfere RNA secondary structure, that be made of positive-sense strand or antisense strand does not excite mammalian cell that has.
Following embodiment only is used for illustrating the present invention, is not to be to limit the invention.
The nucleic acid molecule By-Pass of design target Survivin and Bcl-2 gene
Apoptosis suppressive gene Survivin and Bcl-2, extensive high expression level in malignant cell.Research finds, by crossing Survivin or the Bcl-2 gene of expressing in the inhibition tumor cell, and growth that can inhibition tumor cell.May become the New Policy for the treatment of tumour with the siRNA inhibition Survivin of targeting or the expression of Bcl-2 gene.
Designing nucleic acid molecule By-Pass, it comprises a positive-sense strand and antisense strand 1,2 two antisense strands of antisense strand, and the 3 ' end and 5 ' of antisense strand 1 is held the mRNA that is targeted to the Survivin gene; 5 ' end of antisense strand 2 is targeted to the mRNA of Bcl-2 gene, and 3 ' end is targeted to the mRNA of Survivin gene.The structure of nucleic acid molecule By-Pass as shown in Figure 1.
Sequence SEQ ID NO:1 is the positive-sense strand sequence of nucleic acid molecule By-Pass.
Positive-sense strand: 5 '-GACUUGGCCCAGUGUUUCUCAUGCGUCGGGAUGACUGAGUACCUGAA-3 '
（SEQ ID NO: 1）
Sequence SEQ ID NO:2 is 3 ' the end antisense strand 1 complementary with the 5 ' end of sequence SEQ ID NO:1, sequence SEQ ID NO:3 is 5 ' the end antisense strand 2 complementary with the 3 ' end of SEQ ID NO:1, and the 3 ' end of the 5 ' end of sequence SEQ ID NO:2 and SEQ ID NO:3 is complementary.
Antisense strand 1:5 '-UCCUUUCUGUCAAGAAGCAGUUCAGAAACACUGGGCCAAGUC-3 '
（SEQ ID NO: 2）
Antisense strand 2:5 '-UUCAGGUACUCAGUCAUCCCAACUGCUUCUUGACAGAAAGGA-3 '
（SEQ ID NO: 3）
The siRNA:1 that is used for the contrast experiment) Sur-2 is the siRNA of target Survivin gene, its positive-sense strand sequence is 1～19 Nucleotide of 5 ' end beginning in the sequence (SEQ ID NO:1), and antisense strand is 1～19 Nucleotide of 3 ' end beginning in the sequence (SEQ ID NO:2).2) Bcl-1 is the siRNA of target Bcl-2 gene, and its positive-sense strand sequence is 1～19 Nucleotide of 3 ' end beginning in the sequence (SEQ ID NO:1), and antisense strand is 1～19 Nucleotide of 5 ' end beginning in the sequence (SEQ ID NO:2).
Real-time quantitative PCR detects nucleic acid molecule By-Pass to the reticent effect of Survivin target gene
Cell cultures: liver cancer cell SMMC-7721 in containing the DMEM substratum of 10%FBS (Gibco company), 37 ℃, 5%CO
2Incubator is cultivated.
Cell bed board and transfection: cell is pressed 1 * 10
5/ hole is inoculated in the 96 porocyte culture plates, contains in the DMEM substratum of 10%FBS 37 ℃, 5%CO in nonreactive
2The incubator overnight incubation.Lipofectamin is pressed in transfection
TM2000(Invitrogen company) operation steps is carried out, and the different RNA molecule of experiment is that 10 nM/ holes add by concentration.
Real-time quantitative PCR detects the Survivin gene mRNA expression: extract purification kit TurboCapture mRNA Kit(QIAGEN company with mRNA) extraction purifying cells RNA, operation is undertaken by the test kit specification sheets, without RNase water/hole dissolving RNA, getting 4 μ L RNA is that template is carried out the real-time quantitative PCR reaction with 80 μ L.
