Hairpin nucleic acid composition

Abstract

The present invention addresses the problem of providing a nucleic acid medicine which is capable of inducing nucleic acid immunity in a cell-specific manner and is capable of capturing and / or inactivating a nucleic acid binding protein for which drug development is difficult.　Provided are: a hairpin nucleic acid composition which contains two or more types of hairpin nucleic acids; and a medicinal composition, a protein function-inhibiting composition and a cell death-promoting composition which contain the same as an active ingredient thereof.

Description

Hairpin Nucleic Acid Compositions

[0001] The present invention relates to a hairpin nucleic acid composition, a cell death-inducing composition, and a pharmaceutical composition, a protein function-inhibiting composition, and a cell death-promoting composition containing them as active ingredients.

[0002] Nucleic acid drugs are molecular targeted therapeutic agents that specifically bind to target nucleic acids or proteins and inhibit their function. Nucleic acid drugs have attracted attention as new medicines for diseases that have traditionally been difficult to treat, and in the 2010s, groundbreaking new drugs such as Givlaari, which target the liver as a disease site, were developed both in Japan and overseas (Non-Patent Document 1). However, the practical application of nucleic acid drugs, especially for cancer, has yet to be achieved.

[0003] Nucleic acid drugs that have been marketed to date have primarily been antisense nucleic acids and siRNAs, which inhibit the translation or splicing process of target mRNA by sequence-specific hybridization. In recent years, research into protein-targeting aptamers and decoy nucleic acids has also been actively pursued. Decoy nucleic acids, in particular, are capable of capturing and / or inactivating nucleic acid-binding proteins, such as transcription factors, which are difficult to drug, making them promising anticancer drugs. However, there are several issues with the practical application of decoy nucleic acids as nucleic acid drugs. First, decoy nucleic acids have low cell selectivity, resulting in significant off-target effects and a tendency to be toxic to normal cells. Furthermore, their use is limited to diseases for which target molecules exist.

[0004] Furthermore, in conventional nucleic acid drug discovery, the immunotoxicity of nucleic acid drugs themselves has been a problem. Immunotoxicity occurs when a defense mechanism against foreign nucleic acids, such as viruses (nucleic acid immunity), recognizes nucleic acid drugs as foreign substances, and to avoid this, additional efforts are required, such as chemical modification of nucleic acids and sequence selection (Non-Patent Document 2).

[0005] On the other hand, while efforts have been focused on suppressing nucleic acid immunity with nucleic acid drugs up until now, if it could be induced specifically in cancer cells and other cells, it is believed that effective immunotherapy with few side effects could be realized. Furthermore, if this were to be realized, it would be possible to minimize the need for chemical modifications and sequence considerations to suppress nucleic acid immunity, which would have significant benefits in terms of manufacturing costs and quality control. However, nucleic acid drug discovery from this perspective has been very little done to date, and there have been no successful examples.

[0006] Approved nucleic acid drugs (as of May 2021), Section 2, Department of Genetic Medicine, National Institute of Health Sciences. Shen, W. et al. Nat. Biotechnol. 2019, 37, 640-650.

[0007] An object of the present invention is to provide a nucleic acid drug that can capture and / or inactivate nucleic acid-binding proteins, for which drug discovery is difficult, and that can induce nucleic acid immunity in a cell-specific manner.

[0008] To solve the above problems, the present inventors focused on hybridization chain reaction (HCR), a type of nucleic acid self-assembly technology that has been used primarily for the detection and fluorescent imaging of trace amounts of nucleic acid molecules in vivo. The present inventors induced HCR specifically in cancer cells using a hairpin nucleic acid containing a protein-binding motif for the transcription factor NF-κB, and found that the cancer cells were killed and nucleic acid immunity was induced. The present invention is based on these novel findings and provides the following:

[0009] [1] A hairpin nucleic acid composition comprising two or more hairpin nucleic acids each comprising all or a portion of a protein-binding motif, wherein the hairpin nucleic acids comprise: an initiating hairpin nucleic acid comprising a target RNA-binding cassette and an extended hairpin nucleic acid-binding cassette; and extended hairpin nucleic acids comprising a protruding region cassette and a non-protruding region cassette, wherein each hairpin nucleic acid comprises, in order, a protruding region of 4 to 20 bases, a stem first region comprising 10 to 20 bases, a loop region of 3 to 50 bases, and a stem second region comprising 10 to 20 bases, wherein the stem first region and the stem second region are capable of hybridizing to each other intramolecularly; and in the initiating hairpin nucleic acid, the target RNA-binding cassette comprises all or a portion of the protruding region and the subsequent all or a portion of the stem first region, and is capable of hybridizing to all or a portion of a target RNA; and the extended hairpin nucleic acid-binding cassette comprises all or a portion of the stem second region, and is capable of hybridizing to the protruding region cassette of the extended hairpin nucleic acid.

[0014] The composition of the extended hairpin nucleic acid, wherein the overhang region cassette comprises all or part of the overhang region and all or part of the subsequent stem first region, and is capable of hybridizing with the non-overhang region cassette of another, different extended hairpin nucleic acid, the non-overhang region cassette comprises all or part of the stem second region, and is capable of hybridizing with the overhang region cassette of another, different extended hairpin nucleic acid, the loop region and / or overhang region comprises all or part of the protein-binding motif, and upon binding of the target RNA to the target RNA-binding cassette of the starting hairpin nucleic acid, the entire protein-binding motif becomes double-stranded. [2] The hairpin nucleic acid composition of [1], wherein the starting hairpin nucleic acid comprises an overhang region of 6 to 15 bases. [3] The hairpin nucleic acid composition of [1] or [2], wherein the starting hairpin nucleic acid is capable of hybridizing with the target RNA over a length of 6 or more bases. [4] The hairpin nucleic acid composition according to any one of [1] to [3], wherein the extended hairpin nucleic acid is capable of hybridizing with different extended hairpin nucleic acids over 6 or more bases.[5] The hairpin nucleic acid composition according to any one of [1] to [4], wherein the free energy change of the hairpin structure-forming reaction in the starting hairpin nucleic acid is -20 to -10 kcal / mol. [6] The hairpin nucleic acid composition according to any one of [1] to [5], wherein the hybridizable region comprises any of the following base sequences: (1) a base sequence that is completely complementary to the target sequence to be hybridized; (2) a base sequence in which one or more bases have been deleted, substituted, or added compared to (1); or (3) a base sequence that hybridizes to the target sequence to be hybridized under highly stringent conditions. [7] The hairpin nucleic acid composition according to any one of [1] to [6], wherein the hairpin nucleic acid is composed of DNA and / or RNA nucleotides. [8] The hairpin nucleic acid composition according to [7], wherein the nucleotides include modified nucleotides. [9] The hairpin nucleic acid composition according to any one of [1] to [8], wherein the protein-binding motifs are multiple types.

[10] The hairpin nucleic acid composition according to any one of [1] to [9], wherein the protein-binding motif comprises a transcription factor-binding motif.

[11] The hairpin nucleic acid composition according to any one of [1] to

[10] , wherein the protein capable of binding to the protein-binding motif comprises an apoptosis-related factor, an autophagy-related factor, or an inflammation-related factor.

[12] The hairpin nucleic acid composition according to any one of [1] to

[11] , wherein the target RNA is mRNA or miRNA.

[13] The hairpin nucleic acid composition according to any one of [1] to

[12] , wherein the target RNA is RNA that is expressed in a cell-specific manner.

[14] The hairpin nucleic acid composition according to

[13] , wherein the cells comprise cancer cells, inflammatory cells, or immune cells.

[15] The hairpin nucleic acid composition according to any one of [1] to

[14] , wherein the target RNA-binding cassette is hybridizable with the non-protruding region cassette of the extended hairpin nucleic acid.

[16] The hairpin nucleic acid composition according to any one of [1] to

[15] , wherein the extended hairpin nucleic acid comprises a 5'-end overhanging hairpin nucleic acid and a 3'-end overhanging hairpin nucleic acid.

[17] A protein function-inhibiting composition comprising as an active ingredient the hairpin nucleic acid composition according to any one of [1] to

[16] .

[18] A cell death-promoting composition comprising as an active ingredient the hairpin nucleic acid composition according to any one of [1] to

[16] .

[19] A pharmaceutical composition comprising, as an active ingredient, the hairpin nucleic acid composition according to any one of [1] to

[16] .

[20] The pharmaceutical composition according to

[19] , for treating a disease selected from the group consisting of cancer, immune system diseases, and neurodegenerative diseases.

[0010] (1) A cell death-inducing composition comprising a hairpin nucleic acid having a hairpin structure capable of forming a hybridization linkage structure, the cell death-inducing composition comprising an initiating hairpin nucleic acid that hybridizes with a target RNA, and an extended hairpin nucleic acid that hybridizes with the initiating hairpin nucleic acid or another extended hairpin nucleic acid. (2) The cell death-inducing composition according to (1), wherein the initiating hairpin nucleic acid comprises a target RNA-binding cassette that hybridizes with the target RNA, and an extended hairpin nucleic acid-binding cassette that hybridizes with the extended hairpin nucleic acid, and the extended hairpin nucleic acid comprises a protruding region cassette that hybridizes with the initiating hairpin nucleic acid or another extended hairpin nucleic acid, and a non-protruding region cassette that further hybridizes with another extended hairpin nucleic acid. (3) The cell death-inducing composition according to (1) or (2), wherein binding of the target RNA to the target RNA-binding cassette dissociates the hairpin structure of the initiating hairpin nucleic acid, allowing the extended hairpin nucleic acid-binding cassette to hybridize with the protruding region cassette of the extended hairpin nucleic acid, and this hybridization dissociates the hairpin structure of the extended hairpin nucleic acid, allowing the non-protruding region cassette to hybridize with the protruding region cassette of another extended hairpin nucleic acid, thereby forming a linear double-stranded nucleic acid having the hybridization linkage structure. (4) The cell death-inducing composition according to any of (1) to (3), wherein the extended hairpin nucleic acid comprises a 5'-end overhanging hairpin nucleic acid and a 3'-end overhanging hairpin nucleic acid. (5) The cell death-inducing composition according to any of (1) to (4), wherein the target RNA-binding cassette is further capable of hybridizing with the non-protruding region cassette of the extended hairpin nucleic acid. (6) The cell death-inducing composition according to any one of (1) to (5), wherein the initiation hairpin nucleic acid and / or the extended hairpin nucleic acid further comprise all or part of a protein-binding motif. (7) The cell death-inducing composition according to (6), wherein the loop region and / or the protruding region comprise all or part of the protein-binding motif, and the entire protein-binding motif is double-stranded when the hybridization linkage structure is formed.(8) The cell death-inducing composition according to any one of (1) to (7), wherein the free energy change of the hairpin structure-forming reaction in the initiating hairpin nucleic acid is -20 to -10 kcal / mol. (9) The cell death-inducing composition according to any one of (1) to (8), wherein the hairpin nucleic acid is composed of DNA and / or RNA nucleotides. (10) The cell death-inducing composition according to (9), wherein the nucleotides include modified nucleotides. (11) The cell death-inducing composition according to any one of (1) to (10), wherein the target RNA is mRNA or miRNA. (12) The cell death-inducing composition according to any one of (1) to (11), wherein the target RNA is RNA that is cell-specifically expressed or highly expressed in a cell-specific manner. (13) A pharmaceutical composition comprising the cell death-inducing composition according to any one of (1) to (12) as an active ingredient. (14) The pharmaceutical composition according to (13), for treating a disease selected from the group consisting of cancer, immune system diseases, and neurodegenerative diseases. (15) An anticancer agent comprising a hairpin nucleic acid having a hairpin structure capable of forming a hybridization linkage structure, the anticancer agent comprising an initiating hairpin nucleic acid that hybridizes with a target RNA, and an extended hairpin nucleic acid that hybridizes with the initiating hairpin nucleic acid or another extended hairpin nucleic acid. (16) An anti-inflammatory agent comprising a hairpin nucleic acid having a hairpin structure capable of forming a hybridization linkage structure, the anti-inflammatory agent comprising an initiating hairpin nucleic acid that hybridizes with a target RNA, and an extended hairpin nucleic acid that hybridizes with the initiating hairpin nucleic acid or another extended hairpin nucleic acid. (17) A composition comprising an initiating hairpin nucleic acid and an extended hairpin nucleic acid having a hairpin structure capable of forming a hybridization linkage structure, wherein the initiating hairpin nucleic acid comprises a target RNA-binding cassette that hybridizes to a target RNA and an extended hairpin nucleic acid-binding cassette that hybridizes to the extended hairpin nucleic acid, the extended hairpin nucleic acid comprises a protruding region cassette that hybridizes to the initiating hairpin nucleic acid or another extended hairpin nucleic acid, and a non-protruding region cassette that hybridizes to yet another extended hairpin nucleic acid, and the initiating hairpin nucleic acid and / or extended hairpin nucleic acid further comprise all or a portion of a protein-binding motif.This specification includes the disclosure of Japanese Patent Application No. 2021-128492, from which this application claims priority.

