CN102660543A

CN102660543A - Method, product and application of chemical modification of siRNA sequence using isonucleoside

Info

Publication number: CN102660543A
Application number: CN2012100200282A
Authority: CN
Inventors: 杨振军; 田苗; 黄野; 陈卓; 彭宜红; 张礼和
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2012-01-21
Filing date: 2012-01-21
Publication date: 2012-09-12

Abstract

The invention discloses a method, a product and an application of a chemical modification of a siRNA sequence using an isonucleoside. The related product can be used for disease treatment, especially for the preparation of medicaments for treating tumours and antiviral medicaments. The invention belongs to a filed of biological medicine. A modification strategy provided is characterized in that isonucleosides shown in chemical formulas I and/or II are mixed in a synthesized siRNA sequence so that the siRNA modified by a type of isonucleoside is obtained and the siRNA is stable in property and strong in specificity, and has a better biological activity than the natural siRNA. Compared with the natural siRNA, the siRNA sequence of human MEK-1 gene modified by the said method has better stability and silent activity, and a development prospect in manufacturing medicaments for treating tumours and antiviral medicaments.

Description

A kind of utilize the heteronuclear glycosides to the siRNA sequence carry out chemically modified method, and products thereof and use

Technical field

The present invention relates to a kind of method of siRNA sequence modification, particularly a kind ofly through introducing the heteronuclear glycosides it is carried out the method for chemically modified, the product that is obtained can be used in disease treatment and particularly is applied to prepare in oncotherapy and the antiviral drug.Belong to biomedicine field.

Background technology

1, for melanomatous present Research

Melanoma is a kind of common skin malignant tumour, and its sickness rate is lower, is generally the 1%-3% of whole malignant tumours; But grade of malignancy is high; Being malignant tumour melanoma rather thorny on a kind of clinical treatment, coming from skin mostly, also can be that eye and nasal cavity etc. are located.Melanoma patient's early treatment effect is good, if diagnosed out during knurl diameter≤2mm, the surgical healing rate can reach 90%.But its growth is rapidly, the certain rate variance of early screening effect, and the transfer at positions such as liver, lung, bone and brain will take place in early stage sick sending out, and worsens fast.The curative ratio of malignant melanoma is extremely low at present, and the survival rate in 5 years is lower than 5%, and patient can be dead after the diagnosis several months mostly.The cancer treatment method of various routines (like operation, radiotherapy, chemotherapy and biotherapy etc.) does not all have much obvious effects.Wherein, chemotherapy is must indispensable important means in the malignant tumour complex therapy.But malignant melanoma is insensitive to various chemotherapeutic agents, and shows stronger resistance usually.Some conventional effectively chemotherapeutics are like the alkylating agent class: dicarbazine (DTIC), TM (TMZ); Platinum class: cis-platinum (DDP), carboplatin; Nitrous base class: carmustine, lomustine; Antitubulin: vinca, taxanes medicine etc. are that single therapy or combined chemotherapy all do not have significant curative effect to improving the tumour alleviation and prolonging existence.Interleukin II (Interleukine-2) has certain curative effect to the malignant melanoma patient, but toxicity is big, expense is high, and application is restricted.Also do not set up the treatment means of standard at present for the treatment of metastatic malignant melanoma, remain a very stubborn problem, press for research and development new chemotherapeutics, targeted drug and other treatment means.

The same with other tumours, melanomatous is environmental factors (sunlight exposure etc.) and genetic results of interaction, interrelated generation and the development process that has determined tumour of these factors.Blanket Clark model is divided into MC vicious transformation process benign nevus, makes up 5 steps such as bad mole, radial gorwth phase, vertical growth phase and metastasis melanin tumor.In each vicious transformation stage, RAS, RAF, CDK, p53, AKT, MITF, α the ANOMALOUS VARIATIONS of multiple oncogene, cancer suppressor gene, cyclin, apoptosis regulatory protein, cell adhesion molecule, growth factor have all appearred, for example _vβ ₃, EGF, VEGF or the like.The gene of these variations and albumen possibly become the index of melanoma malignization degree, more possibly become the drug targets that the different steps tumour is effectively treated gradually.These change genes and albumen to the malignization degree of MC tissue and the influence of the rate of transform from the research of molecular mechanism angle, for the announcement melanoma potential source biomolecule cause of disease, carry out disease treatment effectively, provide fundamental basis.

Wherein G albumen RAS and RAF protein serine/threonine are ERK (extracellular signal-regulated kinase: important member in cascade signal path extracellular signal-regulated kinase); The ERK passage is caused infinite multiplication, survival, migration and the invasion and attack of tumour cell by abnormal activation in the tumour cell because RAS and the kinase whose variation of BRAF cause.But greater than finding N-RAS (one type of hypotype of RAS in 90% the malignant melanoma tissue; Account for 15%) or BRAF (one type of hypotype of RAF; Account for 60-80%) variation; Causing the ERK signal path not have control unusually and activate, is to cause one of the infinite multiplication of MC and major reason of fast transferring.The ERK signalling channel is by a mitogen-activated protein kinase (mitogen-activated protein kinase, MAPK) the cascade signal path found the earliest.Under the normal circumstances; Have only after part (growth factor, hormone, cell adhesion molecule etc.) coordination combines outside cell-membrane receptor and the born of the same parents; The G albumen Ras of cell inner membrance just understands exchange GTP and is activated; Activate through kinase whose phosphorylation step by step such as RAF, MEK1/2 and ERK1/2, last activatory ERK1/2 protein kinase is striden film and is got in the nucleus, regulates and control a series of cell vital processes such as for example cell proliferation, survival, migration, deterioration and vasculogenesis.The ERK passage is that normal cell is kept the important requisite signalling channel of its normal vital process, but the abnormal activation of passage (part dependent/non-dependent) also is the one of the main reasons (accounting for 30% human tumor) that causes cell carcinogenesis.Except that melanoma, the overactivity (Fig. 1) of ERK signalling channel is particularly attacked, all detected in the transfer, malignant tumor tissue to primary liver tumor, colon tumor, melanoma, thyroid tumor, lung tumor, ovarian cancer etc.The various kinases of ERK passage upstream and downstream are the important targets of tumor-targeting drug design always, particularly melanomatous medicinal design.