Detect the real-time quantitative PCR primer of Survivin gene:
5 ' forward primer: ACCGCATCTCTACATTCAAG (SEQ ID NO:4)
3 ' reverse primer: CAAGTCTGGCTCGTTCTC (SEQ ID NO:5)
Detect Survivin mrna expression in the sample with above-mentioned Survivin gene-specific primer, the house-keeping gene GAPDH that increases simultaneously contrasts as confidential reference items.Each reaction do 3 parallel.Set up following reaction system: 4 μ L template ribonucleic acids, 12.5 μ L 2 * SensiMix One-Step(Quantance company), 1 μ L, 5 ' forward primer (10 μ M), 1 μ L, 3 ' reverse primer (10 μ M), 0.5 μ L 50 * SYBR Green I uses without RNase water and supplies system to 25 μ L.Reaction conditions: 42 ℃ of reverse transcription 30 min, 95 ℃ of denaturation 7 min, 95 ℃ of sex change 20 sec, 60 ℃ of annealing 30 sec, 72 ℃ are extended 30 sec, circulate 45 times.
With 2
-Δ Δ CtAnalytical method is analyzed experimental result, and make histogram, as shown in Figure 2, nucleic acid molecule By-Pass shows the reticent effect preferably of Survivin gene in the liver cancer cell, compare its suppression efficiency no significant difference with corresponding Sur-2, Sur-10 experimental group and cotransfection experiments group Sur-2+Sur-10+Bcl-1.
Real-time quantitative PCR detects nucleic acid molecule By-Pass to the reticent effect of Bcl-2 target gene
Cell cultures: liver cancer cell SMMC-7721 in containing the DMEM substratum of 10%FBS (Gibco company), 37 ℃, 5%CO
2Incubator is cultivated.
Cell bed board and transfection: cell is pressed 1 * 10
5/ hole is inoculated in the 96 porocyte culture plates, contains in the DMEM substratum of 10%FBS 37 ℃, 5%CO in nonreactive
2The incubator overnight incubation.Lipofectamin is pressed in transfection
TM2000(Invitrogen company) operation steps is carried out, and the different RNA molecule of experiment is that 10 nM/ holes add by concentration.
Real-time quantitative PCR detects the Bcl-2 gene mRNA expression: extract purification kit TurboCapture mRNA Kit(QIAGEN company with mRNA) extraction purifying cells mRNA, operation is undertaken by the test kit specification sheets, without RNase water/hole dissolving RNA, getting 4 μ L RNA is that template is carried out the real-time quantitative PCR reaction with 80 μ L.
Detect the real-time quantitative PCR primer of Bcl-2 gene:
5 ' forward primer: GGCTGGGATGCCTTTGTG (SEQ ID NO:6)
3 ' reverse primer: GCCAGGAGAAATCAAACAGAGG (SEQ ID NO:7)
Detect Bcl-2 mrna expression in the sample with above-mentioned Bcl-2 gene-specific primer, the house-keeping gene GAPDH that increases simultaneously contrasts as confidential reference items.Each reaction do 3 parallel.Set up following 25 μ L reaction systems: 4 μ L template ribonucleic acids, 12.5 μ L 2 * SensiMix One-Step, 1 μ L, 5 ' forward primer (10 μ M), 1 μ L, 3 ' reverse primer (10 μ M), 0.5 μ L 50 * SYBR Green I uses without RNase water and supplies system to 25 μ L.Reaction conditions: 42 ℃ of reverse transcription 30 min, 95 ℃ of denaturation 7 min, 95 ℃ of sex change 20 sec, 60 ℃ of annealing 30 sec, 72 ℃ are extended 30 sec, circulate 45 times.
With 2
-Δ Δ CtAnalytical method is analyzed experimental result, and make histogram, as shown in Figure 3, nucleic acid molecule By-Pass shows the reticent effect preferably of Bcl-2 gene in the liver cancer cell, compare its suppression efficiency no significant difference with cotransfection experiments group Sur-2+Sur-10+Bcl-1 with corresponding Bcl-1 experimental group.
Embodiment 4
Detect ifn response
Cell cultures: liver cancer cell SMMC-7721 in containing the DMEM substratum of 10%FBS (Gibco company), 37 ℃, 5%CO
2Incubator is cultivated.
Cell bed board and transfection: cell is pressed 1 * 10
5/ hole is inoculated in the 96 porocyte culture plates, contains in the DMEM substratum of 10%FBS 37 ℃, 5%CO in nonreactive
2The incubator overnight incubation.Lipofectamin is pressed in transfection
TM2000(Invitrogen company) operation steps is carried out, and the different RNA molecule of experiment is that 10 nM/ holes add by concentration.
