CN103333890A - RNA interference preparation used for treating viral hepatitis B - Google Patents

Abstract

The invention discloses a RNA interference preparation used for treating viral hepatitis B. The RNA interference preparation comprises one or more sense strands and one or more antisense strands, wherein one or more polynucleotide strands are complementary with one or more regions of HBV RNA. An iNA is an iNAs having a structure of double stands. The double strands have structures of linear, loop, similar-loop, hairpin-type, stem-loop, unidirectional or bidirectional. The RNA interference preparation provided by the invention can be used for treating viral hepatitis B.

Description

The RNA for the treatment of hepatitis B disturbs preparation

Technical field

The present invention utilizes RNA to disturb that (RNA interference RNAi) suppresses copying of hepatitis B virus.

Background technology

The expression of double-stranded RNA (dsRNA) arrestin, silencer has widely, the purposes of potential treatment human diseases.DsRNA induced gene silence has three kinds of modes: 1, the inactivation of functional transcription, this phenomenon refer to the DNA of RNA under instructing or histone methylated; 2, small RNA (small interfering RNA, the degraded of the messenger RNA(mRNA) of siRNA) inducing (mRNA); 3, the decay of the functional transcription of RNA mediation.But it is generally acknowledged that the gene that dsRNA induces disturbs (inhibition) to refer to the interior RNA degraded of zooblast.Chemosynthesis microRNA, as siRNA, can be in the sub-micro volumetric molar concentration, the intracellular mRNA of degraded reaches more than 95% under the enzyme catalysis.

The effect of RNAi can last very long, even continues to the several generations cell fission, and to disturb be sequence-specific inhibition to gene simultaneously.So, the specific inhibition of gene expression of RNAi energy, and do not influence its isomer or other mRNA, this species specific signing that is suppressed at research gene function and drug target has the particularly important effect.SiRNA can be used in and is developed to pharmacological agent: 1, because gene overexpression or normal gene of not expressing down; 2, Tu Bian gene and the disease that causes.

Medical use

RNAi can be applied to develop the new class medicine that is different from small molecules and albumen, though long dsRNA causes the ifn response of cell, can not be directly delivered to cell, and the application of siRNA is still compared successful.Clinical trial has also been carried out in the research that is widely used in of success.

Being applied to the treatment aspect, at first is to become and respiratory syncytial virus for senile macula lutea venereal disease.The disease of the treatment of other report comprises antiviral HIV, first, second, hepatitis C, flu and measles etc.; Treatment nervosa degeneration, particularly Huntington's disease also have report as poly glumine disease.RNAi also can contain the division of tumour cell, treatment cancer by the gene that suppresses overexpression.Yet a very important field is the siRNA transmission technology of a safety of exploitation, could guarantee the clinical application of RNAi.

Although the RNAi that studies show that of cell levels is a very promising drug development platform, whether the effect of missing the target of siRNA can cause that some side effects have also caused attention simultaneously, because the effect of missing the target can suppress the gene with the target gene similar sequences.It is calculated that the effect of missing the target can reach 10%.At mammalian cell, but long double-stranded siRNA inducing interferon reaction.Therefore, siRNA or iNA(interferring nucleic acid, interfere RNA) must keep short sequence, to avoid interference plain reaction.

Can designing a siRNA or iNA, not have a best method of ifn response be that curative iNA of design or siRNA can act on one or more target gene, or the different sites of a target gene, its structure is a direction, or different directions, annular, similar annular or linear.This several types iNA or siRNA is different with the structure of the siRNA that uses always.

According to estimates, hepatitis B virus (hepatitis B virus, HBV) population of Gan Raning is approximately up to 1/3rd of 2,000,000,000 people-world population, surpasses 3,500,000 people and can transfer chronic infection to.It is reported that the patient that 15～40% HBV infects can develop into liver cirrhosis, liver failure or liver cancer (HCC) have 500,000 to 1,200,000 people to die from HBV and infect every year.HBV is estimated as about 0.4% in the morbidity of the U.S..Yet, inspect data voluntarily by random samples and show in some foreign minorities colony morbidity and surpass 15%.In the nineties in 20th century, the diagnosis outpatient service that hepatitis B virus is relevant and hospital care number increase several times.Equally, the total expenses of being in hospital is estimated from 3.57 hundred million dollars of 1,500,000,000 dollars of increasing to 2003 of nineteen ninety, is remained on 1,300,000,000 dollars then.

The ultimate aim for the treatment of HBV is to suppress or elimination HBV, relaxes or stops HBV and infect the liver injury that causes, prevents the development of liver failure and liver cancer.Most important short-term and intermediate period treatment target are to improve HBV DNA inhibiting rate to greatest extent.But it is difficult thoroughly eradicating hepatitis B virus, because it is incorporated in the host genome, produces the cccDNA that continues as potential tendency of recurrence.Polyoxyethylene glycol Intederon Alpha-2a (PEG-IFN-α) and Intederon Alpha-2a (IFN-α), nucleoside medicine (lamivudine, Entecavir and Telbivudine) and nucleotide analog (Adefovir and tynofovir) are the medicines of using always on the anti-HBV pharmaceutical market of FDA approval.The main drawback of interferon therapy is its pronounced side effects, limits its life-time service.It is invalid often to decompensated liver cirrhosis and transaminase normal patient.In addition, have only 1/3rd patient antiviral effectively to PEG-IFN-α.Though nucleosides (acid) analogue suppresses hbv replication and the hepatic necrosis inflammation is reduced, and can not eradicate virus fully.In addition, after the drug withdrawal, most of patients is observed the bounce-back of viremia.In addition, long-term treatment produces resistance HBV virus strain, causes the treatment failure.

RNAi is the active process of regulatory gene in the cell.It once was called as other titles, comprise PTGS (PTGS), 2006, Andrew Fire and Craig C.Mello obtains Nobel's physiology or medical science prize jointly, to commend the article that they delivered in 1998, the RNAi phenomenon that they find described in article in nematode research.

Two types micro ribonucleic acid (RNA) molecule-microRNA (miRNA), siRNA (siRNA) are the cores that RNA disturbs.RNA is the direct product of gene, and these microRNAs can be combined with its mRNA specificity and be increased or reduce their activity, for example, and the generation of arrestin matter.RNAi has important effect, and the protection cell is avoided the influence of virus and transposon (transposon).

The RNAi phenomenon is found to be present in many eukaryotic cells, comprises zooblast, is the short-movie section that is called as siRNA of～20 Nucleotide by the Dicer enzyme with long dsRNA molecular degradation.Each siRNA brings down a fever into two strands (ss) RNA, i.e. passenger's chain and drag chain (passenger strand and the guide strand), and passenger's chain is hydrolyzed, and drag chain is combined with the RISC complex body.The catalyst component Argonaute albumen of RISC mixture is induced and activated to drag chain base sequence and mRNA sequence complementary element, and mRNA is degraded.In some organism, this process is transmitted to whole body.

The specificity of the genetic expression of RNAi suppresses, and no matter is in cell cultures with in the animal object, makes it to become a valuable research tool, and synthetic dsRNA transfered cell can be induced the inhibition specific gene.The RNAi technology also can be used for extensive screening, each gene of systematic inhibition cell, and it can help to determine necessary step in the specific cell truck process, as cell fission.Inquiring into signal path is the means of biotechnology and an effective research gene function medically.RNAi has confirmed its specificity and activity in experimentation on animals, have the effect of reticent disease gene and treatment disease.

Existing report, RNAi can suppress HBV in culturing cell and mice infected.But, report is also arranged because the high mutation rate of HBV can not only more effectively suppress HBV by suppressing many sites of HBV, also may stop the HBV transgenation.

Summary of the invention

In brief, this application provides siRNA or the iNA of a kind of method by one or more types, dyes by suppressing the genomic one or more sites of HBV (structure is seen Fig. 1) treatment hepatitis B virus sexuality.

DsRNA and/or contain the double-stranded RNA of two or more fragments is suitable as Dicer and RISC substrate, is used for suppressing HBV genetic expression.In one aspect, the invention provides a kind of method, comprise double-stranded iNA or siRNA, as iNA(siRNA) ID 1-272(table 1), can select the RNA site at one or more HBV.Double-stranded iNA or siRNA are made up of two chains complimentary to one another, the polynucleotide chain of one or more complementary strand can with one or several regional complementarity of HBV RNA.In embodiment, double-stranded iNAs can be (Fig. 1) such as structures of straight chain, annular, class annular, hair fastener type, stem-ring (stem-loop), unidirectional or two-way shape (bidirectional).In other embodiments, double-stranded iNAs(or siRNA) can have two continuous polynucleotide chains, one or more breach (otch) are arranged, between the gap, form the chain of one or more segmentations, as Fig. 1.Double-stranded iNA or the long 10-200 of a siRNA Nucleotide are 15～50 Nucleotide preferably, are more preferably 19-29 Nucleotide.The polynucleotide chain of the complementary region of its double center chain is no less than 10 Nucleotide, is more preferably 19-29 Nucleotide in embodiment further, at least 50%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%iNA, 99%, or any double-stranded region of the Nucleotide in 100% the chain is complementary to another chain.In other embodiments, double-stranded iNA or siRNA can have cohesive end or blunt end.In other embodiments, if iNA or siRNA have cohesive end, the 3' end of iNA or siRNA comprises the length of 1 to 5 nucleosides, can be positioned at one or 3 ' end.

