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WO2011010583A1 - Procédé de criblage d'oligonucléotides, et bibliothèque d'oligonucléotides - Google Patents

Procédé de criblage d'oligonucléotides, et bibliothèque d'oligonucléotides Download PDF

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Publication number
WO2011010583A1
WO2011010583A1 PCT/JP2010/061888 JP2010061888W WO2011010583A1 WO 2011010583 A1 WO2011010583 A1 WO 2011010583A1 JP 2010061888 W JP2010061888 W JP 2010061888W WO 2011010583 A1 WO2011010583 A1 WO 2011010583A1
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oligonucleotide
nucleic acid
analog
mrna
oligonucleotides
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徹 夏目
俊吾 足達
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Galaxy Pharma
GALAXY PHARMA Inc
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Galaxy Pharma
GALAXY PHARMA Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
    • C12N2330/31—Libraries, arrays

Definitions

  • the present invention relates to an oligonucleotide screening method and an oligonucleotide library. More specifically, the present invention relates to an oligonucleotide screening method for obtaining a target oligonucleotide exhibiting a desired biological activity or chemical activity, an oligonucleotide library used in the screening method, and the like.
  • Oligonucleotides in which several to several tens of nucleotides are arranged in a chain by phosphodiester bonds via phosphoric acid are formed between base pairs with respect to a nucleotide chain having a complementary sequence. Are bonded by hydrogen bonds. By utilizing this characteristic, oligonucleotides have been widely used as research reagents and gene diagnostic reagents.
  • Oligonucleotides as research reagents are used, for example, as primers for polymerase chain reaction (PCR) and used to amplify specific gene sequences. Alternatively, it is also used as a probe for detecting the amplified gene sequence. Moreover, as a genetic diagnostic reagent, it is used as a probe immobilized on a DNA chip or a DNA array.
  • PCR polymerase chain reaction
  • nucleic acid pharmaceuticals In recent years, oligonucleotides have been developed as pharmaceuticals called nucleic acid pharmaceuticals.
  • an antisense method that selectively inhibits translation of a sense strand into a protein by introducing an oligonucleotide (antisense oligonucleotide) complementary to a partial sequence of mRNA (sense strand) of a target gene into a cell.
  • an antisense method by binding an antisense oligonucleotide to the mRNA of the target gene and inhibiting the binding of the translation factor complex to the mRNA, the translation of the mRNA into the protein is inhibited and the expression of the target gene product is suppressed. can do.
  • oligonucleotide As an example of an oligonucleotide as a nucleic acid drug, “decoy oligonucleotide” that binds to a specific transcription factor as “bait” and inhibits the function of the transcription factor can also be mentioned. Transcription factors bind to transcriptional control regions such as promoters and enhancers on the genome to control transcription of genes into mRNA. The decoy oligonucleotide binds to this transcription factor as a decoy and competitively inhibits the transcription factor from binding to the transcription control region, thereby inhibiting the transcription of the target gene mRNA and suppressing the expression of the target gene product.
  • LNA locked nucleic acid
  • BNA Bridged Nucleic Acid
  • Nucleosides in natural nucleic acids have two conformations, N-type and S-type. Because of this “fluctuation” between conformations, the double strands formed between DNA-DNA, RNA-RNA strands, and DNA-RNA are not necessarily thermodynamically stable.
  • 2'-O, 4'-C-methano-bridged nucleic acid (2 ', 4'-BNA) bridges the 2' and 4 'positions of ribose (sugar) with "-O-CH2-" It is an artificial nucleic acid whose conformation is fixed to N-type. Since 2 ', 4'-BNA has no fluctuation between conformations, oligonucleotides synthesized with several units of 2', 4'-BNA are compared to oligonucleotides synthesized with conventional natural nucleic acids. It has extremely high binding power and sequence specificity for RNA and DNA, and exhibits excellent heat resistance and nuclease resistance. So far, about 10 types of LNAs have been developed in addition to 2 ′, 4′-BNA (see Patent Documents 1 to 4).
  • the antisense method and the decoy method target only the genes for which the translation factor complex and transcription factor binding region sequences (hereinafter also referred to as “target sequences”) have been clarified. Cannot target unknown genes.
  • an oligonucleotide synthesized as a sequence complementary to the binding region of a translation factor complex or a transcription factor may not necessarily show the expected activity. Therefore, for example, in the antisense method, a plurality of oligonucleotides are synthesized for one translation factor complex-binding region on the target gene mRNA, or a large number of translation factor complex-binding regions on the mRNA are synthesized. It is necessary to synthesize oligonucleotides to find oligonucleotides exhibiting a desired activity by screening.
  • the main object of the present invention is to provide a method for screening an oligonucleotide for efficiently obtaining an oligonucleotide exhibiting biological activity or chemical activity such as target gene expression-suppressing activity.
  • the present invention provides a 7mer basic sequence comprising any nucleic acid analog, any one nucleic acid analog selected from adenine analog, guanine analog, thymine analog or cytosine analog, or adenine, guanine, Prepare two or more oligonucleotide pools composed of oligonucleotides in which one or two nucleic acids selected from thymine or cytosine are bound so that the basic sequences differ between the oligonucleotide pools.
  • An oligonucleotide screening method comprising: a procedure; and a procedure for identifying an oligonucleotide pool containing a target oligonucleotide from the oligonucleotide pool.
  • the screening method may further comprise a procedure for obtaining the target oligonucleotide from the identified oligonucleotide pool.
  • an oligonucleotide pool is prepared by binding one nucleic acid analog or nucleic acid to a basic sequence, an adenine analog, guanine analog, thymine is added to either the 5 ′ end or the 3 ′ end of the basic sequence.
  • Two oligonucleotide pools composed of four kinds of oligonucleotides, each of which binds any one nucleic acid analog selected from analogs or cytosine analogs, or any one nucleic acid selected from adenine, guanine, thymine, or cytosine, respectively. Prepare as above.
