CN104087574B - A method of being used to prepare liver cell targeting transmission siRNA - Google Patents

Abstract

The invention belongs to life sciences, and in particular to a method of it is used to prepare liver cell targeting transmission siRNA.The invention discloses a kind of methods for being used to prepare liver cell targeting transmission siRNA, reaction substrate includes lactobionic acid or galactose derivative, terminal amino group modification RNAi polynucleotide and EDC, wherein, lactobionic acid or galactose derivative are as the carboxyl group donor of coupling reaction and the galactosyl donor of targeting couplings, primary amino group donor of the terminal amino group labeled RNA i polynucleotides as coupling reaction, EDC is as zero-length cross-linkers.The method disclosed in the present is easy to operate, yield is high, activity is good, realizes siRNA and couples with liver cell specific ligand-the covalent of galactosyl, after couplings are absorbed by liver cell specificity, shows the silencing activity for target gene.

Description

A method of being used to prepare liver cell targeting transmission siRNA

Technical field

The invention belongs to life sciences, and in particular to a kind of to be used to prepare liver cell targeting transmission siRNA Method.

Background technique

It is the posttranscriptional gene silencing phenomenon mediated by a kind of siRNA molecule that RNA, which interferes (RNAi), is widely used in Functional identification of genes and gene therapy research field.SiRNA molecule is usually the double-strand knot being made of 19~23 pairs of bases Structure, 13~15kd of molecular weight carry 38~40 net negative charges, therefore the naked molecule of siRNA itself can not pass through negative electrical charge Cell membrane often needs cationic liposome package to carry transfection cell.In addition, siRNA system is in use, its bio distribution ten Divide extensively, it is widely distributed in multi visceras such as liver, kidney, the heart, spleen, intestines, cause RNAi activity in target tissue insufficient, after compensatory dosage Easily there are the side effects such as target alia gene silencing, immunostimulation and miRNA Competitive assays.Therefore, the master of siRNA drug conversion Obstacle is wanted to be to lack ideal high efficiency cell targeting transmission means.

Important metabolic organ of the liver as human body, the infringement of susceptible viral infection, metabolic disease, tumour etc..It improves The naked molecular targeted key that drug conversion when RNAi treats liver disease is to speed up into liver cell of siRNA.Currently, assisting small dry Two class of viral vectors and non-virus carrier can be substantially divided by disturbing RNA liver cell targeting transmission medium.The former transfection efficiency is high, The realization of targeting can be obtained by hepatocyte-specific promoter, but immunological rejection and genetic recombination risk limit it and face Bed conversion.Non-carrier class targeting transmission can by means such as cationic compound (liposome or polymer) or chemical modifications, There to be the aglucon of liver cell specific recognition ability to be achieved in conjunction with siRNA in a manner of covalent bond or non-covalent bond. Wherein, it is that siRNA liver cell targeting passes on the common technique studied that non-covalent bond, which combines,.

Non-covalent bond combination siRNA targeting compound advantage is that method is versatile and flexible, does not change siRNA point Minor structure, results of in vitro studies are ideal.As Oishi (2007) forms glycosyl galactose polyethylene glycol and poly-D-lysine Non-covalent bond complex carries siRNA (particle diameter 110nm) by electrostatic.External tracer shows compound successfully It has been transferred to Huh7 cell.For another example, Kim (2007) is by liposome, cholesterol, the small interference of I electrostatical binding of Genetyping poA- RNA forms siRNA Noncovalent complexes, passes on liver cell in vitro, intake is 3 times of no apoA- I.Sato Utilize glycosyl galactose cholesterol derivative (C4-chol) and neutral liposome DOPE and siRNA non-covalent bond within (2007) The composite particles of 75.3 ± 5.8nm are combined into, experiment in vivo shows that about 80% siRNA compound is distributed in liver parenchyma Cell.

Non-covalent target small-interfering RNA compound deficiency is that non-covalent bond leads to structural instability, degradable；Diameter mistake (> 50nm) greatly, it is difficult to by capillary wall, be easy by liver Kupffer cell clearance；Activating complement system, easily by netted interior Dermal system is removed；Medicine is undesirable for index, and it is little that drug converts space.