To the small-molecule drug of ERK signal conduction pathway, be the emphasis of melanoma medicament research and development always.The clinical study result shows that the small-molecule drug of target sudden change N-RAS, MEK1/2 homologous protein can effectively suppress the melanoma process, and bigger toxic side effect is but arranged.The 60-80% melanoma is found the sudden change of 600 of BRAF kinases, BRAF ^V600EIt is important drug targets.The most effectively clinical development medicine PLX4032 selectivity suppresses BRAF at present ^V600EActivity be kinase whose 3 times of native protein.III phase clinical experiment in 2010 shows that this medicine has extraordinary curative effect to malignant melanoma, can suppress about 80% ERK channel activity, but also has the hazards of medication of activate channel simultaneously.

The small-molecule drug of target ERK passage is remarkable to the proliferation inhibiting effect of malignant melanoma, and this suffices to show that targets such as sudden change NRAS, BRAF and MEK1/2 are for suppressing melanomatous validity.Their toxicity is owing to the specificity recognition differential to the passage target causes.RNA disturbs (RNA interference is called for short RNAi) technological highly selective degraded said target mrna, and the expression of reticent corresponding protein generally has been used for the research of gene and protein function and the diagnoses and treatment research of disease.Maturation development along with the RNA perturbation technique applies to oncotherapy with the siRNA technology, suppresses growth of tumor and transfer thereby reach, and the purpose that finally reaches the treatment tumour is own through becoming possibility.The siRNA perturbation technique is applied to oncotherapy and compared with former therapy of tumor method; Advantage with following uniqueness: 1. specificity is higher; What siRNA caused is the special gene silencing after transcribing, thereby has avoided its interference to the body normal system.2. it is remarkable that high-level efficiency, the effect that RNA disturbs inhibition of gene expression and other gene therapy method are compared effect, so siRNA is a kind of extremely potential oncotherapy strategy.But the siRNAs of natural structure can not satisfy the needs as genomic medicine fully, and necessary chemically modified can improve its body internal stability, pass through characteristics such as film property, bioavailability, improves its reticent effect.Although siRNA body internal stability is poor, because melanoma belongs to the epidermis tumour, independent limbs perfusion is main route of administration, can effectively avoid the weakness of siRNA, is valuable with siRNA as melanomatous treatment tool therefore.In the research process of fs; We adopt specificity to be directed against its function of siRNA blocking-up of MEK1 gene; Investigate the MEK1 gene by silence after downstream ERK albumen and the proteic expression of pERK, thereby proved conclusively the vital role of MEK1 gene in MC propagation.The distinguished sequence of modifying (siMEK1) possibly be applied in the melanomatous genomic medicine of preparation efficacious therapy.But the The Molecular Biology Mechanism more complicated of siRNA effect, chemically modified need be done further discussion in the effect that improves aspect its physico-chemical property and the biological activity.

2, the present Research of relevant virus disease

In recent years, various viruses of wreaking havoc had been brought more and more serious threat to the mankind's life and health, also let people experience more and sought the importance of antiviral safely and effectively.At present, existing antiviral plays a role through different action pathway, such as directly inhibition or kill virus, viral interference absorption, prevention virus penetrate cell, the inhibition viral organism synthesizes, suppresses virus release or enhancing host anti-virus ability etc.Wherein, Most of antiviral mainly are through suppressing duplicating or suppressing virus through regulating body immune system of virus, but present antiviral all selective low, the antiviral scope of ubiquity little, be prone to due to illness poison variation and produce shortcomings such as resistance, toxic side effect are big.The ideal antiviral should be that pair cell is harmless in the dark human host cell of ability, has the selectivity of height to suppress and killing action to virus.But still do not have up to now, through the direct kill virus of host cell and to the less medicine of host cell influence.Therefore, seeking a kind of novel antiviral medicine that antiviral spectrum is wider, can overcome problems such as virus variation and toxic side effect that has, is the current major issue of needing solution badly.

Present antiviral is absorbed in " virus " research own more, and has seriously ignored virus and this important step of host cell interaction.In most virus multiplication process, can pass through activating cells RAF/MEK/ERK signal cascade path, it is synthetic to regulate and control the required albumen of viral self-replication.The ERK path is three grades of kinase cascade paths that are made up of RAF, MEK and three groups of major protein of ERK.In the normal host cell; This path through above-mentioned three histones by preceding to after successively order activate; Finally indicate the activation of whole path with the ERK protein phosphorylation; Activatory ERK albumen is the different substrate of effect further, a series of important physical functions such as performance cell growth, metabolism, propagation, differentiation.And at tumour generation, virus infection with duplicate etc. in the process, this path also discovery can play an important role, and synthesizes closely related with the albumen of tumour amplification and virus replication.

Discoveries such as calendar year 2001 Pleschka, influenza A virus get into when host cell duplicates can cause the activation of RAF/MEK/ERK signal cascade path.Use and act on the suppressor factor U0126 RAF/MEK/ERK signal cascade capable of blocking path activation of MEK1/2, and influenza A virus output is descended more than 90%, prove that the propagation of virus relies on activated host cell RAF/MEK/ERK signal cascade path.The sick virus of influenza A virus, herpes simplex virus I I type (HSV-2), Borna, poxvirus, human immunodeficiency virus type 1, Coxsackie virus, hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis E virus important viruses such as (HEV) duplicate the activation that all relies on RAF/MEK/ERK signal cascade path.

