WO2010005055A1 - Structure oligonucléotidique et procédé de régulation de l’expression génique - Google Patents

Structure oligonucléotidique et procédé de régulation de l’expression génique Download PDF

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WO2010005055A1
WO2010005055A1 PCT/JP2009/062535 JP2009062535W WO2010005055A1 WO 2010005055 A1 WO2010005055 A1 WO 2010005055A1 JP 2009062535 W JP2009062535 W JP 2009062535W WO 2010005055 A1 WO2010005055 A1 WO 2010005055A1
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strand
oligonucleotide
effector
chain
trigger
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Japanese (ja)
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立花亮
田辺利住
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公立大学法人大阪市立大学
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    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to an oligonucleotide structure and a gene expression control method that can be effectively used for RNA interference, RNA interference inhibition, miRNA, miRNA inhibition, decoy nucleic acid, SNP detection, and the like.
  • RNA interference RNA interference
  • RNAi and miRNA mRNA having a sequence homologous to the dsRNA is degraded by introducing dsRNA formed by combining guide strand RNA and passenger strand RNA or miRNA and miRNA * into each other.
  • this refers to the phenomenon of suppressing gene expression (see, for example, Non-Patent Documents 1 to 4).
  • RNAi and miRNA bind to mRNA having a sequence homologous to dsRNA. However, it also binds similarly to mRNA with a single base substitution. For this reason, when there is only a slight sequence difference between the normal gene and the abnormal gene, there is a problem that normal mRNA is degraded in normal cells. Thus, RNAi / miRNA pharmaceuticals have a problem of large side effects.
  • modified polynucleotides are used (for example, see Patent Document 1).
  • RNAs can be incorporated into dicers as single strands or have other functions as single strands in addition to functioning as double-stranded siRNAs (for example, non-patented) Reference 5).
  • iPS cells artificial pluripotent stem cells
  • ES cells embryonic stem cells
  • the differentiated and induced cell group includes undifferentiated stem cells and cells that have not differentiated into desired cells.
  • the presence of various types of cells may cause problems such as canceration.
  • a tumor may be formed when 0.01% of undifferentiated cells are mixed in a cell group transplanted by regenerative medicine. For this reason, attempts have been made to remove undifferentiated stem cells and cells that have not differentiated into desired cells from a group of cells that have been differentiated and induced from stem cells.
  • the present invention has been made in view of the above problems, and an object of the present invention is to distinguish between normal cells and abnormal cells such as cancer cells and function only in abnormal cells or normal cells, and oligonucleotide structures and An object of the present invention is to provide a gene expression control method.
  • Another object of the present invention is to provide an oligonucleotide structure and a gene expression control method capable of easily selecting and separating desired cells from a group of cells containing many types of cells. To do.
  • Another object of the present invention is to provide a new method for detecting a single base substitution product that detects a single base substitution product.
  • the present inventors have found the following oligonucleotide structure and gene expression control method and completed the present invention. That is, the present invention is as follows.
  • the oligonucleotide structure of the present invention is composed of three oligonucleotides, a trigger strand, an effector strand, and a support strand, and each of the three oligonucleotides is composed of the other two strands.
  • the trigger strand has a site for recognizing the sequence of another polynucleotide outside the site to which the support strand is bonded.
  • the oligonucleotide structure of the present invention has a three-way joint (Threee Way Junction, hereinafter also referred to as “TWJ”) structure as described above.
  • the trigger strand has a site for recognizing the sequence of another polynucleotide outside the site to which the support strand is bonded.
  • TWJ Three Way Junction
  • an exchange reaction between the support strand and the other polynucleotide occurs.
  • the three-way joint structure collapses, and the oligonucleotide structure is divided into a trigger strand, a conjugate of another polynucleotide, an effector strand, and a support strand.
  • the effector chain is designed so as to have substantially the same base sequence as the base sequence of at least a part of the target gene or target gene transcript.
  • the strand becomes a guide strand RNA.