  • an oligonucleotide pool by binding two nucleic acid analogs or nucleic acids to a basic sequence, for example, an adenine analog, a guanine analog
  • An oligonucleotide pool consisting of 16 types of oligonucleotides, each of which binds any one nucleic acid analog selected from thymine analogs or cytosine analogs, or any one nucleic acid selected from adenine, guanine, thymine or cytosine Prepare two or more.
  • This screening method preferably includes a procedure for preparing 16384 (4 7) basic sequences consisting of all combinations of seven nucleic acid analogs.
  • oligonucleotide pools By preparing 16384 oligonucleotide pools using 16384 basic sequences, it is theoretically possible to prepare oligonucleotides that can specifically bind to nucleotide chains of any sequence.
  • the screening method according to the present invention for example, in the presence and absence of an oligonucleotide pool, the mRNA is contacted with an mRNA binding protein that binds to the mRNA, and the amount of binding between the mRNA and the mRNA binding protein is measured.
  • an oligonucleotide pool containing oligonucleotides that exhibit an activity of inhibiting the binding between mRNA and mRNA-binding protein By preparing 16384 oligonucleotide pools using 16384 basic sequences, it is theoretically possible to prepare oligonucleotides that can specifically bind to nucleotide chains of any sequence.
  • the mRNA in the presence and absence of an oligonucleot
  • an oligonucleotide pool containing an oligonucleotide exhibiting an activity of enhancing the expression of the protein can be identified it can.
  • the present invention is an oligonucleotide library provided for the above-described oligonucleotide screening method, wherein a 7mer basic sequence comprising any nucleic acid analog is added to an adenine analog, guanine analog, thymine analog or cytosine analog. It is composed of two or more oligonucleotide pools composed of one or two oligonucleotides bound to any one nucleic acid analog selected, or any one nucleic acid selected from adenine, guanine, thymine, or cytosine.
  • the present invention also provides an oligonucleotide library in which the basic sequences are different between the oligonucleotide pools. This oligonucleotide library preferably has 16384 basic sequences consisting of all combinations of seven nucleic acid analogs.
  • the present invention also provides an oligonucleotide exhibiting an activity that inhibits the binding between tumor necrosis factor ⁇ mRNA and RC3H1, and an oligonucleotide exhibiting an activity that enhances the expression of a low density lipoprotein receptor.
  • a “nucleic acid analog” is an artificial nucleic acid obtained by artificially modifying the chemical structure of a ribose or phosphodiester bond of a natural nucleic acid (DNA and RNA), and includes at least one in the oligonucleotide sequence. By being included, the binding affinity and sequence specificity for the complementary strand of the oligonucleotide can be increased as compared to the oligonucleotide consisting only of the natural nucleic acid.
  • the “nucleic acid analog” obtained by modifying the chemical structure of ribose includes at least the above-described bridged nucleic acid (BNA) or locked nucleic acid (LNA).
  • LNA As LNA, 2'-O, 4'-C-methano-bridged nucleic acid (2 ', 4'-BNA) and 3', 4'-BNA, 3'-amino- Conventionally known LNA such as 2 ′, 4′-BNA and 5′-amino-3 ′, 4′-BNA are included.
  • Nucleic acid analogs in which the chemical structure of the phosphodiester bond is modified include phosphorothioate-type artificial nucleic acids (S-oligo) in which the oxygen atom of the phosphate group is replaced with a sulfur atom.
  • the present invention provides an oligonucleotide screening method for efficiently obtaining a target oligonucleotide exhibiting biological activity or chemical activity such as target gene expression suppression activity.
  • the binding affinity and sequence specificity for the complementary strand of an oligonucleotide decreases as the length of the oligonucleotide decreases.
  • Conventional oligonucleotides for research, genetic diagnostic reagents, and nucleic acid pharmaceuticals usually have a binding affinity and sequence specificity for complementary strands of 10 to 40 mer in normal cases, and in most cases around 20 mer. It is said to be long. Since the length of the oligonucleotide is short, the desired activity of the oligonucleotide cannot be obtained unless sufficient binding affinity and sequence specificity for the complementary strand are exhibited.
  • the longer the length of the oligonucleotide the higher the cost for synthesis.
  • the length of the oligonucleotide becomes too long, nonspecific binding to a nucleotide chain having a partially complementary sequence (non-complementary chain) occurs. Therefore, it is desirable that the oligonucleotide be synthesized as short as possible on condition that sufficient binding affinity and sequence specificity for the complementary strand are exhibited.
  • the increase in synthesis cost due to the length of the oligonucleotide requires the synthesis of four types of oligonucleotides for every 1 mer. It will be functional. Therefore, in order to construct an oligonucleotide library at an economically feasible cost, it is desirable that the length of the oligonucleotide be as short as possible.
  • the present inventors added a 1-mer or 2-mer nucleic acid analog or nucleic acid to a nucleic acid analog 7mer. It was found that a total of 8 mer or 9 mer oligonucleotides synthesized in this manner exhibit necessary and sufficient binding affinity and sequence specificity and can express a desired biological activity or chemical activity.
  • the present invention has been completed by the inventors based on this finding, and uses an oligonucleotide library for efficiently obtaining a target oligonucleotide exhibiting a desired biological activity or chemical activity, and this library.
  • the present invention provides a screening method for oligonucleotides.
  • the oligonucleotide library according to the present invention is composed of two or more oligonucleotide pools.
  • Each oligonucleotide pool has a 7-mer “basic sequence” consisting of any nucleic acid analog, any one nucleic acid analog selected from adenine analog, guanine analog, thymine analog or cytosine analog, or adenine, guanine, thymine or It consists of an oligonucleotide in which one or two nucleic acids selected from cytosine are linked.