With the obvious advantage, the couplings molecule of formation that aglucon is passed on siRNA by liver cell after Covalent bonding together Homogeneous, controllability is good, is easy purifying.Aglucon siRNA unimolecule diameter is small, is easy through hepatic sinusoid capillary wall, Pharmacokinetics index is clear.Such as, using cholesterol mainly the liver metabolism the characteristics of, Lorenz and Soutschek (2004) will Cholesterol finds that stability significantly improves (half-life period 95min, plasma clearance after covalently coupling with the end of siRNA positive-sense strand 5 ' Rate 0.5mL/min).It is most with liver and jejunum intake although it is distributed in more histoorgans such as the heart, lung, fat.Again Such as, butyl-amino-vinyl ether polymer is passed through side chain carboxyl group dimethyl using galactosamine as aglucon by Rozema (2007) After acid anhydride key and polyethylene glycol and N- acetylgalactosamine are covalently attached, disulfide bond covalent bond is held with siRNA 5 ', is formed big The small unimolecule compound for 10nm.In-vitro transfection confirms that it is transfected with liver cell identical with cationic liposome siQUEST Efficiency can reduce target gene expression 80%.Be mainly distributed on liver cell in vivo, only on a small quantity by sinus hepaticus nonparenchymal cell and kidney, Splenic uptake.

Aglucon with siRNA Covalent bonding together the relevant technologies are less is seen in report, major obstacle is coupled reaction item Part is more demanding, needs to design more easy, cleverly intermediate bridging molecules.

Summary of the invention

The object of the present invention is to provide a kind of methods for being used to prepare liver cell targeting transmission siRNA, and this method can To realize the covalent linkage of siRNA with liver cell specificity galactose moiety, the glycosyl galactose synthesized using this method is small There is RNA interfering couplings the targeting independent of liposome to pass on liver cell characteristic.

For this purpose, the invention discloses a kind of method for being used to prepare liver cell targeting transmission siRNA, reaction substrate packet Include lactobionic acid or galactose derivative, terminal amino group modification RNAi polynucleotide and 1- ethyl -3- [3- dimethylaminopropyl] Carbodiimides (EDC), wherein the carboxyl group donor and targeting couplings of lactobionic acid or galactose derivative as coupling reaction Galactosyl donor, primary amino group donor of the terminal amino group labeled RNA i polynucleotides as coupling reaction, EDC are handed over as zero-length Join agent.

The method includes the following steps: it is carried out in 0.05M~0.1M2- morpholino b acid sodium (MES) pH of buffer 4.5, Lactobionic acid and terminal amino group labeled RNA i polynucleotides molar ratio are 1:1~2000:1, and EDC and lactobionic acid molar ratio are 1: 1~100:1.

In one embodiment, the method be 25mM lactobionic acid, 12.5 μM of terminal amino group labeled RNA i polynucleotides with 0.1M EDC three is mixed in 0.1M MES pH4.5 buffer, is centrifuged after short term oscillation, in 2h or 4 DEG C of mistake of 25 DEG C of reactions Night makes carboxyl and primary amino group that condensation reaction occur, forms the covalent couplings of glycosyl galactose RNAi polynucleotide.

The method disclosed in the present realizes siRNA and liver cell specific ligand-- galactosyl is covalent Coupling, under the mediation of liver plasma membrane surface asialoglycoprotein receptor, cellular level is shown independent of the positive in vitro The specific liver cell of lipofectamine absorbs.After transmission, glycosyl galactose siRNA couplings can show to be directed to The silencing activity of target gene.Based on similar technology, liver cell targeting transmission small interfering RNA technology established by the present invention may be used also Liver cell targeting for other nucleic acid molecules is passed on.

Detailed description of the invention

Fig. 1 M:5bp DNA Marker；1, naked NH2-siRNA, 4 μ g/100 μ L；2, glycosyl galactose siRNA, 1.5 μ g/100μL

Fig. 2 Huh7 liver cell absorbs the covalent couplings of glycosyl galactose siRNA

Glycosyl galactose siRNA concentration is respectively 1:0 μM；2:1 μM；3:2 μM；4:4 μM；5:0.2 μM of naked siRNA and X-tremeGENE siRNA Transfection Reagent；A:Huh7 cell, B:MDA cell.