[0011] The hairpin nucleic acid composition of the present invention can capture nucleic acid-binding proteins and induce nucleic acid immunity.

[0012] The protein function inhibitor of the present invention can inhibit the function of a protein based on HCR.

[0013] The cell death-promoting composition of the present invention can promote cell death based on HCR in target cells.

[0014] According to the pharmaceutical composition of the present invention, an effect based on HCR can be obtained at the target site.

[0015] 2A is a schematic diagram showing a typical structure of a hairpin nucleic acid of the present invention. A shows the structure of an initiating hairpin nucleic acid. B shows the structures of an extended hairpin nucleic acid with a 5'-end overhang ((I)) and a 3'-end overhang ((II)). C shows the hybridization state between a target RNA and hairpin nucleic acids A and B. In the figure, a indicates the overhang region, b indicates the stem 1 region, b' indicates the stem 2 region, and c indicates the loop region. The thin vertical line in C indicates hybridization of two nucleic acid strands. A is a schematic diagram showing a specific example of HCR. A shows steps 1 to 3 of HCR. B shows the formation of an HCR product by repeating step 3. In the figure, sHP indicates the initiating hairpin nucleic acid, and eHP indicates the extended hairpin nucleic acid. Also, in FIG. 2A, Target indicates the target RNA. This figure shows the HCR efficiency for a set of three hairpin nucleic acids (HP(16), HP(15), and HP(13)) with different minimum free energy changes. This figure shows the abundance of the first hairpin nucleic acid (HP1(16)) and the second hairpin nucleic acid (HP2(16)) of HP(16) over time in the presence of nuclease. This figure shows the NF-κB capture efficiency of the HCR product of HP(16). In the figure, NF-κB / HCR product refers to the complex of NF-κB and the HCR product. This figure shows the relationship between the time after microinjection of HP(16) and the amount of HCR product in HEK293T cells and HeLa cells. In the figure, "**" indicates a p-value of less than 0.01. This figure shows the relationship between the amount of HP(16) introduced and cell viability in HEK293T cells and HeLa cells. In the figure, "**" indicates a p-value of less than 0.01, and "***" indicates a p-value of less than 0.001. This figure shows the amount of IFN-β when poly(dG:dC) and HP(16) were introduced into MCF7 cells. In the figure, NC indicates the negative control. This figure shows a schematic diagram of a specific example of HCR. A shows steps 1 to 3 of HCR. B shows how the HCR product is formed by repeating step 3. In the figure, sHP indicates the initiating hairpin nucleic acid, and eHP indicates the extended hairpin nucleic acid. In Figure 9A, Target indicates the target RNA.This figure shows the amount of HCR product in HEK293T cells, MDA-MB-231 cells, HeLa cells, and A549 cells after microinjection of HP(16). In the figure, "*" indicates a p-value of less than 0.05, and "**" indicates a p-value of less than 0.01. Error bars indicate standard deviation. This figure shows the relative cell viability of HEK293T cells, MDA-MB-231 cells, and HeLa cells after HP(16) transfection. In the figure, "**" indicates a p-value of less than 0.01, and "***" indicates a p-value of less than 0.001. Error bars indicate standard deviation. This figure shows the relative cell viability of HeLa cells after transfection with poly(dA:dT) and HP(16), respectively, and the change in viability following further transfection with STING siRNA. In the figure, "****" indicates a p-value of less than 0.0001. Error bars indicate standard deviation. This figure shows the amount of IFN-β mRNA when HP(16) was introduced into HeLa cells, and the change in this amount when STING siRNA was further introduced. In the figure, "*" indicates a p-value of less than 0.05. Error bars indicate standard deviation. This figure shows the change in tumor volume over time when PBS, poly(dA:dT), and HP(16) were introduced into tumor-bearing mice. In the figure, "**" indicates a p-value of less than 0.01. Error bars indicate standard deviation.

[0016] 1A. Hairpin Nucleic Acid Composition 1A-1. Overview A first aspect A of the present invention is a hairpin nucleic acid composition comprising two or more hairpin nucleic acids capable of forming a hybridization linkage structure. The hairpin nucleic acid composition of the present invention essentially comprises an initiating hairpin nucleic acid and an extended hairpin nucleic acid, and when the initiating hairpin nucleic acid hybridizes to a target RNA, a complex of the target RNA, the initiating hairpin nucleic acid, and the extended hairpin nucleic acid is formed in a linked manner by HCR. The hairpin nucleic acid composition of the present invention can capture nucleic acid-binding proteins to induce nucleic acid immunity, and can also be an active ingredient in a protein function-inhibiting composition, a cell death-promoting composition, and a pharmaceutical composition of the present invention.

[0017] 1A-2. Definitions Terms frequently used in this specification are defined below. As used herein, the term "hairpin nucleic acid" refers to a single-stranded nucleic acid capable of forming a hairpin structure. As used herein, the term "hairpin structure" refers to a secondary structure of a nucleic acid formed by a single-stranded nucleic acid, the secondary structure comprising a set of a stem structure, a loop structure, and a protruding region. Figures 1A and 1B show schematic diagrams of a hairpin nucleic acid. As used herein, a hairpin nucleic acid comprises, in order, a protruding region (a), a stem first region (b), a loop region (c), and a stem second region (b').

[0018] As used herein, "5'-end overhanging type (B(I))" refers to a type of hairpin nucleic acid that includes a protruding region at the 5'-end. A 5'-end overhanging type hairpin nucleic acid includes, in order from the 5'-end, a protruding region, a stem first region, a loop region, and a stem second region. Also, as used herein, "3'-end overhanging type (A and B(II))" refers to a type of hairpin nucleic acid that includes a protruding region at the 3'-end. A 3'-end overhanging type hairpin nucleic acid includes, in order from the 5'-end, a stem second region, a loop region, a stem first region, and a protruding region.

[0019] The "stem structure" is a double-stranded structure formed by two stem regions (b and b') containing hybridizable base sequences.

[0020] The "loop structure" is a loop-shaped structure formed by a loop region (c) consisting of a single-stranded nucleic acid.

[0021] As used herein, "overhanging region (a)" refers to a protruding end in an HCR that recognizes the single-stranded portion of a target RNA or HCR product. A "protruding end" refers to a single-stranded nucleic acid region adjacent to either or both of the free ends of the stem region (ends not adjacent to the loop region). The length of the protruding region is not particularly limited herein, but may be, for example, 4 or more bases, 5 or more bases, 6 or more bases, 7 or more bases, or 8 or more bases. The protruding region may be, for example, 20 or fewer bases, 19 or fewer bases, 18 or fewer bases, 17 or fewer bases, 16 or fewer bases, 15 or fewer bases, 14 or fewer bases, 13 or fewer bases, 12 or fewer bases, 11 or fewer bases, 10 or fewer bases, or 9 or fewer bases. Specifically, the length of the protruding region may be, for example, 4 to 20 bases.

[0022] As used herein, "stem regions (b and b')" refer to nucleic acid regions that hybridize to each other within a molecule to form a stem structure. At least both ends of each stem region consist of complementary bases. The length of each stem region is not particularly limited, and may be, for example, 10 or more bases, 11 or more bases, 12 or more bases, 13 or more bases, or 14 or more bases. The length of each stem region may be, for example, 20 or less bases, 19 or less bases, 18 or less bases, 17 or less bases, 16 or less bases, or 15 or less bases. Specifically, the length of each protruding region may be, for example, 10 to 20 bases. "Stem first region (b)" refers to a stem region adjacent to a protruding region. "Stem second region (b')" refers to a stem region that is not adjacent to a protruding region.

[0023] As used herein, "loop region (c)" refers to a nucleic acid region located between the two stem regions in a single-stranded nucleic acid. The length of the loop region is not particularly limited, and may be, for example, 3 or more bases, 4 or more bases, 5 or more bases, 6 or more bases, 7 or more bases, or 8 or more bases. The loop region may be, for example, 50 or fewer bases, 40 or fewer bases, 30 or fewer bases, 25 or fewer bases, 20 or fewer bases, 19 or fewer bases, 18 or fewer bases, 17 or fewer bases, 16 or fewer bases, 15 or fewer bases, 14 or fewer bases, 13 or fewer bases, 12 or fewer bases, 11 or fewer bases, 10 or fewer bases, or 9 or fewer bases. Specifically, the length of the loop region may be, for example, 3 to 50 bases.

[0024] As used herein, the term "hybridization chain reaction (HCR)" refers to the extension reaction of a double-stranded nucleic acid molecule that occurs through the sequential hybridization of multiple cleaved hairpin nucleic acids. Figures 2 and 9 show a typical HCR process. In step 1, the overhanging region of a starting hairpin nucleic acid (sHP) recognizes and hybridizes with a target RNA to form a target sequence-starting hairpin nucleic acid complex. The hairpin structure of the starting hairpin nucleic acid is cleaved by elongation of hybridization with the target RNA. This formation of the target sequence-starting hairpin nucleic acid complex serves as the starting point for HCR product formation. In step 2, the overhanging region of a first elongating hairpin nucleic acid (eHP1) recognizes and hybridizes with the single-stranded portion of the starting hairpin nucleic acid that has been released by the cleavage, forming a target sequence-starting-elongating hairpin nucleic acid complex. At this time, the hairpin structure of the extended hairpin nucleic acid is cleaved by hybridization extension with the single-stranded portion of the initiating hairpin nucleic acid. In step 3, a second extended hairpin nucleic acid (eHP2) recognizes and cleaves the single-stranded portion of the extended hairpin nucleic acid in the target sequence-initiating extended hairpin nucleic acid complex formed in step 2, and further hybridizes with the target sequence-initiating extended hairpin nucleic acid complex to form a target sequence-initiating (extended)2 hairpin nucleic acid complex. This step 3 is then repeated, allowing successive extended hairpin nucleic acids to hybridize, extending the double-stranded nucleic acid and forming a high-molecular-weight polymer (HCR product) (Figures 2B and 9B). The HCR product forms a linear double-stranded nucleic acid.

[0025] As used herein, the term "hybridization chain structure" refers to a nucleic acid structure contained in the linear double-stranded nucleic acid formed by the above-mentioned HCR. The hybridization chain structure contains a pathogen-associated molecular pattern and induces nucleic acid immunity via pattern recognition receptors.

[0026] As used herein, the term "nucleic acid immunity" refers to an innate immune response based on the recognition of nucleic acids. Nucleic acid immunity is induced by the recognition of non-self nucleic acids and / or damaged self-derived nucleic acids by pattern recognition receptors.

[0027] "Pattern-recognition receptor" is a general term for receptor proteins involved in the induction of the innate immune system, which recognize structural patterns found in non-self molecules and / or damaged self-derived molecules. In this specification, unless otherwise specified, it refers to receptor proteins that recognize nucleic acid molecules. The structural patterns recognized by pattern-recognition receptors are called pathogen-associated molecular patterns (PAMPs).

[0028] The term "pathogen-associated molecular pattern" refers to a molecular pattern that is present in viruses, prokaryotes, and / or protostomes but is not present in the environment in which pattern recognition receptors are present in normal vertebrates. In this specification, the term particularly refers to a structural pattern that is present in the nucleic acid molecules of viruses, prokaryotes, and / or protostomes but is not present in the nucleic acid molecules of vertebrates.

[0029] As used herein, "target RNA" refers to RNA that serves as the starting point for HCR product formation by the hairpin nucleic acid composition of the present invention. The target RNA defines the conditions under which HCR is induced by the hairpin nucleic acid composition of the present invention. The target RNA can specifically hybridize with the initiating hairpin nucleic acid that constitutes the hairpin nucleic acid. The type of target RNA is not particularly limited. Specific examples include mRNA (including, for example, mature mRNA, pre-mRNA, and modified mRNA), non-coding RNA (ncRNA: including microRNA (miRNA) and long non-coding RNA (lncRNA)), and natural antisense RNA.

[0030] The term "complementary" refers to a relationship in which nucleic acid bases can form base pairs with each other via hydrogen bonds, such as Watson-Crick base pairs (natural base pairs) or Hoogsteen base pairs.