  • FIG. 1 schematically shows an oligonucleotide library according to the first embodiment of the present invention.
  • the figure shows an oligonucleotide library L in which each oligonucleotide pool is composed of oligonucleotides in which one nucleic acid analog or nucleic acid is linked to a 7mer basic sequence.
  • A represents an adenine analog
  • G represents a guanine analog
  • T thymine analog
  • C represents a cytosine analog
  • a represents adenine
  • g represents guanine
  • t thymine
  • c represents cytosine.
  • N represents any one nucleic acid analog selected from adenine analog, guanine analog, thymine analog, or cytosine analog
  • n represents any one nucleic acid selected from adenine, guanine, thymine, or cytosine.
  • Oligonucleotide library L is composed of a total of K types of oligonucleotide libraries from pool 1 to pool K.
  • Pool 1 includes four types of oligonucleotides 11, 12, 13, and 14. Oligonucleotides 11, 12, 13, and 14 have a basic sequence 1 represented by “AGTCAGGT” in common, and have a sequence in which A, G, T, and C are linked to the 3 ′ end of the basic sequence 1, respectively. ing. In the pool 1, it is desirable that the oligonucleotides 11, 12, 13, and 14 are contained in the same number of moles, but the concentration of each oligonucleotide is not particularly limited. Hereinafter, the same applies to the pool 2, the pool 3, and the pool K.
  • Pool 2 includes four types of oligonucleotides 21, 22, 23, and 24, and oligonucleotides 21, 22, 23, and 24 have a common basic sequence 2 represented by “AGTGGAGT”.
  • the basic sequence 2 has a sequence in which A, G, T, and C are bonded to the 3 ′ end.
  • the basic sequence 1 of the oligonucleotides 11, 12, 13, and 14 included in the pool 1 and the basic sequence 2 of the oligonucleotides 21, 22, 23, and 24 included in the pool 2 include the fourth nucleic acid analog from the 5 ′ end. “C” and “G” are different.
  • Pool 3 includes four types of oligonucleotides 31, 32, 33, and 34, and oligonucleotides 31, 32, 33, and 34 have a basic sequence 3 represented by “AGTTAGT” in common.
  • the basic sequence 3 has a sequence in which A, G, T, and C are bonded to the 3 ′ end.
  • the basic sequence 1 of the oligonucleotides 11, 12, 13, and 14 included in the pool 1 and the basic sequence 3 of the oligonucleotides 31, 32, 33, and 34 included in the pool 3 include the fourth nucleic acid analog from the 5 ′ end. “C” and “T” are different.
  • the basic sequence 2 of the oligonucleotides 21, 22, 23, and 24 included in the pool 2 and the basic sequence 3 of the oligonucleotides 31, 32, 33, and 34 included in the pool 3 are the fourth nucleic acid from the 5 ′ end.
  • the analogs are different for “G” and “T”, respectively.
  • the pool K (K is an integer of 2 to 16,384) includes four types of oligonucleotides K1, K2, K3, and K4.
  • the oligonucleotides K1, K2, K3, and K4 are indicated by “NNNNNNNN”.
  • the basic sequence K is commonly used, and A, G, T, and C are connected to the 3 ′ end of the basic sequence K, respectively.
  • the basic sequences K of the oligonucleotides K1, K2, K3, and K4 included in the pool K are different from the basic sequences 1 to K-1 of the oligonucleotides included in the pool 1 to the pool (K-1).
  • the oligonucleotide library L includes two or more oligonucleotide pools composed of four types of oligonucleotides in which a nucleic acid analog of A, G, T, or C is bound to a basic sequence that is different between pools. Has been.
  • FIG. 1 illustrates the case where the oligonucleotides K1, K2, K3, and K4 constituting the oligonucleotide pool K are sequences in which A, G, T, and C are bonded to the 3 ′ end of the basic sequence K, respectively.
  • Each nucleic acid analog may be bound to either the 3 ′ end or the 5 ′ end of the basic sequence K, and the oligonucleotides K1, K2, K3, and K4 are respectively A and G at the 5 ′ end of the basic sequence K.
  • T, and C may be combined.
  • FIG. 1 illustrates the case where oligonucleotides K1, K2, K3, and K4 constituting the oligonucleotide pool K are sequences in which the nucleic acid analogs A, G, T, and C are bonded to the ends of the basic sequence K, respectively. did. Either the nucleic acid analog or the nucleic acid may be bound to the end of the basic sequence.
  • the oligonucleotides K1, K2, K3, and K4 have the nucleic acids a, g, t, and c at the ends of the basic sequence K, respectively. A combined sequence may also be used.
  • FIG. 2 schematically shows an oligonucleotide library according to the second embodiment of the present invention.
  • the figure shows an oligonucleotide library L in which each oligonucleotide pool is composed of oligonucleotides in which two nucleic acid analogs or two nucleic acids are linked to a 7mer basic sequence.
  • Oligonucleotide library L is composed of a total of K types of oligonucleotide libraries from pool 1 to pool K.
  • Pool 1 contains 16 types of oligonucleotides 101-116. Oligonucleotides 101 to 116 have a basic sequence 1 represented by “AGTCCAGT” in common, and any one of A, G, T, and C is linked to the 5 ′ end and the 3 ′ end of this basic sequence 1, respectively. It is an array. In the pool 1, it is desirable that the oligonucleotides 101 to 116 are contained in the same number of moles, but the concentration of each oligonucleotide is not particularly limited. Hereinafter, the same applies to the pool 2, the pool 3, and the pool K.
  • the pool 2 includes 16 types of oligonucleotides 201 to 216, and the oligonucleotides 201 to 216 have the basic sequence 2 indicated by “AGTGAGT” in common, and 5 ′ of the basic sequence 2 It is set as the arrangement
  • the fourth nucleic acid analog from the 5 ′ end is “C” and “G”, respectively. And is different.