INA (siRNA) target site of table 1HBV virus

In one aspect, the invention provides the iNAs structure of a linear segmented, its structure has two short chains, and is complementary on the continuous long-chain, and jagged between the short chain (otch) is as iNA ID NO.231-241,249-252, and 269-272.Continuously long-chain comprises two sections, the sequence of each section and HBV RNA complementation or identical, and two short chains of iNA can be identical or be complementary to the different loci of HBV RNA.For example, in embodiment 1, at the site of each HBV RNA, two sections in the long-chain can also can also can in the rear end (3' end) at front end (5' end) on the contrary less than order before and after specific continuously.As iNA ID 231-241,249-252, and 269-272.The length of described continuous chain to the youthful and the elderly be 15 to 80 Nucleotide, be preferably 19～50 Nucleotide.Two short chains are preferably 19 to 27 Nucleotide to 10 to 40 Nucleotide of the youthful and the elderly.In embodiment further, be at least 50%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or any double-stranded region of the Nucleotide in the chain of 100%iNA is complementary to another chain.In other embodiments, double-stranded iNA or siRNA can have cohesive end or blunt end.In other embodiments, if iNA or siRNA have cohesive end, the 3' end of iNA or siRNA comprises 1 to 5 Nucleotide of length, can be positioned at one or

In one aspect, the iNA numbering 242-248 among the present invention has the two strands of two-way ring-type or similar ring texture, as the iNA among the embodiment 2, can act on the different zones of HBV RNA.Two complementary strand RNA or RNA sample oligonucleotide form double-stranded RNA or RNA sample oligonucleotide.At least wherein a chain is two-way, and having a section in this chain at least is that 5' to 3' direction and another section are the directions of 3' to 5', and forms a ring-type or similar ring texture by the complementary nucleotide of these two different directions sections.And the Nucleotide of the strand section in another strand and this ring-type or the two-way chain of similar ring-type forms two strands by complimentary fashion.The length of the two-way chain of annular may be as little to 10 Nucleotide, grows to 1,000 Nucleotide or the length of whole RNA.In one direction the length of section (5' hold to 3' end or 3 ends ' to the 5' end) should between 5 to 80 bases, be preferably 5 to 10 or 15 to 29 Nucleotide.The two-way chain complementation of annular can be any length with another chain, from 10 to 200 Nucleotide, but the double-stranded region that forms should be from 10 to 200 length the Nucleotide, being preferably is 15 to 29 Nucleotide, preferably 19 to 25 Nucleotide.It is 0 Nucleotide (being called otch) that space between the double-stranded section can be lacked as far as possible, and what can grow is 100 or several thousand Nucleotide (breach).Short chain and long-chain at double-stranded region are complementary.Two chains of any double-stranded region can be complimentary to one another wholly or in part.If have only the part complementation, the one or more Nucleotide in chain may be unpaired.Unpaired nucleotide can be at 5'-end or 3'-end, or two ends, or between 5' end and the 3'-end Anywhere.INA among the embodiment 2 namely is example.

In one aspect, the invention provides the iNAs structure with stem-ring structure, the iNA NO.257-266 among the present invention, as shown in the embodiment 4, can be at the RNA different zones of HBV.Two complementary strand RNA or RNA sample oligonucleotide form double-stranded RNA.At least one chain formation stem-ring structure wherein; One section forms ring, and the 5'-end is a part of and form a short double-stranded region between the terminal a part of Nucleotide of the 3'-of same chain.Another chain will with the annular section complementation of non-double-stranded region.The length of similar stem-ring may be as little to 10 Nucleotide, grows to the length of several thousand Nucleotide or one or several mRNA, is preferably 15 to 200 Nucleotide, and being preferably is the length of 19～45 Nucleotide.Similar stem-ring can be any length, but the double-stranded region that forms should be the length of from 10 to 200 Nucleotide, is preferably 15 to 29 Nucleotide.It is 0 Nucleotide (otch) that gap between each double-stranded region can be lacked as far as possible, also can be 100 or several thousand Nucleotide (breach).Article two, any double-stranded region of chain can be complimentary to one another wholly or in part.If have only the part complementation, the one or more Nucleotide in chain may be unpaired.Unpaired nucleotide can be at 5'-end or 3'-end, or two ends, or between 5' end and the 3'-end Anywhere.The iNA that shows among the embodiment 4 namely is example.

This patent disclosed any aspect, provide among some embodiment an iNA molecule contain ribothymidine (ribothymidine) or sulfo-ribothymidine (2 – thioribothymidine) or 2'-O-methyl-5-methyluridine (2 '-O-methyl-5-methyluridine) replace at least one uridine or replace each uridine at one or more chain.In a further embodiment, this iNA further comprises one or more non-standard nucleosides, as deoxyuridylic acid, lock nucleic acid (LNA) molecule or a general Nucleotide (universal nucleotide), or a G clamper (clamp).Universal nucleotide comprises C-phenyl (C-phenyl), C-naphthyl (C-naphthy), inosine (inosine), azoles methane amide (azolecarboxamide), 1-β-D-ribofuranosyl-4-nitroindoline (1-β-D-ribofuranosyl-4-nitroindole), 1-β-D-ribofuranosyl-1-β-D-ribofuranose 5-nitroindoline (1-β-D-ribofuranosyl-5-nitroindole), 1-β-D-ribofuranosyl-1-β-D-ribofuranose 6-nitroindoline (1-β-D-ribofuranosyl-6-nitroindole), or 1-β-D-ribofuranosyl-3-nitro-pyrrole (or1-β-D-ribofuranosyl-3-nitropyrrole).In certain embodiments, the RNA molecule comprises 2'-sugar substitute ingredient, as the 2'-O-methyl (2 '-O-methyl), the 2'-O-methoxy ethyl (2 '-O-methoxyethyl), the 2'-O-2-methoxy ethyl (2 '-O-2-methoxyethyl), the 2'-O-allyl group (2 '-O-allyl) or halogen (the 2'-fluorine, 2 '-fluoro).In certain embodiments, the INA molecule also comprises on the first one or more chains, second chain or the 3rd chain substituted radical on the end cap, can be the deoxynucleotide of alkyl (alkyl), dealkalize base position (abasic), deoxidation dealkalize base (deoxy abasic), glycerine (glyceryl), dinucleotides (dinucleotide), acyclic nucleotide (acyclic nucleotide) or direction independently.In other embodiments; the nucleosides connecting key that further comprises at least one modification; as being thiophosphatephosphorothioate (phosphorothioate) independently; chirality thiophosphatephosphorothioate (chiral phosphorothioate); phosphorodithioate (phosphorodithioate); phosphotriester (; phosphotriester); aminoalkylphosphotriester; methyl-phosphorous acid (methyl phosphonate); alkyl phosphonate (alkyl phosphonate); 3'-alkylene phosphonic acids salt (3 '-alkylene phosphonate); 5'-alkylene phosphonic acids salt (5 '-alkylene phosphonate); chirality phosphonate (chiral phosphonate); phosphono (phosphonoacetate); sulfo-phosphono (thiophosphonoacetate); phosphonic acid ester (phosphinate); phosphoramidate (; phosphoramidate); aminoalkylphosphoramidate; thionophosphoramidate; thionoalkylphosphonate; thionoalkylphosphotriester; selenophosphate, boranophosphate key connecting key.

Description of drawings

Fig. 1, be used for to suppress siRNA or the iNA structural representation of HBV gene.

Fig. 2, at the example of the efficient HBV iNA of HepG2.2.15 cell screening.With iNA RFect transfection iNAs(5nM) to the HepG2.2.15 cell.Two days later, collect and smudge cells, with the variation of real-time quantitative RT-PCR methods analyst genetic expression.

Fig. 3, at the example of the efficient HBV iNA of HepG2.2.15 cell screening.Can act on the iNAs(5nM of two different loci of HBV RNA with the RFect transfection) to the HepG2.2.15 cell.Two days later, collect and smudge cells, with the variation of real-time quantitative RTPCR methods analyst genetic expression.

Fig. 4, iNA suppress the example that copy of HBV in HBV transgenic animal body.The antiviral iNA (siRNA) of an intravenous injection transmission system parcel of hepatitis B virus transgenic mice suppress HBV virogene copying in liver cell specifically, and not copying of the HBV of ApoB siRNA of control group influences.

Fig. 5, HBV1(iNA numbering 267), HBV2(iNA numbering 269) and the iNA intravenously administrable of APO-B reduce the example of HBV DNA in the HBV transgenic mice liver.The HBV transgenic mice is injected iNA totally three times once in a week through the tail vein.The HBV transgenic mice of adefovir ester (ADV) group is oral administration once a day, continuous 14 days (10mg/kg/ days).Tissue and blood are got in administration after three days the last time.A) the Southern blot hybridization is analyzed the variation of HBV DNA in the transgenic mice liver; B) variation of HBV DNA in the pcr analysis transgenic mice liver.* *, P＜0.001(use the multiple comparisons check of One-way ANOVA and Bonferroni method).

Fig. 6, HBV1(iNA numbering 267), HBV2(iNA numbering 269), the iNA intravenously administrable of APO-B reduces the example of HBV transgenic mice blood plasma HBeAg.The HBV transgenic mice is injected iNA totally three times once in a week through the tail vein.The HBV transgenic mice of adefovir ester (ADV) group is adefovir ester (Adefovir dipivoxi) l oral administration 14 days (10mg/kg/ days) once a day.Administration was drawn materials after three days the last time.A) to before the thing, B) give thing after 17 days.* *, P＜0.001(use the multiple comparisons check of One-way ANOVA and Bonferroni method).

Fig. 7, HBV1(iNA numbering 267), HBV2(iNA numbering 269) and the iNA intravenously administrable of APO-B reduce the example of HBV transgenic mice blood plasma HBsAg.The HBV transgenic mice is injected the iNA totally three times of nano colloidal particles transmission system parcel once in a week through the tail vein.The HBV transgenic mice of ADV group is adefovir ester oral administration 14 days (10mg/kg/ days) once a day.Administration was drawn materials after three days the last time.* *, P＜0.001(use the multiple comparisons check of One-way ANOVA and Bonferroni method).