MEK albumen plays the keying action of forming a connecting link in RAF/MEK/ERK signal cascade path reactivation process.MEK albumen has MEK1 and two kinds of hypotypes of MEK2, and the two has incomplete eclipsed physiological function, but the two separately function in virus replication it be unclear that.At present, owing to lack respectively specific inhibitor, so when studying the virus replication function of this path, mainly carry out through using MEK1/2 suppressor factor (U0126 or PD98059) to MEK1 or MEK2.But this type of suppressor factor is to MEK1 and MEK2 non-selectivity, and the two activity is all had restraining effect, be that the antiviral of foundational development is all influential to MEK1 and MEK2 physiological function separately with the MEK1/2 suppressor factor, so this type of medicine has certain toxic side effect.At present, existing people utilizes the RNA perturbation technique on gene level, albumen to be suppressed, and with respect to proteic suppressor factor, has shown clear superiority.

The RNA interference effect makes the especially double-stranded RNA of long-chain change the RNA signaling molecule of about 21 base length into, comprises siRNAs (small interfering RNAs) and miRNAs (microRNAs).SiRNAs and miRNAs participate in the formation of specific albumen composition, suppress transcribing or translating, the perhaps degraded of catalysis mRNA of mRNA.Owing to the blocking gene that the RNA interference effect can be efficient, special is expressed, therefore showed tempting prospect at aspects such as gene function and protein interactions, siRNAs also shows great potential in antitumor antiviral gene therapy simultaneously.The siRNAs of natural structure can not satisfy the needs as genomic medicine fully, and necessary chemically modified can improve its body internal stability, pass through characteristics such as film property, bioavailability, improves its reticent effect.The research of ASONs chemical modification method is very helpful to the structural modification of siRNAs, but because the complicacy of siRNAs mechanism of action and self structure, its chemically modified is also complicated than ASONs, the research that still need go deep into.

Summary of the invention

In order to overcome natural siRNA less stable and nonspecific reticent active function (effect of missing the target) in serum, technical problem to be solved by this invention provides a kind of heteronuclear glycosides that utilizes siRNA is carried out chemically modified to improve its stability and to reduce the method for the effect of missing the target.

A kind of method of utilizing the heteronuclear glycosides siRNA sequence to be carried out chemically modified of the present invention; The positive-sense strand of the siRNA sequence that it is characterized in that modifying in desire and/or one or more sites of antisense strand are mixed the heteronuclear glycoside compound shown in Formula I or the Formulae II and are carried out coupling in the corresponding position to replace the natural nucleus glycoside compound

Wherein, B is thymine base (T), uracil base (U), cytosine(Cyt) base (C), guanyl-(G) or adeninyl (A); Than the natural nucleus glycoside compound; Base is by 2 '-position of 1 ' of sugar ring-be moved to sugar ring, and the heteronuclear glycosides shown in the Formula I is the heteronuclear glycosides of L-configuration, and the heteronuclear glycosides shown in the Formulae II is the heteronuclear glycosides of D-form.

In chemical modification method of the present invention, before carrying out the siRNA modification, the heteronuclear glycoside compound shown in Formula I and/or the Formulae II is prepared into the heteronuclear glycosides phosphoramidite monomer shown in Formulae II I and/or the Formula I V respectively; Described heteronuclear glycosides phosphoramidite monomer and compound method thereof can be referring to document (Yu HW, Zhang LR, Zhuo JC; Ma LT; Zhang LH.Bioorg.Med.Chem.1996,40,609-614.).

Wherein B is guanyl-(G-Bz or G-iBu) or the cytosine(Cyt) base (C-Bz) of benzoyl-protection of adenine base (A-Bz), uridylic base (U), benzoyl-or the protection of different propionyl group of benzoyl-protection.

In the specific embodiment of invention; Use solid phase synthesis; Adopt phosphoramidite method, on dna synthesizer, replace the natural nucleus glycoside phosphoramidite monomer to carry out coupling in the corresponding position to be incorporated in the synthetic sequence one or several Formulae II I and/or the described heteronuclear glycosides of Formula I V phosphoramidite monomer; Nucleosides of every coupling is a circulation, and each circulation comprises four reactions: take off DMT, coupling, sealing, oxidation.

Because the coupling yield under the heteronuclear glycosides normal condition is lower, the coupling time behind the sample introduction number of times that needs to increase heteronuclear glycosides phosphoramidite monomer and the each sample introduction is to guarantee the synthetic yield.The condition of synthetic DNA oligonucleotide chain is to adopt to increase the phosphorus acylated monomeric sample introduction number of times to 3 of heteronuclear glycosides; Coupling time behind each sample introduction be 300 seconds/inferior; The condition of the RNA oligonucleotide chain that synthetic heteronuclear glycosides is modified be the coupling time behind each sample introduction increase to 900 seconds/inferior, coupling 3 times.

Further, the present invention also provides the sequence of the siRNA that modifies according to above each described method synthetic heteronuclear glycosides.

In the specific embodiment of invention, the siRNA sequence that described sequence is modified for the heteronuclear glycosides to people MEK-1 gene.

Sequence before the siRNA of described people MEK-1 gene modifies is:

Positive-sense strand: 5 '-GCAACUCAUGGUUCAUGCUdtdt-3 ' (SEQ ID NO.1)

Antisense strand: 3 '-dtdtCGUUGAGUACCAAGUACGA-5 ' (SEQ ID NO.2).

In the specific embodiment of invention; Wherein mix the heteronuclear glycosides shown in Formula I or the Formulae II on one or the multidigit 1,8,9,11 of the positive-sense strand of the siRNA of above-described people's MEK-1 gene or 12; Replace natural nucleus glycoside to carry out coupling in the corresponding position, preferred, on 8 of the positive-sense strand of siRNA sequence, mix the heteronuclear glycosides shown in Formula I or the Formulae II; Replace natural nucleus glycoside to carry out coupling in the corresponding position; Preferred, on 8 of the positive-sense strand of siRNA sequence, mix the heteronuclear glycosides shown in the Formulae II, replace natural nucleus glycoside to carry out coupling in the corresponding position.

