Novel ribonucleic acid and drug composition based thereon

Abstract

The present invention relates to a heterogeneous structure RNA (hsRNA) comprising ssRNA and dsRNA, wherein the dsRNA has any length and sequence, has perfect complementarity, serves as a TLR3 ligand, and is located in the middle of the hsRNA, and the ssRNA has any sequence (preferably a TLR7 ligand sequence), has a certain length, and is linked to the two 3'-ends of the dsRNA. In addition, the present invention relates to a pharmaceutical composition comprising the same for preventing and treating viral or bacterial infections and cancer.

Description

Technical Field

[0001] The present invention relates to a novel ribonucleic acid (RNA: ribonucleic acid) which overcomes the problems of Poly(I:C), such as length heterogeneity, high toxicity, and inconsistent activity. Specifically, the present invention relates to a so-called hetero-structured RNA (hsRNA: hetero-structured RNA) which includes a homoduplex RNA (dsRNA: double-stranded RNA) in the middle and single-stranded RNAs (ssRNAs: single-stranded RNAs) hanging at each 3'-end of the dsRNA on both sides, wherein both the dsRNA and the ssRNA can have any specific sequence and specific defined length. Although the dsRNA region has perfect complementarity and serves as a TLR3 ligand, the ssRNA sequence is preferably characterized as a TLR7-like ligand. The length of the hsRNA is extremely uniform and has high innate immune activity and stability. The disclosed hsRNA and dsRNA are agents with few side effects and thus can be used as active ingredients of compositions for preventing and treating viral or bacterial infections and cancers. Background Art

[0002] A) TLR3 Ligands as Innate Immune Activators

[0003] TLR3 is typically expressed in dendritic cells (DCs: dendritic cells), B cells, monocyte-derived macrophages, and occasionally in the endosomal compartments of many tumor tissues, and senses dsRNA. The dsRNA can be derived from viruses or virus-infected abnormal cells. The recognition of dsRNA by TLR3 stimulates type I interferons and pro-inflammatory cytokines and DC activation into mature antigen-presenting cells (APCs: antigen-presenting cells), thereby loading antigenic epitopes onto MHC-1 molecules and presenting them to naive T cells. DC activation through TLR3 not only contributes to the innate immune response and adaptive immune response to microbial pathogens, but also activates CD8+ T cells and natural killer (NK: natural killer) cells (Non-Patent Document 1). The minimum length of dsRNA known to be able to induce TLR3 in vitro is 45mer. Although the analysis of in vitro cultured dendritic precursors after in vitro treatment with dsRNA and FLT3 ligand or GM-CSF has been studied, its functional contribution in vivo has not been clearly understood (Non-Patent Document 1).

[0004] There are reports on the administration of Poly(I:C), which is a dsRNA analog, to infections and tumors (Patent Documents 1 to 14). Poly(I:C) stimulates DC, NK, and cytotoxic lymphocytes (CTL: cytotoxic lymphocyte) and inhibits tumor growth (Non-Patent Document 2). However, the clinical application of Poly(I:C) is limited due to its high toxicity and inconsistent activity, which is inherently caused by the extreme heterogeneity in length. Poly(I:C) exhibits different cellular responses depending on its length, where shorter forms activate TLR3, and longer forms activate innate immunity through TLR3 and MDA5 activities, respectively (Non-Patent Document 1).

[0005] B) ssRNA serves as a TLR7 / TLR8 ligand in innate immunity

[0006] SsRNA containing guanosine (G: guanosine) and uridine (U: uridine) (preferably with a repeating "GUU" sequence) stimulates DC and macrophages, which is mediated by NF-κB activation, cytokine secretion, and inflammatory responses through MYD88 and TRAF6 in innate immunity. TLR7 and TLR8 are present on the endosomal membrane and recognize ssRNA. TLR7 is a dual receptor for ssRNA containing guanosine and uridine, where the first binding site in TLR7 preferentially senses small ligands (i.e., G), and the second binding site recognizes the uridine portion of ssRNA.

[0007] C) Extreme length heterogeneity and toxicity of Poly(I:C)-based dsRNA

[0008] The length of dsRNA that activates DC in vivo and in vitro needs to be accurately defined. Several RNA derivatives have been proposed as candidates for vaccine adjuvants (Patent Documents 1 to 18), but due to the inherent limitations in production, identity and homogeneity cannot be guaranteed. For the above reasons, they exhibit extreme heterogeneity in function and safety, thus limiting administration (Patent Documents 1 to 14). The manufacturing process of Poly(I:C)-based substances and their problems are as follows.

[0009] Poly(I:C) is a synthetic dsRNA analog composed of a polyinosine homopolymer chain Poly(I) and a polycytidine homopolymer chain (C), which bind to each other complementarily to form an unnatural dsRNA. Since each chain is enzymatically synthesized separately by adding new bases to the bases at the ends using polynucleotide phosphorylase (PNPase), the length of the resulting homopolymer varies greatly. In addition, when annealing is performed after the synthesis of each chain by matching a long homopolymer (e.g., an average of 400 bases of Poly(C)) complementary to another long homopolymer sequence (e.g., an average of 400 bases of Poly(I)), the upper chain (i.e., Poly(I)) binds at any position along its complementary lower chain (Poly(C)) (strand slippage), which inevitably generates single-stranded regions at the 3' or 5' ends of the two chains. Further binding of the complementary strand to this region again makes the lengths extremely different, ranging from dozens to hundreds of kilobase pairs (kbp) or more (strand extension). In addition, after strand extension, there are an unspecified number of gaps at unspecified positions. Therefore, when treated with RNase T1, which recognizes and cleaves the position where any gap exists, the length randomly decreases (see Figure 8 C).

[0010] Although Poly(I:C)-L-lysine-methylcellulose (Poly(I:C)-LC) is one of the Poly(I:C) derivatives and is less sensitive to degradation by RNase in serum, its preparation process is basically the same as that of Poly(I:C). Therefore, the length is not constant (Patent Documents 1 to 14). Poly(I:C)-LC induces the secretion of IFN-γ and increases CTL responses and antigen-specific antibodies. The toxicity of Poly(I:C)-LC is reduced. PIKA is an alternative name for a Poly(I:C) derivative, which also has a Poly(I:C) form with a non-constant length (Non-Patent Documents 3 and 4).

[0011] Poly(I:C12U) is composed of Poly(I) and Poly(C), where U is added to every 12 C's, resulting in incomplete dsRNA with regularly dispersed mismatches. Due to the presence of multiple nicks, Poly(I:C12U) is more sensitive to degradation by RNase T1, has a shorter half-life, lower side effects than Poly(I:C), and a higher selectivity for TRIF than Myd88. Nevertheless, the length is still highly heterogeneous. Poly(I:C12U) has been approved for human use in Canada and Argentina and was approved as an orphan drug for chronic fatigue syndrome (CFS) in the EU in 2000, but has not been approved by the US FDA. In another case, the synthesis of Poly(I:C) derivatives has been improved, with a more uniform length than Poly(I:C), but the problem of length diversity still exists. For example, Poly(I:C)(100 to 400) is prepared by complementary binding of short chains (e.g., an average of 108 bases of Poly(I)100) with long chains (e.g., an average of 344 bases of Poly(C)400). In this way, dsRNA with an average length close to 400 bp is prepared, but the length range is very wide, from 100 to 2000 bases. When short homopolymer sequences (e.g., Poly(I)100) are complementary bound to long homopolymers (e.g., Poly(C)400), strand slippage and strand extension cannot be prevented. In summary, the length of Poly(I:C)-based dsRNA analogs is not constant, and there are an unspecified number of nicks at unspecified positions within the dsRNA, resulting in low stability against RNase T1 (Patent Document 3, Non-Patent Documents 2 and 5).

[0012] D) Roles of OX40 and PD-1 molecules in the cancer microenvironment

[0013] Both innate and adaptive immune responses are involved in tumor growth and control (Non-Patent Document 7). Immune function is generally suppressed in the tumor microenvironment (TME). Several methods using antagonists for T cell inhibitory receptors or stimulators for T cell receptors to enhance T cell activity have been tested (Non-Patent Document 8).

[0014] OX40, which is a member of the tumor necrosis factor receptor superfamily, is not expressed in naive T cells but is induced in activated T cells upon exposure to antigen (TCR) (Non-Patent Document 9). OX40 is also generally expressed in activated B cells, activated DCs, activated eosinophils, natural killer T (NKT) cells, NK cells, memory T cells, and regulatory T cells (Non-Patent Document 9). OX40L, which is the ligand for OX40, is mainly expressed on APCs. The OX40-OX40L interaction co-stimulates CD4 and CD8 T cells to promote cell proliferation, survival, effector function, and migration. In addition, OX40 can induce the development and function of memory T cells, thereby overcoming the immunosuppressive environment in the TME. When APCs such as dendritic cells mature, the B7 family (e.g., CD80 and CD86) and OX40L on the surface of the APC increase, which helps to determine T cell immune responses and memory differentiation (Non-Patent Document 10).

[0015] The OX40:OX40L binding interaction forms a trimeric receptor (trimer), leading to higher-order aggregation and subsequent signal transduction (Non-Patent Document 11). Antibodies derived from mice stimulated with OX40 (anti-mOX40 rat agonist Ab) can induce tumor rejection in a mouse model (Non-Patent Documents 12 and 13). Antibodies derived from humans stimulated with OX40 (anti-hOX40 mouse agonist Ab) have also been shown to enhance cancer immune function in cancer patients (Non-Patent Document 14). The OX40:OX40L interaction is associated with immune responses in graft-versus-host disease (e.g., organ transplant rejection), asthma / atopy, encephalomyelitis, rheumatoid arthritis, colitis / inflammatory bowel disease, diabetes, and atherosclerosis in non-obese diabetic mice, lupus, inflammation, and autoimmune diseases and disorders (Non-Patent Document 15).

[0016] PD-L1 is overexpressed in many cancers and is generally associated with poor prognosis (Non-Patent Documents 16 and 17). Interestingly, compared to normal tissues and peripheral blood T lymphocytes, most tumor-infiltrating T lymphocytes express PD-1. Upregulation of PD-1 in T cells can constitute an immunosuppressive tumor microenvironment (Non-Patent Document 18). Substantially, the interaction between PD-1 on tumor-infiltrating T cells and PD-L1 on tumor cells attenuates the function of T cells (Non-Patent Documents 19 and 20). Therefore, inhibiting the PD-1:PD-L1 (or PD-L2) interaction can break the immunosuppressive TME. However, in addition to blocking the interaction between the PD-1 receptor and its ligand for optimal treatment, additional immune-enhancing therapies need to be developed.

[0017] Prior Art Documents

[0018] Patent Literature

[0019] (Patent Literature 1) Double-stranded ribonucleic acid for adjuvants; US 20150307884 A1

[0020] (Patent Literature 2) Adjuvant composition; WO 2003028656 A2: Shorter form of Poly(I:C)

[0021] (Patent Literature 3) Clinical methods for the immunomodulatory and vaccine adjuvant use of poly-ICLC and other dsRNAs; US7834064B2

[0022] (Patent Literature 4) dsRNAs as influenza virus vaccine adjuvants or immunostimulants; US 20070224219 A

[0023] (Patent Literature 5) Maturation process of dendritic cells and vaccines; US 7981673 B2

[0024] (Patent Literature 6) Topical active ingredient of mismatched dsRNA; US 5712257 A

[0025] (Patent Literature 7) Hepatitis treatment with mismatched dsRNA; US 5906980 A

[0026] (Patent Literature 8) Double-stranded PolyC: Poly(G / I)RNA for immunostimulation and cancer treatment; EP 3083962A1

[0027] (Patent Literature 9) Methods and formulations for achieving tumor-targeted double-stranded ribonucleic acid-mediated cell death; WO2014165296A1

[0028] (Patent Literature 10) Polyinosinic acid-polycytidylic acid-based adjuvants; US 20070160632 A1

[0029] (Patent Literature 11) In-situ cancer autologous vaccination with intratumorally stable dsRNA virus mimics; US20090088401A1

[0030] (Patent Literature 12) Novel nucleic acids with adjuvant activity and their uses; WO 2012014945 A

[0031] (Patent Literature 13) Improved cancer treatment by combining the use of tlr3 agonists and another therapeutic agent; WO2009130301A1

[0032] (Patent Literature 14) Double-stranded ribonucleic acid conjugates and their uses; US 20170107517 A1

[0033] Double-stranded ribonucleic acid with an uneven physicochemical structure and highly specific biological activity; WO 2010047835A2

[0034] (Patent Document 16) Immunization strategy for preventing H1N1 infection; EP 2477652 A4

[0035] Non-patent literature

[0036] (Non-patent Literature 1) Jelinek I et al., (2011) Journal of Immunology (J Immunol) 186(4): 2422-9

[0037] (Non-patent Literature 2) Mian MF et al., (2013) Journal of Leukocyte Biology (J Leukoc Biol) 94(5): 1025-36

[0038] (Non-patent Literature 3) Zhang Y et al., (2016) Virology 489: 165-72

[0039] (Non-patent Literature 4) Lau YF et al., (2010) Virology 406(1): 80-7

[0040] (Non-patent Literature 5) Nakano T et al., (2018) Bioscience Biotechnology Biochemistry 82(11): 1889-1901

[0041] (Non-patent Literature 6) Matsumoto M et al., (2015) 6: 6280

[0042] (Non-patent Literature 7) Vesely MD et al., (2011) Annual Review of Immunology (Annu Rev Immunol) 29: 235-271

[0043] (Non-patent Literature 8) Mellman I et al., (2011) Nature 480: 480-489

[0044] (Non-patent Literature 9) Sugamura K et al., (2004) Nature Reviews Immunology (Nat Rev Immunol) 4: 420-431

[0045] (Non-patent Literature 10) Soroosh P et al., (2006) Journal of Immunology (J Immunol) 176: 975-5987

[0046] (Non-patent Document 11) Compaan DM et al., (2006) Structure 14: 1321 - 1330

[0047] (Non-patent Document 12) Weinberg AD et al., (2000) Journal of Immunology (J Immunol) 164: 2160 - 2169

[0048] (Non-patent Document 13) Piconese S et al., (2008) Journal of Experimental Medicine (J Exp Med) 205: 825 - 839

[0049] (Non-patent Document 14) Curti BD et al., (2013) Cancer Research 73: 7189 - 7198

[0050] (Non-patent Document 15) Croft M et al., (2009) Immunological Reviews (Immunol Rev) 229(1): 173 - 191

[0051] (Non-patent Document 16) Okazaki T et al., International Immunology (Intern. Immun) 2007 19(7): 813

[0052] (Non-patent Document 17) Thompson RH et al., Cancer Research 2006, 66(7): 3381

[0053] (Non-patent Document 18) Ahmadzadeh M et al., Blood. 2009 Aug 20; 114(8): 1537 - 44

[0054] (Non-patent Document 19) Sharpe et al., Nature Reviews (Nat Rev) 2002

[0055] (Non-patent Document 20) Keir ME et al., 2008 Annual Review of Immunology (Annu Rev Immunol). 26: 677 Summary of the Invention

[0056] Technical Problem

[0057] 1) Overcome the extreme length heterogeneity of Poly(I:C)-type RNA adjuvants

[0058] The pharmaceutical composition should have consistent reproducibility and homogeneity. The most important reasons restricting the administration of Poly(I:C), dsRNA analogs and their derivatives in clinical use are due to the lack of reproducibility in production, including extreme length heterogeneity, unpredictable pharmacokinetics and inconsistent efficacy (Non-Patent Document 3 and Non-Patent Document 6), as well as severe toxicity causing symptoms such as arthralgia, fever, erythema and endotoxin-like shock (Patent Document 15 and Patent Document 17). To date, dsRNA analogs include Poly(I:C), Poly(I:C)-LC and Poly(I:C12U), all of which are composed of two complementary homopolymeric strands. Therefore, the lengths of these derivatives vary greatly and cannot be defined as a specific length.

[0059] The present invention provides a novel sRNA which provides extreme length homogeneity with an accurate molecular formula weight, less toxicity and easy large-scale production. If desired, the hsRNA of the present invention can be treated with RNase T1 to remove the ssRNA region and selectively extract only dsRNA. The hsRNA is designated as having continuous dsRNA in the middle and two ssRNAs hanging at the two 3'-ends on both sides. Both the dsRNA and ssRNA regions are defined as having precise lengths and non-homopolymeric sequences. They can overcome the problems of existing Poly(I:C) and its derivatives by these characteristics.