Fig. 8, HBV1(iNA numbering 267), HBV2(iNA numbering 269) and the iNA intravenously administrable of APO-B reduce the example of HBV RNA in the HBV transgenic mice liver.The HBV transgenic mice is injected iNA totally three times once in a week through the tail vein.The HBV transgenic mice of ADV group is adefovir ester oral administration 14 days (10mg/kg/ days) once a day.Administration was drawn materials after three days the last time.* *, P＜0.001(use the multiple comparisons check of One-way ANOVA and Bonferroni method).

Fig. 9, HBV1(iNA numbering 267), HBV2(iNA numbering 269) and the iNA vein of APO-B give the chemico-analytic example of blood plasma behind the HBV transgenic mice medicine.The HBV transgenic mice is injected iNA totally three times once in a week through the tail vein.The HBV transgenic mice of ADV group is adefovir ester oral administration 14 days (10mg/kg/ days) once a day.Administration was drawn materials after three days the last time.*, P＜0.05(uses the multiple comparisons check of One-way ANOVA and Bonferroni method).

Definition

The definition of used technical term is understood to include these terms and those implications known to the skilled in the art among the present invention, and, and be not intended to and limit the scope of the invention, do not need to repeat statement each time.

Term used herein " one ", " one ", the present invention described in " this " and similar term, and in the claims, will be interpreted as comprising odd number and plural number.Term " comprises "; " have "; " contain " and will be interpreted as open-ended term.Their meaning is for example " to include, but are not limited to ".

Scope that use or set(ting)value refers to each the independent value in this scope, should be equal to the description of any independent value.Here the occurrence that adopts will be understood that exemplary, rather than limit the scope of the invention.

Term interfere RNA (iNA) refers to have the nucleic acid double chain of chain complementation each other as used herein.After entering the RISC mixture, induce the RNAi mechanism of RNA enzyme liberating RNA.In addition, iNA regulates increasing of target gene expression by promoter for RNA a mechanism.In general, each bar chain of iNA is all Nucleotide, mainly is ribonucleotide, but also can be the analogue of RNA, the analogue of RNA and RNA, modified Nucleotide, RNA and DNA, the analogue of RNA and DNA, non-nucleotide, or a chain is DNA fully, and another chain is RNA, so long as can induce the cognate rna degraded by RNAi mechanism, all can be used as the structure of iNA.

Term as used herein " two-way double-stranded iNA " or " two-way double-stranded siRNA " or " two-way iNA " be one through generic term of the present invention, the bi-directional configuration that comprises same chain of interfere RNA (iNAs), it can be cut in cell and form iNA or siRNA.Two strands at least one chain of two-way iNA has the fragment of one or more 5' to 3' directions and the fragment of one or more 3' to 5' directions.One or more parts and its complementation are arranged, to form one or more double-stranded sections on another chain of iNA.Between each double-stranded section, can jagged or otch.Two-way iNA can be linear, stem-annular, or configuration such as circle.The iNA of terminal structure may be blunt or viscosity (outstanding or dangle) end, and its functional objective is the RNA silence.The terminal end construction of viscosity (giving prominence to) has more than and is limited to the 3' protuberance, and the also outstanding structure of 5' is as long as it is to induce the RNAi effect.In addition, the Nucleotide number that dangles be not limited to report 2 or 3, but can be any number, as long as can induce the RNAi effect.For example, may be 1 to 8 or longer, or 2 to 4 bases or longer.

As used herein, term " the iNA two strands of annular or similar annular " is the generic term that the present invention uses, and comprises that interfere RNA (iNAs) has the structural framing of annular or class annular etc., and it can be cut in cell and form iNA or siRNA.The iNA two strands of annular of the present invention or class annular comprises that also expression vector (also abbreviating the iNA expression vector as) can produce iNA iNA double-stranded or that generate after the transit cell record forms and/or transcribes, and induces RNAi in vivo.Positive-sense strand or antisense strand can have one or more otch or breach.The iNA end can be viscosity (giving prominence to) or blunt, as long as its function can make target RNA silence.The end construction of viscosity (giving prominence to) has more than and is limited to the 3' protuberance, also can be the outstanding structure of 5', as long as it is to induce the RNAi effect.In addition, the Nucleotide number that dangles be not limited to report 2 or 3, can be any number, as long as can induce the RNAi effect.For example, dangling may be 1 to 8 base or longer, or 2 to 4 bases.

Loop-stem structure refers to base pairing in the polynucleotide molecule, can occur in single stranded DNA, more commonly RNA.The double-stranded circular structure refers to be complementary to by single or multiple chains the strand position in ring district, the intersegmental jagged or otch of sheet.In addition, any section on stem also can have the complementary strand in one or more strands zone.End structure may be blunt or viscosity (giving prominence to).Can there be one or more chains to be complementary to ring or non-annularity section, the intersegmental jagged or otch of sheet, outstanding with or without 3' or 5' end.

Length refers to the quantity of the Nucleotide among the two-way iNA as used herein, from the 1st base pair of the 5' end of positive-sense strand, ends at terminal last base of 3' of positive-sense strand.

On genetics, microRNA (miRNA) is single stranded RNA, the about 21-23 of a length Nucleotide, the expression of regulatory gene.MiRNA transcribes from DNA, but does not translate into protein (non-coding RNA), and they are to generate premiRNA by the short loop-stem structure of the PRimiRNA that is called as primary transcription, generates miRNA from premiRNA at last.Ripe miRNA molecular moiety is complementary to one or more messenger RNA(mRNA) molecules, and their major function is to reduce expression of gene.

The Nucleotide of iNA molecular modification can be at any chain.For example, modified Nucleotide can have the conformation (for example, in the Northern pseudorotation cycle, seeing Sanger, the principle of nucleic acid construct, SpringerVerlaged., 1984) of a Northern.The example that Nucleotide has the Northern configuration comprises that the Nucleotide of lock nucleic acid (LNA) is (as 2'-O, 4'-C-methylene radical-(D-ribofuranosyl) Nucleotide), (2'-O, 4'-Cmethylene-(Dribofuranosyl) nucleotides), 2'-methoxy ethoxy (MOE) Nucleotide (2'methoxyethoxy (MOE) nucleotides), 2'-methyl-sulfo--ethyl, 2'-deoxidation-2'-fluorine Nucleotide (2'methyl-thio-ethyl, 2'-deoxy-2'-fluoronucleotides), 2'-deoxidation-2'-chlorine Nucleotide (2'-deoxy-2'-chloro nucleotides), the Nucleotide of 2'-azido-(2'-azido nucleotides), 2'-O-methyl nucleotide (2'-Omethyl nucleotides).Keep simultaneously inducing the ability of RNAi and can resisting the Nucleotide of the chemically modified of nuclease degradation.But a kind of conjugated molecule is connected to the ligand of iNA molecule polyoxyethylene glycol, human serum albumin or a cell receptor of chemically modified, but the mediated cell picked-up.Can be connected to chemically modified iNA molecule and by the case description of the contemplated specific conjugated molecule of the present invention, as seen Vargeese, Deng people's United States Patent (USP), publication number is 20030130186 and U.S. Patent Publication No. 20040110296, and its every part all can be incorporated in the present invention.

Several examples are arranged in the present invention, describe sugar, alkali and phosphatic modification can be incorporated into nucleic acid molecule, strengthen stability and the validity of its nuclease.For example, oligonucleotide is modified with enhanced stability and/or is strengthened biological activity, and by modifying the opposing nuclease, for example, the nucleotide base of 2'-amino, 2'-C-allyl group, 2'-fluoro, 2'-O-methyl, 2'-O-allyl group, 2'-H is modified.See Wu Siman and Sai Degelun (treatise of Usman and Cedergren, TIBS17:34,1992; Usman, et al, Nucleic Acids Symp.Ser.31:163,1994; Burgin, et al, Biochemistry35:14090,1996.Broadly described nucleic acid molecule of the prior art is sugar-modified, sees See Ecksteinetal., International Publication PCT No.WO92/07065; Perrault, et al.Nature344:565-568,1990; Pieken, et al.Science253:314-317,1991; Usman and Cedergren, Trends in Biochem.Sci.17:334-339,1992; Usman et al.International Publication PCT No.WO93/15187; Sproat, U.S.Pat.No.5,334,711and Beigelman, et al., J.Biol.Chem.270:25702,1995; Beigelman, et al., International PCT Publication No.WO97/26270; Beigelman, et al., U.S.Pat.No.5,716,824; Usman, et al., U.S.Pat.No.5,627,053; Woolf, et al., International PCT Publication No.WO98/13526; Thompson, et al., Karpeisky, et al, Tetrahedron Lett.39:1131,1998; Earnshaw and Gait, Biopolymers (Nucleic AcidSciences) 48:39-55,1998; Verma and Eckstein, Annu.Rev.Biochem.67:99-134,1998; And Burl ina, et al., Bioorg.Med.Chem.5:1999-2010,1997.Method and strategy that these general Study are described are in order to modify sugar, base and/or phosphoric acid.Similarly modification can be used for iNA double chain acid molecule of the present invention, needs only promoting intracellular RNAi rather than showing the function that suppresses its RNAi of iNA.