  • the pool 3 includes 16 types of oligonucleotides 301 to 316, and the oligonucleotides 301 to 316 have a basic sequence 3 represented by “AGTTAGT” in common. It is set as the arrangement
  • the fourth nucleic acid analogs from the 5 ′ end are “C” and “T”, respectively. And is different.
  • the fourth nucleic acid analog from the 5 ′ end is “G” and “ T ”.
  • the pool K (K is an integer of 2 to 16384) includes 16 types of oligonucleotides K01 to K16, and the oligonucleotides K01 to K16 share the basic sequence K represented by “NNNNNNNN”.
  • the basic sequence K has a sequence in which any one of A, G, T, and C is linked to the 5 ′ end and 3 ′ end.
  • the basic sequences K of the oligonucleotides K01 to K16 included in the pool K are different from the basic sequences 1 to K-1 of the oligonucleotides included in the pools 1 to (K-1).
  • the oligonucleotide library L includes two or more oligonucleotide pools composed of 16 kinds of oligonucleotides in which a nucleic acid analog of A, G, T, or C is bound to a basic sequence that is different between pools. Has been.
  • the oligonucleotides K01 to K16 constituting the oligonucleotide pool K are divided into one of A, G, T, C at the 5 ′ end of the basic sequence K, and A, G, T, at the 3 ′ end.
  • the case where any one of Cs is a sequence in which one is combined has been described.
  • Two nucleic acid analogs may be bound to the 5 ′ end and / or 3 ′ end of the basic sequence K, and both nucleic acid analogs are bound to the 5 ′ end or the 3 ′ end to form 16 types of oligonucleotides. Also good.
  • FIG. 2 illustrates the case where the oligonucleotides K01 to K16 constituting the oligonucleotide pool K have a sequence in which two nucleic acid analogs A, G, T, and C are bonded to the ends of the basic sequence K, respectively. Either the nucleic acid analog or the nucleic acid may be bound to the end of the basic sequence.
  • the oligonucleotides K1, K2, K3, and K4 have the nucleic acids a, g, t, and c at the ends of the basic sequence K, respectively. Two sequences may be combined, or one nucleic acid analog and one nucleic acid may be combined.
  • the oligonucleotide library L shown in FIG. 1 and FIG. 2 contains A, G, T, or C nucleic acid analogs or nucleic acids in a 7mer basic sequence that is different between pools. It comprises two or more oligonucleotide pools consisting of linked 8mer or 9mer oligonucleotides. As described above, a total of 8mer or 9mer oligonucleotide synthesized by adding 1mer or 2mer nucleic acid analog or nucleic acid to nucleic acid analog 7mer such as 2 ', 4'-BNA has the necessary and sufficient binding affinity and It has been shown that it can exhibit sequence specificity and express a desired biological or chemical activity.
  • oligonucleotide library L is composed of 16384 (4 to the 7th power) oligonucleotide pools, which are all combinations of nucleic acid analogs of the basic sequence 7mer, it binds specifically to nucleotide chains of any sequence.
  • An oligonucleotide library can be constructed such that possible 8mer or 9mer oligonucleotides are always included in either oligonucleotide pool.
  • oligonucleotide Screening Method Next, an oligonucleotide screening method according to the present invention will be described. In this screening method, an oligonucleotide exhibiting a desired biological activity or chemical activity is obtained using the oligonucleotide library L described above.
  • FIG. 3 is a flowchart showing the procedure of the oligonucleotide screening method according to the present invention.
  • symbol S 1 is a procedure for preparing a 7mer basic sequence consisting of an arbitrary nucleic acid analog.
  • the basic sequence can be synthesized by combining nucleic acid analogs using a conventional known method.
  • the basic sequence is synthesized using, for example, a known DNA synthesizer.
  • the synthesized basic sequence can be confirmed by purifying using a reverse phase column and then analyzing by reverse phase HPLC or MALDI-TOF-MS.
  • the basic sequence can also be obtained by using a custom oligonucleotide synthesis service.
  • Two or more basic arrays are prepared as different arrays.
  • the base sequence is prepared as 16384 (4 to the 7th power) of all combinations of 7-mer nucleic acid analogs.
  • Step S 2 described below using the basic sequence of 16384 ways, by preparing an oligonucleotide pool 16384 types, theoretically, an oligonucleotide capable of specifically binding to the nucleotide strand of any sequence Can be prepared.
  • Step S 2 designates each basic sequence prepared in Step S 1 one nucleic acid analogs or nucleic acid, or two bonds to, the procedure for preparing oligonucleotide pool It is.
  • oligonucleotide library L is constituted by oligonucleotides in which one nucleic acid analog or nucleic acid is bound to a 7mer basic sequence
  • oligonucleotides included in pool 1 to pool (K-1) A, G, T, and C are respectively linked to the 3 ′ end of the basic sequence K “NNNNNNNN” that is different from the basic sequences 1 to K-1.
  • K is an integer of 2 to 16384
  • the oligonucleotide library L is composed of oligonucleotides in which two nucleic acid analogs or two nucleic acids are linked to a 7mer basic sequence
  • the basic sequence 1 of oligonucleotides contained in pool 1 to pool (K-1) A, G, T, and C are respectively linked to the 5 ′ end and 3 ′ end of the basic sequence K “NNNNNNNN” that is different from the sequence of K ⁇ 1.
  • K is an integer of 2 to 16384
  • Oligonucleotide pools are prepared as two or more pools based on two or more basic sequences having different sequences.
  • the oligonucleotide pool is prepared as 16384 (4 to the 7th power) pools of all combinations of 7mer nucleic acid analogs.
  • 16384 oligonucleotide pools it is possible to prepare an oligonucleotide library L that always contains oligonucleotides that can specifically bind to nucleotide chains of any sequence.