[0031] The terms "hybridize" or "hybridizable" refer to polynucleotides having complementary base sequences base pairing to form a completely or partially complementary double strand.

[0032] In addition, in this specification, the term "plurality" refers to a number of 2 or more. Specifically, for example, it refers to 2 to 60, 2 to 45, 2 to 30, 2 to 14, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3.

[0033] As used herein, the term "hairpin structure-forming reaction" refers to a reaction in which a single-stranded nucleic acid in a hairpin nucleic acid changes from a linear form to a hairpin form, forming a hairpin structure.

[0034] As used herein, the term "free energy change" refers to the net amount of energy supplied from the external environment to a reaction system through a certain reaction under constant temperature and pressure conditions. In this specification, the term particularly refers to the net amount of energy supplied from the external environment in a hairpin structure-forming reaction. For example, if the reaction product is more thermodynamically stable than the starting material, the reaction system will lose energy through the reaction, resulting in a negative free energy change. The free energy change of the present invention includes, for example, both the Gibbs free energy change (ΔG) and the Helmholtz free energy change (ΔF).

[0035] As used herein, the term "protein-binding motif" refers to a double-stranded sequence motif to which a specific protein can bind. For example, a protein-binding motif to which a transcription factor binds is referred to as a "transcription factor-binding motif" herein.

[0036] As used herein, "protein function inhibition" refers to inhibiting the function that the protein can inherently exhibit. The degree of inhibition and the method of inhibition are not particularly limited.

[0037] As used herein, "cell death" refers to the death of a cell. Cell death is broadly classified into programmed cell death and accidental cell death, and cell death as used herein includes both. Examples of programmed cell death include apoptosis, autophagy, and necroptosis. Apoptosis refers to programmed cell death characterized by the formation of aggregates (apoptotic bodies) in which fragmented nuclei are enveloped by the cell membrane. During apoptosis, phenomena such as cell fragmentation, nuclear fragmentation, membrane blebbing, and chromatin condensation are observed. As used herein, autophagy, also known as macroautophagy, refers to programmed cell death that occurs during nutritional stress. During autophagy, phenomena such as widespread vacuolation of the cytoplasm are observed. As used herein, necroptosis refers to programmed cell death in which necrosis-like phenomena, such as the release of cellular contents, are observed. There are several types of programmed cell death in which necrosis-like phenomena are observed, and necroptosis as used herein includes all of them. Accidental cell death refers to cell death caused by mechanical damage to the cell or stress on the outside or inside of the cell. Accidental cell death is also called necrosis.

[0038] As used herein, the term "apoptosis-related factor" refers to any protein associated with a signal pathway that induces apoptosis. The apoptosis-related factor herein includes both apoptosis-promoting factors and apoptosis-suppressing factors. Specific apoptosis-related factors include Fas pathway-related factors, caspases, mitochondrial pathway-related factors, FOXO family proteins, and various kinases (e.g., cyclin-dependent kinases and MAP kinases). The apoptosis-related factor herein includes, for example, proteins known to be associated with diseases such as cancer and neurodegenerative diseases.

[0039] As used herein, the term "autophagy-related factor" refers to any protein involved in a signaling pathway that induces autophagy. Autophagy-related factors herein include both autophagy-promoting factors and autophagy-inhibiting factors. Autophagy-related factors may be associated with any step of autophagy, and examples include factors associated with phagophore development, factors associated with phagophore growth or autophagosome formation, and factors involved in autolysosomes. Autophagy-related factors herein include proteins known to be associated with diseases such as cancer, immune system disorders, and neurodegenerative disorders.

[0040] As used herein, the term "inflammation-related factor" refers to a protein that is affected during inflammation. Inflammation-related factors herein include both pro-inflammatory factors and anti-inflammatory factors. Specific inflammation-related factors include, for example, various interleukins (including, for example, IL-1, TNF-α, and IFN-β), NF-κB family proteins, STAT family proteins, and HIF proteins. Inflammation-related factors herein include, for example, proteins known to be associated with diseases such as cancer and immune system diseases.

[0041] As used herein, the term "immune system disease" refers to a disease characterized by an abnormality in the immune system, specifically including, for example, autoimmune diseases and inflammatory diseases.

[0042] "Immune cells" refer to cells involved in immunity. Specific examples include lymphocytes, macrophages, and dendritic cells. Examples of lymphocytes include T cells, B cells, NK cells, and plasma cells. The immune cells herein are preferably cells that have abnormal activity in immune system diseases.

[0043] "Autoimmune disease" refers to a disease in which an immune response occurs against a self-antigen. Specific examples include Hashimoto's thyroiditis, Graves' disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, type I diabetes, scleroderma, and psoriasis.

[0044] "Inflammatory disease" refers to a disease characterized by high levels of inflammation or degeneration in tissues. Inflammatory diseases as used herein include both chronic and acute inflammatory diseases. Specific examples include celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel syndrome, atherosclerosis, arthritis, ankylosing spondylitis, Crohn's disease, colitis, hepatitis (e.g., viral hepatitis such as chronic active hepatitis and non-viral hepatitis), dermatitis, and psoriasis. "Inflammatory cells" as used herein refer to cells involved in inflammatory responses. Specific examples include eosinophils, neutrophils, basophils, mast cells, and type II innate lymphocytes. Inflammatory cells as used herein are preferably cells exhibiting abnormal activity in inflammatory diseases.

[0045] As used herein, the term "neurodegenerative disease" refers to a disease in which the structure of nervous tissue degenerates over time. Specific examples include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, macular degeneration, multiple sclerosis, muscular dystrophy, Niemann-Pick disease, osteoporosis, and rheumatoid arthritis. Many of these diseases are induced by the accumulation of specific proteins.

[0046] As used herein, "abnormal activity" refers to activity that is markedly or significantly suppressed or promoted compared to activity in normal cells. As used herein, "high expression" of a particular gene refers to a markedly or significantly increased expression level compared to the expression level in normal cells.

[0047] "Statistically significant" refers to a significant difference between the measured value of a test subject and the control value when the difference is statistically processed. For example, the risk rate (significance level) of the obtained value is small, specifically, less than 5% (p<0.05), less than 1% (p<0.01), or less than 0.1% (p<0.001). The "p (value)" shown here indicates the probability that a test statistic will take that value by chance in a distribution based on the null hypothesis in a statistical test. Therefore, the smaller the "p", the lower the probability that the test statistic will take that value, meaning that the null hypothesis is more likely to be rejected. The statistical processing test method can be any known test method capable of determining the presence or absence of significance, and is not particularly limited. For example, the Student's t-test, covariate analysis of variance, etc. can be used.

[0048] 1A-3. Structure The hairpin nucleic acid composition of the present invention comprises two or more hairpin nucleic acids capable of forming a hybridization linkage structure, and the hairpin nucleic acid optionally comprises all or part of a protein-binding motif.

[0049] The hairpin nucleic acid is composed of an initiating hairpin nucleic acid and an extending hairpin nucleic acid, each of which will be specifically described below.

[0050] (1) Initiating Hairpin Nucleic Acid The "initial hairpin nucleic acid (FIG. 1A)" is a hairpin nucleic acid containing a target RNA-binding cassette and an extended hairpin nucleic acid-binding cassette (FIG. 1C).

[0051] The term "target RNA-binding cassette" refers to a nucleic acid region in the starting hairpin nucleic acid that hybridizes with the target RNA. Specifically, for example, the target RNA-binding cassette includes all or part of the overhang region followed by all or part of the stem first region (FIG. 1C). The target RNA-binding cassette can hybridize with all or part of the target RNA, and the base sequence of this cassette is complementary to that of the target RNA at least at both ends of the cassette.

[0052] The term "extended hairpin nucleic acid binding cassette" refers to a nucleic acid region in an initiating hairpin nucleic acid that hybridizes with an extended hairpin nucleic acid. Specifically, for example, the extended hairpin nucleic acid binding cassette comprises all or part of the stem second region (FIG. 1C). The extended hairpin nucleic acid binding cassette is capable of hybridizing with the overhang region cassette of the extended hairpin nucleic acid, and the base sequences of both cassettes are complementary to each other at least at both ends of each cassette.

[0053] The initiating hairpin nucleic acid may contain one or more bases in addition to the target RNA-binding cassette and the extended hairpin nucleic acid binding cassette, for example, as a spacer sequence between the target RNA-binding cassette and the extended hairpin nucleic acid binding cassette, or as a terminal sequence linked to the free ends of the target RNA-binding cassette and the extended hairpin nucleic acid binding cassette.

[0054] In addition, both cassettes of the initiating hairpin nucleic acid may overlap each other by one or more bases.

[0055] (2) Extended Hairpin Nucleic Acid An "extended hairpin nucleic acid (FIG. 1B)" is a hairpin nucleic acid comprising a protruding region cassette and a non-protruding region cassette (FIG. 1C).

[0056] The term "overhang region cassette" refers to a nucleic acid region in an extended hairpin nucleic acid that hybridizes with an initiating hairpin nucleic acid or another extended hairpin nucleic acid. Specifically, for example, an overhang region cassette includes all or part of the overhang region followed by all or part of the stem first region (FIG. 1C). The overhang region cassette can hybridize with an extended hairpin nucleic acid binding cassette of an initiating hairpin nucleic acid or a non-overhang region cassette of another, different extended hairpin nucleic acid, and the base sequences of both cassettes are complementary to each other at least at both ends of each cassette.

[0057] The term "non-overhang region cassette" refers to a nucleic acid region in an extended hairpin nucleic acid that hybridizes with another extended hairpin nucleic acid. Specifically, for example, the non-overhang region cassette comprises all or part of the second stem region ( FIG. 1C ). The non-overhang region cassette can hybridize with the overhang region cassette of another, different extended hairpin nucleic acid, and the base sequences of both cassettes are complementary to each other at least at both ends of each cassette.

[0058] Here, the "other extended hairpin nucleic acid" that hybridizes with the overhang region cassette and the "further extended hairpin nucleic acid" that hybridizes with the non-overhang region cassette are typically different extended hairpin nucleic acid molecules, but these extended hairpin nucleic acid molecules may have the same sequence, i.e., the same type of extended hairpin nucleic acid, or they may have different sequences, i.e., different type of extended hairpin nucleic acid.

[0059] The extended hairpin nucleic acid may contain one or more bases in addition to the overhang and non-overhang region cassettes, such as, for example, a spacer sequence between the overhang and non-overhang region cassettes, or a terminal sequence linked to the free ends of the overhang and non-overhang region cassettes.

[0060] Furthermore, both cassettes of an extended hairpin nucleic acid may overlap by one or more bases. When the target RNA ("Target" in FIG. 9 ) binds to the target RNA binding cassette of the starting hairpin nucleic acid ("sHP" in FIG. 9 ), the hairpin structure (particularly the stem structure) of the starting hairpin nucleic acid dissociates (steps 1 and 2 in FIG. 9A ). This allows the extended hairpin nucleic acid binding cassette of the starting hairpin nucleic acid to hybridize with the protruding region cassette of the extended hairpin nucleic acid ("eHP1" in FIG. 9 ) (step 2 in FIG. 9A ). Hybridization of these two cassettes causes the hairpin structure (particularly the stem structure) of the extended hairpin nucleic acid to dissociate (steps 2 and 3 in FIG. 9A ). This allows the non-protruding region cassette of this extended hairpin nucleic acid to hybridize with the protruding region cassette of another extended hairpin nucleic acid ("eHP2" in FIG. 9 ) (step 3 in FIG. 9A ). This reaction proceeds in a chain reaction, forming a linear double-stranded nucleic acid having a hybridization chain structure (FIG. 9B).

[0061] (3) Cassette The length of each cassette constituting the hairpin nucleic acid is not particularly limited. For example, it can be 4 or more bases, 5 or more bases, 6 or more bases, 7 or more bases, 8 or more bases, 9 or more bases, 10 or more bases, 11 or more bases, 12 or more bases, 13 or more bases, 14 or more bases, 15 or more bases, 16 or more bases, 17 or more bases, 18 or more bases, 19 or more bases, 20 or more bases, 21 or more bases, or 22 or more bases. Furthermore, each cassette can be, for example, 55 or less bases, 50 or less bases, 40 or less bases, 30 or less bases, 25 or less bases, 24 or less bases, or 23 or less bases.

[0062] The sequence of each cassette is not particularly limited as long as it contains a sequence that can hybridize to the target nucleic acid. For example, each cassette may contain one or more bases that are unrelated to the sequence of the target nucleic acid.