1 of above-described positive-sense strand is meant positive-sense strand: 5 ' end beginning first Nucleotide, i.e. " G " in 5 '-GCAACUCAUGGUUCAUGCUdtdt-3 ' nucleotide sequence.All the other positions by that analogy.

The research proof, the siRNA that heteronuclear glycosides provided by the invention is modified has characteristics such as physico-chemical property is stable, combined coefficient height; Product self has the better biological activity than natural siRNA.

The active result that the heteronuclear glycosides is incorporated among the siRNA is applied in the treatment of people's malignant melanoma; The active testing result of protein level and rna level shows; When synthetic; Mix the heteronuclear glycosides phosphoramidite monomer shown in the chemical formula (II) on 8 of the positive-sense strands, replace the natural nucleus glycoside phosphoramidite monomer to carry out link coupled siRNA in the corresponding position and can significantly improve that it is reticent active.

The present invention has utilized the siRNA technical research in MC MAPK signal path, and the kinase whose protein expression situation in downstream behind the specific reticent MEK1 gene has been proved conclusively the vital role of MEK1 gene in melanoma propagation.

Therefore, the present invention proposes the application of described siRNA sequence in preparation treatment human melanin tumor medicine.

The present invention has utilized the siRNA technical research behind specific reticent MEK1 gene; After host cell is depended on the virus infection of ERK path; Downstream kinase whose protein expression situation has been proved conclusively the effect of the anti-virus aspect that siRNA brings into play through blocking-up ERK path.

This result provides foundation for utilizing rational siRNA technology to carry out antiviral research on the one hand; On the other hand; The present invention is incorporated into active result among the siRNA with the heteronuclear glycosides, and to be applied to herpes simplex virus I I type (HSV-2) and enterovirns type 71 (EV71) be in the treatment of the virus of utilizing host ERK path of representative; The active testing result of protein level and rna level shows; The siRNA that the heteronuclear glycosides of 8 D-forms of sense chain is modified can significantly improve its reticent activity, more helps antiviral treatment.

Therefore, the present invention has proposed the application of described siRNA sequence in the preparation antiviral again, and said virus is the virus that dependent cells RAF/MEK/ERK signal path duplicates; Preferably, described virus includes but not limited to influenza A virus, herpes simplex virus I I type (HSV-2), the sick virus of Borna, poxvirus, human immunodeficiency virus type 1, Coxsackie virus, hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis E virus (HEV).

Compared to prior art, the invention has the advantages that:

1, the siRNA of heteronuclear glycosides modification provided by the invention has characteristics such as physico-chemical property is stable, combined coefficient height; Product self has the better biological activity than natural siRNA.

2, through comprehensively investigating D-; L-heteronuclear glycosides mixes siRNA in different loci; Serum stability and the reticent activity of siRNA have been improved; And found to improve the chemically modified strategy of reticent mRNA ability of siRNA and anti-virus ability, for the clinical application of the siRNA of heteronuclear glycosides modification provides certain directive significance.

3, heteronuclear glycoside compound provided by the invention can improve the enzyme stability through the siRNA of its modification significantly; And can keep or improve the gene silencing efficient of oligonucleotide, thereby make siRNA can better play the active effect of silencer through its modification; In addition, the siRNA that positive-sense strand is modified has the effect of the effect that suppresses to miss the target, and wherein carries out the modification of D-heteronuclear glycosides and L-heteronuclear glycosides on 8 on sense chain, and the siRNA sequence of modification is compared the siRNA sequence of natural unmodified, has better stability and reticent active.

Description of drawings:

Fig. 1 is ERK signal conduction pathway (a RAS-RAF-MEK-ERK cascade signal path);

Fig. 2 is the cell growth curve of A375 for malignant melanoma cell;

Fig. 3 is an A375 cell transfecting density test for malignant melanoma cell;

Fig. 4 is the test of A375siRNA transfection after effect time for malignant melanoma cell;

Fig. 5 is protein expression test after the A375 different concns siRNA transfection for malignant melanoma cell;

Fig. 6 is the activity test of the siRNA that natural siRNA and heteronuclear glycosides are modified among the A375 for malignant melanoma cell;

The influence that the siMEK1 sequence 8D/8L that Fig. 7 modifies for the heteronuclear glycosides duplicates HSV-2 under the situation that effectively reduces the MEK1 expression;

The anti-HSV-2 virus activity Western blotting that Fig. 8 modifies siRNA for the heteronuclear glycosides measures the result;

The anti-EV71 virus activity Western blotting that Fig. 9 modifies siRNA for the heteronuclear glycosides measures the result.

List of abbreviations

The disclosed the present invention of this paper uses following chemical name:

The AIBN Diisopropyl azodicarboxylate

Bu ₃SnH tri-n-butyl tin hydrogen

The Bz-benzoyl-

DBU 1,8-diazabicyclo (5.4.0) 11-7-alkene

DMAP 4-Dimethylamino pyridine

The DMTr dimethoxytrityl

DMF N-formyl n n dimetylaniline

The Py pyridine

The TBAF tetrabutyl ammonium fluoride

TBDMS-tertiary butyl dimethylsilyl

The TFA trifluoroacetic acid

TMS-2,4,6-Three methyl Benzene alkylsulfonyl

The Ts-p-toluenesulfonyl

9-BBN 9-boron two ring [3.3.1] nonanes

Embodiment

Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.