The iNA two strands may comprise the iNA molecule of phosphoric acid backbone modification, comprising: one or more thiophosphatephosphorothioates, phosphorodithioate, methylphosphonate, phosphotriester, morpholino base, amidation t-butyl carbamate, carboxymethyl, acetamidate, polymeric amide, sulphonate, sulphonamide, sulfamate, formacetal, thioformacetal, alkyl silyl.The argumentation of oligonucleotide backbone modification referring to, Hunziker and Leumann, Nucleic AcidAnalogues:Synthesis andProperties, in Modern Synthetic Methods, VCH, 1995, pp.331-417, and Mesmaeker, etal., " Novel Backbone Replacements for Oligonucleotides; in CarbohydrateModifications in Antisense Research; " ACS, 1994, pp.24-39.The example of chemically modified can comprise 2'-O-methyl, 2'-deoxidation-2'-fluorine ribonucleotide, " universal base " Nucleotide, " acyclic " Nucleotide, 5-C-methyl nucleotide, the terminal glyceryl ester of connection between thiophosphatephosphorothioate nucleotide, 2'-deoxyribonucleotide, ribonucleotide and/or be inverted deoxidation dealkalize base residue.The antisense district of an iNA molecule can be included in the connection between the thiophosphatephosphorothioate nucleosides of 3'-end in described antisense district.The antisense district can comprise the connection between the terminal thiophosphatephosphorothioate nucleosides of 1-5 5'-.The 3'-terminal nucleotide of the iNA molecule of an annular or similar annular can comprise ribose, the alkali of the chemically modified of ribonucleotide or deoxyribonucleotide.The Nucleotide of 3'-end can comprise one or more universal base ribonucleotides.The Nucleotide of 3'-end can comprise one or more acyclic nucleotides.For example, the iNA of chemically modified can have 1,2,3,4,5,6,7,8, or connects between more thiophosphatephosphorothioate nucleosides.Can have in every chain between 1 to 8 or more thiophosphatephosphorothioate nucleosides and connect.Contact can be the two-way iNA binary that is present in one or two oligonucleotide chain between the thiophosphatephosphorothioate nucleosides, for example, can be at sense strand, antisense strand or two chains.In certain embodiments, the iNA molecule comprises 1,2,3,4,5,6,7,8,9,10, or in the sense strand between more purine sulfo-nucleosides, antisense strand or in two chains.

Can be by the synthesis condition of chemically modified iNA molecule: (1) synthetic two or more RNA at least or the similar oligonucleotide molecules of RNA-and complementary strand; (2) under the condition of Shi Heing, two or more complementary strands are annealed together, obtain the iNA molecule.In certain embodiments, the synthetic of the complementary portion of two-way iNA molecule is synthetic by the solid phase oligonucleotide, or synthetic by the oligonucleotide of solid phase series connection.

Oligonucleotide (for example, the oligonucleotide of some modification or part lack the oligonucleotide of ribonucleotide) can use technology known in the art, Caruthers for example, et al, Methods in Enzymology211:3-19,1992; Thompson, et al., International PCT Publication No.WO99/54459; Wincott, et al., Nucleic AcidsRes.23:2677-2684,1995; Wincott, et al., Methods Mol.Bio.74:59,1997; Brennan, et al., Biotechnol Bioeng.61:33-45,1998; And Brennan, U.S.Pat.No.6,001,311.According to general described program, chemosynthesis RNA sees Usman, et al., J.Am.Chem.Soc.109:7845,1987; Scaringe, et al., Nucleic Acids Res.18:5433,1990; And Wincott, et al, NucleicAcids Res.23:2677-2684,1995; Wincott, et al, Methods Mol.Bio.74:59,1997.Any two or more complementary strands or hairpin RNA or by annular or the annealing of similar annular iNA chain of different complementations can form the duplex structure of complementary iNA.

" overlapping " (overlapping) refers to that to have sequence overlapping when two iNA fragments, and for example, a plurality of Nucleotide (nt) wherein may be less to 5-10 Nucleotide or more of 2-5 Nucleotide as many as.

The iNA that " one or more iNA " refer to has different elementary sequences each other.

" target site " or " target sequence (target) " or " target sequence (targeted) " refer to the sequence (for example RNA) in the target nucleic acid, are the sequences by the siRNA degraded of iNA antisense strand sequence mediation.

Space refers to not connect or jagged or otch between two nucleotide phosphodiesterase diester linkages in the chain.

A kind of mixed type iNA molecule refers to that the double-strandednucleic acid of iNA contains a RNA chain and DNA chain.Preferred RNA chain is the antisense strand of target RNA combination.The complementary portion that DNA and the mixing iNA of RNA chain creation by hybridization contains hybridization can have a 3' viscosity end at least.

For " expression of regulatory gene " used increase here or reduce target gene expression, it can comprise and increases or reduces rna level in the cell, or the translation of RNA, or synthesizing by its encoded protein or protein protomer.

Term " inhibition ", " reduction " or " expression of minimizing " looks like here is expression of gene, or level, the encode RNA molecule of one or more protein or protein subunit, or level or one or more albumen in the activity or the coded protein protomer of target gene of RNA molecule of equivalence, be reduced to and be lower than the level of using before the iNA.

" gene silencing " used herein refers in intracellular portion or the expression of suppressor gene fully, and also can be called as " gene knockout ".The degree of gene silencing can determine that also some of them are summarized among the international publication number WO99/32619 by method as known in the art.

In certain embodiments, the iNA molecule comprises justice and antisense sequences or section, it is characterized in that justice is connected with covalent linkage with Nucleotide or the non-nucleotide in antisense district, or by non covalent bond, ionic linkage interaction, hydrogen bond, Van der Waals interaction, hydrophobic interaction and/or accumulation (stacking) interact.

INAs can be assembled into a two strands by two independent oligonucleotide, wherein a chain is that positive-sense strand is antisense strand with another, wherein, described antisense and positive-sense strand self complementation (nucleotide sequence that is the contained base of each bar chain is mended mutually with the sequence of another chain, has formed a two-phase or duplex structure as wherein antisense strand and sense strand).The base sequence of antisense strand can with the nucleotide sequence complementation of a target nucleic acid molecule or its part, the nucleotide sequence of sense strand can be identical in target nucleic acid sequence or its part.INA can be from single oligonucleotide, and wherein the justice of self complementation of two-way iNA can be connected by the base of nucleic acid or the group of non-nucleic acid with the antisense zone.

INA may contain Nucleotide, and non-nucleotide, or the Nucleotide/non-nucleotide linking group that mixes link together justice and the antisense section of iNA.In certain embodiments, the connection portion of Nucleotide can be 3,4,5,6, the length of 7,8,9 or 10 Nucleotide.In certain embodiments, the Nucleotide link can be a kind of aptamer.As used herein, term " aptamers " or " aptamer " comprise the nucleic acid molecule of specificity binding target molecule, it is characterized in that, described nucleic acid molecule comprises a sequence, and this sequence is the target molecule in its natural surroundings.Fit can be to be attached to target molecule, and wherein said target molecule is not the molecule of the nucleic acid combination of nature.For example, aptamers can be used for being tied to a kind of activity of proteins center, prevents from thus interacting with the naturally occurring ligand of protein.Referring to Gold, et al., Annu.Rev.Biochem.64:763,1995; Brody and Gold, J.Biotechnol.74:5,2000; Sun, Curr.Opin.Mol.Ther.2:100,2000; Kusser, J.Biotechnol.74:27,2000; Hermann and Patel, Science287:820,2000; And Jayasena, Clinical Chemistry45:1628,1999.

The link of a non-nucleotide can be Nucleotide, polyethers, polyamine, polymeric amide, poly-peptide, carbohydrate, the lipid of a dealkalize base, polyhydrocarbon or other polymkeric substance are (for example, polyoxyethylene glycol, having unit number as those is ethylene glycol between 2 and 100).Concrete example is seen Specific examples include those described by Seelaand Kaiser, Nucleic Acids Res.18:6353,1990, and Nucleic Acids Res.15:3113,1987; Cload and Schepartz, J.Am.Chem.Soc.113:6324,1991; Richardson and Schepartz, J.Am.Chem.Soc.113:5109,1991; Ma, et al., Nucleic Acids Res.21:2585,1993, andBiochemistry32:1751,1993; Durand, et al., Nucleic Acids Res.18:6353,1990; McCurdy, et al, Nucleosides﹠amp; Nucleotides10:287,1991; Jschke, et al., Tetrahedron Lett.34:301,1993; Ono, et al., Biochemistry30:9914,1991; Arnold, et al., InternationalPublication No.WO89/02439; Usman, et al., International Publication No.WO95/06731; Dudycz, et al., International Publication No.WO95/11910, and Ferentz and Verdine, J.Am Chem.Soc.113:4000,1991.The unit of the one or more Nucleotide in the nucleic acid chains that A " non-nucleotide connection " refers to be included into comprises group or compound that arbitrary sugar and/or phosphoric acid salt replace, and allows the residue base to show their enzymatic activity.Group or compound can be the dealkalize bases, because it does not contain a kind of nucleotide base of generally acknowledging commonly used, as adenosine, guanine, cytosine(Cyt), uridylic or thymus pyrimidine, also as the C1 position at sugar.

Term used herein " biodegradable connection " refers to the connection molecule of a kind of nucleic acid or non-nucleic acid be designed to another molecule of biodegradable connection.The design of biodegradable connection considers that its stability can for example be delivered to a specific tissue or cell type at specific end use.The stability of the biodegradable connection molecule of nucleic acid can be different or adjustable, for example, ribonucleotide by combination, the Nucleotide of deoxyribonucleotide and chemically modified is as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl group, 2'-O allyl group and other 2'-modified nucleotides.Biodegradable nucleic acid connects the dimer of molecule, tripolymer, the oligonucleotide of the tetramer or longer nucleic acid molecule is about 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more Nucleotide, can comprise that perhaps a phosphorous base connects.For example, phosphoric acid ester or phosphodiester bond.The nucleic acid of biological degradability connects molecule can also comprise nucleic acid backbone, the modification of ribose or nucleic acid base.

