Accompanying drawing explanation
Fig. 1, for suppressing siRNA or the iNA structural representation of HBV gene.
Fig. 2, on HepG2.2.15 cell, screen the example of efficient HBV iNA.By iNA RFect transfection iNAs(5nM) to HepG2.2.15 cell.Two days later, collect and smudge cells, with the change of real-time quantitative RT-PCR methods analyst genetic expression.
Fig. 3, on HepG2.2.15 cell, screen the example of efficient HBV iNA.The iNAs(5nM of HBV RNA two different loci can be acted on RFect transfection) to HepG2.2.15 cell.Two days later, collect and smudge cells, with the change of real-time RT-PCR methods analyst genetic expression.
Fig. 4, iNA suppress the example that copy of HBV in HBV transgenic animal body.The antiviral iNA (siRNA) of a HBV Transgenic Mice intravenous injection transmission system parcel, suppress HBV virogene copying in liver cell specifically, and the ApoB siRNA of control group does not affect on copying of HBV.
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 HBV transgenic mice liver.HBV transgenic mice injects iNA totally three times once in a week through tail vein.The HBV transgenic mice oral administration once a day that adefovir ester (ADV) is organized, continuous 14 days (10mg/kg/ days).Administration got tissue and blood after three days the last time.A) change of HBV DNA in Southern Blot Hybridization Analysis transgenic mice liver; B) change of HBV DNA in pcr analysis transgenic mice liver.* *, P<0.001(use the multiple comparative test 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.HBV transgenic mice injects iNA totally three times once in a week through tail vein.HBV transgenic mice adefovir ester (Adefovir dipivoxi) the l oral administration 14 days (10mg/kg/ days) once a day that adefovir ester (ADV) is organized.Administration was drawn materials after three days the last time.A) to before thing, B) to thing after 17 days.* *, P<0.001(use the multiple comparative test 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.HBV transgenic mice injects the iNA totally three times of nano colloidal particles transmission system parcel once in a week through tail vein.The HBV transgenic mice adefovir ester oral administration 14 days (10mg/kg/ days) once a day of ADV group.Administration was drawn materials after three days the last time.* *, P<0.001(use the multiple comparative test 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 HBV transgenic mice liver.HBV transgenic mice injects iNA totally three times once in a week through tail vein.The HBV transgenic mice adefovir ester oral administration 14 days (10mg/kg/ days) once a day of ADV group.Administration was drawn materials after three days the last time.* *, P<0.001(use the multiple comparative test of One-way ANOVA and Bonferroni method).
Fig. 9, HBV1(iNA numbering 267), HBV2(iNA numbering 269) and the iNA vein of APO-B to the chemico-analytic example of blood plasma after HBV transgenic mice medicine.HBV transgenic mice injects iNA totally three times once in a week through tail vein.The HBV transgenic mice adefovir ester oral administration 14 days (10mg/kg/ days) once a day of ADV group.Administration was drawn materials after three days the last time.*, P<0.05(uses the multiple comparative test of One-way ANOVA and Bonferroni method).
Definition
In the present invention, the definition of technical term used is understood to include these terms and those implications known to the skilled in the art, and, be not intended to limit the scope of the invention, do not need the statement repeated each time.
Term used herein " one ", " one ", " this " and similar term in description the present invention, and in the claims, will be interpreted as comprising odd number and plural number.Term " comprises "; " have "; " contain " and will open-ended term be interpreted as.Being meant to of they, such as, " include, but are not limited to ".
Scope that is that apply or set(ting)value refers to each independent value within the scope of this, should be equal to the description of any independent value.Here adopted occurrence, will be understood to exemplary, instead of limit the scope of the invention.
Interfere RNA (iNA) refers to the nucleic acid double chain with chain complementation each other as the term is employed herein.After entering RISC mixture, the RNAi mechanism of induction RNA enzyme liberating RNA.In addition, iNA is by the RNAa mechanism of promotor, and what regulate target gene to express increases.In general, each chain of iNA is all Nucleotide, mainly 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 completely, and another chain is RNA, as long as by the degraded of RNAi mechanism induction cognate rna, all can be used as the structure of iNA.
Term as used herein " two-way double-strand iNA " or " two-way double-strand siRNA " or " two-way iNA " be one through generic term of the present invention, comprise the bi-directional configuration of the same chain of interfere RNA (iNAs), it can be cut and form iNA or siRNA in cell.Double-strand at least one chain of two-way iNA has the fragment in one or more 5' to 3' direction and the fragment in one or more 3' to 5' direction.Another chain of iNA there is one or more part complementary with it, to form one or more double-strand section.Between each double-strand section, can jagged or otch.Two-way iNA can be linear, stem-annular, or the configuration such as circular.The iNA of terminal structure may be blunt or viscosity (outstanding or pendency) end, and its functional objective is that RNA is reticent.The end construction of viscosity (giving prominence to) end has more than and is limited to 3' protuberance, may also be the structure that 5' is outstanding, as long as it to induce RNAi effect.In addition, the nucleotide number of pendency is not limited to report 2 or 3, but can be any number, as long as can induce RNAi effect.Such as, may be 1 to 8 or longer, or 2 to 4 bases or longer.
As used herein, term " the iNA double-strand of annular or similar annular " is the generic term of the present invention, and comprise the structural framing that interfere RNA (iNAs) has annular or class annular etc., it can be cut and form iNA or siRNA in cell.The iNA double-strand of annular of the present invention or class annular also comprises expression vector (also referred to as iNA expression vector) and can produce iNA double-strand or record the iNA being formed and/or generate after transcribing at transit cell, and induces RNAi in vivo.Positive-sense strand or antisense strand can have one or more otch or breach.INA end can be viscosity (giving prominence to) or blunt, as long as its function can make target RNA reticent.The end construction of viscosity (giving prominence to) has more than and is limited to 3' protuberance, also can be the structure that 5' is outstanding, as long as it to induce RNAi effect.In addition, the nucleotide number of pendency is not limited to report 2 or 3, can be any number, as long as can induce RNAi effect.Such as, pendency may be 1 to 8 base or longer, or 2 to 4 bases.
