WO2012010103A1 - Aptamère et son procédé de préparation - Google Patents

Aptamère et son procédé de préparation Download PDF

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Publication number
WO2012010103A1
WO2012010103A1 PCT/CN2011/077443 CN2011077443W WO2012010103A1 WO 2012010103 A1 WO2012010103 A1 WO 2012010103A1 CN 2011077443 W CN2011077443 W CN 2011077443W WO 2012010103 A1 WO2012010103 A1 WO 2012010103A1
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Prior art keywords
aptamer
target protein
enzyme
ubiquitin
virus
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PCT/CN2011/077443
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English (en)
Chinese (zh)
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蒋宇扬
席真
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刘国平
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Publication of WO2012010103A1 publication Critical patent/WO2012010103A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
    • C12N2330/31—Libraries, arrays

Definitions

  • the present invention relates to an aptamer and a preparation method and application thereof, and more particularly to an aptamer, an aptamer preparation method, a recombinant vector, a transfected virus, and a ubiquitinated target protein removed from an organism.
  • the I protein is an important component of various organisms including humans and participates in various physiological activities.
  • Eukaryotic cells contain between 6,000 and 30,000 protein synthesis genes encoding at least an equal number of proteins. Organisms synthesize large amounts of protein in the body while also consuming or degrading unwanted proteins.
  • the ubiquitin-proteasome pathway is an important pathway for the selective degradation of intracellular proteins.
  • the UPP pathway involves ubiquitin, ubiquitinating enzymes, proteasomes, etc., and the ubiquitinating enzymes include ubiquitin
  • the ubiquitin-activating enzyme (hereinafter referred to as El), the ubiquitin-conjugating enzyme (hereinafter referred to as E2), and the ubiquitin protein ligase (hereinafter referred to as E3).
  • El The ubiquitin-activating enzyme
  • E2 the ubiquitin-conjugating enzyme
  • E3 the ubiquitin protein ligase
  • Ubiquitin is a polypeptide consisting of 76 amino acids.
  • the action of ubiquitin molecule I in El, E2 and E3 combines with the target protein to form a ubiquitin chain in three steps: (1) Ubiquitin (Ub) is activated by El and passes sulfur The ester bond binds to the reactive cysteine (Cys) of El; (2) the activated ubiquitin is transferred to E2 capable of binding to E3; (3) E3 transfers the activated ubiquitin directly from E2 to the target protein, wherein E3 forms a thioester bond with the activated ubiquitin and then transfers it to the target protein.
  • Monoubiquitination of the target protein allows the target protein to be recognized by many ubiquitin recognition domains within the cell, thereby altering the target egg; white activity and location.
  • the formation of a K48-linked polyubiquitin chain on the target protein allows the target protein to be recognized and degraded by the 26S proteasome.
  • the proteasome itself does not have the ability to select proteins. Only proteins that are labeled by ubiquitin molecules and recognized by E3 can be degraded in the proteasome.
  • K63-linked polyubiquitin chains are involved in cellular activities including signaling and DNA repair (Yili Yang et al, Cancer Sci. 2009 January; 100(1): 24-28). Therefore, researchers around the world are studying the use of the UPP pathway to remove proteins associated with disease development, such as El, E2 and E3, to facilitate the ubiquitination process, but
  • the object of the present invention is to provide a technique capable of effectively promoting the removal of a target protein which can be ubiquitinated.
  • the UPP pathway binds ubiquitin to a ubiquitinable target protein by the action of a ubiquitinating enzyme including El, E2 and E2 to identify the target protein as a protein to be degraded, and then recognizes the protein to be degraded by the proteasome. And degrading it, so ubiquitin is very important for the identification of the target protein, and the inventors of the present invention contemplate that ubiquitin can be promoted if a substance capable of effectively binding the ubiquitinating enzyme and the target protein can be found.
  • the enzyme catalyzes the binding of ubiquitin to the target protein, thereby exerting the labeling effect of ubiquitin on the target protein, and effectively promoting the removal of the target protein.
  • the inventors of the present invention have unexpectedly discovered that ubiquitinating enzymes and target proteins can be effectively combined by aptamer technology.