" antisense nucleic acid ", refer to the nucleic acid molecule of non-enzyme, be attached to RNA, DNA or the PNA(protein nucleic acid of target RNA, see Egholm et al., 1993Nature365,566) molecule, and the activity that has changed target RNA (is seen argumentation: Steinand Cheng, 1993Science261,1004and Woolf et al., U.S.Pat.No.5,849-902).Generally, antisense sequences is a single complementary fully sequence of continuous and target sequence.Yet in certain embodiments, antisense molecule can be incorporated into substrate, makes target nucleic acid molecule form ring of antisense molecule itself formation of a ring and/or combination.Therefore, antisense molecule can with the sector sequence complementation of two (or even more) discrete substrates (RNA), the part of the discontinuous sector sequence of antisense molecule, can be complementary to two sections of target sequence.In addition, antisense DNA can be used for target gene RNA, by the interaction of DNA-RNA, and activator RNA enzyme H, degraded target RNA.Antisense oligonucleotide can comprise the active region of one or more RNA enzyme H, can activator RNA enzyme H, and cracking target RNA.Antisense DNA can chemosynthesis, perhaps expression vector that also can be by the single stranded DNA that uses or etc. expression." sense-rna " has complementary target gene RNA sequence, can induce RNA to disturb by being attached to target gene RNA.Sense-rna has the complementary sequence in conjunction with target gene RNA, induces RNA to disturb by the RNA that is attached to target gene.Justice " Yeast Nucleic Acid " has the sequence of complementary sense-rna, and the sense-rna annealing complementary with it forms iNA.These antisenses and just RNA can chemosynthesis.

" nucleic acid " refers to deoxyribonucleotide or ribonucleotide, but their polymkeric substance strand or double chain form.This term comprises and contains by known nucleotide analog or modify framework residue or connect the nucleic acid of son, can chemosynthesis, and natural existence, non-natural exists, and have the similar nucleic acid binding characteristic as benchmark, and similar fashion is by metabolism.The example of this analogue include, but are not limited to thiophosphatephosphorothioate, phosphoramidate, methyl-phosphorous acid, chirality methyl phosphonate, 2'-O-methyl ribonucleotides, peptide nucleic acid(PNA) (peptide nucleic acid(PNA), PNAs).

So-called " RNA " refers to comprise the molecule of at least one ribonucleotide residue." ribonucleotide " refers to that the 2' position of the β D-ribose furanose part of Nucleotide is an oh group.The RNA(that this term comprises double-stranded RNA, single stranded RNA, separation is partially purified RNA for example).Basically the RNA that produces of pure RNA, synthetic RNA, reorganization, and from naturally occurring RNA by adding, disappearance, replacing different and/or change the RNA that one or more Nucleotide change.Such change can comprise adds the non-nucleotide material to the end of iNA or inner.One or more Nucleotide for example.Nucleotide in the RNA molecule in the invention can also comprise non-standard Nucleotide, for example the Nucleotide of non-natural existence or Nucleotide or the deoxynucleotide of chemosynthesis.The RNA of these changes can be described as the analogue of naturally occurring RNA.As used herein, term " Yeast Nucleic Acid " and " RNA " refer to contain the molecule of at least one ribonucleotide residue.Ribonucleotide is the hydroxyl Nucleotide that contains the 2' position of β-D-nuclear benzofuran sugar yl part.The RNA(that these terms comprise double-stranded RNA, single stranded RNA, separation is partially purified RNA for example), basically pure RNA, synthetic RNA, the RNA that reorganization produces, and be different from naturally occurring RNA by adding, lack the RNA modification and change, substitute, revise and/or change one or more Nucleotide.The change of RNA can comprise adds non-nucleotide to the end of iNA or inner.For example the Nucleotide in the RNA molecule comprises one or more non-standard Nucleotide, for example the Nucleotide of non-natural existence or Nucleotide or the deoxynucleotide of chemosynthesis.The RNA of these changes can be described as analogue.

Used term " non-nucleotide " refers to any group or compound, and this compound can be included into the unit of the one or more Nucleotide in the nucleic acid chains, comprises the replacement of arbitrary sugar and/or phosphoric acid salt, replaces and allows residue sequence to show their enzymic activity.Group or compound are the dealkalize bases, because it does not comprise a generally acknowledged nucleotide base, as adenosine, guanine, cytosine(Cyt), uridylic or thymus pyrimidine, therefore lack base in the 1'-position.

" Nucleotide " used herein is admitted in the art, is comprised natural alkali (standard) base, and the modified base that is known in the art.This base analog generally is positioned at the position of nucleotide sugar 1'.Nucleotide generally comprises alkali, sugar and phosphate group.Nucleotide can be at sugar, and phosphoric acid salt and/or base portion are modified and (also can be abbreviated the non-standard Nucleotide of interchangeable nucleotide analog, modified Nucleotide, non-natural nucleotide, other unmodifieds or modification as.Referring to, Usman and McSwiggen, supra; Eckstein, et al., International PCT Publication No.WO92/07065; Usman, etal, International PCT Publication No.WO93/15187; Uhlman﹠amp; Peyman, supra, all are incorporated herein this paper as a reference).Nucleic acids research can be with reference to Limbach as summing up, et al, Nucleic Acids Res.22:2183,1994.The modification group that some non-limiting examples be directed into nucleic acid molecule comprises, inosine, purine, pyridine-4-ketone, pyridin-2-ones, phenyl, pseudouracil, 2,4,6 trimethoxy-benzenes, the 3-6-Methyl Uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines(for example, the 5-methylcytidine), 5-alkyluridines(for example, ribothymidine), 5-halouridine(for example, the 5-broxuridine) or 6-azapyrimidines or 6-alkylpyrimidines(6-methyluridine for example), propine and other group (Burgin, et al., Biochemistry35:14090,1996; Uhlman﹠amp; Peyman, supra).What is called in this respect " modified base " refers to beyond the position of 1' VITAMIN B4, guanine, cytosine(Cyt) and uridylate base or equivalent.

The nucleotide base of base complementrity refers to that forms the base complementrity of hydrogen bond.Uridylic (U) among VITAMIN B4 (A) and thymus pyrimidine (T) or the RNA, guanine (G) and cytosine(Cyt) (C).The complementary segment of nucleic acid or chain (hydrogen bond is passed through in adding) are complimentary to one another." complementation " refers to that nucleic acid can form hydrogen bond with another nucleotide sequence, can close by traditional Watson-Crick or by other non-traditional bonds.

The positive-sense strand of double-stranded iNA molecule can have distal end cap, as an inversion deoxyabasic group, can be at the 3'-of positive-sense strand end, and 5'-end, or two ends.

So-called " cap structure " refers to chemically modified at the arbitrary end of oligonucleotide (referring to Adamic, et al, U.S.Pat.No.5 998,203, is hereby incorporated by).These terminals are modified and can be protected nucleic acid molecule not degraded by excision enzyme, can help to enter in the cell.Can be that 5'-end (5'-cap) or 3'-end (3'-cap) also may be to be present in two ends.The 5'-cap is in non-limiting example, comprise, but be not limited to glyceryl ester, inverted deoxidation dealkalize base residue, 4', 5'-methylene radical Nucleotide, the red furanose of 1-(β-D-) Nucleotide, the 4'-thio nucleotides, the homocyclic nucleus thuja acid, 1,5-anhydrohexitol Nucleotide, L-Nucleotide, α-Nucleotide, the Nucleotide of base modification, phosphorothioate bond, Su Shi-furan pentose yl nucleosides acid (threo-pentofuranosyl nucleotide), acyclic 3', Nucleotide (the acyclic3' of 4'-plug section, 4'-seconucleotide), acyclic 3,4-dihydroxyl butyl Nucleotide (acyclic3,4-dihydroxybutyl nucleotide), acyclic 3,5-dihydroxypentyl Nucleotide (acyclic3,5-dihydroxypentyl nucleotide), the base portion that 3'-3'-falls (3'-3'-inverted nucleotide moiety), the 3'-3'-base section of falling the dealkalize) (3'-3'-inverted abasic moiety), 3'-2'-is inverted nucleotide segment (3'-2'-inverted nucleotidemoiety), 3'-2'-is inverted the phosphoric acid salt of dealkalize base section, 1,4-butyleneglycol; The 3'-phosphoric acid ester (1,4-butanediolphosphate), methyl-phosphorous acid ester group stillben and the pyrene (bridging or non-bridging methylphosphonate moiety stillben andpyrene) of hexylphosphate, amino hexyl phosphoric acid ester (aminohexyl phosphate), 3'-phosphoric acid, 3'-thiophosphatephosphorothioate, phosphorodithioate (phosphorodithioate) or bridge joint or non-bridge joint.

The embodiment of 3'-cap comprises, but be not limited to glyceryl ester, inverted deoxidation dealkalize base residue (part), 4', 5'-methylene radical Nucleotide (4', 5'-methylene nucleotide), the red furanose of 1-(β-D-) Nucleotide (1-(beta-D-erythrofuranosyl) nucleotide), 4', 5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide), 4'-thio nucleotides homocyclic nucleus thuja acid (4'-thionucleotide, carbocyclic nucleotide), 4 '-thio nucleotides (4'-thio nucleotide), 5'-aminoalkyl group phosphoric acid ester (5'-amino-alkyl phosphate), 1,3-diamino-2-propyl phosphate (1,3-diamino-2propylphosphate), 3-Aminopropyphosphinic acid ester (3-aminopropyl phosphate), the amino hexyl phosphoric acid ester (6-aminohexyl phosphate) of 6-, 1, the amino dodecylphosphoric acid hydroxypropyl of 2-phosphoric acid ester (1,2-aminododecylphosphate), 1,5-anhydrohexitol Nucleotide, L-Nucleotide (Lnucleotide), α-Nucleotide (alpha-nucleotide), the Nucleotide of modifying (modified base nucleotide), thiophosphatephosphorothioate (phosphorodithioate), Su Shi-furan pentose yl nucleosides acid (threopentofuranosyl nucleotide), acyclic 3', Nucleotide (the acyclic3' of 4'-plug section, 4'-seco nucleotide), 3,4-dihydroxyl Nucleotide (3,4-dihydroxybutyl nucleotide), 3,5-dihydroxypentyl Nucleotide, the anti-phase nucleotide segment of 5'-5' (5'-5'-inverted nucleotide moiety), 5'-5'-is inverted dealkalize base section (5'-5'-inverted abasicmoiety), 5'-phosphamide (5'-phosphoramidate), 5'-thiophosphatephosphorothioate (5'phosphorothioate), 1,4-butyleneglycol phosphoric acid ester (1,4-butanediol phosphate), 5'-amino (5'-amino), bridge joint and/or 5'-phosphoramidate (bridging and/or non-bridging5'-phosphoramidate) thiophosphatephosphorothioate and/non-bridge or phosphoric acid ester (phosphorothioate and/or phosphorodithioate), non-bridge joint or bridge joint dimethyl methyl phosphonate and 5'-sulfydryl (bridging or non-bridging methylphosphonate and5'mercapto moieties).(for details, with reference to Beaucage and Lyer, Tetrahedron49:1925,1993) and stillben and pyrene (pyrene).