Loop-stem structure refers to base pairing in a polynucleotide molecule, can occur in single stranded DNA, more commonly RNA.Double-stranded circular structure refers to the strand position being complementary to ring district by single or multiple chain, the intersegmental jagged or otch of sheet.In addition, any section on stem also can have the complementary strand of one or more single-stranded regions.End structure may be blunt or viscosity (giving prominence to).One or more chain can be had 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 as used herein refers to the quantity of the Nucleotide in two-way iNA, from the 1st base pair of the 5' end of positive-sense strand, ends at last base of 3' end of positive-sense strand.
On genetics, microRNA (miRNA) is single stranded RNA, and length is about 21-23 Nucleotide, the expression of regulatory gene.MiRNA transcribes from DNA, but does not translate into protein (non-coding RNA), and they generate premiRNA by the short loop-stem structure of the PRimiRNA being called as primary transcription, finally generates miRNA from premiRNA.Ripe miRNA molecule partial complementarity is in one or more messenger RNA molecule, and their major function reduces the expression of gene.
The Nucleotide of iNA molecular modification can at any chain.Such as, modified Nucleotide, can have the conformation of a Northern (such as, in the Northern pseudorotation cycle, seeing Sanger, the principle of nucleic acid construct, SpringerVerlaged., 1984).The example that Nucleotide has Northern configuration comprises the Nucleotide of lock nucleic acid (LNA) (as 2'-O, 4'-C-methylene radical-(D-RIBOSE base) 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 (2'-azido nucleotides) of 2'-azido-, 2'-O-methyl nucleotide (2'-Omethyl nucleotides).The Nucleotide of the ability simultaneously keeping induction RNAi and the chemically modified that nuclease degradation can be resisted.But conjugated molecule is connected to a ligand for the iNA molecule polyoxyethylene glycol of chemically modified, human serum albumin or a cell receptor, can mediated cell picked-up.The example that can be connected to chemically modified iNA molecule and the specific conjugated molecule contemplated by the present invention describes, visible Vargeese, Deng the United States Patent (USP) of people, publication number is 20030130186 and U.S. Patent Publication No. 20040110296, and its every part all can be incorporated in the present invention.
Have several examples in the present invention, describe sugar, alkali and phosphatic modification, can be incorporated into nucleic acid molecule, strengthens stability and the validity of its nuclease.Such as, oligonucleotide is modified with enhanced stability and/or is strengthened biological activity, and by modifying opposing nuclease, such as, 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.The methods and strategies that these general Study describe, in order to modify sugar, base and/or phosphoric acid.Similar modification may be used for iNA double chain acid molecule of the present invention, as long as the promoting intracellular RNAi instead of show the function suppressing its RNAi of iNA.
INA double-strand may comprise the iNA molecule that phosphate backbones is modified, and comprising: one or more thiophosphatephosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino base, amidation t-butyl carbamate, carboxymethyl, acetamidate, polymeric amide, sulphonate, sulphonamide, sulfamate, formacetal, thioformacetal, aIkylsilyl groups.The discussion that oligonucleotide backbone is modified is see, 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 the connection between thiophosphatephosphorothioate nucleotide, 2'-deoxyribonucleotide, the 2'-O-methyl of ribonucleotide, 2'-deoxidation-2'-fluorine ribonucleotide, " universal base " Nucleotide, " acyclic " Nucleotide, 5-C-methyl nucleotide, terminal glyceryl ester and/or be inverted deoxidation and take off base residue.The antisense district of an iNA molecule can be included in the connection between a thiophosphatephosphorothioate nucleosides of the 3'-end in described antisense district.Antisense district can comprise the connection between 1-5 5'-terminal thiophosphate ester nucleosides.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 ribonucleotide.The Nucleotide of 3'-end can comprise one or more acyclic nucleotide.Such as, the iNA of chemically modified can have 1,2,3,4,5,6,7,8, or more thiophosphatephosphorothioate nucleosides between connect.Can have in every bar chain between the thiophosphatephosphorothioate nucleosides of 1 to 8 or more and connect.Between thiophosphatephosphorothioate nucleosides, contact can be the two-way iNA binary being present in one or two oligonucleotide chain, such as, can at sense strand, antisense strand or two chains.In certain embodiments, iNA molecule comprises 1,2,3,4,5,6,7,8,9,10, or more purine thionucleoside between sense strand, in antisense strand or in two chains.
Can by the synthesis condition of chemically modified iNA molecule: (1) synthesis at least similar oligonucleotide molecules of two or more RNA or RNA-and complementary strand; (2) under the condition be applicable to, two or more complementary strands are annealed together, obtain iNA molecule.In certain embodiments, the synthesis of the complementary portion of two-way iNA molecule is synthesized by solid phase oligonucleotide, or the oligonucleotide synthesis of being connected by solid phase.
Oligonucleotide (such as, some oligonucleotide modified or part lack the oligonucleotide of ribonucleotide) can use technology known in the art, such as Caruthers, 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, is shown in 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.Two or more complementary strands any or hairpin RNA or annealed by the annular of different complementations or similar annular iNA chain, can form the duplex structure of complementary iNA.
" overlap " (overlapping) refers to that such as, multiple Nucleotide (nt) wherein may less to 2-5 Nucleotide as many as 5-10 Nucleotide or more when two iNA fragments have overlapping sequences.
The iNA that " one or more iNA " refer to, has different primary series each other.
" target site " or " target sequence (target) " or " target sequence (targeted) " refer to the sequence (such as RNA) in target nucleic acid, are the sequences of the siRNA degraded mediated by iNA antisense strand sequence.