[0060] 2) Reduce the required antigen dose of the antigen in the vaccine

[0061] The present invention also provides hsRNA and dsRNA capable of inducing stronger protective immunity while reducing the required antigen dose. For each predicted virus, each dose of the current seasonal trivalent or quadrivalent vaccine requires at least 15 μg of antigen to be effectively prophylactically immunized. Due to the large amount of antigen required annually, there is a potential risk of insufficient vaccine supply. For FLUAD, the MF59 (squalene oil-in-water emulsion) adjuvant was approved in the United States in 2015 for people over 65 years old. Nevertheless, there is no approved adjuvant vaccine that can significantly reduce the antigen dose without affecting the efficacy.

[0062] 3) Provide a cancer vaccine composition with enhanced anti-cancer efficacy

[0063] The present invention provides a cancer vaccine composition comprising hsRNA and OX40 antibody and / or PD-1 antibody, which continuously and strongly activates dendritic cells in vivo, thereby preventing or treating distant metastatic cancer as well as primary cancer.

[0064] The hsRNA of the present invention promotes cancer cell death and promotes the release of tumor-associated antigens (TAAs) from cancer cells. In this way, the hsRNA or dsRNA in the antibody combination against OX40 agonist or PD-1 may trigger or even synergistically trigger a TAA-dependent immune response to lyse cancer cells.

[0065] Technical solution

[0066] 1) The present invention provides RNA with high length homogeneity and stability through bidirectional synthesis.

[0067] The present invention relates to highly homogeneous hsRNA and dsRNA with a specific length, perfect base pairing, and high stability at room temperature.

[0068] The preparation method of hsRNA is as follows. A DNA fragment capable of guiding the transcription of target RNA includes T7 promoters on both 5'-terminal sides and is inserted into any plasmid vector. If necessary, a fragment with a TLR7 ligand sequence or any length and any sequence can be added to the 5'-terminal of the T7 promoter sequence. The DNA fragment is amplified by PCR and used as a template for in vitro transcription (IVT) by T7 polymerase. Bidirectional IVT generates the top and bottom strands of heteropolymeric ssRNA with perfect complementarity in almost the same molar ratio during IVT and spontaneously forms high-purity hsRNA at room temperature. The hsRNA or dsRNA does not encode a protein, but can optionally encode a protein. In one example, the ssRNA can be a random sequence or include GUU repeat sequences but is not limited thereto, and the ssRNA can be optionally removed by treatment with RNase T1.

[0069] So far, due to technical difficulties in synthesizing dsRNA with a difference of one base, the minimum and optimal lengths of dsRNA for activating DC and the in vivo immune system have not been fully understood. On the contrary, the method of the present invention can specify the correct number of lengths or structures of RNA that shows the optimal immune activation required for TLR3 or TLR7 activation in vivo and in vitro.

[0070] 2) The present invention provides a vaccine composition for preventing and treating infections, which includes RNA that can reduce the dose of vaccine antigen required for infection

[0071] The present invention provides a pharmaceutical composition for preventing or treating viral or bacterial infections, which includes hsRNA or dsRNA.

[0072] RNA with a strong negative charge can effectively receive many large antigens, making the nanocomplex comparable to virus-like particles (VLPs). The nanocomplex encapsulating the antigen in an appropriate formulation is captured by dendritic cells or macrophages, thereby effectively inducing the presentation of the antigen to P cells and T cells. The vaccine composition can be selected from proteins of pathogens, recombinant proteins, subunits, split protein antigens, glycoproteins, peptides, polysaccharides, lipopolysaccharides, polynucleotides, and combinations thereof, but is not limited thereto.

[0073] 3) The present invention provides an anti-cancer vaccine composition comprising the RNA adjuvant of the present invention, and a pharmaceutical composition for preventing or treating a disease comprising the RNA of the present invention and the following antibody.

[0074] The present invention provides a pharmaceutical composition for preventing or treating a disease comprising hsRNA or dsRNA, and a pharmaceutical composition for preventing or treating cancer, which further comprises an OX40 antibody or a PD-1 antibody.

[0075] Beneficial effects

[0076] The hsRNA of the present invention overcomes the extreme length diversity and compositional heterogeneity of Poly(I:C) and provides advantages specified by a specific length and sequence. In addition, by removing the ssRNA overhangs from hsRNA, highly homogeneous dsRNA can be obtained. Furthermore, when the ssRNA overhangs are removed from hsRNA, the hsRNA of the present invention can be prepared into dsRNA with complementary sequences that induces improved immune responses.

[0077] In addition, the antigen complexed with the hsRNA of the present invention can be delivered into APCs to effectively present the antigen to T cells and B cells. Additionally, the hsRNA or dsRNA of the present invention can reduce the antigen dose and can induce protective immune effects against viral or bacterial infections and cancer.

[0078] As a result, the RNA adjuvant of the present invention exhibits the following activities: (1) enhancing adaptive immunity against antigens, (2) reducing the amount of antigen required in the vaccine, (3) improving antigen-specific Th1 polarization cross-protection responses, and (4) enhancing innate immunity when used alone without an antigen.