The solid phase synthesis of the siRNA sequence that heteronuclear glycosides phosphoramidite monomer mixes

The solid phase synthesis of oligonucleotide is initial from its 3 ' end, one by one the coupling nucleoside monomers.The deoxynucleoside that has 5 '-DMTr protection, and its 3 '-position and controlled granulated glass sphere (control pore glass CPG) connects through the succsinic acid bridging, obtains corresponding A, T, and C, G Columns is used for oligonucleotide synthetic solid phase carrier.Usually the nucleoside monomers of appendix on CPG oligonucleotide chain 3 '-terminal nucleosides just, 3 ' the terminal overhang that is characterized as of siRNA strand is the dTdT base, therefore in siRNA synthetic, uses the CPG carrier of end as the T base.

The solid phase synthesis of oligonucleotide chain is to carry out according to the program that configures, and nucleosides of general every coupling is a circulation, and each circulation comprises four reactions: 1. trichoroacetic acid(TCA) solution removes 5 '-DMTr protection; 2. nucleoside coupling reaction, to improve reaction efficiency, the common gradation sample introduction of phosphorus acylated monomer is to improve the coupling yield by 5-second sulfydryl-1H-tetrazolium catalysis; 3. block (capping) reaction is sealed unreacted 5 '-OH with ethanoyl, prevents the formation of short chain; 4. oxidizing reaction is generally used the Py-H of iodine ₂O-THF solution is oxidized to pentavalent phosphorus with three valent phosphors fast.After 5 '-terminal nucleoside coupling is accomplished, can select to remove or keep the DMTr protection base on this nucleosides, to synthesize with the DMT-off mode, the method for the separation and purification of next step oligonucleotide chain adopts anion-exchange column to carry out the separation of oligonucleotide.

The solid phase synthesis of the DNA oligonucleotide chain that the heteronuclear glycosides is modified; The phosphorus acylated monomer that only needs to replace natural nucleus glycoside with the phosphorus acylated monomer of heteronuclear glycosides is in corresponding position sample introduction coupling; But because the coupling yield under the heteronuclear glycosides normal condition is on the low side, the coupling time (300 seconds/inferior) behind the employing increase heteronuclear phosphorus acylated monomeric sample introduction number of times of glycosides (3 times) and the each sample introduction is to guarantee whole oligonucleotide chain synthetic yield.

During without the single stranded RNA oligonucleotide modified, each total coupling time that circulates is 30 minutes (600 seconds/inferior x 3 times) synthetic, and when with the coupling of heteronuclear glycosides monomer, each total coupling time that circulates increases to 45 minutes (900 seconds/inferior x 3 times).

Sense chain and the antisense chain warp of synthetic siRNA are crossed the separation purifying, and the pairing of on the PCR appearance, annealing respectively promptly obtains required double-stranded siRNA, can be used for bioactive test.

According to the present invention, the sequence of the siRNA that natural siRNA of synthetic and heteronuclear glycosides are modified can be but be not limited to the MEK-1 gene to the people.

According to the present invention, the resin of solid phase synthesis is the CPG resin, also can be other known synthetic resin that is applied to oligonucleotide in this area.

According to the present invention; The circulation of oligonucleotide solid phase synthesis comprises deprotection, coupling, sealing and 4 steps of oxidation; The organic reagent that uses can be but be not limited to dichloro acetic acid; 5-second sulfydryl 1-H tetrazolium, the tetrahydrofuran solution of diacetyl oxide pyridine solution and iodine, the organic solvent that uses on the synthesizer are analytically pure anhydrous acetonitrile.

According to the present invention, the cutting condition of oligonucleotide on the solid phase can for but do not limit methylamine alcohol solution or ammoniacal liquor/methylamine solution under 60 ℃, to cut at shaking table, be 30min-60min clipping time.

According to the present invention, the condition that removes of the last protection base TBDMS of 2 '-OH can be but is not limited to TBAF and under 55 ℃, on shaking table, removes protection in the oligonucleotide, and the reaction times is 1.5hr-3.0hr.

According to the present invention, the separation of the chromatographic column of oligonucleotide can be but be not limited to the Dionex-DNAPac-PA200 anion-exchange column that the wash-out ion can be but be not limited to perchlorate.

According to the present invention, the desalting treatment of the oligonucleotide after the separation can be but be not limited to use the Size Exclusion Chromatograph SEC of GE-HiTrap to separate.

In order to understand the present invention better, describe through embodiment.

All solvents, raw material and reagent are analytical pure or CP as not naming especially.The no water treatment of solvent is carried out according to ordinary method.

Instrument and method that product separates, identifies: tlc silica gel GF ₂₅₄, column chromatography silochrom (200-300 order), silica gel H be Haiyang Chemical Plant, Qingdao and produce; TLC develops the color through the 254nm ultraviolet detection or with 5% phosphomolybdic acid ethanol solution; Fusing point uses XT-4A type fusing point appearance to measure, and TM is not proofreaied and correct; Ir spectra uses DE-983G determination of infrared spectroscopy, pressing potassium bromide troche; Syrup is used liquid-film method.FAB and MALDI-TOF are by VG-ZAB-HS and Bruker APEX ^TMII, HR-FAB uses Bruker BIFLEX ^TMThe III mass spectrograph is measured; UV spectrum uses Pharmacia LKB Biochrom 4060 spectrophotometric determinations.Nuclear magnetic resonance spectrum uses Varian VXR-500, JEOL AL300, Bruker Advance 300 nmr determinations, and hydrogen spectrum, carbon spectrum are interior mark with TMS; The phosphorus spectrum is with 85%H ₃PO ₄Be external standard; Ultimate analysis uses the PE-240C elemental analyser to measure.74900 types that syringe pump is produced with Cole-Parmer company.HPLC uses Gilson performance liquid appearance, uses Delter Paker C-18 semipreparative column to separate.The microwave synthesizer uses the full-automatic microwave synthesizer of Sweden Biotage.