  • reference numeral S 3 denotes an oligonucleotide containing an oligonucleotide exhibiting a desired biological activity or chemical activity from among the oligonucleotide pools constituting the oligonucleotide library L. Procedure for identifying nucleotide pools.
  • Each oligonucleotide pool contains 4 types or 16 types of oligonucleotides.
  • each of the oligonucleotide pools is subjected to various assays in the presence and absence thereof to identify an oligonucleotide pool containing the oligonucleotide exhibiting the desired activity (target oligonucleotide pool).
  • the “biological activity or chemical activity” of an oligonucleotide means the amount of organism that can be quantified by experiment, and specifically means the activity of the following oligonucleotide, for example.
  • Antisense activity that inhibits the binding of the translation factor complex to the mRNA by inhibiting the binding of the translation factor complex to the mRNA by binding to the mRNA of the target gene in a complementary manner.
  • Activity that inhibits translation into protein and suppresses expression of target gene product.
  • This activity inhibits the binding of mRNA binding proteins other than translation factor complexes to mRNA, or by inhibiting the binding of mRNA binding proteins to mRNA, resulting in decreased or increased expression of the target gene product. It may be an activity.
  • One example is the activity of stabilizing the mRNA and increasing the expression of the target gene product by inhibiting the binding of the cis element binding factor to the mRNA.
  • this antisense activity may be an activity that binds complementarily to non-coding RNA such as micro-RNA (miRNA) and inhibits or enhances its function.
  • cis element binding factor binds to specific sequences called “cis-elements” present in the 5 ′ and 3 ′ untranslated regions of DNA and RNA.
  • a cis element is involved in regulation of the expression of a gene encoded by its DNA strand or RNA strand.
  • a cis-element binding factor functions as a “trans-acting factor” that binds to a cis-element and positively or negatively controls gene expression.
  • One of the typical mRNA cis elements is “AU-rich element (AU-Rich Element; ARE)”.
  • ARE is a base sequence of about 10 to 150 bps rich in adenosine and uridine, and is abundant in the 3 ′ untranslated region (3 ′ UTR) of mRNA. ARE was initially found as a region where the nucleotide sequence of “AUUUA” frequently overlaps in the 3 ′ UTR of cytokines and lymphokines. ARE is currently estimated to be present in 5-8% of all genes, and ARE is considered to be present in many genes involved in the maintenance of homeostasis (“ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins. ”Nucleic Acids Research, 2001, Vol.29, No.1, p.246-254.).
  • This activity binds to DNA-binding proteins or RNA-binding proteins other than transcription factors as decoys, or binds to DNA-binding proteins or RNA-binding proteins as a decoy, resulting in decreased or increased expression of the target gene product It may be an activity.
  • One example is the activity of stabilizing mRNA and increasing the expression of the target gene product by binding as a decoy to the cis element binding factor and inhibiting the binding of the cis element binding factor to the mRNA.
  • the biological activity or chemical activity of oligonucleotides can be evaluated by conducting various assays in the presence and absence of each oligonucleotide pool.
  • the following assay is performed. First, in the presence of each oligonucleotide pool, the mRNA of a target gene is brought into contact with an mRNA binding protein (here, a translation factor) that binds to the mRNA, and the amount of binding between the mRNA and the mRNA binding protein is measured.
  • an mRNA binding protein here, a translation factor
  • the target mRNA is brought into contact with the mRNA binding protein that binds to the mRNA, and the amount of binding between the mRNA and the mRNA binding protein is measured. Then, the amount of binding in the presence of the oligonucleotide pool is compared with the amount of binding in the absence of the oligonucleotide pool, and the oligonucleotide pool containing the oligonucleotide that exhibits the activity of inhibiting the binding between the mRNA and the mRNA binding protein is identified. .
  • the amount of binding between mRNA and mRNA binding protein can be evaluated, for example, by a pull-down assay using mRNA bait.
  • the following assay is performed. First, the expression level of the target protein is measured for the cells into which each oligonucleotide pool has been introduced. Moreover, the expression level of protein is measured about the cell which has not introduce
  • the expression level of the cells into which the oligonucleotide pool has been introduced is compared with the expression level of the cells into which the oligonucleotide pool has not been introduced, and an oligonucleotide pool containing oligonucleotides that exhibit the activity of increasing the expression of the target gene product is identified.
  • the expression level of the target protein can be evaluated, for example, by Western blot.
  • This assay can identify a target oligonucleotide pool containing decoy oligonucleotides that have the activity of binding to DNA or RNA binding proteins as decoys and increasing the expression of target gene products.
  • the assay is performed for each of two or more oligonucleotide pools constituting the oligonucleotide library L.
  • 16384 kinds of oligonucleotide pools are prepared, and an oligonucleotide library L containing oligonucleotides that can specifically bind to nucleotide chains of any sequence is always prepared.
  • an oligonucleotide library L containing oligonucleotides that can specifically bind to nucleotide chains of any sequence is always prepared.
  • one or more oligonucleotide pools exhibiting the desired activity can be identified.
  • symbol S 4 is a procedure for acquiring an oligonucleotide exhibiting a desired biological activity or chemical activity from the oligonucleotide pool identified in step S 3. is there.
  • the oligonucleotide pools identified in step S 3 contains four or 16 different oligonucleotides.
  • an oligonucleotide exhibiting a desired activity is identified from these oligonucleotides.
  • oligonucleotides can be identified by performing for each of the four or 16 kinds of oligonucleotides were synthesized by the same method as described in Step S 2, the various assays in a manner similar to that described in step S 3.
  • the screening method of an oligonucleotide according to the present invention performs the identification of the object oligonucleotide pool in step S 3, performing a two-step screening procedure of identifying the target oligonucleotide from the object pool of oligonucleotides further in this procedure. Thereby, in the oligonucleotide screening method according to the present invention, an oligonucleotide exhibiting a desired activity can be efficiently obtained.