[0079] In addition, the cancer vaccine composition comprising only the hsRNA of the present invention or comprising hsRNA and an OX40 antibody or a PD-1 antibody exhibits anti-cancer efficacy against both primary cancer and distant cancer. The cancer vaccine composition of the present invention can alone convert refractory cancers that are non-responsive to immune checkpoint inhibitors such as PD-1 antibodies into responsive cancers. <110> NA Vaccine Research Institute Co., Ltd. <120> Novel ribonucleic acid and pharmaceutical composition based thereon <130> NAVI19P-0001-WO <150> KR 10-2019-0079470 <151> 2019-07-02 <150> KR 10-2020-0037711 <151> 2020-03-27 <150> KR 10-2019-0153760 <151> 2019-11-26 <160> 93 <170> KoPatentIn 3.0 <210> 1 <211> 123 <212> RNA <213> Artificial sequence <220> <223> NA1001 <400> 1 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgcccuaua gugagucgua 120 uua 123 <210> 2 <211> 173 <212> RNA <213> Artificial sequence <220> <223> NA1501 <400> 2 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcccuaua gugagucgua 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gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caacccuaua gugagucgua 420 uua 423 <210> 6 <211> 523 <212> RNA <213> Artificial sequence <220> <223> NA5001 <400> 6 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guucccuaua gugagucgua uua 523 <210> 7 <211> 623 <212> RNA <213> Artificial sequence <220> <223> NA6001 <400> 7 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuauccgcu cacaauucca cacaacauac gagccggaag 540 cauaaagugu aaagccuggg gugccuaaug agugagcuaa cucacauuaa uugcguugcg 600 cuccccuaua gugagucgua uua 623 <210> 8 <211> 723 <212> RNA <213> Artificial sequence <220> <223> NA7001 <400> 8 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuauccgcu cacaauucca cacaacauac gagccggaag 540 cauaaagugu aaagccuggg gugccuaaug agugagcuaa cucacauuaa uugcguugcg 600 cucacugccc gcuuuccagu cgggaaaccu gucgugccag cugcauuaau gaaucggcca 660 acgcgcgggg agaggcgguu ugcguauugg gcgcucuucc gcucccuaua gugagucgua 720 uua 723 <210> 9 <211> 823 <212> RNA <213> Artificial sequence <220> <223> NA8001 <400> 9 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuauccgcu cacaauucca cacaacauac gagccggaag 540 cauaaagugu aaagccuggg gugccuaaug agugagcuaa cucacauuaa uugcguugcg 600 cucacugccc gcuuuccagu cgggaaaccu gucgugccag cugcauuaau gaaucggcca 660 acgcgcgggg agaggcgguu ugcguauugg gcgcucuucc gcuuccucgc ucacugacuc 720 gcugcgcucg gucguucggc ugcggcgagc gguaucagcu cacucaaagg cgguaauacg 780 guuauccaca gaaucagggg auacccuaua gugagucgua uua 823 <210> 10 <211> 123 <212> RNA <213> Artificial sequence <220> <223> NA1002 <400> 10 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uuccccuaua gugagucgua 120 uua 123 <210> 11 <211> 173 <212> RNA <213> Artificial sequence <220> <223> NA1502 <400> 11 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aaccccuaua gugagucgua uua 173 <210> 12 <211> 223 <212> RNA <213> Artificial sequence <220> <223> NA2002 <400> 12 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gaucccuaua gugagucgua uua 223 <210> 13 <211> 323 <212> RNA <213> Artificial sequence <220> <223> NA3002 <400> 13 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggacccuaua gugagucgua uua 323 <210> 14 <211> 423 <212> RNA <213> Artificial sequence <220> <223> NA4002 <400> 14 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucccuaua gugagucgua 420 uua 423 <210> 15 <211> 523 <212> RNA <213> Artificial sequence <220> <223> NA5002 <400> 15 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucgcccuaua gugagucgua uua 523 <210> 16 <211> 623 <212> RNA <213> Artificial sequence <220> <223> NA6002 <400> 16 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucguauucuc cuucuugcug cccuuucuga gggaaggacu 540 guguugaca auuuacugag uuuuuuaca ugcuuggc gugaaaaca 600 cuckooa gugagucgua ua 623 <210> 17 <211> 723 <212> RNA <213>人工序列 <220> <223> NA7002 <400> 17 ggggaauucc ggaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaaoug 60 cuaacccca cucuaccuac cucuaccuac cuuaaccuac 120 aaaaaaaaaaaaaaaaaacuccaag aacagaga gagaguggaaaaacugaagu 180 uuuucacaag aauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauccucca aucuuucua aaacaaaag gguccucuug auuchaauuc aucuucuuu 300 ggaucaaga aacuaaccca aauuucagca aaucauaag ggguuuaa gaagaouca 360 cauuuugagggg ugggaggaa caucuuuuuuuuuuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucguauucuc cuucuugcug cccuuucuga gggaaggacu 540 guuguugaca auuuacugag uagugacgac auucauuaca ugcuuggugc guugaaaaca 600 cuuggacuuc auguugaaga ugacaaugaa aaccaacgag caauugugga agguuguggu 660 gggcaguuuc cugucgguaa aaagucugag gaagaaaucc aaccccuaua gugagucgua 720 uua 723 <210> 18 <211> 823 <212> RNA <213> Artificial sequence <220> <223> NA8002 <400> 18 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucguauucuc cuucuugcug cccuuucuga gggaaggacu 540 guuguugaca auuuacugag uagugacgac auucauuaca ugcuuggugc guugaaaaca 600 cuuggacuuc auguugaaga ugacaaugaa aaccaacgag caauugugga agguuguggu 660 gggcaguuuc cugucgguaa aaagucugag gaagaaaucc aacuauuccu uggaaaugca 720 ggaacagcaa ugcguccguu gacagcagca guuacuguag cuggaggaca uucaagauau 780 guucuugaug gaguuccuag gaucccuaua gugagucgua uua 823 <210> 19 <211> 336 <212> RNA <213> Artificial sequence <220> <223> R1 <400> 19 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugcccu auagugaguc guauua 336 <210> 20 <211> 414 <212> RNA <213> Artificial sequence <220> <223> R2 <400> 20 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccuau agugagucgu auua 414 <210> 21 <211> 483 <212> RNA <213> Artificial sequence <220> <223> R3 <400> 21 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccuaua gugagucgua 480 uua 483 <210> 22 <211> 605 <212> RNA <213> Artificial sequence <220> <223> R4 <400> 22 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuacccua uagugagucg 600 uauua 605 <210> 23 <211> 681 <212> RNA <213> Artificial Sequence <220> <223> R5 <400> 23 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccuauagu gagucguauu a 681 <210> 24 <211> 750 <212> RNA <213> Artificial sequence <220> <223> R6 <400> 24 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccuauagug agucguauua 750 <210> 25 <211> 839 <212> RNA <213> Artificial sequence <220> <223> R7 <400> 25 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac ccuauaguga gucguauua 839 <210> 26 <211> 902 <212> RNA <213> Artificial sequence <220> <223> R8 <400> 26 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccuauag ugagucguau 900 ua 902 <210> 27 <211> 1049 <212> RNA <213> Artificial sequence <220> <223> R9 <400> 27 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccugcug aaaccgggug 900 acaccaucau cuucgaagcu aacgguaacc ugaucgcucc gauguacgcu uucgcucugu 960 cucgugguuu cgguucuggu aucaucaccu cuaacgcuuc uaugcacgaa ugcaacacca 1020 aaugccagac ccuauaguga gucguauua 1049 <210> 28 <211> 1170 <212> RNA <213> Artificial sequence <220> <223> R10 <400> 28 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccugcug aaaccgggug 900 acaccaucau cuucgaagcu aacgguaacc ugaucgcucc gauguacgcu uucgcucugu 960 cucgugguuu cgguucuggu aucaucaccu cuaacgcuuc uaugcacgaa ugcaacacca 1020 aaugccagac cccgcugggu gcuaucaacu cuucucugcc guaccagaac auccacccgg 1080 uuaccaucgg ugaaugcccg aaauacguuc guucugcuaa acugcguaug guuaccgguc 1140 ugcguaacau cccuauagug agucguauua 1170 <210> 29 <211> 1665 <212> RNA <213> Artificial sequence <220> <223> R11 <400> 29 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccugcug aaaccgggug 900 acaccaucau cuucgaagcu aacgguaacc ugaucgcucc gauguacgcu uucgcucugu 960 cucgugguuu cgguucuggu aucaucaccu cuaacgcuuc uaugcacgaa ugcaacacca 1020 aaugccagac cccgcugggu gcuaucaacu cuucucugcc guaccagaac auccacccgg 1080 uuaccaucgg ugaaugcccg aaauacguuc guucugcuaa acugcguaug guuaccgguc 1140 ugcguaacau cccgucuauc cagucucgug gucuguucgg ugcuaucgcu gguuucaucg 1200 aaggugguug gaccgguaug aucgacgguu gguacgguua ccaccaccag aacgaacagg 1260 guucugguua cgcugcugac cagaaaucua cccagaacgc uaucaacggu aucaccaaca 1320 aaguuaacac cguuaucgaa aaaaugaaca uccaguucac cgcuguuggu aaagaauuca 1380 acaaacugga aaaacguaug gaaaaccuga acaaaaaagu ugacgacggu uuccuggaca 1440 ucuggaccua caacgcugaa cugcugguuc ugcuggaaaa cgaacguacc cuggacuucc 1500 acgacucuaa cguuaaaaac cuguacgaaa aaguuaaauc ucagcugaaa aacaacgcua 1560 aagaaaucgg uaacgguugc uucgaauucu accacaaaug cgacaacgaa ugcauggaau 1620 cuguucguaa cgguaccuac gacuacccua uagugagucg uauua 1665 <210> 30 <211> 482 <212> RNA <213> Artificial sequence <220> <223> NA,NVT,VP1,VP10 <400> 30 gggcgauaau acaguuuugg acucaggugu gagauuuuau gaucaggacu augaaggaca 60 aauaacccca auggaauaug uaacuggguu guauaacuuu uggucagggc caauagaguu 120 acguuuugau uuuguuucaa augcguuuca cacuggaaca gugauuauau cagcggagua 180 uaaucgauca ucuacuaaua cggaugagug ucagucacac ucaacuuaua cuaaaacguu 240 ccacuuggga gaacaaaaau caguacauuu cacugugccu uauauauaug auacuguuau 300 gcggagaaau acggcuagcg ccuauuuacc gguaacugau uaugauaagg cagauaaugu 360 uaguagggcg caggcuacgg ggauuagagc agaaucuaaa augagaguga aagugagauc 420 gcccuauagu gagucguauu agucgacugc agaggccugc augcaagcuu ggcguaauca 480 ug 482 <210> 31 <211> 493 <212> RNA <213> Artificial sequence <220> <223> NVT IV,NVT 4,VP20 <400> 31 gggcgauaau acaguuuugg acucaggugu gagauuuuau gaucaggacu augaaggaca 60 aauaacccca auggaauaug uaacuggguu guauaacuuu uggucagggc caauagaguu 120 acguuuugau uuuguuucaa augcguuuca cacuggaaca gugauuauau cagcggagua 180 uaaucgauca ucuacuaaua cggaugagug ucagucacac ucaacuuaua cuaaaacguu 240 ccacuuggga gaacaaaaau caguacauuu cacugugccu uauauauaug auacuguuau 300 gcggagaaau acggcuagcg ccuauuuacc gguaacugau uaugauaagg cagauaaugu 360 uaguagggcg caggcuacgg ggauuagagc agaaucuaaa augagaguga aagugagauc 420 gcccuauagu gagucguauu agucgacugc agaggccugc augcaagcuu uguuguuguu 480 guuguuguug uug 493 <210> 32 <211> 123 <212> RNA <213> Artificial sequence <220> <223> NA1001 <400> 32 gggcgggcuu gucugcuccc ggcauccgcu uacagacaag cugugaccgu cuccgggagc 60 ugcauguguc agagguuuuc accgucauca ccgaaacgcg cgacccuaua gugagucgua 120 uua 123 <210> 33 <211> 173 <212> RNA <213> Artificial sequence <220> <223> NA1501 <400> 33 gggcauaguu aagccagccc cgacacccgc caacacccgc ugacgcgccc ugacgggcuu 60 gucugcuccc ggcauccgcu uacagacaag cugugaccgu cuccgggagc ugcauguguc 120 agagguuuuc accgucauca ccgaaacgcg cgacccuaua gugagucgua uua 173 <210> 34 <211> 223 <212> RNA <213> Artificial sequence <220> <223> NA2001 <400> 34 ggguauuuca caccgcauau ggugcacucu caguacaauc ugcucugaug ccgcauaguu 60 aagccagccc cgacacccgc caacacccgc ugacgcgccc ugacgggcuu gucugcuccc 120 ggcauccgcu uacagacaag cugugaccgu cuccgggagc ugcauguguc agagguuuuc 180 accgucauca ccgaaacgcg cgacccuaua gugagucgua uua 223 <210> 35 <211> 323 <212> RNA <213> Artificial sequence <220> <223> NA3001 <400> 35 gggauagcga agaggcccgc accgaucgcc cuucccaaca guugcgcagc cugaauggcg 60 aauggcgccu gaugcgguau uuucuccuua cgcaucugug cgguauuuca caccgcauau 120 ggugcacucu caguacaauc ugcucugaug ccgcauaguu aagccagccc cgacacccgc 180 caacacccgc ugacgcgccc ugacgggcuu gucugcuccc ggcauccgcu uacagacaag 240 cugugaccgu cuccgggagc ugcauguguc agagguuuuc accgucauca ccgaaacgcg 300 cgacccuaua gugagucgua uua 323 <210> 36 <211> 423 <212> RNA <213> Artificial sequence <220> <223> NA4001 <400> 36 ggguuggcac uggccgucgu uuuacaacgu cgugacuggg aaaacccugg cguuacccaa 60 cuuaaucgcc uugcagcaca ucccccuuuc gccagcuggc guaauagcga agaggcccgc 120 accgaucgcc cuucccaaca guugcgcagc cugaauggcg aauggcgccu gaugcgguau 180 uuucuccuua cgcaucugug cgguauuuca caccgcauau ggugcacucu caguacaauc 240 ugcucugaug ccgcauaguu aagccagccc cgacacccgc caacacccgc ugacgcgccc 300 ugacgggcuu gucugcuccc ggcauccgcu uacagacaag cugugaccgu cuccgggagc 360 ugcauguguc agagguuuuc accgucauca ccgaaacgcg cgacccuaua gugagucgua 420 uua 423 <210> 37 <211> 523 <212> RNA <213> Artificial sequence <220> <223> NA5001 <400> 37 gggaacaauu ucacacagga aacagcuaug accaugauua cgaacucguc cucgguuccc 60 ggcgauccuc uggagauaau cguccacuug ccgucaugcu ucuuuggcac uggccgucgu 120 uuuacaacgu cgugacuggg aaaacccugg cguuacccaa cuuaaucgcc uugcagcaca 180 ucccccuuuc gccagcuggc guaauagcga agaggcccgc accgaucgcc cuucccaaca 240 guugcgcagc cugaauggcg aauggcgccu gaugcgguau uuucuccuua cgcaucugug 300 cgguauuuca caccgcauau ggugcacucu caguacaauc ugcucugaug ccgcauaguu 360 aagccagccc cgacacccgc caacacccgc ugacgcgccc ugacgggcuu gucugcuccc 420 ggcauccgcu uacagacaag cugugaccgu cuccgggagc ugcauguguc agagguuuuc 480 accgucauca ccgaaacgcg cgacccuaua gugagucgua uua 523 <210> 38 <211> 623 <212> RNA <213> Artificial sequence <220> <223> NA6001 <400> 38 ggggagcgca acgcaauuaa ugugaguuag cucacucauu aggcacccca ggcuuuacac 60 uuuaugcuuc cggcucguau guugugugga auugugagcg gauaacaauu ucacacagga 120 aacagcuaug accaugauua cgaacucguc cucgguuccc ggcgauccuc uggagauaau 180 cguccacuug ccgucaugcu ucuuuggcac uggccgucgu uuuacaacgu cgugacuggg 240 aaaacccugg cguuacccaa cuuaaucgcc uugcagcaca ucccccuuuc gccagcuggc 300 guaauagcga agaggcccgc accgaucgcc cuucccaaca guugcgcagc cugaauggcg 360 aauggcgccu gaugcgguau uuucuccuua cgcaucugug cgguauuuca caccgcauau 420 ggugcacucu caguacaauc ugcucugaug ccgcauaguu aagccagccc cgacacccgc 480 caacacccgc ugacgcgccc ugacgggcuu gucugcuccc ggcauccgcu uacagacaag 540 cugugaccgu cuccgggagc ugcauguguc agagguuuuc accgucauca ccgaaacgcg 600 cgacccuaua gugagucgua uua 623 <210> 39 <211> 723 <212> RNA <213> Artificial sequence <220> <223> NA7001 <400> 39 gggagcggaa gagcgcccaa uacgcaaacc gccucucccc gcgcguuggc cgauucauua 60 augcagcugg cacgacaggu uucccgacug gaaagcgggc agugagcgca acgcaauuaa 120 ugugaguuag cucacucauu aggcacccca ggcuuuacac uuuaugcuuc cggcucguau 180 guugugugga auugugagcg gauaacaauu ucacacagga aacagcuaug accaugauua 240 cgaacucguc cucgguuccc ggcgauccuc uggagauaau cguccacuug ccgucaugcu 300 ucuuuggcac uggccgucgu uuuacaacgu cgugacuggg aaaacccugg cguuacccaa 360 cuuaaucgcc uugcagcaca ucccccuuuc gccagcuggc guaauagcga agaggcccgc 420 accgaucgcc cuucccaaca guugcgcagc cugaauggcg aauggcgccu gaugcgguau 480 uuucuccuua cgcaucugug cgguauuuca caccgcauau ggugcacucu caguacaauc 540 ugcucugaug ccgcauaguu aagccagccc cgacacccgc caacacccgc ugacgcgccc 600 ugacgggcuu gucugcuccc ggcauccgcu uacagacaag cugugaccgu cuccgggagc 660 ugcauguguc agagguuuuc accgucauca ccgaaacgcg cgacccuaua gugagucgua 720 uua 723 <210> 40 <211> 823 <212> RNA <213> Artificial Sequence <220> <223> NA8001 <400> 40 ggguaucccc ugauucugug gauaaccgua uuaccgccuu ugagugagcu gauaccgcuc 60 gccgcagccg aacgaccgag cgcagcgagu cagugagcga ggaagcggaa gagcgcccaa 120 uacgcaaacc gccucucccc gcgcguuggc cgauucauua augcagcugg cacgacaggu 180 uucccgacug gaaagcgggc agugagcgca acgcaauuaa ugugaguuag cucacucauu 240 aggcacccca ggcuuuacac uuuaugcuuc cggcucguau guugugugga auugugagcg 300 gauaacaauu ucacacagga aacagcuaug accaugauua cgaacucguc cucgguuccc 360 ggcgauccuc uggagauaau cguccacuug ccgucaugcu ucuuuggcac