Specific embodiment

Term " RNA interfere (RNAi) polynucleotides " is the molecule that can induce RNA interference, by with mammalian cell Rnai pathway component part interaction with sequence-specific manner degrade or inhibit transgenosis mRNA (mRNA) turn Record this translation.Two kinds of main RNAi polynucleotides are small (or short) RNA interfering (siRNA) and microRNA (miRNA).RNAi multicore Thuja acid can be selected from the following group: siRNA, microRNA, double-stranded RNA (dsRNA), short hairpin RNA (shRNA) and coding can induce RNA interference Expression cassette.The duplex structure that siRNA includes usually contains 15-50 base-pair, preferably 21-25 base-pair, and have with it is thin The nucleotide of identical (the perfect complementation) of coded sequence or almost the same (partial complementarity) in expressed target gene or RNA intracellular Sequence.SiRNA also has 3' dinucleotide prominent.SiRNA can be by the two annealing polynucleotides or monokaryon of formation hairpin structure Thuja acid composition.SiRNA molecule of the invention includes ariyoshi region and antisense region.In one embodiment, conjugate SiRNA is assembled from two kinds of oligonucleotide fragments, and one of segment includes the nucleotides sequence of the siRNA molecule antisense strand Column, and the second segment includes the nucleotide sequence of the siRNA molecule sense strand.In another embodiment, sense strand passes through Linkers such as polynucleotides joint or non-nucleotide linker connect antisense strand.MicroRNA (miRNA) is about 22 length of nucleotides Non-coding MicroRNA gene product, instruct destruction or the Translational repression of its mRNA target.If between miRNA and said target mrna being portion Divide complementation, then the translation of the said target mrna is suppressed.If complementary extensively, the said target mrna is cut.It is described for miRNA Compound combines and is usually located at mRNA3！The target site of UTR, usually only with the miRNA homeologous." seed region " is One section about seven (7) a continuous nucleotide of the end miRNA5' forms perfect base pairing-in miRNA specificity with its target It plays a crucial role.The combination of RISC/miRNA compound and mRNA can lead to protein translation inhibition or the cutting and degradation of mRNA. If Recent data shows there is perfect homology along overall length miRNA and its target rather than only in the perfect base of seed region display Pairing, then mRNA cutting is preferred occurs (Pillai etc., 2007).

RNAi polynucleotide expression cassette can be transcribed in cell generate children purpura nephritis, can as siRNA, separate have Justice and antisense strand linear siRNA or miRNA function.The DNA of rna plymerase iii transcription contains the promoter selected from following table: U6 promoter, H1 promoter and tRNA promoter.Rna plymerase ii promoter includes that Ul, U2, U4 and U5 promoter, snRNA are opened Mover, microRNA promoter and mRNA promoter.

The list of known miRNA sequence can be found in the database of research organizational protection, such as Wei Erkangmu foundation mulberry Lattice research institute (Wellcome Trust Sanger Institute), Binzhou Bioinformatics Institute (Penn Center ForBioinformatics), this Long Kaitelin cancer commemorates souvenir center (Memorial Sloan Kettering ) and European molecular biosciences laboratory etc. CancerCenter.Known effective siRNA sequence be associated with binding site also in correlation It is sufficiently described in document.RNAi molecule is easy to be designed and produced with techniques known in the art.In addition, calculating instrument can be used for increasing A possibility that adding discovery effectively and specific sequence motif (2004, Khvorova such as 2006, Reynolds such as Pei etc. 2003, 2004, Amarzguioui such as 2005, the Chalk such as 2004, the Heale such as 2003, the U1-Tei such as Schwarz etc. are 2004).

RNAi polynucleotide of the invention can be modified by sulphation.The non-limiting example of the chemical modification includes: thio phosphorus Connection, 2'-O- methyl ribonucleotides, 2'- deoxidation -2'- fluorine ribonucleotide, 2'- dezyribonucleoside between acid esters nucleotide Acid, " universal base " nucleotide, 5-C- methyl nucleotide and the abasic residue incorporation of inversion deoxidation.For various polynucleotides When structure, these chemical modifications, which are shown in cell, keeps polynucleotides activity, while increasing the serum stable of these compounds Property.The siRNA of chemical modification can also make one a possibility that middle interferon activity activation minimum.