The solid phase synthesis of the RNA that embodiment 1. heteronuclear glycosides mix

The synthetic employing Applide Biosystems model 394DNA Synthesizer solid phase synthetic instrument of RNA.The normal phosphorus acylated monomer (rA of nucleosides ^Bz, rC ^Ac, rU, rG ^Ac) buy the phosphorus acylated monomer dT of deoxythymidine, CPG (CPG-dT), CAP-A and CAP-B, oxidation I from Shanghai JiMa pharmacy Technology Co., Ltd ₂Liquid, Cl ₃AudioCodes biotechnology company buys CCOOH from Beijing.5-second sulfydryl 1H-tetrazole solution is bought from ltd of Traditional Chinese Medicine Research & Development Center (Beijing).

Synthetic scale :～1.5 μ mol

The preparation of the phosphorus acylated monomer solution of nucleosides: weighing under the argon shield, add anhydrous acetonitrile, be made into 0.12M solution.

The preparation of 1H-tetrazole solution: weighing under the argon shield, add anhydrous acetonitrile, be made into 0.5M solution

The preparation of the phosphorus acylated monomer solution of heteronuclear glycosides: { [3-O-(4 for 5S-in weighing under the argon shield; 4 '-dimethoxytrityl methyl)-methyl]-4R-O-[(2-cyanoethyl-N; N '-di-isopropyl)-the phosphoramidite base]-3S-(thymine base-1-yl) }-THF (172mg; 0.231mmol), add anhydrous acetonitrile (2.5ml), be made into 0.092M solution; { the 5S-[3-O-(4,4 '-dimethoxytrityl methyl)-methyl]-4R-O-[(2-cyanoethyl-N, N '-di-isopropyl)-phosphoramidite base]-3S-(N of weighing under the argon shield ⁶-benzoyl--adeninyl-9 '-yl) }-(112mg 0.130mmol), adds anhydrous acetonitrile (1.5ml) to THF, is made into 0.087M solution; { the 5S-[3-O-(4,4 '-dimethoxytrityl methyl)-propyl group]-4R-O-[(2-cyanoethyl-N, N '-di-isopropyl)-phosphoramidite base]-3S-(N of weighing under the argon shield ⁶-benzoyl--adeninyl-9-yl) }-(300mg 0.339mmol), adds anhydrous acetonitrile (2.8ml) to THF, is made into 0.121M solution; { [3-O-(4 for 5S-in weighing under the argon shield; 4 '-dimethoxytrityl methyl)-propyl group]-4R-O-[(2-cyanoethyl-N; N '-di-isopropyl)-the phosphoramidite base]-3S-(thymine base-1-yl) }-THF (230mg; 0.298mmol), add anhydrous acetonitrile (2.2ml), be made into 0.135M solution.

Adopt the method for solid phase synthesis to synthesize siRNA sequence and the siRNA sequence that the heteronuclear glycosides mixes of the unmodified of MEK-1 gene; SiRNA sequence before modifying is:

SiMEK1 positive-sense strand (S): 5 '-GCAACUCAUGGUUCAUGCUdtdt-3 '

Antisense strand (AS): 3 '-dtdtCGUUGAGUACCAAGUACGA-5 '

When synthetic, 1,8,9,11 of the positive-sense strand of siRNA sequence or 12 wherein mix on one or the multidigit (I) or (II) shown in heteronuclear glycosides phosphoramidite monomer, replace the natural nucleus glycoside phosphoramidite monomer to carry out coupling in the corresponding position.

Or when synthetic, 1,4,5,11 of the antisense strand of siRNA sequence or 12 wherein mix on one or the multidigit (I) or (II) shown in heteronuclear glycosides phosphoramidite monomer, replace the natural nucleus glycoside phosphoramidite monomer to carry out coupling in the corresponding position.

1 of above-described antisense strand is meant antisense strand: 5 ' end beginning first Nucleotide, i.e. " A " in 3 '-dtdtCGUUGAGUACCAAGUACGA-5 ' nucleotide sequence.All the other positions by that analogy.

Synthesis step: each about 40mg CPG-dT of weighing packs into and synthesizes in the post, sets synthesis program (standard program), each synthetic totally 84 step.Normal nucleoside monomers coupling 3 times, each 600 seconds, heteronuclear glycosides monomer coupling 3 times each 900 seconds, amounted to coupling time 45 minutes.

The cutting of RNA, deprotection: behind the end of synthesis, take off CPG, add strong aqua/methylamine solution, 60 ℃ of constant temperature, shaking table oscillatory reaction 30min cuts down and removes partial protection base, whiz with oligonucleotide from CPG.Add THE/HF/NMP (the solution fluorinion concentration is 1.4M) 150 μ L again, 55 ℃ of temperature controls, shaking table oscillatory reaction 1.5 hours; Reaction finishes the back and adds 3M sodium acetate soln 25 μ L and 1ml propyl carbinol, with mixing solutions at-70 ℃ of freezing 1hr down, then with mixing solutions at 4 ℃ of centrifugal 30min of following 10000r; Carefully remove supernatant, deposition is used 70% washing with alcohol, carefully draws washings; White precipitate is placed on clean cabinet Rio 3-5min, product is placed-70 ℃ preserve down.

The separation and purification of RNA: mixture is with the DEPC water dissolution, HPLC (Sephedax G-25,50%CH ₃CN in H ₂O) desalination, whiz.Then HPLC mode purifying adopts Dionex-DNA Pac-PA200 anion-exchange column gradient elution, 0-40min, 10%A liquid-30%A liquid (B liquid: 0.02M tris-HClO ₄Damping fluid, pH=8.0 contains 10%MeCN; A liquid: 0.4M NaClO ₄In B liquid), flow velocity 1.2ml/min.The lyophilize products therefrom, DEPC water redissolves, HPLC (Sephedax G-25) desalination, lyophilize obtains the target single stranded RNA ,-78 ℃ of preservations.

