Artificial nucleic acids that induce specific three-dimensional structures

Abstract

The present invention addressing the problem of providing: a method for forming a three-dimensional structure dynamically by utilizing non-complementary base pairs in an oligonucleotide therapeutic; a modification mode for a nucleic acid maintaining a three-dimensional structure; and an artificial nucleic acid capable of bonding to a target sequence stably regardless of mutation.　Provided are: an artificial nucleic acid for inducing a specific three-dimensional structure by hybridizing to a nucleic acid of interest that does not form a functional three-dimensional structure; a gene expression inhibitor and a nucleic acid detector including the same as an active ingredient; and a method for producing an artificial nucleic acid.

Description

[Technical Field]

[0001] The present invention relates to an artificial nucleic acid that hybridizes with a target nucleic acid that does not form a functional three-dimensional structure to induce a specific three-dimensional structure, a gene expression inhibitor and a nucleic acid detection agent containing the artificial nucleic acid as an active ingredient, and a method for producing the artificial nucleic acid. [Background technology]

[0002] Nucleic acid molecules such as DNA and RNA are composed of nucleotides, each containing one of four bases: adenine (A), thymine (T) (uracil (U) in RNA), guanine (G), and cytosine (C). A readily pairs with T or U, and C readily pairs with G; this property is called base pair complementarity. In living organisms, various phenomena occur based on this base pair complementarity, such as the formation of the DNA double helix structure, semi-conservative DNA replication, and RNA transcription using DNA as a template. Furthermore, because the rules of this complementarity are clear, it is applied to almost all biotechnology technologies involving nucleic acids, such as PCR, DNA sequencing, gene knockdown, and gene knockout methods. In particular, gene knockdown methods are widely applied as "nucleic acid drugs," which are third-generation pharmaceuticals, and the number of approved drug approvals has been increasing in recent years.

[0003] Gene knockdown methods used for nucleic acid drugs are primarily based on antisense oligonucleotides (ASOs) or siRNAs (small interfering RNAs). While ASOs and siRNAs are easy to design, they have the problem of unstable efficacy if mutations occur in the target sequence. This is because base pairs that ignore complementarity are formed, weakening the binding between the target and these molecules. Therefore, gene regions containing individual-specific diversity variations, such as single nucleotide variants (SNVs), had to be excluded from candidate target sequences.

[0004] In recent years, it has become clear that nucleic acids stably form non-complementary base pairs in living organisms. For example, Non-Patent Literature 1 discloses that in single-stranded nucleic acids that form functional three-dimensional structures, a stable three-dimensional structure is formed by approximately 150 types of non-complementary base pairs.

[0005] By utilizing these non-complementary base pairs and the functional three-dimensional structures they induce, it may be possible to develop nucleic acid drugs that stably act on target sequences containing the mutations described above. However, the use of non-complementary base pairs to solve the problems of conventional nucleic acid drugs has not been considered until now. Moreover, basic knowledge such as methods for actively forming the functional three-dimensional structures necessary for utilizing non-complementary base pairs in nucleic acid drugs, or methods for modifying them while maintaining the three-dimensional structure, has not been obtained. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Leontis, NB et al., Nucleic Acids Res. 2002 Aug 15;30(16):3497-3531. [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to develop a method for inducing the formation of a three-dimensional structure containing non-complementary base pairs, and to provide an artificial nucleic acid that can stably bind to a target sequence without being affected by its mutations. [Means for solving the problem]

[0008] To solve the above problems, the inventors conducted research and development and have succeeded in developing an artificial nucleic acid that hybridizes with target nucleic acids that do not form a three-dimensional structure to induce a specific three-dimensional structure. Furthermore, it has become clear that introducing modifications to this artificial nucleic acid further increases the stability of the double-stranded nucleic acid. The invention is based on the above and other novel findings and provides the following.

[0009] (1) An artificial nucleic acid that hybridizes to a target nucleic acid that does not form a functional three-dimensional structure to induce a specific three-dimensional structure, The artificial nucleic acid includes a structure-forming inducing domain that forms a three-dimensional structure with the target domain of the target nucleic acid. The target domain and the structure-forming induction domain constitute a sequence motif consisting of two strands that form a specific three-dimensional structure. In the aforementioned sequence motif, the three-dimensional structure formation inducing domain and the target domain include complementary regions consisting of complementary sequences. Furthermore, in the sequence motif, the structure-forming induction domain and / or the target domain include one or more non-complementary regions containing sequences that are non-complementary to each other. The aforementioned non-complementary containing region includes non-complementary sequences at both ends. The artificial nucleic acid. (2) The artificial nucleic acid according to (1), wherein the three-dimensional structure-forming-inducing domain and / or the target domain comprises a plurality of complementary regions, and the non-complementary-containing region is located between the plurality of complementary regions. (3) The artificial nucleic acid according to (1) or (2), wherein the non-complementary containing region consists of 2 to 7 bases. (4) The artificial nucleic acid according to any one of (1) to (3), wherein the specific three-dimensional structure comprises one or more selected from the group consisting of a kink turn structure, a bulged G structure, a reverse kink turn structure, a 5S loop E structure, a C loop structure, and a tandem GA structure. (5) The kink turn structure is represented by sequence number 3 (5'-NNNNGAN-3') and sequence number 4 (5'-NGAN-3'), The bulged G structure is represented by sequence number 1 (5'-NNNGUAN-3') and sequence number 2 (5'-NGANNN-3'), The reverse skin turn structure is represented by sequence numbers 5(5'-NNNNAAN-3') and 6(5'-NGAN-3'), The 5S loop E structure is represented by sequence numbers 7(5'-NGUAN-3') and 8(5'-NGAUN-3'), The C-loop structure is represented by sequence numbers 9(5'-NCACU-3') and 10(5'-ANN-3'), or The tandem GA structure is represented by sequence numbers 11(5'-NGAN-3') and 12(5'-NGAN-3'). It consists of, The artificial nucleic acid described in (4), wherein in each base sequence, N is A, C, G, or U. (6) The artificial nucleic acid according to any one of (1) to (5), wherein the target nucleic acid is mRNA or miRNA. (7) The artificial nucleic acid according to any one of (1) to (6), wherein the hybridization is carried out under highly stringent conditions. (8) The artificial nucleic acid according to any one of (1) to (7), wherein the three-dimensional structure-forming-inducing domain contains one or more modified nucleotides. (9) The artificial nucleic acid according to (8), wherein the modified nucleotide is selected from the group consisting of 2'-OMe RNA, 2'-MOE RNA, LNA, 2'-O,5'-N BNA, 2'-deoxy-trans-3',4'-BNA, and DNA. (10) The artificial nucleic acid according to (8) or (9), wherein the modified nucleotide comprises a fluorogroup modification at the 2' position of ribose. (11) The artificial nucleic acid according to any one of (1) to (10), wherein the target domain includes the non-complementary containing region containing mutation. (12) The artificial nucleic acid according to (11), wherein the mutation is a single nucleotide variant, an insertion / deletion mutation, a structural polymorphism, or a combination thereof. (13) The artificial nucleic acid according to any one of (1) to (12), further comprising a hybridize domain consisting of 6 to 120 bases adjacent to one or both of the three-dimensional structure formation inducing domains. (14) A gene expression inhibitor containing any of the artificial nucleic acids described in (1) to (13) as an active ingredient. A nucleic acid detection agent containing, as an active ingredient, the artificial nucleic acid according to any one of (1) to (13). (16) A method for producing an artificial nucleic acid that hybridizes to a nucleic acid for the purpose of not forming a functional three-dimensional structure and induces a specific three-dimensional structure, In the nucleic acid for the purpose, a target domain selection step of searching for sequence information of one of the sequence motifs composed of double strands constituting a specific three-dimensional structure and selecting one or more of them as a target domain, A three-dimensional structure formation induction domain determination step of determining the sequence of the three-dimensional structure formation induction domain so as to constitute the target domain and the sequence motif, and A nucleic acid synthesis step of synthesizing the artificial nucleic acid based on the sequence information determined in the three-dimensional structure formation induction domain determination step comprising In the sequence motif, the target domain and the three-dimensional structure formation induction domain include a complementary region composed of complementary sequences to each other, Furthermore, in the sequence motif, the target domain and / or the three-dimensional structure formation induction domain include a non-complementary-containing region of one or more bases containing non-complementary sequences to each other, The non-complementary-containing region includes non-complementary sequences at both ends thereof, The method. (17) The method according to (16), further comprising a hybridized domain determination step of determining the sequence of a hybridized domain consisting of 6 to 120 bases adjacent to one or both of the three-dimensional structure formation induction domains. This specification includes the disclosure content of Japanese Patent Application No. 2021-126578, which is the basis of the priority of this application.