" asymmetric hair clip " is that linear iNA molecule has one to contain ring-like section, the antisense strand that can be constituted by Nucleotide or non-nucleotide, with a positive-sense strand, wherein, positive-sense strand contains than antisense strand Nucleotide still less, but be enough to be complementary to the antisense strand section, form a two strands that contains ring type structure.

" asymmetric two strands " used herein refers to two independent chains, includes the iNA molecule of positive-sense strand and antisense strand.Wherein, positive-sense strand contains than antisense strand Nucleotide still less, but is enough to be complementary to the antisense strand section, forms a two strands.

INA can be by chemosynthesis or biological production.The method of chemosynthesis can be at Nucleic Acids Research, 624-627, and 1999and Nucleic Acids Research, 3547-3553,1955 find respectively.Molecule clone technology can be used for biological production.

Term siRNA (siRNA) is also sometimes referred to as short interfering rna or reticent RNA, is to be used to refer to the length that double stranded rna molecule has 16-29 Nucleotide, can bring into play the various biological effect.It should be noted that most that siRNA passes through the RNA interference channel, can disturb a special genes to express.Except in addition, siRNA acts on the RNAi correlation function, for example, the formation of antiviral mechanism or chromatin Structure, these complicated approach are illustrated until now.

The phenomenon that the double-stranded RNA activated gene that term RNA a refers to is expressed.This phenomenon is called as " gene activation of little RNA mediation " or RNAa.The promotor of doulbe-chain RNA target tropism gene is induced strong genetic transcription.Recently, RNAa comprises that several other Mammalss non-human primates, mouse, rat are proved to be.

" RNA disturbs the process of the gene silencing of the dependenc RNA that refers to term used herein, is the reticent mixture of being induced by double-stranded RNA in cell, and they are combined with the ARGONAUTE of catalysis unit of RISC there.When double-stranded RNA or the similar iNA of RNA-are external (come self-infection viral rna gene groups or from iNA or the siRNA of transfection), RNA or iNA are imported directly into tenuigenin, cut into short fragment (siRNA) by the Dicer enzyme.DsRNA can be that endogenic (coming from cell) is as the genomic expression of preceding microRNA from the RNA encoding gene.The primary transcript of gene is at first processed, and the pre-miRNA with the stem-ring structure of the feature that forms in nucleus exports to it in tenuigenin then, is cut by Dicer.Therefore, the approach of two dsRNA is arranged, exogenous and endogenous.The activeconstituents of the reticent mixture (RISC) that RNA-induces, endonuclease is called as Argonaute albumen, unties the double-stranded siRNA with they combinations, the RNA of degraded and the complementation of siRNA antisense strand.

Double-stranded RNA after being cut by Dicer, has only drag chain (antisense strand) in conjunction with Argonaute albumen and causes gene silencing.Positive-sense strand is degraded after RISC activates.

Scheme implementation 1

The iNA of segmentation structure comprises the antisense strand at two different HBV RNA sites.Several complementary Nucleotide that do not have are arranged in the middle of continuous chain, for example:

No. 231, iNA sequence (iNA ID#2and#8):

Positive-sense strand: GGGUUUUUCUUGUUGACAAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT

Antisense strand 1:UUGUCAACAAGAAAAACCCCG

Antisense strand 2:UGAAGCGAAGUGCACACGGUC

INA ID#231A (according to the sectional type iNA of iNA ID#2and#8iNA design):

Sense?strand:CCGUGUGCACUUCGCUUCAdTdTuauaGGGUUUUUCUUGUUGACAAdTdT

Ant?i-sense?strand1:UUGUCAACAAGAAAAACCCCG

Anti-sense?strand2:U?GAAGCGAAGU?GCACACGGUC

INA ID#232 (according to the sectional type iNA of iNA ID#2 and #5iNA design)

Positive-sense strand: GGGUUUUUCUUGUUGACAAdTdTuauaGGAUGUGUCUGCGGCGUUUdTdT

Ant?i-sense?strand1:UUGUCAACAAGAAAAACCCCG

Ant?i-sense?strand2:AAACGCCGCAGACACAUCCAG

INA ID#233 (according to the sectional type of iNA ID#2 and #6iNA design)

Positive-sense strand: GGGUUUUUCUUGUUGACAAdTdTuauaUCUUGUUGGUUCUUCUGGAdTdT

Antisense strand 1:UUGUCAACAAGAAAAACCCCG

Antisense strand 2:UCCAGAAGAACCAACAAGAAG

INA ID#234 (according to the sectional type iNA of iNA ID#2 and #7iNA design)

Positive-sense strand: GGGUUUUUCUUGUUGACAAdTdTuauaCGGGGCGCACCUCUCUUUAdTdT

Antisense strand 1:UUGUCAACAAGAAAAACCCCG

Antisense strand 2:UAAAGAGAGGUGCGCCCCGUG

INA ID#235 (according to the sectional type iNA of iNA ID#2 and #9iNA design)

Positive-sense strand: GGGUUUUUCUUGUUGACAAdTdTuauaGGCAGGUCCCCUAGAAGAAdTdT

Antisense strand 1:UUGUCAACAAGAAAAACCCCG

Antisense strand 2:UUCUUCUAGGGGACCUGCCUC

INA ID#236 (according to the sectional type iNA of iNA ID#2 and #11iNA design)

Positive-sense strand: GGGUUUUUCUUGUUGACAAdTdTuauaGCAUGGAGACCACCGUGAAdTdT

Antisense strand 1:UUGUCAACAAGAAAAACCCCG

Antisense strand 2:UUCACGGUGGUCUCCAUGCGA

INA ID#237 is (according to the sectional type iNA of iNA ID#5 and #8iNA design

Positive-sense strand: GGAUGUGUCUGCGGCGUUUdTdTuauaCCGUGUGCACUUCGCUUCAdTdT

Antisense strand 1:AAACGCCGCAGACACAUCCAG

Antisense strand 2:UGAAGCGAAGUGCACACGGUC

INA ID#238 (according to the sectional type iNA of iNA ID#6 and #8iNA design)

Positive-sense strand: UCUUGUUGGUUCUUCUGGAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT

Antisense strand 1:UCCAGAAGAACCAACAAGAAG

Antisense strand 2:UGAAGCGAAGUGCACACGGUC

INA ID#239 (according to the sectional type iNA of iNA ID#7 and #8iNA design)

Positive-sense strand: CGGGGCGCACCUCUCUUUAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT

Antisense strand 1:UAAAGAGAGGUGCGCCCCGUG

Antisense strand 2:UGAAGCGAAGUGCACACGGUC

INA ID#240 (according to the sectional type iNA of iNA ID#9 and #8iNA design):

Positive-sense strand: GGCAGGUCCCCUAGAAGAAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT

Antisense strand 1:UUCUUCUAGGGGACCUGCCUC

Antisense strand 2:UGAAGCGAAGUGCACACGGUC

INA ID#241 (according to the sectional type iNA of iNA ID#11 and #8iNA design):

Positive-sense strand: GCAUGGAGACCACCGUGAAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT

Antisense strand 1:UUCACGGUGGUCUCCAUGCGA

Antisense strand 2:UGAAGCGAAGUGCACACGGUC

Embodiment 2

For the structure of annular or similar annular, each iNA is as follows.Article one, chain comprises two sections polynucleotide, direction opposite (one section 5' is to another section of 3' 3' to 5'), wherein the sequence identical sequence of 5'-3' direction and target RNA.In solution, with the multi-nucleotide hybrid of two ends together, to form a ring structure.Another chain will with the strand regional complementarity of annular, also with target RNA complementation.The example of such sequence, as follows:

INA ID#242 (pressing the class annular HBV iNA of iNA ID#2 design):

Positive-sense strand: GGGCCCGGGUUUUUCUUGUUGACAAUU CCCGGG(part of line is the 3'-5' direction)

Antisense strand: UUGUCAACAAGAAAAACCCCG

INA ID#243 (pressing the class annular HBV iNA of iNA ID#5 design):

Positive-sense strand: GGGCCCGGAUGUGUCUGCGGCGUUUUU CCCGGG(part of line is the 3'-5' direction)

Antisense strand: AAACGCCGCAGACACAUCCAG

INA ID#244 (pressing the class annular HBV iNA6 of iNA ID#6 design):

Positive-sense strand: GGGCCCUCUUGUUGGUUCUUCUGGAUU CCCGGG(part of line is the 3'-5' direction)

Antisense strand: UCCAGAAGAACCAACAAGAAG

INA ID#245 (pressing the class annular HBV of iNA ID#7 design):

Positive-sense strand: GGGCCCCGGGGCGCACCUCUCUUUUU CCCGGG(part of line is the 3'-5' direction)

Antisense strand: UAAAGAGAGGUGCGCCCCGUG

INA ID#246 (pressing the class annular HBV iNA of iNA ID#8 design):

Positive-sense strand: GGGCCCCCGUGUGCACUUCGCUUCAUU CCCGGG(part of line is the 3'-5' direction).