Space refers to not connect or jagged or otch between two nucleotide phosphodiesterase diester linkages in 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 that target RNA combines.Contained the complementary portion of hybridization by the mixing iNA of DNA and the RNA chain creation of hybridization, a 3' viscosity end can be had at least.
In order to " expression of regulatory gene " increase used here or reduce the expression of target gene, it can comprise in cell and increases or reduce rna level, or the translation of RNA, or the synthesis of the albumen of being encoded by it or protein protomer.
Term " suppression ", " reduction " or " expression of minimizing " is meant to the expression of gene here, or level, to encode the RNA molecule of one or more protein or protein subunit, or one or more albumen in the level of the RNA molecule of equivalence or activity or the protein protomer coded by target gene, be reduced to the level before lower than application iNA.
" gene silencing " used herein refers to the expression at intracellular portion or complete suppressor gene, and also can be called as " gene knockout ".The degree of gene silencing, also can be determined by method as known in the art, some of them are summarized in international publication number WO99/32619.
In certain embodiments, iNA molecule comprises justice and antisense sequences or section, it is characterized in that justice is connected with covalent linkage with the Nucleotide in antisense district or non-nucleotide, or interacted by non covalent bond, ionic linkage interaction, hydrogen bond, Van der Waals, hydrophobic interaction and/or accumulation (stacking) interact.
INAs can be assembled into a double-strand by two independent oligonucleotide, wherein chain be positive-sense strand and another be antisense strand, wherein, described antisense and positive-sense strand self-complementary (nucleotide sequence of the base namely contained by each chain is mended mutually with the sequence of another chain, as antisense strand wherein and sense strand define a two-phase or duplex structure).The base sequence of antisense strand can with the nucleotide sequence complementary of a target nucleic acid molecule or its part, the nucleotide sequence of sense strand can in target nucleic acid sequence or its part identical.INA can from single oligonucleotide, and wherein the justice of the self-complementary of two-way iNA can be connected by the group of the base of nucleic acid or non-nucleic acid with antisense region.
INA may contain Nucleotide, non-nucleotide, or the Nucleotide/non-nucleotide linking group of mixing, the justice of iNA and antisense section is linked together.In certain embodiments, the connection portion of Nucleotide can be the length of 3,4,5,6,7,8,9 or 10 Nucleotide.In certain embodiments, Nucleotide link can be a kind of aptamer.As used herein, term " aptamers " or " aptamer " comprise the nucleic acid molecule of specific binding target molecule, it is characterized in that, described nucleic acid molecule, comprise a sequence, and this sequence is the target molecule in its natural surroundings.Fit can be attached to target molecule, and wherein said target molecule is not the molecule that natural nucleic acid combines.Such as, aptamers may be used for the active centre being tied to a kind of protein, prevents from thus interacting with the naturally occurring ligand of protein.See 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 the Nucleotide of a dealkalize base, polyethers, polyamine, polymeric amide, poly-peptide, carbohydrate, lipid, polyhydrocarbon or other polymkeric substance are (such as, polyoxyethylene glycol is ethylene glycol between 2 and 100 as those have unit number).Concrete example is shown in 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 & 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.A " non-nucleotide connection " refers to the unit of the one or more Nucleotide in the nucleic acid chains that can be included into, and comprises group or the compound of arbitrary sugar and/or phosphoric acid salt replacement, and allows residue base to show their enzymatic activity.Group or compound can be dealkalize bases, because it is containing a kind of conventional nucleotide base of generally acknowledging, as adenosine, guanine, cytosine(Cyt), uridylic or thymus pyrimidine, also as in the C1 position of sugar.
Term used herein " biodegradable connection ", refers to the connection molecule of a kind of nucleic acid or non-nucleic acid, is designed to another molecule of biodegradable connection.The design of biodegradable connection considers that its stability for specific end use, such as, can be delivered to specifically tissue or a cell type.The stability of the biodegradable connection molecule of nucleic acid can be different or adjustable, such as, by the ribonucleotide of combination, the Nucleotide of deoxyribonucleotide and chemically modified, as 2'-O-methyl, 2'-fluoro, 2'-is amino, 2'-O-is 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 an or more Nucleotide, or can comprise a phosphorous base and connect.Such as, phosphoric acid ester or phosphodiester bond.The nucleic acid of biological degradability connects molecule and 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-enzymatic, be attached to RNA, DNA or PNA(protein nucleic acid of target RNA, see Egholm et al., 1993Nature365,566) molecule, and change target RNA activity (see discussion: Steinand Cheng, 1993Science261,1004and Woolf et al., U.S.Pat.No.5,849-902).Under normal circumstances, antisense sequences be one single continuously and the sequence of target sequence complete complementary.But in certain embodiments, antisense molecule can be incorporated into substrate, the antisense molecule itself making target nucleic acid molecule define a ring and/or combination forms a ring.Therefore, antisense molecule can the part of, antisense molecule discontinuous sector sequence complementary with the sector sequence of two (or even more) discrete substrate (RNA), can be complementary to two sections of target sequence.In addition, antisense DNA may 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, cracking target RNA.Antisense DNA can chemosynthesis, or also can by the expression vector of single stranded DNA that uses or etc. expression." sense-rna " has complementary target gene RNA sequence, RNA can be induced to disturb by being attached to target gene RNA.Sense-rna has the complementary sequence in conjunction with target gene RNA, induces RNA interference by the RNA being attached to target gene.Justice " Yeast Nucleic Acid " has the sequence of complementary anti-sense RNA, anneals form iNA with the sense-rna of its complementation.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 containing 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 properties as benchmark, and similar fashion is by metabolism.The example of this analogue includes, 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 the molecule comprising at least one ribonucleotide residues." ribonucleotide " refers to that the 2' position of the β D-ribose furanose part of Nucleotide is an oh group.This term comprises the RNA(such as partially purified RNA of double-stranded RNA, single stranded RNA, separation).The RNA of substantially pure RNA, synthesis RNA, generation of recombinating, and pass through interpolation, disappearance, replacement difference from naturally occurring RNA and/or change the RNA of one or more Nucleotide change.Such change can comprise adds non-nucleotide material to the end of iNA or inside.Such as one or more Nucleotide.Nucleotide in RNA molecule in invention can also comprise non-standard nucleotide, the Nucleotide of the Nucleotide that such as non-natural exists or chemosynthesis or deoxynucleotide.These RNA changed can be described as the analogue of naturally occurring RNA.As used herein, term " Yeast Nucleic Acid " and " RNA " refer to the molecule containing at least one ribonucleotide residues.Ribonucleotide is the OH nucleotides of the 2' position containing β-D-nuclear benzofuran sugar yl part.These terms comprise the RNA(such as partially purified RNA of double-stranded RNA, single stranded RNA, separation), substantially pure RNA, synthesis RNA, the RNA that restructuring produces, and being different from naturally occurring RNA by adding, disappearance RNA modifies and changes, substitutes, revises and/or change one or more Nucleotide.The change of RNA can comprise adds non-nucleotide to the end of iNA or inside.Such as, Nucleotide in RNA molecule comprises one or more non-standard nucleotide, the Nucleotide of the Nucleotide that such as non-natural exists or chemosynthesis or deoxynucleotide.These RNA changed can be described as analogue.