Effects of the Invention

[0010] According to the artificial nucleic acid of the present invention, a specific three-dimensional structure can be induced in a nucleic acid that does not form a functional three-dimensional structure. In addition, according to the gene expression inhibitor of the present invention, the expression of a target gene can be suppressed. Furthermore, according to the nucleic acid detection agent of the present invention, a target nucleic acid can be detected. Furthermore, the artificial nucleic acid of the present invention can be produced according to the method for producing artificial nucleic acids of the present invention. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the three-dimensional and secondary structures of the bulge G (BG) structure. A shows an exemplary three-dimensional structure. B shows an exemplary secondary structure. In Figure 1B, the upper chain is the α chain and the lower chain is the β chain, the boxed area represents the consensus sequence, and N represents an arbitrary base. The bars between bases represent complementary pairs, and the white circles represent important non-complementary pairs. α1~α6 and β1~β5 indicate the positions of each base in the α and β chains in the consensus sequence, * indicates the position of a nucleotide that has been shown to adopt the C2'-end conformation, and # indicates the position of a nucleotide in which the hydroxyl group at the 2' position of ribose is involved in hydrogen bonding. [Figure 2] This figure shows the three-dimensional and secondary structures of a kink turn (KT) structure. A shows an exemplary three-dimensional structure. B shows an exemplary secondary structure of the standard motif. C shows an exemplary secondary structure of the non-standard motif. In Figures 2B and 2C, the upper chain is the α chain and the lower chain is the β chain, the boxed area represents the consensus sequence, and N represents an arbitrary base. Thick lines between bases indicate complementary pairs, white circles indicate important non-complementary pairs, and thin lines indicate internucleotide bonds. α1~α7 and β1~β4 indicate the positions of each base in the α and β chains in the consensus sequence, * indicates the position of a nucleotide known to adopt the C2'-end conformation, and # indicates the position of a nucleotide in which the 2' hydroxyl group of ribose is involved in hydrogen bonding. [Figure 3]This figure shows the three-dimensional and secondary structures of a reverse skinn (RKT) structure. A shows an exemplary three-dimensional structure. B shows an exemplary secondary structure. In Figure 3B, the upper chain is the α chain and the lower chain is the β chain, the boxed area represents the consensus sequence, and N represents an arbitrary base. The bars between bases represent complementary pairs, and the white circles represent important non-complementary pairs. α1~α5 and β1~β2 indicate the positions of each base in the α and β chains in the consensus sequence, * indicates the position of a nucleotide that has been shown to adopt the C2'-end conformation, and # indicates the position of a nucleotide in which the 2' hydroxyl group of ribose is involved in hydrogen bonding. [Figure 4] This figure shows the three-dimensional and secondary structures of the 5S loop E(5S) structure. A shows an exemplary three-dimensional structure. B shows an exemplary secondary structure of the simple motif. C shows an exemplary secondary structure of the complex motif. In Figures 4B and 4C, the upper chain is the α chain and the lower chain is the β chain, the boxed area represents the consensus sequence, and N represents an arbitrary base. The bars between bases represent complementary pairs, and the white circles represent important non-complementary pairs. α1~α7 and β1~β7 indicate the positions of each base in the α and β chains in the consensus sequence, and # indicates the position of the nucleotide in which the hydroxyl group at the 2' position of ribose is involved in hydrogen bonding. [Figure 5] This figure shows the three-dimensional and secondary structures of a C-loop (CL) structure. A shows an exemplary three-dimensional structure. B shows an exemplary secondary structure. In Figure 5B, the upper chain is the α chain and the lower chain is the β chain, the boxed area represents the consensus sequence, and N represents an arbitrary base. The bars between bases represent complementary pairs, and the white circles represent important non-complementary pairs. α1~α5 and β1~β3 indicate the positions of each base in the α and β chains in the consensus sequence, * indicates the position of a nucleotide that has been shown to adopt the C2'-end conformation, and # indicates the position of a nucleotide in which the hydroxyl group at the 2' position of ribose is involved in hydrogen bonding. [Figure 6]This figure shows the three-dimensional and secondary structures of a tandem GA (GA) structure. A shows an exemplary three-dimensional structure. B shows an exemplary secondary structure. In Figure 6B, the upper chain is the α chain and the lower chain is the β chain, the boxed area represents the consensus sequence, and N represents an arbitrary base. The bars between bases represent complementary pairs, and the white circles represent important non-complementary pairs. α1~α2 and β1~β2 indicate the positions of each base in the α and β chains in the consensus sequence, and # indicates the position of the nucleotide in which the hydroxyl group at the 2' position of ribose is involved in hydrogen bonding. [Figure 7] This figure shows the base sequence and secondary structure of the double-stranded nucleic acid molecule used in Example 1. A (ROI / RNA-ASO) represents the target RNA (RNA of interest: ROI) and its completely complementary nucleic acid (RNA-ASO), B (ROI / RNA-BG) represents the nucleic acid (RNA-BG) that can form a bulged G structure with the ROI, C (ROI / RNA-KT) represents the nucleic acid (RNA-KT) that can form a kink turn structure with the ROI, D (ROI / RNA-RKT) represents the nucleic acid (RNA-RKT) that can form a reverse kink turn structure with the ROI, E (ROI / RNA-5S) represents the nucleic acid (RNA-5S) that can form a 5S loop E structure with the ROI, F (ROI / RNA-CL) represents the nucleic acid (RNA-CL) that can form a C loop structure with the ROI, and G (ROI / RNA-GA) represents the nucleic acid (RNA-GA) that can form a tandem GA structure with the ROI. In the figure, the dashed box indicates the region containing the sequence that forms each of the three-dimensional structures described above. The lines between bases indicate complementary pairings, while the white circles indicate important non-complementary pairings. [Figure 8] This figure shows the melting curves for each double-stranded nucleic acid molecule shown in Figure 7. In the figure, A to G correspond to the double-stranded nucleic acid molecules A to G shown in Figure 7, respectively. [Figure 9]This figure shows the melting curves of double-stranded nucleic acid molecules of the target nucleic acid and nucleic acid containing 2'-O-methyl (2'-OMe) modification. A (ROI / OMe-ASO) shows the results for double-stranded nucleic acid of ROI and a modified nucleic acid (OMe-ASO) that is completely complementary to it, B (ROI / OMe-BG) shows the results for double-stranded nucleic acid of ROI and a modified nucleic acid (OMe-BG) that can form a bulged G structure, C (ROI / OMe-5S) shows the results for double-stranded nucleic acid of ROI and a modified nucleic acid (OMe-5S) that can form a 5S loop E structure, and D (ROI / OMe-GA) shows the results for double-stranded nucleic acid of ROI and a modified nucleic acid (OMe-GA) that can form a tandem GA structure. [Figure 10] This figure shows nucleic acids that form a bulge G (BG) structure. A shows the secondary structure of the base sequence of the unmodified nucleic acid (unmodified BG) used for crystal structure analysis. B shows the results of crystal structure analysis of nucleic acid crystal I of A. C shows the results of crystal structure analysis of nucleic acid crystal II of A. D shows the secondary structure of the base sequence of the modified nucleic acid (modified BG) used for crystal structure analysis. E shows the results of crystal structure analysis of nucleic acid crystal I of D. F shows the results of crystal structure analysis of nucleic acid crystal II of D. In the figure, the area within the dashed frame indicates the region forming the bulge G structure. In Figures 10A and D, uppercase letters indicate unmodified RNA nucleotides, lowercase letters indicate 2'-OMe modified RNA nucleotides, and italicized lowercase letters indicate DNA nucleotides. Bars between bases indicate complementary pairs, and white circles indicate important non-complementary pairs. [Figure 11]This figure shows nucleic acids that form a bulge G (BG) structure. A shows the secondary structure of the base sequence of unmodified BG. B shows the secondary structure of the base sequence of modified BG. In Figures 11A and B, the GA base pairs enclosed in solid lines indicate the positions of the base pairs shown in C and D below, respectively. C shows the results of crystal structure analysis of the GA base pair in unmodified BG. D shows the results of crystal structure analysis of the GA base pair in modified BG. E shows the secondary structure of the base sequence of unmodified BG. F shows the secondary structure of the base sequence of modified BG. In Figures 11E and F, the GU-A triplets enclosed in solid lines indicate the positions of the base triplets shown in G and H below, respectively. G shows the results of crystal structure analysis of the GU-A triplet in unmodified BG. H shows the results of crystal structure analysis of the GU-A triplet shown in D in modified BG. In Figures 11A, B, E, and F, the bars between bases indicate complementary pairs, and the white circles indicate non-complementary pairs. In Figures 11D and H, 2'-OMeA and 2'-OMeU indicate that A and U are 2'-OMe modified RNA nucleotides, respectively. In Figure 11H, dG indicates that G is a DNA nucleotide. Dashed lines indicate hydrogen bonds. [Figure 12] This figure shows nucleic acids that form a bulge G (BG) structure. A shows the secondary structure of the base sequence of unmodified BG. B shows the secondary structure of the base sequence of modified BG. In Figures 12A and B, the CA base pairs enclosed in solid lines indicate the positions of the base pairs shown in C and D below, respectively. C shows the results of crystal structure analysis of the CA base pair in unmodified BG. D shows the results of crystal structure analysis of the CA base pair in modified BG. E shows the secondary structure of the base sequence of unmodified BG. F shows the secondary structure of the base sequence of modified BG. In Figures 12E and F, the CU base pairs enclosed in solid lines indicate the positions of the base pairs shown in G and H below, respectively. G shows the results of crystal structure analysis of the CU base pair in unmodified BG. H shows the results of crystal structure analysis of the CU base pair in modified BG. In Figures 12A, B, E and F, the bars between bases indicate complementary pairing, and the white circles indicate non-complementary pairing. In Figure 12D, dA indicates that A is a DNA nucleotide. In Figure 12H, 2'-OMeU indicates that U is a 2'-OMe modified RNA nucleotide. [Figure 13]This figure shows nucleic acids that have formed two kink turn (KT) structures (indicated by dashed lines). A shows the secondary structure of the base sequence of the unmodified nucleic acid (unmodified KT) used for crystal structure analysis. B shows the results of the crystal structure analysis of nucleic acid A. C shows the secondary structure of the base sequence of the modified nucleic acid (modified KT) used for crystal structure analysis. D shows the results of the crystal structure analysis of nucleic acid C. In Figures 13A and C, uppercase letters indicate unmodified RNA nucleotides, lowercase letters indicate 2'-OMe modified RNA nucleotides, and italicized lowercase letters indicate DNA nucleotides. Bars between bases indicate complementary pairs, and white circles indicate important non-complementary pairs. [Figure 14] Figure 13 shows a single kink turn (KT) structure for each nucleic acid. A shows the secondary structure of the nucleotide sequence containing a single kink turn structure of an unmodified KT. B shows the results of the crystal structure analysis of nucleic acid A. C shows the secondary structure of the nucleotide sequence containing a single kink turn structure of a modified KT. D shows the results of the crystal structure analysis of nucleic acid C. In Figures 14A and C, uppercase letters indicate unmodified RNA nucleotides, lowercase letters indicate 2'-OMe modified RNA nucleotides, and italicized lowercase letters indicate DNA nucleotides. Bars between nucleotides indicate complementary pairs, and white circles indicate important non-complementary pairs. [Figure 15]This figure shows nucleic acids that have formed a kink turn (KT) structure. A shows the secondary structure of the base sequence of unmodified KT. B shows the secondary structure of the base sequence of modified KT. In Figures 15A and B, the G-AG triplets enclosed in solid lines indicate the positions of the triplets shown in C and D below, respectively. C shows the results of crystal structure analysis of the G-AG triplets in unmodified KT. D shows the results of crystal structure analysis of the G-AG triplets in modified KT. E shows the secondary structure of the base sequence of unmodified KT. F shows the secondary structure of the base sequence of modified KT. In Figures 15E and F, the G-AG triplets enclosed in solid lines indicate the positions of the triplets shown in G and H below, respectively. G shows the results of crystal structure analysis of the G-AG triplets in unmodified KT. H shows the results of crystal structure analysis of the G-AG triplets in modified KT. In the figures, the bases of the nucleotides shown in each figure are indicated by solid lines. In Figures 15A, B, E, and F, the bars between bases represent complementary pairs, and the white circles represent typical non-complementary pairs. In Figures 15D and H, dA and dG indicate that A and G are DNA nucleotides, respectively. Dashed lines indicate hydrogen bonds, and solid circles indicate the functional group at the 2' position of ribose G. [Figure 16] This figure shows nucleic acids that have formed a kink turn (KT) structure. A shows the secondary structure of the base sequence of an unmodified KT. B shows the secondary structure of the base sequence of a modified KT. In Figures 16A and B, the GA base pairs enclosed in solid lines indicate the positions of the base pairs shown in C and D below, respectively. C shows the result of crystal structure analysis of the base at the position shown in A in an unmodified KT. D shows the result of crystal structure analysis of the base at the position shown in B in a modified KT. In Figures 16A and B, the bars between bases indicate complementary pairing, and the white circles indicate non-complementary pairing. In Figure 16D, 2'-OMeG indicates that G is a 2'-OMe modified RNA nucleotide. Also, the dashed lines indicate hydrogen bonds, and the solid circle indicates the functional group at the 2' position of the ribose of G. [Figure 17]This figure shows nucleic acids that form tetraloop (TL) structures and tetraloop receptor (TLR) structures. A shows the secondary structure of the base sequence of the unmodified nucleic acid (unmodified TLR) used for crystal structure analysis. B shows the results of the crystal structure analysis of nucleic acid A. C shows the secondary structure of the base sequence of the modified TLR in which some RNA nucleotides were replaced with DNA nucleotides, used for crystal structure analysis. D shows the results of the crystal structure analysis of nucleic acid C. In Figures 17A and C, uppercase letters indicate unmodified RNA nucleotides, and italicized lowercase letters indicate DNA nucleotides. In Figures 17A and C, bars between bases indicate complementary pairs, and white circles indicate important non-complementary pairs. In addition, solid lines indicate regions that form tetraloop receptor structures, and dashed lines indicate regions that form tetraloop structures. [Figure 18] This figure shows the interaction between tetraloop (TL) structures and tetraloop receptor (TLR) structures. Figure A shows the binding of tetraloop structures to tetraloop receptor structures between unmodified TLR molecules. Figure B shows the binding of tetraloop structures to tetraloop receptor structures between modified TLR molecules. The dashed box shows the tetraloop structure, and the gray image on the right side of the figure shows a space-filling model of the tetraloop receptor structure. [Figure 19] This figure shows the interaction between tetraloop (TL) structures and tetraloop receptor (TLR) structures. A shows the crystal structure analysis results of the interaction. In the figure, the solid lines indicate the positions of the UAA triplets shown in B and C below. B shows the crystal structure analysis results of the UAA triplets in unmodified TLRs. C shows the crystal structure analysis results of the UAA triplets in modified TLRs. D shows the crystal structure analysis results of the interaction. In the figure, the solid lines indicate the positions of the UGA triplets shown in E and F below. E shows the crystal structure analysis results of the UGA triplets in unmodified TLRs. F shows the crystal structure analysis results of the UGA triplets in modified TLRs. In Figures 19C and F, dA and dG indicate that A and G are DNA nucleotides, respectively. Dashed lines indicate hydrogen bonds, and solid circles indicate the functional group at the 2' position of ribose with base G. [Figure 20]This figure shows the interaction between tetraloop (TL) structures and tetraloop receptor (TLR) structures. A shows the crystal structure analysis results of the interaction. In the figure, the solid lines indicate the positions of the CGA triplets shown in B and C below. B shows the crystal structure analysis results of the CGA triplets in unmodified TLRs. C shows the crystal structure analysis results of the CGA triplets in modified TLRs. In Figure 20C, dG indicates that G is a DNA nucleotide. Dashed lines indicate hydrogen bonds, and solid circles indicate the functional group at the 2' position of ribose with base G. [Figure 21] This figure shows the base sequence and secondary structure of the nucleic acid molecule used in Example 6. A(ROI-U / ASO-A) shows the target RNA (ROI-U) with a central base sequence of UUU and its perfectly complementary nucleic acid (ASO-A), B(ROI-A / ASO-A) shows the target RNA (ROI-A) with a central base sequence mutated to AAA and ASO-A, C(ROI-U / KT-SKIP) shows ROI-U and an RNA molecule (KT-SKIP) that induces a kink turn structure so that the above-mentioned mutated portion is included in the bulge structure, and D(ROI-A / KT-SKIP) shows ROI-A and KT-SKIP. In the figure, the dashed box indicates the region containing the sequences that form each of the three-dimensional structures described above. The bar between bases indicates complementary pairing, and the white circle indicates important non-complementary pairing. [Figure 22] Figure 21 shows the melting curves of double-stranded nucleic acids. A shows the melting curve of the target nucleic acid and ASO-A double-stranded nucleic acid. B shows the melting curve of the target nucleic acid and KT-SKIP double-stranded nucleic acid. [Figure 23] This figure shows the base sequence and secondary structure of the nucleic acid molecule used in Example 7. A shows the sequence of the hybridization target region (SO-GFP) and the nucleic acid that is completely complementary to it (ASO-GFP), B shows the RNA molecule that induces a tandem GA structure with SO-GFP (GA-GFP), and C shows the RNA molecule that induces a kink turn structure with SO-GFP (KT-GFP). In the figure, each sequence on the upper side shows the sequence of the specific target region in the mRNA of pUC-frGFP used as the hybridization target region. The dashed box indicates the region containing the sequences that form each of the three-dimensional structures described above. The bar between bases indicates complementary pairing, and the white circle indicates important non-complementary pairing. [Figure 24] This figure shows the results of measuring fluorescence derived from frGFP when various nucleic acids are added. A shows the results when no nucleic acid is added, B shows the results when an RNA molecule with the same sequence as the target region (SO-GFP) is added, C shows the results when ASO-GFP is added, D shows the results when GA-GFP is added, and E shows the results when KT-GFP is added. [Figure 25] This figure shows the state in Example 8 where the fluorescently labeled RNA probe (RNA-2AP: lower sequence) hybridized with the target RNA (upper sequence). In the figure, X represents 2-aminopurine (2AP). White circles indicate non-complementary base pairs (sheared GA base pairs) between guanine (G) and adenine (A), and vertical lines indicate complementary base pairs. [Figure 26] Figure 26A shows the fluorescence spectra of a solution containing RNA-2AP (control) and a solution to which an equimolar amount of target RNA or non-target RNA was added to RNA-2AP, measured using an excitation wavelength of 305 nm. Figure 26B shows the rate of change (ΔF) of fluorescence intensity at a wavelength of 370 nm when target RNA or non-target RNA was added to RNA-2AP. [Figure 27] This figure schematically shows the structure of the artificial nucleic acid of the present invention. A shows the schematic arrangement of each domain of the artificial nucleic acid. B shows the schematic arrangement of the target domain of the target nucleic acid. C shows the hybridized state of the artificial nucleic acid of A and the target nucleic acid of B. In the figure, italics indicate complementary regions of each domain, bold indicates non-complementary regions of each domain, and dashed boxes indicate sequence motifs. [Figure 28]This figure shows the base sequence and secondary structure of the nucleic acid molecule used in Example 7. A shows the sequence of the hybridization target region (SO-GFP) and the nucleic acid that is completely complementary to it (ASO-GFP), B shows the RNA molecule that induces a tandem GA structure with SO-GFP (GA-GFP), and C shows the RNA molecule that induces a kink turn structure with SO-GFP (KT-GFP). In the figure, each sequence on the upper side shows the sequence in the mRNA of pUC-frGFP used as the hybridization target region. The dashed box indicates the region containing the sequences that form the aforementioned three-dimensional structures. The bar between bases indicates complementary pairing, and the white circle indicates important non-complementary pairing. [Modes for carrying out the invention]

[0012] 1.Artificial nucleic acid 1-1. Overview A first aspect of the present invention is an artificial nucleic acid. The artificial nucleic acid of the present invention includes a structure-forming induction domain as an essential component and hybridizes with a target nucleic acid to form a specific three-dimensional structure. The artificial nucleic acid of the present invention can be an active ingredient in the gene expression inhibitor and nucleic acid detection agent of the present invention, which will be described later.

[0013] 1-2.Definition The terms used in this specification are defined below.

[0014] (1) Nucleic acid In this specification, "nucleic acid" or "nucleic acid molecule" refers to a biomacromolecule composed of nucleotides linked together by phosphodiester bonds. While naturally occurring nucleic acids and artificial nucleic acids can be broadly classified, this specification encompasses both.

[0015] In this specification, "natural nucleic acids" refers to nucleic acids that exist in nature. Examples include DNA and RNA. Examples of RNA include mRNA and miRNA.

[0016] In this specification, "miRNA" refers to a single-stranded, non-coding RNA (18-25 nucleotides) that exists in living organisms and regulates the expression of specific genes (target genes).

[0017] In this specification, “artificial nucleic acid” refers to nucleic acid molecules that have been artificially synthesized by biological or chemical methods. Unless otherwise specified, the artificial nucleic acids described herein may consist entirely of unmodified natural nucleotides, or they may contain unnatural or modified nucleotides.

[0018] A "nucleotide" is a molecule in which a phosphate group is covalently bonded to the sugar portion of a nucleoside. In the case of nucleotides containing pentofuranosyl sugars, the phosphate group is usually linked to the hydroxyl group at the 3' or 5' position of the sugar.

[0019] A "nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar is not limited to, but is usually composed of pentofuranosyl sugars. Examples of pentofuranosyl sugars include ribose and deoxyribose. Examples of bases (nucleic acid bases) include adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). In this specification, unless otherwise specified, the base may be a modified or unmodified base.

[0020] In this specification, "conformation of the sugar moiety" refers to the three-dimensional structure adopted by the ribose or deoxyribose of a nucleotide. The main conformations include the C2'-end and C3'-end forms, as shown below with deoxyribose as an example.

[0021] [ka] (In the formula, "Base" indicates the base portion, and the numbers indicate the position of each carbon in the ribose ring.)