uggccgucgu 420 uuuacaacgu cgugacuggg aaaacccugg cguuacccaa cuuaaucgcc uugcagcaca 480 ucccccuuuc gccagcuggc guaauagcga agaggcccgc accgaucgcc cuucccaaca 540 guugcgcagc cugaauggcg aauggcgccu gaugcgguau uuucuccuua cgcaucugug 600 cgguauuuca caccgcauau ggugcacucu caguacaauc ugcucugaug ccgcauaguu 660 aagccagccc cgacacccgc caacacccgc ugacgcgccc ugacgggcuu gucugcuccc 720 ggcauccgcu uacagacaag cugugaccgu cuccgggagc ugcauguguc agagguuuuc 780 accgucauca ccgaaacgcg cgacccuaua gugagucgua uua 823 <210> 41 <211> 123 <212> RNA <213> Artificial sequence <220> <223> NA1002 <400> 41 ggggaaaaug guuaugguag guagauugaa gggagauggg guuaggcaau uaagcagaau 60 ggaguccaau gucagcuuca ccaaccaccu uuugucggaa uuccccuaua gugagucgua 120 uua 123 <210> 42 <211> 173 <212> RNA <213> Artificial sequence <220> <223> NA1502 <400> 42 gggguucuug gaguuguuug aagaccaaau uguuuuuuug agaaaauuuu gaagaaaaug 60 guuaugguag guagauugaa gggagauggg guuaggcaau uaagcagaau ggaguccaau 120 gucagcuuca ccaaccaccu uuugucggaa uuccccuaua gugagucgua uua 173 <210> 43 <211> 223 <212> RNA <213> Artificial sequence <220> <223> NA2002 <400> 43 gggaucugug ccauuucuug ugaaaaacuu caguuuuucc acucucucuc uguguucuug 60 gaguuguuug aagaccaaau uguuuuuuug agaaaauuuu gaagaaaaug guuaugguag 120 guagauugaa gggagauggg guuaggcaau uaagcagaau ggaguccaau gucagcuuca 180 ccaaccaccu uuugucggaa uuccccuaua gugagucgua uua 223 <210> 44 <211> 323 <212> RNA <213> Artificial sequence <220> <223> NA3002 <400> 44 ggguccaaag aagagagaau uugaaacaag aggacccuuu uguguuuuag aaagauugga 60 ggaauucaga cuaagggucu guaucccuug ugccaugcua cuaaucugug ccauuucuug 120 ugaaaaacuu caguuuuucc acucucucuc uguguucuug gaguuguuug aagaccaaau 180 uguuuuuuug agaaaauuuu gaagaaaaug guuaugguag guagauugaa gggagauggg 240 guuaggcaau uaagcagaau ggaguccaau gucagcuuca ccaaccaccu uuugucggaa 300 uuccccuaua gugagucgua uua 323 <210> 45 <211> 423 <212> RNA <213> Artificial sequence <220> <223> NA4002 <400> 45 gggaugcaga aauccuaaaa gaugacuucc ucaccacccu caaaacugaa ucuuucuuaa 60 acaccccuaa ugauuuugcu gaaauuuggg uuaguuucuu ugauccaaag aagagagaau 120 uugaaacaag aggacccuuu uguguuuuag aaagauugga ggaauucaga cuaagggucu 180 guaucccuug ugccaugcua cuaaucugug ccauuucuug ugaaaaacuu caguuuuucc 240 acucucucuc uguguucuug gaguuguuug aagaccaaau uguuuuuuug agaaaauuuu 300 gaagaaaaug guuaugguag guagauugaa gggagauggg guuaggcaau uaagcagaau 360 ggaguccaau gucagcuuca ccaaccaccu uuugucggaa uuccccuaua gugagucgua 420 uua 423 <210> 46 <211> 523 <212> RNA <213> Artificial sequence <220> <223> NA5002 <400> 46 gggcgauugg aaagggauuu cgaaccgggu aauuuaacag uaccagauau aucuuugaug 60 gguucuagca caaucucaug ggguuucucu gcaguagcca cugaugcaga aauccuaaaa 120 gaugacuucc ucaccacccu caaaacugaa ucuuucuuaa acaccccuaa ugauuuugcu 180 gaaauuuggg uuaguuucuu ugauccaaag aagagagaau uugaaacaag aggacccuuu 240 uguguuuuag aaagauugga ggaauucaga cuaagggucu guaucccuug ugccaugcua 300 cuaaucugug ccauuucuug ugaaaaacuu caguuuuucc acucucucuc uguguucuug 360 gaguuguuug aagaccaaau uguuuuuuug agaaaauuuu gaagaaaaug guuaugguag 420 guagauugaa gggagauggg guuaggcaau uaagcagaau ggaguccaau gucagcuuca 480 ccaaccaccu uuugucggaa uuccccuaua gugagucgua uua<s 523 <210> 47 <211> 623 <212> RNA <213> Artificial sequence <220> <223> NA6002 <400> 47 gggaaguguu uucaacgcac caagcaugua augaaugucg ucacuacuca guaaauuguc 60 aacaacaguc cuucccucag aaagggcagc aagaaggaga auacgauugg aaagggauuu 120 cgaaccgggu aauuuaacag uaccagauau aucuuugaug gguucuagca caaucucaug 180 ggguuucu gcaguagcca cugaugcaga aauccuaaaa gaugacuucc ucaccacccu 240 caaaacugaa ucuuucuuaa acaccccuaa ugauuuuugcu gaauuuuggg uauaguuuucuu 300 ugauccaaag aagagagaau uugaacaag aggacccuuu uguguuuuaag aagauugga 360 ggaauucaga cuaagggucu guaucccuug ugccaugcua cuaaucugug ccauuucuug 420 ugaaaaacuu caguuuuucc acucucucuc uguguucuug gaguuguuug aagaccaaau 480 uguuuuuug agaaaauuuu gaaaaaaug guuaugguag guagauugaa gggagauggg 540 guuaggcaau uaagcagaau ggaguccaau gucagcuuca ccaaccaccu uuugucggaa 600 uuccccuaua gugagucgua uua 623 <210> 48 <211> 723 <212> RNA <213> Artificial sequence <220> <223> NA7002 <400> 48 gggguuggau uucuuccuca gacuuuuuac cgacaggaaa cugcccacca caaccuucca 60 caauugcucg uugguuuuca uugucaucuu caacaugaag uccaaguguu uucaacgcac 120 caagcaugua augaaugucg ucacuacuca guaaauuguc aacaacaguc cuucccucag 180 aaagggcagc aagaaggaga auacgauugg aaagggauuu cgaaccgggu aauuuaacag 240 uaccagauau aucuuugaug gguucuagca caaucucaug ggguuucucu gcaguagcca 300 cugaugcaga aauccuaaaa gaugacuucc ucaccacccu caaaacugaa ucuuucuuaa 360 acaccccuaa ugauuuugcu gaaauuuggg uuaguuucuu ugauccaaag aagagagaau 420 uugaaacaag aggacccuuu uguguuuuag aaagauugga ggaauucaga cuaagggucu 480 guaucccuug ugccaugcua cuaaucugug ccauuucuug ugaaaaacuu caguuuuucc 540 acucucucuc uguguucuug gaguuguuug aagaccaaau uguuuuuuug agaaaauuuu 600 gaagaaaaug guuaugguag guagauugaa gggagauggg guuaggcaau uaagcagaau 660 ggaguccaau gucagcuuca ccaaccaccu uuugucggaa uuccccuaua gugagucgua 720 uua 723 <210> 49 <211> 823 <212> RNA <213> Artificial sequence <220> <223> NA8002 <400> 49 gggauccuag gaacuccauc aagaacauau cuugaauguc cuccagcuac aguaacugcu 60 gcugucaacg gacgcauugc uguuccugca uuuccaagga auaguuggau uucuuccuca 120 gacuuuuuac cgacaggaaa cugcccacca caaccuucca caauugcucg uugguuuuca 180 uugucaucuu caacaugaag uccaaguguu uucaacgcac caagcaugua augaaugucg 240 ucacuacuca guaaauuguc aacaacaguc cuucccucag aaagggcagc aagaaggaga 300 auacgauugg aaagggauuu cgaaccgggu aauuuaacag uaccagauau aucuuugaug 360 gguucuagca caaucucaug ggguuucucu gcaguagcca cugaugcaga aauccuaaaa 420 gaugacuucc ucaccacccu caaaacugaa ucuuucuuaa acaccccuaa ugauuuugcu 480 gaaauuuggg uuaguuucuu ugauccaaag aagagagaau uugaaacaag aggacccuuu 540 uguguuuuag aaagauugga ggaauucaga cuaagggucu guaucccuug ugccaugcua 600 cuaaucugug ccauuucuug ugaaaaacuu caguuuuucc acucucucuc uguguucuug 660 gaguuguuug aagaccaaau uguuuuuuug agaaaauuuu gaagaaaaug guuaugguag 720 guagauugaa gggagauggg guuaggcaau uaagcagaau ggaguccaau gucagcuuca 780 ccaaccaccu uuugucggaa uuccccuaua gugagucgua uua 823 <210> 50 <211> 336 <212> RNA <213> Artificial Sequence <220> <223> R1 <400> 50 gggcacaguu uaccguugug agagucuucc agcagguuaa cagagugggu acgguaacg 60 uuuuuuucca gaacgguguc aacggugucg guagagugu uagcguggua accgaugcag 120 auggucag cgucagcagc agccagagcg cacagcagaa ccagcaggu agcuucaug 180 agcucggcuc ccccggaacc gccaccggcc uuucccucua gguaugccac ccgcugggu 240 uucuccagca gugauguuac cauccgacuu accguaguu uugccuuguu uaggcaagcg 300 ugcagguc cgaggucccu auagugaguc guaua 336 <210> 51 <211> 414 <212> RNA <213>人工序列 <220> <223> R2 <400> 51 gggucgcauu ccggguuacc cagcagccaa ccagcgaugu ugcauuuacc cagcugcagc 60 ggagcgauac cuucagacg gcacaguua ccguugugag agcuuccag cagguuaca 120 gagugggua cgguaacguu uuuuuccaga acggugucaa cggugucggu agaguugua 180 gcgugguaac cgaugcagau ggugucagcg ucagcagcag ccagagcgca cagcagaacc 240 agcagguuag cuuucaugag cucggcuccc ccggaaccgc caccggccuu ucccucuagg 300 uaugccacca cgcuggguuu cuccagcagu gauguuacca uccgacuuac cguuaguuuu 360 gccuuguuua ggcaagcgug cagguugucg aggucccuau agugagucgu auua 414 <210> 52 <211> 483 <212> RNA <213> Artificial sequence <220> <223> R3 <400> 52 ggguagcaga uaccguuuuc agaguucggg guuucaacga uguaagacca agaacgaacc 60 ggcagcagcg ggucgcauuc cggguuaccc agcagccaac cagcgauguu gcauuuaccc 120 agcugcagcg gagcgauacc uuucagacgg cacaguuuac cguugugaga gucuuccagc 180 agguuaacag aguggguaac gguaacguuu uuuuccagaa cggugucaac ggugucggua 240 gaguuguuag cgugguaacc gaugcagaug gugucagcgu cagcagcagc cagagcgcac 300 agcagaacca gcagguuagc uuucaugagc ucggcucccc cggaaccgcc accggccuuu 360 cccucuaggu augccaccac gcuggguuuc uccagcagug auguuaccau ccgacuuacc 420 guuaguuuug ccuuguuuag gcaagcgugc agguugucga ggucccuaua gugagucgua 480 uua 483 <210> 53 <211> 605 <212> RNA <213> Artificial sequence <220> <223> R4 <400> 53 ggguaacacc guugguguug ugguucggcc aagaagauuc uuucgggaag auuucgaaac 60 guucgaaaga agaaacagaa gacagcuguu cacgcaguuc uucguagucg augaagucac 120 ccggguagca gauaccguuu ucagaguucg ggguuucaac gauguaagac caagaacgaa 180 ccggcagcag cgggucgcau uccggguuac ccagcagcca accagcgaug uugcauuuac 240 ccagcugcag cggagcgaua ccuuucagac ggcacaguuu accguuguga gagucuucca 300 gcagguuaac agagugggua acgguaacgu uuuuuuccag aacgguguca acggugucgg 360 uagaguuguu agcgugguaa ccgaugcaga uggugucagc gucagcagca gccagagcgc 420 acagcagaac cagcagguua gcuuucauga gcucggcucc cccggaaccg ccaccggccu 480 uucccucuag guaugccacc acgcuggguu ucuccagcag ugauguuacc auccgacuua 540 ccguuaguuu ugccuuguuu aggcaagcgu gcagguuguc gaggucccua uagugagucg 600 uauua 605 <210> 54 <211> 681 <212> RNA <213> Artificial sequence <220> <223> R5 <400> 54 ggguaagaac cuucuuuuuc ggucagccac agcagguuac gguagaaaga agauuuaccu 60 ucgugagagc aagcagcggu aacaccguug guguuguggu ucggccaaga agauucuuuc 120 gggaagauuu cgaaacguuc gaaagaagaa acagaagaca gcuguucacg caguucuucg 180 uagucgauga agucacccgg guagcagaua ccguuuucag aguucggggu uucaacgaug 240 uaagaccaag aacgaaccgg cagcagcggg ucccauccg gguuacccag cagccaacca 300 gcgauguugc auuuacccag cugcagcgga gcgauaccuu ocagacggca caguuaccg 360 uugugagagu ugggacag guaaacagag uggguaacgg 420 gugucaacgg ugucgguag guguuagcg ugguaaccga ugcagauggu guuccguca 480 gcagcagcca gagcgcacag cagaaccagc agguuagcuu ucaugagcuc ggcuccccg 540 gaaccgccac cggccuuucc cucuagguau gccaccacgc uggguuucuc cagcagugau 600 guuaccaucc gacuuaccgu uaguuuugcc uuguuuaggc aagcgugcag guugucgagg 660 uccuauagu gagucguauu a 681 <210> 55 <211> 750 <212> RNA <213>人工序列 <220> <223> R6 <400> 55 ggguggga uaccccacag aaccagaacu ucuuuaccuu uuuuuuaac guagaguuu 60 uucaguuucg gguaagaacc uucuuuuucg gucagccaca gcagguuacg guagaaagaa 120 gauuuaccuu cgugagagca agcagcggua acaccguugg uguugugguu cggccaagaa 180 gauucuuucg ggaagauuuc gaaacguucg aaagaagaaa cagaagacag cuguucacgc 240 aguucuucgu agucgaugaa gucacccggg uagcagauac cguuuucaga guucgggguu 300 ucaacgaugu aagaccaaga acgaaccggc agcagcgggu cgcauuccgg guuacccagc 360 agccaaccag cgauguugca uuuacccagc ugcagcggag cgauaccuuu cagacggcac 420 aguuuaccgu ugugagaguc uuccagcagg uuaacagagu ggguaacggu aacguuuuuu 480 uccagaacgg ugucaacggu gucgguagag uuguuagcgu gguaaccgau gcagauggug 540 ucagcgucag cagcagccag agcgcacagc agaaccagca gguuagcuuu caugagcucg 600 gcucccccgg aaccgccacc ggccuuuccc ucuagguaug ccaccacgcu ggguuucucc 660 agcagugaug uuaccauccg acuuaccguu aguuuugccu uguuuaggca agcgugcagg 720 uugucgaggu cccuauagug agucguauua 750 <210> 56 <211> 839 <212> RNA <213> Artificial sequence <220> <223> R7 <400> 56 gggugaaacg acgguuguag uuagagguaa caacagaaac guaagcguuu ucguucuggu 60 acagguucug cuguucuuua gaguucggcg ggugguggau accccacaga accagaacuu 120 cuuuaccuuu uuuguuaacg uaagaguuuu ucaguuucgg guaagaaccu ucuuuuucgg 180 ucagccacag cagguuacgg uagaaagaag auuuaccuuc gugagagcaa gcagcgguaa 240 caccguuggu guugugguuc ggccaagaag auucuuucgg gaagauuucg aaacguucga 300 aagaagaaac agaagacagc uguucacgca guucuucgua gucgaugaag ucacccgggu 360 agcagauacc guuuucagag uucgggguuu caacgaugua agaccaagaa cgaaccggca 420 gcagcggguc gcauuccggg uuacccagca gccaaccagc gauguugcau uuacccagcu 480 gcagcggagc gauaccuuuc agacggcaca guuuaccguu gugagagucu uccagcaggu 540 uaacagagug gguaacggua acguuuuuuu ccagaacggu gucaacggug ucgguagagu 600 uguuagcgug guaaccgaug cagauggugu cagcgucagc agcagccaga gcgcacagca 660 gaaccagcag guuagcuuuc augagcucgg cucccccgga accgccaccg gccuuucccu 720 cuagguaugc caccacgcug gguuucucca gcagugaugu uaccauccga cuuaccguua 780 guuuugccuu guuuaggcaa gcgugcaggu ugucgagguc ccuauaguga gucguauua 839 <210> 57 <211> 902 <212> RNA <213> Artificial sequence <220> <223> R8 <400> 57 ggguccagua guaguucaua cgaccagccu ggucacgaac uuucggacgu ucagcgauuu 60 ccggggugaa acgacgguug uaguuagagg uaacaacaga aacguaagcg uuuucguucu 120 gguacagguu cugcuguucu uuagaguucg gcggguggug gauaccccac agaaccagaa 180 cuucuuuacc uuuuuuguua acguaagagu uuuucaguuu cggguaagaa ccuucuuuuu 240 cggucagcca cagcagguua cgguagaaag aagauuuacc uucgugagag caagcagcgg 300 uaacaccguu gguguugugg uucggccaag aagauucuuu cgggaagauu ucgaaacguu 360 cgaaagaaga aacagaagac agcuguucac gcaguucuuc guagucgaug aagucacccg 420 gguagcagau accguuuuca gaguucgggg uuucaacgau guaagaccaa gaacgaaccg 480 gcagcagcgg gucgcauucc ggguuaccca gcagccaacc agcgauguug cauuuaccca 540 gcugcagcgg agcgauaccu uucagacggc acaguuuacc guugugagag ucuuccagca 600 gguuaacaga guggguaacg guaacguuuu uuuccagaac ggugucaacg gugucgguag 660 aguuguuagc gugguaaccg augcagaugg ugucagcguc agcagcagcc agagcgcaca 720 gcagaaccag cagguuagcu uucaugagcu cggcuccccc ggaaccgcca ccggccuuuc 780 ccucuaggua ugccaccacg cuggguuucu ccagcaguga uguuaccauc cgacuuaccg 840 uuaguuuugc cuuguuuagg caagcgugca gguugucgag gucccuauag ugagucguau 900 ua 902 <210> 58 <211> 1049 <212> RNA <213> Artificial sequence <220> <223> R9 <400> 58 gggucuggca uuugguguug cauucgugca uagaagcguu agaggugaug auaccagaac 60 cgaaaccacg agacagagcg aaagcguaca ucggagcgau cagguuaccg uuagcuucga 120 agaugauggu gucacccggu uucagcaggg uccaguagua guucauacga ccagccuggu 180 cacgaacuuu cggacguuca gcgauuuccg gggugaaacg acgguuguag uuagagguaa 240 caacagaaac guaagcguuu ucguucuggu acagguucug cuguucuuua gaguucggcg 300 ggugguggau accccacaga accagaacuu cuuuaccuuu uuuguuaacg uaagaguuuu 360 ucaguuucgg guaagaaccu ucuuuuucgg ucagccacag cagguuacgg uagaaagaag 420 auuuaccuuc gugagagcaa gcagcgguaa caccguuggu guugugguuc ggccaagaag 480 auucuuucgg gaagauuucg aaacguucga aagaagaaac agaagacagc uguucacgca 540 guucuucgua gucgaugaag ucacccgggu agcagauacc guuuucagag uucgggguuu 600 caacgaugua agaccaagaa cgaaccggca gcagcggguc gcauuccggg uuacccagca 660 gccaaccagc gauguugcau uuacccagcu gcagcggagc gauaccuuuc agacggcaca 720 guuuaccguu gugagagucu uccagcaggu uaacagagug gguaacggua acguuuuuuu 780 ccagaacggu gucaacggug ucgguagagu uguuagcgug guaaccgaug cagauggugu 840 cagcgucagc agcagccaga gcgcacagca gaaccagcag guuagcuuuc augagcucgg 900 cucccccgga accgccaccg gccuuucccu cuagguaugc caccacgcug gguuucucca 960 gcagugaugu uaccauccga cuuaccguua guuuugccuu guuuaggcaa gcgugcaggu 1020 ugucgagguc ccuauaguga gucguauua 1049 <210> 59 <211> 1170 <212> RNA <213> Artificial sequence <220> <223> R10 <400> 59 gggauguuac gcagaccggu aaccauacgc aguuuagcag aacgaacgua uuucgggcau 60 ucaccgaugg uaaccgggug gauguucugg uacggcagag aagaguugau agcacccagc 120 ggggucuggc auuugguguu gcauucgugc auagaagcgu uagaggugau gauaccagaa 180 ccgaaaccac gagacagagc gaaagcguac aucggagcga ucagguuacc guuagcuucg 240 aagaugaugg ugucacccgg uuucagcagg guccaguagu aguucauacg accagccugg 300 ucacgaacuu ucggacguuc agcgauuucc ggggugaaac gacgguugua guuagaggua 360 acaacagaaa cguaagcguu uucguucugg uacagguucu gcuguucuuu agaguucggc 420 ggguggugga uaccccacag aaccagaacu ucuuuaccuu uuuuguuaac guaagaguuu 480 uucaguuucg gguaagaacc uucuuuuucg gucagccaca gcagguuacg guagaaagaa 540 gauuuaccuu cgugagagca agcagcggua acaccguugg uguugugguu cggccaagaa 600 gauucuuucg ggaagauuuc gaaacguucg aaagaagaaa cagaagacag cuguucacgc 660 aguucuucgu agucgaugaa gucacccggg uagcagauac cguuuucaga guucgggguu 720 ucaacgaugu aagaccaaga