  • strand which can be couple
  • the “substantially identical base sequence” includes a base sequence that is completely complementary to the base sequence of at least a part of the target gene or target gene transcript, a DNA sequence and an RNA sequence, a target It includes a mutant chain of at least a part of the base sequence of the gene or target gene transcript.
  • the trigger strand has a site that recognizes the sequence of another polynucleotide.
  • the base sequence of the portion where the trigger strand and the support strand are bonded is the portion where a strand exchange reaction occurs due to another polynucleotide. Therefore, a part of the trigger strand is designed to have a base sequence that is substantially the same as the base sequence of at least a part of a gene that is expressed in abnormal cells such as cancer cells and not expressed in normal cells. deep. By setting it as such a structure, the oligonucleotide structure of this invention can be disintegrated only in a desired cell.
  • a part of the trigger strand is expressed in a desired cell in a normal cell or a group of cells differentiated from a stem cell, and at least one of genes not expressed in an abnormal cell or a cell that has not differentiated into a desired cell.
  • the three-way joint structure is decomposed only in the abnormal cell, and the single effector chain is formed. It becomes. Even if the effector chain remains single-stranded, it can function as, for example, antisense or single-stranded functional RNA. If there is another single strand that complementarily binds to the effector strand, it becomes a double strand, for example, functions as RNAi. In addition, the other single strand that binds to the trigger strand does not undergo a chain exchange reaction unless it is homologous with the trigger strand. That is, the three-way joint structure is not disassembled.
  • the oligonucleotide structure of this invention can be used also for detection of SNPs.
  • the effector strand may form a double strand with a complementary oligonucleotide. It is good to form a double strand from the effector strands obtained from two types of oligonucleotide structures having trigger strands different in sequence of other polynucleotides to which the trigger strand binds.
  • the sequence of the other polynucleotide is preferably derived from a different gene.
  • the gene expression suppression method of the present invention is composed of three oligonucleotides, a trigger strand, an effector strand, and a support strand, and each of the three oligonucleotides has two other strands.
  • An oligonucleotide structure having a site for recognizing the sequence of another polynucleotide outside the site to which the support strand is bonded; Or it introduce
  • the effector chain has a base sequence substantially identical to at least a part of the base sequence of the target gene or target gene transcript.
  • An oligonucleotide that binds complementarily to the effector strand of the oligonucleotide structure is introduced together with the oligonucleotide structure, and the single-stranded effector produced in the cell or tissue in which the collapse of the three-way joint structure has occurred
  • the strand and the oligonucleotide may form a double strand.
  • Two types of oligonucleotide structures that have the same base sequence of the effector chain and different oligonucleotides recognized by the trigger strand are introduced into cells or tissues, and the other polynucleotides recognized by the trigger strand exist. Only in the cells or tissues to be treated, the three-way joint structure of the two types of oligonucleotide structures may be disrupted, and the resulting effector strands may form a double strand.
  • the method for detecting a single base substitution product of the present invention is composed of three oligonucleotides, a trigger strand, an effector strand, and a support strand, and each of the three oligonucleotides is composed of two other strands.
  • An oligonucleotide structure having a site for recognizing the sequence of another polynucleotide outside the site to which the support strand is bound, Another polynucleotide or a single nucleotide substitution product of another polynucleotide is mixed, and a single nucleotide substitution product of another polynucleotide is detected based on the presence or absence of the collapse of the oligonucleotide structure.
  • the oligonucleotide structure of the present invention When the oligonucleotide structure of the present invention is used, if there is another polynucleotide identical to the site that recognizes the sequence of another polynucleotide outside the site where the support strand of the trigger strand is bound, the oligonucleotide The structure collapses. On the other hand, in the case of a single nucleotide substitution product of another polynucleotide, since it cannot bind to the trigger strand, the oligonucleotide structure does not collapse. Therefore, a single-base substitution product can be detected by confirming the presence or absence of the collapse of the oligonucleotide structure.
  • the oligonucleotide structure of the present invention is composed of three oligonucleotides, a trigger strand, an effector strand, and a support strand, and each of the three oligonucleotides is composed of the other two strands.