[0063] (3-1) Target RNA-Binding Cassette and Overhang Region Cassette As described above, the target RNA-binding cassette of the initiating hairpin nucleic acid and the overhang region cassette of the extended hairpin nucleic acid preferably comprise all or part of the overhang region followed by all or part of the stem first region. For example, these cassettes may be a continuous region comprising the overhang region, the entire stem first region, and the loop region, and part of the stem second region, or a continuous region comprising part of the overhang region and part of the stem first region. For example, the sequence of the overhang region that can hybridize to the target RNA or the non-overhang region cassette of another different extended hairpin nucleic acid may be 4 or more bases, 5 or more bases, 6 or more bases, 7 or more bases, or 8 or more bases. Furthermore, for example, the sequence of the stem first region that can hybridize to the non-protruding region cassette of the target RNA or other different extended hairpin nucleic acids is 2 or more bases, 3 or more bases, 4 or more bases, 5 or more bases, 6 or more bases, 7 or more bases, 8 or more bases, 9 or more bases, 10 or more bases, 11 or more bases, 12 or more bases, 13 or more bases, 14 or more bases, 15 or more bases, or 16 or more bases.

[0064] (3-2) Extended Hairpin Nucleic Acid Binding Cassette and Non-Protruding Region Cassette As described above, the extended hairpin nucleic acid binding cassette of the starting hairpin nucleic acid and the non-protruding region cassette of the extended hairpin nucleic acid preferably comprise all or a portion of the stem second region. For example, these cassettes may be a continuous region comprising a portion of the stem first region, the entire loop region, and the entire stem second region, or a continuous region comprising only a portion of the stem second region. Furthermore, when the hairpin nucleic acid has a protruding end adjacent to the stem second region, the cassette may comprise all or a portion of that protruding end. Specifically, for example, the sequence of the stem second region capable of hybridizing to the non-protruding region cassette of the extended hairpin nucleic acid or the protruding region cassette of another different extended hairpin nucleic acid may be 4 or more bases, 5 or more bases, 6 or more bases, 7 or more bases, 8 or more bases, 9 or more bases, 10 or more bases, 11 or more bases, 12 or more bases, 13 or more bases, 14 or more bases, 15 or more bases, or 16 or more bases.

[0065] (4) Hybridization Linkage Structure The hairpin nucleic acid of the present invention forms a linear double-stranded nucleic acid having a hybridization linkage structure in the presence of a target RNA. The hybridization linkage structure can induce nucleic acid immunity.

[0066] The specific structure of the hybridization chain structure is not particularly limited as long as it is a double-stranded nucleic acid structure capable of inducing nucleic acid immunity. For example, the hybridization chain structure may include a pathogen-associated molecular pattern that is recognized by a pattern recognition receptor present on a biological membrane or in the cytoplasm.

[0067] Examples of pattern recognition receptors that can recognize nucleic acids present on biological membranes include Toll-like receptors such as TLR3, TLR7, TLR8, and TLR9. These receptors are present on the biological membranes of endosomes and lysosomes and recognize nucleic acid molecules that have entered cells.

[0068] Examples of pattern recognition receptors capable of recognizing nucleic acids present in the cytoplasm include RIG-I-like receptors (e.g., RIG-I, MDA5, LGP2, etc.), cGAS, and AIM2.

[0069] The pathogen-associated molecular patterns recognized by each pattern recognition receptor are known in the art. For example, TLR3 is known to recognize double-stranded RNA of 40 base pairs or more, while TLR7 and TLR8 are known to recognize double-stranded RNA rich in polyuracil or guanine and uracil. Furthermore, TLR9 is known to recognize unmethylated single-stranded DNA containing a cytosine- and guanine-rich sequence such as 5'-GTCGTT-3'. Furthermore, RIG-I protein is known to recognize double-stranded RNA containing blunt ends and a triphosphate at the 5' end. Additionally, AIM2 protein is known to recognize double-stranded DNA.

[0070] The hybridization sequence structure may, for example, comprise a pathogen-associated molecular pattern recognized by a pattern recognition receptor present in the cytoplasm, or may comprise a pathogen-associated molecular pattern recognized by a pattern recognition receptor that recognizes double-stranded nucleic acids (e.g., double-stranded DNA and / or double-stranded RNA). The pathogen-associated molecular pattern may, for example, be a double-stranded structure recognized by cGAS or MDA5.

[0071] cGAS (cyclic GMP-AMP synthase) is an enzyme that synthesizes cyclic GMP-AMP. An exemplary amino acid sequence of cGAS is shown in SEQ ID NO: 14. When cGAS binds to foreign double-stranded nucleic acids (e.g., viral nucleic acids) or abnormal self-double-stranded nucleic acids (e.g., nucleic acids leaked from the nucleus in senescent cells), it synthesizes the second messenger 2'-5'-cGAMP from GTP and ATP, and activates the downstream STING (stimulator of interferon genes) pathway via this 2'-5'-cGAMP. The activated STING pathway induces cellular apoptosis and other processes.

[0072] Nucleic acids recognized by cGAS include long double-stranded DNA that does not form chromatin structures and short double-stranded DNA with unpaired guanosine ends. Furthermore, it is known that the cGAS-mediated immune response pathway is easily induced when a large amount of nucleic acid molecules enters a cell or when double-stranded nucleic acids containing oxidized DNA molecules enter the cell.

[0073] MDA5 (melanoma differentiation-associated gene 5) is a cytoplasmic pattern recognition receptor belonging to the RIG-I family. An exemplary amino acid sequence of MDA5 is shown in SEQ ID NO: 15. Upon binding to double-stranded nucleic acids, MDA5 binds to MAVS (Mitochondrial Antiviral Signaling Protein) present on the outer mitochondrial membrane and activates the MAVS pathway. The activated MAVS pathway induces immune responses mediated by type I interferon and the like.

[0074] Nucleic acids recognized by MDA5 include double-stranded RNAs, particularly long double-stranded RNAs and Poly(I:C) molecules.

[0075] For example, the hybridization chain structure has a specific length. Specifically, the median length of the formed HCR product may be, for example, 100 base pairs or more, 150 base pairs or more, 200 base pairs or more, 250 base pairs or more, 300 base pairs or more, 350 base pairs or more, 400 base pairs or more, 450 base pairs or more, 500 base pairs or more, or 550 base pairs or more. It is preferable that a certain amount of double-stranded structures of 500 base pairs or more are formed. For example, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, or 25% or more of the HCR product have a double-stranded structure of 500 base pairs or more.

[0076] (5) Protein-Binding Motif As an optional configuration, the loop region and / or protruding region may contain all or part of a protein-binding motif. In this case, the entire protein-binding motif becomes double-stranded upon binding of the target RNA to the target RNA-binding cassette of the starting hairpin nucleic acid. In the hairpin nucleic acid, all or part of the protein-binding motif is single-stranded, and upon HCR, the entire molecule becomes double-stranded. For example, a single molecule may contain all or part of one or more sequence motifs, or the entire sequence motif may not be contained in a single molecule. When multiple motifs are contained, the types of motifs are not particularly limited. For example, the multiple sequence motifs may all be the same, or may contain multiple types of sequence motifs. Proteins capable of binding to double-stranded nucleic acids and their binding motifs can be identified by known methods. For example, known databases such as Transfac, WordSpy, T-Reg Comparator, MOTIF, TFBIND, TFSEACH, and JASPAR can be used. Also, for example, protein binding motifs can be identified by DNA footprinting, gel mobility shift assays, or other known methods, and / or can be predicted based on known consensus sequence motifs.

[0077] The number of proteins that bind to each sequence motif is not particularly limited. For example, a sequence motif to which one or more types of proteins can bind can be used. Furthermore, the type of protein that binds is not particularly limited. Examples include transcription factors and polymerases. The protein is preferably a transcription factor. In this case, the protein-binding motif includes a transcription factor-binding motif. The protein may be, for example, a protein involved in a specific disease. Specifically, the protein includes, for example, apoptosis-related factors, autophagy-related factors, inflammation-related factors, etc.

[0078] (6) Hairpin Nucleic Acid Compositions Each hairpin nucleic acid constituting the hairpin nucleic acid composition of the present invention may be composed of DNA and / or RNA nucleotides. Furthermore, each hairpin nucleic acid may contain modified nucleotides as constituent nucleotides. The type, number, and position of the modified nucleotides contained are not particularly limited. Specific examples include modifications of internucleotide bonds, analogs with properties and / or structures similar to DNA and RNA, and peptide nucleic acids. The choice of modification may vary depending on the sequence of the target RNA, but those skilled in the art can determine a suitable embodiment by referring to the descriptions in literature related to nucleic acid medicines (e.g., WO 2007 / 143315, etc.). The purpose of the modification is not particularly limited. For example, modifications can be performed for the stabilization, detection, and pharmacological function of hairpin nucleic acids and HCR products.

[0079] The initiating hairpin nucleic acid and the extended hairpin nucleic acid that make up the hairpin nucleic acid composition may contain multiple types of hairpin nucleic acids.

[0080] The initiating hairpin nucleic acid and the extended hairpin nucleic acid contained in the hairpin nucleic acid composition of the present invention may each be either a 5'-end overhang or a 3'-end overhang. Furthermore, the number of 5'-end overhangs and 3'-end overhangs in the initiating hairpin nucleic acid and the extended hairpin nucleic acid is not particularly limited. Preferably, the extended hairpin nucleic acid contains both 5'-end overhangs and 3'-end overhangs.

[0081] The free energy change in the hairpin structure-forming reaction is not particularly limited. Generally, the lower the free energy change in the hairpin structure-forming reaction, the more difficult the hairpin structure is to cleave, and the higher the free energy change, the more easily the hairpin structure is cleaved. The free energy change is, for example, -20 to -10 kcal / mol. Specifically, it is, for example, -20 kcal / mol or less, -19 kcal / mol or less, -18 kcal / mol or less, -17.5 kcal / mol or less, -17 kcal / mol or less, or -16.5 kcal / mol or less. Furthermore, the free energy change is, for example, -10 kcal / mol or more, -11 kcal / mol or more, -12 kcal / mol or more, -12.5 kcal / mol or more, -13 kcal / mol or more, -13.5 kcal / mol or more, -14 kcal / mol or more, -14.5 kcal / mol or more, or -15 kcal / mol or more. The amount of change in free energy can be determined using known software such as NUPAK, which is used to predict the stability of higher-order structures of nucleic acids.

[0082] The length and free energy change of each region in each hairpin nucleic acid do not have to be the same. Generally, among the hairpin nucleic acids involved in a series of HCRs, the cleavage reaction of the hairpin structure of the hairpin nucleic acid that is the most difficult to cleave is considered to be the rate-limiting step of the HCR extension reaction. For example, the free energy change of the hairpin structure formation reaction of the starting hairpin nucleic acid can be lower than the free energy change of the extended hairpin nucleic acid.

[0083] The initiating hairpin nucleic acid is a hairpin nucleic acid capable of hybridizing with a target RNA, and the extended hairpin nucleic acid is a hairpin nucleic acid capable of hybridizing with another hairpin nucleic acid. The terms "initiating hairpin nucleic acid" and "extended hairpin nucleic acid" are named based on their functions, so one hairpin nucleic acid can have the functions of both an initiating hairpin nucleic acid and an extended hairpin nucleic acid. Furthermore, for example, when multiple types of initiating hairpin nucleic acids are included, all of the initiating hairpin nucleic acids may be capable of hybridizing with one type of extended hairpin nucleic acid, or all of the initiating hairpin nucleic acids may be capable of hybridizing with different extended hairpin nucleic acids.

[0084] Preferably, the starting hairpin nucleic acid is capable of hybridizing with one or more extended hairpin nucleic acids of different end-overhangs. For example, if the starting hairpin nucleic acid is a 5'-end overhang, at least one of the extended hairpin nucleic acids of 3'-end overhangs can be hybridized to the starting hairpin nucleic acid. Also, preferably, all extended hairpin nucleic acids are capable of simultaneously hybridizing with two or more extended hairpin nucleic acids of different end-overhangs.

[0085] All or part of each cassette constituting a hairpin nucleic acid may be hybridizable to all or part of multiple types of target RNAs or multiple types of hairpin nucleic acid cassettes. For example, when one hairpin nucleic acid has the functions of both an initiating hairpin nucleic acid and an extended hairpin nucleic acid, all or part of the target RNA binding cassette of that hairpin nucleic acid can hybridize to the non-protruding region cassette of the extended hairpin nucleic acid. In this case, the target RNA binding cassette and the protruding region cassette partially overlap. For example, when the target RNA binding cassette and the protruding region cassette are identical, the target RNA binding cassette can hybridize to the non-protruding region cassette of the extended hairpin nucleic acid. Furthermore, all or part of the cassettes of multiple types of hairpin nucleic acids may be hybridizable to all or part of the same cassette or target RNA.