Real-time quantitative PCR detects the mrna expression of Interferon, rabbit genes involved OAS1: extract purification kit TurboCapture mRNA Kit(QIAGEN company with mRNA) extraction purifying cells RNA, operation is undertaken by the test kit specification sheets, without RNase water/hole dissolving RNA, getting 4 μ L RNA is that template is carried out the real-time quantitative PCR reaction with 80 μ L.
Detect the real-time quantitative PCR primer of Interferon, rabbit genes involved OAS1:
5 ' forward primer: GTGAGCTCCTGGATTCTGCT (SEQ ID NO:8)
3 ' reverse primer: TGTTCCAATGTAACCATATTTCTGA (SEQ ID NO:9)
Detect OAS1 mrna expression in the sample with above-mentioned Interferon, rabbit genes involved OAS1 Auele Specific Primer, the house-keeping gene GAPDH that increases simultaneously contrasts as confidential reference items.Each reaction do 3 parallel.Set up following 25 μ L reaction systems: 4 μ L template ribonucleic acids, 12.5 μ L 2 * SensiMix One-Step, 1 μ L, 5 ' forward primer (10 μ M), 1 μ L, 3 ' reverse primer (10 μ M), 0.5 μ L 50 * SYBR Green I uses without RNase water and supplies system to 25 μ L.Reaction conditions: 42 ℃ of reverse transcription 30 min, 95 ℃ of denaturation 7 min, 95 ℃ of sex change 20 sec, 60 ℃ of annealing 30 sec, 72 ℃ are extended 30 sec, circulate 45 times.
With 2
-Δ Δ CtAnalytical method is analyzed experimental result, and makes histogram, as shown in Figure 4.In the experiment, press simultaneously transfection of 5 ng/ holes Poly(I:C with above-mentioned transfection method) as positive control.Polyinosinic-polycytidylic acid (Poly(I:C)) be a kind of synthetic chemical that is similar to the Yeast Nucleic Acid of infectious virus, immunity system that can irritation cell produces Interferon, rabbit.Compare with normal group, the mRNA of positive controls OAS1 gene has obvious rising, and other different RNA molecule transfection experiment group OAS1 gene does not have high expression level, and this result shows that nucleic acid molecule By-Pass does not cause ifn response.
Embodiment 5
The CCK-8 method detects nucleic acid molecule By-Pass to the inhibiting rate of liver cancer cell SMMC-7721
Cell cultures: liver cancer cell SMMC-7721 in containing the DMEM substratum of 10%FBS (Gibco company), 37 ℃, 5%CO
2Incubator is cultivated.
Cell bed board and transfection: cell is pressed 1 * 10
5/ hole is inoculated in the 96 porocyte culture plates, contains in the DMEM substratum of 10%FBS 37 ℃, 5%CO in nonreactive
2The incubator overnight incubation.Lipofectamin is pressed in transfection
TM2000(Invitrogen company) operation steps is carried out, and the different RNA molecule of experiment is that 10 nM/ holes add by concentration.
CCK-8 measures: every hole adds the CCK-8 solution (colleague's chemistry) of 1/10 culture volume.In cell culture incubator, continue to hatch 0.5～4 h.Measure absorbancy at 450 nm wavelength places with microplate reader.
According to measured A
450Value (OD value) is made growth curve chart, as shown in Figure 5.According to formula: cell proliferation inhibition rate=(l-experimental group average A
450Value/control group average A
450Value) * 100%, calculate inhibitory rate of cell growth, and make histogram, as shown in Figure 6.The result shows that nucleic acid molecule By-Pass has significant inhibition behind effect liver cancer cell 48 h, 72 h, 96 h, compare no significant difference with other experimental group.
Embodiment 6
The CCK-8 method detects nucleic acid molecule By-Pass to the inhibiting rate of liver cancer cell HepG2
Cell cultures: liver cancer cell HepG2 in containing the DMEM substratum of 10%FBS, 37 ℃, 5%CO
2Incubator is cultivated.
Cell bed board and transfection: cell is pressed 1 * 10
5/ hole is inoculated in the 96 porocyte culture plates, contains in the DMEM substratum of 10%FBS 37 ℃, 5%CO in nonreactive
2The incubator overnight incubation.
Transfection is according to Lipofectamin
TM2000(Invitrogen company) specification sheets transfection, RNA presses the 10nM/ hole and adds.