Term " non-nucleotide " used refers to any group or compound, and this compound can be included into the unit of the one or more Nucleotide in nucleic acid chains, comprises arbitrary sugar and/or phosphoric acid salt replacement, replaces and allow residue sequence to show their enzymic activity.Group or compound are 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 1'-position.
" Nucleotide " used herein is admitted in the art, comprises natural alkali (standard) base, and the modified base be known in the art.This base analog is generally positioned at the position of nucleotide sugar 1'.Nucleotide generally comprises alkali, sugar and phosphate group.Nucleotide can at sugar, and phosphoric acid salt and/or base portion are modified (also can referred to as the non-standard nucleotide of interchangeable nucleotide analog, modified Nucleotide, non-natural nucleotide, other unmodifieds or modification.See, Usman and McSwiggen, supra; Eckstein, et al., International PCT Publication No.WO92/07065; Usman, etal, International PCT Publication No.WO93/15187; Uhlman & Peyman, supra, be allly incorporated herein herein as a reference).Nucleic acids research can with reference to Limbach, et al, Nucleic Acids Res.22:2183 as summary, and 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, 3-6-Methyl Uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines(such as, 5-methylcytidine), 5-alkyluridines(such as, ribothymidine), 5-halouridine(such as, 5-broxuridine) or 6-azapyrimidines or 6-alkylpyrimidines(such as 6-methyluridine), propine and other group (Burgin, et al., Biochemistry35:14090, 1996, Uhlman & Peyman, supra).What is called " modified base " in this respect refers to beyond the position VITAMIN B4 of 1', guanine, cytosine(Cyt) and uracil nucleotide base or equivalent.
The nucleotide base of base complementrity refers to that form the base complementrity of hydrogen bond.VITAMIN B4 (A) and the uridylic (U) in thymus pyrimidine (T) or RNA, guanine (G) and cytosine(Cyt) (C).The complementary segment of nucleic acid or chain (being added with hydrogen bond) 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-strand iNA molecule can have end cap, as one is inverted deoxyabasic group, can at the 3'-end of positive-sense strand, and 5'-end, or two ends.
So-called " cap structure " refers to the chemically modified (see Adamic, et al, U.S.Pat.No.5,998,203, be hereby incorporated by) at the arbitrary end of oligonucleotide.These terminals are modified and nucleic acid molecule can be protected not degraded by excision enzyme, can help to enter in cell.Can be 5'-end (5'-cap) or 3'-end (3'-cap) also may be present in two ends.5'-cap, in non-limiting example, includes, but are not limited to glyceryl ester, inverted deoxidation takes off base residue, the acid of 4', 5'-methylene nucleoside, the red furanose of 1-(β-D-) Nucleotide, 4'-thio nucleotides, homocyclic nucleus thuja acid, 1,5-anhydrohexitol Nucleotide, L-Nucleotide, α-Nucleotide, the Nucleotide of base modification, phosphorothioate bond, Su Shi-pentofuranosyl nucleosides acid (threo-pentofuranosyl nucleotide), acyclic 3', 4'-plug section's Nucleotide (acyclic3', 4'-seconucleotide), 3,4-acyclic dihydroxyl butyl Nucleotide (acyclic3,4-dihydroxybutyl nucleotide), acyclic 3,5-dihydroxypentyl Nucleotide (acyclic3,5-dihydroxypentyl nucleotide), the base portion (3'-3'-inverted nucleotide moiety) that 3'-3'-falls, the 3'-3'-base section of falling dealkalize) (3'-3'-inverted abasic moiety), 3'-2'-inverted nucleotide part (3'-2'-inverted nucleotidemoiety), 3'-2'-is inverted the phosphoric acid salt of dealkalize base section, BDO, the methyl-phosphorous acid ester group stillben of 3'-phosphoric acid ester (Isosorbide-5-Nitrae-butanediolphosphate), hexylphosphate, Aminohexyl phosphoric acid ester (aminohexyl phosphate), 3'-phosphoric acid, 3'-thiophosphatephosphorothioate, phosphorodithioate (phosphorodithioate) or bridge joint or non-bridge joint and pyrene (bridging or non-bridging methylphosphonate moiety stillben andpyrene).