  • the trigger strand has a site for recognizing the sequence of another polynucleotide outside the site to which the support strand is bonded. This structure distinguishes between desired cells in a group of cells differentiated from normal cells or stem cells and abnormal cells or cells that have not differentiated into desired cells, and decomposes the three-way joint structure only in one of the cells. Effector chains can be obtained.
  • FIG. 1 is a diagram for explaining the concept of the oligonucleotide structure of the present invention.
  • FIG. 2 is a diagram for explaining the mechanism of collapse of the oligonucleotide structure of the present invention.
  • FIG. 3 is a graph showing the relative amount of KDR mRNA, although no TWJ, TWJ no tail, TWJ mutant tail, siRNA, or nucleic acid component was introduced (“none” in the figure).
  • FIG. 4 is a photograph showing the results of Native-PAGE.
  • FIG. 5 is a photograph showing the results of evaluating the effect of mRNA-corresponding sequences of different lengths on the decay of the TWJ structure.
  • FIG. 1 is a diagram for explaining the concept of the oligonucleotide structure of the present invention.
  • FIG. 2 is a diagram for explaining the mechanism of collapse of the oligonucleotide structure of the present invention.
  • FIG. 3 is a graph showing the relative amount of KDR mRNA, although no
  • FIG. 6 is a photograph showing the results of evaluating the influence of mRNA-corresponding sequences having different lengths on the ability to recognize single nucleotide substitutions.
  • FIG. 7 is a photograph showing the results of evaluating the influence of mRNA-corresponding sequences having different positions for single base substitution on the ability to recognize single base substitution.
  • FIG. 8 is a photograph showing the results of evaluating the influence of a trigger strand having an mRNA-corresponding sequence having a single base substitution position of 3 and a complementary sequence thereto on TEJ decay.
  • FIG. 9 is a graph evaluating the effect of siRNA on caspase in a GFP-expressing strain and a GFP non-expressing strain.
  • FIG. 10 is a diagram illustrating a TWJ structure that reacts with GFP mRNA and releases caspase 3 or 8 siRNA.
  • FIG. 11 is a graph showing an evaluation of the effect of a TWJ structure that releases siRNA against caspase in a GFP-expressing strain and a GFP non-expressing strain.
  • FIG. 1 is a diagram for explaining the concept of the oligonucleotide structure of the present invention.
  • the oligonucleotide constituting the oligonucleotide structure of the present invention may be either DNA or RNA, may be a chimera of DNA and RNA, or may be further chemically modified.
  • the oligonucleotide structure of the present invention is composed of three oligonucleotides, a trigger strand 1, an effector strand 2, and a support strand 3.
  • the trigger chain 1, the effector chain 2, and the support chain 3 chain each form a three-way joint structure by complementary binding to the other two chains.
  • the t2 site of the trigger chain 1 and the e2 site of the effector chain 2 are the e1 site of the effector chain 2 and the s2 site of the support chain 3, and the s1 site of the support chain 3 and the t1 site of the trigger chain 1
  • One site is bonded to each other. These bonds do not have to be complementary bonds, but may be any bonds as long as part of them is bonded.
  • the t1 site of the trigger strand has a homologous sequence with other polynucleotides. This sequence allows other polynucleotides to bind complementarily and disrupt the three-way joint structure of the oligonucleotide structure. Further, at the t1 site of the trigger strand 1, there is a site t1-2 that does not bind to the support strand 3 and recognizes the sequence of another polynucleotide. When a specific polynucleotide binds to t1-2, a chain exchange reaction occurs, and the three-way joint structure of the oligonucleotide structure of the present invention collapses.
  • the t1 site of the trigger strand 1 binds complementarily to a part of mRNA specifically expressed in abnormal cells such as cancer cells.
  • the t1-2 site should be a 6-20 base sequence. That's fine.
  • the t1-2 site may be about 4 bases. Or you may adjust the mRNA base sequence couple
  • the mRNA that binds to the t1 site of the trigger chain 1 may be the same mRNA as the mRNA that the effector chain 2 described later binds, or may be a different mRNA.