[0022] The C2'-endo conformation is a conformation in which the 2' carbon atom protrudes on the base side relative to the ribose ring plane. In a broad sense, it also includes the C2'-endo-C3'-exo conformation and the C3'-exo conformation, and is also called the South conformation or S conformation. Conversely, the C3'-endo conformation is a conformation in which the 3' carbon atom protrudes on the base side relative to the ribose ring plane. In a broad sense, it also includes the C3'-endo-C2'-exo conformation and the C2'-exo conformation, and is also called the North conformation or N conformation. Both ribose and deoxyribose can take on any of these conformations, but typically ribose tends to take on the C3'-endo conformation, while deoxyribose tends to take on the C2'-endo conformation. In this specification, "hydroxyl group at the 2' position of ribose" refers to a hydroxyl group bonded to the carbon atom at the 2' position of ribose.

[0023] In this specification, "modification" refers to the substitution of some or all of a nucleotide, which is a constituent unit of nucleic acids, or a nucleoside, which is a component thereof, with another atomic group, or the addition of a functional group, etc. Specifically, examples include sugar modification, base modification, or modification of phosphodiester bonds.

[0024] In this specification, "modified nucleotide" refers to a nucleotide in which some or all of its atoms are substituted with other atomic groups, or to which functional groups, etc., are added. "Unmodified nucleotide" refers to nucleotides other than modified nucleotides. In principle, most naturally occurring nucleotides fall into this category.

[0025] Modified nucleotides include both artificially constructed and naturally occurring modified nucleotides. This includes artificial nucleotides (nucleotide analogs) that have properties and / or structures similar to unmodified nucleotides, and artificial nucleotides containing modified nucleosides or modified bases that have properties and / or structures similar to unmodified nucleosides or unmodified bases, which are components of unmodified nucleotides. Specific examples of modified nucleosides include debasic nucleosides, arabino nucleosides, 2'-deoxyuridine, α-deoxyribonucleosides, and β-L-deoxyribonucleosides. Specific examples of modified bases include the 2-oxo(1H)-pyridine-3-yl group, the 5-substituted 2-oxo(1H)-pyridine-3-yl group, the 2-amino-6-(2-thiazolyl)purine-9-yl group, the 2-amino-6-(2-thiazolyl)purine-9-yl group, and the 2-amino-6-(2-oxazolyl)purine-9-yl group.

[0026] In this specification, "sugar modification" refers to substitution and / or any change in the sugar portion of a nucleic acid molecule. Specifically, examples include 2'-O-methylribose (2'-OMe) in which the hydroxyl group at the 2' position is substituted with a methoxy group, 2'-O-ethylribose in which it is substituted with an ethoxy group, 2'-O-propylribose in which it is substituted with a propoxy group, or 2'-O-butylribose in which it is substituted with a butoxy group, 2'-deoxy-2'-fluororibose in which the hydroxyl group is substituted with a fluoro group, or 2'-O-methoxyethylribose (2'-MOE) in which the hydroxyl group is substituted with a 2'-O-methoxy-ethyl group. The hydroxyl group may also be substituted with a functional group other than a hydrocarbon. Specifically, examples include substitution with H and halogen elements. Furthermore, the (deoxy)ribose portion of the nucleoside may be substituted with other molecules, such as sugars, morpholino rings, PNA, and XNA. Specifically, examples include substitutions of the ribose portion with arabinose, 2'-fluoro-β-D-arabinose, ribose derivatives in which the hydroxyl group at the 2' position and the carbon atom at the 4' position of ribose are bridged, and ribose derivatives in which the oxygen at the 4' position of the ribose ring is replaced with sulfur. It also includes those in which the oxygen atom on the ribofuranose ring (the oxygen atom at the 4' position of ribose) is replaced with sulfur. In particular, nucleotides having a bridged ribose derivative are called bridged nucleic acids, and examples include 2'-OMe RNA, 2'-MOE RNA, LNA, 2'-O,5'-N BNA, and 2'-deoxy-trans-3',4'-BNA.

[0027] "Modified bases" refer to nucleic acid bases other than naturally occurring adenine, cytosine, guanine, thymine, or uracil, and "base modification" refers to the process of changing a nucleic acid base to one of these. Examples of modified nucleic acid bases include, but are not limited to, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, or N4-methylcytosine; N6-methyladenine or 8-bromoadenine; 2-thio-thymine; N2-methylguanine or 8-bromoguanine; and 5-fluorouracil, 5-bromouracil, 5-iodouracil, or 5-hydroxyuracil.

[0028] In this specification, "non-complementary" means that nucleic acid bases do not form a so-called Watson-Crick base pair (natural base pair). In this specification, "non-complementary pairing" refers to two non-complementary bases facing each other and interacting by hydrogen bonding, and "non-complementary base pair" refers to those two non-complementary bases. Furthermore, the act of two bases facing each other and forming a base pair is referred to as "forming a base pair." Specific examples of non-complementary base pairs include, for example, sheared base pairs.

[0029] In this specification, "sheared base pair" refers to a type of non-complementary base pair formed between two nucleic acid strands, in which the functional group on the shallow groove side of one of the bases is involved in hydrogen bonding.

[0030] [ka]

[0031] In the formula, the numbers within the ring structure indicate the position of each functional group. "Functional groups on the shallow groove side (shallow groove edge)" refer to the functional groups at positions 2-4 in purine bases, and the functional group at position 2 in pyrimidine bases. These shallow groove functional groups are also called sugar edge functional groups. Specific examples of sheared base pairs include GA base pairs, AA base pairs, UC base pairs, and CC base pairs.

[0032] (2) Mutation In this specification, "variation" refers to the diversity of base sequences present in the genome of a biological species population. Typically, polymorphisms are distinguished from variations that occur at a frequency of 1% or more within a population, while variations occur at a frequency of less than 1%. However, in this specification, polymorphisms are included as variations, regardless of their frequency within the population.

[0033] In this specification, a "single nucleotide variant (SNV)" refers to a type of variation in the base sequence present in the genome of a biological species population, where the magnitude of the variation is one nucleotide. Typically, a variation occurring at a frequency of 1% or more within a population is specifically called a single nucleotide polymorphism (SNP). In this specification, the term "single nucleotide variant" is used regardless of the frequency of variation within a population.

[0034] In this specification, "insertion / deletion mutation (in-del mutation)" refers to a type of variation in base sequence present in the genome of a biological species population, with a mutation size of 2 bases or more and less than 50 bases. The type of mutation is not particularly limited. For example, it includes mutations caused by changes in the number of repetitions of short repeat sequences (usually 2 to 7 bases) (short tandem repeat (STR: short tandem repeat; also called microsatellite polymorphism)) and repeat sequence polymorphisms (VNTR: variable number of tandem repeat) with a repeat sequence size of less than 50 bases. The frequency of mutations within a population is irrelevant to the definition of insertion / deletion mutation in this specification.

[0035] In this specification, "structural variant" refers to a type of variation in the base sequence present in the genome of a biological species population, with a magnitude of 50 bases or more. Specific examples of such variations include inversions, translocations, deletions, copy number changes, and insertions. Structural variants also include repetitive sequence polymorphisms (VNTRs) with a repetitive sequence magnitude of 50 bases or more. In this specification, the frequency of variation within a population is irrelevant to the definition of structural variant.

[0036] (3) Secondary structure In this specification, "secondary structure" refers to the formation of base pairs and base protrusions between two nucleic acid strands. In particular, when the term "secondary structure" is used in a narrow sense, it refers to a structure that includes a characteristic shape. Specifically, examples of secondary structures include bulge structures, loop structures, and stem structures. Below, we will describe the bulge structure, which is a representative secondary structure.

[0037] In this specification, "bulge structure" refers to the three-dimensional structure of a secondary structure found in a hybridized double-stranded nucleic acid, specifically the region that forms non-complementary base pairs between the two nucleic acid strands, and the region on one or both strands where bases partially or completely protrude outside the double-stranded nucleic acid molecule. The bulge structure in this specification includes, for example, bulge loops, internal loops, and multi-branch loops. The actual three-dimensional structure of the bulge region does not need to be bulging compared to other regions.

[0038] (4) Three-dimensional structure In this specification, "three-dimensional structure" refers to any three-dimensional structure other than the double helix structure formed by perfectly complementary double-stranded nucleic acid molecules.

[0039] In this specification, "specific three-dimensional structure" refers to the desired three-dimensional structure induced by the artificial nucleic acid of the present invention.

[0040] In this specification, "functional three-dimensional structure" refers to a three-dimensional structure that has at least one function in a living organism. For example, the three-dimensional structures of ribozymes, aptamers, transfer RNA, ribosomal RNA (rRNA), and hairpin structures are functional three-dimensional structures. On the other hand, for example, a three-dimensional structure that single-stranded RNA forms incidentally and is unrelated to the function of that RNA is not a functional three-dimensional structure.

[0041] In this specification, "inducing a three-dimensional structure" refers to the formation of a three-dimensional structure by hybridizing an artificial nucleic acid with the target nucleic acid. For example, the three-dimensional structure observed in rRNA is induced.

[0042] In this specification, "sequence motif" refers to a motif containing sequence information consisting of two strands that can induce a specific three-dimensional structure. In the schematic diagram shown in Figure 27C, this corresponds to the area within the dashed box. Each of the two strands constituting the sequence motif contains a complementary region (italicized in Figure 27C), and one or both nucleic acid strands further contain a non-complementary region (bold in Figure 27C). In this specification, "consensus sequence" refers to sequence information stored in multiple sequences that form the same three-dimensional structure.

[0043] In this specification, "complementary region" refers to a region consisting of mutually complementary sequences. In the schematic diagram shown in Figure 27C, for example, the bases shown in italics correspond to the complementary regions of each nucleic acid chain.

[0044] In this specification, a "non-complementary region" refers to a region of two or more bases containing sequences that are non-complementary to each other. In the schematic diagram shown in Figure 27C, for example, the bases shown in bold correspond to the complementary regions of each nucleic acid chain. Here, the region contains non-complementary sequences at both ends.

[0045] Specific examples of three-dimensional structures include bulged G structures, kink turn structures, reverse kink turn structures, 5S loop E structures, C loop structures, and tandem GA structures. Specific examples of three-dimensional structures and arrangement motifs are described below.

[0046] (i) Bulged G structure In this specification, "bulged-G structure" refers to a three-dimensional structure in which both nucleic acid strands form a bulge structure, characterized in sequence by a triplet of bases (a pair of two bases from one strand and one base from the other) sandwiched between the bulge structure and a sheared-type GA base pair. An example of a bulged-G structure is shown in Figure 1A. The bulge structure included in a bulged-G structure typically consists of 3 to 5 bases each. The bulged-G structure is also known by many other names, such as sarcin / ricin loop structure, α-sarcin and ricin-sensitive loop structure, SRL, or S motif. The sequence motif of the bulged-G structure is not particularly limited, but an example is the motif shown in Figure 1B.

[0047] For example, in motif 1 of Figure 1B (α chain: SEQ ID NO: 1 (5'-NNNGUAN-3': N is A, C, G, or U), β chain: SEQ ID NO: 2 (5'-NGANNN-3': N is A, C, G, or U)), the bases at positions α3, α4, and β3 form a triple base pair, and in motif 2, the bases at positions α4, α5, and β4 form a triple base pair. Furthermore, in motifs 1 and 2, the bases at positions α5 and β4, and α6 and β5, respectively, form a sheared-type GA base pair. In motif 1, for example, the region within the dashed box of each nucleic acid chain can be considered the non-complementary content region of each nucleic acid chain.

[0048] Specific sequence motifs of the bulged G structure include, for example, those described in Correll, CC, et al., Nucleic Acids Research, 2003, Vol. 31, No. 23, 6806-6818; and Correll, CC, et al., Proc. Natl. Acad. Sci., 1998, Vol. 95, pp. 13436-13441. The bulged G structure is recognized by proteins such as α-sarcin and has been reported to be involved in RNA cleavage (see, for example, Gluck, A. and Wool, IG, J. Mol. Biol., 1996, 256: 838-848).

[0049] (ii) Kink turn structure In this specification, "kink turn structure" refers to a three-dimensional structure in which a double-stranded nucleic acid is folded by a bulge structure formed on one strand, and which is characterized by a sheared GA base pair following the bulge structure. An example of a kink turn structure is shown in Figure 2A. The bulge structure typically consists of three or more bases. In the strand forming the bulge structure, guanine (G) is present on the 3' side of the bulge structure, and together with adenine (A) on the other strand, the above-mentioned sheared GA base pair is formed. The sequence motif of the kink turn structure is not particularly limited, but examples include the motifs shown in Figures 2B and C.

[0050] Kink turn structures include standard kink turn structures, such as those shown in Figure 2B (α-chain: SEQ ID NO: 3 (5'-NNNNGAN-3': N is A, C, G, or U), β-chain: SEQ ID NO: 4 (5'-NGAN-3': N is A, C, G, or U)), and non-standard kink turn structures with variations in the sheared GA base pair portion, such as those shown in Figure 2C. However, the kink turn structures described herein encompass all of these. Furthermore, for example, in standard motif 1, the bases at positions α4 and β1, and α5 and β2, form sheared GA base pairs. In standard motif 1, for example, the regions within the dashed boxes of each nucleic acid chain can be considered non-complementary regions of each nucleic acid chain.

[0051] Specific sequence motifs for kink turn structures include, for example, the sequence motifs described in Huang, L. and Lilley, DM, Journal of Molecular Biology, 2016, 428(5Part A): 790-801; and Huang, L. and Lilley, DM, Quarterly Reviews of Biophysics, 2018, 51(e5). Kink turn structures are known to be recognized by proteins belonging to the L7Ae family, for example.

[0052] (iii) Reverse skin structure In this specification, "reverse kink-turn structure" refers to a three-dimensional structure in which a double-stranded nucleic acid is folded in the opposite direction to a kink-turn structure. An example of a reverse kink-turn structure is shown in Figure 3A. The most significant sequence difference from a kink-turn structure is that the bulge structure is adjacent to an AA base pair rather than a sheared GA base pair. The sequence motif of a reverse kink-turn structure is not particularly limited, but an example is the motif shown in Figure 3B.

[0053] The motif in Figure 3B differs from the standard kink turn motif 1 (Figure 2B) in that the base pair at positions α4 and β1 is an AA base pair. In the motif in Figure 3B (α chain: SEQ ID NO: 5 (5'-NNNNAAN-3': N is A, C, G, or U), β chain: SEQ ID NO: 6 (5'-NGAN-3': N is A, C, G, or U)), for example, the region within the dashed box of each nucleic acid chain can be the non-complementary region of each nucleic acid chain. In this specification, the sequence motifs for reverse kink turn structures include a wide range of sequence motifs, similar to kink turn structures, with motif 1 (Figure 3B) being the standard sequence motif. Specific examples of sequence motifs for reverse kink turn structures include, for example, the sequence motif described in Strobel, SA, et al., Rna, 2004, 10(12), 1852-1854.

[0054] (iv) 5S Loop E Structure In this specification, the "5S loop E structure" refers to a structure found in prokaryotic 5S ribosomal RNA, characterized by a partially twisted double helix structure and composed of three or more non-complementary base pairs, including non-complementary AU base pairs and sheared GA base pairs. An exemplary 5S loop E structure is shown in Figure 4A. Typically, the smallest unit of a 5S loop E structure consists of three base pairs. The 5S loop E structure is also called a loop E structure. The sequence motif of a 5S loop E structure may contain multiple sub-motifs, which in this specification are also simply referred to as the sequence motif of the 5S loop E structure. A sub-motif typically consists of three base pairs. The sequence motif of a 5S loop E structure is not particularly limited, but examples include the motifs shown in Figures 4B and 4C.