[0086] The manner in which cassettes hybridize to each other is not particularly limited. Preferably, when the cassettes hybridize to each other, the other cassettes in each hairpin nucleic acid are exposed in different directions, sandwiching the hybridized cassette. For example, when the extended hairpin nucleic acid binding cassette of the starting hairpin nucleic acid and the protruding region cassette of the extended hairpin nucleic acid hybridize, the resulting complex has a double-stranded region sandwiched between two cassettes exposed as single strands (the target RNA binding cassette of the starting hairpin nucleic acid and the non-protruding region cassette of the extended hairpin nucleic acid). Therefore, the cassettes on the 3'-end and the cassettes on the 5'-end can hybridize to each other. The manner in which the starting hairpin nucleic acid hybridizes to the target RNA is not particularly limited.

[0087] The base sequence of the hybridizable region is not particularly limited as long as it is hybridizable. Specific hybridizable base sequences include, for example, the following: (1) a base sequence that is completely complementary to the target sequence to be hybridized; (2) a base sequence in which one or more bases are deleted, substituted, or added compared to (1); or (3) a base sequence that hybridizes to the target sequence to be hybridized under highly stringent conditions.

[0088] Whether or not a second nucleic acid can hybridize can be determined using methods known in the art. For example, it can be determined based on base identity. Typically, a second nucleic acid having a base sequence that is completely complementary to the base sequence of a first nucleic acid and has a certain level of base identity can hybridize with the first nucleic acid. Specifically, for example, hybridization is possible when the base identity is 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. As used herein, "base identity" refers to the percentage (%) of identical bases in one polynucleotide relative to the total number of bases in the other polynucleotide, when the base sequences of two polynucleotides are aligned and, if necessary, gaps are introduced into either base sequence to maximize the degree of base identity between the two. The percent identity can be easily determined using known programs such as the homology search program BLAST (Basic local alignment search tool; Altschul, S. F. et al., J. Mol. Biol., 215, 403-410, 1990). Furthermore, a second nucleic acid having a base sequence completely complementary to the base sequence of a first nucleic acid in which multiple bases have been substituted with other bases is typically capable of hybridizing with the first nucleic acid. Specifically, for example, a second nucleic acid can hybridize when 2 to 60, 2 to 45, 2 to 30, 2 to 14, 2 to 12, or 2 to 10, e.g., 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 bases have been substituted.

[0089] Hybridization conditions are not particularly limited and may include various stringent conditions, such as low stringency conditions and high stringency conditions. "Low stringency conditions" refer to conditions under which nucleic acids hybridize easily. Low stringency conditions refer to low temperature and high salt concentration conditions in post-hybridization washing. For example, post-hybridization washing is performed using a buffer containing 5xSSC and 0.1% SDS at 42°C to 50°C. "High stringency conditions" refer to conditions under which nonspecific hybrids are not formed. Specific examples of low salt concentrations include 15 to 750 mM, preferably 15 to 500 mM, 15 to 300 mM, or 15 to 200 mM. Specific examples of high temperatures include 50 to 68°C or 55 to 70°C. Specific examples of highly stringent conditions include washing at 65°C with 0.1x SSC and 0.1% SDS, where 1x SSC contains 150 mM sodium chloride and 15 mM sodium citrate.

[0090] The expression pattern of the target RNA is not particularly limited. For example, the target RNA may be expressed systemically regardless of the timing or the presence or absence of a stimulus, or may be expressed tissue-specifically, cell-specifically, time-specifically, or in response to a stimulus such as an extracellular signal. The target RNA is preferably RNA that is expressed cell-specifically. In this case, the cells in which the target RNA is expressed are not particularly limited. Specific examples include cancer cells, inflammatory cells, and immune cells. The inflammatory cells and immune cells in which the target RNA is expressed preferably exhibit abnormal activity.

[0091] The expression pattern of the protein that binds to the hybridization linkage structure and protein-binding motif is not particularly limited. For example, the protein may be expressed systemically regardless of timing or the presence or absence of stimulation, or may be expressed in a tissue-specific, cell-specific, or time-specific manner, or in response to stimulation such as an extracellular signal.

[0092] Furthermore, the protein does not necessarily need to be able to stably bind to the sequence motif under any conditions, but only needs to be able to bind under specific conditions. For example, the proteins of the present invention include methylated CpG-binding proteins that do not bind based on the sequence motif alone, but can only bind when the sequence motif contains a specific modification. The conditions that allow the protein to bind to the sequence motif do not need to be met at the time of producing and using the hairpin nucleic acid composition of the present invention, but may be met, for example, through a reaction within a cell.

[0093] 1A-4. Effects The hairpin nucleic acid compositions of the present invention induce HCR in the presence of target RNA and can bind to specific proteins via the formed hybridization chain structure or, further, via protein-binding motifs present in a double-stranded state on the HCR product. The HCR product becomes a polymer, for example, approximately 100 bp to 1000 bp in size, to which proteins such as pattern recognition receptors bind depending on the length and structure of the hybridization chain structure. Furthermore, if the HCR product contains protein-binding motifs, specific proteins further bind to the polymer in large numbers depending on the number of protein-binding motifs. This can lead to, for example, the formation of aggregates and the induction of liquid-liquid phase separation, which can cause stress to cells. Liquid-liquid phase separation, also known as biological phase separation, refers to the change in a homogeneously mixed system from a single-phase liquid state to a two-phase liquid state in response to a change in conditions.

[0094] The hairpin nucleic acid composition of the present invention induces nucleic acid immunity. Nucleic acid immunity refers to an immune response based on nucleic acid recognition by the innate immune system. Nucleic acid immunity is an immune response induced by viral infection, and activates transcription factors of the interferon regulatory factor (IRF) family based on nucleic acid recognition by pattern recognition receptors, including Toll-like receptors. This can induce the expression of type I interferons (INF-α, INF-β, etc.). Alternatively, for example, by capturing specific proteins present in excess, the function of the protein can be inhibited or the amount of free protein can be reduced. This can, for example, inhibit the function of proteins that protect cancer cells from the immune system (e.g., proteins related to the NF-κB signaling pathway).

[0095] These effects can be measured, for example, by administering a test hairpin nucleic acid composition to a subject (e.g., a laboratory animal, cultured cells, etc.) and measuring indicators of the effect of the test hairpin nucleic acid composition after a certain period of time, e.g., 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, or several days (e.g., 1 to 7 or 2 to 7 days). Specifically, for example, when the target is cellular stress, measurement can be performed by measuring indicators of cellular stress, such as cell viability, cell stress markers, and cell death markers. Furthermore, for example, in the case of protein capture, measurement can be performed by measuring the amount of free protein or the amount of reaction products related to the function of the protein (e.g., expression of genes subject to transcriptional regulation, products of enzymatic reactions catalyzed, activity of signaling pathways mediated, etc.).

[0096] For example, the level of an indicator or product expected to be decreased by application of the hairpin nucleic acid may be decreased compared to a negative control (e.g., vehicle administration), or the level of an indicator or product expected to be increased may be increased compared to a negative control. In this case, the amount of change in level is not particularly limited. Specifically, for example, a change of at least 10%, at least 20%, at least 25%, at least 30%, or at least 40% indicates that the test hairpin nucleic acid composition can produce the desired effect. Alternatively, the ability to produce the desired effect may be determined, for example, by confirming whether the change is statistically significant.

[0097] 1B. Cell Death-Inducing Composition 1B-1. Overview A first aspect B of the present invention is a cell death-inducing composition. The cell death-inducing composition of the present invention comprises, as active ingredients, an initiating hairpin nucleic acid and an extended hairpin nucleic acid having a hairpin structure capable of forming a hybridization linkage structure. When the initiating hairpin nucleic acid hybridizes to a target RNA, a complex of the target RNA, the initiating hairpin nucleic acid, and the extended hairpin nucleic acid is formed in a linkage manner by HCR. The cell death-inducing composition of the present invention can capture nucleic acid-binding proteins to induce nucleic acid immunity, and can also be an active ingredient in the cell death-promoting compositions, pharmaceutical compositions, anti-cancer agents, anti-inflammatory agents, and nucleic acid immunity-inducing compositions of the present invention.

[0098] 1B-2. Composition The components of the cell death-inducing composition of this embodiment will be described. The cell death-inducing composition of the present invention contains an initiation hairpin nucleic acid and an extended hairpin nucleic acid as essential components, and a carrier as an optional component. Each component will be described in detail below.

[0099] The initiating hairpin nucleic acid and the extending hairpin nucleic acid are as described in detail in First Aspect A. Therefore, detailed description thereof will be omitted here.

[0100] The cell death-inducing composition of this embodiment comprises an effective amount of an initiator hairpin nucleic acid and an extended hairpin nucleic acid. Except for the desired effect of inducing cell death (e.g., apoptosis), the active ingredients are essentially the same as those of the pharmaceutical composition of the second embodiment described below. Therefore, only the differences will be described here.

[0101] The cell death-inducing composition of the present invention is intended to induce cell death. Therefore, in addition to the hairpin nucleic acid composition described in First Aspect A, the composition may contain one or more active ingredients capable of inducing cell death. Specific active ingredients include, for example, compounds that induce cell death, drugs that promote or suppress gene expression, and drugs that promote or inhibit function at the protein level.

[0102] The cell death-inducing compositions of the present invention do not necessarily induce cell death only in cells containing HCR products. For example, if the hybridization chain structure is recognized by cGAS, the secondary messenger 2'-5'-cGAMP synthesized by cGAS may be transferred through gap junctions to neighboring cells, inducing cell death in those cells as well. Furthermore, for example, an immune response can be induced in association with the cell death of cells containing HCR products. This immune response can promote secondary cell death (e.g., immunogenic cell death) in cells with similar abnormalities that do not contain HCR products.

[0103] 2. Pharmaceutical Composition 2-1. Overview A second aspect of the present invention is a pharmaceutical composition. The pharmaceutical composition of the present invention contains as an active ingredient the hairpin nucleic acid composition and / or cell death-inducing composition described in the first aspect, and induces HCR at a target site containing target RNA. Use of the pharmaceutical composition of the present invention can achieve a desired effect based on HCR at a target site.

[0104] 2-2. Composition The components of the pharmaceutical composition of this embodiment will be described. The pharmaceutical composition of the present invention contains an active ingredient as an essential component and a carrier as an optional component. Each component will be specifically described below.

[0105] (1) Active Ingredients The pharmaceutical composition of the present invention contains an effective amount of the hairpin nucleic acid composition and / or cell death-inducing composition described in the first aspect as an essential active ingredient. The composition of the hairpin nucleic acid composition and the cell death-inducing composition is described in detail in the first aspect, so a detailed description will be omitted here. Depending on the desired effect to be achieved by the pharmaceutical composition, one or more other active ingredients may also be included.

[0106] An "effective amount" refers to the amount of the hairpin nucleic acid composition and / or cell death-inducing composition necessary to exert its function as an active ingredient, and which causes little or no harmful side effects in the subject to which it is administered. This effective amount may vary depending on various conditions, such as information about the subject, the route of administration, and the number of administrations. Ultimately, it is determined by the judgment of the person administering, such as a physician, veterinarian, or pharmacist.

[0107] For example, when the hybridization chain structure includes a structure recognized by cGAS, the effective amount can be determined so that a sufficient amount of hybridization chain structure to activate cGAS is formed in the cell. Specific examples of effective amounts include, but are not limited to, amounts of nucleic acid in target cells of 0.01 nM to 20 nM, 0.05 nM to 15 nM, 0.08 nM to 12 nM, 0.09 nM to 11 nM, 0.1 nM to 10 nM, 0.11 nM to 10 nM, 0.2 nM to 10 nM, 0.5 nM to 10 nM, 0.8 nM to 10 nM, 0.9 nM to 10 nM , 1 nM to 10 nM, 2 nM to 10 nM, 5 nM to 10 nM, 0.1 nM to 10 nM, 0.1 nM to 9 nM, 0.1 nM to 8 nM, 0.1 nM to 6 nM, 0.1 nM to 5 nM, 0.1 nM to 3 nM, 0.1 nM to 2 nM, 0.1 nM to 1 nM, 0.2 nM to 0.9 nM, or 0.3 nM to 0.8 nM.