  • the base sequence may be determined so that the mRNA site binding to the t1 site of the trigger chain 1 and the mRNA site binding to the effector chain 2 are different.
  • the effector chain 2 is an oligonucleotide that is a source of functions such as RNAi after the oligonucleotide structure collapses.
  • the effector chain 2 is an oligonucleotide having about 21 to 30 bases as a whole (e1 site + e2 site).
  • the e1 site or e2 site of the effector chain 2 may be 6 to 7 bases or more.
  • the trigger chain is compared with the sequence of the e2 site of the effector chain 2 so that the bond between the effector chain 2 and the trigger chain 1 is weak enough to be decomposed by the bond between the trigger chain 1 and the specific oligonucleotide. What is necessary is just to design 1 t2 site
  • the effector chain 2 is a sequence that exerts some function on the target gene or target gene transcript after the oligonucleotide structure is disrupted. For this reason, the effector chain 2 needs to have a substantially identical base sequence of at least a part of the target gene or the target gene transcript.
  • the target gene or target gene transcript may be appropriately selected depending on the purpose of using the oligonucleotide structure of the present invention. For example, an oncogene specifically expressed in cancer cells. Alternatively, it may be a gene that is expressed only in normal cells or a gene that is expressed only in desired cells in a group of cells differentiated from stem cells.
  • the support chain 3 is bonded to the effector chain 2 and the trigger chain 1 to form a three-way joint structure.
  • the s1 part of the support chain 3 is complementarily bound to the t1-1 part of the trigger chain 1 to the extent that the three-way joint structure is maintained.
  • the bond between the support chain 3 and the effector chain 2 is a bond having such a weakness that it can be split with the collapse of the three-way joint structure.
  • FIG. 2 (a) when a polynucleotide 4 (mRNA in the example of FIG. 2) having a site that binds complementarily to the trigger strand 1 is present in the vicinity of the oligonucleotide structure of the present invention, the trigger A site t1-2 that recognizes the sequence of another polynucleotide in strand 1 is recognized, and an exchange reaction takes place with support strand 3. This exchange reaction causes the three-way joint structure to collapse.
  • mRNA in the example of FIG. 2 mRNA in the example of FIG. 2
  • the trigger A site t1-2 that recognizes the sequence of another polynucleotide in strand 1 is recognized, and an exchange reaction takes place with support strand 3.
  • This exchange reaction causes the three-way joint structure to collapse.
  • the trigger chain 1, the effector chain 2, and the support chain 3 are separated from each other by the collapse of the above-described three-way joint structure. Thereby, a single-chain effector chain is obtained.
  • the single-stranded effector strand can be used as it is, for example, as an antisense or single-stranded functional RNA.
  • the effector chain has no particular function.
  • the effector strand 2 and the complementary oligonucleotide 2' form a double strand.
  • the effector chain is a chimera of RNA or RNA and DNA
  • the formed double-stranded oligonucleotide degrades mRNA having a sequence homologous to it through the RNAi mechanism as siRNA.
  • the other oligonucleotide 2 'that binds complementarily to the single-stranded effector strand 2 may be added simultaneously with the oligonucleotide structure or may be an oligonucleotide present in the cell.
  • the oligonucleotide 2 ′ that binds complementarily to the effector strand 2 may be derived from the effector strand 2 of another oligonucleotide structure. This is because intracellular RNAi and the like can be performed more effectively.
  • the three-way joint structure does not collapse unless there is a polynucleotide having a site that binds complementarily to the trigger strand 1. Therefore, in the present invention, it is important to select a polynucleotide having a trigger strand 1 and a site that complementarily binds to the trigger strand.
  • a polynucleotide having a site that binds complementarily to the trigger strand for example, only in a specific cell such as a cancer cell, the three-way joint structure can be disrupted only in the specific cell. it can.
  • the three-way joint structure does not collapse.
  • the oligonucleotide structure of the present invention may be used alone, but a plurality of types of oligonucleotide structures may be used.
  • the gene A is included in the trigger strand of one oligonucleotide structure.