[0055] The 5S loop E structure is broadly classified into simple and complex types. The simple type is a structure consisting of three base pairs, as shown in simple motifs 1-3 in Figure 4B. The complex type includes structures containing multiple simple motifs, as shown in complex motifs 1 and 2 in Figure 4C, and structures containing extra base pairs in the middle, as shown in complex motif 3 in Figure 4C. For example, complex motif 1 contains two sub-motifs consisting of bases at positions α1-α3 and β1-β3, and positions α5-α7 and β5-β7. In simple motif 1 (α chain: SEQ ID NO: 7 (5'-NGAUN-3': N is A, C, G, or U), β chain: SEQ ID NO: 8 (5'-NGAUN-3': N is A, C, G, or U)), for example, the region within the dashed box of each nucleic acid chain can be the non-complementary containing region of each nucleic acid chain.

[0056] Specific sequence motifs of the 5S loop E structure include, for example, those described in Correll, CC, et al., Cell, 1997, 91(5), 705-712; Leontis, NB and Westof, E. Rna, 1998, 4(9), 1134-1153, etc. The 5S loop E structure contains Mg +It is known that cations such as these can bind to it, and that double-strand breaks by nucleases are likely to occur near the 5S loop E structure.

[0057] (v) C-loop structure In this specification, a "C-loop structure" refers to a three-dimensional structure in which bulge structures are formed on both nucleic acid strands and the double helix structure is partially twisted, characterized in sequence by a UA base pair following the bulge structure. An exemplary C-loop structure is shown in Figure 5A. Typically, the bulge structures of the two nucleic acid strands are of different sizes; for example, the longer bulge structure contains C. The sequence motif of the C-loop structure is not particularly limited, but an example is the motif shown in Figure 5B.

[0058] A standard C-loop structure is a structure in which a large bulge structure consists of three bases and a small bulge structure consists of one base, as shown in motif 1. However, the small bulge structure may consist of two or more bases, as shown in motif 2, and the large bulge structure may consist of four or more bases, as shown in motif 3. In motif 1 (α-chain; SEQ ID NO: 9 (5'-NCACU-3': N is A, C, G, or U), β-chain: SEQ ID NO: 10 (5'-ANN-3': N is A, C, G, or U)), for example, the region from α1 to α3 of the α-chain and the region at β1 of the β-chain may be the non-complementary content region of the respective nucleic acid chains.

[0059] Specific sequence motifs for the C-loop structure include, for example, those described in Klein, DJ, et al., Journal of molecular biology, 2004, 340(1), 141-177; and Lescoute, A., et al., Nucleic acids research, 2005, 33(8), 2395-2409. Regarding the C-loop structure, it is known that double-stranded nucleic acids possessing the C-loop structure readily form complexes.

[0060] (vi) Tandem GA structure In this specification, "Tandem GA structure" refers to a three-dimensional structure in which bulges are formed on both nucleic acid strands and is characterized in sequence by containing two consecutive sheared GA base pairs. An example of a tandem GA structure is shown in Figure 6A. A tandem GA structure is also called a GA / AG loop. The sequence motif of a tandem GA structure is not particularly limited, but for example, the motif shown in Figure 6B can be cited. In motif 1 (α-chain; SEQ ID NO: 11 (5'-NGAN-3': N is A, C, G, or U), β-chain: SEQ ID NO: 12 (5'-NGAN-3': N is A, C, G, or U)), for example, the region within the dashed box of each nucleic acid strand may be the non-complementary containing region of each nucleic acid strand.

[0061] Specific sequence motifs for tandem GA structures include, for example, those described in Jang, SB, et al., Acta Crystallographica Section D: Biological Crystallography, 2004, 60(5), 829-835.

[0062] 1-3. Structure The artificial nucleic acid of the present invention includes a structure-forming induction domain as an essential component and optionally includes a hybridize domain. The following provides a detailed explanation of each domain.

[0063] 1-3-1. Domains that induce three-dimensional structure formation A "structure-forming-inducing domain" is a domain that forms a three-dimensional structure with the target domain of the nucleic acid in question. In this specification, "target nucleic acid" refers to a nucleic acid that an artificial nucleic acid can hybridize with. Furthermore, "target domain" refers to a region containing the sequence information of one of the double-stranded sequence motifs that constitute a specific three-dimensional structure in the target nucleic acid (Figure 27B). In particular, the target nucleic acid in this specification is a nucleic acid that does not form a functional three-dimensional structure. The target nucleic acid is not particularly limited as long as it is a nucleic acid that does not form a functional three-dimensional structure, but specifically, examples include any RNA such as mRNA and microRNA (miRNA). The target nucleic acid may be a natural nucleic acid or an artificial nucleic acid. Furthermore, there may be multiple types of target nucleic acids. For example, an artificial nucleic acid may be specifically hybridizable to one type of nucleic acid as the target nucleic acid, nonspecifically hybridizable to multiple types of nucleic acids, or simultaneously hybridizable to multiple types of nucleic acids. Moreover, even if it is hybridizable to multiple types of nucleic acids, an artificial nucleic acid that induces a three-dimensional structure only when hybridized with some of those nucleic acids is also included in the artificial nucleic acids of this embodiment.

[0064] The conditions under which the three-dimensional structure is induced are not particularly limited, as long as the target nucleic acid and the artificial nucleic acid can hybridize. For example, it may be induced in vivo or in vitro. Furthermore, the three-dimensional structure can be induced in one or both nucleic acid strands.

[0065] The induced three-dimensional structure is not particularly limited. It may be all or part of a three-dimensional structure found in nucleic acids that form functional three-dimensional structures such as rRNA, or it may be a three-dimensional structure that has not been confirmed to exist in nature. Furthermore, there is no particular limit to the type or number of three-dimensional structures induced. For example, one artificial nucleic acid may induce multiple three-dimensional structures of one type, or multiple three-dimensional structures may be induced in combination. Here, one three-dimensional structure includes one complex three-dimensional structure formed by the combination of multiple three-dimensional structures. For example, an aptamer may be induced as a single three-dimensional structure, or a three-dimensional structure formed by the fusion of multiple aptamers may be induced as a single three-dimensional structure.

[0066] As described above, the target three-dimensional structure induced by the artificial nucleic acid of the present invention is referred to as a specific three-dimensional structure. Specific examples of a specific three-dimensional structure include three-dimensional structures based on double strands (three-dimensional structures other than the "three-dimensional structures based on nucleic acid folding" described below: for example, kink turn structures, bulged G structures, reverse kink turn structures, 5S loop E structures, C loop structures, tandem GA structures, tetraloop receptor structures, hook turn structures, Ω turn structures, and π turn structures, etc.), three-dimensional structures based on nucleic acid folding (three-dimensional structures consisting only of the folded portion of a single-stranded nucleic acid: for example, tetraloop structures including GNRA tetraloop, UNCG tetraloop, and CUUG tetraloop, etc.), or combinations thereof. Preferably, a specific three-dimensional structure includes a three-dimensional structure based on double strands. Preferably, specific examples of a specific three-dimensional structure include one or more selected from the group consisting of kink turn structures, bulged G structures, reverse kink turn structures, 5S loop E structures, C loop structures, and tandem GA structures. The induced three-dimensional structure may be, for example, an unnamed structure, even if its consensus sequence is unknown. Furthermore, the formed three-dimensional structure may be stable or unstable. For example, it may be a structure that is formed only under specific conditions, or the formed three-dimensional structure may change depending on the conditions.

[0067] The three-dimensional structure may be formed asymmetrically with respect to the target nucleic acid and the artificial nucleic acid, in which case a larger three-dimensional structure may be formed with either one. For example, in the case of standard motif 1 (Figure 2B) exemplified as a kink turn structure sequence motif, the α chain may be the target nucleic acid and the β chain may be the artificial nucleic acid, or vice versa.

[0068] Whether or not a target three-dimensional structure can be formed can be determined using methods known in the art. For example, if a sequence motif or consensus sequence known to form a specific three-dimensional structure is used, it can be presumed that the specific three-dimensional structure will be formed. In this case, it is not necessary to actually confirm whether or not the specific three-dimensional structure was formed. If it is necessary to confirm that a specific three-dimensional structure is actually formed by the artificial nucleic acid, this can be confirmed, for example, by in silico analysis, structural analysis, or observation of phenomena that occur specifically for the particular three-dimensional structure. For in silico analysis, for example, known three-dimensional structure prediction programs in the art such as RNAComposer, RNAMotifScan, 3dRNA, ModeRNA, MacroMoleculeBuilder, NAST, iFoldRNA, Vfold3D, SimRNA, or combinations thereof can be used. Alternatively, the three-dimensional structure can be observed by structural analysis of a complex of the nucleic acid and the artificial nucleic acid having the same base sequence as all or part of the target nucleic acid. In this case, the conditions and methods used for crystallization and structural analysis are not particularly limited. Specific methods include, for example, neutron crystallography, small-angle neutron scattering (SANS), nuclear magnetic resonance (NMR), X-ray crystallography, small-angle X-ray scattering, cryo-electron microscopy, or combinations thereof. Furthermore, the formation of a three-dimensional structure can also be confirmed by observing phenomena that occur specifically in relation to a particular three-dimensional structure. For example, in the case of a kink turn structure, it is known that proteins belonging to the L7Ae family bind to it. Therefore, by detecting the binding of this protein, the formation of a kink turn structure can be confirmed. Phenomena that may occur during the formation of each three-dimensional structure are exemplified in the section on the definition of each three-dimensional structure.

[0069] A "structure-forming-inducing domain" is a domain that forms a three-dimensional structure with the target domain of the nucleic acid of interest (Figure 27A). The structure-forming-inducing domain and the target domain constitute a sequence motif of a specific three-dimensional structure (Figure 27C). In the sequence motif, the structure-forming-inducing domain and the target domain contain complementary regions (Figure 27C italics) consisting of complementary nucleotide sequences, and furthermore, the structure-forming-inducing domain and / or the target domain contain a non-complementary-containing region containing one or more non-complementary nucleotide sequences (Figure 27C bold). Here, the non-complementary-containing region contains non-complementary sequences at both ends. In other words, the region is the area between the non-complementary nucleotide located at the 5' end and the non-complementary nucleotide located at the 3' end of the domain to which it belongs. Also, when the structure-forming-inducing domain and / or the target domain contains a non-complementary-containing region, the complementary region is adjacent to one or both of the non-complementary-containing regions in the domain containing the non-complementary-containing region. For example, the structure-forming-inducing domain can be located at the end of an artificial nucleic acid, in which case the base pairs located at the end may be non-complementary.

[0070] The number of bases constituting the complementary region is not particularly limited. Specific base counts include, for example, 1 or more bases, 2 or more bases, 3 or more bases, 4 or more bases, 5 or more bases, 10 or more bases, 20 or more bases, or 30 or more bases. Furthermore, the base sequence of the complementary region is not particularly limited.

[0071] The number of complementary regions in the structure-forming induction domain and the target domain is not particularly limited. The number of complementary regions in the structure-forming induction domain and the number of complementary regions in the target domain do not need to be the same. Typically, complementary regions consist of complementary nucleotide sequences, so the total number of nucleotides in the complementary regions will be the same in the structure-forming induction domain and the complementary regions.

[0072] Typically, when a structure-forming-inducing domain or target domain includes multiple complementary regions, the non-complementary containing region is located between those multiple complementary regions. Preferably, the structure-forming-inducing domain and / or target domain includes multiple complementary regions, and the non-complementary containing region is located between those multiple complementary regions.

[0073] The size of the non-complementary region is not particularly limited as long as it is one base or more. Specific base numbers include, for example, 1-1000 bases, 1-500 bases, 1-400 bases, 1-200 bases, 1-100 bases, 1-80 bases, 1-60 bases, 1-40 bases, 1-30 bases, 2-20 bases, 2-15 bases, 2-10 bases, 2-9 bases, 2-8 bases, or 2-7 bases. Furthermore, the base sequence of the non-complementary region is not particularly limited as long as it contains non-complementary base sequences at both ends. Also, non-complementary regions can contain complementary base sequences. For example, if the non-complementary region of one nucleic acid chain consists of one base, that non-complementary base can be considered to be the non-complementary bases at both ends of that non-complementary region. Furthermore, for example, if the non-complementary region of one nucleic acid chain consists of three bases, the two bases at both ends are non-complementary bases, but the base located between them may be either a non-complementary or complementary base.

[0074] There is no particular limit to the number of non-complementary regions included in the structure-forming induction domain and the target domain. It is sufficient that at least one of the structure-forming induction domain and the target domain contains at least one non-complementary region. For example, in the double-stranded secondary structure of an artificial nucleic acid and a target nucleic acid, if the artificial nucleic acid adopts a bulge structure and the target nucleic acid does not have a base corresponding to that bulge structure, then only the structure-forming induction domain contains one non-complementary region. Since non-complementary regions are the core regions of the three-dimensional structure, the number of non-complementary regions depends on the type and number of three-dimensional structures to be induced.

[0075] The structure-forming induction domain and the target domain constitute a sequence motif that forms a specific three-dimensional structure. The sequence motif does not necessarily constitute the entirety of the structure-forming induction domain and / or the target domain. For example, the entire sequence motif may be contained within a non-complementary content region, or only a part of the sequence motif may be contained within a non-complementary content region. Furthermore, the sequence motifs contained within the structure-forming induction domain and the target domain are not particularly limited. As sequence motifs, for example, a consensus sequence may be used, or a part or all of the base sequence of a nucleic acid that forms a functional three-dimensional structure may be used as is or with some modification.Specific arrangement motifs for each three-dimensional structure include, for example, the kink turn structure with sequence number 3 (5'-NNNNGAN-3': N is A, C, G, or U) and sequence number 4 (5'-NGAN-3': N is A, C, G, or U) (the first N in sequence number 3 and the fourth N in sequence number 4, and the seventh N in sequence number 3 and the first N in sequence number 4 are complementary to each other), and the bulged G structure with sequence number 1 (5'-NNNGUAN-3': N is A, C, G, or U) and sequence number 2 (5' -NGANNN-3':N is A, C, G or U) (the first N in SEQ ID NO: 1 and the sixth N in SEQ ID NO: 2, and the seventh N in SEQ ID NO: 1 and the first N in SEQ ID NO: 2 are complementary to each other), the reverse skinn structure, SEQ ID NO: 5 (5'-NNNNAAN-3':N is A, C, G or U) and SEQ ID NO: 6 (5'-NGAN-3':N is A, C, G or U) (the first N in SEQ ID NO: 5 and the fourth N in SEQ ID NO: 6, and the seventh N in SEQ ID NO: 5 and the first N in SEQ ID NO: 6 are complementary to each other), The 5S loop E structure is such that (each is complementary to the others) and sequence numbers 7 (5'-NGUAN-3':N is A, C, G or U) and 8 (5'-NGAUN-3':N is A, C, G or U) (the first N in sequence number 7 and the fifth N in sequence number 8, and the fifth N in sequence number 7 and the first N in sequence number 8 are complementary to each other), and the C loop structure is such that (5'-NCACU-3':N is A, C, G or U) and 10 (5'-ANN-3':N is A, C, G or The sequence is U) (the first N in sequence number 9 and the fourth N in sequence number 10, and the sixth N in sequence number 9 and the first N in sequence number 10 are complementary to each other), or the tandem GA structure consists of sequence number 11 (5'-NGAN-3':N is A, C, G or U) and sequence number 12 (5'-NGAN-3':N is A, C, G or U) (the first N in sequence number 11 and the fourth N in sequence number 12, and the fourth N in sequence number 11 and the first N in sequence number 12 are complementary to each other).