  • an aptamer comprising a sequence capable of specifically binding to a ubiquitinable target protein and capable of specifically binding to ubiquitination The sequence of the enzyme.
  • a method of preparing an aptamer comprising the steps of:
  • an aptamer obtained by the method of the second aspect of the invention.
  • a recombinant vector comprising the aptamer of the first or third aspect of the invention is provided.
  • a virus transfected with the recombinant vector of the fourth aspect of the invention is provided.
  • a method for removing a ubiquitinable target protein in an organism comprising administering the virus of the fifth aspect of the invention to an organism is provided.
  • a method for the treatment of a disease associated with a ubiquitinable protein of interest comprising administering the virus of the fifth aspect of the invention to an organism.
  • the virus of the fifth aspect of the invention for the preparation of a medicament for the treatment of a disease associated with a ubiquitinable target protein.
  • a pharmaceutical composition for treating a disease associated with a ubiquitinable target protein characterized in that the pharmaceutical composition comprises the virus according to the fifth aspect of the invention Active ingredient.
  • a method for inhibiting gene expression in an organism characterized in that the method comprises administering the virus of the fifth aspect of the invention to an organism, the ubiquitinable target Proteins are key regulatory proteins for the expression of such genes.
  • the virus is transfected with a recombinant vector containing the aptamer, and when the virus is administered to an organism, the virus can replicate in the cells of the organism, and the aptamer is also in the cells of the organism.
  • the replicated aptamer since the aptamer includes a sequence capable of specifically binding to a ubiquitinating enzyme and a sequence capable of specifically binding to a ubiquitinated target protein, the replicated aptamer is capable of efficiently binding to a ubiquitinated target protein simultaneously And a ubiquitinating enzyme to pull the ubiquitinated target protein and the ubiquitinating enzyme protein to promote binding of the ubiquitin to the target protein, and identify the ubiquitinated target protein as a protein to be degraded, and It is then recognized and degraded by the proteasome within the cell.
  • the ubiquitinating enzyme catalyzes the binding of ubiquitin to the ubiquitinated target protein, followed by ubiquitination.
  • the target protein is recognized and degraded by the proteasome in the cell, whereby the aptamer is released from the state of binding to the ubiquitinable target protein, and the released aptamer can recombine the new ubiquitinated target protein and pan. It catalyzes the enzyme and promotes the degradation of the new ubiquitinated target protein. Therefore, the aptamer can be recycled, and even if a small amount of aptamer is administered, it can promote the ubiquitination of the target protein ubiquitin.
  • Figure la is a Western-Blot diagram of an aptamer promoting Thrombin ubiquitination in an embodiment of the present invention
  • Figure lb is another Western-Blot diagram of an aptamer promoting Thrombin ubiquitination in one embodiment of the present invention
  • Electropherogram 2 is a polyacrylamide gel of a Thrombin that degrades ubiquitin by proteasome in an embodiment of the present invention
  • FIG. 3 is a Western-Blot diagram of an aptamer promoting Thrombin ubiquitination according to another embodiment of the present invention
  • FIG. 4 is a view showing another embodiment of the present invention, a proteolytic degradation of ubiquitinated Thrombin polyacrylamide gel Electropherogram. detailed description
  • the aptamers provided by the present invention include a sequence capable of specifically binding to a ubiquitinable target protein and a sequence capable of specifically binding to a ubiquitinating enzyme.
  • the aptamer is a nucleic acid aptamer, and refers to an oligonucleotide capable of specifically binding a target (nucleic acid molecule, or non-nucleic acid molecule such as polypeptide, protein, etc.) with high affinity.
  • the aptamer may be R A or DNA, may be natural or synthetic, and may be unmodified or modified.
  • the aptamer may have a sequence length of 40-500 bases.
  • the aptamer of the present invention contains both a sequence capable of specifically binding to a ubiquitinated target protein and a sequence capable of specifically binding to a ubiquitinating enzyme, and thus can simultaneously be combined with a ubiquitinable target protein and a ubiquitinating enzyme. Binding, thereby linking the ubiquitinated target protein and the ubiquitinating enzyme.
  • the sequence capable of specifically binding to a ubiquitinable target protein can bind to a target protein by any specific binding means, including direct specific binding and indirect specificity.