The embodiment of 3'-cap comprises, but be not limited to glyceryl ester, inverted deoxidation takes off base residue (part), 4', 5'-methylene nucleoside acid (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), 6-Aminohexyl phosphoric acid ester (6-aminohexyl phosphate), 1,2-aminododecane base phosphoric acid hydroxypropyl phosphorate (1,2-aminododecylphosphate), 1,5-anhydrohexitol Nucleotide, L-Nucleotide (Lnucleotide), α-Nucleotide (alpha-nucleotide), the Nucleotide (modified base nucleotide) modified, thiophosphatephosphorothioate (phosphorodithioate), Su Shi-pentofuranosyl nucleosides acid (threopentofuranosyl nucleotide), acyclic 3', 4'-plug section Nucleotide (acyclic3', 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), BDO phosphoric acid ester (Isosorbide-5-Nitrae-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 containing ring-like section, the antisense strand that can be made up of Nucleotide or non-nucleotide, with a positive-sense strand, wherein, positive-sense strand contains the Nucleotide more less than antisense strand, but be enough to be complementary to antisense strand section, form a double-strand containing ring type structure.
" asymmetric double-strand " used herein refers to two independent chains, includes the iNA molecule of positive-sense strand and antisense strand.Wherein, positive-sense strand contains the Nucleotide more less than antisense strand, but is enough to be complementary to antisense strand section, forms a double-strand.
INA can pass through chemosynthesis or biological production.The method of chemosynthesis, can at Nucleic Acids Research, 624-627,1999and Nucleic Acids Research, and 3547-3553,1955 find respectively.Molecule clone technology may be used for biological production.
Term siRNA (siRNA) is also sometimes referred to as short interfering rna or reticent RNA, is used to refer to that double stranded rna molecule has the length of 16-29 Nucleotide, can play various biological effect.It should be noted that most that siRNA is by RNA interference channel, a specific genetic expression can be disturbed.In addition, siRNA acts on RNAi correlation function, and such as, the formation of Antiviral Mechanism or chromatin Structure, the approach of these complexity is illustrated until now.
The phenomenon of the double stranded RNA kinase genetic expression that term RNA a refers to.This phenomenon is called as " tiny RNA mediation gene activation " or RNAa.The promotor of double-stranded RNA targeting gene, the genetic transcription of inducing potent.Recently, RNAa, other Mammalss several, comprises non-human primates, mouse, rat is proved to be.
" RNA disturbs the process of the gene silencing of the dependenc RNA referred to term used herein, is the silencing complex of being induced by double-stranded RNA in cell, and they are combined with the biocatalytic unit ARGONAUTE of RISC there.When double-stranded RNA or the similar iNA of RNA-are external (coming the viral rna gene group of self-infection or iNA or siRNA from transfection), RNA or iNA is imported directly into tenuigenin, cuts into short fragment (siRNA) by Dicer enzyme.DsRNA can be that endogenic (coming from cell) is if front microRNA is from the genomic expression of RNA encoding gene.The primary transcript of gene is first processed, with the pre-miRNA of the stem-ring structure of the feature formed in nucleus, is then exported in tenuigenin, is cut by Dicer.Therefore, there is the approach of two dsRNA, exogenous and endogenous.The activeconstituents of the silencing complex (RISC) of RNA-induction, endonuclease is called as Argonaute albumen, unties the double-strand siRNA combined with them, the RNA of degraded and the complementation of siRNA antisense strand.
Double-stranded RNA, after being cut, only has drag chain (antisense strand) in conjunction with Argonaute albumen and causes gene silencing by Dicer.Positive-sense strand is degraded after RISC activates.
Scheme implementation 1
The iNA of segmentation structure comprises the antisense strand for two different HBV RNA sites.Several Nucleotide not having complementation is had in the middle of continuous print chain, such as:
No. 231, iNA sequence (iNA ID#2and#8):
Positive-sense strand: GGGUUUUUCUUGUUGACAAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT
Antisense strand 1:UUGUCAACAAGAAAAACCCCG
Antisense strand 2:UGAAGCGAAGUGCACACGGUC
INA ID#231A (the sectional type iNA designed according to iNA ID#2and#8iNA):
Sense strand:CCGUGUGCACUUCGCUUCAdTdTuauaGGGUUUUUCUUGUUGACAAdTdT
Ant i-sense strand1:UUGUCAACAAGAAAAACCCCG
Anti-sense strand2:U GAAGCGAAGU GCACACGGUC
INA ID#232 (the sectional type iNA designed according to iNA ID#2 and #5iNA)