[0076] 1-3-2. Hybridized Domains The artificial nucleic acid of the present invention may include hybridize domains adjacent to one or both of the structure-forming induction domains. A hybridize domain is a domain that can hybridize with the target nucleic acid. When multiple hybridize domains exist, each domain is called a hybridize subdomain. For example, a hybridize domain consists of two subdomains adjacent to both of the structure-forming induction domains. The size of a hybridize domain may be between 6 and 120 base pairs. Specifically, for example, the base sequence may be 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, or 22 or more. Alternatively, for example, it may be 120 or less, 110 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, or 30 or less. The base sequence of the hybridize domain is not particularly limited as long as it can hybridize with the target nucleic acid. Preferably, hybridization with the target nucleic acid can be performed under highly stringent conditions.

[0077] In this specification, "hybridize" refers to the formation of a double helix of polynucleotides having completely or partially complementary base sequences. Hybridization conditions are not particularly limited, but can be various stringent conditions, such as low-stringent conditions and high-stringent conditions. "Low-stringent conditions" mean conditions under which nucleic acids readily hybridize. Low-stringent conditions refer to low-temperature and high-salt-concentration conditions during washing after hybridization. For example, washing after hybridization is performed at 42°C to 50°C using a buffer containing, for example, 5×SSC and 0.1% SDS. "High-stringent conditions" mean conditions under which nonspecific hybrids are not formed. Specifically, low salt concentrations refer to, for example, 15 to 750 mM, preferably 15 to 500 mM, 15 to 300 mM, or 15 to 200 mM. Furthermore, the high temperature referred to here specifically means, for example, 50-68°C or 55-70°C. Specific high-stringent conditions include, for example, washing at 65°C with 0.1×SSC and 0.1% SDS. Here, 1×SSC contains 150 mM sodium chloride and 15 mM sodium citrate.

[0078] Whether hybridization is possible can be determined using methods known in the art. For example, it can be determined based on base identity. Generally, a second nucleic acid having a sequence that is completely complementary to the base sequence of a first nucleic acid and a base identity of a certain level or higher is hybridizable with the first nucleic acid. Specifically, for example, hybridization is possible when the base identity is 70% or higher, 80% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%. In this specification, "base identity" means the percentage of identical bases in one polynucleotide to the total number of bases in one polynucleotide when the base sequences of two polynucleotides are aligned and, if necessary, gaps are introduced in either base sequence to maximize the degree of base agreement between the two. % identity can be easily determined using known programs such as the homology search program BLAST (Basic local alignment search tool; Altschul, SF et al, J. Mol. Biol., 215, 403-410, 1990). Furthermore, a second nucleic acid having a nucleotide sequence in which one or more bases are substituted with other bases in a sequence that is normally completely complementary to the nucleotide sequence of the first nucleic acid is hybridizable with the first nucleic acid. In this specification, "multiple" refers to two or more, specifically, for example, 2 to 30, 2 to 14, 2 to 12, 2 to 10, for example, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3.

[0079] Furthermore, the artificial nucleic acid of the present invention may include any additional sequence of any length adjacent to one or both ends of the structure-forming-inducing domain and / or hybridize domain.

[0080] 1-3-3. Modification The artificial nucleic acid of this embodiment may contain one or more modified nucleotides. The modifications introduced are not particularly limited. For example, a phosphodiester bond, a sugar moiety, and / or a base can be modified in the hybridize domain.

[0081] Furthermore, the structure formation-inducing domain may contain one or more modified nucleotides. When the modified nucleotide in the structure formation-inducing domain includes modification of the sugar moiety, it is preferable that the modification does not affect the conformation of the sugar moiety during structure formation. For example, when a modified nucleotide is introduced at the position of a nucleotide whose sugar moiety conforms to the C3'-end type during structure formation, it is preferable that the modified nucleotide can adopt the C3'-end conformation. Specifically, examples of modified nucleotides that can adopt the C3'-end conformation include 2'-OMe RNA, 2'-MOE RNA, LNA, and DNA. Also, for example, when a modified nucleotide is introduced at the position of a nucleotide whose sugar moiety conforms to the C2'-end type during structure formation, it is preferable that the modified nucleotide can adopt the C2'-end conformation. Specifically, examples of modified nucleotides that can adopt the C2'-end conformation include 2'-O,5'-N BNA, 2'-deoxy-trans-3',4'-BNA, and DNA. Furthermore, when modifications such as phosphodiester bonds or bases are made, the conformation of the sugar moiety is generally unaffected.

[0082] Furthermore, when a modified nucleotide is introduced at the position of a nucleotide in which the hydroxyl group at the 2' position of ribose is involved in hydrogen bonding during three-dimensional structure formation, it is preferable that the modified nucleotide is capable of forming hydrogen bonds or has a non-bulky substituent at the 2' position of ribose. The hydroxyl group involved in hydrogen bonding during three-dimensional structure formation may either donate or accept electrons. Specific examples of non-bulky substituents include hydrogen groups, halogen groups (e.g., fluoro groups, chloro groups, and bromo groups), methyl groups, amino groups, and cyano groups.

[0083] The conformation of the sugar moiety and the presence or absence of hydrogen bond formation during the formation of the three-dimensional structure can be determined using methods known in this art. For example, the results of crystal structure analysis of the three-dimensional structure can be downloaded from databases such as the Nucleic Acid Database and Protein Data Bank and examined visually or using software such as 3DNA. Furthermore, examples of the conformation of the sugar moiety and the presence or absence of hydrogen bond formation are provided in the section defining each three-dimensional structure.

[0084] The artificial nucleic acid of this embodiment can stably hybridize with the target nucleic acid while forming a specific three-dimensional structure. In particular, if the three-dimensional structure can be formed without relying on a portion of the base sequence of the non-complementary containing region, that portion of the base sequence may include mutations. According to the artificial nucleic acid of this embodiment, for example, it is possible to form a double helix with equivalent stability to a region of the target nucleic acid where individual differences exist due to mutations, regardless of the individual differences in the base sequence. The type of mutation is not particularly limited, but includes, for example, single nucleotide variants, insertion / deletion mutations, structural polymorphisms, or combinations thereof. Specifically, for example, in the standard type motif 1 of the kink turn structure (Figure 2B), the α chain can be the target domain, and mutations can be included at the α1, α2, α3, or one or more of these positions.

[0085] 2. Gene expression inhibitors 2-1. Overview A second aspect of the present invention is a gene expression inhibitor. The gene expression inhibitor of the present invention contains the artificial nucleic acid described in the first aspect as an active ingredient and has a gene expression inhibitory effect in the target. By using the gene expression inhibitor of the present invention, genes that have been difficult to suppress reliably until now can be inhibited with higher efficiency.

[0086] 2-2. Composition 2-2-1. Components The components of the gene expression inhibitor of the present invention will now be described. The gene expression inhibitor of the present invention includes artificial nucleic acid as an essential component. Each component will be described in detail below.

[0087] In this specification, "gene expression inhibitor" refers to a drug that has a gene expression inhibitory effect.

[0088] In this specification, "gene expression repression" means that the expression of the transcript and / or protein expression of a target gene is suppressed by artificial nucleic acid. In this specification, "transcript" refers to any RNA synthesized from a gene region in DNA by RNA polymerase. Specifically, examples include mRNA transcribed from a gene (e.g., mature mRNA, mRNA precursor, and unmodified mRNA), non-coding RNA (ncRNA) such as miRNA, long non-coding RNA (lncRNA), and natural antisense RNA. The method of expression repression is not particularly limited. For example, it can be carried out in accordance with conventional methods of expression repression, such as methods using RNA molecules or their precursors that have RNA interference (RNAi) activity, such as siRNA, shRNA, and double-stranded RNA; and methods using nucleic acid molecules that suppress translation, such as miRNA, antisense nucleic acids (e.g., antisense DNA and antisense RNA), and ribozymes. The artificial nucleic acids are as described in the first embodiment. Therefore, a detailed explanation is omitted here.

[0089] Since the artificial nucleic acids of the present invention are an improved method of suppressing gene expression using nucleic acids, the gene expression suppression effect described herein includes, for example, all effects known to be obtainable by conventional gene expression suppression methods. Specifically, these include, for example, suppression or reduction of gene expression or transcript expression levels, inhibition of translation, RNA editing, splicing function modification effects (including, for example, splicing switches, exon inclusion, exon skipping, etc.), and degradation of transcripts.

[0090] By using the artificial nucleic acid of the present invention and incorporating the mutated portion of the target nucleic acid into the bulge structure, it is possible to design an artificial nucleic acid that exhibits a similar degree of gene expression suppression regardless of the mutation. Furthermore, the artificial nucleic acid of the present invention can suppress gene expression with higher efficiency compared to conventional ASOs.

[0091] The genes whose expression is suppressed by this embodiment are not particularly limited, but examples include genes whose expression is increased in various diseases. The target diseases are not particularly limited, but examples include those involving the overexpression of normal proteins, the expression of abnormal proteins, and the overexpression of RNA that directly or indirectly regulates protein expression. Overexpression of normal proteins refers to the abnormal production of proteins that are also expressed in normal individuals, and is seen, for example, in inflammatory diseases and autoimmune diseases. The expression of abnormal proteins refers to the production of proteins that are not expressed in normal individuals, at least under certain conditions, and includes cases where the protein itself has an abnormal form, or where the protein itself is normal, but the timing of expression or the cells are different from normal. This abnormality is seen, for example, in neurodegenerative diseases. Overexpression of RNA that directly or indirectly regulates protein expression refers to cases where RNA that positively or negatively regulates protein expression (e.g., miRNA) is overexpressed, resulting in abnormal expression of the regulated protein. This abnormality is seen, for example, in cancer.

[0092] Specific diseases to which the gene expression inhibitor of this embodiment is applied include, for example, muscular dystrophy; cancer; cardiovascular disease; hypertension; infectious diseases; kidney disease; neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, and Creutzfeldt-Jakob disease; autoimmune diseases such as lupus and rheumatoid arthritis; endocarditis; Graves' disease; ALD; respiratory diseases such as asthma or cystic fibrosis; bone diseases such as osteoporosis and joint diseases; liver diseases; skin diseases such as psoriasis or eczema; eye diseases; ear, nose, and throat diseases; other neurological diseases such as Tourette syndrome, schizophrenia, depression, autism, or stroke; or metabolic diseases such as glycogen storage disorders or diabetes.

[0093] The degree of gene expression suppression by the gene expression suppressor in this embodiment is not particularly limited. Specifically, for example, when determined by the expression levels of the gene transcript and / or protein, the gene expression is suppressed by 100%, 90% or more, 75% or more, 60% or more, 50% or more, 40% or more, 30% or more, 25% or more, 20% or more, or 10% or more compared to when the gene expression suppressor is not introduced. Alternatively, it is sufficient if the gene expression is significantly suppressed compared to when the gene expression suppressor is not introduced. The degree of gene expression suppression can be determined by the expression levels of the target gene transcript or protein. For example, the expression level of the transcript can be determined by, for example, Northern hybridization or RT-PCR. The expression level of the protein can be determined by, for example, Western blotting, ELISA, protein activity measurement, or fluorescence intensity from a fluorescent protein.

[0094] 2-2-2. Pharmaceutical Compositions The gene expression inhibitor of this embodiment can be formulated into a pharmaceutical composition by including it as one of the active ingredients together with a carrier or the like.

[0095] The carriers used may include, for example, pharmaceutically acceptable carriers. "Pharmaceutically acceptable carriers" refers to additives commonly used in the pharmaceutical technology field. Examples include solvents, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavorings, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonic agents, sedatives, bulking agents, disintegrants, buffers, coatings, lubricants, colorants, sweeteners, thickeners, flavoring agents, solubilizers, and other additives.

[0096] The solvent may be, for example, water or other pharmaceutically acceptable aqueous solutions, or pharmaceutically acceptable organic solvents (such as vegetable oils). Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffers, and sodium acetate buffers. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low concentrations of nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.

[0097] The above-mentioned carrier is used to avoid or suppress the degradation of the active ingredient, artificial nucleic acid, by enzymes in the body, as well as to facilitate formulation and administration methods, and to maintain the dosage form and efficacy. It should be used as appropriate as needed.

[0098] The dosage form of the pharmaceutical composition is not particularly limited as long as it is a form that can deliver the gene expression inhibitor described in this embodiment, which is the active ingredient, to the target site without inactivating it by decomposition or the like, and that can exert the pharmacological effect (gene expression inhibitory effect) of the active ingredient in the body.

[0099] The specific dosage form will vary depending on the method of administration and / or prescription conditions. Since the methods of administration can be broadly classified into parenteral administration and oral administration, the dosage form should be appropriate for each method of administration.

[0100] The preferred mode of administration for the pharmaceutical composition is not particularly limited and may be either oral or parenteral. Specific examples of parenteral administration include intramuscular, intravenous, intra-arterial, intraperitoneal, subcutaneous (including implantable continuous subcutaneous infusion), intradermal, tracheal / bronchial, rectal, transfusion, intraventricular, intrathecal, nasal, and intramuscular administration.

[0101] If the method of administration is parenteral, the preferred dosage form is a liquid preparation that can be administered directly to the target site or systemically via the circulatory system. An example of a liquid preparation is an injectable preparation. Injectable preparations can be formulated by mixing them with the aforementioned excipients, elixirs, emulsifiers, suspensions, surfactants, stabilizers, pH adjusters, etc., in a unit dose form generally accepted for pharmaceutical production. Other possible forms include ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, eye drops, and suppositories.

[0102] The specific shapes and sizes of each of the above dosage forms are not particularly limited, as long as they fall within the range of dosage forms known in the relevant art. The pharmaceutical composition of the present invention may be manufactured according to conventional methods in the relevant art.

[0103] The amount (content) of gene expression inhibitor contained in a pharmaceutical composition varies depending on the type of artificial nucleic acid, the delivery site, the dosage form of the pharmaceutical composition, the dosage of the pharmaceutical composition, and the type of carrier. Therefore, it should be determined appropriately considering each of these conditions. Usually, the pharmaceutical composition is adjusted so that an effective amount of gene expression inhibitor is contained in a single dose. The "effective amount" refers to the amount necessary for the gene expression inhibitor to exert its function as an active ingredient, and which does not cause little to no harmful side effects to the organism to which it is applied. This effective amount can change depending on various conditions such as the subject's information, the route of administration, and the number of administrations. Ultimately, it is determined by the judgment of a physician, veterinarian, or pharmacist. "Subject's information" refers to various individual information of the organism to which the pharmaceutical composition is applied. For example, if the subject is human, it includes age, weight, sex, diet, health status, disease progression and severity, drug sensitivity, and presence or absence of concomitant drugs.

[0104] 3. Nucleic acid detection agent 3-1. Overview A third aspect of the present invention is a nucleic acid detection agent. The nucleic acid detection agent of the present invention contains the artificial nucleic acid described in the first aspect as an active ingredient and forms a specific three-dimensional structure in the presence of the target nucleic acid. By using the nucleic acid detection agent of the present invention, the target nucleic acid can be detected from a test sample.

[0105] 3-2. Composition 3-2-1. Components The components of the nucleic acid detection agent of the present invention will now be described. The nucleic acid detection agent of the present invention includes an artificial nucleic acid as an essential component and a detectable label as an optional component. Each component will be described in detail.