  • the direct specific binding is, for example but not limited to, the sequence is a ligand of the target protein (specific binding to a protein of interest);
  • the indirect specific binding is, for example but not limited to, the sequence is The ligand of the protein, polypeptide or nucleic acid to which the protein of interest is linked, such that the sequence is indirectly linked to the protein of interest by specific recognition between the sequence and the protein, polypeptide or nucleic acid.
  • the specific binding described in the sequence capable of specifically binding to the ubiquitinating enzyme also includes both the above direct specific binding and indirect specific binding.
  • the sequence capable of specifically binding to the ubiquitinating enzyme is a ligand of platelet-derived growth factor-BB (PDGF-BB), although the ligand cannot be directly directly linked to the ubiquitinating enzyme E3 Binding, however, when the ubiquitinating enzyme is provided in the form of a fusion protein with PDGF-BB, the ligand can achieve indirect specific binding to E3 by recognizing and binding to PDGF-BB.
  • PDGF-BB platelet-derived growth factor-BB
  • the aptamer of the present invention may further comprise other sequences, preferably, the other sequences do not negatively affect Binding of the ubiquitinated target protein or ubiquitinating enzyme, and the other sequence is capable of stabilizing a conformation capable of specifically binding to a ubiquitinated target protein and a conformation capable of specifically binding to a ubiquitinating enzyme sequence In order to ensure that each of them performs the recognition function normally.
  • the aptamer against the ubiquitinating enzyme is not directly used, but the PDGF-BB having the known aptamer apt-1 is fused to the ubiquitinating enzyme, and then Apt-1 is linked to the target protein thrombin (Thrombin) by apt-3 via a linker (L-1), thereby achieving degradation of the target protein.
  • the aptamer comprises apt-l, L1 and apt-3, the sequence of which is shown in SEQ ID No: 1.
  • the ubiquitinating enzyme may be at least one of El, E2 and E3, preferably El, E2 and E3.
  • the aptamer may be a sequence that specifically binds to one of El, E2, and E3, preferably a sequence that specifically binds to E3; and may also include multiples with El, E2, and A sequence in which two or three of E3 specifically bind.
  • the aptamer comprises a plurality of sequences that specifically bind to two or three of El, E2 and E3, respectively; more preferably, the aptamer comprises three and El, E2 and A sequence in which E3 specifically binds.
  • the preparation method of the aptamer provided by the invention comprises the following steps:
  • the ubiquitinating enzyme may be at least one of El, E2 and E3, preferably El, E2 and E3.
  • the aptamer libraries used in the methods of the invention include R A libraries, DNA libraries, and libraries containing modified nucleotides.
  • the aptamer library used in the methods of the invention may be an existing aptamer library, which may be a new aptamer library constructed by the following steps:
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment. It is a large-capacity random aptamer (oligonucleotide) library combined with PCR in vitro amplification technology, which is exponentially rich. The oligonucleotides which specifically bind to the target molecule are subjected to several rounds of repeated in vitro screening and amplification to obtain an oligonucleotide aptamer which specifically binds to the target molecule.
  • An aptamer library typically has at least 10 12 to 10 18 independent aptamers.
  • An aptamer is generally composed of a random sequence of a certain length in the middle and a fixed sequence of the 5' and 3' ends. The fixed sequence at both ends is generally 20 to 25 bases in length, which serves to increase the stability of the library and prepare for amplification. There are four possibilities for each nucleotide position in the random region. If the random sequence length is N, the diversity of the random sequence is 4 N , that is, the library has a library capacity of 4 N .
  • the specific procedure for screening involves incubating the aptamer library and the target molecule at a temperature (usually 37 ° C), and in the first few cycles there may be only a small number (about 0.1% to 0.5%) of the sequence interacting with the target molecule,
  • the bound complex can be separated from the unbound sequence by separation techniques such as affinity chromatography, fiber membrane filtration, and the like.
  • the isolated sequences were re-amplified to produce a secondary library for the next round of screening. This leads to a process of repeated screening and enrichment.
  • the amplification step also includes reverse transcription of the RA sequence, and the resulting double-stranded DNA library is transcribed to generate a secondary RA library. After several rounds of screening and enrichment, the affinity of the isolated random sequence to the target material is no longer increased, and the obtained oligonucleotide aptamer can be screened.