  • oligonucleotide screening method by preparing 16384 oligonucleotide pools, oligonucleotides that can specifically bind to nucleotide chains of any sequence must be in any one or more oligonucleotide pools. Since it can be included, it is possible to obtain an oligonucleotide exhibiting an antisense activity, a decoy activity, etc. even for a target gene for which the target sequence cannot be specified.
  • oligonucleotide screening method it is not always necessary to prepare 16384 oligonucleotide pools.
  • screening can be carried out using a minimum of two or more oligonucleotide pools.
  • Oligonucleotide An oligonucleotide obtained by the oligonucleotide screening method of the present invention exhibits a desired biological activity or chemical activity such as the above-described antisense activity or decoy activity. Therefore, this oligonucleotide can be developed as a nucleic acid medicine, for example.
  • an oligonucleotide exhibiting antisense activity or decoy activity can be used as a nucleic acid drug for enhancing or suppressing the expression of a predetermined target gene. Since the oligonucleotide synthesized by LNA has high heat resistance and excellent nuclease resistance, the obtained oligonucleotide exhibits high stability when used as a nucleic acid pharmaceutical.
  • the oligonucleotide when the expression of a target gene is controlled using the obtained oligonucleotide, the oligonucleotide is added to the cell culture medium and taken into the cell to bind to the target gene mRNA in the cell.
  • the obtained oligonucleotide is introduced into the cell by lipofection or microinjection to bind to the target gene mRNA in the cell.
  • the target of gene expression is a living organ or a living body
  • the oligonucleotide is administered in vivo by an administration route such as oral route, rectal route, nasal route, vascular route, or by direct local administration to the target organ. Alternatively, it is introduced into a living organ and taken into a cell.
  • the base sequence and length of the obtained oligonucleotide, the chemical structure of ribose or the chemical structure of the phosphodiester bond, etc. can be appropriately changed and optimized.
  • optimizing the base sequence and the like it is possible to develop an oligonucleotide exhibiting higher activity or an oligonucleotide having lower toxicity and side effects.
  • the oligonucleotide can be expressed in the cell and bound to the target gene mRNA.
  • plasmids derived from Escherichia coli, Bacillus subtilis or yeast, animal viruses such as bacteriophages, retroviruses, vaccinia viruses, baculoviruses, or those obtained by fusing them with liposomes can be used.
  • the expression vector can be constructed by a known genetic engineering technique.
  • Oligonucleotide or expression vector capable of expressing it is a unit dose required for the practice of a generally accepted formulation together with a pharmaceutically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, etc. It can be produced as a nucleic acid medicine by mixing in the form.
  • This pharmaceutical composition is sterilized, for example, orally as tablets, capsules, elixirs, microcapsules or the like with sugar coating as necessary, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections such as solutions or suspensions.
  • Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavorings such as peppermint, red oil and cherry.
  • the present inventors show that the oligonucleotide represented by the base sequence “CGGAAACA” binds to tumor necrosis factor ⁇ (Tumor Necrosis Factor ⁇ : TNF- ⁇ ) mRNA and this mRNA. It was found to show an activity of inhibiting the binding to the known transcription factor RC3H1.
  • this oligonucleotide or an expression vector capable of expressing this oligonucleotide can be used to regulate the expression of TNF- ⁇ , and can be used as an immunoactivator or anticancer agent.
  • oligonucleotide represented by the base sequence “ATGAATAAA” has an activity of increasing the expression of a low-density lipoprotein receptor (Low-) Density Lipoprotein Receptor: LDLR).
  • this oligonucleotide or an expression vector capable of expressing this oligonucleotide can be used as an LDLR expression enhancer.
  • LDLR has a function of taking LDL cholesterol in plasma into cells by receptor-mediated endocytosis.
  • intracellular uptake of LDL cholesterol by LDLR expressed in the liver plays an important role in the clearance of plasma LDL cholesterol.
  • LDL cholesterol is responsible for cholesterol transport from the liver to peripheral tissues.
  • LDL cholesterol causes vascular injury due to deposition on the inner wall of the blood vessel, causing arteriosclerosis, hypertension, cerebral infarction, myocardial infarction, narrowing. causes heart disease.
  • oligonucleotide can enhance the expression of LDLR, according to the pharmaceutical composition comprising this oligonucleotide or the like as an active ingredient, plasma LDL cholesterol can be applied by enhancing the expression of LDLR, particularly when applied to the liver. It becomes possible to prevent, ameliorate or treat hyperlipidemia and hyperlipidemia-related diseases.
  • prevention here includes not only prevention in the previous stage of disease, but also prevention against recurrence after treatment of the disease.
  • hyperlipidemia-related diseases include diabetes, obesity, and cancer. It is not excluded.
  • the pharmaceutical composition containing the above-mentioned oligonucleotide or the like as an active ingredient has the potential to be applied to prevent, ameliorate, or treat Alzheimer's disease by enhancing the expression of LDLR and controlling the level of plasma LDL cholesterol. There is also.
  • ⁇ Example 1> Screening of oligonucleotides having binding inhibitory activity between TNF- ⁇ mRNA and transcription factor RC3H1
  • TNF- ⁇ tumor necrosis factor ⁇
  • oligonucleotide pools 1N and 2N were composed of 16 types of oligonucleotides in which any one of A, G, T, and C was linked to the 5 'end and 3' end of the basic sequences 1 and 2, respectively.
  • 16 types of oligonucleotides in which any of a, g, t, and c were bonded to the 5 ′ end and 3 ′ end of the basic sequences 1 and 2, respectively, were designated as oligonucleotide pools 1n and 2n.
  • A represents an adenine analog
  • G represents a guanine analog
  • T thymine analog
  • C represents a cytosine analog
  • a represents adenine
  • g represents guanine
  • t thymine
  • c represents cytosine.