Active testing-siQuantTM method of the siRNA that embodiment 2. heteronuclear glycosides are modified

The reticent active testing of the siRNAs that the heteronuclear glycosides is modified; Adopt the siQuantTM method; With Lipofectamine 2000 as transfection reagent; It is as shown in Figure 3 to the inhibition efficient Dual-Luciferase experiment PRELIMINARY RESULTS of people's malignant melanoma cell (A-375) to detect siRNAs, and the siRNA that the heteronuclear glycosides is modified in the experimental example 3 is when heteronuclear glycosides during in siRNA positive-sense strand 5 '-end (S2/As), 3 '-end (S4/As) or mid-way (S5/As) modification; All little to suppressing effectiveness affects, can keep the activity of good reticent said target mrna; And the heteronuclear glycosides is when the siRNA antisense strand is modified; It suppresses efficient and all obviously descends; The heteronuclear glycosides is modified siRNA; Can also keep moderate inhibiting rate (54%) 5 '-terminal (S/As2) when modifying, when (S/As3) modified near the cleavage site in the middle of antisense strand, siRNA had only kept very weak inhibition active (27%).

This shows; SiRNA antisense strand middle part and 5 '-end does not tolerate the modification of heteronuclear glycosides; And these two sites also be unwind with siRNA, site that nicking activity is closely bound up; The heteronuclear glycosides mixes the change that causes double-stranded conformational etc., and the difference of this body structure, possibly produce considerable influence to the 26S Proteasome Structure and Function of RISC.The siRNA positive-sense strand then can tolerate the modification of heteronuclear glycosides largely, and is active unaffected.

Infer that according to preliminary test-results the heteronuclear glycosides is modified in positive-sense strand, do not influence the reticent active of siRNA, and might suppress positive-sense strand and get into RISC and produce " off-target " effect.

Active testing-Western Blot method of the siRNA that embodiment 3. heteronuclear glycosides are modified

The reticent active testing of the siRNAs that the heteronuclear glycosides is modified, as transfection reagent, detect siRNAs is the inhibition efficient of A375 to people's malignant melanoma cell to employing Western Blot method with Lipofectamine 2000.At first investigated the growth curve of A375 cell, found that cell density cell growth tendency when 30%-40% is better, therefore we adopt the culturing cell (see figure 2) of 30%-40% density in follow-up test; Adopt high-density group (H70%), middle density group (M40%) and low density group (L20%) during the siRNA transfectional cell; SiRNA transfection concentration is 30nM; Only add the cell negative contrast of conduct (control) group that transfection reagent is handled; Use concentration as the natural siRNA cells transfected of 30nM as over against according to (30nM), untreated cell is as blank (Blank) group, the running gel result shows in the low density group MEK1 and β-actin protein expression level is low is difficult for detecting; Middle density group and high-density histone expression level are better, have adopted middle density group cell to carry out the transfection (see figure 3) in the follow-up test; After the natural siMEK1 transfection 2,4,6,24, the 48h collecting cell measures, the transfection of the natural siMEK1 up time of 30nM all had effect in the mRNA level in 4 to 24 hours to A375 clone.Prolong in time afterwards, ability to function reduces, and progressively recovers normal level, and MEK1-P1, MEK1-P2 are the PCR result when using different primers.This administration time that just uses siRNA to treat for us provides certain reference data (see figure 4); Investigate different siRNA at 5nM, the proteic expression level of reticent MEK1 under 10nM and the 30nM concentration, it is higher that experimental result is presented under 10nM and the 30nM reticent efficient, and the not good (see figure 5) of reticent efficient under the 5nM; Reticent as a result the time what investigate siRNA sequence that the heteronuclear glycosides modifies, shown in the result of Western Blot method, can see the heteronuclear glycosides in the modification result on 8 on the sense chain, the modification activities of L-configuration heteronuclear glycosides (8L)

Result and natural siRNA sequence (NC) are approaching; And the modification activities result of D-form heteronuclear glycosides (8D) will obviously be superior to the modification of L-configuration heteronuclear glycosides and natural siRNA sequence; Because the heteronuclear glycosides is modified the main space microstructure that changes decorating site, we infer that tentatively the base of 8 on sense chain possibly have certain influence to siRNA performance biological activity near the avtive spot of enzyme in the RISC complex body as a result according to this; In the accompanying drawing; Control: normal cell, Mu: negative control, 8-MeO: the siRNA (see figure 6) that transfection 8-MeO modifies.

The anti-HSV 2 virus activities test of the siRNA that embodiment 4. heteronuclear glycosides are modified

The reticent active testing of the siRNAs protein level that the heteronuclear glycosides is modified, be through Western blotting method measure siMEK 1 pairing MEK 1 albumen and with the expression of the closely-related HSV-2 late protein of ERK path gB.GB gp is a kind of late protein, and content is maximum in the cell that infects, and is the conservative albumen of simplexvirus family camber.

In order to investigate in HEK293 clone; The influence that the siMEK1 sequence 8D/8L that the heteronuclear glycosides is modified duplicates HSV-2 under the situation that effectively reduces the MEK1 expression; We have chosen 30nM is that the final concentration of siRNA transfection subtracts effect to guarantee that tangible MEK1 strikes; The virus quantity that uses is 1.5MOI, has detected the expression of MEK1 albumen and HSV-2 late protein gB through Western blot.