  • the recombinant vector provided by the present invention contains the aptamer of the present invention.
  • the recombinant vector of the present invention can be constructed by inserting the aptamer into a conventional vector by a conventional recombinant vector preparation method, for example, using a restriction enzyme.
  • a recombinant vector is constructed by inserting the aptamer into a plasmid vector.
  • plasmid vectors include, but are not limited to, pBR322 plasmid vector, pUC plasmid vector, pMOB45 plasmid vector, pACYC plasmid vector, pSClO1 plasmid vector, colEl plasmid vector and the like.
  • Methods for constructing a recombinant vector by inserting a stretch of sequence into a conventional vector are well known to those skilled in the art.
  • the virus provided by the present invention is transfected with the above recombinant vector.
  • the above recombinant vector can be transfected into a virus by a conventional transfection method.
  • the virus is preferably a virus which does not cause lethal or irreversible damage to the organism, and may be, for example, an inactivated influenza virus, an adenovirus or the like. Methods for transfecting a stretch of a target sequence into a cell with a viral vector are well known to those skilled in the art and will not be described herein.
  • the present invention provides a method for removing a ubiquitinated target protein in an organism, characterized in that the method comprises applying the above virus to an organism.
  • the target protein may be various proteins to be removed, such as proteins associated with diseases, such as malignant proliferating proteins, key proteins regulating diseases, and the like.
  • the method for inhibiting gene expression in an organism provided by the present invention is characterized in that the method comprises administering the virus to an organism, and the ubiquitinable target protein is a key regulatory protein of the gene expression.
  • the present invention provides a method for treating a disease associated with a ubiquitinable protein of interest, characterized in that the method comprises administering the above virus to an organism.
  • the disease may be a disease caused by malignant proliferation of a target protein, such as a tumor, cancer, or the like, and the disease may also be a disease in which a target protein plays a key regulatory role.
  • diseases include, but are not limited to, articular rheumatism, arteriosclerosis, multiple myeloma, Crohn's disease, brain cancer, tongue cancer, throat cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, biliary tract Cancer, gallbladder cancer, duodenal cancer, colon cancer, liver cancer, uterine cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma, skin cancer, acute myeloid leukemia , acute lymphocytic leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, malignant lymphoma.
  • the organism is preferably a eukaryote, more preferably various animals and humans, and examples of the animal include, but are not limited to, pigs, dogs, cows, sheep, donkeys, horses, and the like.
  • the virus is preferably administered in an amount effective to replicate a target that binds to ubiquitination.
  • the aptamer of the protein does not cause lethal or irreversible damage to the organism, and can be appropriately selected according to actual needs.
  • the virus can be administered to an organism by various conventional administration methods, such as by oral, enteral or parenteral route, by topical application to the skin and mucosa, or by injection (especially subcutaneous injection). Apply.
  • the pharmaceutical composition for treating a disease associated with a ubiquitinable target protein provided by the present invention contains the virus as an active ingredient.
  • the content of the virus is preferably an amount capable of efficiently replicating an aptamer that binds to a ubiquitinable target protein without causing fatal or irreversible damage to the organism, and an appropriate selection can be made according to actual needs.
  • the disease may be a disease caused by malignant proliferation of a target protein, such as a tumor, a cancer, or the like, and the disease may also be a disease in which a ubiquitinated target protein plays a key regulatory role.
  • diseases include, but are not limited to, articular rheumatism, arteriosclerosis, multiple myeloma, Crohn's disease, brain cancer, tongue cancer, throat cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, biliary tract Cancer, gallbladder cancer, duodenal cancer, colon cancer, liver cancer, uterine cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma, skin cancer, acute myeloid leukemia , acute lymphocytic leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, malignant lymphoma.
  • the patient of the disease is preferably a eukaryote, more preferably a variety of animals and humans, examples of which include, but are not limited to, pigs, dogs, cows, sheep, donkeys, horses, and the like.
  • the pharmaceutical composition can be administered by oral, enteral or parenteral route, by topical application to the skin and mucosa, or by injection (especially subcutaneous injection).
  • the pharmaceutical composition may also be administered orally, for example, in the form of a tablet, a coated tablet, a film coating, a granule, a capsule, a soft capsule, a solution, a syrup, an emulsion, a suspension or an aerosol. .