acgaaccggc agcagcgggu cgcauuccgg guuacccagc 780 agccaaccag cgauguugca uuuacccagc ugcagcggag cgauaccuuu cagacggcac 840 aguuuaccgu ugugagaguc uuccagcagg uuaacagagu ggguaacggu aacguuuuuu 900 uccagaacgg ugucaacggu gucgguagag uuguuagcgu gguaaccgau gcagauggug 960 ucagcgucag cagcagccag agcgcacagc agaaccagca gguuagcuuu caugagcucg 1020 gcucccccgg aaccgccacc ggccuuuccc ucuagguaug ccaccacgcu ggguuucucc 1080 agcagugaug uuaccauccg acuuaccguu aguuuugccu uguuuaggca agcgugcagg 1140 uugucgaggu cccuauagug agucguauua 1170 <210> 60 <211> 1665 <212> RNA <213> Artificial Sequence <220> <223> R11 <400> 60 ggguagucgu agguaccguu acgaacagau uccaugcauu cguugucgca uuugugguag 60 aauucgaagc aaccguuacc gauuucuuua gcguuguuuu ucagcugaga uuuaacuuuu 120 ucguacaggu uuuuaacguu agagucgugg aaguccaggg uacguucguu uuccagcaga 180 accagcaguu cagcguugua gguccagaug uccaggaaac cgucgucaac uuuuuuguuc 240 agguuuucca uacguuuuuc caguuuguug aauucuuuac caacagcggu gaacuggaug 300 uucauuuuuu cgauaacggu guuaacuuug uuggugauac cguugauagc guucugggua 360 gauuucuggu cagcagcgua accagaaccc uguucguucu ggugguggua accguaccaa 420 ccgucgauca uaccggucca accaccuucg augaaaccag cgauagcacc gaacagacca 480 cgagacugga uagacgggau guuacgcaga ccgguaacca uacgcaguuu agcagaacga 540 acguauuucg ggcauucacc gaugguaacc ggguggaugu ucugguacgg cagagaagag 600 uugauagcac ccagcggggu cuggcauuug guguugcauu cgugcauaga agcguuagag 660 gugaugauac cagaaccgaa accacgagac agagcgaaag cguacaucgg agcgaucagg 720 uuaccguuag cuucgaagau gaugguguca cccgguuuca gcagggucca guaguaguuc 780 auacgaccag ccuggucacg aacuuucgga cguucagcga uuuccggggu gaaacgacgg B40 uuguaguuag agguaacaac agaaacguaa gcguuuucgu ucugguacag guucugcugu 900 ucuuuagagu ucggcgggug guggauaccc cacagaacca gaacuucuuu accuuuuuug 960 It should be noted that there seems to be a misspelling in "840" which is written as "B40" in the original text. This has been retained as it is in the translation for the sake of consistency with the original.uuaacguaag aguuuuucag uuucggguaa gaaccuucuu uuucggucag ccacagcagg 1020 uuacgguaga aagaagauuu accuucguga gagcaagcag cgguaacacc guugguguug 1080 ugguucggcc aagaagauuc uuucgggaag auuucgaaac guucgaaaga agaaacagaa 1140 gacagcuguu cacgcaguuc uucguagucg augaagucac ccggguagca gauaccguuu 1200 ucagaguucg ggguuucaac gauguaagac caagaacgaa ccggcagcag cgggucgcau 1260 uccggguuac ccagcagcca accagcgaug uugcauuuac ccagcugcag cggagcgaua 1320 ccuuucagac ggcacaguuu accguuguga gagucuucca gcagguuaac agagugggua 1380 acgguaacgu uuuuuuccag aacgguguca acggugucgg uagaguuguu agcgugguaa 1440 ccgaugcaga uggugucagc gucagcagca gccagagcgc acagcagaac cagcagguua 1500 gcuuucauga gcucggcucc cccggaaccg ccaccggccu uucccucuag guaugccacc 1560 acgcuggguu ucuccagcag ugauguuacc auccgacuua ccguuaguuu ugccuuguuu 1620 aggcaagcgu gcagguuguc gaggucccua uagugagucg uauua 1665 <210> 61 <211> 475 <212> RNA <213> Artificial sequence <220> <223> NA,NVT,VP1,VP10 <400> 61 gggcgaucuc acuuucacuc ucauuuuaga uucugcucua auccccguag ccugcgcccu 60 acuaacauua ucugccuuau cauaaucagu uaccgguaaa uaggcgcuag ccguauuucu 120 ccgcauaaca guaucauaua uauaaggcac agugaaaugu acugauuuuu guucucccaa 180 guggaacguu uuaguauaag uugaguguga cugacacuca uccguauuag uagaugaucg 240 auuauacucc gcugauauaa ucacuguucc agugugaaac gcauuugaaa caaaaucaaa 300 acguaacucu auuggcccug accaaaaguu auacaaccca guuacauauu ccauuggggu 360 uauuuguccu ucauaguccu gaucauaaaa ucucacaccu gaguccaaaa cuguauuauc 420 gcccuauagu gagucguauu aggauccgau aucuagaugc auucgcgagg uaccg 475 <210> 62 <211> 499 <212> RNA <213> Artificial Sequence <220> <223> NVT IV,NVT 4,VP20 <400> 62 gggcgaucuc acuuucacuc ucauuuuaga uucugcucua auccccguag ccugcgcccu 60 acuaacauua ucugccuuau cauaaucagu uaccgguaaa uaggcgcuag ccguauuucu 120 ccgcauaaca guaucauaua uauaaggcac agugaaaugu acugauuuuu guucucccaa 180 guggaacguu uuaguauaag uugaguguga cugacacuca uccguauuag uagaugaucg 240 auuauacucc gcugauauaa ucacuguucc agugugaaac gcauuugaaa caaaaucaaa 300 acguaacucu auuggcccug accaaaaguu auacaaccca guuacauauu ccauuggggu 360 uauuuguccu ucauaguccu gaucauaaaa ucucacaccu gaguccaaaa cuguauuauc 420 gcccuauagu gagucguauu aggauccgau aucuagaugc auucgcgagg uaccguuguu 480 guuguuguug uuguuguug 499 <210> 63 <211> 106 <212> RNA <213> Artificial sequence <220> <223> NA1001 <400> 63 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgccc 106 <210> 64 <211> 156 <212> RNA <213> Artificial sequence <220> <223> NA1501 <400> 64 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augccc 156 <210> 65 <211> 206 <212> RNA <213> Artificial sequence <220> <223> NA2001 <400> 65 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccc 206 <210> 66 <211> 306 <212> RNA <213> Artificial Sequence <220> <223> NA3001 <400> 66 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauccc 306 <210> 67 <211> 406 <212> RNA <213> Artificial Sequence <220> <223> NA4001 <400> 67 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaccc 406 <210> 68 <211> 506 <212> RNA <213> Artificial Sequence <220> <223> NA5001 <400> 68 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuccc 506 <210> 69 <211> 606 <212> RNA <213> Artificial Sequence <220> <223> NA6001 <400> 69 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuauccgcu cacaauucca cacaacauac gagccggaag 540 cauaaagugu aaagccuggg gugccuaaug agugagcuaa cucacauuaa uugcguugcg 600 cucccc 606 <210> 70 <211> 706 <212> RNA[[ID=SS]] <213> Artificial Sequence <220> <223> NA7001 <400> 70 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuauccgcu cacaauucca cacaacauac gagccggaag 540 cauaaagugu aaagccuggg gugccuaaug agugagcuaa cucacauuaa uugcguugcg 600 cucacugccc gcuuuccagu cgggaaaccu gucgugccag cugcauuaau gaaucggcca 660 acgcgcgggg agaggcgguu ugcguauugg gcgcucuucc gcuccc 706 <210> 71 <211> 806 <212> RNA <213> Artificial Sequence <220> <223> NA8001 <400> 71 gggucgcgcg uuucggugau gacggugaaa accucugaca caugcagcuc ccggagacgg 60 ucacagcuug ucuguaagcg gaugccggga gcagacaagc ccgucagggc gcgucagcgg 120 guguuggcgg gugucggggc uggcuuaacu augcggcauc agagcagauu guacugagag 180 ugcaccauau gcggugugaa auaccgcaca gaugcguaag gagaaaauac cgcaucaggc 240 gccauucgcc auucaggcug cgcaacuguu gggaagggcg aucggugcgg gccucuucgc 300 uauuacgcca gcuggcgaaa gggggaugug cugcaaggcg auuaaguugg guaacgccag 360 gguuuuccca gucacgacgu uguaaaacga cggccagugc caaagaagca ugacggcaag 420 uggacgauua ucuccagagg aucgccggga accgaggacg aguucguaau cauggucaua 480 gcuguuuccu gugugaaauu guuauccgcu cacaauucca cacaacauac gagccggaag 540 cauaaagugu aaagccuggg gugccuaaug agugagcuaa cucacauuaa uugcguugcg 600 cucacugccc gcuuuccagu cgggaaaccu gucgugccag cugcauuaau gaaucggcca 660 acgcgcgggg agaggcgguu ugcguauugg gcgcucuucc gcuuccucgc ucacugacuc 720 gcugcgcucg gucguucggc ugcggcgagc gguaucagcu cacucaaagg cgguaauacg 780 guuauccaca gaaucagggg auaccc 806 <210> 72 <211> 106 <212> RNA <213> Artificial sequence <220> <223> NA1002 <400> 72 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucccc 106 <210> 73 <211> 156 <212> RNA <213> Artificial sequence <220> <223> NA1502 <400> 73 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacccc 156 <210> 74 <211> 206 <212> RNA <213> Artificial sequence <220> <223> NA2002 <400> 74 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauccc 206 <210> 75 <211> �06 <212> RNA <213> Artificial sequence <220> <223> NA3002 <400> 75 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaccc 306 <210> 76 <211> 406 <212> RNA <213> Artificial sequence <220> <223> NA4002 <400> 76 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug cauccc 406 <210> 77 <211> 506 <212> RNA <213> Artificial Sequence <220> <223> NA5002 <400> 77 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucgccc 506 <210> 78 <211> 606 <212> RNA <213> Artificial sequence <220> <223> NA6002 <400> 78 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucguauucuc cuucuugcug cccuuucuga gggaaggacu 540 guuguugaca auuuacugag uagugacgac auucauuaca ugcuuggugc guugaaaaca 600 cuuccc 606 <210> 79 <211> 706 <212> RNA <213> Artificial Sequence <220> <223> NA7002 <400> 79 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauccucca aucuuucua aaacaaaag gguccucuug auuchaauuc aucuucuuu 300 ggaucaaga aacuaaccca aauuucagca aaucauaag ggguuuaa gaagaouca 360 cauuuugagggg ugggaggaa caucuuuuuuuuuuucug caucaguggc uacugcagag 420 aaccccaug agauugugcu agaacccauc aaaaaaaaaaaaaaacccc 480 gguucgaau cccuuucca ucguauucuc cucuugcug cccuuucuga gggaaggacu 540 guguugaca auuuacugag uuuuuuaca ugcuuggc gugaaaaca 600 shake shake shake shake shake shake shake 660 gggcaguuuc cugucgwaa aaagucugag gagaaaucc aacccc 706 <210> 80 <211> 806 <212> RNA <213>人工序列 <220> <223> NA8002 <400> 80 ggggaauucc gacaaaaggu gguuggugaa gcugacauug gacuccauuc ugcuuaauug 60 ccuaacccca ucucccuuca aucuaccuac cauaaccauu uucuucaaaa uuuucucaaa 120 aaaacaauuu ggucuucaaa caacuccaag aacacagaga gagaguggaa aaacugaagu 180 uuuucacaag aaauggcaca gauuaguagc auggcacaag ggauacagac ccuuagucug 240 aauuccucca aucuuucuaa aacacaaaag gguccucuug uuucaaauuc ucucuucuuu 300 ggaucaaaga aacuaaccca aauuucagca aaaucauuag ggguguuuaa gaaagauuca 360 guuuugaggg uggugaggaa gucaucuuuu aggauuucug caucaguggc uacugcagag 420 aaaccccaug agauugugcu agaacccauc aaagauauau cugguacugu uaaauuaccc 480 gguucgaaau cccuuuccaa ucguauucuc cuucuugcug cccuuucuga gggaaggacu 540 guuguugaca auuuacugag uagugacgac auucauuaca ugcuuggugc guugaaaaca 600 cuuggacuuc auguugaaga ugacaaugaa aaccaacgag caauugugga agguuguggu 660 gggcaguuuc cugucgguaa aaagucugag gaagaaaucc aacuauuccu uggaaaugca 720 ggaacagcaa ugcguccguu gacagcagca guuacuguag cuggaggaca uucaagauau 780 guucuugaug gaguuccuag gauccc 806 <210> 81 <211> 319 <212> RNA <213> Artificial Sequence <220> <223> R1 <400> 81 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccc 319 <210> 82 <211> 397 <212> RNA <213> Artificial sequence <220> <223> R2 <400> 82 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgaccc 397 <210> 83 <211> 466 <212> RNA <213> Artificial sequence <220> <223> R3 <400> 83 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuaccc 466 <210> 84 <211> 588 <212> RNA <213> Artificial Sequence <220> <223> R4 <400> 84 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccc 588 <210> 85 <211> 664 <212> RNA <213> Artificial sequence <220> <223> R5 <400> 85 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 accc 664 <210> 86 <211> 733 <212> RNA <213> Artificial sequence <220> <223> R6 <400> 86 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca ccc 733 <210> 87 <211> 822 <212> RNA <213> Artificial sequence <220> <223> R7 <400> 87 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cc 822 <210> 88 <211> 885 <212> RNA <213> Artificial sequence <220> <223> R8 <400> 88 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gaccc 885 <210> 89 <211> 1032 <212> RNA <213> Artificial Sequence <220> <223> R9 <400> 89 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccugcug aaaccgggug 900 acaccaucau cuucgaagcu aacgguaacc ugaucgcucc gauguacgcu uucgcucugu 960 cucgugguuu cgguucuggu aucaucaccu cuaacgcuuc uaugcacgaa ugcaacacca 1020 aaugccagac cc 1032 <210> 90 <211> 1153 <212> RNA <213> Artificial sequence <220> <223> R10 <400> 90 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccugcug aaaccgggug 900 acaccaucau cuucgaagcu aacgguaacc ugaucgcucc gauguacgcu uucgcucugu 960 cucgugguuu cgguucuggu aucaucaccu cuaacgcuuc uaugcacgaa ugcaacacca 1020 aaugccagac cccgcugggu gcuaucaacu cuucucugcc guaccagaac auccacccgg 1080 uuaccaucgg ugaaugcccg aaauacguuc guucugcuaa acugcguaug guuaccgguc 1140 ugcguaacau ccc 1153 <210> 91 <211> 1648 <212> RNA <213> Artificial sequence <220> <223> R11 <400> 91 gggaccucga caaccugcac gcuugccuaa acaaggcaaa acuaacggua agucggaugg 60 uaacaucacu gcuggagaaa cccagcgugg uggcauaccu agagggaaag gccgguggcg 120 guuccggggg agccgagcuc augaaagcua accugcuggu ucugcugugc gcucuggcug 180 cugcugacgc ugacaccauc ugcaucgguu accacgcuaa caacucuacc gacaccguug 240 acaccguucu ggaaaaaaac guuaccguua cccacucugu uaaccugcug gaagacucuc 300 acaacgguaa acugugccgu cugaaaggua ucgcuccgcu gcagcugggu aaaugcaaca 360 ucgcugguug gcugcugggu aacccggaau gcgacccgcu gcugccgguu cguucuuggu 420 cuuacaucgu ugaaaccccg aacucugaaa acgguaucug cuacccgggu gacuucaucg 480 acuacgaaga acugcgugaa cagcugucuu cuguuucuuc uuucgaacgu uucgaaaucu 540 ucccgaaaga aucuucuugg ccgaaccaca acaccaacgg uguuaccgcu gcuugcucuc 600 acgaagguaa aucuucuuuc uaccguaacc ugcuguggcu gaccgaaaaa gaagguucuu 660 acccgaaacu gaaaaacucu uacguuaaca aaaaagguaa agaaguucug guucuguggg 720 guauccacca cccgccgaac ucuaaagaac agcagaaccu guaccagaac gaaaacgcuu 780 acguuucugu uguuaccucu aacuacaacc gucguuucac cccggaaauc gcugaacguc 840 cgaaaguucg ugaccaggcu ggucguauga acuacuacug gacccugcug aaaccgggug 900 acaccaucau cuucgaagcu aacgguaacc ugaucgcucc gauguacgcu uucgcucugu 960 cucgugguuu cgguucuggu aucaucaccu cuaacgcuuc uaugcacgaa ugcaacacca 1020 aaugccagac cccgcugggu gcuaucaacu cuucucugcc guaccagaac auccacccgg 1080 uuaccaucgg ugaaugcccg aaauacguuc guucugcuaa acugcguaug guuaccgguc 1140 ugcguaacau cccgucuauc cagucucgug gucuguucgg ugcuaucgcu gguuucaucg 1200 aaggugguug gaccgguaug aucgacgguu gguacgguua ccaccaccag aacgaacagg 1260 guucugguua cgcugcugac cagaaaucua cccagaacgc uaucaacggu aucaccaaca 1320 aaguuaacac cguuaucgaa aaaaugaaca uccaguucac cgcuguuggu aaagaauuca 1380 acaaacugga aaaacguaug gaaaaccuga acaaaaaagu ugacgacggu uuccuggaca 1440 ucuggaccua caacgcugaa cugcugguuc ugcuggaaaa cgaacguacc cuggacuucc 1500 acgacucuaa cguuaaaaac cuguacgaaa aaguuaaauc ucagcugaaa aacaacgcua 1560 aagaaaucgg uaacgguugc uucgaauucu accacaaaug cgacaacgaa ugcauggaau 1620 cuguucguaa cgguaccuac gacuaccc 1648 <210> 92 <211> 424 <212> RNA <213> Artificial sequence <220> <223> NA,NVT,VP1,VP10 <400> 92 gggcgauaau acaguuuugg acucaggugu gagauuuuau gaucaggacu augaaggaca 60 aauaacccca auggaauaug uaacuggguu guauaacuuu uggucagggc caauagaguu 120 acguuuugau uuuguuucaa augcguuuca cacuggaaca gugauuauau cagcggagua 180 uaaucgauca ucuacuaaua cggaugagug ucagucacac ucaacuuaua cuaaaacguu 240 ccacuuggga gaacaaaaau caguacauuu cacugugccu uauauauaug auacuguuau 300 gcggagaaau acggcuagcg ccuauuuacc gguaacugau uaugauaagg cagauaaugu 360 uaguagggcg caggcuacgg ggauuagagc agaaucuaaa augagaguga aagugagauc 420 gccc 424 <210> 93 <211> 424 <212> RNA <213> Artificial sequence <220> <223> NVT IV, NVT 4, VP20 <400> 93 gggcgauaau acaguuuugg acucaggugu gagauuuuau gaucaggacu augaaggaca 60 aauaacccca auggaauaug uaacuggguu guauaacuuu uggucagggc caauagaguu 120 acguuuugau uuuguuucaa augcguuuca cacuggaaca gugauuauau cagcggagua 180 uaaucgauca ucuacuaaua cggaugagug ucagucacac ucaacuuaua cuaaaacguu 240 ccacuuggga gaacaaaaau caguacauuu cacugugccu uauauauaug auacuguuau 300 gcggagaaau acggcuagcg ccuauuuacc gguaacugau uaugauaagg cagauaaugu 360 uaguagggcg caggcuacgg ggauuagagc agaaucuaaa augagaguga aagugagauc 420 gccc 424 Description of the Drawings