  • N represents any one nucleic acid analog selected from adenine analog, guanine analog, thymine analog, or cytosine analog
  • n represents any one nucleic acid selected from adenine, guanine, thymine, or cytosine. Represents.
  • TNF- ⁇ mRNA was synthesized by in vitro translation.
  • MEGAscriptT7 kit by amplifying the 869-1652th nucleotide sequence from the 5 ′ end of TNF- ⁇ mRNA (GenBank Accession No.NM_000594.2, SEQ ID NO: 1) by PCR using a primer having T7 promoter sequence at the 5 ′ end (Ambion) was used to synthesize RNA according to the attached protocol.
  • a reaction for covalently binding Flag-hydrazide to the 3 ′ end of the synthesized TNF- ⁇ mRNA was performed, and the 3 ′ end of the TNF- ⁇ mRNA was labeled with Flag.
  • the labeled mRNA was purified using RNeasy Mini Kit from Qiagen.
  • mRNA flag labeling is performed according to a known method (see “Programmable ribozymes for mischarging tRNA with nonnatural amino acids and their applications to translation.” Methods, 2005, Vol, 36, No.3, p.239-244). It was.
  • TNF- ⁇ mRNA-Flag The purified Flag-labeled TNF- ⁇ mRNA (hereinafter referred to as “TNF- ⁇ mRNA-Flag”) is mixed with cell-extracted protein extracted from 293T cells, and oligonucleotide pools 1N, 1n, 2N, or 2n are mixed with each oligonucleotide. The reaction was carried out by adding the final concentration to 100 nM. After immunoprecipitation, the eluted sample was subjected to Western blotting using an anti-RC3H1 antibody (Bethyl Laboratories).
  • FIG. 4 Western blot results are shown in “FIG. 4”.
  • RC3H1 was detected in samples (lanes 3 and 4) obtained by mixing the cell extract protein with 10 pmol of TNF- ⁇ mRNA-Flag and oligonucleotide pool 1N or 1n and immunoprecipitating with anti-Flag antibody.
  • the cell extract protein itself is mixed.
  • the cell extract protein is mixed with 10 pmol TNF- ⁇ mRNA-Flag and oligonucleotide solvent (water), and a sample obtained by immunoprecipitation with anti-Flag antibody is obtained. It is flowing.
  • RC3H1 was detected in samples (lanes 5 and 6) obtained by mixing the cell extract protein with 10 pmol of TNF- ⁇ mRNA-Flag and oligonucleotide pool 2N or 2n and immunoprecipitating with anti-Flag antibody. It was revealed that the binding of RC3H1 to TNF- ⁇ mRNA-Flag was inhibited.
  • Oligonucleotides showing activity to inhibit the binding between TNF- ⁇ mRNA and RC3H1 were obtained from the oligonucleotide pools 2N and 2n.
  • Immunoprecipitation and Western blotting were performed on 16 kinds of oligonucleotides constituting the oligonucleotide pool 2N or 2n by the same procedure as described above.
  • oligonucleotides 2N1 and 2n1 were obtained from each of the oligonucleotide pools 2N and 2n as oligonucleotides exhibiting binding inhibitory activity between TNF- ⁇ mRNA and RC3H1.
  • each screening step was performed with the oligonucleotide library as 2 (oligonucleotide pools 1N and 2N, or oligonucleotide pools 1n and 2n), which is the minimum number of oligonucleotide pools.
  • the oligonucleotides shown in “Table 3” were obtained as oligonucleotides showing the binding inhibitory activity between the target TNF- ⁇ mRNA and RC3H1.
  • the 9mer oligonucleotide in which two nucleic acid analogs or nucleic acids are bound to the basic sequence of the nucleic acid analog 7mer can exhibit a desired activity (in this case, the binding inhibitory activity between TNF- ⁇ mRNA and RC3H1). It was revealed.
  • This result shows that oligonucleotides that can specifically bind to nucleotide chains of any sequence are prepared by preparing 16384 (4 to the 7th power) oligonucleotide pools that are all combinations of 7-mer nucleic acid analogs.
  • oligonucleotide library that always contains an oligosaccharide that exhibits a desired activity such as an antisense activity or a decoy activity even for a target gene for which a target sequence cannot be specified by using this library. It shows that nucleotides can be obtained.
  • Example 2 Screening for oligonucleotides having an activity of inhibiting the binding of ZFP36L1 to LDLR mRNA and increasing the expression of LDLR
  • a 9-mer oligonucleotide in which two nucleic acid analogs or nucleic acids were bound to the basic sequence of nucleic acid analog 7-mer It has been clarified that the activity of increasing the expression of a low-density lipoprotein receptor (LDLR) is exhibited.
  • LDLR low-density lipoprotein receptor
  • oligonucleotide pool 3N Sixteen types of oligonucleotides in which A, G, T, and C were bonded to the 5 ′ end and 3 ′ end of basic sequence 3 were designated as oligonucleotide pool 3N. In addition, 16 types of oligonucleotides in which a, g, t, and c were bonded to the 5 ′ end and 3 ′ end of the basic sequence 3, respectively, were designated as an oligonucleotide pool 3n.
  • oligonucleotide pools 3N or 3n were transfected into cells by lipofection (Darmafect 4, Thermo Scientific). Oligonucleotide pools 3N and 3n were used after being diluted so that the final concentration of each oligonucleotide was 40 nM.
  • FIG. 5 Western blot results are shown in “FIG. 5”. An increase in LDLR expression level was confirmed in cells transfected with oligonucleotide pools 3N and 3n (lanes 2 and 3), compared to cells not transfected with the oligonucleotide pool (lane 1). Note that ⁇ -actin was used as an internal standard for expression level evaluation. The expression level of ⁇ -actin was not significantly changed regardless of the presence or absence of transfection.