Positive-sense strand: GGGUUUUUCUUGUUGACAAdTdTuauaGGAUGUGUCUGCGGCGUUUdTdT
Ant i-sense strand1:UUGUCAACAAGAAAAACCCCG
Ant i-sense strand2:AAACGCCGCAGACACAUCCAG
INA ID#233 (sectional type designed according to iNA ID#2 and #6iNA)
Positive-sense strand: GGGUUUUUCUUGUUGACAAdTdTuauaUCUUGUUGGUUCUUCUGGAdTdT
Antisense strand 1:UUGUCAACAAGAAAAACCCCG
Antisense strand 2:UCCAGAAGAACCAACAAGAAG
INA ID#234 (the sectional type iNA designed according to iNA ID#2 and #7iNA)
Positive-sense strand: GGGUUUUUCUUGUUGACAAdTdTuauaCGGGGCGCACCUCUCUUUAdTdT
Antisense strand 1:UUGUCAACAAGAAAAACCCCG
Antisense strand 2:UAAAGAGAGGUGCGCCCCGUG
INA ID#235 (the sectional type iNA designed according to iNA ID#2 and #9iNA)
Positive-sense strand: GGGUUUUUCUUGUUGACAAdTdTuauaGGCAGGUCCCCUAGAAGAAdTdT
Antisense strand 1:UUGUCAACAAGAAAAACCCCG
Antisense strand 2:UUCUUCUAGGGGACCUGCCUC
INA ID#236 (the sectional type iNA designed according to iNA ID#2 and #11iNA)
Positive-sense strand: GGGUUUUUCUUGUUGACAAdTdTuauaGCAUGGAGACCACCGUGAAdTdT
Antisense strand 1:UUGUCAACAAGAAAAACCCCG
Antisense strand 2:UUCACGGUGGUCUCCAUGCGA
INA ID#237 (the sectional type iNA designed according to iNA ID#5 and #8iNA
Positive-sense strand: GGAUGUGUCUGCGGCGUUUdTdTuauaCCGUGUGCACUUCGCUUCAdTdT
Antisense strand 1:AAACGCCGCAGACACAUCCAG
Antisense strand 2:UGAAGCGAAGUGCACACGGUC
INA ID#238 (the sectional type iNA designed according to iNA ID#6 and #8iNA)
Positive-sense strand: UCUUGUUGGUUCUUCUGGAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT
Antisense strand 1:UCCAGAAGAACCAACAAGAAG
Antisense strand 2:UGAAGCGAAGUGCACACGGUC
INA ID#239 (the sectional type iNA designed according to iNA ID#7 and #8iNA)
Positive-sense strand: CGGGGCGCACCUCUCUUUAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT
Antisense strand 1:UAAAGAGAGGUGCGCCCCGUG
Antisense strand 2:UGAAGCGAAGUGCACACGGUC
INA ID#240 (the sectional type iNA designed according to iNA ID#9 and #8iNA):
Positive-sense strand: GGCAGGUCCCCUAGAAGAAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT
Antisense strand 1:UUCUUCUAGGGGACCUGCCUC
Antisense strand 2:UGAAGCGAAGUGCACACGGUC
INA ID#241 (the sectional type iNA designed according to iNA ID#11 and #8iNA):
Positive-sense strand: GCAUGGAGACCACCGUGAAdTdTuauaCCGUGUGCACUUCGCUUCAdTdT
Antisense strand 1:UUCACGGUGGUCUCCAUGCGA
Antisense strand 2:UGAAGCGAAGUGCACACGGUC
Embodiment 2
For structure that is annular or similar annular, each iNA is as follows.Article one, chain comprises two sections of polynucleotide, direction contrary (one section of 5' to 3' another section of 3' to 5'), the wherein sequence identical sequence of 5'-3' direction and target RNA.In the solution, by the multi-nucleotide hybrid of two ends together, to form a ring structure.Another chain is by complementary with the single-stranded regions of annular, also complementary with target RNA.The example of such sequence, as follows:
INA ID#242 (the class annular HBV iNA designed by iNA ID#2):
Positive-sense strand: GGGCCCGGGUUUUUCUUGUUGACAAUUcCCGGG(part of line is 3'-5' direction)
Antisense strand: UUGUCAACAAGAAAAACCCCG
INA ID#243 (the class annular HBV iNA designed by iNA ID#5):
Positive-sense strand: GGGCCCGGAUGUGUCUGCGGCGUUUUUcCCGGG(part of line is 3'-5' direction)
Antisense strand: AAACGCCGCAGACACAUCCAG
INA ID#244 (the class annular HBV iNA6 designed by iNA ID#6):
Positive-sense strand: GGGCCCUCUUGUUGGUUCUUCUGGAUUcCCGGG(part of line is 3'-5' direction)
Antisense strand: UCCAGAAGAACCAACAAGAAG
INA ID#245 (the class annular HBV designed by iNA ID#7):
Positive-sense strand: GGGCCCCGGGGCGCACCUCUCUUUUUcCCGGG(part of line is 3'-5' direction)
Antisense strand: UAAAGAGAGGUGCGCCCCGUG
INA ID#246 (the class annular HBV iNA designed by iNA ID#8):
Positive-sense strand: GGGCCCCCGUGUGCACUUCGCUUCAUUcCCGGG(part of line is 3'-5' direction).
Antisense strand: UGAAGCGAAGUGCACACGGUC
INA ID#247 (the class annular HBV iNA designed by iNA ID#9):
Positive-sense strand: GGGCCCGGCAGGUCCCCUAGAAGAAUUcCCGGG(part of line is 3'-5' direction)
Antisense strand: UUCUUCUAGGGGACCUGCCUC
INA ID#248 (the class annular HBV iNA designed by iNA ID#11):
Positive-sense strand: GGGCCCGCAUGGAGACCACCGUGAAUUcCCGGG(part of line is 3'-5' direction)
Antisense strand: UUCACGGUGGUCUCCAUGCGA
Embodiment 3
Chemically modified:
INA ID#249 (the class annular HBV iNA designed by iNA ID#2 and #8)
Positive-sense strand: mGGGUUUUUCUUGUUGAmCAAdTdTuauamCCGmUGUGmCACUUCGCUUmCAdTdT
Antisense strand 1:UUGUCAACAAGAAAAACCCCG
Antisense strand 2:UGAAGCGAAGUGCACACGGUC
(" m " preceding base refers to 2 ' methoxyl group (2 '-OMethyl) RNA base. and " d " preceding base refers to DNA base)
INA ID#250 (sectional type designed according to iNA ID#2 and #8iNA):
Positive-sense strand: mGGGUUUUUCUUGUUGAmCAAdTdTuauamCCGmUGUGmCACUUCGCUUmCAdTdT