[0080] Figure 1 Shows the in vivo screening results of an RNA-based adjuvant for activating DCs (Example 1).

[0081] Figure 1 Shows that the next adjuvant (abbreviated as NA, alias NVT, VP1 and VP10) increases the expression of stimulatory cofactors (CD40 and CD86) and MHCII in CD11c+ DC cells as measured by mean fluorescence intensity (MFI). 10 μg of NA is superior to 100 μg of Poly(I:C).

[0082] Figure 1 Shows that NA more strongly induces the secretion of IL-6, IL-12, and TNF-α compared to Poly(I:C).

[0083] Figure 1 Is the qRT-PCR result, which shows that NA induces the Th1 cell marker T-bet and Th1 cytokine, interferon gamma (IFN-γ) mRNA without inducing Th2 cytokine (IL-4) and Th17 cytokine (IL-17A). Each data is the average of three independent analyses.

[0084] Figure 2 Shows the in vivo activity of DCs according to the length of hsRNA of the present invention (Example 2).

[0085] Figure 2 A is the in vitro synthesis result of hsRNA series 1 prepared from an artificial sequence (skeleton group 1), which shows hsRNAs with lengths of 140 bases (NA1001), 190 bases (NA1501), 240 bases (NA 2001), 340 bases (NA3001), 440 bases (NA4001), 540 bases (NA5001), 640 bases (NA6001), and 840 bases (NA8001), respectively.

[0086] Figure 2B is the in vitro synthesis result of the hsRNA series 2 derived from the tomato sequence (scaffold group 2), which respectively shows the lengths of 140 bases (NA1002), 190 bases (NA1502), 240 bases (NA2002), 340 bases (NA3002), 440 bases (NA4002), 540 bases (NA5002), 640 bases (NA6002), 740 bases (NA7002), and 840 bases (NA8002).

[0087] Figure 2 C shows the MFI of CD40 and CD86 surface expression after injecting 5 μg of Figure 2 each hsRNA shown in A. Each data is the average of three independent analyses.

[0088] Figure 2 D shows the MFI of CD40 and CD86 surface expression after injecting 5 μg of Figure 2 each hsRNA shown in B. Each data is the average of three independent analyses.

[0089] Figure 3 Shows the innate immune activity index and immune response results of the hsRNA of the present invention (Example 3).

[0090] Figure 3 A is a 1% agarose gel electrophoresis map showing the lengths determined after preparing the hsRNA (R1 to R11, VP10, see Table 2) of the present invention.

[0091] Figure 3 B shows the increase in IFN-β in response to Figure 3 the hsRNA in A and the transient co-transfection of luciferase driven by the interferon-β promoter in HEK 293.

[0092] Figure 3 C shows that intramuscular (i.m.) administration of hsRNA-adjuvanted ovalbumin (OVA: ovalbumin) in mice induces anti-OVA IgG1 (Th2 polarization) and IgG2a (Th1 polarization) in serum. Compared with R3, R5, and R10, NAR7 (R7) of the present invention enhances the Th1 (IgG2a) and Th2 (IgG1) responses.

[0093] Figure 4 Shows the physicochemical properties of NA (NVT) of one of the hsRNAs of the present invention (Example 4).

[0094] Figure 4Panel A shows that the NA length with size homogeneity after RNase T1 treatment is consistently constant in HPLC analysis, indicating that the dsRNA sites are well-preserved.

[0095] Figure 4 Panel B shows that dsRNA is stable for 150 days or longer at 45 °C.

[0096] Figure 4 Panel C shows that the half-life of NA in 100% calf serum is less than 10 minutes.

[0097] Figure 4 Panel D shows that the NA-adjuvanted antigen has a longer shelf life.

[0098] Figure 5 Shows the pattern of innate immune activity indices after subcutaneous injection (s.c.) of hsRNA and dsRNA of the present invention (Example 5).

[0099] Figure 5 Panel A shows that hsRNA (NVT 4-T1) elicits a lower pro-inflammatory cytokine response than dsRNA (NVT 4+T1). This study used NVT 4 (NVT IV, identical to VP20, see Table 2).

[0100] Figure 5 Panel B shows that subcutaneous injection of hsRNA stimulates higher DC activation (CD86 induction) than dsRNA within 24 hours.

[0101] Figure 5 Panel C shows the immune cell profiles in the draining lymph nodes (dLN) at 24 hours and 48 hours. After 48 hours, dsRNA was found to have higher immune activity than hsRNA.

[0102] Figure 5 Panel D shows that dsRNA highly induces TNF-α, IFN-β, and IL-6 compared to hsRNA. This indicates that dsRNA more strongly induces innate immunity, while hsRNA is a substance beneficial for safety.

[0103] Figure 6 Shows that the OVA vaccine adjuvanted with NA (NVT) more strongly induces a Th1-polarized immune response than other adjuvants (Example 6).

[0104] Figure 6 Panel A shows intramuscular injection of adjuvanted or non-adjuvanted vaccines in BalB / c mice on day 0, followed by a boost on day 14. Peripheral blood and splenocytes were harvested on day 21 for flow cytometry and IgG quantification.

[0105] Figure 6B and 6C show that NA (NVT) activates the Th1 response (IgG2c) to a higher level than other adjuvants and increases the number of CD8 T cells and CD4 T cells secreting IFN-γ in the spleen, but does not increase the Th2 immune response (IgG1).

[0106] Figure 7 Comparison of NA (NVT) and NVT+SE (squalene emulsion) (NVTⅡ) in innate and adaptive immune induction (Example 7).

[0107] Figure 7 A is the method for verifying the activity by intramuscular injection of NVT and NVTⅡ.

[0108] Figure 7 B shows that NVTⅡ induces DC activity in the inguinal lymph node (iLN) through NVT and phosphate-buffered saline (PBS) 24 hours after injection.

[0109] Figure 7 C shows that both NVT and NVT II induce neutralizing antibodies in the serum, but NVT II strongly induces 2-fold or more compared to NVT.

[0110] Figure 8 Structure of the hsRNA of the present invention is shown (Example 8). Each ssRNA with overhangs at the 3'-end is located on both sides of the heteropolymeric dsRNA in the middle. Both dsRNA and ssRNA can be synthesized with single-base differences.

[0111] Figure 8 A is a schematic diagram of hsRNA. The heteropolymeric dsRNA in the middle of hsRNA has any specific length and any specific sequence and has perfect complementarity. The two ssRNAs with overhangs at the two 3'-ends of the dsRNA have any specific length but no complementarity capable of serving as a potential TLR7 / 8 ligand. The dsRNA can be an artificial sequence or can be a natural sequence, but does not encode a protein. After treatment with RNaseIII, the dsRNA is cleaved into dsRNA fragments with an average length of 20 to 25bp. However, RNase T1, which acts only on the ssRNA overhangs and not on the dsRNA, completely degrades the ssRNA overhangs. The present invention provides the advantage that dsRNA with a length (>100bp) can be mass-produced in vitro using T7 RNA polymerase in a molar ratio similar to that of two complementary ssRNAs derived from DNA strands.

[0112] In the present invention, the ssRNA overhang is characterized by having a specific length and sequence (a sequence including a large number of GUs, a TLR7 ligand sequence) and being able to act as a specific TLR7 / 8 ligand, rather than a simple overhang. Additionally, the ssRNA can have the function of preventing the degradation of the dsRNA ends.

[0113] Bidirectional transcription from a single template DNA generates two complementary top and bottom ssRNAs with nearly the same molar ratio. During IVT, the two strands then spontaneously anneal to form hsRNA, which has perfect dsRNA in the middle and two ssRNA overhangs at both 3' ends. Importantly, the two ssRNA overhangs are designed not to be complementary to each other to prevent base pairing or strand extension.

[0114] Figure 8 Panel B shows the results of digesting hsRNA with RNase A, RNaseIII, and RNaseIII + RNase T1. RNase A, which can degrade both dsRNA and ssRNA, degrades all the RNA. When treated with RNaseIII, which can cleave dsRNA into fragments of 20 to 25 bp in length, the fragment bands in the range of about 25 bp appear most abundantly. RNase III does not cleave the ssRNA overhangs (51 and 58 bases respectively) of the proposed hsRNA (NVT4). Thus the ssRNA overhangs remain undegraded and they overlap by about 25 bp. When treated with RNaseIII and RNase T1 simultaneously, the ssRNA fragments (51 and 58 bases) are completely degraded and disappear, and only a single band of 25 bp clearly appears.

[0115] Figure 8 Panel C shows the separation of NA (533 - base hsRNAs, dsRNA site 424 bp) in an agarose gel before and after treatment with DNase I and RNase T1, and then a constant result with a specific length appears. After IVT, the residual DNA template is removed by treatment with DNase I, and then only hsRNA remains. When RNase T1 removes the ssRNA overhangs present in the hsRNA, only the dsRNA site remains. If there is one or more nicks in the dsRNA site, RNase T1 will cleave the nick site, resulting in two or more dsRNA fragments. In contrast, only a single band in the gel indicates that there are no nicks in the dsRNA site and the ssRNA overhangs.

[0116] Figure 8 Panel D shows that Poly(I:C), a competitive drug of another invention, has heterogeneity due to the extreme length diversity generated during the preparation stage, which cannot be overcome even after fractionation is completed after preparation (Non - Patent Document 2).

[0117] Figure 8 E shows the basic structure of Poly(I:C) as a competing drug of another invention, which can only be represented as an average length, rather than a specific length. For example, homopolymeric Poly(I) with an average of about 389 bases and Poly(C) with an average of 344 bases are synthesized separately, annealed to be complementary to each other, and then dsRNA is prepared. However, since strand slippage (random-site base pairing) occurs at unpredictable and unspecifiable sites where complementary association occurs, it is inevitable to form multiple nicks with variable gaps at unpredictable positions. In addition, random-site base pairing should produce unspecifiable Poly(I) and poly(C) tail base extensions at two positions, regardless of the 5' or 3' ends, and the complementary strands are associated with them again, resulting in strand extension. Therefore, the length extends to hundreds of kilobases or more (Non-Patent Document 2) (Non-Patent Document 5). Additionally, since PNPase is used in the preparation of Poly(I:C), a phosphate group (P) is always attached to the 5' end, resulting in a low yield (Non-Patent Document 2 and Non-Patent Document 5).

[0118] In contrast, the hsRNA of the present invention can be designed to form dsRNA, which includes ssRNA tail overhangs on both sides, having a definite length only at the two 3' ends and having no complementary sequences between them to prevent strand extension, in contrast to Poly(I:C) and its derivatives.

[0119] Figure 9 Shows the immune response after intranasal (i.n.: intranasal) administration of the whole inactivated influenza vaccine (iPR8) with (iPR8+NVT) or without (iPR8) NA (NVT) as an adjuvant or the NVT of the present invention alone (Example 9).

[0120] Figure 9 A is a schematic diagram of the test method.

[0121] Figure 9 B shows the distribution maps of alveolar macrophages, neutrophils, and natural killer cells in the bronchoalveolar lavage fluid from mice in the NVT, iPR8, and iPR8+NVT treatment groups.

[0122] Figure 9 C shows a significant increase in IL-6, IL-12, and TNF-α after 24 hours.

[0123] Figure 9 D shows an increase in germinal center B (GCB) cells and follicular helper T cells (T FH ) in the mediastinal lymph node (mLN) on the 7th day.

[0124] Figure 9 Panel E shows an increase in serum anti-iPR8 IgG in the iPR8+NVT group after 21 days.

[0125] Figure 9 Panel F shows an increase in IgA in nasal lavage fluid on day 21.

[0126] Figure 10 Effect of the adjuvant of the present invention on the efficacy of a commercial influenza vaccine and antigen dose sparing. ((4IV (Vaxigrip)) via NA (NVT), hsRNA of the present invention (Example 10).

[0127] Figure 10 Panel A is a schematic diagram of the test method.

[0128] Figure 10 Panel B shows that the adjuvant NA (NVT) or NA+SE (NVT II) can save the antigen amount required to induce IgG to about 1 / 5 and about 1 / 25 or less, respectively. Vaxigrip (2018 / 2019 seasonal quadrivalent influenza vaccine 4IIV) was used as a model antigen.

[0129] Figure 10 Panel C shows that a higher amount of total IgG in the NVTI and NVTⅡ groups was maintained up to 17 weeks after vaccination.

[0130] Figure 10 Panel D shows that the hemagglutination inhibition (HAI) titer ≥40 was maintained for at least more than 40 up to week 17, proportional to the amount of neutralizing antibody. HAI≥40 is the correlate of protection (COP), where 50% of the vaccinated individuals are protected. Figure 11 Shows the induction of antibody IgG against a meningococcal vaccine (Menactra, Sanofi) with NVTⅡ of the present invention as an adjuvant (Example 11).

[0131] Figure 11 Panel A is a schematic diagram of the test method.

[0132] Figure 11 Panels B and 11C show an increase in antibodies against total conjugate antigen (Menactra) and serogroup A antigen on days 14 and 21 after priming, respectively.

[0133] Figure 12 Shows the effect of NVTⅡ adjuvant on immunogenicity restoration (Example 12).

[0134] Figure 12 Panel A is a schematic diagram of the test method. Vaxigrip was heat-treated at 37°C for 5 weeks, and used NVTⅡ or PBS as an adjuvant, followed by an intramuscular vaccination schedule for IgG response.

[0135] Figure 12 B shows that NVTII restored the immunogenicity of Vaxigrip as assessed by IgG antibody levels. Note that Vaxigrip alone completely lost its immunogenicity.

[0136] Figure 12 C shows that NVTII-mediated IgG levels were associated with increased HAI titers ≥50 against IAV H1N1 and IBV at week 18.

[0137] Figure 13 The effect of the RNA of the present invention alone on melanoma growth inhibition is shown (Example 13).

[0138] Figure 13 A is a schematic diagram of the test method. Six days after B16F10-OVA melanoma cells were implanted into seven-week-old female C57BL / 6 mice, NA was administered via intratumoral (it) route (NVT) on days 6, 8, and 10, and then tumor mass and mouse survival were measured.

[0139] Figure 1 B shows that the hsRNA of the present invention significantly inhibits melanoma growth.

[0140] ​ C shows that hsRNA prolonged survival.

[0141] ​ The RNA of the present invention alone has been shown to inhibit the growth of colorectal cancer and lung cancer (Example 14).

[0142] ​ The RNA of the present invention alone was shown to inhibit the growth and metastasis of triple-negative breast cancer (TNBC) (Example 15).

[0143] ​ A is a schematic diagram of the testing method. Triple-negative breast cancer (TNBC) 4T1 cells were subcutaneously implanted into the mammary adipose tissue of seven-week-old female C57BL / 6 mice. NA (NVT) RNA was administered intratumorally (IT) once every two days starting on day 8 for a total of nine doses, and tumor mass was measured on the designated days.

[0144] ​ B shows that NA(NVT) inhibited the growth of primary mammary glands by 60%.

[0145] ​ C shows that NVT inhibits metastasis of primary breast cancer to the lung.

[0146] ​ Shows the inhibitory effect of intraperitoneal administration of NVT-adjuvant OVA cancer vaccine on melanoma cell metastasis. (Example 16).

[0147] ​ A is a schematic diagram of the test method.

[0148] ​ B shows the morphology of the spleen and liver on day 18, where the size and metastatic nodules of the liver can be clearly seen.

[0149] ​ C shows the average weights of the spleen and liver on day 18.

[0150] ​ D shows the number of tumor nodules metastasized to the liver. In the separate NA or NA+OVA treatment groups, liver metastasis was significantly inhibited.

[0151] ​ Shows the inhibitory effect of a cancer vaccine comprising hsRNA and OX40 antibody on primary and distant cancer growth. (Example 17).

[0152] ​ A is a schematic diagram of the test method. The melanoma cell line B16F10 OVA was subcutaneously implanted on the left side of 7-week-old female C57BL / 6 mice. On day 6 after implantation, the designated regimen (i.e., IR+NVT+OX40 Ab) was administered 4 times every 2 days (primary cancer). On day 15, the mice received B16F10-Ova or EG7 / OVa cells on the right side (distant cancer) as well, and then the tumor mass was measured on the designated dates.

[0153] ​ B shows that the radioresistant cancer (IR) cells + NVT+OX40 Ab composition significantly inhibits the growth of primary cancer.

[0154] ​ C shows that NVT+OX40 Ab without IR cells completely inhibits the growth of primary melanoma.