  • a sequence that complementarily binds to at least a part of mRNA is provided, and a sequence that complementarily binds to at least a part of mRNA of gene B is provided on the trigger strand of another oligonucleotide structure.
  • a duplex can be formed from the effector chain only when genes A and B are expressed.
  • the oligonucleotide structure of the present invention can be introduced into a cell, tissue, or individual as long as the target gene can be transcribed into RNA or translated into protein within the cell. Also good.
  • the introducer that is the subject of the present invention means a cell, tissue, or individual.
  • the cells used in the present invention include germline cells, somatic cells, differentiated totipotent cells, multipotent cells, divided cells, non-divided cells, parenchymal tissue cells, epithelial cells, immortalized cells, and transformed cells. It may be. Specific examples include undifferentiated cells such as stem cells, cell groups differentiated and derived from stem cells, cells derived from organs or tissues, or differentiated cells thereof.
  • Tissues include single-cell embryos or constitutive cells, multi-cell embryos, fetal tissues and the like.
  • the differentiated cells include adipocytes, fibroblasts, muscle cells, cardiomyocytes, endothelial cells, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, Examples include basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and endocrine or exocrine gland cells.
  • the individual used as the recipient in the present invention include those belonging to plants, animals, protozoa, viruses, bacteria, or fungal species.
  • the plant may be monocotyledonous, dicotyledonous or gymnosperm, and the animal may be a vertebrate or invertebrate.
  • Preferred microorganisms as recipients of the present invention are those used in agriculture or by industry and are pathogenic to plants or animals.
  • Fungi include organisms in both mold and yeast forms.
  • vertebrates include mammals including fish, cattle, goats, pigs, sheep, hamsters, mice, rats, monkeys and humans, and invertebrates include nematodes and other reptiles, gyro Drosophila, and other insects are included.
  • a method for introducing an oligonucleotide structure into a recipient when the recipient is a cell or tissue, a calcium phosphate method, an electroporation method, a lipofection method, a virus infection, or a double-stranded polynucleotide solution. Soaking or transformation method is used. Examples of the method for introduction into an embryo include microinjection, electroporation, and virus infection.
  • a method by injection or perfusion into the body cavity or stromal cells of the plant or spraying is used.
  • an animal individual In the case of an animal individual, it is introduced systemically by oral, topical, parenteral (including subcutaneous, intramuscular and intravenous administration), vaginal, rectal, nasal, ophthalmic, intraperitoneal administration, etc.
  • the method, electroporation method, virus infection or the like is used.
  • the oligonucleotide structure can be mixed directly with the biological food.
  • it when introduced into an individual, it can be administered, for example, as an implanted long-term release preparation or by ingesting an introduced body into which an oligonucleotide structure has been introduced.
  • the oligonucleotide structure of the present invention can be used for any purpose as long as it obtains a desired oligonucleotide at a predetermined location and utilizes the desired oligonucleotide.
  • it can be used for the following purposes.
  • RNAi is caused as double-stranded RNA. be able to.
  • the functions of siRNA and miRNA can be inhibited as a single-stranded modified RNA.
  • the obtained single-stranded effector strand 2 is DNA, it can function as a decoy nucleic acid as a double-stranded DNA.
  • a fluorescent dye that develops color when the three-way joint structure collapses may be added to the end of the effector chain or support chain, and used for detection of single base substitution. it can. Or it can also be used for the detection of single base substitution by confirming the presence or absence of collapse of the three-way joint structure by electrophoresis or the like.
  • the three-way joint structure does not collapse in the oligonucleotide with a single base substitution.
  • a specific oligonucleotide can be functioned only in a desired cell.
  • normal cells and abnormal cells can be distinguished.
  • only cells differentiated into desired cells from a group of cells differentiated / derived from stem cells can be selected.
  • the oligonucleotide structure of the present invention has a structure in which three simple short sequences are combined. As a result, it is considered non-toxic.
  • the use of the oligonucleotide structure of the present invention can suppress the collapse of the three-way joint structure in normal cells. For this reason, the side effect of the RNAi medicine considered as a conventional problem can be reduced.