CCK-8 measures: every hole adds the CCK-8 solution (colleague's chemistry) of 1/10 culture volume.In cell culture incubator, continue to hatch 0.5～4 h.Measure absorbancy at 450 nm wavelength places with microplate reader.
Interpretation of result: according to measured A
450Value (OD value) is made growth curve chart, as shown in Figure 7.According to formula: cell proliferation inhibition rate=(l-experimental group average A
450Value/control group average A
450Value) * 100%, calculate inhibitory rate of cell growth, and make histogram, as shown in Figure 8.The result shows that nucleic acid molecule By-Pass has significant inhibition behind effect liver cancer cell 48 h, 72 h, 96 h, compare no significant difference with other experimental group.
＜110〉Biomics Bioisystech Co., Ltd
＜120〉a kind of interfere RNA molecule and application
<130> BIOMICS20110203
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 47
<212> RNA
＜213〉artificial sequence
<400> 1
gacuuggccc aguguuucuc augcgucggg augacugagu accugaa 47
<210> 2
<211> 42
<212> RNA
＜213〉artificial sequence
<400> 2
uccuuucugu caagaagcag uucagaaaca cugggccaag uc 42
<210> 3
<211> 42
<212> RNA
＜213〉artificial sequence
<400> 3
uucagguacu cagucaucca aggaaagaca guucuucguc aa 42
<210> 4
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 4
accgcatctc tacattcaag 20
<210> 5
<211> 18
<212> DNA
＜213〉artificial sequence
<400> 5
caagtctggc tcgttctc 18
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<211> 18
<212> DNA
＜213〉artificial sequence
<400> 6
ggctgggatg cctttgtg 18
<210> 7
<211> 22
<212> DNA
＜213〉artificial sequence
<400> 7
gccaggagaa atcaaacaga gg 22
<210> 8
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 8
gtgagctcct ggattctgct 20
<210> 9
<211> 25
<212> DNA
＜213〉artificial sequence
<400> 9
tgttccaatg taaccatatt tctga 25
Claims (2)
1. interfere RNA molecule, it is characterized in that, formed by a positive-sense strand and two antisense strands, the length of every chain is at least 30 Nucleotide, at least two parts in its positive-sense strand or the antisense strand are distinguished the different loci of the heterogeneic RNA of target or same gene RNA, and this nucleic acid molecule can not cause the Interferon, rabbit effect after entering cell; And every positive-sense strand and antisense strand all contain 5 ' end and 3 ' end, wherein: 3 ' end complementation of 5 ' end of positive-sense strand and article one antisense strand; 3 ' end of positive-sense strand is complementary with 5 ' end of second antisense strand; Article one, 3 ' end of 5 ' of antisense strand end and second antisense strand is complementary, after annealing, forms the nucleotide double with secondary structure; Described positive-sense strand is:
5’-GACUUGGCCCAGUGUUUCUCAUGCGUCGGGAUGACUGAGUACCUGAA-3’，
Article one, antisense strand is:
5’-UCCUUUCUGUCAAGAAGCAGUUCAGAAACACUGGGCCAAGUC-3’，
The second antisense strand is:
5’-UUCAGGUACUCAGUCAUCCCAACUGCUUCUUGACAGAAAGGA-3’。
2. interfere RNA molecule claimed in claim 1 is regulated application in the medicine of Survivin and Bcl-2 gene function in the cell in preparation.
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| US13/415,600 US9115167B2 (en) | 2011-03-10 | 2012-03-08 | Multi-targets interfering RNA molecules and their applications |
| US14/805,746 US10443057B2 (en) | 2011-03-10 | 2015-07-22 | Multi-targets interfering RNA molecules and their applications |
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| Title |
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| 戴大鹏 等.一种快速构建单靶或双靶基因位点RNAi质粒载体的方法.《基础医学与临床》.2009,第29卷(第4期),全文. * |
| 李曙芳 等.RNA 干扰技术的应用新进展.《中国实验动物学报》.2009,第17卷(第1期),全文. * |
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Address after: 226016 Jiangsu city of Nantong Province Economic and Technological Development Zone No. 76 Chang Xing Lu Patentee after: Study on molecular medicine 100 Kechuang source (Nantong) Co., Ltd. Address before: 226016 Jiangsu city of Nantong Province Economic and Technological Development Zone No. 76 Chang Xing Lu Patentee before: Biomics Branch Institute of molecular medicine (Nantong) Co., Ltd. |