[0155] ​ D and 17E show that only NVT+OX40 Ab completely inhibits distant cancers B16FO-OVA identical to the primary tumor and heterologous distant cancer EG7 / OVA.

[0156] ​ Shows the inhibitory effect of hsRNA complexed with PD-1 antibody or OX40 antibody on melanoma growth. (Example 18).

[0157] ​ A is a schematic diagram of the test method.

[0158] ​ Panel B shows that the tumor inhibitory effect of the NA+PD-1 composition is generally similar to that of the NA+OX40 Ab composition.

[0159] ​ Panel C shows that the two compositions have similar inhibitory effects on tumors of distant cancers.

[0160] Best Mode

[0161] Hereinafter, embodiments and examples of the present application with respect to the drawings will be described in detail so that those of ordinary skill in the art to which the present invention pertains can easily practice. However, the present application can be implemented in various forms and is not limited to the embodiments and examples described herein.

[0162] Throughout this specification, when a specific part "includes" a specific component, unless otherwise stated, it means that the part may also include other components, rather than excluding other components.

[0163] The present invention provides hsRNA (140 to 1682 bases), which includes dsRNA (106 - 1648 bp) located in the middle and ssRNA hanging at both 3'-ends on both sides of the dsRNA. Among them, both the dsRNA and the ssRNA can have any specific length and any specific sequence. The dsRNA is defined as having a certain length, which is composed of two completely complementary heteropolymeric chains. The ssRNA tail can serve as a TLR7-like ligand or a TLR8-like ligand, and can have a non-complementary G / U sequence as a TLR7 ligand, preferably a GUU repeat sequence, and can have a length of 10 bases or more, preferably a length of 15 to 80 bases, more preferably a length of 17 to 75 bases.

[0164] The hsRNA of the present invention should be prepared to uniformly have the desired length and high stability.

[0165] Compared with hsRNA, Poly(I:C) and its derivatives cannot be defined as a specific molecular formula weight because there are chain slippage and chain extension between the homopolymeric chains.

[0166] In addition, the present invention provides a method for preparing hsRNA, which includes the following steps:

[0167] 1) Insert a template DNA fragment into any plasmid vector, where T7 promoter sequences are added at both 5' positions;

[0168] 2) Use T7 RNA polymerase to transcribe (IVT) RNA bidirectionally from the template DNA in vitro to generate two heteropolymeric complementary chains with almost the same molar ratio, and then form hsRNA through spontaneous strand-strand annealing during IVT;

[0169] 3) Removal of template DNA; and

[0170] 4) Harvesting and purification of hsRNA.

[0171] If needed, additional sequences can be added to the 5'-end of the promoter sequence. The present invention also provides the following methods for adding other sequences:

[0172] 1) Inserting any TLR7 / 8 ligand-like sequence at the 5'-end of the promoter sequence; and

[0173] 2) Performing bidirectional IVT from template DNA using T7 polymerase, during which two complementary top and bottom ssRNA strands with almost the same molar ratio are generated, and then annealing to form hsRNA.

[0174] The hsRNA of the present invention can serve as a TLR3 ligand, with a base length of 140 to 1682 bases, preferably 200 to 1500 bases, more preferably 300 to 1000 bases, and most preferably 600 to 900 bases, but not limited thereto.

[0175] As the base length increases, the hsRNA tends to exhibit a higher level of dendritic cell activation. However, as the base length increases, the cost of bulk manufacturing also increases.

[0176] Similarly, the base length of the dsRNA part of the present invention can be 106 to 1648 bp, preferably 200 to 1500 bp, more preferably 300 to 1000 bp, more preferably 400 to 900 bases, and most preferably 600 to 900, but not limited thereto.

[0177] The base length of hsRNA or dsRNA can be increased or decreased within a range of 20%.

[0178] The present invention provides a dsRNA, characterized by comprising any one of the base sequences selected from the group consisting of SEQ ID NO: 63 to 93 and serving as a TLR3 ligand. The base length of the dsRNA can be 106, 156, 206, 306, 319, 397, 406, 424, 466, 506, 588, 606, 664, 706, 733, 806, 822, 885, 1032, 1153, or 1648 bp.

[0179] The names and necessary information of the RNAs used in the present invention are shown in Tables 1 and 2 below. As shown in Table 2, in the present invention, parts having the same length and sequence but described by multiple names, such as NA, NVT (abbreviation for Next Adjuvant), VP1, VP10, and VP11. However, VP11 is a dsRNA in which VP10 hsRNA is treated with RNase T1. In addition, NVT IV (NVT 4) and VP20 represent the same RNA.

[0180] The present invention provides homogeneous dsRNA obtained by removing ssRNA from hsRNA.

[0181] The hsRNA or dsRNA may be composed of the base sequences of SEQ ID NO shown in Table 1 below. Table 1 below shows the correspondence between each hsRNA or dsRNA and the sequences described in the examples of the present invention.

[0182]

Table 1

[0183]

[0184]

[0185] The hsRNA or dsRNA can activate immune cells, including dendritic cells, neutrophils, B cells, macrophages, T cells, mast cells, and natural killer cells.

[0186] The present invention provides a pharmaceutical composition comprising hsRNA, wherein the hsRNA includes heteropolymeric dsRNA in the middle and two ssRNAs hanging at the two 3'-ends, and wherein both the dsRNA and the ssRNA can have any specific length and any specific sequence.

[0187] The present invention provides a pharmaceutical composition comprising homogeneous dsRNA obtained by removing ssRNA from hsRNA. The dsRNA includes any one of the base sequences selected from the group consisting of SEQ ID NO: 63 to 93 and their complementary sequences.

[0188] The present invention provides a vaccine adjuvant comprising hsRNA or dsRNA.

[0189] The present invention provides a homogeneous pharmaceutical agent comprising hsRNA or dsRNA.

[0190] The present invention provides a cancer therapeutic agent comprising hsRNA or dsRNA.

[0191] The present invention provides an anticancer agent comprising the hsRNA according to claim 1 or the dsRNA according to claim 7 as an active ingredient.

[0192] The present invention provides a pharmaceutical composition for preventing or treating viral or bacterial infections, cancer or cancer-related diseases, characterized in that it comprises hsRNA or dsRNA, and further comprises an additional adjuvant or immunogen. The "pharmaceutical composition" includes a vaccine composition for preventing or treating viral or bacterial infections such as influenza virus or meningococcus.

[0193] The immunogen can be a trivalent seasonal influenza vaccine, a quadrivalent seasonal influenza vaccine, an inactivated influenza vaccine or a meningococcal (Neisseria meningitidis groups A, C, Y and W-135) vaccine. It can exhibit protective effects against homologous or heterologous subtypes of viruses.

[0194] Compared with a vaccine composition that does not include hsRNA or dsRNA, the vaccine composition for preventing or treating viral or bacterial infections can reduce the dose of immunogen (antigen) required. The necessary dose can be reduced to, for example, 1 / 2 or less, 1 / 3 times or less, 1 / 5 or less, 1 / 10 or less, 1 / 25 or less, 1 / 50 or less or 1 / 100 or less.

[0195] The vaccine composition for preventing or treating viral or bacterial infections can enhance, maintain or restore the antigenicity of the immunogen.

[0196] The vaccine composition for preventing or treating viral or bacterial infections may include an additional adjuvant. The additional adjuvant can be selected from one or more of an oil-in-water emulsion adjuvant, an aluminum salt, Freund's adjuvant and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), and the oil-in-water emulsion adjuvant can be a squalene oil-in-water emulsion.

[0197] The present invention provides a pharmaceutical composition for preventing or treating cancer or cancer-related diseases, characterized in that it comprises hsRNA or dsRNA, and may further comprise an additional adjuvant or immunogen. The "pharmaceutical composition" includes a vaccine composition for preventing or treating cancer.

[0198] The present invention provides a vaccine composition for preventing or treating cancer, which comprises hsRNA or dsRNA and an immunogen.

[0199] The above immunogen can be selected from, but is not limited to, peptides, whole protein antigens, protein antigen domains, inactivated or attenuated live organisms, cell-derived organisms, inactivated cancer tissues, cancer cell cytoplasm, cancer-related antigens or immunodeterminant (epitope) DNA or mRNA encoding proteins.

[0200] Compared with a vaccine composition that does not include hsRNA or dsRNA, the vaccine composition for preventing or treating cancer can reduce the dose of immunogen required. The necessary dose can be reduced to, for example, 1 / 2-fold or less, 1 / 3 or less, 1 / 5 or less, 1 / 10 or less, 1 / 25 or less, 1 / 50 or less, or 1 / 100 or less.

[0201] The vaccine composition for preventing or treating cancer can enhance, maintain, or restore the antigenicity of the immunogen.

[0202] The vaccine composition for preventing or treating cancer can include additional adjuvants. The additional adjuvants can be selected from one or more of an oil-in-water emulsion adjuvant, an aluminum salt, Freund's adjuvant, and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), and the oil-in-water emulsion adjuvant can be a squalene oil-in-water emulsion.

[0203] Cancer includes malignant epithelial tumors, lymphomas, leukemias, blastomas, and sarcomas, but is not limited thereto. More specific examples of cancer include squamous cell carcinoma, myeloma, skin cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, colorectal cancer, glioma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, head and neck cancer, or metastatic cancer thereof, but is not limited thereto.

[0204] The vaccine composition for preventing or treating cancer can exhibit an inhibitory effect on cancer metastasis.

[0205] The present invention provides a pharmaceutical composition for preventing or treating cancer, which includes hsRNA or dsRNA, and an OX40 antibody or a PD-1 antibody.

[0206] The OX40 antibody can be an OX40 agonist antibody, and the PD-1 antibody can be a PD-1 antagonist antibody. The OX40 antibody can be a monoclonal antibody or an antigen-binding fragment thereof, particularly against OX40. The PD-1 antibody can be a monoclonal antibody or an antigen-binding fragment thereof, particularly against PD-1. The OX40 antibody or the PD-1 antibody can be a human antibody, a humanized antibody, a chimeric antibody or a murine antibody, and can comprise a human constant region. The human constant region is selected from IgG1, IgG2, IgG3 and IgG4 constant regions, preferably IgG1 or IgG4 constant region. In one embodiment, the antigen-binding fragment is selected from Fab, Fab'-SH, F(ab')2, scFv and Fv fragments. Examples of specific human PD-1 monoclonal antibodies that can be used as PD-1 antagonist antibodies include pembrolizumab, nivolumab, avelumab, pidilizumab, etc., but are not limited thereto.

[0207] Table 2 lists hsRNAs derived from four different backbone vectors used in the examples of the present invention. Each hsRNA is referred to by each name in the following cases.

[0208] [Table 2]

[0209]

[0210]

[0211] Hereinafter, the present invention will be described in more detail by working examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto. In most (but not all) examples, RNase T1-treated dsRNAs, such as NA (alias NVT, VP1 and VP10), were tested. NA represents dsRNA, otherwise separately referred to as hsRNA.

[0212] [Example 1]

[0213] Activation of in vivo dendritic cells (DCs) by the next adjuvant based on hsRNA

[0214] This example demonstrated the in vivo innate immune activity ( ​ ) by intraperitoneal (i.p.) injection into C57BL / 6 mice with the next adjuvant based on hsRNA (abbreviated as NA).

[0215] One embodiment of NA (also known as NVT or VP10) (Table 2) of the present invention induces the differentiation of dendritic cell markers (CD40, CD86, and MHC-II) to levels equal to or higher than those of 100 μg of Poly(I:C) ( ​ ). NA further induces type I interferon-β (IFN-β), which is associated with the skewing of naïve CD4 T cells to Th1 CD4 T cells and the secretion of IL-6 and IL-12 ( ​ ). In addition, NA induces T-bet (Th1 cell marker) and IFN-γ, but does not induce Th2 cytokines (IL-4) and Th17 cytokines (IL-17A) ( ​ ). Compared with the Poly(I:C) positive control group, NA strongly activates DC.

[0216] [Example 2]

[0217] In vivo DC activation depends on the length of hsRNA rather than the sequence

[0218] hsRNA was prepared from two independent backbones into different lengths, where the dsRNA region varied from 106 to 806 bp, but the ssRNA overhangs at both ends in this Example 2 were constant at 17 bases ( ​ A and ​ B), and spleen DC activation was tested intraperitoneally in mice.

[0219] The level of DC activation increased with the increase in hsRNA length, as evaluated by the induction of CD40 and CD86, showing high activity at 400 to 900 bases ( ​ C and ​ D).

[0220] All hsRNAs showed better activity than the positive control group Poly(I:C).

[0221] Since all ssRNA overhangs are non-complementary, with 17 bases at each end, a total of 34 bases, the difference in DC activation from hsRNA must be generated with the increase in dsRNA length. Therefore, dsRNAs in the range of approximately 406 to 806 bp showed high activity.

[0222] When reexamining 540 hsRNA (506 bp dsRNA) that showed high activity at various concentrations (0.5 to 20 μg), the maximum activity was observed at 10 μg. Compared with in vivo Poly(I:C), its activity was 10 to 20 times higher.

[0223] [Example 3]

[0224] The antigen - specific antibody response depends on the length of hsRNA

[0225] More hsRNAs with a wide range (353 to 1682 bases) are derived from the third backbone vector, which is different from the first two vectors shown in Example 2( ​ A). After transiently co - transfecting these RNAs with an IFN - β promoter - driven vector into HEK 293 cells, a surrogate reporter of innate immune activation was determined( ​ B). The IFN - β promoter activity increased proportionally with the length of hsRNA and dsRNA, reaching a maximum in the range of 406bp to 806bp. Notably, this activity was independent of the specific RNA sequence, which was consistent with Example 2.

[0226] Some of them (R3, R5, R7, and R10) were complexed with the model antigen OVA, and their ability to induce anti - OVA IgG in mice was tested in vivo.

[0227] Consistent with the above, high levels of anti - OVA IgG1 and Th1 - mediated anti - OVA IgG2 were observed near R5 (dsRNA length of 664bp)( ​ C).

[0228] In summary, hsRNAs (140 to 1682 bases) and dsRNAs (106 to 1648bp) showed excellent effects on innate and adaptive responses.

[0229] Therefore, the present invention claims hsRNAs (112 to 2018 bases) that are shortened or extended within a 20% range from each of the above - tested hsRNAs (140 to 1682bp). In addition, in addition to each dsRNA selected from SEQ ID NO: 63 to 93, the present invention also includes dsRNAs (85 to 1977bp) that are shortened or extended within a 20% range.

[0230] [Example 4]

[0231] Physicochemical property analysis of hsRNA and dsRNA

[0232] HPLC analysis and agarose gel electrophoresis demonstrated the highly uniform - sized hsRNAs and dsRNAs of the present invention( ​ A), which was in contrast to Poly(I:C)( ​ C).

[0233] NA was stable for 150 days or longer at 45°C( ​B), but has a very short half-life in media supplemented with RNase A-rich serum, which reflects a hit and run as a possible targeting mode ( ​ C). Antigens complexed with NA can be stored for longer periods at room temperature ( ​ D).

[0234] [Example 5]

[0235] Common and distinct immunoassays between hsRNA and dsRNA

[0236] After subcutaneous (s.c.) treatment of (i) RNase T1-free hsRNA (NVT4-T1) and (ii) T1-treated dsRNA (NVT 4+T1) into BalB / C mice, innate immune parameters were compared in vivo using NVT 4 (VP20) at 24 and 48 hours as follows.

[0237] Although both hsRNA and dsRNA strongly induced DC activation, dsRNA increased more immune cells (especially B cells and neutrophils) compared to hsRNA, which was further associated with a proportional increase in TNF-α, IFN-β, and IL-6 ( ​ ). Collectively, dsRNA more strongly induced innate immunity. hsRNA elicited neutrophils, IL-6, and TNF-α at lower levels, which may be beneficial for safety concerns.

[0238] [Example 6]

[0239] Comparison of Th1-polarized immune responses between NA and other adjuvants

[0240] OVA was complexed with NA, Alum, squalene (SE), IFA, and Poly(I:C) and then intramuscularly injected ( ​ A) to compare immune response induction.

[0241] Although the Th2 response (IgG1) was similar, the Th1 response (IgG2c) induced by OVA with the NA adjuvant was higher than the other responses ( ​ B). Additionally, more IFN-γ-secreting splenic CD8 and CD4 T cells rapidly proliferated in response to the OVA vaccine formulation with the NA adjuvant ( ​ C). When administered intranasally, NA induced stronger mucosal IgA and mucosal IgG in body fluids (tears, nasal, and draining fluids) and serum compared to other adjuvants (data not shown).