In one embodiment, chemical modification RNAi polynucleotide of the invention includes the duplex with two chains, One or both can chemical modification, wherein each chain is about 19~about 29 nucleotide.In one embodiment, RNAi of the invention Polynucleotides include the nucleotide of one or more modifications, while keeping mediate rna i in the cell or rebuilding in an in vitro system Ability.RNAi polynucleotide can through modifying, wherein chemical modification include it is one or more (for example, about 1,2,3,4,5,6,7,8, 9,10 or more) nucleotide.RNAi polynucleotide of the invention may include the nucleotide of modification, account for institute in RNAi polynucleotide There are a certain percentages of total nucleotide number.Equally, RNAi polynucleotide of the invention usually can be about 5~about 100% (such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% nucleotide position) nucleotide position on include modification nucleotide.It is given The actual percentage of the acid of modified nucleoside present in RNAi polynucleotide depends on nucleosides present in the RNAi polynucleotide Sour sum.If RNAi polynucleotide be it is single-stranded, modifying percentage can be based on nucleotide present in single stranded RNA i polynucleotides Sum.Equally, if RNAi polynucleotide is double-strand, sense strand, antisense strand or sense and antisense chain can be based on by modifying percentage Total nucleotide number present in the two.In addition, the actual percentage of modified nucleoside acid present in given RNAi polynucleotide is also It may depend on purine present in the RNAi polynucleotide and pyrimidine nucleotide sum.For example, wherein in RNAi polynucleotide Existing all pyrimidine nucleotides and/or all purine nucleotides are by modification.

The rna expression of RNAi polynucleotide controlling gene coding.Since several genes can share a degree of sequence each other Column homology, so RNAi polynucleotide may be designed as the genoid that targeting has enough sequence homologies.Therefore, RNAi is more Nucleotide may include the shared or sequence complementary to the unique sequence of specific gene target between different genes target.Therefore, RNAi polynucleotide may be designed as the conservative region that targeting has the RNA sequence of homology between Several gene, to target base Because of the Several gene (such as different genes isotype, splice variant, mutated gene) in family.In another embodiment, RNAi polynucleotide may be designed as targeting to the unique sequence of specific RNA sequences in term single gene.

Term " complementation " refers to that polynucleotides pass through conventional Watson Crick with another polynucleotide sequence or other are unconventional The ability of type formation hydrogen bond.When being related to polynucleotide molecule of the invention, polynucleotide molecule and its target (effective bound site Point) or the combination ionization energy of complementary series be enough to promote the correlation function of the polynucleotides, as enzyme mRNA cutting or turn over Translate inhibition.The measurement of nucleic acid molecules combination ionization energy is well known in the art that (1986, Turner such as Frier etc. is 1987).It is complementary Percentage indicates the base percentage for adjoining chain in the first polynucleotide molecule, and the molecule can be with the second polynucleotide sequence shape At Watson-Crick base pairing.All bases and the second multicore glycosides in the perfect complementary connecting chain for indicating polynucleotide sequence The continuous base of identical quantity forms hydrogen bond in acid sequence.

Inhibit, lower or strike and subtracts gene expression expression such as the rna level with the genetic transcription or from RNA translation Measured by polypeptide, albumen or protein protomer level, the expression of the gene is lower than without blocking polynucleotides conjugate of the present invention When the level observed.The gene expression inhibition that is carried out with present composition institute delivery of polynucleotides is lowered or is struck and subtracts, preferably When lower than in the presence of control inactivation nucleic acid (the mixed and disorderly nucleic acid of sequence or the nucleic acid for being passivated mispairing) or the polynucleotides are not coupled and cover The level observed when covering polymer.

RNAi polynucleotide conjugate

RNAi polynucleotide conjugate provides delivering in the RNAi polynucleotide body of improvement.Described in Functional delivery expression RNAi polynucleotide is delivered to cell and the expected sequence-specific with biological activity, gene expression inhibits.Give lactation Many molecules (including polynucleotides) of animal vascular system are usually removed from body by liver.Liver is to the clear of polynucleotides Degradation occurs for polynucleotides described in removing or in addition processing is with the removal from body and wherein the polynucleotides do not cause base Because the sequence-specific of expression inhibits, the removing is not regarded as Functional delivery.

RNAi polynucleotide conjugate is formed by being covalently attached the RNAi polynucleotide and the targeting ligand part. Can synthesize or modify the polynucleotides makes it contain reactive group A.Can synthesize or modify the targeting moiety contains it There is reactive group B.Reactive group A and B are selected, so that it can be connected with methods known in the art by covalent bond.