Antisense strand: UGAAGCGAAGUGCACACGGUC

INA ID#247 (pressing the class annular HBV iNA of iNA ID#9 design):

Positive-sense strand: GGGCCCGGCAGGUCCCCUAGAAGAAUU CCCGGG(part of line is the 3'-5' direction)

Antisense strand: UUCUUCUAGGGGACCUGCCUC

INA ID#248 (pressing the class annular HBV iNA of iNA ID#11 design):

Positive-sense strand: GGGCCCGCAUGGAGACCACCGUGAAUU CCCGGG(part of line is the 3'-5' direction)

Antisense strand: UUCACGGUGGUCUCCAUGCGA

Embodiment 3

Chemically modified:

INA ID#249 (pressing the class annular HBV iNA of iNA ID#2 and #8 design)

Positive-sense strand: mGGGUUUUUCUUGUUGAmCAAdTdTuauamCCGmUGUGmCACUUCGCUUmCAdTdT

Antisense strand 1:UUGUCAACAAGAAAAACCCCG

Antisense strand 2:UGAAGCGAAGUGCACACGGUC

(" m " the preceding base refer to 2 ' methoxyl group (2 '-OMethyl) RNA base. " d " base the preceding refers to the DNA base)

INA ID#250 (according to the sectional type of iNA ID#2 and #8iNA design):

Positive-sense strand: mGGGUUUUUCUUGUUGAmCAAdTdTuauamCCGmUGUGmCACUUCGCUUmCAdTdT

Antisense strand 1:UUGUmCAAmCAAGAAAAACCCCG

Antisense strand 2:UGAAGCGAAGUGCACACGGUC

(" m " the preceding base refer to 2 ' methoxyl group (2 '-O-Methyl) RNA base. " d " base the preceding refers to the DNA base)

INA ID#250A (according to the sectional type of iNA ID#2 and #8iNA design):

Positive-sense strand: mCCGmUGUGmCACUUCGCUUmCAdTdTuauamGGGUUUUUCUUGUUGAmCAAdTdT

Antisense strand 1:UUGUmCAAmCAAGAAAAACCCCG

Antisense strand 2:UGAAGCGAAGUGCACACGGUC

(" m " the preceding base refer to 2 ' methoxyl group (2 '-O-Methyl) RNA base. " d " base the preceding refers to the DNA base)

INA ID#251 (according to the sectional type of iNA ID#2 and #8iNA design):

Positive-sense strand: mGGGUUUUUCUUGUUGAmCAAdTdTuauamCCGmUGUGmCACUUCGCUUmCAdTdT

Antisense strand 1:UUGUmCAAmCAAGAAAAACCCCG

Antisense strand 2:UGAAGCGAAGUGmCAmCACGGUC

(" m " the preceding base refer to 2 ' methoxyl group (2 '-O-Methyl) RNA base. " d " base the preceding refers to the DNA base)

INA ID#252 (according to the sectional type of iNA ID#2 and #8iNA design):

Positive-sense strand: mGGGUUUUUCUUGUUGAmCAAdTdTuauamCCGmUGUGmCACUUCGCUUmCAdTdT

Antisense strand 1:UUGUCAACAAGAAAAACCCCG

Antisense strand 2:UGAAGCGAAGUGmCAmCACGGUC

(" m " the preceding base refer to 2 ' methoxyl group (2 '-O-Methyl) RNA base. " d " be base isDNA base the preceding)

INA ID#253 (according to the sectional type of iNA ID#2iNA design):

Positive-sense strand: mGGGUUUUUCUUGUUGAmCAAdTdT

Antisense strand: UUGUmCAAmCAAGAAAAACCCCG

(" m " the preceding base refer to 2 ' methoxyl group (2 '-OMethyl) RNA base. " d " base the preceding refers to the DNA base)

INA ID#254 (according to the sectional type of iNA ID#2iNA design):

Positive-sense strand: mGGGUUUUUCUUGUUGAmCAAdTdT

Antisense strand: UUGUCAACAAGAAAAACCCCG

(" m " the preceding base refer to 2 ' methoxyl group (2 '-OMethyl) RNA base. " d " base the preceding refers to the DNA base)

INA ID#255 (according to the sectional type of iNA ID#8iNA design):

Positive-sense strand: CCGmUGUGmCACUUCGCUUmCAdTdT

Antisense strand: UGAAGCGAAGUGmCAmCACGGUC

(" m " the preceding base refer to 2 ' methoxyl group (2 '-OMethyl) RNA base. " d " base the preceding refers to the DNA base)

INA ID#256 (according to the sectional type of iNA ID#8iNA design):

Positive-sense strand: CCGmUGUGmCACUUCGCUUmCAdTdT

Antisense strand: UGAAGCGAAGUGCACACGGUC

(" m " the preceding base refer to 2 ' methoxyl group (2 '-O-Methyl) RNA base. " d " base the preceding refers to the DNA base)

Embodiment 4

For similar stem-ring structure, each iNA is that the polynucleotide of a chain can form an annular, the duplex structure of the combination by the principle of Watson-Crick by 5'-terminal nucleotide chain and 3'-terminal nucleotide.Another chain will by complementation to the ring the strand section to form double-stranded iNA.As follows:

INA ID#257 (according to the stem-ring-like iNA of iNA ID#2iNA design):

Positive-sense strand: GGGCCCGGGUUUUUCUUGUUGACAAUUGGGCCC_

Antisense strand: UUGUCAACAAGAAAAACCCCG

INA ID#258 (according to the stem-ring-like iNA of iNA ID#5iNA design):

Positive-sense strand: GGGCCCGGAUGUGUCUGCGGCGUUUUUGGGCCC_

Antisense strand: AAACGCCGCAGACACAUCCAG

INA ID#259 is (according to the stem-ring-like iNA of iNA ID#6iNA design

Positive-sense strand: GGGCCCUCUUGUUGGUUCUUCUGGAUUGGGCCC_

Antisense strand: UCCAGAAGAACCAACAAGAAG

INA ID#260 (according to the stem-ring-like iNA of iNA ID#7iNA design):

Positive-sense strand: GGGCCCCGGGGCGCACCUCUCUUUUUGGGCCC_

Antisense strand: UAAAGAGAGGUGCGCCCCGUG

INA ID#261 (according to the stem-ring-like iNA of iNA ID#8iNA design):

Positive-sense strand: GGGCCCCCGUGUGCACUUCGCUUCAUUGGGCCC_

Antisense strand: UGAAGCGAAGUGCACACGGUC

INA ID#262 (according to the stem-ring-like iNA of iNA ID#9iNA design):

Positive-sense strand: GGGCCCGGCAGGUCCCCUAGAAGAAUUGGGCCC

Antisense strand: UUCUUCUAGGGGACCUGCCUC

INA ID#263 (according to the stem-ring-like iNA of iNA ID#11iNA design):

Positive-sense strand: GGGCCCGCAUGGAGACCACCGUGAAUUGGGCCC

Antisense strand: UUCACGGUGGUCUCCAUGCGA

INA ID#264 (according to the stem-ring-like iNA of iNA ID#2iNA design):

Positive-sense strand: GGGCCCmGGGUUUUUCUUGUUGAmCAAUUGGGCCC_

Antisense strand: UUGUCAACAAGAAAAACCCCG

(" m " be 2 ' methoxyl group (2 '-O-Methyl) the RNA base that refers to of base the preceding." d " base the preceding refers to the DNA base).

INA ID#265 (according to the stem-ring-like iNA of iNA ID#8iNA design):

Positive-sense strand: GGGCCC mCCGmUGUGmCACUUCGCUUmCAUUGGGCCC_

Antisense strand: UGAAGCGAAGUGCACACGGUC

(" m " base the preceding refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " base the preceding refers to the DNA base).

INA ID#266 (according to the stem-ring-like iNA of iNA ID#8iNA design):

Positive-sense strand: GGGCCC mCCGmUGUGmCACUUCGCUUmCAUUGGGCCC_

Antisense strand: UGAAGCGAAGUGmCAmCACGGUC

(" m " base the preceding refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " base the preceding refers to the DNA base).

Scheme implementation 5: more iNAs

INA ID#267 (according to iNA ID#8HBV iNA):

Positive-sense strand: [mC] CG[mU] GUG[mC] ACUUCGCUU[mC] A[dT] [dT]

Antisense strand: UGAAGCGAAGUG[mC] A[mC] ACGGUC

(" m " base the preceding refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " base the preceding refers to the DNA base).

INA ID#268 (according to iNA ID#2HBV iNA):

Positive-sense strand: [mG] GGUUUUUCUUGUUGA[mC] AA[dT] [dT]

Antisense strand: UUGUCAACAAGAAAAACCCCG

(" m " base the preceding refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " base the preceding refers to the DNA base).

INA ID#269 (according to iNA ID#2 and #8HBV iNA):

Positive-sense strand: [mG] GGUUUUUCUUGUUGA[mC] AA[dT] [dT] UAUA[mC] CG[mU] GUG[mC] ACUUCGCUU[mC] A[dT] [dT]

Antisense strand 1:UUGUCAACAAGAAAAACCCCG

Antisense strand 2:UGAAGCGAAGUG[mC] A[mC] ACGGUC

(" m " base the preceding refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " base the preceding refers to the DNA base).

INA ID#270 (according to iNA ID#2 and #8HBV iNA):

Positive-sense strand: [mC] CG[mU] GUG[mC] ACUUCGCUU[mC] A[dT] [dT] UAUA[mG] GGUUUUUCUUGUUGA[mC] AA[dT] [dT]

Antisense strand 1:UUGUCAACAAGAAAAACCCCG

Antisense strand 2:UGAAGCGAAGUG[mC] A[mC] ACGGUC

(" m " base the preceding refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d base the preceding refers to the DNA base).