[0108] As used herein, the term "subject" refers to a target to which the hairpin nucleic acid composition, cell death-inducing composition, pharmaceutical composition, protein function-inhibiting composition, cell death-promoting composition, and nucleic acid immunity-inducing composition of the present invention are applied. Subjects include not only individuals, but also organs, tissues, and cells. When the subject is an individual, it may be any animal, including humans. Examples of non-human subjects include various livestock, poultry, pets, and laboratory animals. Without being limited thereto, the subject may be an individual with abnormal protein expression or abnormal cells, or an individual in need of disease treatment or prevention.

[0109] As used herein, "subject information" refers to various individual information of the living body to which the method is applied. For example, if the subject is a human, this includes age, weight, sex, diet, health condition, disease progression and severity, drug sensitivity, and the presence or absence of concomitant medications.

[0110] As used herein, the term "target site" refers to a biological site containing a target cell. As used herein, the term "target cell" refers to a cell on which the effects of the hairpin nucleic acid composition and / or cell death-inducing composition of the present invention are expected to be exerted. Specifically, the target cell is a cell that expresses a target RNA and / or a cell that highly expresses a target RNA. For example, the target cell may be a cell that further expresses a pattern recognition receptor or a cell that further expresses a protein capable of binding to a protein-binding motif.

[0111] (2) Carrier The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to an additive commonly used in the pharmaceutical formulation field. Examples include solvents, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavorings, excipients, vehicles, preservatives, binders, diluents, isotonicity agents, sedatives, buffers, coating agents, lubricants, colorants, thickeners, solubilizers, and other additives.

[0112] The solvent may be, for example, water or other pharmaceutically acceptable aqueous solutions, or pharmaceutically acceptable organic solvents (e.g., vegetable oils, etc.). Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.

[0113] The above-mentioned carriers are used to avoid or suppress the degradation of the active ingredients, the hairpin nucleic acid composition and / or cell death-inducing composition, by enzymes, etc. in the body, as well as to facilitate formulation and administration methods and maintain the dosage form and efficacy, and may be used appropriately as needed.

[0114] (3) Dosage Form The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it is a form that can deliver the active ingredient, the hairpin nucleic acid composition and / or cell death-inducing composition described in the first aspect, to the target site without inactivating it by degradation or the like, and can exert the pharmacological effect of the active ingredient in the body.

[0115] The specific dosage form varies depending on the administration mode and / or formulation conditions. The administration mode of the pharmaceutical composition of the present invention can be broadly divided into oral administration and parenteral administration. If the administration method is parenteral administration, a preferred dosage form is a liquid that can be administered directly to the target site or systemically via the circulatory system. An example of a liquid is an injection. An injection can be formulated by appropriately combining the above-mentioned excipients, elixirs, emulsifiers, suspending agents, surfactants, stabilizers, pH adjusters, etc., and mixing them in a unit dosage form required for generally accepted pharmaceutical practice. Other examples include ointments, plasters, cataplasms, transdermal agents, lotions, inhalants, aerosols, eye drops, and suppositories.

[0116] The specific shape and size of each of the above dosage forms are not particularly limited as long as they are within the range of dosage forms known in the art. The pharmaceutical composition of the present invention may be formulated according to a conventional method in the art.

[0117] The hairpin nucleic acid composition and / or cell death-inducing composition of the present invention has excellent solubility in water, Japanese Pharmacopoeia Dissolution Test Fluid 2, or Japanese Pharmacopoeia Disintegration Test Fluid 2; excellent pharmacokinetics (e.g., blood drug half-life, brain transferability, metabolic stability, CYP inhibition); low toxicity (e.g., superior as a pharmaceutical in terms of acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, drug interactions, carcinogenicity, phototoxicity, etc.); and few side effects (e.g., suppression of sedation, avoidance of lamellar necrosis), thus possessing excellent properties as a pharmaceutical.

[0118] (4) Dosage Form and Dose In the present specification, there is no particular limitation on the preferred dosage form of the pharmaceutical composition of the present invention. For example, it may be administered orally or parenterally. Parenteral administration is usually used.

[0119] Specific examples of parenteral administration include intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration (including continuous subcutaneous administration by implantation), intradermal administration, tracheal / bronchial administration, rectal administration, administration by transfusion, intratumoral administration, juxtatumoral administration (e.g., intradermal or subcutaneous administration in the vicinity of a tumor), intraventricular administration, intrathecal administration, intranasal administration, and intramuscular administration.

[0120] The pharmaceutical composition of the present invention exerts an additive inhibitory effect within cells even when administered repeatedly. Furthermore, in the case of repeated administration, the efficacy can be improved by leaving a certain interval between administrations (e.g., half a day or more).

[0121] (5) Target Diseases The target diseases for the pharmaceutical composition of the present invention are not particularly limited. For example, the pharmaceutical composition of the present invention can be used for diseases associated with the abnormal expression of specific proteins or the presence of abnormal cells, such as neurological diseases, central nervous system diseases, metabolic diseases, tumors (e.g., malignant tumors (cancer)), infectious diseases, immune system diseases (e.g., autoimmune diseases, allergic diseases, and inflammatory diseases), and abnormal protein storage diseases. The pharmaceutical composition of the present invention can be used for the treatment of diseases selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, and abnormal protein storage diseases.

[0122] Malignant tumors (cancers) in this specification include, for example, leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, prostate cancer, uterine cancer, endometrial cancer, cervical cancer, ovarian cancer, adrenal cancer, thyroid cancer, skin cancer, head and neck cancer, gastrointestinal cancer, pancreatic cancer, breast cancer, and lung cancer, as well as metastases thereof.

[0123] For example, when cancer is the target disease, an anticancer drug can be prepared containing the hairpin nucleic acid composition and / or cell death-inducing composition of the present invention as an active ingredient, using a configuration similar to that of the pharmaceutical composition of this embodiment.

[0124] In this case, the target cancer may be any of the cancers described above, and the target RNA may be an RNA that is specifically expressed in the target cancer cells and / or an RNA that is specifically highly expressed in the target cancer cells.

[0125] The anticancer agent of the present invention can optionally contain other anticancer agents. For example, when the target disease is an inflammatory disease, an anti-inflammatory agent can be prepared containing the hairpin nucleic acid composition and / or cell death-inducing composition of the present invention as an active ingredient, in a similar manner to the pharmaceutical composition of this embodiment.

[0126] In this case, the target inflammatory disease may be any of the inflammatory diseases described above in the definition section. Furthermore, the target RNA may be RNA specifically expressed in the inflammatory cells of the target application and / or RNA specifically highly expressed in the inflammatory cells of the target application. Furthermore, for example, if there are cells that cause the inflammatory response, the target RNA may be RNA specifically expressed and / or highly expressed in the cells that cause the inflammatory response. The anti-inflammatory agent of the present invention may optionally further contain another anti-inflammatory agent.

[0127] 3. Protein Function-Inhibiting Composition 3-1. Overview The third aspect of the present invention is a protein function-inhibiting composition. The protein function-inhibiting composition of the present invention contains the hairpin nucleic acid composition described in First Aspect A as an active ingredient, and induces HCR at a target site containing target RNA. By using the protein function-inhibiting composition of the present invention, it is possible to inhibit protein function based on HCR in, for example, a target cell.

[0128] 3-2. Composition The components of the protein function-inhibiting composition of this embodiment will be described. The protein function-inhibiting composition of the present invention contains an active ingredient as an essential component and a carrier as an optional component. Each component will be specifically described below.

[0129] The protein function-inhibiting composition of the present invention contains, as an essential active ingredient, an effective amount of the hairpin nucleic acid composition described in First Aspect A. Preferably, it contains an effective amount of the hairpin nucleic acid composition described in First Aspect A, which contains a protein-binding motif. Except for the desired effect of inhibiting protein function, the active ingredient is essentially the same as that of the pharmaceutical composition described in Second Aspect. Therefore, only the differences will be described here.

[0130] The protein function-inhibiting composition of the present invention is intended to inhibit protein function. Therefore, in addition to the hairpin nucleic acid composition described in First Aspect A, the composition may contain one or more active ingredients capable of inhibiting protein function. Specific active ingredients include, for example, gene expression inhibitors (e.g., antisense nucleic acids and short hairpin nucleic acids) and protein-level function inhibitors (e.g., antibodies, aptamers, and competitive inhibitors).

[0131] The basic details of the carrier, dosage form, etc. used in the protein function-inhibiting composition of the present invention are the same as those of the pharmaceutical composition of the second aspect, and therefore detailed explanations thereof will be omitted here.

[0132] The protein function-inhibiting composition of the present invention does not necessarily have to inhibit only proteins that directly bind to the protein-binding motif, but may also inhibit, for example, proteins that indirectly bind to the HCR product via proteins that bind to the protein-binding motif.

[0133] 4. Cell Death-Promoting Composition 4-1. Overview The fourth aspect of the present invention is a cell death-promoting composition. The cell death-promoting composition of the present invention contains the hairpin nucleic acid composition described in First Aspect A as an active ingredient, and induces HCR in target cells containing target RNA. Use of the cell death-promoting composition of the present invention can promote cell death based on HCR in target cells.

[0134] 4-2. Composition The components of the cell death-promoting composition of this embodiment will be described. The cell death-promoting composition of the present invention contains an active ingredient as an essential component and a carrier as an optional component. Each component will be described in detail below.

[0135] The cell death-promoting composition of the present invention contains an effective amount of the hairpin nucleic acid composition described in First Aspect A as an essential active ingredient. Except for the desired effect of promoting cell death, the active ingredient is essentially the same as that of the pharmaceutical composition described in Second Aspect A. Therefore, only the differences will be described here.

[0136] The cell death-promoting composition of the present invention is intended to promote cell death. Therefore, in addition to the hairpin nucleic acid composition described in First Aspect A, the composition may contain one or more active ingredients capable of promoting cell death. Specific active ingredients include, for example, compounds that induce cell death, drugs that suppress gene expression, and drugs that inhibit protein function.

[0137] The basic details of the carriers and dosage forms used in the cell death-promoting composition of the present invention are the same as those of the pharmaceutical composition of the second aspect, and therefore a detailed description thereof will be omitted here.

[0138] The cell death-promoting compositions of the present invention do not necessarily induce cell death only in cells containing an HCR product. As shown in the examples below, the cell death-promoting compositions of the present invention can induce an immune response in association with cell death of cells containing an HCR product. This immune response can secondarily promote cell death in cells with a similar abnormality that do not contain an HCR product. In this specification, the promoted cell death is, for example, immunogenic cell death.

[0139] 5. Nucleic Acid Immunity-Inducing Composition 5-1. Overview The fifth aspect of the present invention is a nucleic acid immunity-inducing composition. The nucleic acid immunity-inducing composition of the present invention contains the hairpin nucleic acid composition and / or cell death-inducing composition described in the first aspect as an active ingredient, and induces HCR at a target site containing target RNA. By using the nucleic acid immunity-inducing composition of the present invention, it is possible to induce nucleic acid immunity based on HCR in, for example, a target cell.

[0140] 5-2. Composition The components of the nucleic acid immunity-inducing composition of this embodiment will be described. The nucleic acid immunity-inducing composition of the present invention contains a hairpin nucleic acid composition and / or a cell death-inducing composition as active ingredients as essential components, and contains a carrier as an optional component. Each component will be described in detail below.

[0141] The nucleic acid immunity-inducing composition of the present invention contains, as an essential active ingredient, an effective amount of the hairpin nucleic acid composition and / or cell death-inducing composition described in the first aspect. Except for the desired effect of inducing nucleic acid immunity, the active ingredients are essentially the same as those of the pharmaceutical composition described in the second aspect. Therefore, only the differences will be described here.

[0142] The nucleic acid immunity-inducing composition of the present invention is intended to induce nucleic acid immunity. Therefore, in addition to the hairpin nucleic acid composition and / or cell death-inducing composition described in the first aspect, the composition may contain one or more active ingredients capable of inducing nucleic acid immunity as active ingredients. Specific active ingredients include, for example, molecules that increase the activity of pattern recognition receptors and / or their downstream factors (e.g., their agonists, expression promoters, etc.), nucleic acid molecules recognized by other types of pattern recognition receptors, and lipid molecules and sugar molecules recognized by pattern recognition receptors that recognize substances other than nucleic acids.

[0143] The basic details of the carrier, dosage form, etc. used in the nucleic acid immunity-inducing composition of the present invention are the same as those of the pharmaceutical composition of the second aspect, and therefore detailed explanations thereof will be omitted here.

[0144] The nucleic acid immunity-inducing composition of the present invention may induce nucleic acid immunity in cells other than cells containing HCR products. For example, if the hybridization sequence is recognized by cGAS, 2'-5'-cGAMP, a second messenger synthesized by cGAS, may be transferred through gap junctions to neighboring cells, thereby inducing nucleic acid immunity in those cells.