  • oligonucleotide structure of the present invention by selecting an effective trigger chain and an effector chain and designing a support chain that can bind to this, it is possible to prevent diseases involving genes and diseases affected by individual genotypes. But it can respond flexibly.
  • Example 1 [Evaluation of RNAi generation in cells] The occurrence of RNAi in human breast cancer-derived cells (MCF-7 cells) was evaluated. Specifically, a DNA corresponding to a part of mRNA of vascular endothelial growth factor (VEGF) is used as an effector chain, and KDR (2), which is one of VEGF receptors, is used as a trigger chain. Type receptor) DNA was used. VEGF is a factor that is produced by many cancer cells and induces blood vessels to cancer tissue. Furthermore, it is known that cancer cells themselves proliferate in response to this growth factor. KDR is present on the surface of cells such as vascular endothelial cells and cancer cells, and binds to VEGF.
  • VEGF vascular endothelial growth factor
  • TWJ structure The oligonucleotide structure having a three-way joint structure shown in FIG. 1 (hereinafter referred to as “TWJ structure”), and the sequence of the site t1-2 where the oligonucleotide structure recognizes the trigger chain is changed to a sequence that cannot recognize the trigger chain.
  • An oligonucleotide structure having a three-way joint structure (hereinafter referred to as “TWJ mutant tail”), and an oligonucleotide structure having a three-way joint structure in which the oligonucleotide structure does not have a site t1-2 for recognizing a trigger strand A body (hereinafter referred to as “TWJ no tail”) was prepared. Table 1 shows the sequences of the prepared oligonucleotides.
  • siRNA oligonucleotide structure and effector chain
  • siRNA siRNA prepared using the RNA shown in SEQ ID NO: 4
  • MCF-7 cell human breast cancer-derived cell
  • KDR cDNA was prepared, and the relative amount of mRNA was examined by real-time PCR (polymerase chain reaction, polymerase chain reaction). The results are shown in FIG. FIG. 3 is a graph showing the relative amount of KDR mRNA, although no TWJ, TWJ no tail, TWJ mutant tail, siRNA, or nucleic acid component was introduced (“none” in the figure).
  • FIG. 3 shows that TWJ clearly causes RNAi and decreases KDR mRNA.
  • TWJ no tail and TWJ mutant tail do not reduce KDR mRNA and cause almost no RNAi. From this, it was found that RNAi can be caused by using the oligonucleotide structure of the present invention.
  • Example 2 [Evaluation of decay of oligonucleotide structure in electrophoresis] Whether or not the oligonucleotide structure having the three-way joint structure shown in FIG. 1 (hereinafter referred to as “TWJ structure”) was made and whether or not the structure was destroyed were examined. Table 2 shows the sequences of the prepared oligonucleotides.
  • the experiment is as follows from the effector chain, support chain, trigger chain, chain that disrupts the structure (corresponding to mRNA), and chain complementary to the effector chain (strand that generates siRNA-corresponding duplex together with the effector chain)
  • the combinations were mixed and analyzed by Native-PAGE after 30 minutes.
  • the results are shown in FIG.
  • FIG. 4 is a photograph showing the results of Native-PAGE.
  • each lane shows the following.
  • Lane a Effector chain, support chain, trigger chain b
  • Lane c complementary to effector chain Effector chain, support chain, trigger chain, effector Strand
  • d lane complementary to strand effector strand, strand complementary to effector strand
  • the band consisting of the mRNA equivalent chain and the trigger chain is strongly detected at the position of C (region surrounded by a white ellipse in the figure), and the structure is collapsed. I understood.
  • a siRNA-equivalent duplex consisting of the released effector strand and a strand complementary to the effector strand was strongly detected at the position A (region surrounded by a white ellipse in the figure).
  • the lane d shows the positive control in the case where the effector strands added in the lanes b and c and the strand complementary to the effector strands generate a total amount of siRNA equivalent duplexes.