  • a product in the form of an aqueous ethanol solution or a dry form.
  • the pharmaceutical composition may also be administered by the rectal route, for example in the form of a suppository; or by parenteral administration, for example in the form of injectable solutions or infusions, microcapsules or implants; or by transdermal routes.
  • Administration for example in the form of an ointment, solution, pigment or colorant; or by a transdermal route (skin patch) or by other routes suitable for use, for example, as an aerosol or nasal spray.
  • compositions are preferably prepared by a conventional method in which a pharmaceutically inert, organic or inorganic excipient is added to the composition obtained according to the present invention.
  • compositions may be in the form of a solid or a liquid, in any pharmaceutical form usually used for pharmaceuticals, such as ordinary tablets or sugar-coated tablets, pills, troches, capsules, drops, granules prepared by standard methods. , an injectable preparation, an ointment, a cream or a gel.
  • coated tablets and hard gelatin capsules for example, lactose, corn starch or derivatives thereof, talc and stearic acid or stearate can be used.
  • Suitable carriers for soft capsule carriers or suppositories include, for example, fats, waxes, semi-solid or liquid polyols, natural or modified oils, and the like.
  • Excipients for suitable solution formulations include, for example, water, Alcohol, glycerin, polyol, sucrose, invert sugar, glucose, vegetable oil, and the like.
  • Suitable carriers for the microcapsules or implants include, for example, copolymers of glyoxylic acid and lactic acid.
  • the active ingredient as defined above may be admixed with excipients commonly used in pharmaceutical compositions such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous forming. Fatty substances, paraffin derivatives, glycols, various wetting agents, dispersing or emulsifying agents and preservatives of the agent, animal or plant origin.
  • the pharmaceutical compositions may also contain additives such as diluents, disintegrating agents, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners.
  • additives such as diluents, disintegrating agents, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners.
  • a coloring agent, a flavoring or flavoring agent, a thickening agent and a buffering agent and may also contain a solvent or a solubilizing agent, a retarder, a salt which changes the osmotic pressure, a coating material or an antioxidant.
  • the virus provided by the present invention is transfected with a recombinant vector containing the aptamer, and when the virus is administered to an organism, the virus can replicate in the cells of the organism.
  • the aptamer is also replicated in the cell of the organism, since the aptamer includes a sequence capable of specifically binding to the ubiquitinating enzyme and a sequence capable of specifically binding to the ubiquitinated target protein, thus replicating the aptamer It can effectively combine the ubiquitinated target protein and ubiquitinating enzyme to bring the ubiquitinated target protein and ubiquitinating enzyme protein closer together to promote the binding of ubiquitin to the ubiquitinated target protein.
  • the ubiquitinated target protein is identified as a protein to be degraded and subsequently recognized and degraded by the intracellular proteasome.
  • the ubiquitinating enzyme catalyzes the binding of ubiquitin to the ubiquitinated target protein, followed by the ubiquitinated target protein. It is recognized and degraded by the proteasome in the cell, so that the aptamer is released from the state of binding to the ubiquitinable target protein, and the released aptamer can recombine the new ubiquitinated target protein and ubiquitination.
  • the enzyme promotes the degradation of the new ubiquitinated target protein. Therefore, the aptamer can be recycled, and even if a small amount of aptamer is administered, it can promote ubiquitination of the target protein which can be ubiquitinated. effect.
  • E1 and E2 were purchased from Enzo Life's ubiquitin activation kit (UW0400), wherein E1 is a human-derived ubiquitin-activating enzyme El, and E2 is a human recombinant ubiquitin-binding enzyme UbcH5a.
  • E3 is a VHL (von Hippel-Lindau disease tumor suppressor) purchased from Enzo Life Company.
  • the 10x ubiquitination reaction buffers used in the examples of the present invention, DTT, Mg-ATP, IPP and Ub-biotin are components of the above kit.
  • the fusion protein of ubiquitinase E3 and PDGF-BB, and the fusion protein of ubiquitinating enzyme E3 and VEGF-165 were expressed in E. coli BL21 by constructing the gene fragment into pET28 vector, and then using Ni-NTA affinity column. (purchased from Qiagen) obtained by purification.