[0106] In this specification, "nucleic acid detection agent" refers to a drug used to detect a target nucleic acid from a test sample. According to the nucleic acid detection agent of the present invention, by including the mutated portion of the target nucleic acid in the bulge structure, the target nucleic acid can be detected to the same degree regardless of the mutation.

[0107] A "test sample" is a substance whose presence or absence or quantity of the target nucleic acid is to be tested. The test sample is not particularly limited as long as it may contain the target nucleic acid. Specific examples of test samples include, for example, blood, serum, blood cells, urine, feces, sweat, saliva, oral mucosa, sputum, lymph, cerebrospinal fluid, tears, breast milk, amniotic fluid, semen, tissue, biopsy, and cultured cells, as well as environmental substances collected from the environment, artificially synthesized nucleic acids, and mixtures thereof. Before the nucleic acid detection agent of this embodiment is applied, the test sample may undergo known pretreatments such as chopping, homogenization, and extraction. The artificial nucleic acids are as described in the first embodiment. Therefore, a detailed explanation is omitted here.

[0108] The nucleic acid detection agent of this embodiment includes optionally detectable labels. The type of detectable label is not particularly limited and may be appropriately determined depending on the detection method. Specific detectable labels include, for example, fluorescent dyes (e.g., FITC, Texas, Cy3, Cy5, Cy7, FAM, HEX, VIC, JOE, Rox, TET, Bodipy493, NBD, and TAMRA), luminescent substances (e.g., acridinium esters), uncolored small molecules that act as enzyme substrates or antigens (e.g., biotin and DIG), and radioisotopes (e.g., 32 P, 3 H and 14 C, etc. are included.

[0109] Additionally, fluorescent bases can be used as labels. The type of fluorescent base is not particularly limited, as long as it is a nucleic acid base that emits fluorescence. Specific fluorescent bases are listed, for example, in the Glen Research catalog (https: / / www.glenresearch.com / media / / folio3 / productattachments / product_catalog / Glen_Product_Catalog_2021.pdf). Examples of fluorescent bases, though not limited to those listed, include 2-aminopurine, pyrrolocytosine, 9-aminoethyl-1,3-diaza-2-oxofenoxazine, and 1,N 6 Examples include etenoadenine, 5-(1-pyrenyl-ethinyl)uracil, 1,3-diaza-2-oxofhenothiazine, and 1,3-diaza-2-oxofenoxazine. The fluorescent base may be bound to the 1' position of the sugar, for example.

[0110] The nucleotides having a fluorescent base may, for example, be nucleotides having 2-aminopurine or pyrrolocytosine.

[0111] 3-2-2. Detection By using the nucleic acid detection agent of this embodiment, the target nucleic acid can be detected from the test sample.

[0112] The detection method is not particularly limited as long as the target nucleic acid hybridized with the nucleic acid detection agent can be detected. For example, detection can be performed without a detectable label, via a detectable label, via a detectable structure recognition molecule, or a combination thereof.

[0113] The nucleic acid detection agent of this embodiment allows for the detection of target nucleic acids without the need for detectable labels or the like. The detection method in this case is not particularly limited as long as it is a method for detecting nucleic acids. Specifically, examples include methods using electrophoresis, methods using nucleases, and methods using the melting curve of double-stranded nucleic acids. The artificial nucleic acid of the present invention and the target nucleic acid hybridize with a specific three-dimensional structure. Therefore, the electrophoretic mobility of the double-stranded nucleic acids of the artificial nucleic acid and the target nucleic acid differs from that of ordinary complementary double-stranded nucleic acids with a similar number of bases. Accordingly, for example, the target nucleic acid can be detected by detecting a double-stranded nucleic acid with the same electrophoretic mobility as expected when a specific three-dimensional structure is formed. Furthermore, the double-stranded nucleic acids of the artificial nucleic acid and the target nucleic acid may contain single-stranded portions having a specific base sequence in their target domain and / or structure-forming-inducing domain. Therefore, for example, the target nucleic acid can be detected by treating the double-stranded nucleic acid obtained from the test sample with a nuclease that specifically cleaves it, and then detecting the nucleic acid having the aforementioned specific base sequence. Furthermore, the melting curves of the artificial nucleic acid of the present invention and the target nucleic acid differ from those of ordinary complementary double-stranded nucleic acids with a similar number of bases. Therefore, for example, the target nucleic acid can be detected by detecting a double-stranded nucleic acid that exhibits the same melting curve as expected when a specific three-dimensional structure is formed.

[0114] The nucleic acid detection agent of this embodiment can be detected via a detectable label. The detection method is not particularly limited, but is usually determined by the type of label used and the properties of the sample to be tested. When a fluorescent dye or luminescent substance is used, it can be detected, for example, by visual inspection, using a microscope (e.g., a fluorescence microscope), using a detector (e.g., fluorescence-activated cell sorting (FACS), a photometer, a spectrophotometer, etc.), or a combination thereof. When a non-colored low-molecular-weight substance that acts as an enzyme substrate or antigen is used as the label, it can be detected, for example, after processing such as enzyme treatment, using the same detection method as when a fluorescent dye or luminescent substance is used. When a radioisotope is used as the label, it can be detected, for example, by autoradiography, a scintillation counter, positron emission tomography (PET), or a combination thereof.

[0115] The nucleic acid detection agent of this embodiment can be detected via recognition molecules during three-dimensional structure formation. The artificial nucleic acid of the present invention hybridizes with the target nucleic acid by forming a specific three-dimensional structure. Therefore, the target nucleic acid can be detected by observing phenomena that specifically occur during the formation of a specific three-dimensional structure. For example, the target nucleic acid can be detected by detecting the binding of a specific protein (e.g., an antibody against a specific three-dimensional structure), the binding of a specific type of nucleic acid, or the cleavage of double-stranded nucleic acids at a specific position relative to the three-dimensional structure. Phenomena that may occur during the formation of each three-dimensional structure are exemplified in the section on the definition of each three-dimensional structure. The detection method used should be appropriately determined according to the phenomenon to be detected.

[0116] The nucleic acid detection agent according to this embodiment can be provided as a composition mixed with other molecules and reagents necessary for detection, or as a kit together with other molecules and reagents necessary for detection. The other molecules necessary for detection may include some or all of the molecules necessary for detection, depending on the detection method described above.

[0117] The nucleic acid detection agent of the present invention can also be used in Southern blotting, Northern blotting, and in situ hybridization, etc.

[0118] 4. Method for producing artificial nucleic acids 4-1. Overview A fourth aspect of the present invention is a method for producing artificial nucleic acids. The method for producing artificial nucleic acids according to this aspect includes a target domain selection step, a structure formation induction domain determination step, and a nucleic acid synthesis step as essential steps, and includes a hybridize domain determination step as an optional step. According to this method, the artificial nucleic acid described in the first aspect can be synthesized.

[0119] 4-2. Method The method of this embodiment includes a target domain selection step, a structure formation induction domain determination step, and a nucleic acid synthesis step as essential steps, and includes a hybridize domain determination step as an optional step. Each step will be described in detail below.

[0120] 4-2-1. Target Domain Selection Process The "target domain selection process" is a process of searching for the sequence information of one of the double-stranded sequence motifs that constitute a specific three-dimensional structure in the target nucleic acid, and selecting one or more of them as target domains.

[0121] In the target domain selection process, the base sequence of the target nucleic acid and the sequence motif of a specific three-dimensional structure are compared to select a target domain that can form the specific three-dimensional structure.

[0122] The target nucleic acid and specific three-dimensional structure can be appropriately selected in accordance with the description in the first embodiment.

[0123] As described in the first embodiment, in the sequence motif, the target domain and the structure-forming induction domain include complementary regions consisting of complementary sequences, and further, the target domain and / or the structure-forming induction domain include a non-complementary region containing one or more non-complementary sequences, the non-complementary region containing non-complementary sequences at both ends thereof. The complementary region may be adjacent to one or both of the non-complementary regions if they exist, but the selection criteria are not particularly limited. For example, they may be determined randomly or based on past reports.

[0124] In this process, a region in the target nucleic acid having a base sequence that matches part or all of the sequence information of one of the sequence motifs of a specific three-dimensional structure is searched. Here, "sequence information of one of the sequence motifs" refers to using the sequence information of a double-stranded sequence motif separated into single strands for the search. For example, the search may be performed using only the sequence information of one strand of the sequence motif, or the sequence information of both strands may be used separately for the search. The method of searching for the base sequence used in this case is not particularly limited. For example, a commonly used identity search program such as the identity search program BLAST (Basic local alignment search tool; Altschul, SF et al, J. Mol. Biol., 215, 403-410, 1990) can be used for the search. In this case, there may be one specific three-dimensional structure to be searched for or multiple specific structures.

[0125] The sequence motifs used in this process are not particularly limited. For example, a consensus sequence may be used as a sequence motif, or a sequence whose entire base sequence has been determined may be used. In particular, when using a consensus sequence, regions in the target nucleic acid that match the conserved bases and their arrangement are searched for. When using a sequence whose entire base sequence has been determined, not only regions that are completely identical to that sequence, but also regions containing sequences that have a certain level of identity with that sequence can be selected as candidate target domains. For example, a sequence with 1, 2, 3, 4, 5, or 6 base substitutions can be considered a candidate target domain. Alternatively, for example, a sequence can be considered a candidate target domain (hereinafter referred to as a candidate target domain) if its identity is 80% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.5% or higher, 99.9% or higher, or 100%. Furthermore, the number of sequence motifs used in the search is not limited. For example, a search can be performed on multiple types of sequence motifs at once.

[0126] If multiple target domain candidates are obtained, one or more of them can be selected as the target domain. The selection criteria are not particularly limited. For example, selection can be made randomly, based on the gene region containing the target domain candidate, based on the three-dimensional structure that can be induced by each target domain candidate, based on sequence identity with the sequence motif, based on information regarding the ease of forming the nucleotide sequence and three-dimensional structure, or a combination thereof. For example, the sequence of the selected target domain candidate may be a sequence that has already been shown to form a three-dimensional structure. Also, although not limited, the target domain candidate is preferably located in a region where the desired effect is expected to be obtained by hybridizing the artificial nucleic acid. Furthermore, if the target nucleic acid contains the mutation described in the first embodiment, a target domain candidate corresponding to a nucleotide position in the sequence motif where the location of the mutation is not uniquely determined can be selected preferentially. Also, for example, if multiple target domain candidates exist consecutively with some overlap, these consecutive small target domain candidates may be combined into one large target domain.

[0127] The order in which sequence motif searching and target domain selection are performed is not particularly limited, but preferably, target domain selection is performed simultaneously with or after sequence motif searching.

[0128] 4-2-2. Determination process for domains that induce three-dimensional structure formation The "three-dimensional structure formation induction domain determination step" is a step in which the sequence of three-dimensional structure formation induction domains is determined so as to constitute the sequence motif with the target domain. This step can be performed simultaneously with or after the target domain selection step described above.

[0129] The sequence of the structure-forming induction domain is determined according to the sequence information of the strand corresponding to the artificial nucleic acid in the sequence motif containing the sequence information of the target domain.

[0130] If the base sequence of a sequence motif is not uniquely determined, those bases can be determined arbitrarily. Furthermore, if, for example, an unspecified sequence is uniquely determined by the bases at corresponding positions on the other strand, that determination can be followed. There are no particular limitations on the criteria for base selection. For example, if RNA forming a specific three-dimensional structure frequently has a particular base at that position, that base may be selected.

[0131] In this process, it is possible to decide to make some or all of the nucleotides in the structure-forming induction domain modified nucleotides. The introduction of modified nucleotides in the structure-forming induction domain follows the method described in the first embodiment.

[0132] If this process is performed simultaneously with the target domain selection process, for example, the sequence of structure-forming-inducing domains can be determined for all target domain candidates, and then a target domain can be selected from the target domain candidates.

[0133] The order in which the sequence determination and the determination of modified nucleotide introduction are carried out is not particularly limited, but preferably, the determination of modified nucleotide introduction is carried out at the same time as or after the sequence determination.

[0134] 4-2-3. Hybridization Domain Determination Process The "hybridize domain determination step" is a step in which the base sequence of a hybridize domain consisting of 6 to 120 bases adjacent to one or both of the structure formation induction domains is determined. This step is optional and can be performed simultaneously with or after the structure formation induction domain determination step described above.

[0135] This step involves determining the base sequence of the hybridization domain in the artificial nucleic acid. The hybridization domain can be adjacent to one or both of the structure-forming induction domains, and either can be arbitrarily selected. For example, if the hybridization domain is adjacent to both of a single structure-forming induction domain, the hybridization domain will contain two subdomains. The base sequence of the hybridization domain can be determined according to the base sequence of a region of the target nucleic acid other than the target domain. The method of determining the base sequence is not particularly limited. Furthermore, the degree of base identity of the base sequence is not particularly limited as long as hybridization is possible.

[0136] Furthermore, the method of this embodiment may include steps to determine any additional sequences to be included in modifications or artificial nucleic acids. The timing of these steps is not particularly limited. Details regarding these steps are as described in the first embodiment, and the method of determination is not particularly limited.

[0137] 4-2-4. Nucleic acid synthesis process The "nucleic acid synthesis step" is a step in which artificial nucleic acids are synthesized based on the sequence information determined in the three-dimensional structure formation induction domain determination step. This step can be performed simultaneously with or after the three-dimensional structure formation induction domain determination step described above. Furthermore, if the hybridize domain determination step is performed, it can be performed simultaneously with or after the aforementioned step.

[0138] The artificial nucleic acids of the present invention can be manufactured by those skilled in the art by appropriately selecting known methods. Based on the nucleotide sequence information of the artificial nucleic acid determined in the structure formation induction domain determination step, the artificial nucleic acid can be synthesized using, for example, a commercially available automated nucleic acid synthesizer from GE Healthcare, Thermo Fisher Scientific, Beckman Coulter, etc. When the hybridize domain determination step is performed, the artificial nucleic acid can be synthesized based on the structure formation induction domain determination step and the nucleotide sequence information of the artificial nucleic acid determined in the structure formation induction domain determination step. After synthesis, post-processing may be performed, for example, by purifying the obtained artificial nucleic acid using a reversed-phase column. [Examples]

[0139] <Example 1: Evaluation of the stability of RNA molecules and nucleic acid molecules that hybridize to them and induce a three-dimensional structure> (the purpose) We will confirm whether a three-dimensional structure can be actively formed between two nucleic acid strands and investigate the stability of the double-stranded nucleic acid whose three-dimensional structure has been induced.

[0140] (method) 1. Design and Synthesis of Nucleic Acid Molecules An RNA molecule with the base sequence shown in SEQ ID NO: 13 was designed as the target nucleic acid (ROI: upper part of Figures 7A-G). As artificial nucleic acids, based on the consensus sequence of three-dimensional structure, RNA molecules that induce a bulged G structure (RNA-BG; SEQ ID NO: 14: lower part of Figure 7B), RNA molecules that induce a kink turn structure (RNA-KT; SEQ ID NO: 15: lower part of Figure 7C), RNA molecules that induce a reverse kink turn structure (RNA-RKT; SEQ ID NO: 16: lower part of Figure 7D), RNA molecules that induce a 5S loop E structure (RNA-5S; SEQ ID NO: 17: lower part of Figure 7E), RNA molecules that induce a C loop structure (RNA-CL; SEQ ID NO: 18: lower part of Figure 7F), and RNA molecules that induce a tandem GA structure (RNA-GA; SEQ ID NO: 19: lower part of Figure 7G) were designed. In addition, as a control, an RNA molecule with a sequence complementary to the target nucleic acid (RNA-ASO; SEQ ID NO: 20: lower part of Figure 7A) was designed.