  • Example 3 [Evaluation of length of polynucleotide binding to trigger strand of TWJ structure]
  • a TWJ structure having a three-way joint structure shown in FIG. 1 was prepared, and the influence of the length of the mRNA equivalent sequence on the decay of the TWJ structure was examined.
  • a DNA corresponding to a part of mRNA of vascular endothelial growth factor (VEGF) is used as an effector chain
  • KDR (2) which is one of VEGF receptors
  • Type receptor Type receptor
  • FIG. 5 is a diagram showing the results of evaluating the influence of mRNA-corresponding sequences having different lengths on the collapse of the TWJ structure.
  • FIG. 5 is a photograph showing the results of Native-PAGE.
  • M lane is a standard substance
  • TWJ lane is a TWJ structure itself
  • Example 4 Evaluation of single base substitution recognition ability
  • a TWJ structure having a three-way joint structure shown in FIG. 1 was prepared, and the influence of the length of the mRNA-corresponding sequence on the ability to recognize a single base substitution was examined.
  • a DNA corresponding to a part of mRNA of vascular endothelial growth factor (VEGF) is used as an effector chain
  • KDR (2) which is one of VEGF receptors, is used as a trigger chain.
  • Type receptor Type receptor
  • FIG. 6 is a photograph showing the results of evaluating the influence of mRNA-corresponding sequences of different lengths on the ability to recognize single nucleotide substitutions.
  • the mRNA equivalent sequence and the TWJ structure differ in the length n of the mRNA equivalent sequence (n is the site of the mRNA binding to the t1-2 site of the trigger chain 1) and the position of the single base substitution.
  • n is the site of the mRNA binding to the t1-2 site of the trigger chain 1
  • the results are shown in FIG.
  • FIG. 6 is a photograph showing the results of Native-PAGE.
  • M lane indicates the standard substance
  • TWJ lane indicates the TWJ structure itself, in each lane
  • the upper number indicates the length of n
  • the lower number indicates the position of the single base substitution.
  • “-” indicates that no single base substitution is included.
  • the number indicating the position of the single base substitution where the positive number indicates that there is a single base substitution at the site of the mRNA that binds to the t1-2 site of the trigger strand 1, and the negative number indicates the t1- It means that there is a single base substitution at the site of mRNA that binds to one site. Positive / negative is calculated from the boundary between the trigger chain 1 t1-1 site and t1-2 site.
  • Example 5 [Evaluation of position specificity of single base substitution recognition ability] A TWJ structure having a three-way joint structure shown in FIG. 1 was prepared, and the influence of the position of single base substitution contained in the mRNA-corresponding sequence on the single base substitution recognition ability was examined. Specifically, a DNA corresponding to a part of mRNA of vascular endothelial growth factor (VEGF) is used as an effector chain, and KDR (2), which is one of VEGF receptors, is used as a trigger chain. Type receptor) DNA was used. Table 5 shows the sequences of the prepared oligonucleotides. The underline in each sequence means a single strand part of the TWJ structure or a complementary strand of the single strand part.
  • VEGF vascular endothelial growth factor
  • FIG. 7 is a photograph showing the results of evaluating the influence of mRNA-corresponding sequences with different positions for single base substitution on the ability to recognize single base substitution.
  • match is a sequence corresponding to mRNA that complementarily binds to the t1 site of the trigger strand
  • 1 to 8 are single nucleotide substitutions shown in Table 5
  • 2 ′, 4 ′ are 2
  • 4 A single base substitution product in which a different ribonucleic acid is introduced at the same position as the single base substitution product is shown.
  • a complementary trigger chain was prepared at position 3 of the mRNA-corresponding sequence as shown in Table 6, and the same experiment as described above was performed.
  • the underline in the sequence means a single-stranded part of the TWJ structure.
  • FIG. 8 is a photograph showing the results of evaluating the influence of the mRNA corresponding sequence with the position of single base substitution 3 and the trigger strand having a sequence complementary thereto on TEJ structure collapse.
  • M lane indicates a standard substance
  • lane 1 indicates a TWJ structure
  • lane 2 indicates a mixture of a TWJ structure and an mRNA-corresponding sequence that are reacted. It can be seen from FIG. 8 that the TWJ structure has collapsed. That is, it was found that the use of the TWJ structure has a single base substitution recognition ability.