[0242] [Example 7]

[0243] Innate and antigen-specific antibody responses between hsRNA and hsRNA plus additional adjuvants (i) Two formulations of NVT; dsRNA alone (NA), (ii) NVT II; Preparation of NVT(NA)+SE (squalene emulsion)( ​ A). After intramuscular (i.m.) injection into BalB / C mice, DC activity was measured by flow cytometry. DC stimulation in the inguinal lymph nodes (iLN) in NVT II was higher than that in NVT( ​ B).

[0244] In addition, model vaccines were prepared, including (i) OVA alone, (ii) OVA+NVT, and (iii) OVA+NVT II, and anti-OVA IgG1 (Th2 response) and IgG2a (Th1 response) were tested in the same mice.

[0245] Therefore, the antibody response induced by OVA+NVT was about 100-fold stronger than that of OVA alone. OVA+NVTII induced an antibody response 2-fold or more stronger than OVA+NVT( ​ C).

[0246] [Example 8]

[0247] Homogeneity of the structure and length of hsRNA

[0248] The hsRNA of the present invention has a dsRNA in the middle and two ssRNA overhangs at both 3' ends. Both the dsRNA and ssRNA have non-homopolymeric sequences and can be defined to have precise lengths, thus preventing homopolymer-mediated strand slippage and extension. The dsRNA region has an arbitrary length and an arbitrary sequence and is fully complementary. The sequence can be an artificial or natural sequence but does not encode a protein. The two ssRNA overhangs at the two 3' ends of the dsRNA have any specific length and any specific sequence and serve as potential TLR7 / 8-like ligands. Since the two ssRNA tails are not complementary, no base pairing occurs between the sequences within and between the two overhangs, thus inherently preventing strand extension but corresponding to potential TLR7 / 8 ligands ​A). RNase T1 can remove the two ssRNA regions at both ends, thereby generating blunt-ended dsRNA. The presence of the intermediate dsRNA was verified by RNaseIII treatment. The dsRNA was short-cut by RNaseIII into fragments with an average length of 20 to 25 bp. Longer treatment of dsRNA resulted in fragments with a length of 12 bp or shorter. The results are consistent with the mode of action of various RNases. After RNaseIII treatment of hsRNA with 51-base and 58-base ssRNA overhangs at the two 3'-ends, 20- to 25-bp dsRNA fragments and ssRNA fragments of approximately 50 bases were identified as expected. After RNaseIII and RNase T1 treatment of the same hsRNA, the ssRNA was degraded, and only 20- to 25-bp dsRNA remained. When treated with RNase A, all the RNA was degraded ( ​ B).

[0249] In addition, when hsRNA was treated with RNase T1, the complementarity and stability of the dsRNA site were verified from the single band on the agarose gel before and after treatment ( ​ C).

[0250] Any specific hsRNA has extremely high length homogeneity, down to a single base unit ( ​ A, 3A, and 4A).

[0251] As another specific example, any specific single strand (≥7 bases) containing the polyuridine (U) base can be added to both sides of the dsRNA. In addition, the single strand can be designed to have stability by adding a specific sequence, thereby forming an internal dsRNA such as a hairpin structure or a palindromic loop structure or acting as a TLR7-like ligand.

[0252] In contrast, Poly(I:C) is a competitive drug with extreme length diversity and cannot be prepared with a constant length. Instead, it is an artificial homopolymeric dsRNA with non-designable incisions inside and no ssRNA tail at the designated site such as the 3'-end of the dsRNA. The length diversity of Poly(I:C) is caused by in-chain slippage and chain extension. As a result, multiple incisions are formed at non-designable sites in the dsRNA site, and these incisions are cut into various lengths by RNase T1 ( ​ D). In addition, due to the uncontrolled chain extension in Poly(I:C), the number of upper and lower strands forming the dsRNA cannot be specified. Therefore, Poly(I:C) is easily digested by RNase T1. The extreme heterogeneity of Poly(I:C) was published in one of the original articles (Non-Patent Document 2) ( ​ Left side of D).

[0253] RNaseIII digestion of Poly(I:C)-L (long species) produces small species similar to Poly(I:C)-S (short species). ​ D). (Non-patent Document 2). By complementary binding of short PolyC (P100 with an average of 100 bases) and long PolyI (P400 with an average of 400 bases) to reduce chain extension, this extreme heterogeneity was partially improved. Nevertheless, the lengths vary in a very different range from 100 bp to 100 kbp (Non-patent Document 5).

[0254] The RNA of the present invention is essentially different from the existing inventions in the following aspects.

[0255] Compared with Poly(I:C), the RNA of the present invention: 1) has a high degree of length homogeneity, is a non-homopolymer of the sequence, and has no gap in the dsRNA due to complete complementarity, and 2) can serve as a TLR3 ligand and a TLR7-like ligand.

[0256] Compared with siRNA, the RNA of the present invention: 1) is essentially different from synthetic siRNA during preparation, 2) does not have the function of targeting and inhibiting specific genes possessed by siRNA, 3) since the RNA of the present invention has a length of 45 bp or more, it can serve as a TLR3 ligand that exerts sufficient activity, while the range of siRNA is as short as 21 to 25 bp, so siRNA cannot stimulate TLR3 (Non-patent Document 1), and 4) the ssRNA of the present invention has a specific sequence of a specific length or longer (for example, 17 bases or more) and acts as a TLR7 / 8-like ligand, while a very short 3'-overhang at one end of siRNA corresponds to a simple residual sequence without a function. Compared with dsRNA, hsRNA with an ssRNA overhang may exhibit a low toxicity index in vivo (Example 5).

[0257] [Example 9]

[0258] Immune response to dsRNA-adjuvanted whole inactivated influenza vaccine (iPR8) by intranasal administration

[0259] When NA (NVT) was administered to BalB / c mice together with iPR8, the numbers of mDC and rDC cells in the mediastinal lymph nodes increased after 24 hours, and DC (CD80 and CD86) were activated, and then decreased to the baseline level after 96 hours (data not shown). In addition, the total cells, alveolar macrophages, neutrophils, and natural killer cells in the bronchoalveolar lavage fluid increased. ​B), which is proportional to the increase in IL-6, IL-12, and TNF-α, where NVT alone also showed a positive effect on the elevation of those pro-inflammatory cytokines( ​ C). This increase in innate immune markers was accompanied by an increase in germinal center B (GCB) cells, follicular helper T cells (T FH ) as well as IgG and IgA in serum and nasal lavage fluid( ​ D and ​ E).

[0260] In the absence of antigen, the NA adjuvant alone also has a protective effect against a lethal live virus infection. After multiple intranasal administrations of NA to mice, the lifespan was extended by an average of 4 days or more, and the mortality associated with virus infection (viremia) was reduced by approximately 20% (data not shown). Again, the NA adjuvant alone of the present invention without antigen exhibits a strong immune enhancing effect.

[0261] [Example 10]

[0262] Effect of hsRNA on dose sparing of commercial influenza split vaccine

[0263] The adjuvant effect in a commercial vaccine antigen was tested. The vaccines tested included (i) quadrivalent inactivated influenza vaccine (4IIV, Vaxigrip, Sanofi Pasteur), (ii) 4IIV + NVT (NA), and (iii) 4IIV + NVTⅡ (NA + SE). IgG levels and hemagglutination inhibition (HAI) titers were measured within 18 weeks after priming after a 2-week interval between priming and boosting by intramuscular (i.m.) injection in female Balb / c mice( ​ A). As a result, compared to the non-adjuvanted antigen alone, the antigen dose could be reduced to approximately 1 / 5 level and 1 / 25 in the NVT- and NVTⅡ-adjuvant groups, respectively( ​ B). This effect persisted for at least 17 weeks after injection( ​ C). The NA adjuvant of the present invention can spare antigen dose; the amount of vaccine antigen required to ensure the desired immunity can be greatly reduced.

[0264] The increase in binding antibodies was closely associated with an increase in HAI titers of 40 or higher, which is the correlation of protection (COP) against influenza virus and may be able to provide sufficient protective immunity for 50% of the challenged or infected individuals. Although both adjuvants significantly increased the HAI titers, NVTⅡ particularly caused a 3- to 5-fold increase in HAI titers against influenza A virus (IAV subtype H1N1) and influenza B virus (IBV)( ​ D).

[0265] [Example 11]

[0266] Dose-sparing effect of hsRNA on commercial meningitis vaccines

[0267] When administering the Menactra vaccine to mice, NA was used as an adjuvant ( ​ A).

[0268] The Menactra vaccine is a CRM197 conjugate vaccine against Neisseria meningitidis groups (A, C, Y, and W-135) that are meningococcal. Specifically, the vaccine antigen is covalently linked to the diphtheria toxin-derived carrier protein CRM197. In the present invention, Menactra with NVTⅡ strongly induced Menactra-specific antibodies 5-fold or more ( ​ B and 11C).

[0269] [Example 12]

[0270] Immunogenicity restoration effect of NVTⅡ

[0271] The influenza vaccine (2018 / 2019 QIV Vaxigrip) was treated at 37 °C for five weeks to eliminate immunogenicity and then used with NVT II as an adjuvant and intramuscularly (i.m.) injected. IgG ELISA and HAI assays were performed at the designated time points until 18 weeks after initiation ( ​ A).

[0272] As a result, the binding antibodies increased significantly and the immunogenicity seemed to be restored ( ​ B). In addition, there was at least a 10-fold antigen-sparing effect. The increase in binding antibodies should be related to the increase in neutralizing antibodies because the HAI titers against H1N1 and IBV increased to 40 or more at the 18th week ( ​ C).

[0273] In another experiment, when Vaxigrip was pre-mixed with NVTII and placed at 37 °C for 5 weeks, the antigenicity was completely maintained and the HAI titer increased to 40 or more (data not shown).

[0274] [Example 13]

[0275] Antitumor effect of hsRNA on melanoma growth in vivo

[0276] After subcutaneously implanting B16F10-OVA melanoma cells into 7-week-old female C57BL / 6 mice, NA was injected intratumorally (i.t.) or intramuscularly three times at two-day intervals starting from the sixth day ( ​A). Tumor growth in mice administered with NA was retarded, and the mice survived longer compared to the control group ( ​ B and 13C).

[0277] [Example 14]

[0278] Inhibitory effect of hsRNA on the growth of colorectal cancer and lung cancer in vivo

[0279] After subcutaneous implantation of CT26 colorectal cancer and LL / 2 lung cancer in seven-week-old female C57BL / 6 mice, NVT was administered subcutaneously, intratumorally (i.t.) or intramuscularly (i.m.) alone every two days for a total of eight times. NA treatment retarded the growth of colon cancer and lung cancer in the mice ( ​ ).

[0280] [Example 15]

[0281] Inhibitory effect of single hsRNA on the growth and lung metastasis of triple-negative breast cancer (TNBC) in vivo

[0282] After subcutaneous implantation of 4T1 triple-negative breast cancer (TNBC) cell line into the mammary fat tissue of seven-week-old female C57BL / 6 mice, single NA was injected into the tumor every two days starting from the eighth day for a total of nine times ( ​ A). As a result, the primary breast cancer was delayed by 60% ( ​ B), and this growth inhibition was consistent with a significant reduction in distant metastasis to the lungs ( ​ C).

[0283] [Example 16]

[0284] Anti-hepatic metastasis of single hsRNA or hsRNA complexed with TAA vaccine in vivo

[0285] On day zero, the vaccine was pre-administered intraperitoneally (i.p) (i) PBS, (ii) OVA, (iii) NA, (iv) OVA + NA. On day three, B16F10-OVA melanoma was implanted into the spleen of seven-week-old female C57BL / 6 mice. In the following three days, the same vaccine was injected intraperitoneally, and then the degree of metastasis from the spleen to the liver was studied ( ​ A).

[0286] Metastasis in the NA- or OVA + NA treatment group was lower than that in the untreated or OVA-alone treatment group ( ​ B and 16C). Liver metastasis on day eighteen was reduced by approximately 50% in single NA and by 90% or more in the OVA + NA group ( ​D). As for the survival rate, starting from the fourteenth day, all mice in all other groups died within eighteen days, while the mice in the OVA+NA treatment group started to die from the twenty-fourth day and all died within twenty-eight days.

[0287] [Example 17]

[0288] Inhibitory effect of the OX40 Ab complex with hsRNA adjuvant on primary and distant melanoma growth

[0289] After subcutaneous implantation of B16F10-OVA melanoma cells on the left side of seven-week-old female C57BL / 6 mice, the cancer vaccines (i) PBS, (ii) PD-1Ab, (iii) IR cells (irradiated cancer cells), and (iv) IR cells + NA + OX40 Ab were administered intratumorally (i.t.) into the primary cancer tissue every two days for a total of four times (on days 6, 8, 10, and 12) after tumor implantation. ​ A). The growth of the cancer was measured until the twenty-second day.

[0290] As a result, although the growth of the primary cancer was inhibited by approximately 50% and 20% in the PD-1 antibody and IR cells respectively, 90% growth inhibition was observed in the IR+NVT+OX40 Ab group. ​ B).

[0291] In addition, NA+OX40Ab without IR cells inhibited the growth of the primary cancer almost to the same extent as above, and the TAA in the vaccine composition was not required. ​ C).

[0292] On the fifteenth day, two types of allogeneic cancers (B16F10 / OVA) and T cell lymphoma (EG7 / OVA) were implanted at a second site on the right side opposite to the first implantation. The growth was measured for the next sixteen days without further administration of the therapeutic vaccine.

[0293] As a result, almost no growth of the cancer at the second site occurred in all mice injected with NA+OX40 Ab. In contrast, all mice in the NA+isotype Ab group died before the eleventh day. Therefore, the size of the tumor mass is not shown in the figure. ​ D and 17E).

[0294] In summary, regardless of IR, NA+OX40 Ab inhibits the growth of 90% or more of primary melanoma. In addition, distant cancers implanted at other sites are also captured by NA+OX40 Ab.

[0295] [Example 18]

[0296] Inhibitory effects of PD-1 antibody or OX40 complex with hsRNA adjuvant on primary and distant melanoma After subcutaneous implantation of B16F10-OVA melanoma cells into the left and right sides of 7-week-old female C57BL / 6 mice, the cancer vaccines (i) PBS, (ii) NA, (iii) NA + OX40 antibody (OX40 Ab) (clone OX-86, rat IgG1, InvivoGen), and (iv) NA + PD-1 antibody (PD-1 Ab) (clone RMP1-14, rat IgG2a / λ, InvivoGen) were administered intratumorally (i.t.) into the left primary cancer tissues once every two days after tumor implantation, for a total of four times (on days 6, 8, 10, and 12). The growth of cancer in the left (inoculated tumor) and right (distant tumor) was measured until the 20th day ( ​ A).

[0297] Therefore, the inhibitory effect of NA + PD-1 Ab on primary cancer was similar to or better than that of NA + OX40 Ab. The same results were observed in the non-vaccinated distant tumors ( ​ B and 18C).

Claims

1. An RNA of a heterogeneous structure (hsRNA) or an RNase T1 cleavage product of the hsRNA, wherein the hsRNA comprises heteropolymeric double-stranded RNA (dsRNA) in the middle and is flanked by single-stranded RNA (ssRNA) overhangs at both 3'-ends, and wherein the hsRNA comprises dsRNA formed as a result of complementary binding of the base sequence of SEQ ID NO: 30 and the base sequence of SEQ ID NO: 61 and two ssRNA overhangs.

2. The hsRNA or the RNase T1 cleavage product of the hsRNA according to claim 1, characterized in that: The dsRNA serves as a TLR3 ligand.

3. The hsRNA or the RNase T1 cleavage product of the hsRNA according to claim 1, characterized in that: The ssRNA serves as a TLR7-like ligand or a TLR8-like ligand.

4. A pharmaceutical composition comprising the hsRNA according to claim 1 or an RNase T1 cleavage product of the hsRNA.

5. The pharmaceutical composition according to claim 4, wherein, The pharmaceutical composition is for treating viral or bacterial infections, cancer or cancer-related diseases, and further comprises an additional adjuvant or immunogen, wherein the viral infection is an influenza virus infection, the bacterial infection is Neisseria meningitidis, and the cancer is skin cancer, breast cancer, lung cancer or colorectal cancer.

6. The pharmaceutical composition according to claim 4, wherein: The pharmaceutical composition comprises an OX40 antibody or a PD-1 antibody.

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