  • Example 1 The E3-ALBP type aptamer Aptamer-1 was constructed.
  • E3 represents the E3 portion of the ubiquitinating enzyme
  • P represents the target protein portion
  • A represents the E3 ligand (a sequence capable of specifically binding to the ubiquitinating enzyme)
  • B represents the target protein ligand (which is capable of specifically binding to the target protein)
  • L is the linker.
  • the target protein portion P is Thrombin
  • the aptamer Aptamer-1 is composed of the ligand apt-1 (as A), L-1 (as L) and B Thrombin of the ubiquitinating enzyme E3 moiety.
  • the composition of the apt-3 (as B), the specific sequence is shown in Table 1, and the sequence of Aptamer-1 is shown in SEQ ID No: 1.
  • the ubiquitinating enzyme E3 moiety is a fusion-expressed ubiquitinating enzyme E3 and PDGF-BB (VHL-PD), and the ligand apt-1 of the ubiquitinating enzyme E3 moiety is a specific ligand of PDGF-BB. body.
  • the components required for the in vitro ubiquitination reaction were mixed for in vitro ubiquitination, and the amounts of the components were as shown in Table 2. After incubating at 37 ° C for 24 hours, the reacted product was subjected to polyacrylamide gel electrophoresis and detected with an antibody against Thrombin, and the results are shown in the second lane of Fig. 1. The reaction system in which Aptamer-1 was not added was used as a control, and the results are shown in the first lane of Fig. 1.
  • Figure 1 shows the sequence of the E3 ligand, linker and target protein ligand used in each example.
  • VHL-PD 1000 ⁇
  • Table 2 shows the amount of each component added in the in vitro ubiquitination reaction system.
  • Fig. la in Example 1 in which Aptamer-1 was added, Thrombin ubiquitination was remarkable, whereas in Comparative Example 1 in which Aptamer-1 was not added, no significant Thrombin ubiquitination was observed. This indicates that the aptamer of the present invention contributes to the ubiquitination of the target protein.
  • lane 1 is the result of a control without the addition of Aptamer-1
  • lane 2 is the result of the reaction product of Example 1.
  • Fig. 1b no lane was observed in lane 1 without addition of Aptamer-1, and in response to ubiquitin in lane 2 corresponding to the band of ubiquitinated Thrombin of panel la The band of antibodies, which further indicates that Thrombin has indeed been ubiquitinated.
  • Example 2 To the reaction product obtained in Example 1, l g proteasome Proteasome 26S was added for degradation, and the degradation product was subjected to polyacrylamide gel electrophoresis, and the reaction product without the proteasome was used for comparison. The results of the assay are shown in Fig. 2.
  • each ubiquitinated Thrombin in the product not added to the proteasome is still in (lane 1), whereas in the product added to the proteasome, ie, lane 2, only Thrombin that is not ubiquitinated is in The ubiquitinated Thrombin has been degraded.
  • the aptamer of the present invention is effective in promoting ubiquitination of the target protein and promoting its degradation.
  • the in vitro ubiquitination reaction was carried out according to the method of Example 1, wherein, in the present example, the target protein portion P is Thrombin, and the aptamer Aptamer-2 is a ligand of the ubiquitinating enzyme E3 moiety apt-2 (as A), L-2
  • E3 moiety apt-2 (as A)
  • L-2 The composition of the ligand apt-3 (as B) of L) and T Thrombin is shown in Table 1, and the sequence of Aptamer-2 is shown in SEQ ID No: 2.
  • the ubiquitinating enzyme E3 portion is a fusion protein (E3-VE) of the fusion-expressed ubiquitinating enzyme E3 and vascular endothelial growth factor (VEGF-165), and the ligand apt- of the ubiquitinating enzyme E3 moiety.
  • E3-VE fusion protein
  • VEGF-165 vascular endothelial growth factor
  • 2 is a specific ligand for VEGF-165.
  • Example 3 in which Aptamer-2 was added, Thrombin ubiquitination was remarkable (lane 2), whereas in Comparative Example 1 in which Aptamer-2 was not added, no significant Thrombin ubiquitination was observed. Phenomenon (lane 1). This demonstrates that the aptamers of the invention help to assist in the ubiquitination of the protein of interest.