  • Fas ligand binds to cell surface receptor Fas and activates caspase-8.
  • Caspase 8 then activates caspase 3 and causes apoptosis.
  • caspase 8 or 3 is not present in the cell, this cell becomes resistant to apoptosis by Fas ligand.
  • siRNAs against caspases 8 and 3 shown in Table 7 siCasp8 And siCasp3 (each 33 microM), siRNA against GFP as a control (siGFP, 33 microM), transfection agent alone (Lipo), and no addition (none) were transfected into each cell line.
  • Fas ligand 25 ng / ml was added.
  • cell viability was assayed by WST in a conventional manner.
  • FIG. 9 is a graph evaluating the effect of siRNA on caspases in GFP-expressing strains and GFP non-expressing strains.
  • FasL + indicates that Fas ligand was added
  • FasL ⁇ indicates that Fas ligand was not added
  • the vertical axis indicates WST activity of the cells. From FIG. 9, the WST activity of the cells containing Fas ligand was less than half that of the cells not containing GFP expression and non-expressing strains.
  • the case where siRNA against caspase was added was almost the same as the case where Fas ligand was not added regardless of the GFP expression or non-expression strain. That is, it was suggested that siRNA for caspase knocks down caspase mRNA in cells.
  • TWJ structure A TWJ structure having a trigger chain having a site recognizing GFP mRNA and an effector chain that releases siRNA for caspase 8 or 3 (“guide chain” shown in Table 8 below) was designed.
  • the TWJ structure is disrupted by GFP mRNA, siRNA against caspase 8 or 3 is released, and caspase 8 or 3 is knocked down. This makes it resistant to apoptosis induction by Fas ligand.
  • GFP mRNA does not exist in GFP non-expressing cells, the TWJ structure does not collapse. That is, siRNA is not released and is sensitive to apoptosis. For this reason, cell death occurs by Fas ligand.
  • TWJcasp8 219 means a TWJ structure that reacts with GFP mRNA 219- and releases cRNA 8 siRNA as shown in FIG.
  • Cell selective survival by TWJ structure Using human cultured cell line HEK293T (GFP non-expressing strain) and a strain in which GFP is constitutively expressed (GFP expressing strain), four types of TWJ structures + passenger chains (each 33 microM), and only a transfection agent as a control ( Lipo) and none (none) were transfected into each cell line. Two days later, FasLigand (25 ng / ml) was added. One day later, cell viability was assayed by WST in a conventional manner. The results are shown in FIG.
  • FIG. 11 is a graph evaluating the effect of a TWJ structure that releases siRNA against caspase in a GFP-expressing strain and a non-GFP-expressing strain.
  • FasL + indicates that Fas ligand was added
  • FasL ⁇ indicates that Fas ligand was not added
  • the vertical axis indicates WST activity of the cells.
  • the TWJ structure of the present invention when used, it has cell selectivity that it survives if GFP mRNA is present, and that apoptosis is induced and death occurs if there is GFP mRNA.

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Abstract

La présente invention concerne une structure oligonucléotidique qui peut faire la différence entre une cellule normale et une cellule anormale comme une cellule cancéreuse et qui peut agir uniquement dans une cellule anormale. La structure oligonucléotidique comprend trois chaînes oligonucléotidiques, c’est-à-dire, une chaîne de déclenchement, une chaîne effectrice et une chaîne de soutien, chacune des trois chaînes oligonucléotidiques étant liée aux deux autres chaînes afin de former une structure ressemblant à un raccord à trois voies, et la chaîne de déclenchement possédant un site pouvant reconnaître les séquences des autres chaînes polynucléotidiques à l’extérieur d’un site qui est lié à la chaîne de soutien.
PCT/JP2009/062535 2008-07-09 2009-07-09 Structure oligonucléotidique et procédé de régulation de l’expression génique WO2010005055A1 (fr)

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Publication number Priority date Publication date Assignee Title
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