Antisense strand 1:UUGUmCAAmCAAGAAAAACCCCG
Antisense strand 2:UGAAGCGAAGUGCACACGGUC
(" m " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base. and " d " preceding base refers to DNA base)
INA ID#250A (sectional type designed according to iNA ID#2 and #8iNA):
Positive-sense strand: mCCGmUGUGmCACUUCGCUUmCAdTdTuauamGGGUUUUUCUUGUUGAmCAAdTdT
Antisense strand 1:UUGUmCAAmCAAGAAAAACCCCG
Antisense strand 2:UGAAGCGAAGUGCACACGGUC
(" m " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base. and " d " preceding base refers to DNA base)
INA ID#251 (sectional type designed according to iNA ID#2 and #8iNA):
Positive-sense strand: mGGGUUUUUCUUGUUGAmCAAdTdTuauamCCGmUGUGmCACUUCGCUUmCAdTdT
Antisense strand 1:UUGUmCAAmCAAGAAAAACCCCG
Antisense strand 2:UGAAGCGAAGUGmCAmCACGGUC
(" m " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base. and " d " preceding base refers to DNA base)
INA ID#252 (sectional type designed according to iNA ID#2 and #8iNA):
Positive-sense strand: mGGGUUUUUCUUGUUGAmCAAdTdTuauamCCGmUGUGmCACUUCGCUUmCAdTdT
Antisense strand 1:UUGUCAACAAGAAAAACCCCG
Antisense strand 2:UGAAGCGAAGUGmCAmCACGGUC
(" m " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base. " d " preceding base isDNA base)
INA ID#253 (sectional type designed according to iNA ID#2iNA):
Positive-sense strand: mGGGUUUUUCUUGUUGAmCAAdTdT
Antisense strand: UUGUmCAAmCAAGAAAAACCCCG
(" m " preceding base refers to 2 ' methoxyl group (2 '-OMethyl) RNA base. and " d " preceding base refers to DNA base)
INA ID#254 (sectional type designed according to iNA ID#2iNA):
Positive-sense strand: mGGGUUUUUCUUGUUGAmCAAdTdT
Antisense strand: UUGUCAACAAGAAAAACCCCG
(" m " preceding base refers to 2 ' methoxyl group (2 '-OMethyl) RNA base. and " d " preceding base refers to DNA base)
INA ID#255 (sectional type designed according to iNA ID#8iNA):
Positive-sense strand: CCGmUGUGmCACUUCGCUUmCAdTdT
Antisense strand: UGAAGCGAAGUGmCAmCACGGUC
(" m " preceding base refers to 2 ' methoxyl group (2 '-OMethyl) RNA base. and " d " preceding base refers to DNA base)
INA ID#256 (sectional type designed according to iNA ID#8iNA):
Positive-sense strand: CCGmUGUGmCACUUCGCUUmCAdTdT
Antisense strand: UGAAGCGAAGUGCACACGGUC
(" m " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base. and " d " preceding base refers to 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 combined by the principle of Watson-Crick by 5'-terminal nucleotide chain and 3'-terminal nucleotide.Another chain by by complementation to the single stranded regions of ring to form double-strand iNA.As follows:
INA ID#257 (stem-ring-like iNA designed according to iNA ID#2iNA):
Positive-sense strand: GGGCCCGGGUUUUUCUUGUUGACAAUUGGGCCC_
Antisense strand: UUGUCAACAAGAAAAACCCCG
INA ID#258 (stem-ring-like iNA designed according to iNA ID#5iNA):
Positive-sense strand: GGGCCCGGAUGUGUCUGCGGCGUUUUUGGGCCC_
Antisense strand: AAACGCCGCAGACACAUCCAG
INA ID#259 (stem-ring-like the iNA designed according to iNA ID#6iNA
Positive-sense strand: GGGCCCUCUUGUUGGUUCUUCUGGAUUGGGCCC_
Antisense strand: UCCAGAAGAACCAACAAGAAG
INA ID#260 (stem-ring-like iNA designed according to iNA ID#7iNA):
Positive-sense strand: GGGCCCCGGGGCGCACCUCUCUUUUUGGGCCC_
Antisense strand: UAAAGAGAGGUGCGCCCCGUG
INA ID#261 (stem-ring-like iNA designed according to iNA ID#8iNA):
Positive-sense strand: GGGCCCCCGUGUGCACUUCGCUUCAUUGGGCCC_
Antisense strand: UGAAGCGAAGUGCACACGGUC
INA ID#262 (stem-ring-like iNA designed according to iNA ID#9iNA):
Positive-sense strand: GGGCCCGGCAGGUCCCCUAGAAGAAUUGGGCCC
Antisense strand: UUCUUCUAGGGGACCUGCCUC
INA ID#263 (stem-ring-like iNA designed according to iNA ID#11iNA):
Positive-sense strand: GGGCCCGCAUGGAGACCACCGUGAAUUGGGCCC
Antisense strand: UUCACGGUGGUCUCCAUGCGA
INA ID#264 (stem-ring-like iNA designed according to iNA ID#2iNA):
Positive-sense strand: GGGCCCmGGGUUUUUCUUGUUGAmCAAUUGGGCCC_
Antisense strand: UUGUCAACAAGAAAAACCCCG
(2 ' methoxyl group (2 '-O-Methyl) the RNA base that " m " preceding base refers to." d " preceding base refers to DNA base).
INA ID#265 (stem-ring-like iNA designed according to iNA ID#8iNA):
Positive-sense strand: GGGCCC mCCGmUGUGmCACUUCGCUUmCAUUGGGCCC_
Antisense strand: UGAAGCGAAGUGCACACGGUC
(" m " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " preceding base refers to DNA base).
INA ID#266 (stem-ring-like iNA designed according to iNA ID#8iNA):
Positive-sense strand: GGGCCC mCCGmUGUGmCACUUCGCUUmCAUUGGGCCC_
Antisense strand: UGAAGCGAAGUGmCAmCACGGUC
(" m " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " preceding base refers to 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 " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " preceding base refers to DNA base).