  • Example 4
  • the in vitro ubiquitination reaction was carried out according to the method of Example 1, wherein, in the present embodiment, the target protein portion P is VEGF-165, and the aptamer Aptamer-3 is a ligand of the ubiquitinating enzyme E3 moiety apt-1 (As A), L-3 (as L) and the ligand apt-2 (as B) of VEGF-165, as shown in Table 1, the sequence of Aptamer-3 is shown in SEQ ID No: 3, The ubiquitinating enzyme E3 moiety is E3-PD.
  • the target protein portion P is VEGF-165
  • the aptamer Aptamer-3 is a ligand of the ubiquitinating enzyme E3 moiety apt-1 (As A), L-3 (as L) and the ligand apt-2 (as B) of VEGF-165, as shown in Table 1, the sequence of Aptamer-3 is shown in SEQ ID No: 3,
  • ubiquitination components and conditions are shown in Table 2, except that an equivalent amount of Aptamer-3 was used instead of Aptamer-1, and an equal weight of VEGF-165 was used instead of Thrombin for 24 hours at 37 °C.
  • the reacted system was subjected to acrylamide gel electrophoresis and detected with an antibody against VEGF-165, and the results were similar to those of Example 1, and a band of ubiquitinated VEGF-165 was observed. This indicates that the aptamer of the present invention contributes to the ubiquitination of the target protein.
  • the lg proteasome Proteasome 26S was added to the reaction product obtained in Example 5 for degradation, and the degradation product was detected by polyacrylamide gel electrophoresis, and the reaction product without the proteasome was used for comparison. Similar to Example 1: each ubiquitinated VEGF-165 in the product not added to the proteasome is still present, whereas in the product added to the proteasome, only VEGF-165 not ubiquitinated is in the ubiquitin The VEGF-165 has been degraded. This fully demonstrates that the aptamer of the present invention is effective in promoting ubiquitination of a target protein and promoting its degradation.

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un aptamère et son procédé de préparation, l'aptamère comprenant une séquence protéique cible d'ubiquitine susceptible d'établir une liaison spécifique et une séquence d'ubiquitinase susceptible d'établir une liaison spécifique. L'invention concerne en outre un vecteur recombinant de l'aptamère et son virus de transfection, ainsi que des composés pharmaceutiques contenant ledit virus.
PCT/CN2011/077443 2010-07-22 2011-07-21 Aptamère et son procédé de préparation WO2012010103A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201010237448 2010-07-22
CN201010237448.7 2010-07-22

Publications (1)

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WO2012010103A1 true WO2012010103A1 (fr) 2012-01-26

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PCT/CN2011/077443 WO2012010103A1 (fr) 2010-07-22 2011-07-21 Aptamère et son procédé de préparation

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WO (1) WO2012010103A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005044840A2 (fr) * 2003-10-17 2005-05-19 The Cbr Institute For Biomedical Research, Inc. Modulation d'anergie et procedes pour l'isolement de composes de modulation d'anergie
CN1890263A (zh) * 2003-12-10 2007-01-03 路德维希癌症研究院 肿瘤抑制蛋白
WO2008156561A1 (fr) * 2007-06-12 2008-12-24 Beth Israel Deaconess Medical Center Procédés et compositions pour le traitement et le diagnostic de la myopathie induite par la statine
WO2009004484A2 (fr) * 2007-07-03 2009-01-08 Board Of Regents, The University Of Texas System Modulateur de p53 et cible cancereuse

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005044840A2 (fr) * 2003-10-17 2005-05-19 The Cbr Institute For Biomedical Research, Inc. Modulation d'anergie et procedes pour l'isolement de composes de modulation d'anergie
CN1890263A (zh) * 2003-12-10 2007-01-03 路德维希癌症研究院 肿瘤抑制蛋白
WO2008156561A1 (fr) * 2007-06-12 2008-12-24 Beth Israel Deaconess Medical Center Procédés et compositions pour le traitement et le diagnostic de la myopathie induite par la statine
WO2009004484A2 (fr) * 2007-07-03 2009-01-08 Board Of Regents, The University Of Texas System Modulateur de p53 et cible cancereuse

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