[0145] Example 1: Evaluation of HCR efficiency in a cell-free system (Objective) To investigate the relationship between hairpin nucleic acid structure and HCR efficiency in a cell-free experimental system. (Method) 1. Design of hairpin nucleic acids A set of three hairpin nucleic acids targeting miR-21 (SEQ ID NO: 1) was designed using the online software NUPACK (http: / / www.nupack.org / ). The sequence of each hairpin nucleic acid and the free energy change in the hairpin structure formation reaction are shown in Table 1.

[0146]

[0147] In the set of three hairpin nucleic acids (HP(16), HP(15), and HP(13)), the first hairpin nucleic acid (HP1) had the lowest free energy change. HP1(16), HP1(15), and HP1(13) had free energy changes of -16.4 kcal / mol, -15.3 kcal / mol, and -13.5 kcal / mol, respectively. Each designed hairpin nucleic acid was chemically synthesized using an automated nucleic acid synthesizer.

[0148] 2. Evaluation of HCR efficiency For each hairpin nucleic acid set, miR-21 was added to a TE buffer solution containing HP1 and the second hairpin nucleic acid (HP2) at a concentration of 1 μM each, at a final concentration of 0.1 μM, and the mixture was allowed to stand at room temperature. As a control, a mixture without miR-21 was used. After standing, the reaction solution was analyzed by 1% agarose gel electrophoresis. SYBR was used to stain the gel. TM Gold (Thermo Fisher Scientific) was used.

[0149] (Results) The results are shown in Figure 3. HCR was induced with all three hairpin nucleic acid sets evaluated. In all cases, monomeric hairpin nucleic acid molecules were abundant without the addition of miR-21, and the amount of monomers was significantly reduced with the addition of miR-21. In particular, with the hairpin nucleic acid sets with HP1 free energy changes of -16.4 kcal / mol and -15.3 kcal / mol, almost no HCR product was observed without the addition of miR-21, indicating that the reaction proceeds in a miR-21-dependent manner, as is the target RNA. In the following experiments, we used the hairpin nucleic acid set HP(16), which showed the highest HCR efficiency.

[0150] Example 2: Evaluation of nuclease resistance of hairpin nucleic acids (Objective) The relationship between the length of the protruding region of a hairpin nucleic acid and its nuclease resistance was investigated. (Method) HP1(16) or HP2(16) was added to a DMEM solution (Gibco) containing 10% fetal bovine serum (FBS; Biowest) and 0.5% penicillin-streptomycin (Nacalai Tesque) at a final concentration of 0.5 μM, and the mixture was allowed to stand at 37°C. The internucleotide bonds in the protruding regions of the HP1(16) and HP2(16) used were phosphorothioate-modified. Aliquots (20 μL) of the reaction mixture were sampled at regular intervals, and the reaction was stopped by adding formamide (10 μL). Analysis was then performed by 5% polyacrylamide gel electrophoresis. SYBR was used to stain the gel. TM Gold (Thermo Fisher Scientific) was used.

[0151] (Results) The results are shown in Figure 4. It was found that both hairpin nucleic acids remained in sufficient amounts for several hours, even in FBS containing a large amount of nuclease. Furthermore, after one day, most of the hairpin nucleic acids had been degraded by nuclease, indicating that unreacted hairpin nucleic acids were degraded by nuclease and appropriately eliminated. Furthermore, although HP1(16) and HP2(16) have overhangs of 8 and 10 bases, respectively, there was no significant difference in the nuclease resistance between the two, indicating that the length of the overhang does not significantly affect nuclease activity.

[0152] Example 3: Evaluation of Protein Capture Efficiency of HCR Products (Objective) The protein capture efficiency of protein-binding motifs on HCR products was investigated. (Method) HP1(16) and HP2(16) each contain a recognition sequence for NF-κB. Therefore, miR-21 was added to a final concentration of 10 nM in a TE buffer solution containing HP1(16) fluorescently modified with FAM (FAM-HP1(16); Table 2, SEQ ID NO: 8) and HP2(16) at a concentration of 0.1 μM, respectively, and the solution was allowed to stand at room temperature. Recombinant NF-κB (Cayman Chemical) was added to the reaction solution at a final concentration of 50 ng / μL and allowed to stand. A reaction solution without NF-κB was used as a control. NF-κB binding was detected using the gel shift method. First, the reaction solution was subjected to 5% polyacrylamide gel electrophoresis. Bands were detected using Gel Doc. TM This was performed by detecting FAM fluorescence using an EZ Imager (BioRad; excitation wavelength 430 nm to 460 nm).

[0153] The sequences of the hairpin nucleic acids used in Examples 3 and 4 are shown below. As shown in Table 2 below, in FAM-HP1(16) (SEQ ID NO: 8), the internucleoside linkage from the first base to the ninth base from the 5' end is a phosphorothioate linkage, and the 21st nucleotide from the 5' end is the thymidine analog dT-FAM (3'). In TAMRA-HP2(16) (SEQ ID NO: 9), the internucleoside linkage from the 36th base to the 46th base from the 5' end is a phosphorothioate linkage, and the 46th nucleotide from the 5' end is a cytidine to which 5-TAMRA is linked via a C6 amino linker (catalog number: 26-6418, Gene Link).

[0154]

[0155] (Results) The results are shown in Figure 5. It was found that HCR production occurred normally when NF-κB was not added. Nucleic acid chains of various lengths were detected as HCR products (arrowheads in Figure 5). When NF-κB was added, the band of the HCR product alone was barely detectable, and a band with very low mobility was observed. This indicates that the complex between the HCR product and NF-κB (NF-κB / HCR product) was formed with high efficiency, regardless of the length of the HCR product.

[0156] Example 4: Evaluation of HCR efficiency in cell lines (Objective) To confirm that HCR occurs in human cells and to examine its efficiency. (Method) 4 HEK293T cells and HeLa cells were seeded in 3.5 cm glass-bottom dishes and cultured in 200 μL of DMEM containing 10% FBS and 0.5% penicillin-streptomycin until approximately 90% confluent. A TE buffer solution containing 0.1 μg / μL of FAM-HP1(16) and TAMRA-fluorescently modified HP2(16) (TAMRA-HP2(16); Table 2, SEQ ID NO: 9) was microinjected into HEK293T cells and HeLa cells using Femtotips (Eppendorf). The fluorescence resonance energy transfer (FRET) reaction, which occurs when FAM and TAMRA are brought into close proximity, was detected by confocal microscopy. FRET efficiency was calculated as the ratio of the fluorescence intensities at 521 nm and 575 nm when excited at 488 nm. The relative FRET efficiency at each time point was calculated as the relative value, with the FRET efficiency at the start of the reaction set to 1. The experiment was performed four times.

[0157] (Results) The results are shown in Figure 6. No change in FRET efficiency was observed in human embryonic kidney HEK293T cells, which barely express miR-21 (Figure 6, open circles). On the other hand, when the hairpin nucleic acid was introduced into human cervical cancer HeLa cells, which express miR-21, an increase in the FRET signal was observed over time (Figure 6, filled circles). These results demonstrate that the designed HP(16) selectively induces HCR in cells expressing miR-21. Furthermore, HCR was found to initiate immediately after the hairpin nucleic acid was introduced into the cells and to be completed within approximately 90 minutes.

[0158] Example 5: Evaluation of the efficiency of cell death promotion by HCR (Objective) To confirm that HCR promotes cell death in human cells and to investigate its efficiency. (Method) 5 HEK293T cells and HeLa cells were seeded into 24-well multiwell plates and cultured in 500 μL of DMEM containing 10% FBS and 0.5% penicillin-streptomycin until approximately 90% confluent. To a mixture of 50 μL of OPTI-MEM (Thermo Fisher Scientific) and 1 μL of Lipofectamine Plus Reagent (Thermo Fisher Scientific), containing HP1(16) and HP2(16) at concentrations of 0 μg / μL, 0.5 μg / μL, and 1 μg / μL, Lipofectamine LTX (Thermo Fisher Scientific) and 50 μL of OPTI-MEM were added and incubated at room temperature. The reaction solution was then added to the dish. The medium was then replaced with 500 μL of DMEM containing 10% FBS and 0.5% penicillin-streptomycin. After 24 hours, the medium was replaced with 400 μL of DMEM solution containing 9% FBS, 0.45% penicillin-streptomycin, and 10% Presto Blue (Invitrogen). Cell death was detected by measuring Presto Blue fluorescence. Relative cell viability was calculated for each cell type relative to the cell viability without hairpin nucleic acid, which was set at 100%. Experiments were performed in quadruplicate.

[0159] (Results) The results are shown in Figure 7. In HEK293T cells, which barely express miR-21, no significant decrease in cell viability was observed even with increasing concentrations of hairpin nucleic acid (white bars in Figure 7). In contrast, in HeLa cells, which express miR-21, the introduction of hairpin nucleic acid significantly decreased cell viability, indicating that cell death was induced depending on the amount of hairpin nucleic acid introduced (black bars in Figure 7). This indicates that the introduction of hairpin nucleic acid can promote cell death via HCR specifically in cells containing target RNA. Furthermore, observation with a fluorescence microscope revealed that the introduction of hairpin nucleic acid led to the formation of aggregates and the induction of liquid-liquid phase separation.

[0160] Since simple induction of HCR is known not to induce cell death, it was suggested that the formation of aggregates due to the capture of NF-κB promotes cell death.

[0161] Example 6: Evaluation of the Efficiency of Immune Response Induction by HCR (Objective) To confirm the induction of an immune response by HCR in human cells and to examine its efficiency. (Method) Lipofection was performed as described in Example 5, except that MCF7 cells were used as cultured cells. 24 hours after the medium change following lipofection, 5 μL of medium was collected, and IFN-β was quantified using ELISA (PBL Assay Science). A negative control (NC) was performed without the hairpin nucleic acid HP(16), and a positive control (PC) was performed using poly(dG:dC) (InvivoGen) instead of HP(16). The relative IFN-β level was calculated relative to the IFN-β level in NC, which was defined as 1.

[0162] (Results) The results are shown in Figure 8. Poly(dG:dC), which is known to have strong immune activating properties, was introduced into miR-21-expressing human breast cancer MCF7 cells. Approximately twice as much INF-β was produced in the PC cells compared to the NC cells. Furthermore, when the same cells were introduced with HP(16), high levels of INF-β were observed, similar to those in the PC cells. These results demonstrate that HCR products induce nucleic acid immunity.

[0163] It is known that NF-κB normally protects cancer cells from immune responses. Therefore, we demonstrated that the hairpin nucleic acid HP(16) induces cell death via HCR specifically in cells expressing the target RNA miR-21 by both inhibiting NF-κB function through NF-κB capture and inducing nucleic acid immunity.

[0164] Example 7: Evaluation of HCR efficiency for each cell type (Objective) The relationship between cell type and the efficiency of HCR was investigated.

[0165] (Method) Introduction of HP(16) into cells and evaluation of HCR efficiency were performed using the same procedures as in Example 4, except for the cells used. Human embryonic kidney (HEK293T) cells, human cervical cancer (HeLa) cells, human breast cancer (MDA-MB-231) cells, and human alveolar basal adenocarcinoma (A549) cells were used as cells to introduce HP(16). As in Example 4, the relative FRET efficiency was calculated as the ratio of the fluorescence intensities at 521 nm and 575 nm when excited at 488 nm.

[0166] 5.0×10 4 HEK293T cells, HeLa cells, MDA-MB-231 cells, and A549 cells were seeded in 24-well multiwell plates and cultured in 500 μL of Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and 0.5% penicillin-streptomycin. After 24 hours of culture, the medium was replaced with Opti-MEM (Invitrogen).

[0167] Hairpin nucleic acids were transfected into each cell line at 0.5 μg per well using Lipofectamine LTX (Invitrogen). The hairpin nucleic acids used for transfection were a set of HP1(16) fluorescently modified with Cy5 (Cy5-HP1(16); Table 3, SEQ ID NO: 10) and HP2(16) fluorescently modified with FAM (FAM-HP2(16); Table 3, SEQ ID NO: 11) (HP(16)), or a set of hairpin nucleic acids with a scrambled sequence (scrambled hairpin nucleic acids).

[0168] After 3 hours of incubation, the FRET reaction that occurs when HCR occurs and FAM and Cy5 come into close proximity was observed. Observations were performed by confocal microscopy. FRET efficiency (relative value) indicates the FRET signal intensity when HP(16) was introduced relative to the FRET signal intensity when a scrambled hairpin nucleic acid was introduced. The FRET signal intensity for each condition was calculated as the ratio of the fluorescence intensity of Cy5 to the fluorescence intensity of FAM observed when excited at 488 nm. Experiments were performed four times. Statistical significance was determined by unpaired t-test.