The targeting moiety connects the end 3' of the RNAi polynucleotide.For siRNA polynucleotides, the targeting portion Sense strand or antisense strand can be connected by dividing, although it is preferred that sense strand.In some embodiments, the siRNA passes through containing reaction The short alkyl chain of group A (such as primary amine group) connects targeting moiety described in connecting.Then, reactive group A is coupled on targeting moiety Reactive group B (such as carboxylic group).

For the intrahepatic liver cell of target, preferred targeting ligand is galactose cluster.Galactose cluster includes to have 2~4 kinds The molecule of terminal galactose derivative.As used herein, term galactose derivative includes galactolipin and to asialoglycoprotein sugar egg The compatibility of polymeric immunoglobulin receptor equals or exceeds the galactose derivative of galactolipin.Terminal galactose derivative is connected by its C-1 carbon Molecule.The galactolipin terminal glycoprotein of asialoglycoprotein receptor (ASGPr) simultaneously conjugate branch unique to liver cell.Preferably Galactose cluster has respectively affinity to asialoglycoprotein receptor 3 kinds of terminal galactose amine or galactosamine derivative Object.Preferred galactose cluster has 3 kinds of end N- acetyl-galactosamines.Other generic terms of this field include three feeler types (tr1-antennary) galactolipin, trivalent galactolipin and galactolipin tripolymer.Known three feelers type galactose derivative cluster with than Two feeler types or the stronger compatibility combination ASGPr of single-tap angle-style galactose derivative structure (Baenziger and Fiete, 1980, Cell, 22,611-620；ConnoIIy etc., 1982, J.Biol.Chem., 257,939-945).Multivalence is needed to obtain NM compatibility.When giving altogether with delivery polymer, more feeler type galactolipins can be simulated by improving single galactose derivative concentration Activity that the receptor of derivative is affine.

Three kind galactose derivatives of the galactose cluster containing respective connection central fascicle point.The galactose derivative passes through institute State the C-1 carbon combination central fascicle point of sugar.Galactose derivative preferably passes through connector or introns connect the branch point.It is preferred that Introns be flexible hydrophilic spacer (United States Patent (USP) 5885968；The J.Med.Chem.1995Vol.39 such as Biessen ρ .1538-1546).Preferred flexibly hydrophilic spacer is PEG introns.Preferred PEG introns are PEG3 introns.Branch Point can be any small molecule, allow three kinds of galactose derivatives to combine, also allow the branch point combination RNAi multicore Thuja acid.Exemplary branch point group is two lysines.Two lysine molecules (can combine three kinds of galactolipins by it containing three kinds of amidos Derivative) and carboxyl reactive group (two lysines can combine RNAi polynucleotide by it).Branch point and RNAi polynucleotide Combination can pass through connector or introns and occur.Preferred introns are flexible hydrophilic spacers.Preferred flexibly hydrophilic Every son be PEG introns.Preferred PEG introns are PEG3 introns (three ethylene units).The galactose cluster also can be used Means known in the art connect the end 3' or 5' of the RNAi polynucleotide.For the RNAi polynucleotide with 2 chains, example Such as siRNA, the galactose cluster can connect remaining any end except the end antisense strand 5'.

Preferred galactose derivative is N- acetyl-galactosamine (GalNAc).Have to asialoglycoprotein receptor Other sugar of compatibility can be selected from containing list below: galactolipin, galactosamine, N- formoxyl galactosamine, N- acetyl group Galactosamine, N- propiono galactosamine, the positive bytyry galactosamine of N- and N- isobutyryl galactosamine.Study a large amount of half Lactose derivatives to the compatibility of asialoglycoprotein receptor (see, for example, 1bst, S.T. and Drickamer, K.J.B.C.1996,271,6686) or its easy-to-use this field conventional method determination.

Administered in vivo

In pharmacology and toxicology, administration route is to make the approach of drug, liquid, toxic agent or other substances contact body. The drug and nucleic acid administration way for being commonly used for treatment mammal are well known in the art, and can be used for giving combination of the present invention Object.The compounds of this invention can be applied in the preparation according to the approach appropriate adjustment by any suitable pathways, most preferably intestines Outside stomach.Therefore, can by injection, such as intravenously, intramuscular, intradermal, subcutaneous or intraperitoneal injection give the compounds of this invention.Cause This, the present invention also provides the pharmaceutical compositions containing pharmaceutically acceptable carrier or excipient.