[0141] For the synthesis of RNA molecules, the nucleic acid automatic synthesizer NTS-M2-MX (Nihon Techno Service Co, Ltd) was used. The synthesized samples were purified by gel filtration using NAP-10 columns (Cytiva). After purification, 20% denaturing polyacrylamide gel electrophoresis containing 7 M urea was performed to confirm that the samples were correctly synthesized with high purity.

[0142] 2. Measurement of the thermal stability of double-stranded RNA For temperature control, a spectrophotometer V-630 (Jasco) connected to a temperature controller PAC-743R (Jasco) was used. The thermal melting of double-stranded nucleic acids was measured by measuring the absorbance of ultraviolet light at a wavelength of 260.0 nm in the temperature range of 20 to 100 °C (measurement interval 1 °C, heating rate 1 °C / min). As samples, test solutions were prepared by adding ROI and artificial nucleic acids or RNA-ASO to a solution containing 10 mM sodium cacodylate (pH 7.0) and 100 mM sodium chloride so that the final concentration was 1 μM each.

[0143] To create a thermal melting curve, the relative absorbance (A) was calculated from the measured absorbance using the following formula. A=(A t -A min ) / (A max -A[[ID=,22]] min ) (A t : Absorbance at t °C, A min : Minimum value of absorbance, A max : Maximum value of absorbance)

[0144] The inflection point of the thermal melting curve created as a sigmoid curve was approximately calculated as the point where the straight line approximated by the least squares method in the linear region of the temperature change data intersects that linear region, and that temperature was taken as the double-stranded melting temperature (T m ).

[0145] (Results) The results are shown in Figure 8 and Table 1.

[0146] [Table 1]

[0147] In all cases where nucleic acids were used, the melting curve was sigmoid-shaped, indicating that stable double strands could be formed with the target nucleic acid regardless of which nucleic acid was used (Figure 8). Compared with the thermal melting curves of the target nucleic acid and the control double strand nucleic acid (Figure 8A), when six types of artificial nucleic acids were used (Figures 8B-G), the thermal melting curves of the double strand nucleic acid with the target nucleic acid shifted to the lower temperature side. m The values ​​were also low (Table 1). However, it was found that the double strands were sufficiently stable at temperatures close to body temperature (below 40°C). Among the six types of artificial nucleic acids, RNA-GA was the most stable (Figure 8G), and RNA-BG was the least stable (Figure 8B).

[0148] <Example 2: Evaluation of the effect of modification on the stability of double-stranded nucleic acids> (the purpose) This study investigates the stability of double-stranded nucleic acids when modified nucleotides are used.

[0149] (method) The experiment was conducted according to the method of Example 1. The nucleic acids used were as follows: As the target nucleic acid, an RNA molecule (ROI) having the nucleotide sequence shown in SEQ ID NO: 13 was used, as in Example 1. As artificial nucleic acids, three types of nucleic acid molecules were used that had the same nucleotide sequence as in Example 1, but in which all nucleotides were modified with 2'-O-methyl (2'-OMe). These were a nucleic acid molecule that induces a bulged G structure (OMe-BG; SEQ ID NO: 21), a nucleic acid molecule that induces a 5S loop E structure (OMe-5S; SEQ ID NO: 22), and a nucleic acid molecule that induces a tandem GA structure (OMe-GA; SEQ ID NO: 23). In addition, as a control, a nucleic acid molecule (OMe-ASO; SEQ ID NO: 24) that had the same nucleotide sequence as in Example 1, but in which all nucleotides were modified with 2'-OMe was used.

[0150] (result) The results are shown in Figure 9 and Table 2.

[0151] [Table 2]

[0152] Unlike the control (Figure 9A), in all cases where artificial nucleic acids were used, the melting curve shifted to the high-temperature side compared to Example 1 which used unmodified RNA (Figures 9B-D), m The value also increased (ΔT in Table 2) m (modi) (of the column). In particular, the T of OMe-GA m The values ​​were higher compared to the control, indicating that structures induced were sometimes more stable than those that were perfectly complementary to the target nucleic acid. The improvement in stability due to modification of artificial nucleic acids was particularly pronounced in the relatively low-temperature range, and all artificial nucleic acids were more stable than ROI / OMe-ASO in the temperature range of approximately 50°C or below (Figure 9). Among the three types of artificial nucleic acids, RNA-GA was the most stable after modification (Figure 9D), and RNA-BG was the least stable (Figure 9B). This indicates that the order of stability of artificial nucleic acids does not change significantly before and after modification when similar modifications are performed.

[0153] From these results, it was confirmed that artificial nucleic acids can form stable double helix structures with the target nucleic acid, even though they possess multiple bases that are not complementary to the target nucleic acid. Furthermore, it was found that nucleotide modification with 2'-OMe, etc., is an effective means of improving the stability of the double helix structure.

[0154] <Example 3: Evaluation of the effect of nucleotide modification on three-dimensional structure (bulged G structure)> (the purpose) We will investigate the effects of nucleotide modifications on bulge G structure.

[0155] (method) 1. Design and Synthesis of Nucleic Acid Molecules We used an RNA molecule that forms a bulge G structure (unmodified BG; Figure 10A; SEQ ID NO: 25: PDB-ID=1Q9A) and an RNA molecule in which the nucleotides in one of the nucleic acid strands of the double-stranded region were modified as follows (modified BG; Figure 10D; SEQ ID NO: 26). • 2'-OMe modification was introduced to all nucleotides in which the sugar moiety conformation is C3'-endo type and the hydroxyl group at the 2' position is not involved in hydrogen bonding (Figure 10D, lowercase). • All nucleotides with a C2'-end conformation in the sugar portion were replaced with DNA (Figure 10D, lowercase italics).

[0156] Each nucleic acid molecule was chemically synthesized using an automated nucleic acid synthesizer. The molecules were then purified by 20% denatured polyacrylamide gel electrophoresis (30 cm × 40 cm) containing 7 M urea, and further desalted by gel filtration using NAP-10 columns.

[0157] 2.X-ray crystal analysis Crystallization was performed using the hanging drop vapor diffusion method. Drops were prepared by mixing 0.2 μL each of 1 mM or 2 mM crystallization oligonucleotide and crystallization buffer, and then equilibrated in the reservoir solution. Crystallization was performed for each nucleic acid under two different crystallization conditions. Details of the crystallization conditions are shown in Table 3.

[0158] [Table 3]

[0159] Single crystals were scooped up using a CryoLoop (Hampton Research) and immersed in a 40% 2-methyl-2,4-pentadiol (MPD) solution for several seconds. These crystals were then rapidly frozen in liquid nitrogen and used for X-ray diffraction experiments.

[0160] X-ray diffraction experiments were performed using Photon Factory's structural biology beamlines BL-17A and ARNE-3A. The XDS program was used to process the diffraction data. Initial phase determination was performed using the molecular substitution method with the Phaser program (Phenix). Atomic parameter refinement was performed using the phenix.refine program (Phenix).

[0161] (result) The results are shown in Tables 4 and 5, and Figures 10-12.

[0162] [Table 4]

[0163] [Table 5]

[0164] Crystals were obtained for both unmodified and modified BG under two different crystallization conditions (Table 3: referred to as Crystal I and Crystal II, respectively), and structural analysis was successful for all of them (Tables 4 and 5).

[0165] Two three-dimensional structures were obtained from two types of crystals. Crystal I and Crystal II showed no difference in three-dimensional structure, indicating that they form a constant three-dimensional structure regardless of the crystallization conditions (Figure 10). In particular, the three-dimensional structure of unmodified BG was in complete agreement with that revealed in a previous study (Correll, CC, et al., Nucleic Acids Research, 2003, 31(23), 6806-6818) (Figures 10B and C). Furthermore, it was found that a bulged G structure was formed in modified BG regardless of the crystallization conditions (Figures 10E and F). When the nucleotide interactions were observed for each base pair, it was found that the nucleotide interactions of unmodified BG and modified BG were exactly the same (Figures 11 and 12).

[0166] This indicates that the modification method used in this example does not affect the three-dimensional structure. In other words, if the conformation of the sugar portion does not change before and after modification, there is basically no effect on the three-dimensional structure. Furthermore, it was found that the 2' position of the C3'-endo type ribose can be modified without issue if its hydroxyl group is not involved in hydrogen bonding.

[0167] <Example 4: Evaluation of the effect of nucleotide modification on three-dimensional structure (kink turn structure)> (the purpose) We will investigate the effect of nucleotide modifications on kink turn structure.

[0168] (method) 1. Design and Synthesis of Nucleic Acid Molecules We used two RNA molecules that hybridize to form two kink turn structures (unmodified KT; Figure 13A; Sequence ID 27: PDB-ID=4CD1) and two RNA molecules in which the nucleotides in one of the nucleic acid strands of each double-stranded region forming the kink turn structure were modified as follows (modified KT; Figure 13C; Sequence ID 28). • In all nucleotides where the sugar moiety conformation is C3'-endo type and the hydroxyl group at the 2' position is not involved in hydrogen bonding, a 2'-OMe modification was introduced (Figure 13C lowercase). • In a single nucleotide where the sugar moiety conforms to the C3'-end type and the hydroxyl group at the 2' position is involved in hydrogen bonding, a bulky OMe modification was introduced at the 2' position (Figure 13C lowercase). • All nucleotides with a C2'-end conformation in the sugar portion were replaced with DNA (Figure 13C, lowercase italics). The synthesis and purification of nucleic acid molecules were carried out in accordance with Example 3.

[0169] 2.X-ray crystal analysis Crystallization and structural analysis were performed for each nucleic acid under one crystallization condition, in accordance with Example 3. Details of the crystallization conditions are shown in Table 6.

[0170] [Table 6]

[0171] (result) The results are shown in Table 7 and Figures 13-16.

[0172] [Table 7]

[0173] Crystals were obtained for both unmodified and modified KT, and structural analysis was successful for both (Table 7).

[0174] The three-dimensional structure of unmodified KT was in complete agreement with that revealed in a previous study (McPhee, SA, et al., Nature communications, 2014, 5(1), 1-6), and two kink turn structures were formed (Figures 13A and B). Since the crystallization conditions used in this example differed from those in the previous study, it was found that, like the bulged G structure, the kink turn structure is a stable three-dimensional structure regardless of the crystallization conditions. Furthermore, no significant differences in three-dimensional structure were observed between modified KT and unmodified KT (Figures 13 and 14). In addition, the interactions between nucleotides were observed for each base pair in one of the two formed kink turn structures (Figures 15 and 16). As a result, the hydrogen bonds between bases were basically maintained (Figure 15). When the hydroxyl group at the 2' position of ribose was involved in the hydrogen bond, it was found that some hydrogen bonds were cleaved by substitution in DNA nucleotides or 2'-OMe modification (circles in Figures 15C and D, and Figures 16C and D). In particular, it was found that bulky 2'-OMe modifications can sometimes partially break hydrogen bonds between bases (Figures 16C and D).

[0175] From this, it was found that the modification method used in this example does not have a significant effect on the stereostructure, but it can affect stability. In other words, from the standpoint of stability, it was found that for ribose having a C3'-end conformation, it is preferable not to modify the 2' position if its hydroxyl group is involved in hydrogen bonding, or to modify it with a substituent that is not bulky and does not cleave hydrogen bonds between bases.

[0176] <Example 5: Evaluation of the effect of nucleotide modification on three-dimensional structure (tetraloop receptor structure)> (the purpose) We will investigate the effects of nucleotide modifications on the structure of tetraloop receptors.

[0177] (method) 1. Design and Synthesis of Nucleic Acid Molecules In an RNA molecule that forms a tetraloop receptor structure (unmodified TLR; Figure 17A; SEQ ID NO: 29: PDB-ID=4FNJ), a nucleic acid molecule (modified TLR; Figure 17C; SEQ ID NO: 30) was used in which a nucleotide in one of the nucleic acid strands of the double-stranded region was replaced with DNA (Figure 17D, italicized lowercase). The synthesis and purification of nucleic acid molecules were carried out in accordance with Example 3.

[0178] 2.X-ray crystal analysis Crystallization and structural analysis were carried out in accordance with Example 3. Crystallization was performed using two different crystallization conditions. Details of the crystallization conditions are shown in Table 8.

[0179] [Table 8]

[0180] (result) The results are shown in Table 9 and Figures 17-20.

[0181] [Table 9]

[0182] Crystals were obtained from the modified TLR under two different crystallization conditions (referred to as Crystal I and Crystal II, respectively), and structural analysis was successful for both (Table 9 and the three-dimensional structure of Crystal I are shown in Figure 17D).

[0183] The secondary and stereochemical structures of the unmodified TLR are shown in Figures 17A and 17B, respectively (Coonrod, LA, et al., Biochemistry, 2012, 51(42), 8330-8337). In comparison, no significant difference was observed in the stereochemical structure of the modified TLR (Figure 17D). Furthermore, since there was no difference in the stereochemical structure between crystal I and crystal II, it was found that the tetraloop receptor structure, like the bulged G structure and kink turn structure, is a stable stereochemical structure regardless of the crystallization conditions. In addition, when the interaction between the tetraloop structure (within the dashed box in Figure 18) and the tetraloop receptor structure (space-filling model in Figure 18) between two molecules was observed, no significant difference in interaction was found between the modified TLR and the unmodified TLR (Figure 18).

[0184] When the conformation of each nucleotide was observed, the conformation of the sugar moiety of each nucleotide was maintained between modified and unmodified TLRs. This is thought to be because both DNA and RNA can adopt both C3'-end and C2'-end conformations. However, even when RNA that relatively easily adopts the C3'-end conformation was replaced with DNA that relatively easily adopts the C2'-end conformation, the conformation remained constrained, demonstrating the importance of maintaining the conformation of the sugar moiety before and after modification.

[0185] Furthermore, we observed the nucleotide-nucleotide interactions observed in the interaction between the tetraloop (TL) structure and the tetraloop acceptor (TLR) structure (Figures 19 and 20). We found that some hydrogen bonds were cleaved when the hydroxyl group at the 2' position of ribose, which was involved in hydrogen bonding, was substituted with hydrogen (circles in Figures 19E and F and Figure 20). However, there was no effect on the hydrogen bonds between bases, and the interaction between the tetraloop structure and the tetraloop acceptor structure was maintained. This indicates that even when the hydroxyl group at the 2' position of ribose is involved in hydrogen bonding, substitution with a substituent that is not bulky, such as hydrogen, does not significantly affect the structure or function.

[0186] <Example 6: Evaluation of the stability of RNA molecules containing mutations and nucleic acid molecules that hybridize to them and induce a three-dimensional structure> (the purpose) This study investigates the stability of double-stranded nucleic acids whose three-dimensional structure is induced by hybridization when an RNA molecule contains mutations.

[0187] (method) 1. Design and Synthesis of Nucleic Acid Molecules The experiment was conducted according to the method of Example 1. The nucleic acids used were as follows: As target nucleic acids containing mutations, we designed an RNA molecule with a UUU base sequence near the center (ROI-U; upper part of Figures 21A and C; SEQ ID NO: 31) and an RNA molecule with a mutated AAA base sequence near the center (ROI-A; upper part of Figures 21B and D; SEQ ID NO: 32). As an artificial nucleic acid, we designed an RNA molecule (KT-SKIP; lower part of Figures 21C and D; SEQ ID NO: 34) that induces a kink turn structure so that the above-mentioned mutated portion is included in the bulge structure, based on the consensus sequence of the three-dimensional structure. In addition, as a control, we designed an RNA molecule (ASO-A; lower part of Figures 21A and B; SEQ ID NO: 33) that has a sequence complementary to ROI-U.