INA ID#271 (according to iNA ID#2 and #8HBV iNA):

Positive-sense strand: [mG] GGUUUUUCUUGUUGA[mC] AA[dT] [dT] NNNNN[mC] CG[mU] GUG[mC] ACUUCGCUU[mC] A[dT] [dT]

Antisense strand 1:UUGUCAACAAGAAAAACCCCG

Antisense strand 2:UGAAGCGAAGUG[mC] A[mC] ACGGUC

(" m " base the preceding refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " base the preceding refers to the DNA base.The n-quadrant of being decorated with line refers at the regional nt number of justice can not wait (1-1000), and also the RNA of chemically modified or DNA base or different connections are good for).

INA ID#272 (according to iNA ID#2 and #8HBV iNA):

Positive-sense strand: [mC] CG[mU] GUG[mC] ACUUCGCUU[mC] A[dT] [dT] NNNN[mG] GGUUUUUCUUGUUGA[mC] AA[dT] [dT]

Antisense strand 1:UUGUCAACAAGAAAAACCCCG

Antisense strand 2:UGAAGCGAAGUG[mC] A[mC] ACGGUC

(" m " base the preceding refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " base the preceding refers to the DNA base.The nt number that the n-quadrant of being decorated with line refers in the justice zone can not wait (1-1000), also RNA or the DNA base of chemically modified, or different connections is strong).

Scheme implementation example 6

The siRNA pharmaceutical formulation.

DOTMA is dissolved in the ethanol, mixes with siRNA to produce water-insoluble throw out, separate and dry sediment after, throw out is dissolved in chloroform or the similar solvent, and further mixes at chloroform with other lipids, as technology as described in (WO/2010/135207).After removing organic solvent, drying agent and 9% sucrose water hydration get final product animals administer.

Scheme implementation example 7

Experimental technique

Cell transfecting: the HepG2.2.15 cell inoculation is in 96 orifice plates, at CO ₂In (5%CO ₂), overnight incubation in 37 ℃ of incubators.Second day morning, cell covered～40% orifice plate area.Dilute 0.5 μ L siRNA(1 μ M stock solution with 10 μ L DMEM nutrient solutions).DMEM nutrient solution with 10 μ L dilutes 0.4 μ L RFect(, hundred biography biotechnologies), and at room temperature kept 5 minutes.The siRNA of mixed diluting and the RFect of dilution, vortex concussion 10 seconds, and kept at room temperature 20 minutes.Add 20 μ L transfection complex bodys in the hole that contains 80 μ LDMEM nutrient solutions.At CO ₂In (5%CO ₂), continue in 37 ℃ of incubators to cultivate.

All programs of using in the research of laboratory animal in the body are mechanism's the care of animal and the use council (IACUC) approval, and according to the locality, state and federal regulations carry out.The HBV transgenic mice is by the prescription of 0.2 milliliter of injection of tail vein injection siRNA.The tissue of results and blood are used for the analyzing gene change of Expression.

The separation of mRNA: after the transfection two days, cell is washed once with 100 μ L PBS, add 100 μ L(Turbocapture test kits then, the Qiagen corporate system) lysis buffer.The cellular products (80 μ L) of dissolving is transferred to the seizure plate of the mRNA in 96 holes, at room temperature hatches 1 hour.For mouse tissue, administration two days later, mouse is gathered the mouse liver tissue.Use the Polytron(Turbocapture test kit, the Qiagen corporate system) in lysis buffer homogenate.Shift the seizure plate of the mRNA in 80 μ L to, 96 hole then, at room temperature hatched 1 hour.With 100 μ L lavation buffer solutions washing three times, the elution buffer of 80 μ L joined in each hole then, 65 ℃ of following incubations 5 minutes.Elute soln (containing mRNA's) is transferred to a new clear plate in 96 holes.

Real-time RT-PCR: the mRNA that 3 μ L separate is used for real-time RT-PCR.The RT-PCR method adopts SYBR Green one step real-time RT-PCR test kit, and (SensiMix one goes on foot SYBR Green test kit, BIOLINE).Mix 11 μ L master mix(and contain reversed transcriptive enzyme), the forward of 1 μ L and reverse primer (6 μ M), the SYBR Green of 0.3 μ L50X and 2.7 μ L water.The temperature of reverse transcription reaction is at 42 ° of C, and after 30 minutes, 95 ° of C afterwards were used for activating the Tag polysaccharase in 15 minutes; The temperature and time of PCR circulation is 95 ° of C, 15 seconds, and 60 ° of C, 30 seconds, 72 ° of C, 20 seconds.Variation with the genetic expression of Δ Δ CT methods analyst.

The Southern blot hybridization is analyzed liver HBV DNA: liver tissue homogenate is in lysate.Hepatic tissue (about 0.1 gram) is containing lysis buffer (EDTA of 1mM, the Tris of 10mM, among the 10mM NaCl, 0.5%SDS, Proteinase K) with grinding pestle homogenate.In order to extract DNA, homogenate was hatched under 55 ℃ 12 hours, then, add isopyknic phenol (Phenol).With sample mix, and with 12,000xg centrifugal 10 minutes., then chloroform is added supernatant liquor, and recentrifuge.Go out DNA with NaCl and ethanol sedimentation then.The DNA precipitation that is settled out is dissolved in the TE damping fluid (pH8.0) that contains ribonuclease A. the DNA of some amount (containing 40 micrograms of DNA usually), (New England Biolabs company, MA) being proved to be the HBV gene order in 3 hours 37 ° of C digestion is not contain HindIII inscribe site with the HindIII digestive ferment.The DNA of digestion, extraction again, and by the separation of 1%TAE agarose gel electrophoresis.DNA is transferred to the positively charged nylon membrane of BioDyne B then.DNA is fixed on the film after the UV irradiation.The hybridization use [ ³²P] the CTP-mark, probe made with the HBV genome of being cloned into the pBluescript plasmid of Hae III digestion.Hybridization is containing 10%PEG-8000, the 0.05M sodium phosphate, and 0.33 mg/ml salmon sperm dna spends the night under 60 ℃ in the solution of 7% SDS and carries out.Use phosphorus formation method (Optiquant) is measured radiated signal and is measured the density of radioactive bands.

Viral DNA is to determine with respect to the ratio of transgenosis DNA band with the viral DNA band with respect to the amount of host mouse DNA.Calculating is that the cell according to each host mouse contains 1.3 parts HBV transgenosis and carries out.

Sxemiquantitative pcr analysis liver HBV DNA:PCR method: 95 ℃ following 2 minutes, 40 circulations then: 95 ℃ following 10 seconds, following 30 seconds at 60 ℃.Use the method for typical curve to measure.

Blood plasma HBeAg and HBsAg analyze: with explanation (international immunodiagnosis, Foster City, CA) mensuration of ELISA method according to the manufacturer.

Claims (16)

1. be used for suppressing the iNA that copies or breed of hepatitis B virus, comprise one or more positive-sense strands and one or more antisense strand, one or more regional complementarities of one or more polynucleotide chain and HBV RNA.

2. the iNA described in claim 1 is characterized in that, described iNA is continuous two strands or discontinuity duplex structure, and this continuous two strands or discontinuity duplex structure are the structure of straight chain, annular, class annular, hair fastener type, stem-ring, unidirectional or two-way shape.

3. the iNA described in claim 2 is characterized in that, the double-stranded iNAs of discontinuity has two or many polynucleotide, and one or more breach or otch are arranged, and forms the double stranded region of one or more complementations between breach or otch.

4. the iNA described in claim 2 is characterized in that, long 10～200 Nucleotide of double-stranded iNA are 15～50 Nucleotide preferably, are more preferably 19-29 Nucleotide.

5. the iNA described in claim 2 is characterized in that, the polynucleotide chain of double-stranded complementary region is no less than 10 Nucleotide, is more preferably 19-29 Nucleotide.

6. the iNA described in claim 2 is characterized in that, at least 50%, 70%, and 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%iNA, 99%, or any double-stranded region of the Nucleotide in 100% the chain is complementary to another chain.

7. the iNA described in claim 2 is characterized in that, double-stranded iNA or siRNA have cohesive end or blunt end, and wherein, cohesive end is positioned on one or two chains, the terminal outstanding long 1-5 of a base nucleosides.

8. the iNA described in claim 1 is characterized in that, comprises the antisense strand of a long positive-sense strand and two weak points, two different positions complementations of antisense strand and HBV RNA.

9. the iNA described in claim 1 is characterized in that, comprises a long antisense strand, and antisense strand comprises the antisense fragment with two different positions complementations of HBV RNA; With the positive-sense strand of two weak points, the sequence of two different positionss of the sequence of two positive-sense strands and HBV RNA is the same or approaching.

10. iNA as claimed in claim 8 or 9, it is characterized in that: the length of long-chain is 15 to 80 Nucleotide, is preferably 19～50 Nucleotide; Every long 10 to 40 Nucleotide of two short chains are preferably 19 to 27 Nucleotide.

11. iNA as claimed in claim 1 is characterized in that: have the arbitrary structure among the following structure 1-31:

12. the iNA described in claim 1 is characterized in that, comprises any one or its combination among the iNA ID No.1-272, or any one or its combination among iNA ID NO.1-11 and the 231-272.

13. the iNA described in arbitrary claim among the claim 1-12 is characterized in that: one or more Yeast Nucleic Acid with chemically modified or thymus nucleic acid replace.

14. the iNA described in arbitrary claim among the claim 1-12 is characterized in that: include the Nucleotide that thiophosphatephosphorothioate or 2'-modify.

15. the purposes of the iNA described in the arbitrary claim of claim 1-14, it is used in mammalian cell inhibition HBV genetic expression and copies.

16. a formula of medicine, comprise in the claim 1 to 14 in arbitrary claim iNA and medicine carrier.

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