The parenteral route given include in vascular (intravenous, intra-arterial), intramuscular, in essence, under intradermal, corium (subdermal), subcutaneous (subcutaneous), in tumour, peritonaeum is interior, injects in intrathecal, Subdural space, Epidural cavity and lymph, Use syringe and needle or conduit.It indicates to be known as in the tubular structure of vascular in vascular described herein, connects intracorporal tissue Or organ.The tubular structure it is intracavitary, body fluid flow direction or flow from body part.The example of body fluid includes blood, cerebrospinal fluid (CSF), lymph or bile.The example of vascular include coronary artery, arteriole, capillary, veinlet, sinusoid, vein, Lymphatic vessel, bile duct and salivary gland duct or other exocrine glandular tubes.Approach includes by blood vessels such as artery or veins in vascular Delivering.Blood circulation system provides the whole body diffusion of drug.

The RNAi polynucleotide targeting moiety conjugate is pharmaceutically injected in acceptable carriers solution.It can pharmaceutically connect By those characteristics of mammal acceptable and/or substance on finger pharmacology/toxicological point.The pharmaceutically acceptable finger of phrase Pharmaceutical formulation and allergy or other unfavorable or toxic reaction molecular entities, combination are not generated usually when giving mammal Object and characteristic.Terms used herein " pharmaceutically acceptable " preferably refers to be ratified or Chinese Pharmacopoeia or other generally acknowledged pharmacopeia by SFDA It is listed to be used for animal applications, it is more particularly for people.

Therapeutic effect

RNAi polynucleotide targeting moiety conjugate is for research purposes or for generating variation in treatment cell.RNAi Polynucleotides targeting moiety conjugate is for investigational agent and various treatments, diagnosis, target validation, genome exploitation, gene work Journey transformation and Drug Discovery application.

Liver is the most important target tissue of gene therapy, because it is in metabolism (the rouge egg in such as various hypercholesterolemias White metabolism) and circulating protein (coagulation factor in such as hemophilia) secretion in play a crucial role.In addition, common acquired disease is such as Chronic hepatitis and hardening and may also be treated with the liver therapy based on polynucleotides.It influences or by many diseases of hepatic effects Or illness can subtract in (inhibition) liver gene expression and treat by striking.The liver diseases and illness can be selected from containing column below Table: liver cancer (including hepatocellular carcinoma, HCC), virus infection (including hepatitis), metabolic disorder (including hyperlipidemia and diabetes), Cystic fibrosis and acute liver damage.

The amount (dosage) of RNAi polynucleotide conjugate to be administrated can be empirically determined.With 0.1~10mg/kg animal body The siRNA- conjugate of weight and the delivery polymer of 5~60mg/kg the weight of animals can effectively strike and subtract gene expression.It is excellent in mouse Choosing amount is 0.25~2.5mg/kg siRNA- conjugate.The amount of RNAi polynucleotide conjugate is easy to improve, because it usually exists It is nontoxic under large dosage.

It is target that the present invention, which selects people's glyceraldehyde phosphate dehydrogenase (GAPDH) gene, and observation glycosyl galactose siRNA is total Valence couplings pass on ability, and the RNAi silencing effect for target gene to the targeting of liver cell line Huh7.Below with reference to The invention will be further described for drawings and examples.

1 lactobionic acid of embodiment and terminal amino group mark siRNA coupling reaction

S1:0.4M EDC is prepared in 0.2M MES buffer, pH4.5, -20 DEG C save, and use forward horizontal stand to room temperature；It prepares S2:0.1M lactose acid solution uses forward horizontal stand to room temperature in 0.2M MES pH of buffer 4.5, -20 DEG C of preservations；Prepare S3:2OD Terminal amino group label siRNA (6nmol, 80 μ g) is dissolved in the Rnase inactivation water of DEPC processing, -80 DEG C of preservations, before use It balances to room temperature.100 μ l S1,100 μ l S2 are mixed with 200 μ l S3, are slightly centrifuged, and each component is total in 400 μ l reaction Final concentration in volume is respectively 0.1M EDC, 25mM lactobionic acid, 12.5 μM of terminal amino groups label siRNA (positive-sense strand: guaugacaacagccucaagt-NH2；Antisense strand: cuugaggcuguugucauactt), reaction system is 0.1M MES buffering Liquid, pH4.5.Reaction solution is in 25 DEG C of reaction 2h.With miRNAOUT column desalination, returns and receive total siRNA.The small interference of glycosyl galactose The covalent couplings connection product of RNA is after desalting processing, the row identification of 20% polyacrylamide gel electrophoresis over long distances.Fig. 1 shows, The RNA molecule amount of swimming lane 1 has more about 1 base size compared with 2 amino labeled siRNA of swimming lane, shows the small interference of glycosyl galactose The covalent couplings of RNA correctly synthesize.