[0188] (result) The results are shown in Figure 22. In all cases where nucleic acids were used, the melting curve was sigmoid-shaped, indicating that double-stranded nucleic acids could be formed with the target nucleic acid regardless of which nucleic acid was used (Figure 22). When the control ASO-A was used, the thermal melting curve of the double-stranded nucleic acid with ROI-A shifted significantly to the low-temperature side compared to the double-stranded nucleic acid with ROI-U (Figure 22A). m The values ​​also showed that the thawing temperature was 86°C for double-stranded nucleic acids with ROI-U, while it was 78°C for double-stranded nucleic acids with ROI-A, indicating that the mutation significantly affects the stability of double-stranded nucleic acids. When using KT-SKIP, in which the three-dimensional structure was induced to include the mutated portion in the bulge structure, the thawing curves for double-stranded nucleic acids with ROI-U and those with ROI-A were not significantly different (Figure 22B), and both were T m The value was 75°C.

[0189] This indicates that by inducing a three-dimensional structure and incorporating the mutated region into the bulge structure, it is possible to design artificial nucleic acids that hybridize to the same extent with the target nucleic acid regardless of the mutation.

[0190] <Example 7: Suppression of protein expression using nucleic acid molecules that hybridize and induce three-dimensional structure> (the purpose) We will confirm that protein expression can be suppressed using nucleic acid molecules that hybridize and induce a three-dimensional structure, and compare this suppression effect with that of antisense nucleic acids.

[0191] (method) As the target nucleic acid, we used mRNA, which is the transcript of pUC-frGFP DNA (SEQ ID NO: 35) included with the protein synthesis kit "Cellless-kun N Mini" (Taiyo Nippon Sanso). A specific region (positions 277-301 of SEQ ID NO: 35; upper part of Figures 23A-C and upper part of Figures 28A-C; SEQ ID NO: 36) was used as the target region for hybridization. As artificial nucleic acids, we designed RNA molecules that induce a tandem GA structure by hybridizing with the target region based on the consensus sequence of three-dimensional structure (GA-GFP; lower part of Figures 23B and 28B; SEQ ID NO: 38) and RNA molecules that induce a kink turn structure (KT-GFP; lower part of Figures 23C and 28C; SEQ ID NO: 39). Furthermore, as control RNA molecules, we designed an RNA molecule with the same sequence as the target region (SO-GFP; upper part of Figures 23A-C and upper part of Figures 28A-C; SEQ ID NO: 36) and an RNA molecule with a sequence complementary to the target region (ASO-GFP; lower part of Figures 23A and lower part of Figure 28A; SEQ ID NO: 37). Nucleic acid synthesis was carried out in accordance with Example 1.

[0192] Protein synthesis was performed either without the addition of the designed nucleic acid molecule or with the addition of the designed nucleic acid molecule. Protein synthesis was carried out using the protein synthesis kit "Cellless-Kun N Mini" (Taiyo Nippon Sanso) according to the manufacturer's recommended protocol. The outline is as follows. A 50 μl reaction solution was prepared by mixing the "Cellless-Kun N Mini" reaction solution premix, pUC-frGFP DNA (final concentration 0.5 nM), and designed RNA molecules (SO-GFP, ASO-GFP, GA-GFP, KT-GFP: each at a final concentration of 20 μM). A reaction solution without RNA was also prepared as a negative control. These reaction solutions were incubated at 30°C for 90 minutes in a PCR Thermal Cycler Dice Touch (Takara) to perform protein synthesis, and then incubated at 4°C for 5 minutes to stop the reaction. The reaction solutions were used in the following experiments.

[0193] To investigate the protein synthesis inhibitory effect, fluorescence spectroscopy was performed. Fluorescence spectra were measured using an FP-8300 (Jasco) with an excitation wavelength of 480 nm, capturing fluorescence spectra in the 500-600 nm range. The above experiment was conducted at room temperature.

[0194] (result) The results are shown in Figure 24. In the reaction mixture without the designed RNA molecule, a clear peak derived from frGFP was observed at 510 nm (Figure 24A). When SO-GFP was added, a similar peak was observed at the same wavelength (Figure 24B). This confirmed that the addition of the RNA molecule itself did not affect protein synthesis or fluorescence detection. When ASO-GFP was added, the peak height decreased, indicating that protein translation was inhibited (Figure 24C). When GA-GFP and KT-GFP were used, almost no peak was observed at 510 nm in either case (Figures 24D and E).

[0195] This indicates that using nucleic acids that induce three-dimensional structure can inhibit protein synthesis with higher efficiency compared to using conventional ASOs.

[0196] <Example 8: Detection of target RNA using a fluorescently labeled RNA probe containing 2-aminopurine (a fluorescently labeled nucleic acid probe with RNA as its main constituent nucleotide)> We used target RNAs with sequence numbers 40-43 as target RNAs and non-target RNAs (Table 10), and investigated the detection of target RNA using a fluorescently labeled RNA probe containing 2-aminopurine.

[0197] [Table 10]

[0198] As shown in Figure 25, the base sequence of the fluorescently labeled RNA probe was designed to hybridize with the target RNA (SEQ ID NO: 40) to form a kink turn structure, and to include a 2'-deoxyribonucleotide containing the fluorescent base 2-aminopurine (2AP) at position L3 in the kink turn motif (X in the sequence in Figure 25) (Figure 25). All nucleotides in the fluorescently labeled RNA probe except for the 2'-deoxyribonucleotide containing the above-mentioned fluorescent base were RNA (hereinafter, the fluorescently labeled RNA probe containing 2AP designed in this way will be referred to as RNA-2AP). The base sequence of the designed RNA-2AP is shown below. 5'-ACA UGA UGA UGA AGA GGA XGA GAG GAG AGU-3' (Sequence No. 44) (where X = 2AP)

[0199] The above-mentioned fluorescently labeled RNA probe (RNA-2AP), target RNA, and three types of non-target RNAs (non-target RNA1-3) were chemically synthesized using the phosphoramidite method with the NTS-M2-MX automated nucleic acid synthesizer (Nippon Techno Service Co., Ltd.). 2-Aminopurine-CE Phosphoramidite (Glen Research) was used as the nucleotide phosphoramidite containing 2-aminopurine. The samples obtained by the above chemical synthesis were purified by gel filtration using NAP-10 columns (Cytiva), and the high purity and correct synthesis of the target RNA were confirmed by electrophoresis using a 20% denatured polyacrylamide gel containing 7 M urea.

[0200] The ability of the synthesized fluorescently labeled RNA probe (RNA-2AP) to specifically detect the target RNA was investigated by fluorescence spectroscopy, as described below.

[0201] A solution (test solution) was prepared by adding RNA-2AP and 0.01 mM of target RNA or non-target RNA to a solution containing 10 mM sodium cacodylate (pH 7) and 100 mM sodium chloride so that the final concentration of each was 0.01 mM. Also, as a control, a solution (control solution) was prepared by adding only 0.01 mM of RNA-2AP to a solution containing 10 mM sodium cacodylate (pH 7) and 100 mM sodium chloride.

[0202] The 3D fluorescence spectrum of the control solution was measured using FP-8300 (Jasco), and the maximum excitation wavelength and the maximum fluorescence wavelength were determined. Based on the results, the measurement conditions (the range of the excitation wavelength and the fluorescence wavelength to be detected) used for subsequent fluorescence spectrum measurements were determined.

[0203] The fluorescence spectrum of the test solution was measured using FP-8300 (Jasco) under the measurement conditions determined as described above. Specifically, the fluorescence spectrum in the range of 330 to 450 nm was measured using an excitation wavelength of 305 nm.

[0204] Furthermore, the change rate ΔF of the fluorescence intensity when target RNA or non-target RNA was added to RNA-2AP was calculated by the following formula, and the target RNA detection ability of RNA-2AP was evaluated. ΔF = (F - F0) / F (F: Fluorescence intensity at a specific wavelength of the test solution, F0: Fluorescence intensity at the same wavelength of the control solution) The above experiment was conducted at room temperature. The results are shown in Figure 26.

[0205] The fluorescence intensity of RNA-2AP showed almost no change when any of the three non-target RNAs were added, but the fluorescence intensity increased significantly when the target RNA was added (Figure 26A). When the percentage change in fluorescence intensity at a wavelength of 370 nm was calculated when target RNA or non-target RNA was added to RNA-2AP, the percentage change in fluorescence intensity when target RNA was added was ΔF = 0.59, which was significantly higher than the percentage change in fluorescence intensity when non-target RNA was added (ΔF = -0.05 to 0.07) (Figure 26B). These results demonstrate that RNA-2AP can detect target RNA in a sequence-specific manner. All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. An artificial nucleic acid that hybridizes with a target nucleic acid that does not form a functional three-dimensional structure to induce a specific three-dimensional structure, The artificial nucleic acid includes a structure-forming inducing domain that forms a three-dimensional structure with the target domain of the target nucleic acid. The target domain and the structure-forming induction domain constitute a sequence motif consisting of two strands that form a specific three-dimensional structure. In the aforementioned sequence motif, the three-dimensional structure formation inducing domain and the target domain include complementary regions consisting of complementary sequences. Furthermore, in the sequence motif, the structure-forming inducing domain and / or the target domain include one or more non-complementary regions containing sequences that are non-complementary to each other. The aforementioned non-complementary containing region includes non-complementary sequences at both ends, The aforementioned structure-forming inducing domain contains one or more modified nucleotides, Modified nucleotides are one or more of the following modified nucleotides: Modified nucleotides capable of adopting a C3'-end conformation, in which the conformation of the sugar portion is introduced at the position of a nucleotide that is C3'-end type during three-dimensional structure formation; Modified nucleotides capable of adopting a C2'-end conformation, in which the conformation of the sugar portion is introduced at the position of a nucleotide that is C2'-end type during three-dimensional structure formation; and The modified nucleotide includes a modified nucleotide in which the hydroxyl group at the 2' position of ribose is introduced to the position of a nucleotide involved in hydrogen bonding during three-dimensional structure formation, and which is capable of forming hydrogen bonds or has a non-bulky substituent at the 2' position of ribose. The aforementioned non-bulky substituent is selected from the group consisting of a hydrogen group, a halogen group, a methyl group, an amino group, and a cyano group. The three-dimensional structure-forming inducing domain and / or the target domain includes a plurality of complementary regions, and the non-complementary containing region is located between the plurality of complementary regions. The aforementioned specific three-dimensional structure includes one or more selected from the group consisting of a kink turn structure, a bulged G structure, a reverse kink turn structure, a 5S loop E structure, a C loop structure, a tandem GA structure, a tetraloop receptor structure, and a tetraloop structure. The artificial nucleic acid.

2. The artificial nucleic acid according to claim 1, wherein the structure-forming-inducing domain further comprises a modified nucleotide having a phosphodiester bond and / or a base modification.

3. The artificial nucleic acid according to claim 1, further comprising a hybridize domain consisting of 6 to 120 bases adjacent to one or both of the three-dimensional structure formation inducing domains.

4. The artificial nucleic acid according to claim 1, wherein the non-complementary containing region consists of 2 to 7 bases.

5. The kink turn structure is 5'-NNNNGAN-3' and 5'-NGAN-3', The bulged G structure consists of 5'-NNNGUAN-3' and 5'-NGANNN-3', The reverse skin turn structure is 5'-NNNNAAN-3' and 5'-NGAN-3', The 5S loop E structure is 5'-NGUAN-3' and 5'-NGAUN-3', The C-loop structure is 5'-NCACU-3' and 5'-ANN-3', or The tandem GA structure is 5'-NGAN-3' and 5'-NGAN-3' It consists of, In each base sequence, N is A, C, G, or U. The artificial nucleic acid according to claim 1.

6. The artificial nucleic acid according to claim 1, wherein the target nucleic acid is mRNA or miRNA.

7. The artificial nucleic acid according to claim 1, wherein the hybridization is carried out under highly stringent conditions.

8. The artificial nucleic acid according to claim 1, wherein the modified nucleotide capable of adopting the C3'-end conformation is selected from the group consisting of 2'-OMe RNA, 2'-MOE RNA, LNA, and DNA.

9. The artificial nucleic acid according to claim 1, wherein the modified nucleotide capable of adopting the C2'-end conformation is selected from the group consisting of 2'-O,5'-N BNA, 2'-deoxy-trans-3',4'-BNA, and DNA.

10. The artificial nucleic acid according to claim 1, wherein the target domain includes the non-complementary containing region containing mutations.

11. The artificial nucleic acid according to claim 10, wherein the mutation is a single nucleotide variant, an insertion / deletion mutation, a structural polymorphism, or a combination thereof.

12. A gene expression inhibitor comprising the artificial nucleic acid described in any one of claims 1 to 11 as an active ingredient.

13. A nucleic acid detection agent comprising an artificial nucleic acid as described in any one of claims 1 to 11 as an active ingredient.

14. A method for producing artificial nucleic acids that induce a specific three-dimensional structure by hybridizing with a target nucleic acid that does not form a functional three-dimensional structure, A target domain selection step involves searching for sequence information of one of the double-stranded sequence motifs constituting a specific three-dimensional structure in the nucleic acid of the objective, and selecting one or more of them as target domains. A step of determining a structure-forming-inducing domain to determine the arrangement of structure-forming-inducing domains so as to constitute the target domain and sequence motif, and Nucleic acid synthesis step, which synthesizes the artificial nucleic acid based on the sequence information determined in the three-dimensional structure formation induction domain determination step. Includes, In the sequence motif, the target domain and the structure-forming induction domain include complementary regions consisting of sequences that are complementary to each other. Furthermore, in the sequence motif, the target domain and / or the structure-forming induction domain include one or more non-complementary regions containing sequences that are non-complementary to each other. The aforementioned non-complementary containing region includes non-complementary sequences at both ends, The aforementioned structure-forming inducing domain contains one or more modified nucleotides, Modified nucleotides are one or more of the following modified nucleotides: Modified nucleotides capable of adopting a C3'-end conformation, in which the conformation of the sugar portion is introduced at the position of a nucleotide that is C3'-end type during three-dimensional structure formation; Modified nucleotides capable of adopting a C2'-end conformation, in which the conformation of the sugar portion is introduced at the position of a nucleotide that is C2'-end type during three-dimensional structure formation; and Modified nucleotides in which the hydroxyl group at the 2' position of ribose is introduced to the position of a nucleotide involved in hydrogen bonding during three-dimensional structure formation, and which are capable of forming hydrogen bonds or have a non-bulky substituent at the 2' position of ribose; Includes, The aforementioned non-bulky substituent is selected from the group consisting of a hydrogen group, a halogen group, a methyl group, an amino group, and a cyano group. The three-dimensional structure-forming inducing domain and / or the target domain includes a plurality of complementary regions, and the non-complementary containing region is located between the plurality of complementary regions. The aforementioned specific three-dimensional structure includes one or more selected from the group consisting of a kink turn structure, a bulged G structure, a reverse kink turn structure, a 5S loop E structure, a C loop structure, a tandem GA structure, a tetraloop receptor structure, and a tetraloop structure. The aforementioned method.

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