  • Oligonucleotides having activity to increase the expression of LDLR were acquired from the oligonucleotide pools 3N and 3n.
  • oligonucleotide pools 3N and 3n As a result, from the oligonucleotide pools 3N and 3n, the following oligonucleotides 3N1 and 3n1 were obtained as oligonucleotides showing LDLR expression increasing activity.
  • Example 3 the mechanism by which the oligonucleotides 3N1, 3n1 obtained in Example 2 increase the expression of LDLR was examined.
  • LDLR mRNA was synthesized by in vitro translation. Amplification of the 2677-3585bp region of LDLR mRNA (SEQ ID NO: 2, GenBank Accession No.NM_000527) was performed by PCR using a primer having a T7 promoter sequence at the 5 'end, and MEGAscript T7 kit (Cat.No.1333, Ambion) was used to synthesize RNA. A reaction for covalently binding Flag-hydrazide to the 3 ′ end of the synthesized LDLR mRNA was performed, and the 3 ′ end of the LDLR mRNA was labeled with Flag. The labeled mRNA was purified using RNeasy Mini Kit (Cat. No. 74106) manufactured by Qiagen.
  • Flag-labeled LDLR mRNA was mixed with anti-Flag antibody beads (Cat. No. F2426, Sigma) and reacted at 4 ° C for 1 hour. Thereafter, 3 mg of cell-extracted protein extracted from 293T cells cultured in DMEM medium containing 10% FBS ⁇ ⁇ was added, and further reacted at 4 ° C for 1 hour. After washing away unbound protein, RNA and RNA binding protein were eluted with Flag peptide.
  • ZFP36L1 and ZFP36L2 were identified by LC-MS / MS.
  • ZFP36L1 and ZFP36L2 form one family of ARE binding factors (hereinafter referred to as “ZFP36 family”) together with ZFP36 (also known as TTP).
  • ZFP36 family has been reported to bind to ARE and destabilize mRNA and function to promote degradation (“Tristetraprolin and its family members can promote the cell-free deadenylation of AU-rich element-containing mRNAs by poly (A) ribonuclease. ”Molecular Cell Biology, 2003, Vol.23, No.11, p.3798-812).
  • LDLR mRNA was synthesized by in vitro translation and labeled with Flag peptide.
  • 3′UTR 2677-3585 bps from the 5 ′ end of LDLR mRNA (see SEQ ID NO: 2) was used.
  • LDLR mRNA -Flag Purified Flag-labeled LDLR mRNA
  • FIG. 6 Western blot results are shown in “FIG. 6”.
  • ZFP36L1 was detected in a sample (lane 2) obtained by mixing cell extract protein containing ZFP36L1 / ZFP36L2 with LDLR mRNA-Flag and immunoprecipitating with anti-Flag antibody.
  • lane 1 the cell extracted protein itself flows.
  • ZFP36L1 can bind to LDLR mRNA-Flag.
  • ZFP36L2 the same applies to the present embodiment hereinafter.
  • oligonucleotides 3N1, 3n1 can remarkably inhibit the binding of ZFP36L1 and ZFP36L2 to LDLR mRNA.
  • the result of this example is that the 9mer oligonucleotide in which two nucleic acid analogs or nucleic acids are bound to the base sequence of the nucleic acid analog 7mer inhibits the binding of the desired activity (here, ZFP36L1 and ZFP36L2 to LDLR mRNA) As a result, it is clarified that the activity of increasing the expression of LDLR can be exhibited.
  • the desired activity here, ZFP36L1 and ZFP36L2 to LDLR mRNA
  • the oligonucleotides 3N1, 3n1 inhibit the binding of ZFP36L1 and ZFP36L2 to LDLR mRNA, thereby inhibiting the mRNA degradation promoting function of ZFP36L1 and ZFP36L2 and stabilizing LDLR mRNA. It was strongly suggested that the expression of LDLR mRNA was increased by promoting. This is very significant in that it is revealed for the first time that ZFP36L1 and ZFP36L2 are involved in the expression control mechanism of LDLR mRNA in vivo.
  • the oligonucleotide screening method according to the present invention can contribute to elucidating a new control mechanism in the living body, starting from the obtained oligonucleotide.

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Abstract

La présente invention concerne un procédé de criblage d'un oligonucléotide, qui peut être utilisé pour obtenir de manière efficace un oligonucléotide ayant des activités biologiques ou chimiques y compris une activité d'inhibition de l'expression d'un gène cible. L'invention concerne spécifiquement un procédé de criblage d'un oligonucléotide, qui comprend : une étape S2 consistant à préparer au moins deux ensembles d'oligonucléotides ; et une étape S3 consistant à identifier un ensemble d'oligonucléotides comprenant un oligonucléotide ayant une activité biologique ou chimique parmi les ensembles d'oligonucléotides. Dans l'étape S2, chacun des ensembles d'oligonucléotides comprend un oligonucléotide composé d'une séquence de bases de 7 motifs monomères comprenant un analogue d'acide nucléique et un ou plusieurs composants qui sont liés à la séquence de bases et qui sont indépendamment choisis parmi un analogue d'acide nucléique et un acide nucléique, l'analogue d'acide nucléique étant choisi parmi des analogues d'adénine, des analogues de guanine, des analogues de thymine et des analogues de cytosine et l'acide nucléique étant choisi parmi l'adénine, la guanine, la thymine et la cytosine, et les séquences de bases des ensembles d'oligonucléotides étant différentes l'une de l'autre.
PCT/JP2010/061888 2009-07-22 2010-07-14 Procédé de criblage d'oligonucléotides, et bibliothèque d'oligonucléotides Ceased WO2011010583A1 (fr)

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JP2002521310A (ja) 1997-09-12 2002-07-16 エクシコン エ／エス オリゴヌクレオチド類似体
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