Compared to document report, glycosyl galactose siRNA couplings synthesized by the present invention are completed using one-step method, are obtained Rate height (> 90%), is shown in Table 1.Connection galactose moiety in the end of siRNA positive-sense strand 3 ' leads to end melting temperature reduction, according to Schwarz rule, interference activity are improved.

1 three kinds of table sugared ammonia coupling reactions compare

Note: document 1:Shinsuke S, et al.Facile preparation of DNA-tagged carbohydrates.Bioorganic and Medicinal Chemistry Letters2003,13:2633-6.

Document 2:Dalpathado DS, et al.Reductive amination of carbohydrates using NaBH(OAc)3.Anal Bioanal Chem2005,381:1130-7.

Embodiment 2Huh7 liver cell absorbs the covalent couplings test of glycosyl galactose siRNA

The every hole culture medium total volume of 24 orifice plates is the 450 μ l of DMEM complete medium without serum and antibiotic, is added half The covalent couplings proper volume of lactosylation siRNA, making final concentration is respectively 0 μM, 1 μM, 2 μM and 4 μM.After 5h, remove multiple Object is closed, replacement contains the DMEM complete medium of 10% fetal calf serum and twin antibiotic.After being incubated for 72h, RIPA cracks Huh7 cell, It collects total protein and detects target protein expression amount for Western Blot.Set up the non-liver source cell of MDA, liposome transfection, with And blanc cell control.Fig. 2 shows, 2 μM and 4 μM of glycosyl galactose siRNA be incubated for Huh7 cell GAPDH expression quantity relative to Blanc cell has dropped 17% and 39% respectively, and MDA cell is not shown out to the active intake of glycosyl galactose siRNA.

Various aspects of the present invention have been described above.However, it should be understood that without departing from spirit of that invention Under the premise of, those skilled in the art can carry out equivalent change and modification to it, and the change and modification equally fall into the application The coverage area of appended claims.

Claims (3)

1. a kind of method for being used to prepare liver cell targeting transmission siRNA, reaction substrate includes that lactobionic acid or galactolipin spread out Biology, terminal amino group modification RNAi polynucleotide and EDC, wherein the carboxylic of lactobionic acid or galactose derivative as coupling reaction The galactosyl donor of base donor and targeting couplings, primary amino group of the terminal amino group labeled RNA i polynucleotides as coupling reaction Donor, EDC is as zero-length cross-linkers, it is characterised in that the method in 0.05M~0.1M MES pH of buffer 4.5 into Row, lactobionic acid and terminal amino group labeled RNA i polynucleotides molar ratio are 1: 1~2000: 1, EDC and lactobionic acid molar ratio It is 1: 1~100: 1, the RNAi polynucleotide is siRNA.

2. the method as described in claim 1, it is characterised in that the method is 25mM lactobionic acid, 12.5 μM of terminal amino group labels RNAi polynucleotide and 0.1M EDC three are mixed in 4.5 buffer of 0.1M MES pH, are centrifuged after short term oscillation, in 25 DEG C 2h or 4 DEG C of reaction overnight, makes carboxyl and primary amino group that condensation reaction occur, and forms glycosyl galactose RNAi polynucleotide and covalently couples Object.

3. the method as described in claim 1, it is characterised in that the RNAi polynucleotide can be modified by sulphation, the chemical modification It include: connection between phosphorothioate nucleotide, 2'-O- methyl ribonucleotides, 2'- deoxidation -2'- fluorine ribonucleotide, 2'- de- Oxygen ribonucleotide, 5-C- methyl nucleotide are inverted the abasic residue incorporation of deoxidation.