As shown in Figure 7,24h behind the cell infection compares with HSV-2 group, Mutant group, and the CPE phenomenon of natural siMEK1,8D, 8L group is lighter.As shown in Figure 8, the MEK1 that Western blotting result is presented at 8D, 8L sequence set under the final concentration of 30nM expresses has obvious reduction than control group, and 8D subtracts effect to striking of MEK1 and slightly is superior to natural siMEK1,8L sequence.The proteic variation tendency basically identical of the variation of viral protein and MEK1.Because viral protein expression reduces than control group, explains that under the final concentration of 30nM natural siMEK1,8D, 8L sequence all can effectively suppress duplicating of HSV2, and antiviral activity is relatively preferably revealed in the 8D sequence table.SiQuantTM method gained experimental result is consistent in this experimental result and the instance 2, and more deep.

The anti-EV71 virus activity test of the siRNA that embodiment 5 heteronuclear glycosides are modified

According to the method identical, promptly utilize Western blotting to measure the activity that the heteronuclear glycosides is modified the anti-EV71 virus of siRNA, result such as Fig. 9 with embodiment 3.It is thus clear that anti-EV71 virus result and the anti-HSV2 result of heteronuclear glycosides modification siRNA are consistent.

This paper shows and the information described in detail is enough to realize above-mentioned purpose of the present invention, so the preferred embodiments of the invention represent theme of the present invention, and this themes as the present invention and extensively contains.Scope of the present invention contains other conspicuous for a person skilled in the art embodiment fully; Therefore; Scope of the present invention is not limited by any content except that accompanying claims; Wherein except offering some clarification on, the singulative of used element is not meant " one with unique ", and is meant " one or more ".Concerning persons skilled in the art, it is for referencial use that therefore the Equivalent on structure, composition and the function of all known above-mentioned embodiment preferred and additional embodiment part introduces this paper, and attempt to be contained by claim of the present invention.

In addition, do not need certain equipment or method to express each problem that the present invention solves, because they all have been included within the claim of the present invention.In addition, all parts, the composition in the open fact of the present invention no matter, perhaps method steps whether in claim by clearly narration, they all not have contribution to the public.But, concerning those of ordinary skills, clearly under the prerequisite that does not deviate from the essence of the present invention liking enclosed in the claim to be illustrated and scope, can on form, reagent and synthetic details, make various changes and modification.

Sequence table

< 110>Peking University

< 120>a kind of utilize the heteronuclear glycosides to the siRNA sequence carry out chemically modified method, and products thereof and use

<130>KLPI110886

<170>PatentIn?3.5

<210>1

<211>21

<212>RNA

<213>siMEK1?sense?strand(S)

<400>1

GCAACUCAUGGUUCAUGCUdtdt

<210>2

<211>21

<212>RNA

<213>siMEK1?antisense?strand(AS)

<400>2

AGCAUGAACCAUGAGUUGCdtdt

Claims

1. method of utilizing the heteronuclear glycosides siRNA sequence to be carried out chemically modified; The positive-sense strand of the siRNA sequence that it is characterized in that modifying in desire and/or one or more sites of antisense strand are mixed the heteronuclear glycoside compound shown in Formula I or the Formulae II and are carried out coupling in the corresponding position to replace the natural nucleus glycoside compound

2. method according to claim 1 is characterized in that before carrying out the siRNA modification, the heteronuclear glycoside compound shown in Formula I and/or the Formulae II being prepared into the heteronuclear glycosides phosphoramidite monomer shown in Formulae II I and/or the Formula I V respectively,

3. method according to claim 2; It is characterized in that using solid phase synthesis; Adopt phosphoramidite method, on dna synthesizer, replace the natural nucleus glycoside phosphoramidite monomer to carry out coupling in the corresponding position to be incorporated in the synthetic sequence the described heteronuclear glycosides of one or several claim 2 phosphoramidite monomer; Nucleosides of every coupling is a circulation, and each circulation comprises four reactions: take off DMT, coupling, sealing, oxidation.

4. compound method according to claim 3, the condition that it is characterized in that the synthetic DNA oligonucleotide chain are to adopt to increase the phosphorus acylated monomeric sample introduction number of times to 3 of heteronuclear glycosides; Coupling time behind each sample introduction be 300 seconds/inferior; The condition of the RNA oligonucleotide chain that synthetic heteronuclear glycosides is modified be the coupling time behind each circulation sample introduction increase to 900 seconds/inferior, coupling 3 times.

5. the sequence of the siRNA that modifies according to each described method synthetic heteronuclear glycosides of claim 1-4.

6. the sequence of siRNA according to claim 5 is characterized in that the siRNA sequence to people MEK-1 gene that described sequence is modified for the heteronuclear glycosides.

7. the sequence of siRNA according to claim 6, the siRNA sequence before it is characterized in that modifying is:

Positive-sense strand: 5 '-GCAACUCAUGGUUCAUGCUdtdt-3 '

Antisense strand: 3 '-dtdtCGUUGAGUACCAAGUACGA-5 '.

8. siRNA sequence according to claim 7; It is characterized in that wherein mixing the heteronuclear glycosides shown in Formula I or the Formulae II on one or the multidigit 1,8,9,11 of the positive-sense strand of siRNA sequence or 12; Replace natural nucleus glycoside to carry out coupling in the corresponding position, preferred, on 8 of the positive-sense strand of siRNA sequence, mix the heteronuclear glycosides shown in Formula I or the Formulae II; Replace natural nucleus glycoside to carry out coupling in the corresponding position; Preferred, on 8 of the positive-sense strand of siRNA sequence, mix the heteronuclear glycosides shown in the Formulae II, replace natural nucleus glycoside to carry out coupling in the corresponding position.

9. the application of each described siRNA sequence of claim 6-8 in preparation treatment human melanin tumor medicine.

10. the application of each described siRNA sequence of claim 6-8 in the preparation antiviral; Said virus is the virus that dependent cells RAF/MEK/ERK signal path duplicates; Preferably, said virus comprises influenza A virus, herpes simplex virus I I type (HSV-2), the sick virus of Borna, poxvirus, human immunodeficiency virus type 1, Coxsackie virus, hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis E virus (HEV).