[0169] The sequences of the hairpin nucleic acids used in Example 7 are shown below. As shown in Table 3 below, in Cy5-HP1(16) (SEQ ID NO: 10), the internucleoside bond from the first base to the ninth base from the 5' end is a phosphorothioate bond, and the 46th nucleotide from the 5' end is a thymidine linked to Cy5 via a C6 amino linker similar to that used in Example 4. In FAM-HP2(16) (SEQ ID NO: 11), the internucleoside bond from the 36th base to the 46th base from the 5' end is a phosphorothioate bond, and the 21st nucleotide from the 5' end is the thymidine analog dT-FAM(3').

[0170]

[0171] (Results) The results are shown in FIG.

[0172] In human embryonic kidney HEK293T cells, which barely express miR-21, the FRET signal intensity was equivalent to that obtained when scrambled hairpin nucleic acids were used (approximately 100%) (Figure 10). This is consistent with the results of Example 4 (open circles in Figure 6). On the other hand, an increase in FRET signal intensity was observed in all of the miR-21-expressing cells: human cervical cancer HeLa cells, human breast cancer MDA-MB-231 cells, and human alveolar basal epithelial adenocarcinoma A549 cells (Figure 10).

[0173] These results demonstrate that the designed HP(16) selectively induces HCR in cells that express miR-21, and that it induces HCR in cells that express miR-21, regardless of the type of cancer.

[0174] Example 8: Evaluation of cell death promotion efficiency for each cell type (Objective) The effect of cell type on the efficiency of HCR in promoting cell death was investigated.

[0175] (Method) 5.0×10 4 A549, MDA-MB-231, and B16 cells were seeded into 24-well multiwell plates and cultured in 500 μL of DMEM containing 10% FBS and 0.5% penicillin-streptomycin until the cells reached approximately 80% confluence. The medium was then replaced with OPTI-MEM. HP(16) was transfected into each well at a dose of 0.5 μg per well using Lipofectamine LTX (Invitrogen).

[0176] After 48 hours, the medium was replaced with a standard growth solution. Cell death was detected by measuring the fluorescence of Presto Blue (Invitrogen). Fluorescence was measured using a multi-well Cytation tube as in Example 5. TM The measurements were performed using a plate reader (BioTek Instruments).

[0177] Relative cell viability was calculated for each cell type relative to the cell viability without hairpin nucleic acid, which was defined as 100%. Experiments were performed four times. Statistical significance was determined by unpaired t-test.

[0178] (Results) The results are shown in Figure 11. In MDA-MB-231 cells and mouse malignant melanoma B16 cells, the cell viability was significantly reduced by HP(16) transfection (Figure 11). In contrast, the cell viability was not significantly reduced by HP(16) transfection in A549 cells.

[0179] Although A549, MDA-MB-231, and B16 cells all express miR-21, the differences in cell viability between cell types suggested that factors other than the expression of miR-21 may affect the efficiency of promoting cell death.

[0180] Example 9: Evaluation of the effect of the cGAS-STING pathway on the efficiency of promoting cell death (Purpose) The effect of the cGAS-STING pathway on the efficiency of promoting cell death in HCR was investigated.

[0181] (Method) 5.0×10 5 HeLa cells were seeded in a 6-well multiwell plate and cultured in 500 μL of DMEM containing 10% FBS and 0.5% penicillin-streptomycin until they reached approximately 80% confluence. The medium was then replaced with OPTI-MEM.

[0182] STING siRNA (human TMEM173 siRNA, catalog number: AM16708; Silencer select siRNA, Thermo Fisher Scientific) was added to the medium to a final concentration of 5 nM and transfected into each cell. After 24 hours, the medium was replaced with standard growth solution. After another 24 hours of culture, the cells were seeded onto a 24-well multiwell plate and cell death was detected. Cell death detection was essentially performed as in Example 8. However, HeLa cells with or without STING siRNA transfection were used. In addition, HP(16) or poly(dA:dT) (Invitrogen) was used as the transfected nucleic acid. Experiments were performed in quadruplicate. Statistical significance was determined by unpaired t-test.

[0183] (Results) The results are shown in FIG.

[0184] In HeLa cells not transfected with STING siRNA, HP(16) significantly reduced cell viability compared with poly(dA:dT), which is known to have strong immune-activating properties (Figure 12). This indicates that HP(16) can induce cell death more efficiently than poly(dA:dT).

[0185] On the other hand, the induction of cell death by HP(16) was significantly suppressed in HeLa cells transfected with STING siRNA (Figure 12).

[0186] These results indicate that HP(16) induces cell death via the cGAS-STING pathway, which is consistent with the lack of HP(16)-induced cell death in A549 cells, which are known not to express STING protein (Fig. 11).

[0187] Example 10: Evaluation of the efficiency of HCR in inducing an immune response (Objective) The efficiency of HCR in inducing an immune response in human cells was investigated.

[0188] (Method) Cell culture and lipofection were performed as described in Example 9, except that HP(16) was used as the nucleic acid to be introduced. Eighteen hours after lipofection and medium change, the cells were suspended, and RNA was extracted using NucleoSpin RNA XS (Macherey-Nagel). The extracted RNA was analyzed by qRT-PCR using the One Step TB Green PrimeScript PLUS RT-PCR Kit (TaKaRa Bio) and a Rotor-Gene 3000 (QIAGEN). The following primers were used for IFN-β mRNA: forward primer: 5'-ACAGGTTACCTCCGAAACTGAAGA-3' (SEQ ID NO: 12); reverse primer: 5'-TTAGCCATCAGTCACTTAAACAGCA-3' (SEQ ID NO: 13). The relative IFN-β mRNA level was calculated as a relative value of the normalized IFN-β mRNA level under each condition, with the normalized IFN-β mRNA level in the control group normalized to GAPDH mRNA level set at 1. Statistical significance was determined by unpaired t-test.

[0189] (Results) The results are shown in FIG.

[0190] In HeLa cells without HP(16) transfection, INF-β mRNA was barely detectable (data not shown). Similar to the results in Example 6, INF-β mRNA was significantly elevated by HP(16) transfection (Figure 13). However, this elevation was significantly suppressed in HeLa cells transfected with STING siRNA (Figure 13).

[0191] This indicates that the induction of nucleic acid immunity by HCR products occurs via the cGAS-STING pathway.

[0192] Example 11: Evaluation of cell death induction efficiency in cancer cells in vivo (Objective) The efficiency of cell death induction by HCR in cancer cells in vivo was investigated.

[0193] (Method) First, mice were loaded with cancer cells. 1.5 × 10 cells were suspended in 100 μL of PBS. 5B16 cells were administered subcutaneously to 6-week-old C57BL / 6 mice. After that, tumor volumes increased to 100 mm. 3 Mice that reached this age were subjected to a drug administration experiment.

[0194] Medication was administered peritumorally, three times every four days. 8.4 μg of HP(16) or poly(dA:dT) was administered using AteloGene Local Use Quick Gelation (KOKEN). PBS was administered as a control.

[0195] The tumor volume and body weight were measured on days 0, 4, 6, 8, 9, 12 and 15 after the start of administration.

[0196] These experiments were conducted with the approval of the Ethics Committee of the University of Tokyo. The endpoint was a tumor volume of 2,000 mm 3 At this stage, the mice were euthanized. The tumor volume was calculated using the following formula: (tumor volume) = (longest diameter of tumor) × (short diameter of tumor). 2 / 2

[0197] (Results) The results are shown in FIG.

[0198] In the poly(dA:dT)-treated group, tumor volume increase was suppressed from 6 days after the start of treatment compared with the PBS-treated control group (Figure 14(i) and (ii)). On the other hand, in the HP(16)-treated group, tumor volume increase was significantly suppressed compared with the control and poly(dA:dT)-treated groups from the first measurement after treatment, and tumor volume 15 days after treatment was significantly smaller than that of the control group (Figure 14(iii)). Furthermore, mouse weight remained almost unchanged in both groups. These results demonstrate that induction of tumor cell death by HCR products is effective in vivo and useful for cancer treatment. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A cell death-inducing composition comprising a hairpin nucleic acid having a hairpin structure capable of forming a hybridization chain structure, the cell death-inducing composition comprising a starting hairpin nucleic acid that hybridizes with a target RNA, and an extension hairpin nucleic acid that hybridizes with the starting hairpin nucleic acid or another extension hairpin nucleic acid.

2. The starting hairpin nucleic acid comprises a target RNA-binding cassette that hybridizes with the target RNA, and an extension hairpin nucleic acid-binding cassette that hybridizes with the extension hairpin nucleic acid, The extension hairpin nucleic acid comprises a protruding region cassette that hybridizes with the starting hairpin nucleic acid or another extension hairpin nucleic acid, and a non-protruding region cassette that hybridizes with yet another extension hairpin nucleic acid. The cell death-inducing composition according to claim 1.

3. When the target RNA binds to the target RNA-binding cassette, the hairpin structure of the starting hairpin nucleic acid dissociates, enabling the extension hairpin nucleic acid-binding cassette and the protruding region cassette of the extension hairpin nucleic acid to hybridize. Further, upon such hybridization, the hairpin structure of the extension hairpin nucleic acid dissociates, enabling the non-protruding region cassette and the protruding region cassette of another extension hairpin nucleic acid to hybridize, thereby forming a linear double-stranded nucleic acid having the hybridization chain structure. The cell death-inducing composition according to claim 1 or 2.

4. The extension hairpin nucleic acid comprises 5'-end protruding and 3'-end protruding hairpin nucleic acids. The cell death-inducing composition according to claim 1 or 2.

5. The target RNA-binding cassette is further capable of hybridizing with the non-protruding region cassette of the extension hairpin nucleic acid. The cell death-inducing composition according to claim 1 or 2.

6. The starting hairpin nucleic acid and / or the extension hairpin nucleic acid further comprises all or part of a protein-binding motif. The cell death-inducing composition according to claim 1 or 2.

7. The loop region and / or the protruding region comprises all or part of the protein-binding motif, When the hybridization chain structure is formed, all of the protein-binding motif is composed of a double strand, The cell death-inducing composition according to claim 6.

8. The cell death-inducing composition according to claim 1 or 2, wherein in the starting hairpin nucleic acid, the free energy change amount of the hairpin structure-forming reaction is -20 to -10 kcal / mol.

9. The cell death-inducing composition according to claim 1 or 2, wherein the hairpin nucleic acid is composed of DNA and / or RNA nucleotides.

10. The cell death-inducing composition according to claim 9, wherein the nucleotide contains a modified nucleotide.

11. The cell death-inducing composition according to claim 1 or 2, wherein the target RNA is mRNA or miRNA.

12. The cell death-inducing composition according to claim 1 or 2, wherein the target RNA is an RNA that is specifically expressed or highly expressed specifically in cells.

13. A pharmaceutical composition comprising the cell death-inducing composition according to claim 1 or 2 as an active ingredient.

14. The pharmaceutical composition according to claim 13, for treating a disease selected from the group consisting of cancer, immune system diseases, and neurodegenerative diseases.

15. An anticancer agent comprising a hairpin nucleic acid having a hairpin structure capable of forming a hybridization chain structure, comprising a starting hairpin nucleic acid that hybridizes with a target RNA, and an extension hairpin nucleic acid that hybridizes with the starting hairpin nucleic acid or another extension hairpin nucleic acid.

16. An anti-inflammatory agent comprising a hairpin nucleic acid having a hairpin structure capable of forming a hybridization chain structure, comprising a starting hairpin nucleic acid that hybridizes with a target RNA, and an extension hairpin nucleic acid that hybridizes with the starting hairpin nucleic acid or another extension hairpin nucleic acid.

17. A composition comprising a starting hairpin nucleic acid and an extension hairpin nucleic acid having a hairpin structure capable of forming a hybridization chain structure, wherein the starting hairpin nucleic acid comprises a target RNA-binding cassette that hybridizes with a target RNA and an extension hairpin nucleic acid-binding cassette that hybridizes with the extension hairpin nucleic acid, the extension hairpin nucleic acid comprises a protruding region cassette that hybridizes with the starting hairpin nucleic acid or another extension hairpin nucleic acid, and a non-protruding region cassette that hybridizes with yet another extension hairpin nucleic acid, the composition, wherein the starting hairpin nucleic acid and / or the extension hairpin nucleic acid further comprises all or part of a protein-binding motif.