INA ID#268 (according to iNA ID#2HBV iNA):
Positive-sense strand: [mG] GGUUUUUCUUGUUGA [mC] AA [dT] [dT]
Antisense strand: UUGUCAACAAGAAAAACCCCG
(" m " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " preceding base refers to 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 " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " preceding base refers to 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 " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." the preceding base of d refers to 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 " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " preceding base refers to DNA base.The n-quadrant being decorated with line refers to can not wait (1-1000) at the nt number of sense region, and RNA or the DNA base or the different connections that may also be chemically modified 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 " preceding base refers to 2 ' methoxyl group (2 '-O-Methyl) RNA base." d " preceding base refers to DNA base.The n-quadrant being decorated with line refers to can not wait (1-1000) at the nt number of sense region, may also be RNA or the DNA base of chemically modified, or different connections is good for).
Solution Embodiments 6
SiRNA pharmaceutical formulation.
DOTMA is in ethanol molten, mixes produce water-insoluble throw out with siRNA, is separated and after dry sediment, by precipitate dissolves in chloroform or similar solvent, and mixes at chloroform with other lipids further, technique as described in (WO/2010/135207).After removing organic solvent, drying agent and 9% sucrose water hydration, get final product animals administer.
Solution Embodiments 7
Experimental technique
Cell transfecting: HepG2.2.15 cell is inoculated in 96 orifice plates, at CO2in (5%CO2), overnight incubation in 37 DEG C of incubators.Morning cell covers ~ the orifice plate area of 40%.0.5 μ L siRNA(1 μM of stock solution is diluted) with 10 μ L DMEM nutrient solutions.Dilute 0.4 μ L RFect(hundred with the DMEM nutrient solution of 10 μ L and pass biotechnology), and at room temperature keep 5 minutes.The siRNA of mixed diluting and the RFect of dilution, vortex shakes 10 seconds, and keeps at room temperature 20 minutes.Add 20 μ L transfecting complexes in the hole containing 80 μ LDMEM nutrient solutions.At CO2in (5%CO2), continue in 37 DEG C of incubators to cultivate.
The all programs used in the research of experiment in vivo animal are that the Institutional Animal management and council (IACUC) ratifies, and according to locality, state and federal regulations carry out.HBV transgenic mice, by the formula of tail vein injection 0.2 milliliter injection siRNA.The tissue of results and blood are used for the change that analyzing gene is expressed.
Cell 100 μ L PBS are washed once, then add 100 μ L(Turbocapture test kits, Qiagen Inc. by the separation of mRNA: after transfection two days) lysis buffer.The cellular products (80 μ L) of dissolving transfers to the seizure plate of the mRNA in 96 holes, at room temperature hatches 1 hour.For mouse tissue, two days later, mouse gathers mouse liver tissue in administration.Use Polytron(Turbocapture test kit, Qiagen Inc.) in lysis buffer homogenate.Then shift the seizure plate of 80 μ L to the mRNA in 96 holes, at room temperature hatch 1 hour.Wash three times with 100 μ L lavation buffer solutions, then the elution buffer of 80 μ L joins in each hole, incubation 5 minutes at 65 DEG C.Elute soln (containing mRNA's) is transferred to the 96 new clear plates in hole.
Real-time RT-PCR: the mRNA that 3 μ L are separated is used for real-time RT-PCR.RT-PCR method adopts SYBR Green mono-step real-time RT-PCR test kit (SensiMix mono-step SYBR Green test kit, BIOLINE).Mix 11 μ L master mix(containing reversed transcriptive enzyme), the forward of 1 μ L and reverse primer (6 μMs), the SYBR Green of 0.3 μ L50X and 2.7 μ L water.The temperature of reverse transcription reaction at 42 ° of C, after 30 minutes, 95 ° of C afterwards, 15 minutes for activating Tag polysaccharase; The temperature and time of PCR circulation is 95 ° of C, 15 seconds, 60 ° of C, 30 seconds, 72 ° of C, 20 seconds.With the change of Δ Δ CT methods analyst genetic expression.
Southern Blot Hybridization Analysis liver HBV DNA: liver tissue homogenate is in lysate.Hepatic tissue (about 0.1 gram) is using the homogenate of grinding pestle containing lysis buffer (in the Tris of the EDTA of 1mM, 10mM, 10mM NaCl, 0.5%SDS, Proteinase K).In order to extract DNA, homogenate being hatched 12 hours at 55 DEG C, then, adding isopyknic phenol (Phenol).By sample mix, and with 12,000xg centrifugal 10 minutes., then chloroform is added supernatant liquor, and recentrifuge.Then DNA is gone out with NaCl and alcohol settling.The DNA be settled out is precipitated the TE damping fluid (pH8.0) be dissolved in containing ribonuclease A. the DNA (usually containing 40 micrograms of DNA) of some amount, to digest at 37 ° of C with HindIII digestive ferment (New England Biolabs company, MA) that within 3 hours, to be proved to be HBV gene sequence be not containing HindIII inscribe site.The DNA of digestion, then extract, and be separated by 1%TAE agarose gel electrophoresis.Then DNA is transferred to the positively charged nylon membrane of BioDyne B.DNA is fixed on film after UV irradiates.Hybridization uses [32p] CTP-mark, make probe by the HBV gene group being cloned into pBluescript plasmid that Hae III digests.Hybridize containing 10%PEG-8000,0.05M sodium phosphate, 0.33 mg/ml salmon sperm dna, spend the night at 60 DEG C in the solution of the SDS of 7% and carry out.Phosphorus formation method (Optiquant) is used to measure radiated signal and measure the density of radioactive bands.
Viral DNA is determine with the ratio of viral DNA band relative to transgenosis DNA band relative to the amount of host mouse DNA.Calculating is that the HBV transgenosis containing 1.3 parts according to the cell of each host mouse is carried out.
Semi quantitative PCR analysis liver HBV DNA:PCR method: at 95 DEG C 2 minutes, then 40 circulations: at 95 DEG C 10 seconds, at 60 DEG C 30 seconds.The method of typical curve is used to measure.
Blood plasma HBeAg and HBsAg analyzes: measure according to the explanation (international immunodiagnosis, Foster City, CA) of manufacturer by ELISA method.